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Enterobacterial common antigen integrity is a checkpoint for flagellar biogenesis in Serratia marcescens.

ABSTRACT: Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in the wec cluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in the wec mutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation of flhDC expression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that in S. marcescens, its biosynthesis conditions the expression of the flagellar regulon.

SUBMITTER: Castelli ME

PROVIDER: S-EPMC2223741 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Enterobacterial common antigen integrity is a checkpoint for flagellar biogenesis in Serratia marcescens.

Castelli María E ME Fedrigo Griselda V GV Clementín Ana L AL Ielmini M Verónica MV Feldman Mario F MF García Véscovi Eleonora E

Journal of bacteriology 20071102 1

Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expre ...[more]

PMID: 17981971

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Project description:The Gram-negative cell envelope is a complex structure delineating the cell from its environment. Recently, we found that enterobacterial common antigen (ECA) plays a role maintaining the outer membrane (OM) permeability barrier, which excludes toxic molecules including many antibiotics. ECA is a conserved carbohydrate found throughout Enterobacterales (e.g., Salmonella, Klebsiella, and Yersinia). There are two OM forms of ECA (phosphoglyceride-linked ECAPG and lipopolysaccharide-linked ECALPS) and one periplasmic form of ECA (cyclic ECACYC). ECAPG, found in the outer leaflet of the OM, consists of a linear ECA oligomer attached to phosphoglyceride through a phosphodiester linkage. The process through which ECAPG is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECAPG biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between elyC and ECAPG biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found ΔelyC was lethal specifically in strains producing ECAPG without other ECA forms, suggesting ECAPG biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECAPG synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECACYC. Our data demonstrate ECACYC can regulate ECAPG synthesis in strains wild type for elyC. Overall, our data demonstrate ElyC and ECACYC act in a novel pathway that regulates the production of ECAPG, supporting a model in which ElyC provides feedback regulation of ECAPG production based on the periplasmic levels of ECACYC. IMPORTANCE Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales, including Klebsiella pneumoniae, Salmonella enterica, and Yersinia pestis. As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery. Here, we elucidated, in E. coli K-12, a new pathway for the regulation of biosynthesis of one cell surface form of ECA, ECAPG. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECACYC, genetically interact to inhibit the synthesis of ECAPG, potentially through feedback regulation based on ECACYC levels. This is the first insight into the pathway responsible for synthesis of ECAPG and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECAPG levels.

Project description:Sulfur is an essential nutrient that contributes to cellular redox homeostasis, transcriptional regulation, and translation initiation when incorporated into different biomolecules. Transport and reduction of extracellular sulfate followed by cysteine biosynthesis is a major pathway of bacterial sulfur assimilation. For the opportunistic pathogen Serratia marcescens, function of the cysteine biosynthesis pathway is required for extracellular phospholipase activity and flagellum-mediated surface motility, but little else is known about the influence of sulfur assimilation on the physiology of this organism. In this work, it was determined that an S. marcescens cysteine auxotroph fails to differentiate into hyperflagellated and elongated swarmer cells and that cysteine, but not other organic sulfur molecules, restores swarming motility to these bacteria. The S. marcescens cysteine auxotroph further exhibits reduced transcription of phospholipase, hemolysin, and flagellin genes, each of which is subject to transcriptional control by the flagellar regulatory system. Based on these data and the central role of cysteine in sulfur assimilation, it was reasoned that environmental sulfur availability may contribute to the regulation of these functions in S. marcescens Indeed, bacteria that are starved for sulfate exhibit substantially reduced transcription of the genes for hemolysin, phospholipase, and the FlhD flagellar master regulator. A global transcriptomic analysis further defined a large set of S. marcescens genes that are responsive to extracellular sulfate availability, including genes that encode membrane transport, nutrient utilization, and metabolism functions. Finally, sulfate availability was demonstrated to alter S. marcescens cytolytic activity, suggesting that sulfate assimilation may impact the virulence of this organism.IMPORTANCESerratia marcescens is a versatile bacterial species that inhabits diverse environmental niches and is capable of pathogenic interactions with host organisms ranging from insects to humans. This report demonstrates for the first time the extensive impacts that environmental sulfate availability and cysteine biosynthesis have on the transcriptome of S. marcescens The finding that greater than 1,000 S. marcescens genes are differentially expressed depending on sulfate availability suggests that sulfur abundance is a crucial factor that controls the physiology of this organism. Furthermore, the high relative expression levels for the putative virulence factors flagella, phospholipase, and hemolysin in the presence of sulfate suggests that a sulfur-rich host environment could contribute to the transcription of these genes during infection.

Project description:A clinical isolate of Serratia marcescens (TN9106) produced a metallo beta-lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum beta-lactams. The blaIMP gene providing imipenem resistance was cloned and expressed in Escherichia coli HB101. The IMP-1 was purified from E. coli HB101 that harbors pSMBNU24 carrying blaIMP, and its apparent molecular mass was calculated to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of IMP-1 against various beta-lactams revealed that this enzyme hydrolyzes not only various broad-spectrum beta-lactams but also carbapenems. However, aztreonam was relatively stable against IMP-1. Although clavulanate or cloxacillin failed to inhibit IMP-1, Hg2+, Fe2+, or Cu2+ blocked the enzyme's activity. Moreover, the presence of EDTA in the reaction buffer resulted in a decrease in the enzyme's activity. Carbapenem resistance was not transferred from S. marcescens TN9106 to E. coli CSH2 by conjugation. A hybridization study confirmed that blaIMP was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, blaIMP was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the metallo beta-lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of blaIMP was 39.4%. Four consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial metallo beta-lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum beta-lactams.

Project description:UnlabelledMany polarly flagellated bacteria require similar two-component regulatory systems (TCSs) and σ(54) to activate transcription of genes essential for flagellar motility. Herein, we discovered that in addition to the flagellar type III secretion system (T3SS), the Campylobacter jejuni flagellar MS ring and rotor are required to activate the FlgSR TCS. Mutants lacking the FliF MS ring and FliG C ring rotor proteins were as defective as T3SS mutants in FlgSR- and σ(54)-dependent flagellar gene expression. Also, FliF and FliG required each other for stability, which is mediated by atypical extensions to the proteins. A FliF mutant that presumably does not interact with the T3SS protein FlhA did not support flagellar gene transcription, suggesting that FliF-T3SS interactions are essential to generate a signal sensed by the cytoplasmic FlgS histidine kinase. Furthermore, the flagellar T3SS was required for FlgS to immunoprecipitate with FliF and FliG. We propose a model whereby the flagellar T3SS facilitates FliF and FliG multimerization into the MS ring and rotor. As a result, these flagellar structures form a cytoplasmic complex that interacts with and is sensed by FlgS. The synthesis of these structures appears to be a regulatory checkpoint in flagellar biogenesis that the FlgS kinase monitors to initiate signal transduction that activates σ(54) and expression of genes required for the next stage of flagellation. Given that other polar flagellates have flagellar transcriptional hierarchies that are organized similarly as in C. jejuni, this regulatory checkpoint may exist in a broad range of bacteria to influence similar TCSs and flagellar gene transcription.ImportanceDespite the presence of numerous two-component regulatory systems (TCSs) in bacteria, direct signals sensed by TCSs to activate signal transduction are known for only a minority. Polar flagellates, including Pseudomonas, Vibrio, Helicobacter, and Campylobacter species, require a similar TCS and σ(54) for flagellar gene transcription, but the activating signals for these TCSs are unknown. We explored signals that activate the Campylobacter jejuni FlgSR TCS to initiate σ(54)-dependent flagellar gene transcription. Our discoveries suggest that the FlgS histidine kinase monitors the formation of the flagellar type III secretion system and the surrounding MS and C rings. The synthesis of these structures creates a regulatory checkpoint in flagellar biogenesis that is sensed by FlgS to ensure proper transcription of the next set of genes for subsequent steps in flagellation. Given the conservation of flagellar-associated TCSs and transcriptional cascades in polar flagellates, this regulatory checkpoint in flagellar biogenesis likely impacts flagellation in a broad range of bacteria.

Dataset Information

Enterobacterial common antigen integrity is a checkpoint for flagellar biogenesis in Serratia marcescens.

Publications

Enterobacterial common antigen integrity is a checkpoint for flagellar biogenesis in Serratia marcescens.

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