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Over the course of the study, the average score on the MMSE questionnaire significantly increased, from 8.7 to 10.6, out of a maximum of 30, in the group receiving probiotics, but not in the control group, where it fell from 8.5 to 8.0.

Even though this increase is moderate, and all patients remained severely cognitively impaired, the results are the first to show that probiotics can improve human cognition.

Walter Lukiw, Professor of Neurology, Neuroscience and Ophthalmology and Bollinger Professor of Alzheimer's disease at Louisiana State University, who reviewed the study but was not involved in the research, said: "This early study is interesting and important because it indicates that probiotics can in principle improve human cognition.

"This is in line with some of our recent studies which indicate that the gastrointestinal tract microbiome in Alzheimer's is significantly altered in composition when compared to age-matched controls."

Dr Rosa Sancho, Head of Research at Alzheimer's Research UK, said: "The brain is often viewed as being separate from the rest of the body but scientists are understanding more about how changes in the body can impact upon the brain too. This new study raises interesting questions about the link between the gut and the brain, and their association with Alzheimer's disease.

"The improvements in memory and thinking seen in people with Alzheimer's disease in this study will need to be repeated in much larger studies before we can understand the real benefits of probiotics for the brain. We don't fully understand how changes in the gut could be affecting the brain, and Alzheimer's Research UK is funding research in this area to improve our understanding of this link."

The study was published in the journal Frontiers in Aging Neuroscience.

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Probiotics can help thinking and memory for people with Alzheimer's disease

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Over the course of the study, the average score on the MMSE questionnaire significantly increased, from 8.7 to 10.6, out of a maximum of 30, in the group receiving probiotics, but not in the control group, where it fell from 8.5 to 8.0.

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Bacteriocin: An approach to Bio preservation

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Bacteriocin: An approach to Bio preservation

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Probiotics: Uses and side effects of Lactobacillus

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Probiotics: Uses and side effects of Lactobacillus

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If you are an avid yogurt fan, then you must have surely come across the name Lactobacillus bulgaricus by now. After all, this probiotic component is becoming very popular nowadays in all sorts of health-boosting products from around the world.

But how much do you actually know about Lactobacillus bulgaricus and its contribution to human wellness? Whether you consider yourself a nutritional expert or just a casual 'dabbler' in the vastly alternative ways of improving your overall wellbeing, it is important to know exactly what probiotics are and how different strains can help you achieve a better lifestyle over time.

Probiotics and Lactobacillus bulgaricus

Did you know that your gut can contain up to 100 billion probiotics at any given time? Or that almost 80% of a person's immunity can be found at the intestinal level? If not, then you probably only found out about probiotics from yogurt advertisements or by skimming through various health articles online.

In simple terms, probiotics represent 'good' bacteria that develop a symbiotic relationship with your gastrointestinal system. This means that such colonies thrive within the mouth, stomach, small intestines, and colon, where they help with food decomposition, nutrient absorption, and regular bowel movements.

So, while these beneficial microorganisms only take up a minute part of your substance intake for their own survival, their overall contribution to health and digestion are often indispensable to an individual's welfare.

Probiotics also display good antimicrobial properties, which come in handy in the case of both airborne and ingested pathogens (fungi, yeasts, microbes, etc.). By producing lactic acid and small doses of hydrogen peroxide, probiotics are able to protect your organism against infections and illnesses before they even start developing.

Not to mention that this 'pro-life' bacteria (as the etymology translates) has been more than once connected with meliorated mental stability , fewer episodes of eczemas and skin breakouts, as well as improved respiratory functions.

Probiotics are generally classified according to their family, genus, and species. Their presence within the human body or in certain products is measured in CFUs or colony-forming units, which designate the number of live bacteria colonies found in that specific environment at that particular moment.

The most well-known names of probiotics are Lactobacillus , Bifidobacterium , Streptococcus , , etc., each genus having its own trademark properties in terms of general human health.

It pertains to Lactobacillus genus and Delbrueckii species respectively, having two distinct potential sources. One is of animal origin (mammals) and the other one is of a vegetable nature, as extracted from the Bulgarian snowdrop flower ( Galanthus nivalis ).

The geographical specificity of Lactobacillus bulgaricus is obviously reflected into its name because it was first isolated by the Bulgarian doctor Stamen Grigorov at the beginning of the 20 th century. Just a few years afterwards, pioneering immunologist Élie Metchnikoff drew our attention to the significance of a balanced intestinal microbiota (or microflora) to local and general wellness alike.

The good and the bad of Lactobacillus bulgaricus

One of the main advantages brought about by Lactobacillus bulgaricus is its influence on the gastrointestinal system all throughout its length. As some points out, Lactobacillus bulgaricus supplementation can be employed to boost oral health in the sense of improved dental protection, reduced instances of periodontal diseases, and suppression of Streptococcus bacteria (which are known to cause both tooth decay and throat infections).

In addition, this probiotic augments digestive patterns by meliorating food decomposition and ensuring proper nutrient absorption within the gut. This is done through complex fermentation processes and accelerated lactic acid production.

Another gastrointestinal issue many people suffer from nowadays is that of irregular bowel movements. From episodes of diarrhea and constipation to IBS (irritable bowel syndrome), IBD (inflammatory bowel disorder), and Crohn's disease, having a 'temperamental' gut can really affect your quality of life.

Luckily enough, specialists have already tapped into the very feasible employment of probiotics such as Lactobacillus bulgaricus for these gastrointestinal imbalances and have concluded that a regularized internal microbiota does influence correct digestive patterns and eliminates the indigestion-like symptoms of these illnesses on a long-term basis.

With regards to immune shielding, Lactobacillus bulgaricus has been seen to display a series of antiseptic and antimicrobial properties. Its major inhibitory capacities were spotted in the case of Salmonella and Escherichia coli pathogens, which are otherwise notorious for their damaging action inside the gastrointestinal tract.

Furthermore, some studies indicate that this probiotic can be used to combat the onset and subsequent effects of enterocolitis in children, which is caused by the harmful pathogen called Enterobacter sakazakii .

It turns out that probiotics of the Lactobacillus bulgaricus type can also positively impact cardiovascular health. A series of experiments from 2010 suggests that probiotics have the potential to reduce cholesterol readings and arterial stiffness, as well as constitute a safer alternative to traditional options (since medication often comes packed with side effects or interactions).

Take, for example, this 2013 that used various probiotic strains ( Lactobacillus bulgaricus included) to demonstrate how 'friendly' microorganisms of this sort act as brain modulators for emotions and sensations.

The most common side effects induced by this probiotic include bloating, flatulence, nausea, and disruptions in your bowel motility patterns (namely diarrhea or constipation). In fact, these are normal reactions caused by your organism trying to adapt to the new bacterial numbers inside the intestinal microflora, meaning that your abdominal discomfort should subside within a couple of days to a week tops.

Although this probiotic is non-motile (it does not normally 'travel' within the body), such health exceptions make it easier for the microorganisms to become displaced and cause a number of unwanted complications.

Why Lactobacillus bulgaricus?

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Lactobacillus Bulgaricus | Benefits & Side Effects - ProbioticsAmerica.com

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If you are an avid yogurt fan, then you must have surely come across the name Lactobacillus bulgaricus by now. After all, this probiotic component is becoming very popular nowadays in all sorts of health-boosting products from around the world. But how much do you actually know about Lactobacillus bulgaricus and its contribution to human wellness?

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Brewery: Fast souring with Lactobacillus

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Brewery: Fast souring with Lactobacillus

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Fast Souring with Lactobacillus - Best Practices, Sensory, & Science

Hello Sour Brewers!

There are a number of brewing situations and recipe designs where both craft and home brewers would like to establish a nice clean baseline level of lactic acidity in their beers within a short period of time. In order to accomplish this, brewers turn to the Lactobacillus family of lactic acid producing bacteria because these bacteria are both easy to work with and readily available both as pure cultures and naturally on many base malts. In order to accomplish this goal, several methods have been developed to allow Lactobacillus to multiply and ferment lactic acid in the wort before it undergoes a standard fermentation by Saccharomyces (brewer's yeast) or Brettanomyces. There are several process based issues that arise when attempting to give Lactobacillus sole reign over your wort, and for many brewers the results of such attempts have been lackluster or tainted by off-flavors. Despite this, many brewers have successfully used these methods to produce well-made sour beers with clean levels of lactic acidity.

The goal of the first part of this article is to review the various methods used to produce fast souring in beer and to discuss the best practices to follow when employing these methods. The second component of this article will be a discussion of how to analytically taste your beers in order to detect the various common off-flavors that can arise from fast-souring processes. Finally, a third element of this discussion with center on the science behind these fermentations, with a focus on the various unwanted bacteria that can compete with Lactobacillus. We will review why these bacteria produce the off-flavors that they do and why the best-practices reviewed in part one help to ensure that these bacteria are kept at bay.

Part 1 - Fast Souring Methods & Best Practices

The Sour Mash

Sour mashing is a process in which a typical mash is conducted, but is then allowed to remain warm for up to several days after an inoculation with Lactobacillus. This inoculation can come from either a pure culture of Lactobacillus, a Lactobacillus starter, or from the addition of a small portion of un-mashed base grain, which will naturally have one or more wild stains of Lactobacillus on its husks. After a certain level of souring has been achieved, the mash is sparged as normal and followed by standard boiling and cool-side fermentation practices. The benefit of this method is that Lactobacillus and any other bacteria present in the mash will be killed by the boil and only sanitized beer will move forward into fermentation equipment. The downside of this process is that, of the three methods we will discuss, this one is the most difficult to control and the one most likely to yield off-flavors. The following practices will help you gain the best results from a sour mash, but as we will discuss in Part 3, are not a guarantee of success.

Best Practices when Sour Mashing

Conduct either a multistep or single infusion mash as dictated by your equipment or recipe.

After your saccharification rest is complete, reduce the pH of the wort to 4.5 by addition of food grade lactic or phosphoric acid. This can also be achieved by the addition of acidulated malt (added after sacc rest). Acidulated malt drops the pH on average 0.06 to 0.1 per % of the total grist. This equates to about 7-10% of the total grist for any mash that has a pH of 5.2

Verify your pH using a meter or test strips and adjust as needed before continuing on.

Make sure to include a mash-out step which raises the temperature above 168° F for at least 10 minutes to ensure enzyme inactivation and a locking-in of your fermentable to non-fermentable sugar ratio.

Cool the mash to below 120° F, then add Lactobacillus via a pure culture or by adding a small portion of un-mashed base grain. In any of these processes, using a pure culture is likely to yield more predictable results and reduce your risk of contamination by off-flavor producing bacteria.

Remove oxygen from the mash tun by flushing the vessel with a layer of carbon dioxide or nitrogen. The mash tun should be sealed as best as possible to keep oxygen out during the sour mash. These inert gases can be bubbled through the mash itself or added via a port above the liquid layer as they are both heavier than oxygen and will displace it. If your equipment allows, a slow trickle of inert gas can be used to continually ensure an anaerobic environment during sour mashing.

If possible, the temperature of the sour mash should be maintained between 112° to 120° F throughout the sour mashing process.

Check the progress of your Lactobacillus fermentation via a pH meter every 12 to 24 hours. a pH around 3.6 generally equates with a tartness appropriate for styles like Berliner Weisse or Gose. A pH around 3.3 will be strongly sour, on par with many young lambic style beers. Keep in mind that at a pH below 3.4 to 3.5, some strains of Saccharomyces will have difficulty producing a healthy fermentation. In these cases it may be best to use a mixed or 100% Brettanomyces fermentation, as this family of yeast can remain healthy at pH levels down to and possibly below 3.0

After your wort reaches the desired pH, raise the mash to 170° to 180° and sparge as usual.

While a starting pH of 4.5 will generally eliminate the risk of bacteria that can cause food poisoning, use caution when tasting the results of a sour mash. Only a low pH and the presence of alcohol from yeast fermentation can ensure that your fermented product is safe to drink.

Sour Kettling

Very similar to the sour mashing process, sour kettling allows you to produce a sour wort that can then be boiled for sanitation before moving it into your fermentation equipment. Overall though, souring in the boil kettle allows for greater versatility and as a process can often be more easily regulated. This method also allows you to choose whether to kill the Lactobacillus before fermentation by yeast or to allow its continued activity in your aging beer. Sour kettling begins with the use of a completely standard mashing and sparging procedure. If you want to kill Lactobacillus before the rest of your fermentation, then you will perform the steps of sour kettling before the boil. Oppositely, if you want Lactobacillus to survive into your final product, then the boil is conducted before the sour kettling procedure.

Best Practices when Souring in the Kettle

If choosing to boil at the end, the first step will be to adjust the pH of your wort to 4.5 using either food grade lactic or phosphoric acid.

If choosing to boil before souring, make sure that your recipe either does not include hop additions or that those additions produce less than 10 IBUs in the finished beer. Too much alpha acid, or hop oils in general, will inhibit Lactobacillus and prevent souring via its fermentation. After boiling, adjust the pH to 4.5 as previously discussed.

Cool the wort to below 120° F before pitching your culture of Lactobacillus or adding a sack of base malt to the kettle. As in sour mashing, pure cultures are more predictable and less likely to yield contamination by off-flavor producing bacteria.

Flush the kettle with CO₂ or Nitrogen gas and seal it up as best as possible. Consider scrubbing oxygen from the wort by bubbling inert gas through the wort for several minutes. As before, if your equipment allows, these gases can be slowly trickled into the sealed kettle throughout the sour fermentation to maintain an anaerobic environment.

Maintain the wort at a temperature between 112° and 120° F, if possible.

Just like sour mashing, check the pH of your wort every 12 to 24 hours.

When the desired pH has been reached, either conduct a normal boil procedure, or if you have already boiled and wish to maintain Lactobacillus in your final beer, transfer to fermentation vessels using sanitary practices.

Sour Worting in a Fermentation Vessel

This third process assumes that you wish for Lactobacillus to survive and remain active in your fermenting and finished beer. On both a homebrew and commercial scale, it is best to use either pure laboratory-sourced cultures or cultures collected and maintained from starters which have proven to be free of off-flavors and aromas. Doing so, in combination with the other best practices discussed, will virtually ensure that your beer remains free of unwanted microorganisms and their off-flavors. For commercial brewers who cannot heat their fermenters , this process is identical to sour-kettling after a standard boil. When developing recipes for sour-wort beers, the alpha-acid level from hop additions must be restrained to less than 10 IBUs to prevent the inhibition of Lactobacillus.

Best Practices When Souring in a Fermentation Vessel

Mash and Boil your wort as normal. During the boil, adjust the pH to 4.5 using food grade lactic or phosphoric acid.

Cool the wort to below 120° F and transfer into a sanitized fermentation vessel. Ensure proper closed & sanitary transfer practices. I have run 110° F wort into glass carboys without incident, but I do not recommend that this be done routinely. It is far better to use a stainless vessel (for homebrewers, either 5 gallon Cornelius or half-barrel Sankey kegs work well) for these hot wort transfer and fermentation procedures.

Purge both the wort and the fermentation vessel with carbon dioxide using either the line out on a corny keg or by using a sintered stone in other fermentation vessels.

Pitch a pure culture of Lactobacillus or a Lactobacillus starter that is free of off-aromas.

Maintain a fermentation temperature between 112° to 120° F. This can be achieved on the homebrew scale by the use of a ferm-wrap or other fermentation heating element and an insulated chamber such as a small chest freezer or a large insulated picnic cooler.

As in previous methods, check the pH every 12 to 24 hours.

When the desired pH is reached, cool the wort to the proper yeast pitching and fermentation temperature (between 45° to 55° F for lager Saccharomyces or between 65° to 75° F for ale Saccharomyces or Brettanomyces)

Oxygenation at this point is very beneficial for the yeast and will not harm further Lactobacillus flavor development

Pitch one or more Saccharomyces or Brettanomyces strains and proceed through a standard alcoholic fermentation from this point forward.

The ultimate goal of these three processes is the same: Produce a beer with a pleasant level of lactic acidity that is free of off-flavors in a short period of time. Lets now look at how to smell and taste the beer to detect the most common off-flavors that can arise from these fast souring methods.

Part 2 - Detecting Common Off-Flavors and Aromas and Understanding Their Cause.

As a brewer myself, it is always my goal to produce the best beer possible and it is from this perspective that I will launch into a discussion about off-flavors and aromas. That being said, I also know that, for many brewers who are first starting to experiment with sour beer, it can sometimes be difficult to distinguish between "bad funk" and "good funk" in these beers.

In general, Lactobacillus in isolation (the goal of these methods) should produce a far simpler flavor profile than produced by either traditional brewers yeast or the common sour beer yeast Brettanomyces. The primary product of Lactobacillus is lactic acid, which is a simple organic acid with a soft pleasant sourness. Common foods that feature this acid include Greek yogurt, kefir, sauerkraut, and sourdough bread. For the candy fans, the addition of lactic acid is what differentiates "Sour Patch Kids Extreme" from the classic "Sour Patch Kids".

The fermentation of Lactobacillus will also give its beers a fairly subtle yet classic mustiness. This can smell a bit like a damp basement or the forest after a rainstorm. Another aroma commonly associated with Lactobacillus is a floury smell associated with sourdough bread and sourdough starters. These are light aromas easily overpowered by other additions to the beer such as hops or fruit. Additionally, the aroma and flavor compounds produced by brewer's yeast strains or Brettanomyces will easily mask these characteristics. Despite this, I believe it is the presence of these non-acidic characteristics that differentiate beers actually fermented with Lactobacillus from those that have simply received a large addition of food-grade lactic acid. In my opinion, this latter process creates an inferior product which tastes somehow artificial.

Lactobacillus beers, when smelled at room temperatures, should not "stink". This is especially true after a primary fermentation by Saccharomyces or Brettanomyces. Immediately after the Lactobacillus fermentation, it is common for the wort to smell:

Musty or Floury (This would be considered "good funk")

Sugary, Syrupy, & Sweet (Because Lactobacillus produces only a minute level of attenuation in comparison to yeast)

A little Buttery (Occasionally, because certain strains of Lactobacillus can produce diacetyl in their fermentation)

Lactobacillus species are divided into two groups, homofermentative and heterofermentative. Homofermentative species will only produce lactic acid during their fermentations. Alternatively, heterofermentative species will primarily produce lactic acid but can also produce ethanol, carbon dioxide, and acetic acid during their fermentations. I bring this up because, in very low levels, this acetic acid will add flavor complexity and should not be considered an off-flavor when using these species / strains. On the other hand, if there is a strong and distinct aroma or flavor of vinegar, this would generally be considered undesirable and is likely due to contamination of the wort by Acetobacter or other unwanted aerobic microbes.

If using a clean ale strain such as White Labs WLP001, Wyeast 1056, or Safale US-05 to ferment your wort after souring with Lactobacillus, you would expect the fully attenuated beer to have only a slightly musty and fruity aroma, as well as a clean and simple sour flavor.

Lets now take a look at the aromas and flavors that we do not want to detect in either the wort or finished beer:

Parmesan Cheese, Rancid Milk, or Stinky Feet (Isovaleric Acid)

Vomit or Bile (Butyric Acid)

Fecal, Manure, Poopy Diaper (Aromatic Indoles)

A strong presence of Vinegar (Acetic Acid)

Bandaid, Liquid Smoke, Medicinal (Phenolic Compounds)

When I am brewing a batch of sour beer using a Lactobacillus-first fermentation, the presence of any of these characteristics in my beer would lead to the batch being dumped. This is because I am not willing to spend time aging a beer with off-flavors in the hope that they will eventually become better. While personally I'm not willing to age such batches, three of the chemical compounds that are listed above can be either reduced or turned into flavor positive compounds by the yeast Brettanomyces. These compounds include isovaleric acid, butyric acid, and certain phenolic compounds. If you detect these compounds in very low levels, aging with Brettanomyces can eliminate them over time. However, if any of these compounds are intensely strong, it is best to dump the batch and review your processes. A good rule of thumb would be that if a flavor or aroma is off-putting enough that you would not drink several pints of the beer, then save your efforts and dump it.

When tasting your beer for off-flavors, it is sometimes very useful to drink another commercial example of the style that is known to be free of defects. This side-by-side comparison can really help to highlight flavors in your beer that you may not have otherwise noticed. Additionally, because strong lactic acid can be overwhelming to some taster's palates, it can be useful to adjust your palate to intense acidity before analyzing the flavors in your beer. This can be done with a favorite bottle of strongly sour beer, a glass of water blended with lemon-juice concentrate, or by eating a few sour candies like Sour Patch Kids or Warheads.

A few more general tips for tasting and smelling your beers include:

Pour the beer into a tulip glass or other similar aroma-promoting glass.

Smell and taste the beer when it is at cellar temperature and at room temperature. Tasting or smelling the beer at cold temperatures will mask or reduce many flavor and aroma compounds.

When smelling the beer, don't be afraid to get your nose deep in the glass and take several good whiffs in a row. If your nose needs a reset, smell the skin of your clean dry arm for several seconds.

When tasting the beer, pull some air into your mouth with the sip. This helps to aerate the sample and bring out both flavor and aroma compounds.

Avoid tasting the beer after smoking, chewing tobacco, or eating any strongly spiced or otherwise potently flavored foods.

If you are hunting for subtleties, don't be afraid to ask for help. Genetically we all have a very wide range of sensitivities to different chemical compounds. A group of good analytic tasters can be the most useful tool in a brewer's arsenal.

At this point we have covered both the best-practices to be used while fast-souring with Lactobacillus and how to analyze our beers for both positive and negative results. For the practical brewer, you now have all the tools you need to go forth and brew delicious fast-soured beers. However, for the inquisitive brewers, I will now wrap up this article with a discussion of the science behind these practices.

Part 3 - Fast Souring Science

Before jumping into a discussion of the organisms responsible for producing off-flavors in these fermentations, I need to mention a caveat to this information: Sour mashing, sour kettling, and Lactobacillus-only fermentations are relatively recent products of the American craft brewing community. There have been no detailed scientific studies directly looking at the organisms that are in play when these processes fail. However we can make fairly educated guesses as to the identity of the organisms involved based upon the chemicals and off-flavors that they leave behind. Many of these organisms have been studied in other food products or in other processes related to industrial brewing (practically all well-funded beer science has been performed in the arena of industrial lager brewing). Deducing the identity of the organisms that commonly compete with Lactobacillus in these fermentations allows us to re-engineer the process to reduce or eliminate the risk of these unwanted microbes.

Butyric Acid Bacteria

The easiest of these organisms to identify are members of the Clostridium family. These bacteria, especially Clostridium butyricum, are well known in the brewing industry as producers of butyric acid due to their propensity to infect sugar syrups in bulk storage containers. Clostridium are obligate anaerobic (they cannot survive in the presence of oxygen), endospore forming, gram positive, rod shaped bacteria which metabolize simple sugars to produce butyric acid. Butyric acid both smells and tastes like vomit, bile, and rancid milk or cheeses. This chemical is present in low doses in goat and sheep milk, butter, and parmesan cheese. It is also present in human vomit.

Clostridium, and other less common butyric acid bacteria, while requiring the same oxygen free environment that Lactobacillus prefers, cannot tolerate the same pH and temperature ranges as Lactobacillus. While Lactobacillus can comfortably thrive between 115° and 120° F, Clostridium dies at temperature above 112° F. Additionally, while Lactobacillus purposely acidifies its environment in order to outcompete acid-intolerant bacteria, Clostridium prefers an optimum pH of 6.3. As the pH of its environment drops near 5.0, Clostridium loses its ability to produce butyric acid, and at a pH of 4.7 or below, these bacteria become completely inactivated. These tolerances produce our recommendations to always pre-acidify the wort to a pH of 4.5 and maintain a temperature above 112° F when possible.

Indole Producing Bacteria

The presence of fecal aromas or flavors in a sour beer are an indication of an infection by bacteria known as coliform bacteria. These bacteria include families such as Citrobacter, Klebsiella, Enterobacter, and Escherichia and are found throughout nature. One species, E. coli, is a common bacteria found in the large intestines of most humans. This species also garnishes media attention from time to time due to its occasional ability to become pathogenic and cause a toxic diarrhea. Many species of these bacteria metabolize various amino acids to produce the chemical indole, a chemical which smells strongly of feces. These bacteria are rod shaped facultative anaerobes (preferring oxygen free environments but able to survive in its presence), which do not form spores. Certain groups of these bacteria can survive at temperatures above those preferred by Lactobacillus. Luckily for use, these bacteria, while able to survive a low pH environment, do not reproduce or metabolize well as the pH approaches 4.4. Therefore, our recommendation of lowering the pH to 4.5 before fermentation will help to prevent these bacteria from producing off-flavors in our fermentations.

Bacterial Production of Isovaleric Acid

Unfortunately, this narrative becomes more complicated when we look at bacterial sources for isovaleric acid. One definitive source of this off-flavor is metabolism of the amino acid leucine by a bacterium called Bacillus subtilis. This bacterium is present on human skin and is responsible, in part, for the production of foot odor. This rod shaped bacteria is known for its ability to form a protective endosperm which allows it to survive a wide range of environmental conditions. Luckily, this bacteria is an obligate aerobe (requiring oxygen to survive) and therefore can be prevented from affecting our sour fermentations when we maintain an oxygen free environment.

A second, and less controllable source of this off-flavor may be an interaction between a bacteria commonly found in yogurt cultures, Streptococcus thermophilus, and the very Lactobacillus which we are utilizing in our souring fermentations. Lactobacillus, in isolation, does not possess the metabolic enzymes needed to convert leucine into isovaleric acid. However, it seems that when S. thermophilus is present, it can potentially supply Lactobacillus with the chemical intermediates needed to perform this metabolism. The bad news regarding this interaction is the fact that S. thermophilus thrives in all of the same environmental conditions as Lactobacillus. Therefore the only process control that can prevent its influence in our sour fermentations is the use of good sanitation practices.

This potential interaction is one of the reasons why I strongly prefer to use only pure cultures of Lactobacillus or starters which have already proven to be free of off-flavors. The temperatures of mashing will generally sanitize our wort and the process of boiling definitely will. (2015 Update: Always perform at least a 15 minute boil before beginning a fast souring process) As long as from these points forward only known cultures are introduced, there will be little to no risk of contamination. In fact, while I have used Lactobacillus first fermentations dozens of times, I have never experienced any of these common off-flavors in my beers.

Another reason to consider only using known cultures is the fact that bacteria can and do frequently swap genetic material, allowing certain strains to develop properties that they previously lacked. Therefore, the only way we can ever really know what is fermenting in our beers is to ensure that we put only the bacteria that we want into them. With that said, following the best practices outlined here will almost certainly yield positive results in your breweries.

Hopefully, this review has been interesting, informative, and helpful to brewers looking to develop a quick and simple acidity in their beers. These processes alone work very well when producing styles like Berliner Weisse and Gose, and can be very useful for establishing a baseline acidity in other more complex styles as well. As always, please email me at matt@sourbeerblog.com with any questions.

Have fun and brew sour!

Cheers!
Matt "Dr. Lambic" Miller
November 2015 Update: For those looking to expand their fast souring knowledge with techniques such as Lactobacillus pitch rate, starters, and recipe design, check out our new follow up article: Lactobacillus 2.0 - Advanced Techniques for Fast Souring Beer!
References:

Ghoddusi, Hamid B., Richard E. Sherburn, and Olusimbo O. Aboaba. "Growth Limiting PH, Water Activity, and Temperature for Neurotoxigenic Strains of Clostridium Butyricum." ISRN Microbiology 2013 (2013): 1-6. Web.

Goodwin, Jay, and Scott Moskowitz. " The Sour Hour / Episode 4." The Sour Hour. The Brewing Network. Concord, CA, 20 Nov. 2014. Radio. (Features guest brewer Khris Johnson of Green Bench Brewing in St. Petersburg, Florida. Khris is the first brewer that I've heard make the direct connection between certain sour mash/sour kettle off-flavors and isovaleric acid)

Helinck, S., D. Le Bars, D. Moreau, and M. Yvon. "Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids." Applied and Environmental Microbiology 70.7 (2004): 3855-861. Web.

Klocker, Alb. Fermentation Organisms; a Laboratory Handbook. London: Longmans, Green, 1903. Print.

Sakamoto, Kanta, and Wil N. Konings. "Beer Spoilage Bacteria and Hop Resistance." International Journal of Food Microbiology 89.2-3 (2003): 105-24. Web.

Vriesekoop, Frank, Moritz Krahl, Barry Hucker, and Garry Menz. "125Anniversary Review: Bacteria in Brewing: The Good, the Bad and the Ugly." Journal of the Institute of Brewing 118.4 (2012): 335-45. Web.

Zhang, Chunhui, Hua Yang, Fangxiao Yang, and Yujiu Ma. "Current Progress on Butyric Acid Production by Fermentation." Current Microbiology 59.6 (2009): 656-63. Web.

Zigová, J., and E. Šturdík. "Advances in Biotechnological Production of Butyric Acid." Journal of Industrial Microbiology and Biotechnology 24.3 (2000): 153-60. Web.

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Fast Souring with Lactobacillus - Best Practices, Sensory, & Science Hello Sour Brewers! There are a number of brewing situations and recipe designs where both craft and home brewers would like to establish a nice clean baseline level of lactic acidity in their beers within a short period of time.

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Health and nutritional benefit from lactic acid bacteria

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Health and nutritional benefit from lactic acid bacteria

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Research 6: Qualitative characteristics of Lactobacillus bulgaricus and methods of its quantitative determination

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Research 5: Effects of two strains of Lactobacillus Bulgaricus on hemopoiesis after irradiation

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Research 5: Effects of two strains of Lactobacillus Bulgaricus on hemopoiesis after irradiation

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Research 4: RAY-PROTECTIVE EFFECT OF THE PROBIOTIC BIOMILK

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Research 4: RAY-PROTECTIVE EFFECT OF THE PROBIOTIC BIOMILK

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Research 3: STUDY OF THE ANTIOXIDANT ACTIVITY OF THE PROBIOTICBIOMILK UNDER EXPERIMENTAL CONDITIONS 

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Research 3: STUDY OF THE ANTIOXIDANT ACTIVITY OF THE PROBIOTICBIOMILK UNDER EXPERIMENTAL CONDITIONS 

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Research 2: RADIOPROTECTIVE EFFECT OF THE PROBIOTIC BIOMILK CONTAININGLACTOBACILLUS BULGARICUS 

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Research 2: RADIOPROTECTIVE EFFECT OF THE PROBIOTIC BIOMILK CONTAININGLACTOBACILLUS BULGARICUS 

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Research 1: Analyses of Synbiosis between Lactobacillus Bulgaricus and (Oat & Onion) Prebiotic

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Research 1: Analyses of Synbiosis between Lactobacillus Bulgaricus and (Oat & Onion) Prebiotic

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Formation of yogurt from milk aided by lactic acid fermentation at molecular level

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Formation of yogurt from milk aided by lactic acid fermentation at molecular level

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An animation reveals the microscopic and molecular changes during yogurt making. It was created in collaboration with Dr. Harold McGee, author of On Food and Cooking: The Science and Lore of the Kitchen. For more information, please go to l2xy2.com

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Home - Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365

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Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365

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Powerpoint slides about Lactobacillus bulgaricus in yogurt

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Powerpoint slides about Lactobacillus bulgaricus in yogurt

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which in turn produces formic acid and carbon dioxide ... formic acid CO2. proteins. peptides. Lactobacillus thermophilus. Lactobacillus bulgaricus ... - A free PowerPoint PPT presentation (displayed as a Flash slide show) on PowerShow.com - id: 102cee-OGZhN

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Facts video about Lactobacillus bulgaricus

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Facts video about Lactobacillus bulgaricus

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Lactobacillus bulgaricus GLB44 Top  #24 Facts

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Uploaded by Rishika Janaki on 2016-01-28.

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An almost perfect digestive booster, immune protector, and mental health aid – what is not to love about Lactobacillus bulgaricus? In all truthfulness, its few drawbacks do not seem to overpower its more obvious advantages in all areas of human health. Quite literally, this probiotic has the potential to ensure head-to-toe wellness when used accordingly.

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Unfortunately, there are also a few things which make Lactobacillus bulgaricus a less desired addition to one’s overall wellbeing.

The most common side effects induced by this probiotic include bloating, flatulence, nausea, and disruptions in your bowel motility patterns (namely diarrhea or constipation). In fact, these are normal reactions caused by your organism trying to adapt to the new bacterial numbers inside the intestinal microflora, meaning that your abdominal discomfort should subside within a couple of days to a week tops.

Nevertheless, some people have been shown to go through more serious cases of allergies when trying out Lactobacillus bulgaricus in higher concentrations of bacteria. This is why it is important to check by your doctor or personal physician before taking any sort of Lactobacillus bulgaricus supplementation, since your medical history – both past and current – plays a huge part in how you process this probiotic to begin with.

For instance, people diagnosed with Short-Bowel Syndrome (SBS) or with a severely weakened immune system cannot benefit from Lactobacillus bulgaricus in any shape or form because they run the risk of developing sepsis (blood poisoning).

Although this probiotic is non-motile (it does not normally ‘travel’ within the body), such health exceptions make it easier for the microorganisms to become displaced and cause a number of unwanted complications.

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Lactobacillus bulgaricus comes through as a highly resistant probiotic, one that can withstand higher temperatures and unfriendly environments (bile, gastric acid, etc.). As a consequence, it can form long-lasting colonies within the gut microflora and hence preserve the integrity of the intestinal lining in optimal conditions.

One of the main advantages brought about by Lactobacillus bulgaricus is its influence on the gastrointestinal system all throughout its length. As some research points out, Lactobacillus bulgaricus supplementation can be employed to boost oral health in the sense of improved dental protection, reduced instances of periodontal diseases, and suppression of Streptococcus bacteria (which are known to cause both tooth decay and throat infections).

In addition, this probiotic augments digestive patterns by meliorating food decomposition and ensuring proper nutrient absorption within the gut. This is done through complex fermentation processes and accelerated lactic acid production.

Consequently, Lactobacillus bulgaricus can also help those struggling with lactose intolerance by facilitating the digestion of dairy and doing away with the most obvious symptoms of this condition (visceral cramping, gas, discomfort, etc.).

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Milk is fermented to make yogurt.   To set up yogurt production milk is heated, this helps kill other microorganisms that would compete with the starter culture.  Lactobacillus bulgaricus interacts with  Streptococcus thermophilus to form a symbiotic starter culture to ferment milk. By creating the starter culture it increases the production of lactic acid in the fermentation process. Fermentation of the milk usually happens when the milk is around 45 degrees Celsius.  Streptococcusthermophilus starts to grow first dropping the milks pH from 6.6 to 5.0, and produces carbon dioxide and lactic acid. These products stimulate Lactobacillus bulgaricus to grow and further drop the pH to 4.2. The sugar found in milk is lactose, when the starter culture is added to milk it breaks the lactose into glucose.  After the sugar glucose is formed it then is fermented into lactic acid by the microorganisms.  Acetaldehyde is also produced, this chemical compound creates the tart and sour taste of plain yogurt. The lactic acid decreases the pH of the milk and causes the casein, protein found in milk, molecules to denature and stick together.  The milk then curdles to produce yogurt. To enhance the flavor a wide variety of fruits are mixed in or are found at the bottom of the finished product.

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ANTIBACTERIAL ACTIVITY OF LACTOBACILLUS SAKE ISOLATED FROM MEAT

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ANTIBACTERIAL ACTIVITY OF LACTOBACILLUS SAKE ISOLATED FROM MEAT

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Federal Centre for Meat Research, Institute of Microbiology, Toxicology, and Histology, Federal Republic of Germany.

ABSTRACT

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.

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Antibacterial activity of Lactobacillus sake isolated from meat.

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Federal Centre for Meat Research, Institute of Microbiology, Toxicology, and Histology, Federal Republic of Germany. A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide.

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I found an interesting article regarding our chosen bacterium. Take a look! Many things I have learnt through this article.

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History and Origin of Lactobacillus bulgaricus

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History and Origin of Lactobacillus bulgaricus

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The Legend

The legend of the first dicovery of Lactobacillus Bulgaricus is placed in the country of Bulgaria. This is where it's name comes from. The story is of a shepherd in the Balkan Mountain. When the shepherd was up in the mountains he ran out of vessels for his goat's milk so, he made a bag from fresh lamb's hide to hold the extra milk. When he woke up the next morning he found that the lambs hides no longer held milk but yogurt, which was caused by Lactobacillus Bulgaricus.

The Facts

Even though people use lactobacillus for many years unknowingly, the bacterium itself was discovered in 1905 by Stamen Grigorov. Three years later a microbiologisy named

Ellis Metchinoff  noticed that a peasant group in Bulgaria was

thriving and bore children that lived past the age of 100. He studied this group when he realized that the people would frequently break from their work to eat a mixture of yogurt, onions and nuts. He realized that Lactobacillus bulgaricus was able to combat with common intestinal disease that cause toxins which would speed up the aging process. So Ellis basically discovered a trick for one to live longer.

http://emilddie.pbworks.com/w/page/10227630/History%20and%20Origin

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History

Lactobacillus bulgaricus was first discovered in Bulgaria by Stamen Grigorov, who made the discovery in 1905. He extracted it from yogurt and found out about its benefits. He also named the bacteria after his country. A couple of years later in 1908,  Ellie Metchnikoff did research on Lactobacillus bulgaricus.  He started drinking fermented milk and yoghourt  and he experienced improved health and well-being. He concluded his research and proved that it helps your digestive system and also allows you to live longer. Today large companies produce yogurt and milk with Lactobacillus bulgaricus to help people live a healthy life.
LegendsThere is two legends about how it was discovered. They both took place in Bulgaria and thats where the name of the bacteria comes from.Legend one says that a Bulgarian shepherd in the Balkan Mountains didn’t enough vessels to hold all the milk from his sheep so he made a bag from a fresh lamb's hide and put the extra milk there. The next morning he found yoghurt in the bag caused by Lactobacillus bulgaricus.Another legend is that the Bulgars who were nomadic that lived in Bulgaria, knew about and utilized Lactobacillus bulgaricus for their food to live a healthy life.

https://sites.google.com/site/lactobulgaricus/histroy

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Lactobacillus surface layer proteins: structure, function and applications

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Lactobacillus surface layer proteins: structure, function and applications

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<Abstract>
Bacterial surface (S) layers are the outermost proteinaceous cell envelope structures found on members of nearly all taxonomic groups of bacteria and Archaea. They are composed of numerous identical subunits forming a symmetric, porous, lattice-like layer that completely covers the cell surface. The subunits are held together and attached to cell wall carbohydrates by non-covalent interactions, and they spontaneously reassemble in vitro by an entropy-driven process. Due to the low amino acid sequence similarity among S-layer proteins in general, verification of the presence of an S-layer on the bacterial cell surface usually requires electron microscopy. In lactobacilli, S-layer proteins have been detected on many but not all species. Lactobacillus S-layer proteins differ from those of other bacteria in their smaller size and high predicted pI. The positive charge in Lactobacillus S-layer proteins is concentrated in the more conserved cell wall binding domain, which can be either N- or C-terminal depending on the species. The more variable domain is responsible for the self-assembly of the monomers to a periodic structure. The biological functions of Lactobacillus S-layer proteins are poorly understood, but in some species S-layer proteins mediate bacterial adherence to host cells or extracellular matrix proteins or have protective or enzymatic functions. Lactobacillus S-layer proteins show potential for use as antigen carriers in live oral vaccine design because of their adhesive and immunomodulatory properties and the general non-pathogenicity of the species.

and please check on this page.
-Reference-
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3666127/

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RESEARCH ARTICLE: Rerouting of pyruvate metabolism during acidadaptation in Lactobacillus bulgaricus

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RESEARCH ARTICLE: Rerouting of pyruvate metabolism during acidadaptation in Lactobacillus bulgaricus

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Enter your email address below. If your address has been previously registered, you will receive an email with instructions on how to reset your password. If you don't receive an email, you should register as a new user

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Development of the Human Gastrointestinal Microbiota and Insights From High-Throughput Sequencing 

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Development of the Human Gastrointestinal Microbiota and Insights From High-Throughput Sequencing 

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Check out this scientific article!

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Development of the Human Gastrointestinal Microbiota and Insights From High-Throughput Sequencing

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Little was known about the development of the gastrointestinal (GI) tract microbiota, until recently, because of difficulties in obtaining sufficient ...

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Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation

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Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation

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Abstract

Background

The broad ecological distribution of L. casei makes it an insightful subject for research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of L. casei, we performed comparative analysis of 17 L. casei genomes representing strains collected from dairy, plant, and human sources.

Results

Differences in L. casei genome inventory revealed an open pan-genome comprised of 1,715 core and 4,220 accessory genes. Extrapolation of pan-genome data indicates L. casei has a supragenome approximately 3.2 times larger than the average genome of individual strains. Evidence suggests horizontal gene transfer from other bacterial species, particularly lactobacilli, has been important in adaptation of L. casei to new habitats and lifestyles, but evolution of dairy niche specialists also appears to involve gene decay.

Conclusions

Genome diversity in L. casei has evolved through gene acquisition and decay. Acquisition of foreign genomic islands likely confers a fitness benefit in specific habitats, notably plant-associated niches. Loss of unnecessary ancestral traits in strains collected from bacterial-ripened cheeses supports the hypothesis that gene decay contributes to enhanced fitness in that niche. This study gives the first evidence for a L. casei supragenome and provides valuable insights into mechanisms for genome evolution and lifestyle adaptation of this ecologically flexible and industrially important lactic acid bacterium. Additionally, our data confirm the Distributed Genome Hypothesis extends to non-pathogenic, ecologically flexible species like L. casei.

Background

Lactic acid bacteria (LAB) constitute a group of Gram-positive, non-sporulating, nutritionally fastidious, and strictly fermentative bacteria that produce lactic acid as the major end product from carbohydrate []. Lactobacillus, which currently holds 177 species http://www.bacterio.cict.fr/ , is by far the largest and most diverse genus of LAB. Different species of lactobacilli are important components of the oral cavity as well as the gastrointestinal and reproductive tracts of vertebrates, while others are indigenous to milk, plant material, and meat environments []. Many play important roles in both traditional and commercial-scale food and feed fermentations, or food spoilage.

A few species of Lactobacillus show remarkable ecological adaptability and may be recovered from a variety of diverse habitats []. Lactobacillus casei, for example, has been isolated from raw and fermented dairy (especially cheese, where they often emerge as the dominant adventitious or "nonstarter" species during ripening []) and plant materials (e.g., wine, silage, pickles, and kimchi), as well as the oral cavity and gastrointestinal tracts of humans and animals []. As an aside, debate over the taxonomy of L. casei in recent years has led to interchangeable use of L. casei and L. paracasei in the literature. All of the strains included in this study show >99% identity to the 16S rRNA sequence of L. casei ATCC 334, which is the current type strain for L. paracasei[].

The broad ecological distribution of L. casei reflects a metabolic flexibility that has fueled widespread application of the species in the food and health industries; different strains are employed as acid-producing starter cultures for milk fermentation, as adjunct cultures to accelerate or intensify flavor development in bacterial-ripened cheeses, and as probiotics to enhance human or animal health [-]. The Food and Agriculture Organization and the World Health Organization of the United Nations define probiotics as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" []. Strains used as starter cultures or probiotics must, of course, be nonpathogenic to humans even when consumed in very high numbers (e.g., 10 ⁹-10 ¹⁰ cfu/dose). Thus, the wide ecological distribution of L. casei makes this species a particularly interesting and relevant subject for research on genetic diversity, genome evolution, and lifestyle adaptation.

Bacterial genome evolution and adaptation are thought to occur via three major processes: 1) modification of existing genes by mutation with vertical inheritance [-]; 2) acquisition of exogenous genes or gene clusters by bacteria through horizontal gene transfer (HGT) that impart a fitness benefit [-]; and 3) deletion or decay of genes that no longer confer a fitness benefit [-]. Comparative genome analysis of lactobacilli and other LAB has shown that all three mechanisms have contributed to the adaptation of these microbes to new habitats and lifestyles, but gene decay and acquisition by HGT appear to be especially dominant forces [-]. In L. casei and many other LAB, genome evolution is expected to reflect adaptation of the species to dynamic, nutritionally variable and ancient environments, such as the gastrointestinal tract and plant materials, as well as relatively recent expansion into a more constant and nutrient-rich milk-based niche. Not surprisingly, comparative genome analysis between L. casei ATCC 334 and other sequenced lactobacilli confirmed L. casei has features, including a relatively high number of carbohydrate-related genes and IS elements [, ], which are consistent with a metabolically and genetically versatile lifestyle [, ].

The evolutionary history of L. casei has been viewed through its population structure; multi-locus sequence typing (MLST) of 40 L. casei strains isolated from different niches indicated the species had diverged into three lineages []. Separation of two major clusters occurred approximately 1.5 million years ago; one large cluster contained strains recovered from human, plant, and dairy sources, while the second and smaller cluster was predominantly comprised of corn silage isolates. In contrast, the third major cluster included 15 strains that had been isolated from cheese in the United States, Australia, and Europe, and was predicted to reflect recent divergence of strains that were highly specialized for this relatively new ecological niche (manufacture of cheese is thought to have begun ~8,000 years ago) [, ]. This cluster of cheese specialists was also revealed in a follow up study that explored L. casei genetic diversity by comparative genome hybridization (CGH) of 21 strains against an ATCC 334 whole genome microarray []. That work further suggested that adaptation to the cheese environment had been accompanied by extensive decay of genes involved in carbohydrate utilization and transcriptional regulation []. Metabolic simplification via the loss of genes for carbohydrate metabolism has been associated with adaptation of other LAB species to the nutrient-rich milk environment [-]. Intriguingly, many of the genes that L. casei cheese specialists lacked were associated with genomic islands postulated to function in lifestyle adaptation of other L. casei strains [].

CGH data for the 21 strains as well as comparative genome analysis between strains ATCC 334 and BL23 identified numerous hypervariable regions and genomic islands, respectively, some of which showed an atypical base composition that is commonly associated with gene acquisition by HGT []. The diversity and distribution of these regions among different strains indicated that: i) there was a large pool of accessory genes in the population; ii) most L. casei strains were likely niche generalists that could exploit a variety of habitats and tolerate a wide range of environmental conditions; and iii) HGT has played a significant role in the evolution, lifestyle adaptation, and metabolic diversity of L. casei[]. However, these conclusions were softened by the knowledge that microarray-based CGH analysis cannot identify genes that may be present in the test strain but absent from the reference strain, and because the ecological origin of strain BL23 is unclear [].

In recent years, the availability of multiple genome sequences for a single species has demonstrated that a number of pathogenic bacteria possess an extensive pan-genome or "supragenome" that may be several fold larger than the genome of any single strain [, , , -]. This discovery has given rise to the Distributed Genome Hypothesis (DGH), which postulates that access to a supragenome through homologous recombination allows individual strains to rapidly shuffle their genetic information and overcome host defense responses []. Although the DGH has been almost exclusively applied to describe genome evolution in pathogens, the circumstances upon which it is founded (i.e., cells aggregate into polyclonal or polymicrobial biofilms which facilitates HGT) [] are widespread in the microbial world. Baumdicker et al. [] recently showed the DGH extended to nonpathogenic bacteria, and it was our hypothesis that a distributed supragenome would drive genome evolution and lifestyle adaptation in an ecologically flexible species such as L. casei.

To test this hypothesis, we collected draft sequences for 12 strains that had been isolated from dairy (n = 5), plant (n = 5), or human (n = 2) sources that appear to provide broad representation for the genetic diversity in L. casei[, ], and performed comparative genome analysis between these strains plus the complete sequences from 5 additional L. casei strains (ATCC 334, BL23, Zhang, BD-II, and LC2W) in the public database [, -].

Results and discussion

L. casei genome features

Genome features of the 17 L. casei strains included in the study, which provide a broad representation of genetic, ecological, and geographical diversity in the species, are presented in Tables  and . The 12 new draft sequences had a range of coverage between 17X and 133X (mean = 31.6X) and ranged in total contig number from 28-167 (mean = 99 contigs) (Table ). Using a set of only three closed genome references (ATCC 334, BL23, and Zhang), we were able to determine orientation and order the contigs of each draft. The contig numbers listed in Table  were obtained after contigs were ordered and oriented in Mauve and the draft was aligned end-to-end based on the advanced order, which reduced the number of contigs obtained from the de novo assembly by an average of 20% (data not shown). Each of the genomes was assembled into scaffolds based on the placed and unplaced contigs from Mauve, with the unplaced contigs being ordered by name at the end of the scaffold.

Although the final contig order was not independently validated for all 12 draft genomes, an optical restriction map [] was used as a reference to validate assembly of the Lpc-37 genome by this approach (see Additional file : Figure S1 in supplementary online material). The overall arrangement of the contig order for Lpc-37 was done using progressive Mauve, and alignment of the contigs to the optical map demonstrates that the method of assembly and ordering yielded a well ordered draft, with only a few major gaps. The optical map gave an estimated genome size of 3,014,302 bp with a total concatenated ordered draft length of 2,916,119 bp (~97% coverage). Based on the alignment between optical map and draft assembly, the size difference is likely derived from 3 major sequence gaps approximately 30, 70, and 10kb in size (Additional file : Figure S1).

The number of predicted CDS features in each genome ranged from 2,643 to 3,262, with an overall GC content of 46.1-46.6% (Table ). Comparative genomics of the resulting architecture revealed synteny was relatively high across the L. casei genomes, with several large blocks of highly conserved gene content across all 17 L. casei strains (Figure ). These data also indicate that genome size differences were not due to major chromosomal insertions, deletions or re-arrangements.

Sequence homology was determined even more accurately when strains were aligned based on gene content (Figure ), so that most of the diversity is observed as indels. As expected, the number of Locally Collinear Blocks (LCBs) detected using the progressive Mauve alignment increased with the number of genomes compared. Hierarchical clustering of strains based on overall gene content yielded a dendrogram with 6 clusters, designated A-F (Figure ). When members of each cluster were aligned to each other or to their closest reference genome, the number of LCBs was reduced, and the overall sequence homology detected between the strains was apparent based on the height of the similarity profile within each LCB (Figure ). No major genomic rearrangements were detected within any of the draft sequences. Content conservation was even higher within clusters (Figure ), most notably for clusters E and F. Even within clusters B and C, which contain 7 and 3 strains, respectively, overall synteny was high with mostly localized polymorphic content (Figure ).

Comparison of the gene content-based dendrogram (Figure ) to an MLST-based phylogenetic tree for the 17 L. casei strains examined in this study (Figure ) revealed similar clustering for strains in gene content clusters A, E, F, and parts of B and C. However, MLST-derived relationships among the remaining strains did not resemble those derived from overall gene content. The basis for this observation is well understood; MLST-based phylogeny reflects relatively slow genome evolution caused by point mutations and selective pressure, whereas the gene content dendrogram captures more rapid (and unpredictable) large-scale insertion and deletion events. Thus, variations between gene content- and MLST-based dendrograms are expected in bacterial species like L. casei that display frequent intra-species recombination [, , , ].

Characterization of the L. casei core and supragenome

Comparative genome analysis between the five complete and 12 draft genomes provided new insights to the genetic diversity of L. casei. The L. casei genome encodes an average of 2,800 (±151) orthologous clusters, of which 1,715 were common to all 17 strains analyzed ("core genome"). Graphing the numbers of core and total features ("pan-genome", or total number of different genes found within a species) as a function of the number of strains sequenced revealed the slope for core gene number was approaching an asymptote, whereas the pan-genome continued to expand even after compilation of 17 genomes (Figure A). Overall, differences in L. casei genome inventory reveal an open pan-genome comprised of 1,715 core and 4,220 accessory orthologous clusters identified to date, including a large number of unique orthologous clusters in each strain (range = 19 to 326, with an average of 119) (Table  and Figure B). Extrapolation of these data using the binomial mixture model described by Snipen et al. [] yields an estimated core genome of 1,600 orthologous clusters and a pan-genome of 9,072 total orthologous clusters. These findings indicate L. casei has a supragenome that is approximately 3.2 times larger than the average genome of individual strains, and support the hypothesis that the relative size and content of the pan-genome is an indicator of genetic plasticity and potential for environmental adaptation within a species [].

Strain-specific features of the L. casei supragenome

Protein homology searches with strain-specific accessory gene products (i.e., unique to a particular strain) revealed 8-87% (mean = 54%) of these proteins had highest similarity to gene products found in other strains of L. casei or Lactobacillus paracasei (see Additional file : Table S1 in the online supplementary material). The lowest fractions (<50%) were found in the two wine isolates (A2-362 and UCD174) and three dairy strains (Lc-10, ATCC 334, and Zhang). Expansion of the analysis to include gene products with greatest homology to proteins from other species of lactobacilli accounted for 45-95% (mean = 77%) of the strain-specific accessory genes products. For example, different proteins with very high amino acid identity (E value < 1e⁻⁸⁰) to orthologs in the closely related species Lactobacillus rhamnosus were found in all L. casei strains except three dairy isolates (M36, BD-II, and BL23). Additionally, strain-specific accessory genes encoding orthologs with high homology to proteins from a broad range of other Lactobacillus species including L. plantarum, L. fermentum, L. brevis, L. buchneri, L. coryniformis, L. coleohominis, L. farciminis, L. helveticus, L. hilgardii, L. jensenii, L. kefiranofaciens, L. ois, L. pentosus, and L. salivarius were sporadically distributed among the 17 L. casei strains examined (Additional file : Table S1). Many of these lactobacilli are only distantly related to L. casei, but all share at least one ecological niche with this species and theoretically might have contributed to the diversity of the L. casei supragenome. There were, for example, clear relationships between ecological co-localization with particular Lactobacillus species and the unique accessory gene content of individual L. casei strains. L. coryniformis, for example, is commonly found in fermented plant material (e.g., silage) [] and accessory gene products with very high homology scores to various proteins from this species were found within each of the five silage and wine isolates, but not in any of the human or dairy strains (Additional file : Table S1). Similarly, L. casei strains of dairy origin had a greater prevalence of accessory genes whose products gave very high homology scores to different proteins from L. fermentum (Additional file : Table S1), a species commonly found in milk products [].

Overall, L. casei plant isolates showed the most diverse repertoire of strain-specific accessory genes. Genes encoding orthologs with high amino acid identity to different proteins from L. plantarum, L. buchneri, and L. pentosus, for example, were most prevalent in plant isolates, as were proteins with orthologs in distantly related bacteria such as Enterococcus, Streptococcus, Bacillus, and Clostridium species (Additional file : Table S1). Finally, accessory genes with very high homology scores to various proteins from Listeria sp. were detected in each of the human L. casei isolates and in the three dairy strains, but not in any of the plant isolates, even though Listeria are commonly recovered from vegetation [].

If niche-associated gene exchange contributes to the composite nature of the L. casei supragenome, and this process is important to L. casei strain evolution and lifestyle adaptation, then one might expect to find evidence of relatively recent events among different strains. Indeed, several insertion sequence elements in L. casei strains shared at least 99% nucleotide sequence identity with elements that have been identified in the ge nomes of L. rhamnosus, L. brevis, L. buchneri, L. fermentum, Oenococcus oeni and even Listeria innocua (see Additional file : Table S2 in the online supplementary material). More interestingly, L. casei strains UCD174, BD-II, and UW1 each possess a unique polycistronic region encoding features associated with lifestyle adaptation [] that share very high (98-99%) nucleotide sequence identity with genomic regions in L. plantarum or L. brevis (see Additional file : Table S2 and Additional file : Figure S2 in the supplementary online material). Only one strain of L. brevis has been sequenced to date, but nucleotide BLAST searches showed each of the clusters with homology to L. plantarum was common among sequenced strains of that species.

The four-gene cluster found in L. casei UW1 is virtually identical to a locus in L. brevis ATCC 367 that encodes an ABC sugar transport system of unknown specificity (Additional file : Figure S2A). Tests on subset of nine stains, including UW1, for the ability to grow in CDAA supplemented with one of 60 different substrates did not reveal any capability that was unique to strain UW1 (see Additional file : Figure S3 in supplementary online material), so the function of this gene cluster remains unclear. In both species, the cluster is flanked on one side by a gene for transposase (Additional file : Figure S2A), and the G+C content of the cluster (ORFs range from 37-39%) is considerably lower than that of either species' genome (46%) (Table  and Additional file : Table S2), suggesting the cluster may have been acquired by one or both species from a third donor.

The polycistronic cluster identified in L. casei BD-II is plasmid borne, and carries five of the six genes that comprise the L. plantarum lar operon [] (Additional file : Figure S2B). Goffin and coworkers [] showed lar is required for lactate racemization activity in L. plantarum, but the function of most lar-encoded proteins, or even whether all six genes are required for this activity, remains unknown. D-lactate is a component of the cell wall in L. plantarum and L. casei, and Goffin et al. [] suggested Lar activity may provide the cell with a rescue pathway for D-lactate production under conditions that inhibit D-lactate dehydrogenase activity. The lar genes of L. casei BD-II show 98-99% nucleotide sequence identity to their counterparts in L. plantarum, and the BD-II locus is flanked on each side by transposase genes (Additional file : Figure S2B). There are no transposase genes in the immediate vicinity of the lar operon in L. plantarum. Collectively, these observations provide good evidence that the lar locus in L. casei BD-II was acquired via HGT from L. plantarum, but its role, if any, in lifestyle adaptation must yet be determined.

The third and most compelling example of lifestyle evolution via HGT was found in the wine isolate L. casei UCD174. This bacterium contains a polycistronic cluster for L(+)-tartrate catabolism and malate transport that was previously thought to be one of three defined L. plantarum-specific gene clusters []. Tartaric and malic acids are the primary acids in grapes and therefore the strongest acids in wine. Tartrate dehydratase allows cells to convert tartrate to oxaloacetic acid, an important metabolic intermediate, and malate transport and metabolism are known to enhance acid tolerance in L. casei and other lactic acid bacteria [-]. Thus, acquisition of this cluster by UCD174 very likely promoted survival and adaptation of this strain to the acid environment of wine. Features of the UCD174 tartrate dehydratase cluster, including 99% nucleotide sequence identity over the cluster and associated aroAB genes with the corresponding L. plantarum locus, and the presence of flanking transposase genes in UCD174 (Additional file : Figure S2C), provide strong evidence that the locus was acquired by HGT from L. plantarum.

Finally, we have noted L. rhamnosus might be an important source of genetic diversity in L. casei, but also found evidence that the reverse may be true. Of the nine publicly available complete or draft genome sequences for L. rhamnosus, only two strains, GG (ATCC 53103) and LMS2-1, contain a genomic region that encodes three secreted LPXTG-like pilin proteins (SpaCBA) plus a dedicated sortase for their export []. Functional genomic studies showed the SpaCBA pilus promotes adhesion to intestinal epithelial cells, and may function to modulate interleukin-8 expression that is induced by lipoteichoic acid or other surface molecules []. While the spaCBA locus and associated sortase are clearly uncommon among L. rhamnosus strains, it is fully conserved in L. casei strains ATCC 334, BL23, Zhang, and T71499, and present but probably inactive (due to frameshifts or deletions) in L. casei strains 21/1, M36, CRF28, UW4, A2-362, 32G, Lc-10, and Lpc-37. Collectively, these strains span the major MLST-defined genetic lineages for L. casei[, ], which suggests the genes were not recently acquired. The spaCBA and sortase genes of L. casei ATCC 334 show 95-99% nucleotide sequence identity to their counterparts in L. rhamnosus, whose locus is also flanked by transposase genes that are virtually identical to elements found in L. casei. There are no transposase genes in the immediate vicinity of the spaCBA cluster in the L. casei strains sequenced to date. Collectively, these observations provide compelling evidence that the spaCBA locus in L. rhamnosus GG and LMS2-1 may have originally been acquired via HGT from L. casei.

Overall, the composite nature of the L. casei strain-specific accessory gene pool and the presence of gene clusters in some strains that appear to have been recently acquired support our hypothesis that evolution of the L. casei supragenome has been heavily influenced by ecological co-localization with other bacterial species. Placed within the greater context of the DGH, we propose that L. casei, and probably other ecologically flexible species, have access to a supragenome whose composition is not exclusive to the species, but instead might be viewed as a subset of the microbial metagenome within a particular niche. While the primary mechanism(s) for supragenome access by L. casei are unknown, natural transformation has never been demonstrated in lactobacilli, and the prevalence of IS elements and plasmid-linked traits among genes that appear to have been recently acquired (see Additional file : Table S2) suggest that conjugation may be a key driver of HGT in L. casei. However, the widespread distribution of phage-related proteins among the L. casei accessory gene pool (Additional file : Table S1) suggest transduction could also be an important mechanism for genome evolution in this species.

Adaptive immunity against invasive DNA

Although conjugation and transduction may be important mechanisms for HGT in L. casei, these and other bacteria have also acquired sophisticated mechanisms to combat invasive bacteriophage and plasmid DNA. One key example is the CRISPR-Cas adaptive immunity system, which consists of clustered regularly interspaced short palindromic repeats (CRISPR) adjacent to cas (CRISPR-associated) genes. The CRISPR loci are comprised of partially palindromic repeats separated by short stretches of "spacer" DNA that are acquired from invasive bacteriophage or plasmid DNA. Once present, these spacer sequences allow cells to recognize and cleave invasive DNA that contains those sequences [-]. Two distinct types of CRISPR loci were identified in the L. casei genomes. These two loci are typically characterized by idiosyncratic CRISPR repeats: 5'-GTCTCAGGTAGATGTCGAATCAATCAGTTCAAGAGC-3' for the Type II-A (Lsal1 family) locus, and 5'-GTTTTCCCCGCACATGCGGGGGTGATCCC-3' for the Type I-E (Ldbu1 family) locus. Such repeats have been previously identified in a variety of lactobacilli, including L. salivarius, L. casei and L. rhamnosus for Lsal1, and L. casei, L. delbrueckii, L. fermentum, L. acidophilus and L. brevis for Ldbu1 []. Overall, CRISPR repeats were highly conserved, with >97% typical repeats across Lsal1, and >96% typical repeats for Ldbu1 (data not shown).

Lsal1-type CRISPR loci were identified in 11 strains, while Ldbu1-type loci were identified in 3 strains, with both types present in the M36 genome (Figure A). Only four strains (32G, A2-362, 12A, and UW4) did not possess CRISPR loci. The cas content for the Lsal1 loci is typical of Type II-A systems [], with the universal cas1 and cas2, in combination with the cas9 signature gene. Also, a tracrRNA homologous to those found in Type II systems [] was identified in the intergenic region between cas9 and cas1. Though universal genes were highly conserved, up to 19% nucleotide polymorphism was observed for the cas9 signature gene. The genomic location of these loci across the 13 strains was consistent, and occurred immediately following LCABL_23790 homologs (Figure A). The cas content for Ldbu1 is typical of Type I-E systems [], with the universal cas1 and cas2, in combination with the cas3 signature gene, and Cascade-encoding genes, notably cas6e[, ].

As expected, the spacer content was hypervariable across CRISPR loci from different strains, with spacer numbers ranging from 4 to 44 for Lsal1, and between 21 and 60 for Ldbu1 (Figure B). Of note, identical sets of spacers were conserved across cluster F strains (BL23, BD-II and LC2W) indicating very close genetic relatedness, which is also reflected in strain clustering based on overall gene content and MLST analysis (Figures  and ). Several sets of contiguous spacers were also conserved between cluster F strains and strains Lc-10 and Zhang, which suggest common ancestry or HGT inheritance of this CRISPR locus. In contrast, spacer content showed more typical hypervariability across the other strains, with only 1 spacer shared between UW1 and UCD174 (Figure B). For Ldbu1, however, several sets of contiguous spacers were conserved between the dairy strains Lpc-37 and M36 (Figure B).

Analysis of spacer sequences revealed that numerous spacers show homology to Lactobacillus phages (Lc-Nu, A2, Lrm1, and J1) and plasmids (pYIT356, pREN, and pLgLA39) (Figure ). Analysis of sequence conservation in the direct vicinity of proto-spacers that showed similarity or identity to CRISPR spacers revealed the presence of proto-spacer associated motifs (PAM) [-], namely, a conserved TGAAA immediately downstream of the Lsal1 protospacers, and AAY immediately upstream of the Ldbu1 protospacers (Figure ). The TGAAA pentamer is homologous to the AGAAW PAM previously identified downstream of protospacers in Streptococcus thermophilus Type II systems [, , ].

Overall, CRISPR locus hypervariability across strains, in terms of occurrence, locus type and spacer content illustrates their functional value in response to environmental pressure, notably in providing resistance against phages. This polymorphism indicates CRISPR loci are desirable targets for typing of L. casei strains, in disagreement with a previous report []. The critical role that CRISPR-Cas systems play in resistance to viruses has been documented in dairy cultures [, , , , ], as well as environmental samples [, -]. The occurrence of numerous L. casei CRISPR spacers with homology to Lactobacillus phages (notably Lc-Nu, Lrm1, A2 and phi AT3) that prey upon closely related species (Figure ) combined with the larger numbers of spacer sequences in strains isolated from commercial cheese production environments (Lc-10, Lpc-37 and M36) further underscores the selective pressure against phage infection that exists in industrial dairy manufacturing environments. The propensity of hypervariable CRISPR loci for HGT [] is also illustrated within L. casei by the co-occurrence of various IS elements for both types of loci (Figure A), the sharing of contiguous spacer sets across strains that belong to different phylogenetic clusters (Figures  and B), and the skewed GC content of cas genes (50-63% for Ldbu1 cas genes versus 46.5% genome-wide content). Overall, these results highlight the reliance of L. casei strains on CRISPR-Cas systems to provide immunity against invasive elements, as previously shown in bacteria and archaea [, , , , ].

Evolution via genome decay

Our results and previous studies [, ] have indicated HGT is a dominant force in genome evolution of L. casei, but a prior CGH experiment also provided evidence for a genetically distinct and geographically distributed cluster of L. casei cheese specialists whose evolution was accompanied by extensive decay of genes involved in carbohydrate utilization and transcriptional regulation []. This hypothesis is supported by the fact that energy production in L. casei is primarily derived from carbohydrate fermentation, so niche adaptation should be heavily predicated by the ability of strains to utilize available carbohydrate. Fermenting plant material, for example, can contain a diverse array of simple and complex carbohydrates as well as sugar alcohols [], and many of these substrates will also be encountered in the GI tract as a consequence of diet. Thus, the ability to utilize C5 sugars and certain C5 and C6 sugar alcohols is more prevalent in L. casei isolated from plant material and the human GI tract than in cheese isolates []. The overlap in carbohydrate availability and use by L. casei associated with plants or the gastrointestinal tract also supports the hypothesis that many strains from these environments should be viewed as ecological generalists, whereas adaptation to cheese has been accompanied by extensive genome decay that, ultimately, resulted in niche specialization [].

To explore the role of genome decay in the relationship between niche adaptation and substrate utilization, we tested a subset of nine stains (ATCC 334, 21/1, 32G, M36, CFR28, T71499, 12A, UW1 and UW4) distributed across the major MLST-defined L. casei lineages [, ] for the ability to grow in CDAA supplemented with one of 60 different substrates associated with plant, gut, or dairy niches. Growth was detected on a total of 30 substrates, with individual strains able to utilize as few as 17 to as many as 26 different substrates (Additional file : Figure S3). Results showed the two cheese specialists UW1 and UW4, which share the same MLST lineage (Figure ), had the most restricted substrate profile with growth on 18 and 17 different substrates, respectively. Each of the other strains was able to utilize at least 20 different carbohydrates, while two of the corn silage isolates (32G and 12A) each grew on 26 substrates, although their profiles were not identical (Additional file : Figure S3).

The genetic basis for utilization of many of these substrates is unknown, and efforts to detail the impact of genome decay on different substrate profiles is further challenged by the fact that most of L. casei genome sequences used for this study are incomplete. Nonetheless, evidence for genome decay in the evolution of the cheese specialist strains UW1 and UW4 was observed in regard to genes for inulin, sucrose and cellobiose utilization. In L. casei, the ability to ferment sucrose and inulin is encoded by an operon for fructooligosaccharide utilization ( fos) [, ] that is present in the other seven strains tested, which were all sucrose- and inulin-positive, but completely absent in UW1 and UW4.

In contrast to the single fos operon, we identified nine distinct gene clusters among the 17 L. casei genomes studied here that may function in cellobiose utilization (Figure ). Cellobiose is a disaccharide formed by enzymatic or acid hydrolysis of cellulose that, like sucrose and inulin, may be encountered in plant material or in the human gastrointestinal tract but not in milk or cheese. Each of the nine strains analyzed in this part of the study possessed single copies of one (UW1) to eight (21/1 and 12A) of these gene clusters (Figure ), and all but UW1 were able to ferment cellobiose (Additional file : Figure S3). While the function of each cluster in cellobiose utilization (as opposed to other β-glucosides) has not been demonstrated, data for strains ATCC 334 and UW4 show at least two of these clusters must enable cellobiose fermentation in L. casei (Figures  and S3). Cross comparison of the distribution of clusters 1 to 6 across the MLST lineages for the nine L. casei strains tested (Figures  and ) provides clear evidence of genome decay; many clusters are entirely absent, presumably due to deletion events, and all but 12A contained at least one cluster that was predicted to be nonfunctional due to frameshift mutations (Figure ). The single cluster in the cellobiose-negative strain UW1, for example, was predicted to be nonfunctional (Figure ). Finally, examination of all 17 genomes included in this study confirmed the cheese specialists UW1 and UW4 had the fewest total cellobiose clusters, and were the only strains lacking cluster 5.

Conclusions

In bacteria, the concept of species is traditionally anchored to features that are encoded by the core genome, but which often do not adequately describe the genetic diversity that is characteristic of a particular species [, ]. Thus, there is increasing awareness that a large number of strains, preferably of disparate origin, must be sequenced to gain an accurate understanding of the evolution and biology of a particular bacterial species. To our knowledge, this is the first study to explore genome evolution and diversity in an ecologically flexible lactic acid bacterium using genome sequences from a large number of ecologically divergent strains.

As is depicted in Figure , the 17 L. casei genomes included in the study were not sufficient to capture the full pan-genome of this remarkable species. Indeed, mathematical extrapolation of these data indicated only 2/3 of the actual L. casei pan-genome has been sequenced to date (5,935 orthologous clusters found versus an estimated 9,072 total). Evidence for a much larger, open pan-genome was also provided by hierarchical clustering of strains based on overall gene content, since 2 of the 6 clusters (A and D) currently contain single isolates. Overall, our findings indicate L. casei has an open and distributed supragenome that is approximately 3.2 times larger than the average genome size for individual strains.

Characterization of the L. casei supragenome suggested its composition has been influenced by ecological co-localization with other bacterial species, especially lactobacilli. These findings provide additional confirmation that the DGH extends to non-pathogenic species [], and indicate ecologically flexible bacteria like L. casei have access to a supragenome whose composition might be viewed as a subset of the microbial metagenome within a particular ecological niche. Finally, our results also provide support for the hypothesis that HGT has been a dominant force in adaptation of L. casei to new habitats and lifestyles, and that evolution of a genetically distinct and geographically distributed cluster of L. casei cheese specialists has been accompanied by extensive decay of genes associated with carbohydrate utilization [].

Methods

Genome sequencing and assembly

Genomic DNA was extracted using a MasterPure Gram-Positive DNA Purification Kit (Epicentre Biotechnologies, Madison, WI). All 12 of the genome drafts were sequenced using a whole-genome shotgun strategy by pyrosequencing (GS FLX Titaninum 454 Life Sciences). The Lpc-37 and Lc-10 genomes were sequenced by University of Illinois-Urbana Champaign under contract services. The remaining 10 genomes were sequenced as a contract service by the Génome Québec Innovation Centre at McGill University (Montreal, Canada). Resulting sequences were assembled de novo using NGen SeqMan 2.0 (DNAstar, Madison, WI). The draft contigs for each genome assembly were compared to all five published genomes (Table ) using progressive Mauve genome alignment software [] and visually inspected for the best overall matching Local Collinear Blocks (LCBs). Based on the best matches, each draft was individually ordered and contigs oriented, but unlocated contigs were not scaffolded. Using the new contig orientation and order, the drafts assemblies were improved by hand curation in silico using SeqMan 8.1.2 (DNAstar). For strain Lpc-37, the draft genome assembly was also validated by comparison to a NheI optical restriction map (OpGen Inc, Gaithersburg MD) (see Additional file : Figure S1 in supplementary online material).

Initial annotations were generated using the RAST annotation server () with subsequent manual inspection and curation, including comparative analyses to improve consistency among orthologous groups. Pseudogene assignments were not exhaustive and were based on the available sequence; they may be influenced by sequencing artifacts due to the draft nature of the genomes.

Ortholog predictions

Ortholog predictions were made based on reciprocal-best Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi ) hits using a cutoff of 80% identity over 80% of the length. Confirmation of the reciprocal BLAST data and additional ortholog predictions were generated from whole-genome Mauve alignment data using a custom script and the A Systematic Annotation Package (ASAP) for Community Analysis of Genomes database ( https://asap.ahabs.wisc.edu ) []. This script uses synteny to determine whether features should be confirmed as orthologs, and can add orthologs based on genome context in cases where the BLAST results are ambiguous (e.g. paralogs) or that fail the cutoffs. This allows the initial BLAST assessment to be more strict, preventing the inclusion of homologs in the ortholog table and providing a one-to-one ortholog relationship between genomes.

CDS and MLST dendrograms

A present/absent matrix of the CDS in all 17 strains of L. casei was created by a custom script and used to generate a dendrogram by the Ward method of hierarchical clustering (JMP version 9). Multiple sequence alignments were performed using molecular evolutionary genetic analysis (MEGA) software version 4 ( http://www.megasoftware.net ). A minimum evolution (ME) tree for all 17 L. casei strains was constructed using MEGA 4 based on the results of a bootstrapping test (1000 replicates) of strain phylogeny []. The phylogenetic tree was linearized assuming equal evolutionary rates in all lineages []. The evolutionary distances were computed using the modified Nei-Gojobori method [] and are presented in the units of the number of synonymous substitutions per synonymous site. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option).

To estimate the divergence time among different L. casei clusters, the concatenated sequences of 6 MLST loci ( ftsZ, metRS, mutL, pgm, nrdD, and polA) were analyzed as described previously []. Divergence times between different clusters were indicated in a scale of millions of years. Calculations were based on the number of single nucleotide substitutions in each strain, and the estimated rate of single nucleotide substitutions between Escherichia coli and Salmonella enterica of 4.7 × 10⁻⁹ per site per year [, ].

Estimation of the L. casei core and pan-genome size

Protein coding features in each genome were grouped using OrthoMCL [] with default settings and the resulting data were uploaded to the ASAP database. These data were retrieved from the database with a custom script that organized the feature IDs into a tab-delimited matrix allowing for identification of accessory genes (all features not included in the core genome) and strain-specific genes (unique to a single isolate) by inspection of this table. The best hits of the strain-specific features from a BLAST [] analysis against the nr database using default parameters were collected and are presented in Additional file : Table S1. Graphical depictions of L. casei pan and core genome data, including the heat map, were generated using a custom php-based script that polls these data from the ASAP database and builds a present/absent matrix. Pan and core genome size estimates were derived with an R-package using a binomial mixture model [].

CRISPR identification and characterization

CRISPR loci were identified using a combination of homology to previously indentified repeats [], de novo identification using CRISPRFinder [] and repeat region identification using Dotter []. Spacers were visualized as previously described [], and homologies to foreign genetic elements were assessed using BLAST []. CRISPR loci sequences were independently confirmed by Sanger sequencing of PCR amplicons. Nucleotide conservation between CRISPR spacers and corresponding proto-spacers in phages, plasmids and chromosomal sequences were visualized using WebLogo [].

Carbohydrate utilization

L. casei strains 21/1, 12A, M36, UCD174, A2-362, 32G, T71499, CRF28, UW1, UW4, and ATCC 334 were transferred from -80C freezer stocks into MRS broth (Difco Laboratories, Detroit, MI) and grown overnight (16-18 h) at 37°C. Strains were inoculated at 1% (v/v) into a filter-sterilized chemically defined amino acid medium (CDAA) with 25 mM galactose and incubated 16-18 h at 37°C. The CDAA was comprised of 114 mg sodium acetate, 171 mg sodium citrate, 171 mg ammonium chloride, 343 mg potassium phosphate (monobasic), 343 mg potassium phosphate (dibasic), 114 mg magnesium sulfate tetrahydrate, 6 mg iron sulfate hexahydrate, 6 mg manganese sulfate tetrahydrate, 4 g sodium chloride, 228 mg L-phenylalanine, 455 mg L-tyrosine, 6 mg L-adenine, 6 mg L-guanine, 6 mg L-uracil, 6 mg L-xanthine, 351 mg DL-aspartate, 245 mg L-glutamate, 545 mg L-tryptophan, 443 mg L-alanine, 312 mg L-arginine, 746 mg L-asparagine, 857 mg L-cysteine, 816 mg L-glutamine, 341 mg glycine, 900 mg L-histidine, 923 mg L-isoleucine, 326 mg L-leucine, 428 mg L-lysine, 148 mg DL-methionine, 93 mg L-proline, 946 mg DL-serine, 404 mg DL-threonine, 651 mg L-valine, 24 mg L-cystine, 1 mL trace elements solution [], 0.4 mL Tween 80, 0.4 mL Tween 20, 0.4 mL Tween 60, plus 10 mL RPMI 1640 vitamin solution (100X; added prior to experimentation), and pH adjusted to 5.5.

Cells were collected by centrifugation at 13,000 x g for 5 min at 4°C, then suspended in CDAA lacking carbohydrate. Samples of each strain were then added to a final absorbance at 595nm (A ₅₉₅) of 0.1 into 1 mL CDAA adjusted to pH 5.5 that contained 2 mM galactose as a growth booster plus one of the following substrates: 25 mM meso-erythritol, D-xylose, D-ribose, D-arabinose, D-adonitol, D-arabitol, D-xylitol, D-glucose, D-mannose, D-galactose, D-fructose, lactone, D-mannitol, D-galactitol, D-sorbitol, myo-inositol, D-glucosamine, n-acetyl D-glucosamine, sialic acid (Indofine Chemical Company, Inc., Hillsborough, NJ), lactulose, D-lactose, D-sucrose, D-turanose, D-maltose, D-cellobiose, D-trehalose, D-maltitol, D-lactitol, D-raffinose, or D-melezitose; or 4.5 mg/mL (which is equivalent to 25 mM glucose) of heparin, N-acetyl-D-galactosamine, fucose, panose, galactosamine, amylopectin, high methylated pectin, stachyose, pectin, arabinogalactan, rhamnose, inulin, mucin, or phytic acid; or 12.5 mg/mL isomaltose, galacturonic acid, polydextrose, glucuronic acid, amygdalin, maltotriose, pullulan, amylopectin, carboxymethyl cellulose, xylan, lignin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, dextrin, or amylose. Unless noted, substrates were purchased from Sigma-Aldrich Co. (St. Louis, MO). Strains A2-362 and UCD174 were not included in these studies because they were unable to grow well in CDAA.

Inoculated mixtures were incubated at 37°C under static conditions. Aliquots (50 μl) were periodically collected over a 48 h period, placed in a 96-well microtiter plate, and A ₅₉₅ was determined using a 96 well plate-reader (Bio-Rad, Hercules, CA). Uninnoculated mixtures containing individual substrates were used as blanks for the plate reader. Carbohydrates that produced a turbid sample at time 0 (amylopectin, mucin, carboxymethyl cellulose, xylan, lignin, α-cyclodextrin, γ-cyclodextrin, β-cyclodextrin, dextrin, and amylose) were diluted in a 0.9% sterile saline solution and plated on MRS agar (Difco Laboratories) using the drop plate method []. Plates were incubated at 37°C for 48 h prior to colony enumeration.

All growth experiments were performed in triplicate, and the ability to utilize a particular substrate was determined by two-tailed student's t-test comparison (α = 0.05) between the mean A ₅₉₅ values from cells incubated in CDAA containing no carbohydrate versus CDAA containing the substrate of interest. A dendrogram showing the relationships between strains in regard to substrate utilization was created using the Ward method in hierarchical clustering (JMP version 9, SAS Institute Inc., Cary, NC).

Identification of cellobiose gene clusters

Putative gene clusters for cellobiose utilization were identified by keyword screening of gene annotations for "β-glucosidase", and by BLAST homology searches of each genome using β-glucosidase protein sequences from the Carbohydrate-Active enZYmes database (). Neighboring genes were then examined for support functions such as cellobiose transport and transcription activators or terminators.

Abbreviations

Declarations

Acknowledgements

We thank Anne-Claire Coûté-Monvoisin for technical assistance and Lars Snipen for providing a pre-release version of his R-package software. This work was supported by Danisco, Inc., the United States Department of Agriculture, the U.S. Department of Energy Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494), and the Utah Agricultural Experiment Station (UAES). This manuscript was approved by the UAES as journal paper number 8424.

Electronic supplementary material

Competing interests

Authors' contributions

JRB, JLS, RB, BS, ECN-E, NP, HC, and PH conceived and designed the study; JRB, ECN-E, BS, KT, JH, WM, and RB performed experiments and bioinformatic analyses; JRB, JLS, KT, JH, ECN-E, HC, RB, BS, NP, and WM contributed to data interpretation; PH contributed reagents, materials and analytical/technical expertise; JRB, JLS, ECN-E, RB, and BS wrote the paper. All authors read and approved the final manuscript.

BS, WM, PH, RB, and JLS received salary and/or funding from DuPont, a supplier of bacterial cultures to the food industry. Peggy Steele, a member of JLS's family, is also employed by DuPont.

Below are the links to the authors' original submitted files for images.

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Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation

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The broad ecological distribution of L. casei makes it an insightful subject for research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of L. casei, we performed comparative analysis of 17 L. casei genomes representing strains collected from dairy, plant, and human sources.

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The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution

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The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution

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Abstract

Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. ( i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; ( ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and ( iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

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Results and Discussion

Primary Sequence Characteristics.

The size of the L. bulgaricus genome was estimated at ≈1.8 Mbp on the basis of results obtained after pulsed-field gel electrophoresis of chromosomal DNA digests (data not shown). This estimation was confirmed in the present whole-genome sequencing project, where a circular chromosomal sequence of 1,864,998 bp has been assembled. The primary characteristics of this sequence are presented in Table 1.

The overall GC content (49.7%) differs significantly from that of the closely related species L. acidophilus (34.7%; ref. 10) and L. johnsonii (34.6%; ref. 11), a difference that is mainly due to very important differences at codon position 3 (GC3): 65.0% GC in L. bulgaricus as compared with 25.0% and 24.4% in L. acidophilus and L. johnsonii, respectively. Whereas the latter values fit the strong correlation that can be observed between GC3 and overall GC content in bacteria ( Fig. 1), the L. bulgaricus GC3 value (65.0%) strongly deviates from the expected value (54.0%). Because the evolution at codon position 3 is generally much faster than at positions 1 and 2, the high GC3 value suggests that the L. bulgaricus genome is in an active phase of evolution toward a higher GC content or that in L. bulgaricus the correlation between GC3 and overall GC content has been lost or changed.

In the L. bulgaricus genome, a large number of coding sequences (CDS) have been annotated as "fragments" on the basis of blastp and blastx ( 12) results. These fragments correspond to 270 different pseudogenes (of which 43 are remnants of transposase coding genes) that are regularly distributed over the genome, with a slight underrepresentation in the region from 283 kbp to 672 kbp ( Fig. 2). This high number of pseudogenes suggests that the genome is in an active state of gene elimination and concomitant size reduction. Not counting pseudogenes, only 73% of the genome consists of (putative) coding sequences. A distinct 2.5-kbp noncoding region (starting at position 764 kbp) was identified that has all of the features of a CRISPR region (clustered regularly interspaced short palindromic repeats) ( 13). CRISPRs have been interpreted as traces of past invasions by extrachromosomal elements and have been hypothesized to provide immunity against foreign DNA expression by coding antisense RNA ( 14).

L. bulgaricus contains a relatively high number of rRNA and tRNA genes. In the firmicutes, a clear correlation can be observed between the numbers of rRNA and tRNA genes and between each of these and genome size ( Fig. 3 and Fig. 5, which is published as supporting information on the PNAS web site). Although in L. bulgaricus the first correlation is respected, the numbers of tRNA and rRNA genes are ≈50% higher than the average and 20-30% higher than the highest values observed so far for this genome size, corresponding to values found for genomes of 3-4 Mbp in size. Whereas variation in rRNA and tRNA gene copy numbers may be related to the capacity to respond to changing environmental conditions ( 15, 16), the exceptionally high copy numbers found in L. bulgaricus likely indicate that the genome has undergone a recent phase of size reduction.

Genome Structure and Organization: Replication Terminus with a 47.5-kbp Inverted Repeat and Duplicated dif Sites.

A sharp change in the sign of the GC skew coincides with the location of the dnaA and dnaN genes (data not shown), indicating the likely presence of the origin of replication in this region. The presence of several DnaA boxes in this region confirms this hypothesis ( 17). On the opposite side of the genome, a second change in the sign of the GC skew locates the replication terminus in the region between positions 940000 and 946000 (data not shown). An intriguing feature of the replication terminus region of the ATCC11842 genome is that it contains an inverted repeat of 47.5 kbp (positions 918952-966484) enclosing a unique central region of 1.4 kbp between the repeated sequences (23 kbp each; see Fig. 6, which is published as supporting information on the PNAS web site). In bacterial genomes, inverted repeats of this size are extremely rare. To our knowledge, they have been observed only in the replication terminus region of certain Streptomyces chromosomes that have circularized after telomere deletion ( 18). Preliminary results of one primer PCR amplification in 30 different L. bulgaricus strains show that an inverted repeat is conserved in most or all strains (data not shown). The size of the unique central sequence and/or the size of the repeat may largely vary between strains, however, and this feature may represent a transient stage in the evolution of the L. bulgaricus genome.

In L. bulgaricus the repeated sequence includes the putative dif site (Figs. 6 and 7, which are published as supporting information on the PNAS web site), which is the recombination site involved in the resolution of chromosome dimers ( 19). Consequently, L. bulgaricus contains two putative dif sites in opposite orientation at a distance of 2.5 kbp (0.55 kbp of repeated sequence at either side of the unique central sequence of 1.4 kbp) instead of the unique site found in other microbial genomes, such as Bacillus subtilis (Fig. 7). One may hypothesize that this configuration could interfere with the efficient segregation of dimeric chromosomes that may be formed during replication, because recombination between two dif sites of opposite orientation in a chromosome dimer would result in the inversion of a part of the dimer rather than resolution of the dimer. Such FtsK XerCD-dependent inversions have been observed in plasmids containing dif sites of opposite orientation in vitro ( 20). The results of PCR analysis (data not shown) suggest that the 1.4-kbp unique sequence enclosed by the repeated sequences may be present in either of two orientations in a population of strain ATCC11842 (Fig. 6). This result would be expected as a consequence of such a site-specific recombination between two dif sites of opposite orientation or, alternatively, of homologous recombination between the inverted repeats.

Nonrandom Distribution of Mobile Elements.

The L. bulgaricus ATCC11842 genome contains a large number of transposases and remnants thereof (Table 2, which is published as supporting information on the PNAS web site), most of which have been described earlier in Lactobacillus delbrueckii insertion sequence (IS) elements (ISL3, ISL4, ISL5, ISL4-5, ISL6, ISL7, and ISldl1) ( 21- 23). In the present work, seemingly intact transposases of two new types could be detected [named ISL8 (Ldb0326 and Ldb2033) and ISL9 (Ldb1987)], as well as remnants of several other types.

Intriguingly, a region of 415 kbp (22% of the genome) between positions 279 kbp and 694 kbp is completely exempt of IS elements, whereas (remnants of) the more abundant IS elements seem randomly distributed over the remaining 78% of the genome ( Fig. 2). This discontinuity in the distribution of IS elements coincides with the region, mentioned above, where pseudogenes are less abundant. The absence of IS elements does not, however, explain the difference in the frequency of pseudogenes, because in the whole genome inactivation of only 9 of 227 nontransposase pseudogenes occurred from insertion of IS elements. The positions of transposases often coincide with regions of locally elevated A+T content ( Fig. 2 and Fig. 8, which is published as supporting information on the PNAS web site), often considered as possible signs of horizontal gene transfer.

In contrast to many other lactic acid bacteria, L. bulgaricus ATCC11842 does not contain any prophage. Only four pseudogenes of putative phage origin were detected in a large region around the replication terminus, where the attB sites of the elsewhere-described bacteriophages mv4 ( 24) and JCL1032 (K.-A. Riipinen, personal communication) are found.

Synteny Among L. bulgaricus, L. acidophilus, and L. johnsonii Genomes.

A clear global synteny is observed among the similarly sized genomes of L. bulgaricus and L. acidophilus or L. johnsonii ( Fig. 4 and Fig. 9, which is published as supporting information on the PNAS web site). The most important perturbations of synteny are found in a region of ≈300 kbp around the replication terminus of the L. bulgaricus genome and in two smaller regions at ≈400 kbp at either side of the replication origin. By using the criterion of blast score ratio with a cutoff value of 0.4 ( 25) or the results of a blastp reciprocal best-hit analysis, ≈55-60% of the L. bulgaricus proteins can be considered as having homologues in L. acidophilus and L. johnsonii and constitute the common backbone of these three closely related genomes. Between 25% and 35% of the proteins appear unique to L. bulgaricus (Fig. 10 and Tables 3-5, which are published as supporting information on the PNAS web site). Although the differences between these genomes are likely to be of importance in their respective ecological niches, they mainly concern unknown functions. The most marked differences among known functions comprise the presence of complete pathways for the biosynthesis of folate and saturated fatty acids (see below) in L. bulgaricus, both of which are partially lacking in L. acidophilus and L. johnsonii. Likewise, L. bulgaricus contains all or most of the enzymes necessary for the biosynthesis of purines and pyrimidines, respectively, of which several are absent from L. acidophilus and L. johnsonii and can grow in a purine-free medium (data not shown). L. bulgaricus contains ppx and ppk genes that are involved in the turnover of polyphosphate and thereby may play a role in adaptation to stress conditions ( 26). Inversely, proteins of known function that are present in L. acidophilus or L. johnsonii but not in L. bulgaricus are mainly involved in sugar transport and metabolism (data not shown).

Restriction Modification Systems.

The L. bulgaricus genome contains a complete type I restriction modification (R/M) system (Ldb1051, Ldb1052, and Ldb1053), an additional type I specificity subunit (Ldb1055), and a putative Mrr type restriction endonuclease (Ldb0474). The presence of these R/M systems may explain part of the difficulties encountered previously to transform this strain. A type III restriction modification system appears inactive as a consequence of mutations in the gene encoding the endonuclease (Ldb1227-Ldb1229), whereas the corresponding methyltransferase (Ldb1230) seems intact.

Regulatory Functions: Two SigA Homologues and Relatively Few Regulators.

A unique feature of the L. bulgaricus genome is that it encodes two 58% identical SigA homologues in two genes that are organized in tandem and apparently cotranscribed. While one of the two encoded proteins (Ldb1246) shows −35 and −10 recognition domains that are 100% identical to those in B. subtilis SigA, the other (Ldb1245) shows some important differences in these domains (Fig. 11, which is published as supporting information on the PNAS web site). It is unknown whether this second copy is active and recognizes different promoters. If so, it may be considered a new σ factor developed after gene duplication. Apart from these SigA homologues, two putative extracytoplasmic function (ECF)-type σ factors (Ldb0066 and Ldb1881), one putative σ factor of unidentified type (Ldb1677), and one anti-σ factor (Ldb1880) were found.

Relatively few genes encoding transcriptional regulators were identified in L. bulgaricus, and the difference with Lactobacillus plantarum ( 27) was especially striking (53 and 234 predicted genes, respectively; Table 6, which is published as supporting information on the PNAS web site), even when considering the smaller genome size of L. bulgaricus (1.9 Mb as opposed to 3.3 Mb for L. plantarum). Both these features likely reflect adaptations to the stable and nutritionally rich milk environment, where fewer biosynthetic functions and less adaptive regulation are required. The largely different sugar metabolic capacities of these two species are reflected by the difference in the number of LacI type regulators. Notable differences in MarR-, MerR-, TetR-, and ArsR-type regulators may indicate significantly different levels of resistance to a variety of compounds and stress conditions or differing strategies to cope with them. Regulatory circuits may have a less complicated structure in L. bulgaricus, as exemplified by the predicted HrcA regulon (Table 7, which is published as supporting information on the PNAS web site) ( 28). In B. subtilis, dnaK and groESL on one hand and clp on the other hand are under the control of two different regulators, HrcA and CtsR, respectively. L. bulgaricus encodes only a HrcA homologue. CIRCE boxes, the operators for HrcA ( 29), are found upstream of dnaK and groESL and upstream of clpP and clpE, suggesting a broader role for HrcA in L. bulgaricus. A similar organization was found in L. acidophilus and L. johnsonii (Table 7) ( 10, 11).

Pseudogenes and Specialization Through Loss of Function.

L. bulgaricus strain ATCC11842 was originally isolated from bulgarian yogurt. Traditional yogurt-making involves the sequential transfer of samples of yogurt cultures to fresh milk. With the first records of yogurt (kisim) dating to 3200 before Christ ( 30), one would logically predict that L. bulgaricus adapted to this environment over time. Traces of such an adaptation process were found throughout the L. bulgaricus genome in the form of pseudogenes and incomplete metabolic pathways.

A total of 270 pseudogenes have been detected in the L. bulgaricus genome, including 43 transposases. The remaining 227 genes represent 12% of the total number of protein-coding genes. This percentage of pseudogenes is remarkably high ( 31) and may indicate a recent and ongoing process of specialization. A function, albeit inactivated, could be assigned to 81 pseudogenes. For 30 of these genes, intact paralogues are present in the genome whereas for the remaining 51 genes no intact paralogues could be detected, indicating that the corresponding functions are completely lost (Table 8, which is published as supporting information on the PNAS web site). These include genes implicated in carbohydrate metabolism, amino acid and cofactor biosynthesis, and competence development. For 146 pseudogenes it was difficult to determine whether their function could be assumed by a paralogue as only a general function (e.g., "ABC transporter") or no function at all could be attributed to these genes.

Complete transport systems for the milk sugar lactose (lactose permease), mannose/glucose [phosphotransferase system (PTS) IIABCD], fructose (PTS IIABC), and glycerol (glycerol uptake facilitator) are present, but several other transport systems appeared incomplete. Remnants were found of cellobiose (PTS subunit IIC), sucrose (PTS subunit IIA), maltose (partial subunits of a dedicated ABC transporter), and some other sugar transport systems of undetermined specificity (several isolated PTS IIA subunits). Together with remnants (pseudogenes) of key enzymes in carbohydrate-specific metabolic pathways (glycerol kinase, 6-phospho-β-glucosidase/galactosidase, α-amylase, and mannitol-1-phosphate 5-dehydrogenase), these reveal the prior existence of metabolic capacities that may notably have served in a plant-associated environment.

Apart from the intrinsic evidence for specialization through loss of function provided by the presence of pseudogenes, the complete absence of a large number of enzymes involved in the biosynthesis of amino acids (incomplete or lacking pathways) also suggests an adaptation to the protein-rich milk environment. Here the presence of an extracellular protease, several amino acid transport systems, two complete peptide transport systems, and numerous peptidases (Tables 9 and 10, which are published as supporting information on the PNAS web site) would render most amino acid biosynthesis pathways superfluous. As a consequence, the extracellular protease has become essential for growth in milk ( 32).

Interestingly, only part of this adaptation to the milk environment is shared by S. thermophilus. Whereas L. bulgaricus has lost most of its amino acid biosynthesis capacity, S. thermophilus retains its ability to synthesize all amino acids, except histidine ( 33). This difference may be explained by the fact that S. thermophilus does not generally possess an extracellular protease to exploit the rich source of milk proteins. Perhaps S. thermophilus either adapted to the milk environment independently or coevolved with the protease producing L. bulgaricus but retained an advantage in conserving its capacity for biosynthesis of amino acids.

Central Carbohydrate Metabolism.

As a consequence of the (partial) inactivation of several sugar transport and degradation pathways and inactivation of the LacR repressor ( 22), carbohydrate metabolism in L. bulgaricus appears to prefer the milk sugar lactose. After transport and hydrolysis, this organism selectively metabolizes the glucose moiety, because no enzymes are present to use galactose, which is known to accumulate in the culture medium ( 6, 34). Whereas glucose, fructose, and mannose can also be metabolized, utilization of lactose results in higher growth rates ( 35). A similar accumulation of galactose in the culture medium is observed when S. thermophilus is grown on lactose, albeit for a different reason. In contrast to L. bulgaricus, S. thermophilus does possess the necessary genes to direct catabolism of galactose, but these genes are generally repressed in the presence of lactose ( 36).

Although a putative transporter for ribose and a ribokinase are present, L. bulgaricus cannot grow on this sugar as the only carbon source (data not shown), presumably as a consequence of an incomplete pentose-phosphate pathway (see below). A complete glycolytic pathway is present leading to the production of pyruvate, which can subsequently be converted to l- or d-lactate to regenerate NAD⁺.

Three particular features are noted relating to carbohydrate metabolism (Fig. 12, which is published as supporting information on the PNAS web site).

The first substrate level phosphorylation step of glycolysis, in which glyceraldehyde-3-P is converted to 3-P-glycerate via 1,3-bi-P-glycerate, yielding NADH and ATP, can be bypassed by a nonphosphorylating NADP⁺-dependent glyceraldehyde-3P dehydrogenase (GapN). This would result in glycolysis yielding pyruvate and NADPH with no net ATP production. The GapN protein is not present in other sequenced Lactobacillus species but is very common among streptococci. In Streptococcus mutans, the role of GapN has been suggested to be the production of NADPH because this bacterium lacks the NADPH-generating enzymes in the oxidative branch of the pentose phosphate pathway ( 37). GapN may also be implicated in an additional level of regulation of glycolysis, allowing the adjustment of metabolic and energy fluxes ( 38).

L. bulgaricus contains an apparently intact oxidative, NADPH-generating branch of the pentose phosphate pathway. However, distinct from the related species L. acidophilus and L. johnsonii, the nonoxidative branch lacks a transketolase. Because pentose-phosphate cycling is generally regarded as a mechanism enabling the efficient synthesis of NADPH ( 39), the interruption of this cycle would support the hypothesis of an NADPH-producing role for GapN under conditions where the oxidative pentose-phosphate pathway is not able to meet the demand.

Similar to L. acidophilus and L. johnsonii, no pyruvate dehydrogenase complex is present, nor are other enzymes that could generate acetyl-CoA directly from pyruvate under anaerobic conditions. The most likely alternative route would convert the pentose phosphate pathway intermediate d-xylulose-5-P to acetyl-P, which can subsequently be used to generate acetyl-CoA. Acetyl-P may also be produced from acetate, which has been found to be an essential component of a chemically defined medium for L. bulgaricus ( 35). One may speculate that acetate fulfills this role under conditions where the pentose-phosphate pathway would be unable to supply sufficient substrates for acetyl-CoA generation.

Acetyl-CoA may subsequently be used for the production of saturated fatty acids. It is not clear whether L. bulgaricus would be able to produce unsaturated fatty acids, however, because a FabA or FabM homologue is missing ( 40). Failure to produce acetyl-CoA or the inability to produce unsaturated fatty acids may explain the need for Tween 80 in laboratory media for L. bulgaricus. Oleic acid, the unsaturated fatty acid in Tween 80, is also available in milk.

Protocooperation Between L. bulgaricus and S. thermophilus.

L. bulgaricus and S. thermophilus have long been known to stimulate each other's growth and product acidification during milk fermentation in a process called protocooperation. Several factors have been shown or postulated to be responsible for this effect, of which the most obvious is that L. bulgaricus possesses an extracellular cell wall-bound proteinase ( 32) which, through degradation of milk proteins, can supply peptides and amino acids to S. thermophilus, which generally does not possess such a protease. Among the other protocooperative factors are formate and CO ₂ produced by S. thermophilus, which stimulate the growth of L. bulgaricus ( 42).

Analysis of the L. bulgaricus genome revealed additional factors that may play a role in protocooperation. ( i) The genome sequence encodes a full set of genes for the biosynthesis of folate (Fig. 13, which is published as supporting information on the PNAS web site), a cofactor in many metabolic reactions and an essential component of the human diet ( 42). L. bulgaricus does not, however, possess a means to produce p-aminobenzoic acid (PABA) which feeds into this pathway. S. thermophilus does possess the necessary enzymes to produce PABA and folate, and as a consequence yogurt is generally regarded as a source of PABA. L. bulgaricus may thus benefit from elevated levels of PABA (and folate) when cocultured with S. thermophilus. ( ii) Two features in the genome suggest that L. bulgaricus has a particular need for polyamines, which participate in many cellular processes and may play a role in tolerance to oxidative stress ( 43, 44). First, two ABC transporters are found that could serve the uptake of putrescine and/or spermidine from the culture medium. One of these is encoded by a potABCD operon (52% GC) in which the genes are arranged in the same order as found in most other bacteria (ERGO). The other is encoded by atypical, AT-rich (38% GC) genes that are present in the order potBCAD found in very few bacteria and probably were acquired by horizontal transfer. Second, two genes are found that encode ornithine decarboxylases that catalyze the conversion of ornithine to putrescine. These enzymes are rare in Gram-positive bacteria, except for lactobacilli of the acidophilus group. Because L. bulgaricus does not possess the genes necessary to produce ornithine, one may hypothesize that during yogurt fermentation ornithine is provided by S. thermophilus, which does possess the enzymes necessary to produce ornithine, but no ornithine decarboxylase (Fig. 14, which is published as supporting information on the PNAS web site). Interestingly, S. thermophilus does also possess an ABC transporter dedicated to the uptake of spermidine/putrescine, and, thus, it could be speculated that L. bulgaricus and S. thermophilus mutually benefit from an exchange of ornithine and putrescine.

A prediction of protein localization (Tables 11 and 12, which are published as supporting information on the PNAS web site) revealed a limited number of putative cell wall-bound and extracellular proteins. Perhaps some of these could contribute to direct contacts between L. bulgaricus and S. thermophilus as visualized by Bolotin et al. ( 9). From a fermentation viewpoint, the exopolysaccharide (eps) gene clusters present in L. bulgaricus (Table 13, which is published as supporting information on the PNAS web site) and S. thermophilus appear highly diverse. Variation of these eps clusters among strains is likely important to the texture characteristics of yogurt and merits further investigation.

Stress Resistance.

The L. bulgaricus genome sequence reveals few of the genes that are known to be involved in the resistance to oxidative stress or low pH, even though this lactic acid bacterium acidifies the culture medium and can exhibit optimal growth under microaerobic conditions ( 45). The only enzyme that could eliminate oxygen appears to be pyruvate oxidase, but no catalase is present to detoxify the H ₂O ₂ produced in this reaction. Strain ATCC11842 does not appear to possess the NADH oxidase described by Marty-Teysset et al. ( 46) in another L. bulgaricus strain. Other genes involved in oxidative stress resistance are two thioredoxins, two thioredoxin reductases, and possibly a homologue of the regulatory RNA polymerase-binding protein Spx ( 47). The uptake of polyamines or their precursors may also provide an alternative means of improving oxidative stress tolerance (see above). Acid stress caused by the production of lactic acid may be countered primarily by the action of an H+ transporting ATPase. The presence of two ornithine decarboxylases and several cation:proton antiporters may also assist in the stabilization of intracellular pH, as in L. acidophilus ( 48).

Conclusions

The L. bulgaricus genome sequence reveals a number of features that support the hypothesis of a genome in a rapid phase of evolution. Among the lactobacilli of the acidophilus complex, the species L. delbrueckii is often regarded as atypical because of its strongly differing GC content. This difference has been the reason to rename what was originally called the L. delbrueckii group of lactobacilli and give it the name of a more representative member, L. acidophilus ( 49). The present genome sequence reveals that the observed difference in GC content is mainly due to a difference in GC content at codon position 3, which may be interpreted as the result of recent evolution and justify the conclusion that L. bulgaricus is less atypical than once thought. This conclusion is corroborated by the clear global synteny among the genomes of L. bulgaricus, L. acidophilus, and L. johnsonii, reflecting the presence of a common backbone.

The large number of pseudogenes and other features suggest that the L. bulgaricus genome is in a relatively recent state of evolution and adaptation to the dairy environment. For example, it has been reported that L. bulgaricus will deplete available folate sources rather than produce folate ( 50), an essential component of the human diet. The present genome sequence shows that all of the genes of the folate pathway are present and seemingly intact in L. bulgaricus ATCC11842.

In the light of a new turn in modern microbiological research with an increasing interest for the metagenome of the human gastrointestinal tract, the functional comparison of the industrial bacterium L. bulgaricus and the closely related bacteria from the gastrointestinal tract, L. acidophilus and L. johnsonii, offers an interesting perspective, particularly because L. bulgaricus has evolved away from the other members of the acidophilus complex by its rapid and specialized adaptation to an environment created by man, fermented milk.

Materials and Methods

Sequencing.

The genome sequence of the L. bulgaricus type strain ATCC11842 was determined by using a shotgun sequencing and assembly strategy followed by multiplex long accurate PCR ( 51) and gap filling. Two types of libraries with inserts of 5 kbp (obtained by mechanical shearing) and 10 kbp (obtained by enzymatic digestion) were constructed in pcDNA2.1 (Invitrogen) and pBeloBAC11 (California Institute of Technology), respectively. Plasmid insert ends and PCR products were sequenced by using dye-primer and dye-terminator chemistries on LICOR4200L and ABI3700/ABI3730 sequencers. The genome sequence has been submitted to the European Molecular Biology Laboratory database under accession no. CR954253.

Annotation.

A draft annotation of the genome sequence was generated by using self-training gene detection software (show, http://ssb2.jouy.inra.fr/ssb/SHOW) and an interface for annotation developed at Institut National de la Recherche Agronomique (Agmial: K.B., V.L., R.B., P.N., S. Chaillou, M.v.d.G., S.P., E.M., M. Hoebeke, P.B., and J.-F.G., unpublished data). This draft annotation was manually checked and was updated where appropriate by using the tools provided in the Institut National de la Recherche Agronomique interface and the ergo suite developed by Integrated Genomics ( 52).

Comparative Genome Analysis.

Comparison of predicted L. bulgaricus proteins with predicted proteins from L. acidophilus and L. johnsonii and analysis of global synteny between the genomes of these bacteria were performed by using blast score ratio analysis software provided by Rasko et al. ( 25).

More details on the methods used are presented as Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Acknowledgments

We thank A. Sorokin, P. Serror, A. Fernandez, C. Chervaux, and T. Smokvina for useful discussions and N. Galleron, B. Quinquis, V. Brachet, M.-C. Beaussart, J. Musset, and L. Prieux for technical assistance. The work of S.P. and C.G. was in part financed by Danone Vitapole.

Footnotes

†To whom correspondence should be addressed. E-mail: maarten.vandeguchte{at}jouy.inra.fr

↵¶ Present address: Department of Computer Science, University College London, London WC1E 6BT, United Kingdom.

Author contributions: M.v.d.G., S.P., C.G., V.B., C.R., S.O., S.M., R.D., S.D.E., and E.M. designed research; M.v.d.G., S.P., C.G., V.B., C.R., S.O., S.M., and R.D. performed research; K.B., P.N., A.C., V.L., R.B., J.-M.B., T.W., J.-F.G., P.B., and J.W. contributed new reagents/analytic tools; M.v.d.G., S.P., C.G., V.B., P.N., C.R., S.O., S.M., R.D., A.B., S.D.E., and E.M. analyzed data; and M.v.d.G. and E.M. wrote the paper.

↵ †† Sasaki, T., Ito, Y. & Sasaki, Y. (1993) FEMS Microbiol. Rev. 12, P8.

Conflict of interest statement: No conflicts declared.

Data deposition: The L. bulgaricus genome sequence has been submitted to the European Molecular Biology Laboratory database (accession no. CR954253).

Abbreviations:

Abbreviations:

CDS,

coding sequences;

IS,

insertion sequence.

View Abstract

# data[].attributes.attachment_link.title
The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution

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Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus ) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions.

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Animal feed and Lactobacillus bulgaricus

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Animal feed and Lactobacillus bulgaricus

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Hi all . Check this research article. They use Lactobacillus bulgaricus in animal feed! Share your thought !

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Voges-Proskauer Test - Amrita University

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▶ This video channel is developed by Amrita University's CREATE http://www.amrita.edu/create ▶ Subscribe @ https://www.youtube.com/user/amritacreate http://www.youtube.com/amritavlab ▶ Like us @ https://www.facebook.com/CREATEatAmrita ▶ For more Information @ http://vlab.amrita.edu/index.php?sub=3&brch=76&sim=215&cnt=1 ▶ Amrita Virtual Lab Project website http://vlab.amrita.edu The Voges-Proskauer test determines the capability of some organisms to produce non acidic or neutral end products, such as acetyl methyl carbinol, from the organic acids that result from glucose metabolism.

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Biochemical tests of bacteria (Oxidase Test, Indole Production, Coagulase test, MR-VP test, citrate utilization test, urease test, catalase production, motility test, fermentation of carbohydrates, nitraye reduction test)

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Hydrogen sulphide test

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Voges- Proskauer test

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NaCl tolerance test

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Methyl red test

http://www.vumicro.com/vumie/help/VUMICRO/Methyl_red_Test.htm

Gram stain test

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Each of the members do our parts and share the information as mind map in the Popplet.

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Popplet is the best app for sharing visual ideas.

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Zoom meeting with the members

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First zoom meeting with the group members. Introduction , expalanation about apps that we are going to use and our parts.