Rick W. Ye

Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon

In response to sublethal concentrations of antibiotics, bacteria often induce an adaptive response that can contribute to antibiotic resistance. We report the response of Bacillus subtilis to bacitracin, an inhibitor of cell wall biosynthesis found in its natural environment. Analysis of the global transcriptional profile of bacitracin‐treated cells reveals a response orchestrated by two alternative σ factors (σB and σM) and three two‐component systems (YvqEC, YvcPQ and BceRS). All three two‐component systems are located next to target genes that are strongly induced by bacitracin, and the corresponding histidine kinases share an unusual topology: they lack about 100 amino acids in their extracellular sensing domain, which is almost entirely buried in the cytoplasmic membrane. Sequence analysis indicates that this novel N‐terminal sensing domain is a characteristic feature of a subfamily of histidine kinases, found almost entirely in Gram‐positive bacteria and frequently linked to ABC transporters. A systematic mutational analysis of bacitracin‐induced genes led to the identification of a new bacitracin‐resistance determinant, bceAB, encoding a putative ABC transporter. The bcrC bacitracin resistance gene, which is under the dual control of σX and σM, was also induced by bacitracin. By comparing the bacitracin and the vancomycin stimulons, we can differentiate between loci induced specifically by bacitracin and those that are induced by multiple cell wall‐active antibiotics.

Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon

The Bacillus subtilis ferric uptake repressor (Fur) protein coordinates a global transcriptional response to iron starvation. We have used DNA microarrays to define the Fur regulon and the iron starvation stimulon. We identify 20 operons (containing 39 genes) that are derepressed both by mutation of fur and by treatment of cells with the iron chelator 2,2′‐dipyridyl. These operons are direct targets of Fur regulation as judged by DNase I footprinting. Analyses of lacZ reporter fusions to six Fur‐regulated promoter regions reveal that repression is highly selective for iron. In addition to the Fur regulon, iron starvation induces members of the PerR regulon and leads to reduced expression of cytochromes. However, we did not find any evidence for genes that are directly activated by Fur or repressed by Fur under iron‐limiting conditions. Although genome searches using the 19 bp Fur box consensus are useful in identifying candidate Fur‐regulated genes, some genes associated with Fur boxes are not demonstrably regulated by Fur, whereas other genes are regulated from sites with little apparent similarity to the conventional Fur consensus.

Antibiotics that inhibit cell wall biosynthesis induce expression of the Bacillus subtilisσW and σM regulons

Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors. The σ W regulon includes functions involved in detoxification and protection against antimicrobials, whereas σ M is essential for growth at high salt concentrations. We now report that antibiotics that inhibit cell wall biosynthesis induce both σ W and σ M regulons as monitored using DNA microarrays. Induction of selected σ W ‐dependent genes was confirmed using lacZ reporter fusions and Northern blot analysis. The ability of vancomycin to induce the σ W regulon is dependent on both σ W and the cognate anti‐ σ , RsiW, but is independent of the transition state regulator AbrB. These results suggest that the membrane‐localized RsiW anti‐ σ W factor mediates the transcriptional response to cell wall stress. Our findings are consistent with the idea that one function of ECF σ factors is to coordinate antibiosis stress responses and cell envelope homeostasis.

Global Gene Expression Profiles of Bacillus subtilis Grown under Anaerobic Conditions

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.

Applications of DNA microarrays in microbial systems.

DNA microarray technology allows a parallel analysis of RNA abundance and DNA homology for thousands of genes in a single experiment. Over the past few years, this powerful technology has been used to explore transcriptional profiles and genome differences for a variety of microorganisms, greatly facilitating our understanding of microbial metabolism. With the increasing availability of complete microbial genomes, DNA microarrays are becoming a common tool in many areas of microbial research, including microbial physiology, pathogenesis, epidemiology, ecology, phylogeny, pathway engineering and fermentation optimization.

Response of Bacillus subtilis to Nitric Oxide and the Nitrosating Agent Sodium Nitroprusside

We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B. subtilis to NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress. Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur. In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II). Aerobically, NO gas induced hmp, the sigmaB general stress regulon, and, to a lesser extent, the Fur and PerR regulons. Surprisingly, NO gas induced the sigmaB regulon via the energy branch of the sigmaB regulatory cascade while induction by SNP was mediated by the environmental stress branch. This emphasizes that NO and SNP elicit genetically distinct stress responses.

Genetic and physiological responses of Bacillus subtilis to metal ion stress.

Metal ion homeostasis is regulated principally by metalloregulatory proteins that control metal ion uptake, storage and efflux genes. We have used transcriptional profiling to survey Bacillus subtilis for genes that are rapidly induced by exposure to high levels of metal ions including Ag(I), Cd(II), Cu(II), Ni(II) and Zn(II) and the metalloid As(V). Many of the genes affected by metal stress were controlled by known metalloregulatory proteins (Fur, MntR, PerR, ArsR and CueR). Additional metal‐induced genes are regulated by two newly defined metal‐sensing ArsR/SmtB family repressors: CzrA and AseR. CzrA represses the CadA efflux ATPase and the cation diffusion facilitator CzcD and this repression is alleviated by Zn(II), Cd(II), Co(II), Ni(II) and Cu. CadA is the major determinant for Cd(II) resistance, while CzcD protects the cell against elevated levels of Zn(II), Cu, Co(II) and Ni(II). AseR negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. Our results extend the range of identified effectors for the As(III)‐sensor ArsR to include Cd(II) and Ag(I) and for the Cu‐sensor CueR to include Ag(I) and, weakly, Cd(II) and Zn(II). In addition to systems dedicated to metal homeostasis, specific metal stresses also strongly induced pathways related to cysteine, histidine and arginine metabolism.

Functional Analysis of the Bacillus subtilis Zur Regulon

The Bacillus subtilis zinc uptake repressor (Zur) regulates genes involved in zinc uptake. We have used DNA microarrays to identify genes that are derepressed in a zur mutant. In addition to members of the two previously identified Zur-regulated operons (yciC and ycdHI-yceA), we identified two other genes, yciA and yciB, as targets of Zur regulation. Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation. Zur binds to an approximately 28-bp operator upstream of the yciA gene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons, yciC and ycdHI-yceA. Analysis of a yciA-lacZ fusion indicates that this operon is induced under zinc starvation conditions and derepressed in the zur mutant. Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway. Mutation of yciA or yciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of the ycdH mutant under conditions of zinc limitation. Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system. Mutant strains lacking all three identified zinc uptake systems (yciABC, ycdHI-yceA, and zosA) are dependent on micromolar levels of added zinc for optimal growth.

Stationary-Phase Quorum-Sensing Signals Affect Autoinducer-2 and Gene Expression in Escherichia coli

ABSTRACT Quorum sensing via autoinducer-2 (AI-2) has been identified in different strains, including those from Escherichia, Vibrio, Streptococcus, and Bacillus species, and previous studies have suggested the existence of additional quorum-sensing signals working in the stationary phase of Escherichia coli cultures. To investigate the presence and global effect of these possible quorum-sensing signals other than AI-2, DNA microarrays were used to study the effect of stationary-phase signals on the gene expression of early exponential-phase cells of the AI-2-deficient strain E. coli DH5α. For statistically significant differential gene expression (P < 0.05), 14 genes were induced by supernatants from a stationary culture and 6 genes were repressed, suggesting the involvement of indole (induction of tnaA and tnaL) and phosphate (repression of phoA, phoB, and phoU). To study the stability of the signals, the stationary-phase supernatant was autoclaved and was used to study its effect on E. coli gene expression. Three genes were induced by autoclaved stationary-phase supernatant, and 34 genes were repressed. In total, three genes (ompC, ptsA, and btuB) were induced and five genes (nupC, phoB, phoU, argT, and ompF) were repressed by both fresh and autoclaved stationary-phase supernatants. Furthermore, supernatant from E. coli DH5α stationary culture was found to repress E. coli K-12 AI-2 concentrations by 4.8-fold ± 0.4-fold, suggesting that an additional quorum-sensing system in E. coli exists and that gene expression is controlled as a network with different signals working at different growth stages.

The global transcriptional response of Bacillus subtilis to manganese involves the MntR, Fur, TnrA and σB regulons

We have used DNA microarrays to monitor the global transcriptional response of Bacillus subtilis to changes in manganese availability. Mn(II) leads to the MntR‐dependent repression of both the mntH and mntABCD operons encoding Mn(II) uptake systems. Mn(II) also represses the Fur regulon. This repression is unlikely to be a direct effect of Mn(II) on Fur as repression is sensitive to 2,2′‐dipyridyl, an iron‐selective chelator. We suggest that elevated Mn(II) displaces iron from cellular‐binding sites and the resulting rise in free iron levels leads to repression of the Fur regulon. Many of the genes induced by Mn(II) are activated by σB or TnrA. Both of these regulators are controlled by Mn(II)‐dependent enzymes. Induction of the σB‐dependent general stress response by Mn(II) is largely dependent on RsbU, a Mn(II)‐dependent phosphatase that dephosphorylates RsbV, ultimately leading to release of active σB from its antisigma, RsbW. The activity of TnrA is inhibited when it forms an inactive complex with feedback‐inhibited glutamine synthetase. Elevated Mn(II) reduces the sensitivity of glutamine synthetase to feedback inhibitors, and we suggest that this leads to the observed increase in TnrA activity. In sum, three distinct mechanisms can account for most of the transcriptional effects elicited by manganese: (i) direct binding of Mn(II) to metalloregulators such as MntR, (ii) perturbation of cellular iron pools leading to increased Fur activity and (iii) altered activity of Mn(II)‐dependent enzymes that regulate the activity of σB and TnrA.