Resilience in the Face of Uncertainty: Sigma Factor B Fine-Tunes Gene Expression To Support Homeostasis in Gram-Positive Bacteria

ABSTRACTGram-positive bacteria are ubiquitous and diverse microorganisms that can survive and sometimes even thrive in continuously changing environments. The key to such resilience is the ability of members of a population to respond and adjust to dynamic conditions in the environment. In bacteria, such responses and adjustments are mediated, at least in part, through appropriate changes in the bacterial transcriptome in response to the conditions encountered. Resilience is important for bacterial survival in diverse, complex, and rapidly changing environments and requires coordinated networks that integrate individual, mechanistic responses to environmental cues to enable overall metabolic homeostasis. In many Gram-positive bacteria, a key transcriptional regulator of the response to changing environmental conditions is the alternative sigma factor σB. σBhas been characterized in a subset of Gram-positive bacteria, including the generaBacillus,Listeria, andStaphylococcus. Recent insight from next-generation-sequencing results indicates that σB-dependent regulation of gene expression contributes to resilience, i.e., the coordination of complex networks responsive to environmental changes. This review explores contributions of σBto resilience inBacillus,Listeria, andStaphylococcusand illustrates recently described regulatory functions of σB.

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Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00026-16 ◽

2016 ◽

Vol 80 (4) ◽

pp. 1029-1057 ◽

Cited By ~ 19

Author(s):

Ruben A. T. Mars ◽

Pierre Nicolas ◽

Emma L. Denham ◽

Jan Maarten van Dijl

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Regulation Of Gene Expression ◽

Regulatory Rna ◽

Gram Positive ◽

Content Type ◽

Rna Molecules ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

The Stability

SUMMARYBacteria can employ widely diverse RNA molecules to regulate their gene expression. Such molecules includetrans-acting small regulatory RNAs, antisense RNAs, and a variety of transcriptional attenuation mechanisms in the 5′ untranslated region. Thus far, most regulatory RNA research has focused on Gram-negative bacteria, such asEscherichia coliandSalmonella. Hence, there is uncertainty about whether the resulting insights can be extrapolated directly to other bacteria, such as the Gram-positive soil bacteriumBacillus subtilis. A recent study identified 1,583 putative regulatory RNAs inB. subtilis, whose expression was assessed across 104 conditions. Here, we review the current understanding of RNA-based regulation inB. subtilis, and we categorize the newly identified putative regulatory RNAs on the basis of their conservation in other bacilli and the stability of their predicted secondary structures. Our present evaluation of the publicly available data indicates that RNA-mediated gene regulation inB. subtilismostly involves elements at the 5′ ends of mRNA molecules. These can include 5′ secondary structure elements and metabolite-, tRNA-, or protein-binding sites. Importantly, sense-independent segments are identified as the most conserved and structured potential regulatory RNAs inB. subtilis. Altogether, the present survey provides many leads for the identification of new regulatory RNA functions inB. subtilis.

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Small-Molecule Antibiotics Inhibiting tRNA-Regulated Gene Expression Is a Viable Strategy for Targeting Gram-Positive Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01247-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01247-20 ◽

Cited By ~ 1

Author(s):

Ville Y. P. Väre ◽

Ryan F. Schneider ◽

Haein Kim ◽

Erica Lasek-Nesselquist ◽

Kathleen A. McDonough ◽

...

Keyword(s):

Gene Expression ◽

Small Molecule ◽

Bacterial Infections ◽

Gram Positive ◽

Content Type ◽

T Box ◽

Gram Positive Bacteria ◽

Bactericidal Effects ◽

Low Levels ◽

Regulated Gene Expression

ABSTRACTBacterial infections and the rise of antibiotic resistance, especially multidrug resistance, have generated a clear need for discovery of novel therapeutics. We demonstrated that a small-molecule drug, PKZ18, targets the T-box mechanism and inhibits bacterial growth. The T-box is a structurally conserved riboswitch-like gene regulator in the 5′ untranslated region (UTR) of numerous essential genes of Gram-positive bacteria. T-boxes are stabilized by cognate, unacylated tRNA ligands, allowing the formation of an antiterminator hairpin in the mRNA that enables transcription of the gene. In the absence of an unacylated cognate tRNA, transcription is halted due to the formation of a thermodynamically more stable terminator hairpin. PKZ18 targets the site of the codon-anticodon interaction of the conserved stem I and reduces T-box-controlled gene expression. Here, we show that novel analogs of PKZ18 have improved MICs, bactericidal effects against methicillin-resistant Staphylococcus aureus (MRSA), and increased efficacy in nutrient-limiting conditions. The analogs have reduced cytotoxicity against eukaryotic cells compared to PKZ18. The PKZ18 analogs acted synergistically with aminoglycosides to significantly enhance the efficacy of the analogs and aminoglycosides, further increasing their therapeutic windows. RNA sequencing showed that the analog PKZ18-22 affects expression of 8 of 12 T-box controlled genes in a statistically significant manner, but not other 5′-UTR regulated genes in MRSA. Very low levels of resistance further support the existence of multiple T-box targets for PKZ18 analogs in the cell. Together, the multiple targets, low resistance, and synergy make PKZ18 analogs promising drugs for development and future clinical applications.

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Redefining the Clostridioides difficile σB Regulon: σB Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide

mSphere ◽

10.1128/msphere.00728-20 ◽

2020 ◽

Vol 5 (5) ◽

Cited By ~ 1

Author(s):

Ilse M. Boekhoud ◽

Annika-Marisa Michel ◽

Jeroen Corver ◽

Dieter Jahn ◽

Wiep Klaas Smits

Keyword(s):

Hydrogen Peroxide ◽

Stress Response ◽

Sigma Factor ◽

Gene Activation ◽

Luciferase Reporter ◽

Alternative Sigma Factor ◽

Gram Positive ◽

Content Type ◽

Gram Positive Bacteria ◽

Sigma B

ABSTRACT In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation. IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.

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The Listeria monocytogenes σB Regulon and Its Virulence-Associated Functions Are Inhibited by a Small Molecule

mBio ◽

10.1128/mbio.00241-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 27

Author(s):

M. Elizabeth Palmer ◽

Soraya Chaturongakul ◽

Martin Wiedmann ◽

Kathryn J. Boor

Keyword(s):

Bacillus Subtilis ◽

Listeria Monocytogenes ◽

Small Molecule ◽

Sigma Factor ◽

Pathogen Transmission ◽

Bile Salt Hydrolase ◽

Alternative Sigma Factor ◽

Bacterial Survival ◽

Gram Positive ◽

Content Type

ABSTRACTThe stress-responsive alternative sigma factor σBis conserved across diverse Gram-positive bacterial genera. InListeria monocytogenes, σBregulates transcription of >150 genes, including genes contributing to virulence and to bacterial survival under host-associated stress conditions, such as those encountered in the human gastrointestinal lumen. An inhibitor ofL. monocytogenesσBactivity was identified by screening ~57,000 natural and synthesized small molecules using a high-throughput cell-based assay. The compound fluoro-phenyl-styrene-sulfonamide (FPSS) (IC50= 3.5 µM) downregulated the majority of genes previously identified as members of the σBregulon inL. monocytogenes10403S, thus generating a transcriptional profile comparable to that of a 10403S ΔsigBstrain. Specifically, of the 208 genes downregulated by FPSS, 75% had been identified previously as positively regulated by σB. Downregulated genes included key virulence and stress response genes, such asinlA,inlB,bsh,hfq,opuC, andbilE. From a functional perspective, FPSS also inhibitedL. monocytogenesinvasion of human intestinal epithelial cells and bile salt hydrolase activity. The ability of FPSS to inhibit σBactivity in bothL. monocytogenesandBacillus subtilisindicates its utility as a specific inhibitor of σBacross multiple Gram-positive genera.IMPORTANCEThe σBtranscription factor regulates expression of genes responsible for bacterial survival under changing environmental conditions and for virulence; therefore, this alternative sigma factor is important for transmission ofL. monocytogenesand other Gram-positive bacteria. Regulation of σBactivity is complex and tightly controlled, reflecting the key role of this factor in bacterial metabolism. We present multiple lines of evidence indicating that fluoro-phenyl-styrene-sulfonamide (FPSS) specifically inhibits activity of σBacross Gram-positive bacterial genera, i.e., in bothListeria monocytogenesandBacillus subtilis. Therefore, FPSS is an important new tool that will enable novel approaches for exploring complex regulatory networks inL. monocytogenesand other Gram-positive pathogens and for investigating small-molecule applications for controlling pathogen transmission.

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CovS Inactivates CovR and Is Required for Growth under Conditions of General Stress in Streptococcus pyogenes

Journal of Bacteriology ◽

10.1128/jb.186.12.3928-3937.2004 ◽

2004 ◽

Vol 186 (12) ◽

pp. 3928-3937 ◽

Cited By ~ 106

Author(s):

Tracy L. Dalton ◽

June R. Scott

Keyword(s):

Gene Expression ◽

Streptococcus Pyogenes ◽

Sigma Factor ◽

Regulatory System ◽

Stress Conditions ◽

Mild Stress ◽

Gram Positive ◽

Gram Positive Bacteria ◽

General Stress ◽

Two Component

ABSTRACT The gram-positive human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) causes diseases ranging from mild and often self-limiting infections of the skin or throat to invasive and life-threatening illnesses. To cause such diverse types of disease, the GAS must be able to sense adverse environments and regulate its gene expression accordingly. The CovR/S two-component signal transduction regulatory system in GAS represses about 15% of the GAS genome, including many genes involved in virulence, in response to the environment. We report that CovR is still able to repress transcription from several promoters in the absence of the putative histidine kinase sensor for this system, CovS. We also show that a phosphorylation site mutant (D53A) of CovR is unable to repress gene expression. In addition, we report that a strain with a nonpolar mutation in CovS does not grow at a low pH, elevated temperature, or high osmolarity. The stress-related phenotypes of the CovS mutant were complemented by expression of covS from a plasmid. Selection for growth of a CovS mutant under stress conditions resulted in isolation of second-site mutations that inactivated covR, indicating that CovR and CovS act in the same pathway. Also, at 40°C in the wild-type strain, CovR appeared to be less active on the promoter tested, which is consistent with the hypothesis that it was partially inactivated by CovS. We suggest that under mild stress conditions, CovS inactivates CovR, either directly or indirectly, and that this inactivation relieves repression of many GAS genes, including the genes needed for growth of GAS under stress conditions and some genes that are necessary for virulence. Growth of many gram-positive bacteria under multiple-stress conditions requires alteration of promoter recognition produced by RNA polymerase association with the general stress response sigma factor, σB. We provide evidence that for GAS, which lacks a sigB ortholog, growth under stress conditions requires the CovR/S two-component regulatory system instead. This two-component system in GAS thus appears to perform a function for which other gram-positive bacteria utilize an alternative sigma factor.

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Linking transcription, RNA polymerase II elongation and alternative splicing

Biochemical Journal ◽

10.1042/bcj20200475 ◽

2020 ◽

Vol 477 (16) ◽

pp. 3091-3104 ◽

Cited By ~ 1

Author(s):

Luciana E. Giono ◽

Alberto R. Kornblihtt

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Rna Polymerase Ii ◽

Environmental Changes ◽

Transcription Elongation ◽

Single Gene ◽

Regulation Of Gene Expression ◽

Regulation Of Transcription ◽

Stepping Stones ◽

Eukaryotic Transcription

Gene expression is an intricately regulated process that is at the basis of cell differentiation, the maintenance of cell identity and the cellular responses to environmental changes. Alternative splicing, the process by which multiple functionally distinct transcripts are generated from a single gene, is one of the main mechanisms that contribute to expand the coding capacity of genomes and help explain the level of complexity achieved by higher organisms. Eukaryotic transcription is subject to multiple layers of regulation both intrinsic — such as promoter structure — and dynamic, allowing the cell to respond to internal and external signals. Similarly, alternative splicing choices are affected by all of these aspects, mainly through the regulation of transcription elongation, making it a regulatory knob on a par with the regulation of gene expression levels. This review aims to recapitulate some of the history and stepping-stones that led to the paradigms held today about transcription and splicing regulation, with major focus on transcription elongation and its effect on alternative splicing.

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Antimicrobial Nodule-Specific Cysteine-Rich Peptides Induce Membrane Depolarization-Associated Changes in the Transcriptome of Sinorhizobium meliloti

Applied and Environmental Microbiology ◽

10.1128/aem.01791-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6737-6746 ◽

Cited By ~ 64

Author(s):

Hilda Tiricz ◽

Attila Szűcs ◽

Attila Farkas ◽

Bernadett Pap ◽

Rui M. Lima ◽

...

Keyword(s):

Stress Responses ◽

Sinorhizobium Meliloti ◽

Antimicrobial Agents ◽

Cellular Functions ◽

Gram Positive ◽

Content Type ◽

Gram Positive Bacteria ◽

Peptide Family ◽

Wide Range ◽

Natural Target

ABSTRACTLeguminous plants establish symbiosis with nitrogen-fixing alpha- and betaproteobacteria, collectively called rhizobia, which provide combined nitrogen to support plant growth. Members of the inverted repeat-lacking clade of legumes impose terminal differentiation on their endosymbiotic bacterium partners with the help of the nodule-specific cysteine-rich (NCR) peptide family composed of close to 600 members. Among the few tested NCR peptides, cationic ones had antirhizobial activity measured by reduction or elimination of the CFU and uptake of the membrane-impermeable dye propidium iodide. Here, the antimicrobial spectrum of two of these peptides, NCR247 and NCR335, was investigated, and their effect on the transcriptome of the natural targetSinorhizobium melilotiwas characterized. Both peptides were able to kill quickly a wide range of Gram-negative and Gram-positive bacteria; however, their spectra were only partially overlapping, and differences were found also in their efficacy on given strains, indicating that the actions of NCR247 and NCR335 might be similar though not identical. Treatment ofS. meliloticultures with either peptide resulted in a quick downregulation of genes involved in basic cellular functions, such as transcription-translation and energy production, as well as upregulation of genes involved in stress and oxidative stress responses and membrane transport. Similar changes provoked mainly in Gram-positive bacteria by antimicrobial agents were coupled with the destruction of membrane potential, indicating that it might also be a common step in the bactericidal actions of NCR247 and NCR335.

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Restoration of Bioactive Lantibiotic Suicin from a RemnantlanLocus of Pathogenic Streptococcus suis Serotype 2

Applied and Environmental Microbiology ◽

10.1128/aem.03213-13 ◽

2013 ◽

Vol 80 (3) ◽

pp. 1062-1071 ◽

Cited By ~ 17

Author(s):

Jian Wang ◽

Yong Gao ◽

Kunling Teng ◽

Jie Zhang ◽

Shutao Sun ◽

...

Keyword(s):

Gene Promoter ◽

Streptococcus Suis ◽

Gene Clusters ◽

Disulfide Bridge ◽

Gram Positive ◽

Content Type ◽

Gram Positive Bacteria ◽

System A ◽

Bactericidal Activities

ABSTRACTLantibiotics are ribosomally synthesized, posttranslationally modified antimicrobial peptides. Their biosynthesis genes are usually organized in gene clusters, which are mainly found in Gram-positive bacteria, including pathogenic streptococci. Three highly virulentStreptococcus suisserotype 2 strains (98HAH33, 05ZYH33, and SC84) have been shown to contain an 89K pathogenicity island. Here, on these islands, we unveiled and reannotated a putative lantibiotic locus designatedsuiwhich contains a virulence-associated two-component regulator,suiK-suiR. In silicoanalysis revealed that the putative lantibiotic modification genesuiMwas interrupted by a 7.9-kb integron and that other biosynthesis-related genes contained various frameshift mutations. By reconstituting the intactsuiMinEscherichia colitogether with a semi-in vitrobiosynthesis system, a putative lantibiotic named suicin was produced with bactericidal activities against a variety of Gram-positive strains, including pathogenic streptococci and vancomycin-resistant enterococci. Ring topology dissection indicated that the 34-amino-acid lantibiotic contained two methyllanthionine residues and one disulfide bridge, which render suicin in an N-terminal linear and C-terminal globular shape. To confirm the function ofsuiK-suiR, SuiR was overexpressed and purified.In vitroanalysis showed that SuiR could specifically bind to thesuiAgene promoter. Its coexpression withsuiKcould activatesuiAgene promoter inLactococcus lactisNZ9000. Conclusively, we obtained a novel lantibiotic suicin by restoring its production from the remnantsuilocus and demonstrated that virulence-associated SuiK-SuiR regulates its production.

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Attenuator LRR – a regulatory tool for modulating gene expression in Gram‐positive bacteria

Microbial Biotechnology ◽

10.1111/1751-7915.13797 ◽

2021 ◽

Author(s):

Xia Cai ◽

Qian Wang ◽

Yu Fang ◽

Die Yao ◽

Yunda Zhan ◽

...

Keyword(s):

Gene Expression ◽

Gram Positive ◽

Gram Positive Bacteria

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ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522179113 ◽

2016 ◽

Vol 113 (12) ◽

pp. E1710-E1719 ◽

Cited By ~ 98

Author(s):

Rebecca M. Corrigan ◽

Lauren E. Bellows ◽

Alison Wood ◽

Angelika Gründling

Keyword(s):

Stringent Response ◽

Ribosome Assembly ◽

Bacterial Survival ◽

Positive Bacterium ◽

Gram Positive ◽

Guanosine Tetraphosphate ◽

Target Proteins ◽

Gram Positive Bacteria ◽

Ribosome Inactivation

The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacteriumStaphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases—RsgA, RbgA, Era, HflX, and ObgE—as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with anrsgAdeletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs fromBacillus subtilisandEnterococcus faecalisretain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.

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