Aspects of oral streptococcal metabolic diversity: Imagining the landscape beneath the fog.

It is widely acknowledged that the human-associated microbial community influences host physiology, systemic health, disease progression, and even behavior. There is currently an increased interest in the oral microbiome, which occupies the entryway to much of what the human initially encounters from the environment. In addition to the dental pathology that results from a dysbiotic microbiome, microbial activity within the oral cavity exerts significant systemic effects. The composition and activity of the oral microbiome is influenced by (1) host-microbial interactions, (2) the emergence of niche-specific microbial "ecotypes," and (3) numerous microbe-microbe interactions, shaping the underlying microbial metabolic landscape. The oral streptococci are central players in the microbial activity ongoing in the oral cavity, due to their abundance and prevalence in the oral environment and the many interspecies interactions in which they participate. Streptococci are major determinants of a healthy homeostatic oral environment. The metabolic activities of oral Streptococci, particularly the metabolism involved in energy generation and regeneration of oxidative resources vary among the species and are important factors in niche-specific adaptations and intra-microbiome interactions. Here we summarize key differences among streptococcal central metabolic networks and species-specific differences in how the key glycolytic intermediates are utilized.

Disruption of the adh (Acetoin Dehydrogenase) Operon Has Wide-Ranging Effects on Streptococcus mutans Growth and Stress Response.

The agent largely responsible for initiating dental caries, Streptococcus mutans, produces acetoin dehydrogenase that is encoded by the adh operon. The operon consists of the adhA and B genes (E1 dehydrogenase), adhC (E2 lipoylated transacetylase), adhD (E3 dihydrolipoamide dehydrogenase), and lplA (lipoyl ligase). Evidence is presented that AdhC interacts with SpxA2, a redox-sensitive transcription factor functioning in cell wall and oxidative stress responses. In-frame deletion mutations of adh genes conferred oxygen-dependent sensitivity to slightly alkaline pH (pH 7.2-7.6), within the range of values observed in human saliva. Growth defects were also observed when glucose or sucrose served as major carbon sources. A deletion of the adhC orthologous gene, acoC gene of Streptococcus gordonii, did not result in pH sensitivity or defective growth in glucose and sucrose. The defects observed in adh mutants were partially reversed by addition of pyruvate. Unlike most 2-oxoacid dehydrogenases, the E3 AdhD subunit bears an N-terminal lipoylation domain nearly identical to that of E2 AdhC. Changing the lipoyl domains of AdhC and AdhD by replacing the lipoate attachment residue, lysine to arginine, caused no significant reduction in pH sensitivity but the adhDK43R mutation eliminating the lipoylation site resulted in an observable growth defect in glucose medium. The adh mutations were partially suppressed by a deletion of rex, encoding an NAD+/NADH-sensing transcription factor that represses genes functioning in fermentation. spxA2 adh double mutants show synthetic growth restriction at elevated pH and upon ampicillin treatment. These results suggest a role for Adh in stress management in S. mutans. IMPORTANCE Dental caries is often initiated by Streptococcus mutans, which establishes a biofilm and a low pH environment on tooth enamel surfaces. The current study has uncovered vulnerabilities of S. mutans mutant strains that are unable to produce the enzyme complex, acetoin dehydrogenase (Adh). Such mutants are sensitive to modest increases in pH to 7.2-7.6, within the range of human saliva, while a mutant of a commensal Streptococcal species is resistant. The S. mutans adh strains are also defective in carbohydrate utilization and are hypersensitive to a cell wall-acting antibiotic. The studies suggest that Adh could be a potential target for interfering with S. mutans colonization of the oral environment.

Increased Oxidative Stress Tolerance of a Spontaneously Occurring perR Gene Mutation in Streptococcus mutans UA159.

The ability of bacteria, such as the dental pathogen Streptococcus mutans, to coordinate a response against damage-inducing oxidants is a critical aspect of their pathogenicity. The oxidative stress regulator SpxA1 has been demonstrated to be a major player in the ability of S. mutans to withstand both disulfide and peroxide stresses. While studying spontaneously occurring variants of an S. mutans ΔspxA1 strain, we serendipitously discovered that our S. mutans UA159 host strain bore a single-nucleotide deletion within the coding region of perR, resulting in a premature truncation of the encoded protein. PerR is a metal-dependent transcriptional repressor that senses and responds to peroxide stress such that loss of PerR activity results in activation of oxidative stress responses. To determine the impact of loss of PerR regulation, we obtained a UA159 isolate bearing an intact perR copy and created a clean perR deletion mutant. Our findings indicate that loss of PerR activity results in a strain that is primed to tolerate oxidative stresses in the laboratory setting. Interestingly, RNA deep sequencing (RNA-Seq) and targeted transcriptional expression analyses reveal that PerR offers a minor contribution to the ability of S. mutans to orchestrate a transcriptional response to peroxide stress. Furthermore, we detected loss-of-function perR mutations in two other commonly used laboratory strains of S. mutans, suggesting that this may be not be an uncommon occurrence. This report serves as a cautionary tale regarding the so-called domestication of laboratory strains and advocates for the implementation of more stringent strain authentication practices.IMPORTANCE A resident of the human oral biofilm, Streptococcus mutans is one of the major bacterial pathogens associated with dental caries. This report highlights a spontaneously occurring mutation within the laboratory strain S. mutans UA159 found in the coding region of perR, a gene encoding a transcriptional repressor associated with peroxide tolerance. Though perR mutant strains of S. mutans showed a distinct growth advantage and enhanced tolerance toward H2O2, a ΔperR deletion strain showed a small number of differentially expressed genes compared to the parent strain, suggesting few direct regulatory targets. In addition to characterizing the role of PerR in S. mutans, our findings serve as a warning to laboratory researchers regarding bacterial adaptation to in vitro growth conditions.

Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis.

Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP α subunit (αCTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in αCTD severely impaired Spx-activated transcription. Attempts to substitute alanine for αCTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where "Y263C" indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound αCTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli αD259, which has been implicated in αCTD-σ70 interaction (σ70 R603, corresponding to R362 of B. subtilis σA). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while αCTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-αCTD complex.IMPORTANCE Though extensively studied in Escherichia coli, the role of αCTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis Here, we conduct phenotypic analyses of putatively lethal αCTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-αCTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several αCTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

Use of Highly Specific Molecular Markers Reveals Positive Correlation between Abundances of Mesodinium cf. major and Its Preferred Prey, Teleaulax amphioxeia, During Red Water Blooms in the Columbia River Estuary.

In a previous study, Teleaulax amphioxeia-the preferred prey of Mesodinium in the Columbia River estuary-were undetectable within intense annual blooms, suggesting blooms are prey-limited or prey are acquired outside of bloom patches. We used a novel molecular approach specifically targeting the prey (i.e., Unique Sequence Element [USE] within the ribosomal RNA 28S D2 regions of T. amphioxeia nucleus and nucleomorph) in estuarine water samples acquired autonomously with an Environmental Sample Processor integrated within a monitoring network (ESP-SATURN). This new approach allowed for both more specific detection of the prey and better constraint of sample variability. A positive correlation was observed between abundances of M. cf. major and T. amphioxeia during bloom periods. The correlation was stronger at depth (> 8.2 m) and weak or nonexistent in the surface, suggesting that predator-prey dynamics become uncoupled when stratification is strong. We confirmed exclusive selectivity for T. amphioxeia by M. cf. major and observed the incorporation of the prey nucleus into a 4-nuclei complex, where it remained functionally active. The specific biomarker for T. amphioxeia was also recovered in M. cf. major samples from a Namibian coastal bloom, suggesting that a specific predator-prey relationship might be widespread between M. cf. major and T. amphioxeia.

Evidence that Oxidative Stress Induces spxA2 Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation.

Bacillus anthracis possesses two paralogs of the transcriptional regulator, Spx. SpxA1 and SpxA2 interact with RNA polymerase (RNAP) to activate the transcription of genes implicated in the prevention and alleviation of oxidative protein damage. The spxA2 gene is highly upregulated in infected macrophages, but how this is achieved is unknown. Previous studies have shown that the spxA2 gene was under negative control by the Rrf2 family repressor protein, SaiR, whose activity is sensitive to oxidative stress. These studies also suggested that spxA2 was under positive autoregulation. In the present study, we show by in vivo and in vitro analyses that spxA2 is under direct autoregulation but is also dependent on the SpxA1 paralogous protein. The deletion of either spxA1 or spxA2 reduced the diamide-inducible expression of an spxA2-lacZ construct. In vitro transcription reactions using purified B. anthracis RNAP showed that SpxA1 and SpxA2 protein stimulates transcription from a DNA fragment containing the spxA2 promoter. Ectopically positioned spxA2-lacZ fusion requires both SpxA1 and SpxA2 for expression, but the requirement for SpxA1 is partially overcome when saiR is deleted. Electrophoretic mobility shift assays showed that SpxA1 and SpxA2 enhance the affinity of RNAP for spxA2 promoter DNA and that this activity is sensitive to reductant. We hypothesize that the previously observed upregulation of spxA2 in the oxidative environment of the macrophage is at least partly due to SpxA1-mediated SaiR repressor inactivation and the positive autoregulation of spxA2 transcription. IMPORTANCE: Regulators of transcription initiation are known to govern the expression of genes required for virulence in pathogenic bacterial species. Members of the Spx family of transcription factors function in control of genes required for virulence and viability in low-GC Gram-positive bacteria. In Bacillus anthracis, the spxA2 gene is highly induced in infected macrophages, which suggests an important role in the control of virulence gene expression during the anthrax disease state. We provide evidence that elevated concentrations of oxidized, active SpxA2 result from an autoregulatory positive-feedback loop driving spxA2 transcription.

Adaptor bypass mutations of Bacillus subtilis†spx suggest a mechanism for YjbH-enhanced proteolysis of the regulator Spx by ClpXP.

The global regulator, Spx, is under proteolytic control exerted by the adaptor YjbH and ATP-dependent protease ClpXP in Bacillus subtilis. While YjbH is observed to bind the Spx C-terminus, YjbH shows little affinity for ClpXP, indicating adaptor activity that does not operate by tethering. Chimeric proteins derived from B. subtilis AbrB and the Spx C-terminus showed that a 28-residue C-terminal section of Spx (AbrB28), but not the last 12 or 16 residues (AbrB12, AbrB16), was required for YjbH interaction and for ClpXP proteolysis, although the rate of AbrB28 proteolysis was not affected by YjbH addition. The result suggested that the YjbH-targeted 28 residue segment of the Spx C-terminus bears a ClpXP-recognition element(s) that is hidden in the intact Spx protein. Residue substitutions in the conserved helix α6 of the C-terminal region generated Spx substrates that were degraded by ClpXP at accelerated rates compared to wild-type Spx, and showed reduced dependency on the YjbH activity. The residue substitutions also weakened the interaction between Spx and YjbH. The results suggest a model in which YjbH, through interaction with residues of helix α6, exposes the C-terminus of Spx for recognition and proteolysis by ClpXP.

spxA2, encoding a regulator of stress resistance in Bacillus anthracis, is controlled by SaiR, a new member of the Rrf2 protein family.

Spx, a member of the ArsC (arsenate reductase) protein family, is conserved in Gram-positive bacteria, and interacts with RNA polymerase to activate transcription in response to toxic oxidants. In Bacillus anthracis str. Sterne, resistance to oxidative stress requires the activity of two paralogues, SpxA1 and SpxA2. Suppressor mutations were identified in spxA1 mutant cells that conferred resistance to hydrogen peroxide. The mutations generated null alleles of the saiR gene and resulted in elevated spxA2 transcription. The saiR gene resides in the spxA2 operon and encodes a member of the Rrf2 family of transcriptional repressors. Derepression of spxA2 in a saiR mutant required SpxA2, indicating an autoregulatory mechanism of spxA2 control. Reconstruction of SaiR-dependent control of spxA2 was accomplished in Bacillus subtilis, where deletion analysis uncovered two cis-elements within the spxA2 regulatory region that are required for repression. Mutations to one of the sequences of dyad symmetry substantially reduced SaiR binding and SaiR-dependent repression of transcription from the spxA2 promoter in vitro. Previous studies have shown that spxA2 is one of the most highly induced genes in a macrophage infected with B. anthracis. The work reported herein uncovered a key regulator, SaiR, of the Spx system of stress response control.

Management of oxidative stress in Bacillus.

The spore-forming bacterium and model prokaryotic genetic system, Bacillus subtilis, is extremely useful in the study of oxidative stress management through proteomic and genome-wide transcriptomic analyses, as well as through detailed structural studies of the regulatory factors that govern the oxidative stress response. The factors that sense oxidants and induce expression of protective activities include the PerR and OhrR proteins, which show acute discrimination for their peroxide stimuli, whereas the general stress control factor, the RNA polymerase sigma(B) subunit and the thiol-based sensor Spx, govern the protective response to oxidants under multiple stress conditions. Some specific and some redundant protective mechanisms are mobilized at different stages of the Bacillus developmental cycle to deal with vulnerable cells in stationary-phase conditions and during spore germination and outgrowth. An important unknown is the nature and influence of the low-molecular-weight thiols that mediate the buffering of the redox environment.

Residue substitutions near the redox center of Bacillus subtilis Spx affect RNA polymerase interaction, redox control, and Spx-DNA contact at a conserved cis-acting element.

Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP α subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at −44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP σA subunit from a −35-like element upstream of the trxB (thioredoxin reductase) promoter to positions −36 and −11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP.

Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis.

Spx activates transcription initiation in Bacillus subtilis by directly interacting with the C-terminal domain of the RNA polymerase (RNAP) holoenzyme α subunit, which generates a complex that recognizes the promoter regions of genes within the Spx regulon. Many Gram-positive species possess multiple paralogs of Spx, suggesting that two paralogous forms of Spx could simultaneously contact RNAP. The composition of Spx/RNAP was examined in vitro using an Spx variant (SpxΔCHA) bearing a 12-amino-acid deletion of the C terminus (SpxΔC) and a hemagglutinin (HA) epitope tag and Spxc-Myc, a full-length Spx with a C-terminal myelocytomatosis oncoprotein (c-Myc) epitope tag. All Spx/RNAP complexes bearing deletion or C-terminal-tagged variants were transcriptionally active in vivo and in vitro. Reaction mixtures containing SpxΔCHA and Spxc-Myc combined with RNAP were applied to either anti-HA or anti-c-Myc affinity columns. Eluted fractions contained RNAP with only one of the epitope-tagged Spx derivatives. The resin-bound RNAP complex bearing a single epitope-tagged Spx derivative was transcriptionally active. In vivo production of SpxΔC and SpxΔCHA followed by anti-HA affinity column chromatography of a cleared lysate resulted in retrieval of Spx/RNAP with only the SpxΔCHA derivative. Binding reactions that combined active Spxc-Myc, inactive Spx(R60E)ΔCHA, and RNAP, when applied to the anti-HA affinity column, yielded only inactive Spx(R60E)ΔCHA/RNAP complexes. The results strongly argue for a model in which a single Spx monomer engages RNAP to generate an active transcriptional complex.

Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP.

Proteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His-Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His-Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant Spx(DD) (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxΔC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx-ClpX interaction, resulting in rapid degradation.

YjbH-enhanced proteolysis of Spx by ClpXP in Bacillus subtilis is inhibited by the small protein YirB (YuzO).

The Spx protein of Bacillus subtilis is a global regulator of the oxidative stress response. Spx concentration is controlled at the level of proteolysis by the ATP-dependent protease ClpXP and a substrate-binding protein, YjbH, which interacts with Spx. A yeast two-hybrid screen was carried out using yjbH as bait to uncover additional substrates or regulators of YjbH activity. Of the several genes identified in the screen, one encoded a small protein, YirB (YuzO), which elevated Spx concentration and activity in vivo when overproduced from an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible yirB construct. Pulldown experiments using extracts of B. subtilis cells producing a His-tagged YirB showed that native YjbH interacts with YirB in B. subtilis. Pulldown experiments using affinity-tagged Spx showed that YirB inhibited YjbH interaction with Spx. In vitro, YjbH-mediated proteolysis of Spx by ClpXP was inhibited by YirB. The activity of YirB is similar to that of the antiadaptor proteins that were previously shown to reduce proteolysis of a specific ClpXP substrate by interacting with a substrate-binding protein.

The YjbH protein of Bacillus subtilis enhances ClpXP-catalyzed proteolysis of Spx.

The global transcriptional regulator Spx of Bacillus subtilis is controlled at several levels of the gene expression process. It is maintained at low concentrations during unperturbed growth by the ATP-dependent protease ClpXP. Under disulfide stress, Spx concentration increases due in part to a reduction in ClpXP-catalyzed proteolysis. Recent studies of Larsson and coworkers (Mol. Microbiol. 66:669-684, 2007) implicated the product of the yjbH gene as being necessary for the proteolytic control of Spx. In the present study, yeast two-hybrid analysis and protein-protein cross-linking showed that Spx interacts with YjbH. YjbH protein was shown to enhance the proteolysis of Spx in reaction mixtures containing ClpXP protease but not ClpCP protease. An N-terminal truncated form of YjbH with a deletion of residues 1 to 24 (YjbH(Delta1-24)) showed no proteolysis enhancement activity. YjbH is specific for Spx as it did not accelerate proteolysis of the ClpXP substrate green fluorescent protein (GFP)-SsrA, a GFP derivative with a C-terminal SsrA tag that is recognized by ClpXP. Using inductively coupled plasma atomic emission spectroscopy and 4-(2-pyridylazo) resorcinol release experiments, YjbH was found to contain zinc atoms. Zinc analysis of YjbH(Delta1-24) revealed that the N-terminal histidine-rich region is indispensable for the coordination of at least one Zn atom. A Zn atom coordinated by the N-terminal region was rapidly released from the protein upon treatment with a strong oxidant. In conclusion, YjbH is proposed to be an adaptor for ClpXP-catalyzed Spx degradation, and a model of YjbH redox control involving Zn dissociation is presented.

Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis-acting element required for transcriptional activation.

The Spx protein of Bacillus subtilis interacts with RNA polymerase (RNAP) to activate transcription initiation in response to thiol-oxidative stress. Protein-DNA cross-linking analysis of reactions containing RNAP, Spx and trxA (thioredoxin) or trxB (thioredoxin reductase) promoter DNA was undertaken to uncover the organization of the Spx-activated transcription initiation complex. Spx induced contact between the RNAP sigma(A) subunit and the -10 promoter sequence of trxA and B, and contact of the betabeta' subunits with core promoter DNA. No Spx-DNA contact was detected. Spx mutants, Spx(C10A) and Spx(G52R.), or RNAP alpha C-terminal domain mutants that impair productive Spx-RNAP interaction did not induce heightened sigma and betabeta' contact with the core promoter. Deletion analysis and the activity of hybrid promoter constructs having upstream trxB DNA fused at positions -31, -36 and -41 of the srf (surfactin synthetase) promoter indicated that a cis-acting site between -50 and -36 was required for Spx activity. Mutations at -43 and -44 of trxB abolished Spx-dependent transcription and Spx-induced cross-linking between the sigma subunit and the -10 region. These data are consistent with a model that Spx activation requires contact between the Spx/RNAP complex and upstream promoter DNA, which allows Spx-induced engagement of the sigma and large subunits with the core promoter.

Regulation of quinone detoxification by the thiol stress sensing DUF24/MarR-like repressor, YodB in Bacillus subtilis.

Recently, we showed that the MarR-type repressor YkvE (MhqR) regulates multiple dioxygenases/glyoxalases, oxidoreductases and the azoreductase encoding yvaB (azoR2) gene in response to thiol-specific stress conditions, such as diamide, catechol and 2-methylhydroquinone (MHQ). Here we report on the regulation of the yocJ (azoR1) gene encoding another azoreductase by the novel DUF24/MarR-type repressor, YodB after exposure to thiol-reactive compounds. DNA binding activity of YodB is directly inhibited by thiol-reactive compounds in vitro. Mass spectrometry identified YodB-Cys-S-adducts that are formed upon exposure of YodB to MHQ and catechol in vitro. This confirms that catechol and MHQ are auto-oxidized to toxic ortho- and para-benzoquinones which act like diamide as thiol-reactive electrophiles. Mutational analyses further showed that the conserved Cys6 residue of YodB is required for optimal repression in vivo and in vitro while substitution of all three Cys residues of YodB affects induction of azoR1 transcription. Finally, phenotype analyses revealed that both azoreductases, AzoR1 and AzoR2 confer resistance to catechol, MHQ, 1,4-benzoquinone and diamide. Thus, both azoreductases that are controlled by different regulatory mechanisms have common functions in quinone and azo-compound reduction to protect cells against the thiol reactivity of electrophiles.

Proteomic signatures uncover thiol-specific electrophile resistance mechanisms in Bacillus subtilis.

Proteomic and transcriptomics signatures are powerful tools for visualizing global changes in gene expression in bacterial cells after exposure to stress, starvation or toxic compounds. Based on the global expression profile and the dissection into specific regulons, this knowledge can be used to predict the mode of action for novel antimicrobial compounds. This review summarizes our recent progress of proteomic signatures in the model bacterium for low-GC Gram-positive bacteria Bacillus subtilis in response to the antimicrobial compounds phenol, catechol, salicylic acid, 2-methylhydroquinone (2-MHQ) and 6-brom-2-vinyl-chroman-4-on (chromanon). Catechol, 2-MHQ and diamide displayed a common mode of action, as revealed by the induction of the thiol-specific oxidative stress response. In addition, multiple dioxygenases/glyoxalases, azoreductases and nitroreductases were induced by thiol-reactive compounds that are regulated by two novel thiol-specific regulators, YodB and MhqR (YkvE), both of which contribute to electrophile resistance in B. subtilis. These novel thiol-stress-responsive mechanisms are highly conserved among Gram-positive bacteria and are thought to have evolved to detoxify quinone-like electrophiles.

Regulation of respiratory genes by ResD-ResE signal transduction system in Bacillus subtilis.

Successful respiration in Bacillus subtilis using oxygen or nitrate as the terminal electron acceptor requires the ResD-ResE signal transduction system. Although transcription of ResDE-controlled genes is induced at the stationary phase of aerobic growth, it is induced to a higher extent upon oxygen limitation. Furthermore, maximal transcriptional activation requires not only oxygen limitation, but also nitric oxide (NO). Oxygen limitation likely results in conversion of the ResE sensor kinase activity from a phosphatase-dominant to a kinase-dominant mode. In addition, low oxygen levels promote the production and maintenance of NO during nitrate respiration, which leads to elimination of the repression exerted by the NO-sensitive transcriptional regulator NsrR. ResD, after undergoing ResE-mediated phosphorylation, interacts with the C-terminal domain of the alpha subunit of RNA polymerase to activate transcription initiation at ResDE-controlled promoters.

Factors affecting the bacterial community composition and heterotrophic production of Columbia River estuarine turbidity maxima.

Estuarine turbidity maxima (ETM) function as hotspots of microbial activity and diversity in estuaries, yet, little is known about the temporal and spatial variability in ETM bacterial community composition. To determine which environmental factors affect ETM bacterial populations in the Columbia River estuary, we analyzed ETM bacterial community composition (Sanger sequencing and amplicon pyrosequencing of 16S rRNA gene) and bulk heterotrophic production (3 H-leucine incorporation rates). We collected water 20 times to cover five ETM events and obtained 42 samples characterized by different salinities, turbidities, seasons, coastal regimes (upwelling vs. downwelling), locations, and particle size. Spring and summer populations were distinct. All May samples had similar bacterial community composition despite having different salinities (1-24 PSU), but summer non-ETM bacteria separated into marine, freshwater, and brackish assemblages. Summer ETM bacterial communities varied depending on coastal upwelling or downwelling conditions and on the sampling site location with respect to tidal intrusion during the previous neap tide. In contrast to ETM, whole (>0.2 µm) and free-living (0.2-3 µm) assemblages of non-ETM waters were similar to each other, indicating that particle-attached (>3 µm) non-ETM bacteria do not develop a distinct community. Brackish water type (ETM or non-ETM) is thus a major factor affecting particle-attached bacterial communities. Heterotrophic production was higher in particle-attached than free-living fractions in all brackish waters collected throughout the water column during the rise to decline of turbidity through an ETM event (i.e., ETM-impacted waters). However, free-living communities showed higher productivity prior to or after an ETM event (i.e., non-ETM-impacted waters). This study has thus found that Columbia River ETM bacterial communities vary based on seasons, salinity, sampling location, and particle size, with the existence of three particle types characterized by different bacterial communities in ETM, ETM-impacted, and non-ETM-impacted brackish waters. Taxonomic analysis suggests that ETM key biological function is to remineralize organic matter.

Control of gene expression in Gram-positive bacteria: extensions of and departures from enteric paradigms.

Our introduction to prokaryotic gene expression has always focused on the operon and regulatory mechanisms that operate within enteric bacteria such as Escherichia coli, Salmonella species, and their phages. While operon organization and many of the components of regulatory networks are conserved in Gram-positive species, there exists unique features that set these organisms apart from the enterics. Two examples are presented herein: carbon catabolite control and regulation of RNA polymerase sigma subunit activity, are presented. The accompanying reviews highlight the diversity and novel aspects of genetic control in Gram-positive bacteria, with descriptions of quorum-sensing systems, transcriptional control, and RNA processing mechanisms.