GlnR-Mediated Regulation ofectABCDTranscription Expands the Role of the GlnR Regulon to Osmotic Stress Management

ABSTRACTEctoine and hydroxyectoine are excellent compatible solutes for bacteria to deal with environmental osmotic stress and temperature damages. The biosynthesis cluster of ectoine and hydroxyectoine is widespread among microorganisms, and its expression is activated by high salinity and temperature changes. So far, little is known about the mechanism of the regulation of the transcription ofectgenes and only two MarR family regulators (EctR1 in methylobacteria and the EctR1-related regulator CosR inVibrio cholerae) have been found to negatively regulate the expression ofectgenes. Here, we characterize GlnR, the global regulator for nitrogen metabolism in actinomycetes, as a negative regulator for the transcription of ectoine/hydroxyectoine biosynthetic genes (ectoperon) inStreptomyces coelicolor. The physiological role of this transcriptional repression by GlnR is proposed to protect the intracellular glutamate pool, which acts as a key nitrogen donor for both the nitrogen metabolism and the ectoine/hydroxyectoine biosynthesis.IMPORTANCEHigh salinity is deleterious, and cells must evolve sophisticated mechanisms to cope with this osmotic stress. Although production of ectoine and hydroxyectoine is one of the most frequently adopted strategies, the in-depth mechanism of regulation of their biosynthesis is less understood. So far, only two MarR family negative regulators, EctR1 and CosR, have been identified in methylobacteria andVibrio, respectively. Here, our work demonstrates that GlnR, the global regulator for nitrogen metabolism, is a negative transcriptional regulator forectgenes inStreptomyces coelicolor. Moreover, a close relationship is found between nitrogen metabolism and osmotic resistance, and GlnR-mediated regulation ofecttranscription is proposed to protect the intracellular glutamate pool. Meanwhile, the work reveals the multiple roles of GlnR in bacterial physiology.

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CosR Is a Global Regulator of the Osmotic Stress Response with Widespread Distribution among Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00120-20 ◽

2020 ◽

Vol 86 (10) ◽

Cited By ~ 2

Author(s):

Gwendolyn J. Gregory ◽

Daniel P. Morreale ◽

E. Fidelma Boyd

Keyword(s):

Phylogenetic Analysis ◽

Stress Response ◽

Osmotic Stress ◽

Vibrio Parahaemolyticus ◽

Compatible Solutes ◽

Global Regulator ◽

Content Type ◽

Widespread Distribution ◽

Low Salinity ◽

Osmotic Stress Response

ABSTRACT Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2. Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX. Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria. IMPORTANCE Vibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria. Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.

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Role of GlnR in Controlling Expression of Nitrogen Metabolism Genes in Listeria monocytogenes

Journal of Bacteriology ◽

10.1128/jb.00209-20 ◽

2020 ◽

Vol 202 (19) ◽

Author(s):

Rajesh Biswas ◽

Abraham L. Sonenshein ◽

Boris R. Belitsky

Keyword(s):

Listeria Monocytogenes ◽

Nitrogen Metabolism ◽

Transcriptional Regulator ◽

Nitrogen Sources ◽

Negative Regulator ◽

Ammonium Uptake ◽

Ammonium Transport ◽

Growth Media ◽

Content Type

ABSTRACT Listeria monocytogenes is a fastidious bacterial pathogen that can utilize only a limited number of nitrogen sources for growth. Both glutamine and ammonium are common nitrogen sources used in listerial defined growth media, but little is known about the regulation of their uptake or utilization. The functional role of L. monocytogenes GlnR, the transcriptional regulator of nitrogen metabolism genes in low-G+C Gram-positive bacteria, was determined using transcriptome sequencing and real-time reverse transcription-PCR experiments. The GlnR regulon included transcriptional units involved in ammonium transport (amtB glnK) and biosynthesis of glutamine (glnRA) and glutamate (gdhA) from ammonium. As in other bacteria, GlnR proved to be an autoregulatory repressor of the glnRA operon. Unexpectedly, GlnR was most active during growth with ammonium as the nitrogen source and less active in the glutamine medium, apparently because listerial cells perceive growth with glutamine as a nitrogen-limiting condition. Therefore, paradoxically, expression of the glnA gene, encoding glutamine synthetase, was highest in the glutamine medium. For the amtB glnK operon, GlnR served as both a negative regulator in the presence of ammonium and a positive regulator in the glutamine medium. The gdhA gene was subject to a third mode of regulation that apparently required an elevated level of GlnR for repression. Finally, activity of glutamate dehydrogenase encoded by the gdhA gene appeared to correlate inversely with expression of gltAB, the operon that encodes the other major glutamate-synthesizing enzyme, glutamate synthase. Both gdhA and amtB were also regulated, in a negative manner, by the global transcriptional regulator CodY. IMPORTANCE L. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

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Major Compatible Solutes and Structural Adaptation of Proteins in Extremophiles

10.4018/978-1-7998-9144-4.ch008 ◽

2022 ◽

pp. 161-186

Author(s):

Hardik Shah ◽

Khushbu Panchal ◽

Amisha Panchal

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Osmotic Stress ◽

Amino Acid Composition ◽

Scientific Community ◽

Compatible Solutes ◽

Organic Solutes ◽

Structural Adaptation ◽

Extreme Habitats

Extremophiles are the most ancient microbes on the Earth and also a center of attraction for the scientific community for research because of their ability to adapt to extreme habitats. Compatible solutes are among those factors which enable these microorganisms to thrive in such extreme habitats. Under osmotic stress, the majority of extremophiles accumulate specific organic solutes such as amino acids, sugars, polyols, and their derivatives. In addition, proteins in extremophiles are found to be evolved by changing their amino acid composition to alter the hydrophobicity of its core and surface charge to maintain activity. This chapter encompasses a comprehensive study about the role of various compatible solutes in the endurance of microorganisms under extremophilic conditions, synthesis of compatible solutes, nature of extremophilic proteins, and their applications. Furthermore, an attempt has been made to cover various strategies adopted by the scientific community while pursuing research on compatible solutes.

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Reassessing the Role of the Type II MqsRA Toxin-Antitoxin System in Stress Response and Biofilm Formation: mqsA Is Transcriptionally Uncoupled from mqsR

mBio ◽

10.1128/mbio.02678-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 11

Author(s):

Nathan Fraikin ◽

Clothilde J. Rousseau ◽

Nathalie Goeders ◽

Laurence Van Melderen

Keyword(s):

Oxidative Stress ◽

Stress Response ◽

Biofilm Formation ◽

Bile Salts ◽

Regulatory Networks ◽

Constitutive Expression ◽

Toxin Gene ◽

Global Regulator ◽

Bacterial Physiology

ABSTRACT Toxin-antitoxin (TA) systems are broadly distributed modules whose biological roles remain mostly unknown. The mqsRA system is a noncanonical TA system in which the toxin and antitoxins genes are organized in operon but with the particularity that the toxin gene precedes that of the antitoxin. This system was shown to regulate global processes such as resistance to bile salts, motility, and biofilm formation. In addition, the MqsA antitoxin was shown to be a master regulator that represses the transcription of the csgD, cspD, and rpoS global regulator genes, thereby displaying a pleiotropic regulatory role. Here, we identified two promoters located in the toxin sequence driving the constitutive expression of mqsA, allowing thereby excess production of the MqsA antitoxin compared to the MqsR toxin. Our results show that both antitoxin-specific and operon promoters are not regulated by stresses such as amino acid starvation, oxidative shock, or bile salts. Moreover, we show that the MqsA antitoxin is not a global regulator as suggested, since the expression of csgD, cspD and rpoS is similar in wild-type and ΔmqsRA mutant strains. Moreover, these two strains behave similarly in terms of biofilm formation and sensitivity to oxidative stress or bile salts. IMPORTANCE There is growing controversy regarding the role of chromosomal toxin-antitoxin systems in bacterial physiology. mqsRA is a peculiar toxin-antitoxin system, as the gene encoding the toxin precedes that of the antitoxin. This system was previously shown to play a role in stress response and biofilm formation. In this work, we identified two promoters specifically driving the constitutive expression of the antitoxin, thereby decoupling the expression of antitoxin from the toxin. We also showed that mqsRA contributes neither to the regulation of biofilm formation nor to the sensitivity to oxidative stress and bile salts. Finally, we were unable to confirm that the MqsA antitoxin is a global regulator. Altogether, our data are ruling out the involvement of the mqsRA system in Escherichia coli regulatory networks.

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Metabolic Switching of Mycobacterium tuberculosis during Hypoxia Is Controlled by the Virulence Regulator PhoP

Journal of Bacteriology ◽

10.1128/jb.00705-19 ◽

2020 ◽

Vol 202 (7) ◽

Author(s):

Prabhat Ranjan Singh ◽

Anil Kumar Vijjamarri ◽

Dibyendu Sarkar

Keyword(s):

Mycobacterium Tuberculosis ◽

Nitrogen Metabolism ◽

Electron Acceptor ◽

Latent Infection ◽

Critical Role ◽

Hypoxic Stress ◽

Content Type ◽

Metabolic Switching ◽

Virulence Regulator

ABSTRACT Mycobacterium tuberculosis retains the ability to establish an asymptomatic latent infection. A fundamental question in mycobacterial physiology is to understand the mechanisms involved in hypoxic stress, a critical player in persistence. Here, we show that the virulence regulator PhoP responds to hypoxia, the dormancy signal, and effectively integrates hypoxia with nitrogen metabolism. We also provide evidence to demonstrate that both under nitrogen limiting conditions and during hypoxia, phoP locus controls key genes involved in nitrogen metabolism. Consistently, under hypoxia a ΔphoP strain shows growth attenuation even with surplus nitrogen, the alternate electron acceptor, and complementation of the mutant restores bacterial growth. Together, our observations provide new biological insights into the role of PhoP in integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR. The results have significant implications on the mechanism of intracellular survival and growth of the tubercle bacilli under a hypoxic environment within the phagosome. IMPORTANCE M. tuberculosis retains the unique ability to establish an asymptomatic latent infection. To understand the mechanisms involved in hypoxic stress which play a critical role in persistence, we show that the virulence regulator PhoP is linked to hypoxia, the dormancy signal. In keeping with this, phoP was shown to play a major role in M. tuberculosis growth under hypoxia even in the presence of surplus nitrogen, the alternate electron acceptor. Our results showing regulation of hypoxia-responsive genes provide new biological insights into role of the virulence regulator in metabolic switching by sensing hypoxia and integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR.

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Role of Toll Interleukin-1 Receptor (IL-1R) 8, a Negative Regulator of IL-1R/Toll-Like Receptor Signaling, in Resistance to Acute Pseudomonas aeruginosa Lung Infection

Infection and Immunity ◽

10.1128/iai.05695-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 100-109 ◽

Cited By ~ 30

Author(s):

Tania Véliz Rodriguez ◽

Federica Moalli ◽

Nadia Polentarutti ◽

Moira Paroni ◽

Eduardo Bonavita ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Interleukin 1 ◽

Bacterial Load ◽

Lung Infection ◽

Negative Regulator ◽

Current Evidence ◽

Toll Like Receptor ◽

Content Type ◽

Deficient Mice

ABSTRACTToll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulatorin vivounder different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused byPseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acuteP. aeruginosainfection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense againstP. aeruginosaacute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8−/−IL-1RI−/−double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused byP. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

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Silencing the Honey Bee (Apis mellifera) Naked Cuticle Gene (nkd) Improves Host Immune Function and Reduces Nosema ceranae Infections

Applied and Environmental Microbiology ◽

10.1128/aem.02105-16 ◽

2016 ◽

Vol 82 (22) ◽

pp. 6779-6787 ◽

Cited By ~ 22

Author(s):

Wenfeng Li ◽

Jay D. Evans ◽

Qiang Huang ◽

Cristina Rodríguez-García ◽

Jie Liu ◽

...

Keyword(s):

Apis Mellifera ◽

Immune Function ◽

Honey Bee ◽

Honey Bees ◽

Nosema Ceranae ◽

Negative Regulator ◽

Mrna Levels ◽

Content Type ◽

Bee Disease

ABSTRACTNosema ceranaeis a new and emerging microsporidian parasite of European honey bees,Apis mellifera, that has been implicated in colony losses worldwide. RNA interference (RNAi), a posttranscriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling infections of parasites and pathogens in honey bees. While previous studies have focused on the silencing of parasite/pathogen virulence factors, we explore here the possibility of silencing a host factor as a mechanism for reducing parasite load. Specifically, we used an RNAi strategy to reduce the expression of a honey bee gene,naked cuticle(nkd), which is a negative regulator of host immune function. Our studies found thatnkdmRNA levels in adult bees were upregulated byN. ceranaeinfection (and thus, the parasite may use this mechanism to suppress host immune function) and that ingestion of double-stranded RNA (dsRNA) specific tonkdefficiently silenced its expression. Furthermore, we found that RNAi-mediated knockdown ofnkdtranscripts inNosema-infected bees resulted in upregulation of the expression of several immune genes (Abaecin,Apidaecin,Defensin-1, andPGRP-S2), reduction ofNosemaspore loads, and extension of honey bee life span. The results of our studies clearly indicate that silencing the hostnkdgene can activate honey bee immune responses, suppress the reproduction ofN. ceranae, and improve the overall health of honey bees. This study represents a novel host-derived therapeutic for honey bee disease treatment that merits further exploration.IMPORTANCEGiven the critical role of honey bees in the pollination of agricultural crops, it is urgent to develop strategies to prevent the colony decline induced by the infection of parasites/pathogens. Targeting parasites and pathogens directly by RNAi has been proven to be useful for controlling infections in honey bees, but little is known about the disease impacts of RNAi silencing of host factors. Here, we demonstrate that knocking down the honey bee immune repressor-encodingnkdgene can suppress the reproduction ofN. ceranaeand improve the overall health of honey bees, which highlights the potential role of host-derived and RNAi-based therapeutics in controlling the infections in honey bees. The information obtained from this study will have positive implications for honey bee disease management practices.

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Metabolic and Community Synergy of Oral Bacteria in Colorectal Cancer

mSphere ◽

10.1128/msphere.00102-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 62

Author(s):

Kaitlin J. Flynn ◽

Nielson T. Baxter ◽

Patrick D. Schloss

Keyword(s):

Colorectal Cancer ◽

Fusobacterium Nucleatum ◽

Oral Bacteria ◽

Oral Microbiota ◽

Community Members ◽

Content Type ◽

Bacterial Physiology ◽

Oral Biofilms ◽

Two Factors

ABSTRACT The oral periodontopathic bacterium Fusobacterium nucleatum has been repeatedly associated with colorectal tumors. Molecular analysis has identified specific virulence factors that promote tumorigenesis in the colon. However, other oral community members, such as members of the Porphyromonas spp., are also found with F. nucleatum on colonic tumors, and thus, narrow studies of individual pathogens do not take community-wide virulence properties into account. A broader view of oral bacterial physiology and pathogenesis identifies two factors that could promote colonization and persistence of oral bacterial communities in the colon. The polymicrobial nature of oral biofilms and the asaccharolytic metabolism of many of these species make them well suited to life in the microenvironment of colonic lesions. Consideration of these two factors offers a novel perspective on the role of oral microbiota in the initiation, development, and treatment of colorectal cancer.

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The Anti-Anti-Sigma Factor BldG Is Involved in Activation of the Stress Response Sigma Factor σH in Streptomyces coelicolor A3(2)

Journal of Bacteriology ◽

10.1128/jb.00828-10 ◽

2010 ◽

Vol 192 (21) ◽

pp. 5674-5681 ◽

Cited By ~ 19

Author(s):

Beatrica Sevcikova ◽

Bronislava Rezuchova ◽

Dagmar Homerova ◽

Jan Kormanec

Keyword(s):

Stress Response ◽

Osmotic Stress ◽

Sigma Factor ◽

Streptomyces Coelicolor ◽

Direct Interaction ◽

Morphological Differentiation ◽

Osmotic Stress Response ◽

Its Gene ◽

Two Hybrid

ABSTRACT The alternative stress response sigma factor σH has a role in regulation of the osmotic stress response and in morphological differentiation in Streptomyces coelicolor A3(2). Its gene, sigH, is located in an operon with the gene that encodes its anti-sigma factor UshX (PrsH). However, no gene with similarity to an anti-anti-sigma factor which may have a role in σH activation by a “partner-switching” mechanism is located in the operon. By using a combination of several approaches, including pull-down and bacterial two-hybrid assays and visualization of the complex by native polyacrylamide electrophoresis, we demonstrated a direct interaction between UshX and the pleiotropic sporulation-specific anti-anti-sigma factor BldG. Osmotic induction of transcription of the sigHp2 promoter that is specifically recognized by RNA polymerase containing σH was absent in an S. coelicolor bldG mutant, indicating a role of BldG in σH activation by a partner-switching-like mechanism.

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FNR Regulates Expression of Important Virulence Factors Contributing to Pathogenicity of Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.02315-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 5086-5098 ◽

Cited By ~ 28

Author(s):

Nicolle L. Barbieri ◽

Bryon Nicholson ◽

Ashraf Hussein ◽

Wentong Cai ◽

Yvonne M. Wannemuehler ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Factors ◽

Bacterial Infections ◽

Uropathogenic Escherichia Coli ◽

Type I ◽

Wild Type ◽

Global Regulator ◽

Content Type ◽

Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) is responsible for the majority of urinary tract infections (UTIs), which are some of the world's most common bacterial infections of humans. Here, we examined the role of FNR (fumarate andnitratereduction), a well-known global regulator, in the pathogenesis of UPEC infections. We constructed anfnrdeletion mutant of UPEC CFT073 and compared it to the wild type for changes in virulence, adherence, invasion, and expression of key virulence factors. Compared to the wild type, thefnrmutant was highly attenuated in the mouse model of human UTI and showed severe defects in adherence to and invasion of bladder and kidney epithelial cells. Our results showed that FNR regulates motility and multiple virulence factors, including expression of type I and P fimbriae, modulation of hemolysin expression, and expression of a novel pathogenicity island involved in α-ketoglutarate metabolism under anaerobic conditions. Our results demonstrate that FNR is a key global regulator of UPEC virulence and controls expression of important virulence factors that contribute to UPEC pathogenicity.

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