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

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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Overexpression of the waaZ Gene Leads to Modification of the Structure of the Inner Core Region of Escherichia coli Lipopolysaccharide, Truncation of the Outer Core, and Reduction of the Amount of O Polysaccharide on the Cell Surface

Journal of Bacteriology ◽

10.1128/jb.185.5.1659-1671.2003 ◽

2003 ◽

Vol 185 (5) ◽

pp. 1659-1671 ◽

Cited By ~ 29

Author(s):

Emilisa Frirdich ◽

Buko Lindner ◽

Otto Holst ◽

Chris Whitfield

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Inner Core ◽

Gene Clusters ◽

Outer Core ◽

Core Region ◽

Resonance Spectroscopy ◽

E Coli ◽

K 12 ◽

Escherichia Coli Lipopolysaccharide

ABSTRACT The waa gene cluster is responsible for the biosynthesis of the lipopolysaccharide (LPS) core region in Escherichia coli and Salmonella. Homologs of the waaZ gene product are encoded by the waa gene clusters of Salmonella enterica and E. coli strains with the K-12 and R2 core types. Overexpression of WaaZ in E. coli and S. enterica led to a modified LPS structure showing core truncations and (where relevant) to a reduction in the amount of O-polysaccharide side chains. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the predominant LPS structures in an E. coli isolate with an R1 core (waaZ is lacking from the type R1 waa gene cluster) with a copy of the waaZ gene added on a plasmid. Novel truncated LPS structures, lacking up to 3 hexoses from the outer core, resulted from WaaZ overexpression. The truncated molecules also contained a KdoIII residue not normally found in the R1 core.

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LamB, OmpC, and the Core Lipopolysaccharide of Escherichia coli K-12 Function as Receptors of Bacteriophage Bp7

Journal of Virology ◽

10.1128/jvi.00325-20 ◽

2020 ◽

Vol 94 (12) ◽

Cited By ~ 1

Author(s):

Peipei Chen ◽

Huzhi Sun ◽

Huiying Ren ◽

Wenhua Liu ◽

Guimei Li ◽

...

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Inner Core ◽

Phage Infection ◽

Broad Host Range ◽

Content Type ◽

E Coli ◽

Phage Adsorption ◽

Specific Receptors ◽

K 12

ABSTRACT Bp7 is a T-even phage with a broad host range specific to Escherichia coli, including E. coli K-12. The receptor binding protein (RBP) of bacteriophages plays an important role in the phage adsorption process and determines phage host range, but the molecular mechanism involved in host recognition of phage Bp7 remains unknown. In this study, the interaction between phage Bp7 and E. coli K-12 was investigated. Based on homology alignment, amino acid sequence analysis, and a competitive assay, gp38, located at the tip of the long tail fiber, was identified as the RBP of phage Bp7. Using a combination of in vivo and in vitro approaches, including affinity chromatography, gene knockout mutagenesis, a phage plaque assay, and phage adsorption kinetics analysis, we identified the LamB and OmpC proteins on the surface of E. coli K-12 as specific receptors involved in the first step of reversible phage adsorption. Genomic analysis of the phage-resistant mutant strain E. coli K-12-R and complementation tests indicated that HepI of the inner core of polysaccharide acts as the second receptor recognized by phage Bp7 and is essential for successful phage infection. This observation provides an explanation of the broad host range of phage Bp7 and provides insight into phage-host interactions. IMPORTANCE The RBPs of T4-like phages are gp37 and gp38. The interaction between phage T4 RBP gp37 and its receptors has been clarified by many reports. However, the interaction between gp38 and its receptors during phage adsorption is still not completely understood. Here, we identified phage Bp7, which uses gp38 as an RBP, and provided a good model to study the phage-host interaction mechanisms in an enterobacteriophage. Our study revealed that gp38 of phage Bp7 recognizes the outer membrane proteins (OMPs) LamB and OmpC of E. coli K-12 as specific receptors and binds with them reversibly. HepI of the inner-core oligosaccharide is the second receptor and binds with phage Bp7 irreversibly to begin the infection process. Determining the interaction between the phage and its receptors will help elucidate the mechanisms of phage with a broad host range and help increase understanding of the phage infection mechanism based on gp38.

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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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Characterization of the flexible genome complement of the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31)

Microbiology ◽

10.1099/mic.0.27469-0 ◽

2005 ◽

Vol 151 (2) ◽

pp. 385-398 ◽

Cited By ~ 33

Author(s):

Jana Hejnova ◽

Ulrich Dobrindt ◽

Radka Nemcova ◽

Christophe Rusniok ◽

Alojz Bomba ◽

...

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Gene Clusters ◽

Coli Strain ◽

Bac Clone ◽

Sequence Comparisons ◽

Multiple Sequence ◽

E Coli ◽

Gut Colonization ◽

K 12

Colonization by the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31) has proved to be safe and efficient in the prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants in Czech paediatric clinics over the past three decades. In searching for traits contributing to this beneficial effect related to the gut colonization capacity of the strain, the authors have analysed its genome by DNA–DNA hybridization to E. coli K-12 (MG1655) genomic DNA arrays and to ‘Pathoarrays’, as well as by multiplex PCR, bacterial artificial chromosome (BAC) library cloning and shotgun sequencing. Four hundred and ten E. coli K-12 ORFs were absent from A0 34/86, while 72 out of 456 genes associated with pathogenicity islands of E. coli and Shigella were also detected in E. coli A0 34/86. Furthermore, extraintestinal pathogenic E. coli-related genes involved in iron uptake and adhesion were detected by multiplex PCR, and genes encoding the HlyA and cytotoxic necrotizing factor toxins, together with 21 genes of the uropathogenic E. coli 536 pathogenicity island II, were identified by analysis of 2304 shotgun and 1344 BAC clone sequences of A0 34/86 DNA. Multiple sequence comparisons identified 31 kb of DNA specific for E. coli A0 34/86; some of the genes carried by this DNA may prove to be implicated in the colonization capacity of the strain, enabling it to outcompete pathogens. Among 100 examined BAC clones roughly covering the A0 34/86 genome, one reproducibly conferred on the laboratory strain DH10B an enhanced capacity to persist in the intestine of newborn piglets. Sequencing revealed that this BAC clone carried gene clusters encoding gluconate and mannonate metabolism, adhesion (fim), invasion (ibe) and restriction/modification functions. Hence, the genome of this clinically safe and highly efficient colonizer strain appears to harbour many ‘virulence-associated’ genes. These results highlight the thin line between bacterial ‘virulence’ and ‘fitness' or ‘colonization’ factors, and question the definition of enterobacterial virulence factors.

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A SitABCD homologue from an avian pathogenic Escherichia coli strain mediates transport of iron and manganese and resistance to hydrogen peroxide

Microbiology ◽

10.1099/mic.0.28682-0 ◽

2006 ◽

Vol 152 (3) ◽

pp. 745-758 ◽

Cited By ~ 85

Author(s):

Mourad Sabri ◽

Simon Léveillé ◽

Charles M. Dozois

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Metal Ion ◽

Growth Promotion ◽

Coli Strain ◽

Transport Systems ◽

Virulence Plasmid ◽

E Coli ◽

K 12 ◽

Iron And Manganese

An operon encoding a member of the family of ATP-binding cassette (ABC) divalent metal ion transporters, homologous to Salmonella enterica SitABCD, has been identified in the avian pathogenic Escherichia coli (APEC) strain χ7122. The sitABCD genes were located on the virulence plasmid pAPEC-1, and were highly similar at the nucleotide level to the chromosomally encoded sitABCD genes present in Shigella spp. A cloned copy of sitABCD conferred increased growth upon a siderophore-deficient E. coli strain grown in nutrient broth supplemented with the chelator 2,2′-dipyridyl. Ion rescue demonstrated that Sit-mediated growth promotion of this strain was due to the transport of iron. SitABCD mediated increased transport of both iron and manganese as demonstrated by uptake of 55Fe, 59Fe or 54Mn in E. coli K-12 strains deficient for the transport of iron (aroB feoB) and manganese (mntH) respectively. Isotope uptake and transport inhibition studies showed that in the iron transport deficient strain, SitABCD demonstrated a greater affinity for iron than for manganese, and SitABCD-mediated transport was higher for ferrous iron, whereas in the manganese transport deficient strain, SitABCD demonstrated greater affinity for manganese than for iron. Introduction of the APEC sitABCD genes into an E. coli K-12 mntH mutant also conferred increased resistance to the bactericidal effects of hydrogen peroxide. APEC strain χ7122 derivatives lacking either a functional SitABCD or a functional MntH transport system were as resistant to hydrogen peroxide as the wild-type strain, whereas a Δsit ΔmntH double mutant was more sensitive to hydrogen peroxide. Overall, the results demonstrate that in E. coli SitABCD represents a manganese and iron transporter that, in combination with other ion transport systems, may contribute to acquisition of iron and manganese, and resistance to oxidative stress.

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Human Neutrophil Chemotaxis Is Modulated by Capsule and O Antigen from an Extraintestinal Pathogenic Escherichia coli Strain

Infection and Immunity ◽

10.1128/iai.71.11.6435-6445.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6435-6445 ◽

Cited By ~ 9

Author(s):

Thomas A. Russo ◽

Bruce A. Davidson ◽

Diana M. Topolnycky ◽

Ruth Olson ◽

Stacy A. Morrill ◽

...

Keyword(s):

Escherichia Coli ◽

Ex Vivo ◽

Specific Antigen ◽

Lavage Fluid ◽

Coli Strain ◽

Neutrophil Chemotaxis ◽

Gram Negative ◽

E Coli ◽

O Antigen

ABSTRACT Gram-negative enteric bacilli, such as Escherichia coli, are common causes of nosocomial pneumonia. The interaction between pulmonary neutrophils and the infecting pathogen is a critical step in determining the outcome. Previous studies from our laboratory, for which a rat model of pneumonia was used, established that pulmonary neutrophil recruitment was modulated by the E. coli virulence factors capsule and O-specific antigen. To begin to understand the mechanism by which this recruitment occurs, we conducted in vitro and ex vivo chemotaxis assays, for which we used a clinically relevant E. coli isolate (CP9) and isogenic derivatives that were deficient in only the O antigen (CP921) or capsule (CP9.137) as chemoattractants with or without the high-affinity N-formylmethionyl-leucyl-phenylalanine receptor antagonist N-tert-butoxycarbonyl-methionine-leucine-phenylalanine (N-t-BOC). Given that only live E. coli was used for the initial in vitro chemotaxis assays, it was predicted that only N-t-BOC-sensitive chemotaxis would occur. However, both N-t-BOC-sensitive and -insensitive chemotaxis was observed. N-t-BOC-insensitive chemotaxis was mediated in part by interleukin 8, which was produced by neutrophils that had migrated toward E. coli. N-t-BOC-insensitive chemotaxis was only observed when live E. coli bacteria, not cell-free E. coli culture supernatants, were used as chemoattractants, suggesting that a direct E. coli-neutrophil interaction was necessary. The presence of both capsule and O antigen diminished total, N-t-BOC-sensitive, and N-t-BOC-insensitive neutrophil chemotaxis in vitro. The presence of capsule significantly decreased total, N-t-BOC-sensitive, and N-t-BOC-insensitive neutrophil chemotaxis ex vivo when cell-free bronchoalveolar lavage fluid from infected rats was used as the source of chemotactic factors. These effects of E. coli capsule and O antigen on neutrophil chemotaxis are novel, and they expand our understanding of the mechanisms by which these virulence traits contribute to the pathogenesis of gram-negative pneumonia and other extraintestinal infections.

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Structural Determination and Genetic Identification of the O-Antigen from an Escherichia coli Strain, LL004, Representing a Novel Serogroup

International Journal of Molecular Sciences ◽

10.3390/ijms222312746 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12746

Author(s):

Jing Wang ◽

Yujuan Xu ◽

Chunjun Qin ◽

Jing Hu ◽

Jian Yin ◽

...

Keyword(s):

Escherichia Coli ◽

Nmr Spectra ◽

Coli Strain ◽

Genetic Identification ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

O Antigen ◽

Antigen Structure ◽

Dependent Pathway

The O-antigen is the outermost component of the lipopolysaccharide layer in Gram-negative bacteria, and the variation of O-antigen structure provides the basis for bacterial serological diversity. Here, we determined the O-antigen structure of an Escherichia coli strain, LL004, which is totally different from all of the E. coli serogroups. The tetrasaccharide repeating unit was determined as →4)-β-d-Galp-(1→3)-β-d-GlcpNAc6OAc(~70%)-(1→3)-β-d-GalpA-(1→3)-β-d-GalpNAc-(1→ with monosaccharide analysis and NMR spectra. We also characterized the O-antigen gene cluster of LL004, and sequence analysis showed that it correlated well with the O-antigen structure. Deletion and complementation testing further confirmed its role in O-antigen biosynthesis, and indicated that the O-antigen of LL004 is assembled via the Wzx/Wzy dependent pathway. Our findings, in combination, suggest that LL004 should represent a novel serogroup of E. coli.

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Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.152.3.1138-1146.1982 ◽

1982 ◽

Vol 152 (3) ◽

pp. 1138-1146

Author(s):

L J Lee ◽

J B Hansen ◽

E K Jagusztyn-Krynicka ◽

B M Chassy

Keyword(s):

Escherichia Coli ◽

Lactobacillus Casei ◽

Specific Activity ◽

Protein Product ◽

Coli Strain ◽

Cloning And Expression ◽

E Coli ◽

Lactose Metabolism ◽

Gene Coding ◽

K 12

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.

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Identification of an Escherichia coli Operon Required for Formation of the O-Antigen Capsule

Journal of Bacteriology ◽

10.1128/jb.187.15.5259-5266.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5259-5266 ◽

Cited By ~ 57

Author(s):

Adi Peleg ◽

Yulia Shifrin ◽

Ophir Ilan ◽

Chen Nadler-Yona ◽

Shani Nov ◽

...

Keyword(s):

Escherichia Coli ◽

Secretion System ◽

Side Chain ◽

E Coli ◽

O Antigen ◽

Group 4 ◽

K 12 ◽

Polysaccharide Capsules ◽

Polysaccharide Secretion ◽

Group 1

ABSTRACT Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.

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A Commensal Gone Bad: Complete Genome Sequence of the Prototypical Enterotoxigenic Escherichia coli Strain H10407

Journal of Bacteriology ◽

10.1128/jb.00710-10 ◽

2010 ◽

Vol 192 (21) ◽

pp. 5822-5831 ◽

Cited By ~ 121

Author(s):

Lisa C. Crossman ◽

Roy R. Chaudhuri ◽

Scott A. Beatson ◽

Timothy J. Wells ◽

Mickael Desvaux ◽

...

Keyword(s):

Escherichia Coli ◽

Genomic Sequence ◽

Epidemiological Data ◽

Coli Strain ◽

Enterotoxigenic Escherichia Coli ◽

E Coli ◽

Extraintestinal Disease ◽

K 12 ◽

Genetic Context ◽

Complete Genomic

ABSTRACT In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.

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