APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2005, p. 354–362 Vol. 71, No. 1


Interactions between Oral Bacteria: Inhibition of Streptococcus mutans
Bacteriocin Production by Streptococcus gordonii

Bing-Yan Wang and Howard K. Kuramitsu

First, let me get this off my chest: I'm very excited about this paper, even before I read it. I wish, I really really wish, they had used colicins instead of gram + bacteriocins, but still ... this is a project I've been considering a long time, whether I can demonstrate that bacteriocins are modulated by quorum sensing.

Clearly, I had missed:
Kleerebezem, M., and L. E. Quadri. 2001. Peptide pheromone-dependent
regulation of antimicrobial peptide production in Gram-positive bacteria: a
case of multicellular behavior. Peptides 22:1579–1596.

So, on with the paper...

The Big Picture:
Bacteriocins, specifically colicins, are a topic I talk and write about often, so I'm providing an 'attenuated' version of the topic here. These are antibiotics, but different from the normal antibiotics we use in the pharacopiea.
1. They are produced by bacteria or eubacteria, not fungi or streptomycetes. While yeast and paramecium both have things that may be ecologically analagous (killing factors) the bacteriocins proper are not produced by them.
2. They are proteins. The classical antibiotics are small organic molecules. Many other antagonistic agents are inorganic molecules (peroxide, acid, etc).
3. They are narrow spectrum. While acidifying the environment may kill lots of things, and penicillin targets a broad range of bacteria, the bacteriocins target a small group.
4. This group is related to the organism producing the colicin, and thus is a relatively direct competitor.
5. Many of the bacteriocins are born on plasmids. This used to be a significant trait but is no longer, because we keep discovering them in the chromosome. Of the plasmids, there are large and small plasmids, so there is no particular rhyme or reason to that trait.
6. The bacteriocins require immunity proteins to keep from killing other cells that carry the bacteriocin genes.
7. The bacteriocins require some sort of bacteriocin release protein, otherwise known as BRP or the lysis protein. This kills the cell when the bacteriocin is released.
8. Bacteriocins are generally regulated by the SOS DNA damage response pathway, among others.
9. There are several kinds of bacteriocins with respect to structure. The classic colicins have three domains: one for target acquisition (the receptor binding domain), one for molecular judo (the translocation domain), and one for the coup de grace (the killing domain). Other bacteriocins may be too small for all this structure (like the microcins).
10. Naming a bacteriocin means taking the target species/genus name and roughly adding -cin to it.

So, with that background out of the way... we have suicidal cells that produce toxins that kills closely related cells, expect those that happen to share the toxin genes. Pretty complicated. How does the bacteria know when to release it? Suicide is pretty drastic if there is not threat around! Also, why would one single bacteria want to commit suicide, if there aren't relatives around to use the newly reserved resources?
So, it would make sense that possibly the bacteriocins are under some sort of quorum sensing control. After all, the current known controls for colicins (SOS) are pretty rudimentary. They only turn on in pretty much certain cell death circumstances. And yet, this doesn't square with experience. Colicins are produced by about 1% of the population all the time in a lab culture, and it goes up in stationary phase, to maybe 3%... but which 1%/3%? And how and why? Random? Lethal mutants? Additional control pathways?

This paper sets out to demonstrate that a particular bacteriocin of Stept. mutans (an important resident of the dental flora) is regulated by quorum sensing. They use the classic methods, both on agar and in broth, to estimate bacteriocin production. They also use sequencing and mutagenesis to demonstrate the involvement of the particular quorum sensing pathway.
The use of these mutants allowed them to figure out which genes were necessary for stimulating the system and which for detecting the quorum sensing molecule. It also allowed them to separate production of the molecule from production of the bacteriocin, so they could add back the chemical in a controlled fashion. They proved their point beyond a shadow of a doubt. Bacteriocin production is positively regulated by quorum sensing in Strept. mutans.

Interestingly, some of the bacteria involved secrete something into broth that is heat liable and destroys the quorum sensing molecule, not the bacteriocin. This is an interesting observation for any number of reasons. It isn't something included in that many models of competition, but it makes sense - if reliable communication is an advantage, disrupting it removes that advantage. In fact, by making the communication unreliable, it changes the entire course of selective pressures, possibly adding a need for back-up communication. Another way to make it unreliable would be to generate false signals... clearly, there is a significant lack of understanding of the difficult environment that bacteria navigate with respect to their intercellular communication.
They also found that the communication was very robust. Large amounts of the molecule were produced but only small amounts were needed to stimulate full bacteriocin production (or, maximally stimulated, at least). This indicates that the protease must be very effective. Attempts to knock out one protease did not eliminate the full proteolysis, presumably because there are others working as well. The system gets more and more complex as you look longer and harder.

As an aside, effectively, there was also some methods development as they studied the impact of different media on the bacteriocin production. What this indicates (to me) is less of in vivo importance and more about the difficulty in designing good experimental systems for in vitro bacteriocin studies, given the huge impact of relatively minor changes to the media.

There are also issues because biofilm formation is regulated by quorum sensing, and so things may be different in broth or on agar, or on other surfaces. This was briefly explored, but quickly becomes a whole other set of studies. The findings of this paper were more or less insensitive to biofilms, but then, the populations weren't mixed either, nor was it an oral model system.

This paper was extremely provacative and interesting, adding new factors to any decent model of bacteriocin production, regulation, and ecological function. One wonders whether the proteases eating the quorum sensing molecules would be important in limiting bacteriocin production. One option is that the cells producing the quorum sensing molecules are so close together and the production level is so high relative to the saturation levels for detection that the degredation doesn't matter. What it might do is insulate neighboring populations from each other's signals, making the whole system much MORE robust but not effectively limiting bacteriocin production (especially given that sensitive cells may not approach the producing colonies due a halo of toxin).
Some theories in the past suggested that bacteriocins were limited in effectiveness because of other exogenous proteases, in the intestines or mouth, for example, but this was largely a response to flawed in vivo studies that couldn't detect bacteriocin effectiveness, and so that hypothesis has been set aside.

All told, this paper is an important contribution to the field of bacteriocin research. It is thorough, within its scope, and seems to be very useful. I hope to see it built upon in the near future.

Scarce Evidence of Yogurt Lactic Acid Bacteria in Human Feces after Daily Yogurt Consumption by Healthy Volunteers
Rosa del Campo, Daniel Bravo, Rafael Cantón, Patricia Ruiz-Garbajosa, Raimundo García-Albiach, Alejandra Montesi-Libois, Francisco-Javier Yuste, Victor Abraira, and Fernando Baquero
Applied and Environmental Microbiology, January 2005, p. 547-549, Vol. 71, No. 1

Following the same theme as the last post, I'm discussing this article about probiotic yogurt bacteria that has a much less rosy take on the impact of the probiotics themselves. It also is a very differently run study.

First, the study is a paragon of design. A huge number of volunteers 114, with all sorts of medical tests prior to the experiment. Pasteurized and unpasteurized yogurts done double blind, and, after a 2 week 'no yogurt' period, reversed in the same patients. A control group with no yogurt. Admittedly, there were only three samples per patient - one to start and one after each time period - but there is also a much simpler question being asked: can we detect the yogurt bacteria in the feces?

This is less important then it might sound, given that the lactobacilli are probably most important in the small intestine, and most will be digested in the large instestine should they wash out of the small intestine and die. However, in the case of a successful colonization, there would be expected to be some shedding (after all, how would lactobacilli get from person to person, otherwise?) and they are looking for 'detectable presence' rather than expecting overwhelming numbers.

They used all sorts of molecular and culture means to detect the bacteria. Most of these detection methods failed. PCR detected the bacteria not a single time, despite being able to detect them in a mixture of yogurt and feces 100,000:1 (I said this was good research, not fun research). Concentrations down to 10^3/gm feces would have been detected via culture - they were not detected either. This is 100-1000 times less than the typical E. coli concentration, and easily 1 part in 10^9 of the total population. As for DNA hybridization, the most sensitive method, it detected the DNA in less than 10 patients for each round. The pasteurized yogurt contributed 1 of the positive signals for each type of lactic acid bacteria under study, the others contributed about 7 or so - many times more, but not a good percentage of the 114 volunteers, given their daily consumption of the yogurt.

In short, if the bacteria are colonizing the patients, they are doing so in a way that does not result in stable fecal shedding at normally detectable levels. It is considered probable that the DNA hybridization was due to the DNA already present in the yogurt, not the reproduction of the bacteria.

The authors wisely do say they don't know whether the bacteria reproduce in the small intestine. However, they have made their point, that these "probiotic bacteria" are not replicating extensively. If the yogurt is beneficial, it is not because it is acting like an inoculum to the large intestine, certainly, and probably not to the small intestine either. They speculate it might be a 'prebiotic' - giving nutrients to existing bacteria.


The results would possibly be quite different in an antibiotic treated host. However, this study punches a hole or two in common hypotheses about the healthful effects of yogurt and how they inoculate the host. It was a great study, but must have been quite a bit of work for all those negative PCR products. Good job!

Monitoring of Antibiotic-Induced Alterations in the Human Intestinal Microflora and Detection of Probiotic Strains by Use of Terminal Restriction Fragment Length Polymorphism

Cecilia Jernberg, Åsa Sullivan, Charlotta Edlund, and Janet K. Jansson
Applied and Environmental Microbiology, January 2005, p. 501-506, Vol. 71, No. 1

The Big Picture:
Antibiotics are a great tool for treating bacterial infections, but they are something of an atomic bomb. Broad spectrum antibiotics kill all sorts of things, intended and unintended. Some of the unintended victims are happy symbiotic Lactobacillus and other lactic acid bacteria that love the pH 5 and rich nutrient soup of the small intestine. They help us digest, enrich our lives with vitamins, and so on. People who have taken antibiotics often get various forms of diahrrea, as well as other GI distress, because they have killed their nice symbiotes.

To address this, scientists have recommended investigating "probiotics." These are organisms that help out around the body. The simplest case are some nice lactic acid bacteria to enhance our normal flora, or even replace those wiped out by antibiotics. These lactic acid bacteria are found in sources like kefir, yogurt, sourdough, saurkraut, pickles, etc (only in unpasturized ones!).

Probiotics are not to be confused with prebiotics, which are nutrients meant to encourage the growth of symbiotic bacteria particularly, or biochemical precursors to good things that symbiotes produce for us. Prebiotics include various starches and sugars.

The fields of prebiotics and probiotics have been notoriously sketchy. Sometimes its a quack trying to prove how his "special blend" of bacteria is good for you. Scientists, too, take shortcuts in this field, getting some uncharacterized mess of soil bacteria or food bacteria and trying to demonstrate benefits. Industry hasn't stayed out of the fray, and the natural/bioactive food people are always making unsubstantiated claims about their products. The 'real' dairy industry is happy enough to glom on - not that their products aren't healthful, but just that they haven't necessarily found ideal bacteria for any form of probiotic therapy. If those lactic acid bacteria do you any good at all, it is pretty much dumb luck.

So, recently, there have been some efforts to improve the science behind the probiotics. Some of the big problems: the intestinal flora is hard to sample, hard to observe, and difficult to characterize. Most of the relevant bacteria are not cultured, their properties are unknown, and they change in frequency and growth state rapidly as they move around in the intestines, not to mention in to the field and laboratory. As a result, things like fecal samples are possibly irrelevent, maybe even misleading, when studying the small intestine - and further, even if the samples were good, the means by which they are studied (culturing) hits a relatively small number of the strains, not necessarily the important ones.

Some of these things are changing, particularly with the advent of molecular techniques to characterize strains and communities. Not that these techniques don't have an amazing number of problems... just that they have fewer, potentially, than the culture techniques.

This particular study uses 8 patients. All were given clindamycin, four were given the probiotic in addition. Molecular methods and culture methods were both employed to track the populations in the patients. The molecular method chosen was T-RFLP.
This method basically uses the rRNA as the marker for populations and creates an easy to read fingerprint of the rRNAs to track the populations in a quantifiable fashion.
Samples were taken on three days, first: before antibiotic treatment second: during antibiotic treatment (7 days) third: after all the therapy was over (21 days)
The yogurt was consumed for the first 14 days (both during and after the antibiotic administration).

This is one of the weaknesses with the study... only three sample points per patient. It would have been very interesting to inspect the variance with a little more detail, say, two or three points during treatment, as well as two or three afterwards and two or three before. Also, the yogurt was given for a full week after the antibiotic treatment ended. It would have been very interesting to see a sample taken the day that concluded, rather than merely a week afterward.

Regardless, their statistical analysis appears very impressive, with principal componants and little three element dendrograms. What they are trying to demonstrate with all that is that the probiotic helped the community come back to normal, more or less, after the antibiotic administration ended.

Unfortunately, I am willing to presume that the populations changed - but I don't feel convinced of that by the data. Were I skeptical, all I'd have to say is that they haven't given any sense of the baseline variance in the populations. They would have needed day -14, -7, and 0 or something like that, to give a sense of what their principal componants would do without any perturbation at all.


"The results of the present study demonstrate that T-RFLP is
a useful tool for investigating the human intestinal microflora."

This is the striking result of all this sampling and data analysis. Come on, guys! Can't we at least hear about the probiotics? Well, none of the volunteers had diahrea... so there wasn't much to say about the relative rates of negative side effects from the clindamycin. And big changes in fecal flora due to clindamycin had already been published elsewhere, so that couldn't be their point. And they didn't take the appropriate samples to make a claim about the probiotic, even though they hint at such a claim in the Results section.
Thus, their lack of an impressive conclusion really may spring from the fact that the experiment was not well designed in the above respect. Their entire discussion hinges on the question of whether the molecular methods were better than the culture based ones (they were) and whether they were able to differentiate populations (they were) and whether the relative quantification is good enough to be worth the trouble, given that absolute numbers are obtained via culture methods (yes, relative is useful). So, it becomes, in the end, a methods paper with a hint of medical application.

Frustrating, but still nice to have in the literature. Unfortunate, though, because if they had done even 1 more sample (to get a baseline), they might have been able to demonstrate a statistically significant change in the population, etc.

Mercury Adaptation among Bacteria from a Deep-Sea Hydrothermal Vent
Costantino Vetriani, Yein S. Chew, Susan M. Miller, Jane Yagi, Jonna Coombs, Richard A. Lutz, and Tamar Barkay
Appl. Environ. Microbiol. 2005. 71: 220-226.

The Big Picture:
Mercury contamination from industrial sources is a huge issue, as are, in fact, all forms of heavy metal contamination of surface waters. Lead, cadmium, mercury, copper... they are all pretty toxic to people drinking the water, to fish, etc, and they tend to be biologically magnified as they are concentrated up the trophic levels. That is, they persist in an animal, and are passed to the thing that eats that animal. Man, at the top of many food chains, gets a whopping dose.

So, one idea that is popular is bioremediation, with plants or bacteria taking the heavy metals out of the water or soil and 'sequestering' them in some easily harvested or bio-inavailable form. If harvested, then the waste can be contained. If bio-inavailable, then the material becomes inert and no longer an immediate threat.

Anyway, the hunt for plants and bacteria that do things with heavy metals has been long. Along the way, we have learned much about the processing and cycling of metals in biological systems. This paper picks up there, with the realization that there are some cool things happening at the deep sea hydrothermal vents.

These vents are like mini-volcanoes on the ocean floor, where hot water comes out of the crust into the otherwise frigid, lightless, and pretty lifeless ocean floor. Bacteria do all sorts of cool things with the chemicals down there to get energy, and they pass that energy to little animals, eventually to bigger and bigger animals. For all sorts of information about symbiosis down there, look to the works of Cavanaugh.

Anyway, one interesting thing is that the hot water sucks cinnabar out of the rocks. Cinnabar is a red mercuric sulfide compound. The hot water sucks out the mercury and then swirls it into the ocean. There is a distinct gradient of mercury in this fashion.

For the non-microbial evolutionary biologist, this seems mildly interesting. However, thinking about enrichment cultures... when you want a bacteria to learn to digest a toxic chemical, you need to start at low concentrations (so it can survive) and steadily increase the concentration to keep challenging it, until it can handle the high concentrations necessary. In nature, by having a steady gradient of mercury in a spatially structured environment, also aligned with a chemo-nutrient gradient, there will be competition for the other chemicals leading the bacteria up the mecury gradient. Ideal conditions for the evolution of mercury processing/decontamination.

Well, that's pretty much what these researchers found. They found that merA was present in a whole bunch of bacteria, even when their isolations didn't select for mercury resistance. They found that mercury resistance was related to the temperature at which the bacteria grew... which makes sense given the gradient of temperature going up towards the vent. They did nice physiological tests and demonstrated the bacteria making the mercury less bioavailable. Then they sequenced a bunch of merA genes and tested the MR protein for optimum temperature. They found that the high temperature bacteria had high temperature MR.

They also found that one of their merA was in another cluster in their tree. This is consistent with putting the root of the merA at the deep sea vents. However, this small tree that they constructed with other known examples of merA was not terribly convincing in and of itself.
Personally, I take issue with both the size of the tree and the method of building it... neighbor joining alone, with some moderate bootstraps (85 at a key node), and not too many merA sequences outside of their experimental samples. I'd prefer to have seen a larger tree with maximum likelihood backing up their conclusions. However, this would require going and isolating other mercury resistant samples, sequencing them, and adding them to the tree. It is pretty far afield for an otherwise sturdy paper.

One place this might be useful is in an industrial setting, where a high temperature bioreactor could be used in a mercury containing waste stream. It might be possible to feed the bacteria and keep the stream at a temperature that discourages outside contamination. Mercury could be pulled out in a particular stage of waste decontamination.

I'd like to see them isolate some more strains and get a bigger merA tree before they really speculate about the evolution. At the same time, this was a fascinating paper on the ecology of the system, and poses some interesting issues. For one, a person might be able to selectively isolate bacteria from different regions of the plume by using crossed mercury/temperature/pressure gradients. This could be a real boon to vent ecologists.

Jolly fun read!