Defective O-Antigen Polymerization in tolA and pal Mutants of Escherichia coli in Response to Extracytoplasmic Stress

ABSTRACT We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, ΔtolA and Δpal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both ΔtolA and Δpal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in ΔtolA and Δpal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110ΔtolA and W3110Δpal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol + and pal + genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

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The Small Noncoding DsrA RNA Is an Acid Resistance Regulator in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.186.18.6179-6185.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6179-6185 ◽

Cited By ~ 65

Author(s):

Richard A. Lease ◽

Dorie Smith ◽

Kathleen McDonough ◽

Marlene Belfort

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Resistance Genes ◽

Acid Resistance ◽

Dna Arrays ◽

E Coli ◽

Specific Mrnas ◽

Steady State Levels ◽

Control Translation ◽

K 12

ABSTRACT DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (σs), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.

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The yliA, -B, -C, and -D Genes of Escherichia coli K-12 Encode a Novel Glutathione Importer with an ATP-Binding Cassette

Journal of Bacteriology ◽

10.1128/jb.187.17.5861-5867.2005 ◽

2005 ◽

Vol 187 (17) ◽

pp. 5861-5867 ◽

Cited By ~ 61

Author(s):

Hideyuki Suzuki ◽

Takashi Koyanagi ◽

Shunsuke Izuka ◽

Akiko Onishi ◽

Hidehiko Kumagai

Keyword(s):

Escherichia Coli ◽

Atp Binding ◽

Sulfur Source ◽

Dependent Manner ◽

Oxygen Species ◽

E Coli ◽

Living Organisms ◽

K 12 ◽

Atp Binding Cassette

ABSTRACT Glutathione protects cells and organisms from oxygen species and peroxides and is indispensable for aerobically living organisms. Moreover, it acts against xenobiotics and drugs by the formation and excretion of glutathione S conjugates. In this study, we show that the yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a glutathione transporter with the ATP-binding cassette. The transporter imports extracellular glutathione into the cytoplasm in an ATP-dependent manner. This transporter, along with γ-glutamyltranspeptidase, has an important role in E. coli growth with glutathione as a sole sulfur source.

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Structure and genetics of Escherichia coli O antigens

FEMS Microbiology Reviews ◽

10.1093/femsre/fuz028 ◽

2019 ◽

Vol 44 (6) ◽

pp. 655-683 ◽

Cited By ~ 7

Author(s):

Bin Liu ◽

Axel Furevi ◽

Andrei V Perepelov ◽

Xi Guo ◽

Hengchun Cao ◽

...

Keyword(s):

Escherichia Coli ◽

Genetic Basis ◽

Structural Diversity ◽

Gene Clusters ◽

Current Standard ◽

E Coli ◽

Antigen Gene ◽

O Antigen ◽

Or Groups ◽

O Antigens

ABSTRACT Escherichia coli includes clonal groups of both commensal and pathogenic strains, with some of the latter causing serious infectious diseases. O antigen variation is current standard in defining strains for taxonomy and epidemiology, providing the basis for many serotyping schemes for Gram-negative bacteria. This review covers the diversity in E. coli O antigen structures and gene clusters, and the genetic basis for the structural diversity. Of the 187 formally defined O antigens, six (O31, O47, O67, O72, O94 and O122) have since been removed and three (O34, O89 and O144) strains do not produce any O antigen. Therefore, structures are presented for 176 of the 181 E. coli O antigens, some of which include subgroups. Most (93%) of these O antigens are synthesized via the Wzx/Wzy pathway, 11 via the ABC transporter pathway, with O20, O57 and O60 still uncharacterized due to failure to find their O antigen gene clusters. Biosynthetic pathways are given for 38 of the 49 sugars found in E. coli O antigens, and several pairs or groups of the E. coli antigens that have related structures show close relationships of the O antigen gene clusters within clades, thereby highlighting the genetic basis of the evolution of diversity.

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KatP contributes to OxyR-regulated hydrogen peroxide resistance in Escherichia coli serotype O157 : H7

Microbiology ◽

10.1099/mic.0.031435-0 ◽

2009 ◽

Vol 155 (11) ◽

pp. 3589-3598 ◽

Cited By ~ 29

Author(s):

Gaylen A. Uhlich

Keyword(s):

Escherichia Coli ◽

Oxidative Damage ◽

Physiological Role ◽

Deletion Strain ◽

Lag Phase ◽

Dependent Manner ◽

E Coli ◽

Mutant Strains ◽

Catalase Peroxidase ◽

K 12

Escherichia coli K-12 defends itself against peroxide-mediated oxidative damage using two catalases, KatG and KatE, and the peroxiredoxin, alkyl hydroperoxide reductase, encoded by ahpC. In E. coli O157 : H7 strain ATCC 43895 (EDL933), plasmid pO157 carries an additional catalase-peroxidase gene, katP. KatP has been shown to be a functional catalase-peroxidase. However, deletion of pO157 does not alter the peroxide resistance of strain EDL933, leaving the physiological role of katP unclear. To examine the individual roles of peroxide-resistance genes in E. coli O157 : H7, mutant strains of ATCC 43895 were constructed bearing individual deletions of katG, katE, katP and ahpC, as well as double, triple and quadruple deletions encompassing all possible gene combinations thereof. The wild-type and all 15 mutant strains were compared for differences in aerobic growth, ability to scavenge exogenous H2O2 and resistance to exogenous peroxides. Although KatG scavenged the most exogenous H2O2, KatP scavenged statistically greater amounts than either KatE or AhpC during exponential growth. However, katG and ahpC together were sufficient for full peroxide resistance in disc diffusion assays. Strains with only katG or ahpC were the only triple deletion strains with significantly shorter generation times than the quadruple deletion strain. ahpC was the only gene that could allow rapid transition from lag phase to exponential phase in a triple deletion strain. Gene expression studies revealed that katP is an OxyR-regulated gene, but its expression is suppressed in stationary phase by RpoS. These studies indicate that pO157-borne katP contributes to the complex gene network protecting strain 43895 from peroxide-mediated oxidative damage in an OxyR-dependent manner.

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Overlapping stimulons arising in response to divergent stresses in Escherichia coli

10.1101/2021.12.17.473201 ◽

2021 ◽

Author(s):

Huijing Wang ◽

GW McElfresh ◽

Nishantha Wijesuriya ◽

Adam Podgorny ◽

Andrew D Hecht ◽

...

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Stress Responses ◽

Differential Expression Analysis ◽

Stringent Response ◽

Coli Strain ◽

Growth Media ◽

Overflow Metabolism ◽

Rna Seq ◽

E Coli

Cellular responses to stress can cause a similar change in some facets of fitness even if the stresses are different. Lactose as a sole carbon source for Escherichia coli is an established example: too little causes starvation while excessive lactose import causes toxicity as a side-effect. In an E. coli strain that is robust to osmotic and ionic differences in growth media, B REL606, the rate of antibiotic-tolerant persister formation is elevated in both starvation-inducing and toxicity-inducing concentrations of lactose in comparison to less stressful intermediate concentrations. Such similarities between starvation and toxification raise the question of how much the global stress response stimulon differs between them. We hypothesized that a common stress response is conserved between the two conditions, but that a previously shown threshold driving growth rate heterogeneity in a lactose-toxifying medium would reveal that the growing fraction of cells in that medium to be missing key stress responses that curb growth. To test this, we performed RNA-seq in three representative conditions for differential expression analysis. In comparison to nominally unstressed cultures, both stress conditions showed global shifts in gene expression, with informative similarities and differences. Functional analysis of pathways, gene ontology terms, and clusters of orthogonal groups revealed signatures of overflow metabolism, membrane component shifts, and altered cytosolic and periplasmic contents in toxified cultures. Starving cultures showed an increased tendency toward stringent response-like regulatory signatures. Along with other emerging evidence, our results show multiple possible pathways to stress responses, persistence, and possibly other phenotypes. These results suggest a set of overlapping responses that drives emergence of stress-tolerant phenotypes in diverse conditions.

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CS5 Pilus Biosynthesis Genes from Enterotoxigenic Escherichia coli O115:H40

Journal of Bacteriology ◽

10.1128/jb.181.18.5847-5851.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5847-5851 ◽

Cited By ~ 3

Author(s):

Thomas G. Duthy ◽

Lothar H. Staendner ◽

Paul A. Manning ◽

Michael W. Heuzenroeder

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Surface Expression ◽

Open Reading Frames ◽

Enterotoxigenic Escherichia Coli ◽

Cell Surface Expression ◽

E Coli ◽

Pilus Biogenesis ◽

K 12 ◽

And Function

ABSTRACT We have sequenced the entire region of DNA required for the biosynthesis of CS5 pili from enterotoxigenic Escherichia coli O115:H40 downstream of the major subunit gene, designatedcsfA (for coli surface factor five A). Five more open reading frames (ORFs) (csfB, csfC,csfE, csfF, and csfD) which are transcribed in the same direction as the major subunit and are flanked by a number of insertion sequence regions have been identified. T7 polymerase-mediated overexpression of the cloned csf ORFs confirmed protein sizes based on the DNA sequences that encode them. The expression of only the csf region in E. coli K-12 resulted in the hemagglutination of human erythrocytes and the cell surface expression of CS5 pili, suggesting that the cluster contains all necessary information for CS5 pilus biogenesis and function.

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IraL Is an RssB Anti-adaptor That Stabilizes RpoS during Logarithmic Phase Growth in Escherichia coli and Shigella

mBio ◽

10.1128/mbio.01043-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 12

Author(s):

Andrew J. Hryckowian ◽

Aurelia Battesti ◽

Justin J. Lemke ◽

Zachary C. Meyer ◽

Rodney A. Welch

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Stress Response ◽

Sigma Factor ◽

Logarithmic Phase ◽

Phase Growth ◽

Content Type ◽

General Stress Response ◽

E Coli ◽

K 12

ABSTRACTRpoS (σS), the general stress response sigma factor, directs the expression of genes under a variety of stressful conditions. Control of the cellular σSconcentration is critical for appropriately scaled σS-dependent gene expression. One way to maintain appropriate levels of σSis to regulate its stability. Indeed, σSdegradation is catalyzed by the ClpXP protease and the recognition of σSby ClpXP depends on the adaptor protein RssB. Three anti-adaptors (IraD, IraM, and IraP) exist inEscherichia coliK-12; each interacts with RssB andinhibitsRssBactivity under different stress conditions, thereby stabilizing σS. Unlike K-12, someE. coliisolates, including uropathogenicE. colistrain CFT073, show comparable cellular levels of σSduring the logarithmic and stationary growth phases, suggesting that there are differences in the regulation of σSlevels amongE. colistrains. Here, we describe IraL, an RssB anti-adaptor that stabilizes σSduring logarithmic phase growth in CFT073 and otherE. coliandShigellastrains. By immunoblot analyses, we show that IraL affects the levels and stability of σSduring logarithmic phase growth. By computational and PCR-based analyses, we reveal thatiraLis found in manyE. colipathotypes but not in laboratory-adapted strains. Finally, by bacterial two-hybrid and copurification analyses, we demonstrate that IraL interacts with RssB by a mechanism distinct from that used by other characterized anti-adaptors. We introduce a fourth RssB anti-adaptor found inE. colispecies and suggest that differences in the regulation of σSlevels may contribute to host and niche specificity in pathogenic and nonpathogenicE. colistrains.IMPORTANCEBacteria must cope with a variety of environmental conditions in order to survive. RpoS (σS), the general stress response sigma factor, directs the expression of many genes under stressful conditions in both pathogenic and nonpathogenicEscherichia colistrains. The regulation of σSlevels and activity allows appropriately scaled σS-dependent gene expression. Here, we describe IraL, an RssB anti-adaptor that, unlike previously described anti-adaptors, stabilizes σSduring the logarithmic growth phase in the absence of additional stress. We also demonstrate thatiraLis found in a large number ofE. coliandShigellaisolates. These data suggest that strains containingiraLare able to initiate σS-dependent gene expression under conditions under which strains withoutiraLcannot. Therefore, IraL-mediated σSstabilization may contribute to host and niche specificity inE. coli.

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O-Antigen-Dependent Colicin Insensitivity of UropathogenicEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00545-18 ◽

2018 ◽

Vol 201 (4) ◽

Cited By ~ 12

Author(s):

Connor Sharp ◽

Christine Boinett ◽

Amy Cain ◽

Nicholas G. Housden ◽

Sandip Kumar ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Growth Conditions ◽

Membrane Receptors ◽

Cell Surface Receptor ◽

Dominant Factor ◽

Content Type ◽

E Coli ◽

O Antigen ◽

K 12

ABSTRACTThe outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted byEscherichia coli, can target otherE. colicells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of variousE. colistrains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why doE. colistrains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenicE. colisequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen intoE. coliK-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizingE. colitoward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coliinfections can be a major health burden, especially with the organism becoming increasingly resistant to “last-resort” antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenicE. colistrain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity amongE. coliorganisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenicE. colisuch as uropathogenicE. coli(UPEC).

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The lipopolysaccharide of the mastitis isolate Escherichia coli strain 1303 comprises a novel O-antigen and the rare K-12 core type

Microbiology ◽

10.1099/mic.0.046912-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1750-1760 ◽

Cited By ~ 13

Author(s):

Katarzyna A. Duda ◽

Buko Lindner ◽

Helmut Brade ◽

Andreas Leimbach ◽

Elżbieta Brzuszkiewicz ◽

...

Keyword(s):

Escherichia Coli ◽

Inner Core ◽

Bovine Mastitis ◽

Outer Core ◽

Coli Strain ◽

Core Oligosaccharide ◽

2D Nmr Spectroscopy ◽

E Coli ◽

O Antigen ◽

K 12

Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[→4)-β-d-Quip3NAc-(1→3)-α-l-Fucp2OAc-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli 1303 was also determined. This cluster, located between the gnd and galF genes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal ld-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core – namely terminal ld-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolate E. coli 1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.

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Structure of the O-antigen of Salmonella O66 and the genetic basis for similarity and differences between the closely related O-antigens of Escherichia coli O166 and Salmonella O66

Microbiology ◽

10.1099/mic.0.037325-0 ◽

2010 ◽

Vol 156 (6) ◽

pp. 1642-1649 ◽

Cited By ~ 10

Author(s):

Bin Liu ◽

Andrei V. Perepelov ◽

Dan Li ◽

Sof'ya N. Senchenkova ◽

Yanfang Han ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Gene Clusters ◽

Coding Region ◽

E Coli ◽

Antigen Gene ◽

O Antigen ◽

New Genes ◽

O Antigens ◽

Antigen Structure

O-antigen is a component of the outer membrane of Gram-negative bacteria and is one of the most variable cell surface constituents, leading to major antigenic variability. The O-antigen forms the basis for bacterial serotyping. In this study, the O-antigen structure of Salmonella O66 was established, which differs from the known O-antigen structure of Escherichia coli O166 only in one linkage (most likely the linkage between the O-units) and O-acetylation. The O-antigen gene clusters of Salmonella O66 and E. coli O166 were found to have similar organizations, the only exception being that in Salmonella O66, the wzy gene is replaced by a non-coding region. The function of the wzy gene in E. coli O166 was confirmed by the construction and analysis of deletion and trans-complementation mutants. It is proposed that a functional wzy gene located outside the O-antigen gene cluster is involved in Salmonella O66 O-antigen biosynthesis, as has been reported previously in Salmonella serogroups A, B and D1. The sequence identity for the corresponding genes between the O-antigen gene clusters of Salmonella O66 and E. coli O166 ranges from 64 to 70 %, indicating that they may originate from a common ancestor. It is likely that after the species divergence, Salmonella O66 got its specific O-antigen form by inactivation of the wzy gene located in the O-antigen gene cluster and acquisition of two new genes (a wzy gene and a prophage gene for O-acetyl modification) both residing outside the O-antigen gene cluster.

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