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February 18, 2013

Bacterial Antidepressants: Avoiding Stationary Phase Stress

by S. Marvin Friedman

In bacterial quorum sensing, the absolute number of cells is irrelevant; only the number of bacteria in a given volume plays a role. (Credit: Copyright Wiley-VCH) Source.

High on the list of the exciting manners bacteria communicate with one another is quorum sensing (QS), a population-dependent gene regulation system that operates within a wide range of species. The general scheme of QS is as follows: at high population densities, signal molecules called autoinducers reach threshold levels, at which point they initiate a signal transduction pathway leading to transcription of specific genes. This altered gene expression allows the bacterial community to behave in a cooperative manner so as to achieve a common goal. In a sense, the bacterial population now functions like a multicellular organism. In many Proteobacteria, such as vibrios and pseudomonads, autoinducers are acyl-homoserine lactones (AHLs), compounds synthesized by the LuxI family of signal synthases and detected by the LuxR family of transcriptional regulators. Cellular activities regulated by AHLs include bioluminescence, biofilm formation, motility, and the making of virulence factors, among others. A large body of studies has illuminated the molecular mechanisms underlying QS, but identifying what’s in it for the species has not always been easy. In the paper to be discussed here, Goo and collaborators show how QS enables bacteria to avoid the perils of entry into stationary phase.

Death in the Stationary Phase

The researchers studied three closely-related species of the proteobacteria Burkholderia: the rice pathogen B. glumae, the opportunistic human pathogen B. pseudomallei, and the saprophyte, B. thailandensis. Each of these species has an N-octanoyl homoserine lactone (C8-HSL) signaling system. The basic experiment was to grow these three Burkholderia strains and their corresponding QS signal synthesis mutants in rich LB broth. (Elio pipes in: LB broth is actually a nutrient-scarce medium that supports exponential growth only at low bacterial density. Growth ceases soon, thus this medium may be an OK choice for studies of the stationary phase, all the more so because LB is scarcely buffered, which is relevant to this work. But for studies using growing cultures, LB is an abomination. For an erudite treatment of the limitations of LB medium, see here) The wild type strains survived long periods in stationary phase but the QS mutants died shortly after entering the stationary phase. These crashes were avoided by adding C8-HSL to the culture medium, indicating that QS was somehow involved in preventing death in the stationary phase.

Bacterial panicle blight of rice caused by B. glumae. Source.

So, what causes these stationary phase crashes? Could it be something as simple sounding as changes in the pH of the culture? Sure enough, these strains all die at high pH. To some extent, they bring this upon themselves because as the cultures enter the stationary phase, the pH of the medium rises from 7.0 to 7.5 to 8.0. However, the wild type handles this very well by eventually lowering the pH, but QS mutants can't do that. The pH of their cultures remains high and they therefore die out. Addition of pH 7 HEPES buffer spares the QS mutants from death.

Countering High pH

What makes the pH rise? Other bacteria are known to produce ammonia by the deamination of amino acid in rich media, so could that be it? The authors found that ammonia was continuously produced in both wild type and QS mutant strains. Thus, returning to neutral pH the medium depends on some metabolite that lowers the pH even in the presence of elevated ammonia concentrations. That becomes the name of the game. To find out what is involved, the investigators compared the RNAseq transcriptomes of wild types and QS mutants. The genes activated by QS included a two-gene operon that is responsible for making oxalate (HOOC-COOH) via the enzymes ObcA and ObcB. The researchers hypothesized that the acidic oxalate neutralizes the alkalization caused by ammonia. Indeed, the level of oxalate produced was dramatically lower in the QS mutants than that in the wild type strains. It follows that obcA and obcB mutants should be subject to ammonia-induced toxicity and, as expected, both such mutants exhibited massive population crashes as the pH rose in the stationary phase.

Cell viability and culture medium pH of B. glumae, B. pseudomallei, and B. thailandensis grown in LB broth. A. colony count. B. pH of the culture supernates. Open blue circles: wild type strains of three Burkholderia species. Open orange triangles: C8-HSL synthase mutants. Open green squares: QsmR mutants. Filled red triangles: C8-HSL synthase mutants grown in media containing 1 μm or 4 μm C8-HSL.

Regulation of Quorum Sensing

Is activation of the obc operon by QS direct or indirect? Transcriptome analysis showed that in addition to a functional C8-HSL QS system, a regulator called QsmR is required for turning on obcA and obcB. Thus, the obc genes belong to the QsmR regulon, which is activated by QS. They confirmed it by measuring the expression levels of obcA and obcB from a chromosomal obcAB-gusA transcriptional fusion in B. glumae. Indeed, QsmR directly interacts with the obcA-B promoter. Since low levels of obcAB expression were found in both qsmR and QS mutants, it follows that qsmR mutants must fail to accumulate oxalate. Indeed, qsmR mutant population crashes were found to correlate with elevated pH and the absence of oxalate production.

The results presented in this study show that QS in Burkholderia controls a battery of genes, including qsmR, and that the qsmR product directly activates oxalate production by obcAB. Oxalate, a dianionic acid formed by the condensation of acetyl-CoA and oxaloacetate derived from the TCA cycle, is an ideal agent for neutralizing the ammonia formed by the deamination of amino acids in the stationary phase. Regulating the pH also matters for another reason: AHLs are degraded above pH 8.0, thus prevention of alkalization is needed to protect these QS signal molecules. Convincing though these results may be, the question can be asked: do these organisms ever find themselves in a natural high pH environment where oxalate production would be protective? A good guess is that at least B. glumae, being a plant pathogen, encounters alkaline conditions in the host’s apoplast (the space that we would call the periplasm in bacteria). Plants respond to infection by locally raising the pH. Interestingly, of 200 isolates of B. glumae, all the 180 pathogenic strains made oxalate, whereas the remaining non-virulent 20 did not. This suggests that oxalate formation is important for survival in the host and therefore required for the organisms to be pathogenic.

Is This a General Phenomenon?

It should be noted that this remarkable regulation system that both anticipates and counteracts stationary phase stress is not universal: many bacteria are endowed with QS-independent homeostatic mechanisms for controlling pH fluctuations. Furthermore, alkalization is not always the determining factor for initiating population crashes in the stationary phase. It would be interesting to know when the Burkholderia system emerged during the course of bacterial evolution, its genetic origin (horizontal gene transfer?), and how widespread it is in the microbial world.

Friedman, Marvin_sm

S. Marvin Friedman is Professor Emeritus, Department of Biological Sciences, Hunter College of CUNY, New York City and an Associate Blogger for Small Things Considered.

Goo E, Majerczyk CD, An JH, Chandler JR, Seo YS, Ham H, Lim JY, Kim H, Lee B, Jang MS, Greenberg EP, & Hwang I (2012). Bacterial quorum sensing, cooperativity, and anticipation of stationary-phase stress. Proceedings of the National Academy of Sciences of the United States of America, 109 (48), 19775-80 PMID: 23150539


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