Mammals are complex assemblages of mammalian and bacterial cells organized into functional organs, tissues, and cellular communities. Human biology can no longer concern itself only with human cells: Microbiomes at different body sites and functional metagenomics must be considered part of systems biology. The emergence of metagenomics has resulted in the generation of vast data sets of microbial genes and pathways present in different body habitats. The profound differences between microbiomes in various body sites reveal how metagenomes contribute to tissue and organ function. As next-generation DNA-sequencing methods provide whole-metagenome data in addition to gene-expression profiling, metaproteomics, and metabonomics, differences in microbial composition and function are being linked to health and disease states in different organs and tissues. Global parameters of microbial communities may provide valuable information regarding human health status and disease predisposition. More detailed knowledge of the human microbiome will yield next-generation diagnostics and therapeutics for various acute, chronic, localized, and systemic human diseases.

We identify ef1090 (renamed ebpR) and show its importance for the transcriptional regulation of expression of the Enterococcus faecalis pilus operon, ebpABC. An ebpR deletion (⌬ebpR) mutant was found to have reduced ebpABC expression with loss of pilus production and a defect in primary adherence with, as a consequence, reduced biofilm formation.Enterococcus faecalis is a gram-positive bacterium that is part of the normal intestinal flora of most humans but is also a major cause of opportunistic infection (9). A major regulator of virulence in E. faecalis OG1RF is the Fsr system (15). In a previous microarray analysis, we identified several Fsr-regulated genes encoding putative regulators, one of which was ef1090 (2). EF1090 contains two putative helix-turn-helix DNA-binding domains and shares 42% similarity with AcpB from Bacillus anthracis, a member of the AtxA/MgaA family (4). Upstream and in the opposite orientation of ef1090 is the ebpABC operon, which encodes pilus components shown to be important for virulence in the rat endocarditis model and the mouse urinary tract infection model (13,17).To test whether the putative EF1090 regulator was involved in the regulation of the pili genes, we first performed semiquantitative reverse transcription-PCR (qRT-PCR) (2). The ebpA, ebpB, and ebpC transcripts were strongly reduced in an ef1090 transposon insertion mutant (5) compared to the parent strain (data not shown). Based on these results, ef1090 was renamed ebpR for its role as an endocarditis-and biofilmassociated pilus regulator. To prevent any effects caused by the transposon insertion from complicating our analysis, we then created an unmarked in-frame ebpR deletion mutant (Table 1) of OG1RF by using the recently published PheS* system (7). The ⌬ebpR mutant had no growth defects compared to OG1RF in BHI, TSBG (biofilm medium), or BHI-40% horse serum (binding assay medium).To assess transcriptional differences between OG1RF and the ⌬ebpR mutant, we used microarray analysis with slide preparation (1), probe labeling, hybridization, data acquisition, and statistical analysis performed as described previously (2). RNA was extracted at late log growth phase from TSBG-grown cultures (2). Within the level of detection of the microarray analysis, the ebpABC transcript was undetectable in the ⌬ebpR mutant compared to OG1RF, indicating that ebpR is important for ebpABC expression at the transcriptional level. srtC was not significantly altered in these conditions. We confirmed these results by using qRT-PCR (12). The amount of transcript for each gene of interest was normalized against gyrB transcripts. qRT-PCR showed that ebpA transcripts levels were decreased 119-fold (P ϭ 0.0018) in the ebpR mutant versus the wild type, whereas srtC transcripts were only 1.7-fold decreased (P ϭ 0.028) (Fig. 1A). The slight effect of ebpR on srtC is likely related to the presence of an independent srtC promoter and the low level of readthrough from the ebpA promoter described previously (13). Since basal ebpABC expression was detec...

Bacillus anthracis shares many regulatory loci with the nonpathogenic Bacillus species Bacillus subtilis. One such locus is sinIR, which in B. subtilis controls sporulation, biofilm formation, motility, and competency. As B. anthracis is not known to be motile, to be naturally competent, or to readily form biofilms, we hypothesized that the B. anthracis sinIR regulon is distinct from that of B. subtilis. A genome-wide expression microarray analysis of B. anthracis parental and sinR mutant strains indicated limited convergence of the B. anthracis and B. subtilis SinR regulons. The B. anthracis regulon includes homologues of some B. subtilis SinR-regulated genes, including the signal peptidase gene sipW near the sinIR locus and the sporulation gene spoIIE. The B. anthracis SinR protein also negatively regulates transcription of genes adjacent to the sinIR locus that are unique to the Bacillus cereus group species. These include calY and inhA1, structural genes for the metalloproteases camelysin and immune inhibitor A1 (InhA1), which have been suggested to be associated with virulence in B. cereus and B. anthracis, respectively. Electrophoretic mobility shift assays revealed direct binding of B. anthracis SinR to promoter DNA from strongly regulated genes, such as calY and sipW, but not to the weakly regulated inhA1 gene. Assessment of camelysin and InhA1 levels in culture supernates from sinR-, inhA1-, and calY-null mutants showed that the concentration of InhA1 in the culture supernatant is inversely proportional to the concentration of camelysin. Our data are consistent with a model in which InhA1 protease levels are controlled at the transcriptional level by SinR and at the posttranslational level by camelysin.

Regulating gene expression during infection is critical to the ability of pathogens to circumvent the immune response and cause disease. This is true for the group A Streptococcus (GAS), a pathogen that causes both invasive (e.g., necrotizing fasciitis) and noninvasive (e.g., pharyngitis) diseases. The control of virulence (CovRS) two-component system has a major role in regulating GAS virulence factor expression. The regulator of cov (RocA) protein, which is a predicted kinase, functions in an undetermined manner through CovRS to alter gene expression and reduce invasive disease virulence. Here, we show that the ectopic expression of a truncated RocA derivative, harboring the membrane-spanning domains but not the dimerization or HATPase domain, is sufficient to complement a rocA mutant strain. Coupled with a previous bioinformatic study, the data are consistent with RocA being a pseudokinase. RocA reduces the ability of serotype M1 GAS isolates to express capsule and to evade killing in human blood, phenotypes that are not observed for M3 or M18 GAS due to isolates of these serotypes naturally harboring mutant rocA alleles. In addition, we found that varying the RocA concentration attenuates the regulatory activity of Mg 2ϩ and the antimicrobial peptide LL-37, which positively and negatively regulate CovS function, respectively. Thus, we propose that RocA is an accessory protein to the CovRS system that influences the ability of GAS to modulate gene expression in response to host factors. A model of how RocA interacts with CovRS, and of the regulatory consequences of such activity, is presented.

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.

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Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment, V. cholerae must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of V. cholerae to increases in environmental osmolarity. Optimal growth of V. cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted V. cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted V. cholerae displays no growth advantage when competed against high-osmolarityadapted cells in low-osmolarity medium. These results may have implications for V. cholerae population dynamics when seawater and freshwater and their attendant microbes mix.

Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed "nutritional immunity." Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCEThe mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood-hemoglobin and serum albumin-can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for bacterial pathogens. There are three broad, necessary, and common elements inherent in most if not all bacterial infections of vertebrate hosts. They are (i) the initial entry of the bacterial invader and adherence to host tissues, (ii) the production of virulence factors or toxins (usually proteins) that subvert host innate and acquired immune defense mechanisms, and (iii) the acquisition and construction of biomolecules to drive cellular metabolism for the production of progeny. There has been intense investigation, and knowledge of the biochemistry, cell biology, and contribution to disease of a diverse array of bacterial adhesins and virulence factors (points i and ii identified above) is growing. However, our knowledge of nutrient acquisition during pathogenesis has lagged, perhaps because it is viewed as being a necessary and somewhat mundane process that may lack the elegant sophistication observed with virulence factors. Considering that all systemic infections are absolutely dependent on the replication of bacterial cells, identifying the mechanisms by which ba...