Isolation of Additional Bacteriophages with Genomes of Segmented Double-Stranded RNA

ABSTRACT Eight different bacteriophages were isolated from leaves ofPisum sativum, Phaseolus vulgaris,Lycopersicon esculentum, Daucus carota sativum,Raphanus sativum, and Ocimum basilicum. All contain three segments of double-stranded RNA and have genomic-segment sizes that are similar but not identical to those of previously described bacteriophage φ6. All appear to have lipid-containing membranes. The base sequences of some of the viruses are very similar but not identical to those of φ6. Three of the viruses have little or no base sequence identity to φ6. Two of the viruses, φ8 and φ12, contain proteins with a size distribution very different from that of φ6 and do not package genomic segments of φ6. Whereas φ6 attaches to host cells by means of a pilus, several of the new isolates attach directly to the outer membrane. Although the normal hosts of these viruses seem to be pseudomonads, those viruses that attach directly to the outer membrane can establish carrier states in Escherichia coli or Salmonella typhimurium. One of the isolates, φ8, can form plaques on heptoseless strains of S. typhimurium.

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Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability

Journal of Bacteriology ◽

10.1128/jb.00498-06 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5385-5392 ◽

Cited By ~ 191

Author(s):

Amanda J. McBroom ◽

Alexandra P. Johnson ◽

Sreekanth Vemulapalli ◽

Meta J. Kuehn

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Cell Envelope ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Fold Increase ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Physiological Basis ◽

Gram Negative

ABSTRACT It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the σE cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.

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Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli

Microorganisms ◽

10.3390/microorganisms8081184 ◽

2020 ◽

Vol 8 (8) ◽

pp. 1184 ◽

Cited By ~ 1

Author(s):

Alexia N. Torres ◽

Nayaret Chamorro-Veloso ◽

Priscila Costa ◽

Leandro Cádiz ◽

Felipe Del Canto ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Shiga Toxin ◽

Outer Membrane Proteins ◽

Protein A ◽

Subcellular Location ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Virulence Determinants ◽

T Lymphocyte Proliferation

Shiga toxin-producing Escherichia coli (STEC) causes outbreaks and sporadic cases of gastroenteritis. STEC O157:H7 is the most clinically relevant serotype in the world. The major virulence determinants of STEC O157:H7 are the Shiga toxins and the locus of enterocyte effacement. However, several accessory virulence factors, mainly outer membrane proteins (OMPs) that interact with the host cells may contribute to the virulence of this pathogen. Previously, the elongation factor thermo unstable (EF-Tu), l-asparaginase II and OmpT proteins were identified as antigens in OMP extracts of STEC. The known subcellular location of EF-Tu and l-asparaginase II are the cytoplasm and periplasm, respectively. Therefore, we investigate whether these two proteins may localize on the surface of STEC and, if so, what roles they have at this site. On the other hand, the OmpT protein, a well characterized protease, has been described as participating in the adhesion of extraintestinal pathogenic E. coli strains. Thus, we investigate whether OmpT has this role in STEC. Our results show that the EF-Tu and l-asparaginase II are secreted by O157:H7 and may also localize on the surface of this bacterium. EF-Tu was identified in outer membrane vesicles (OMVs), suggesting it as a possible export mechanism for this protein. Notably, we found that l-asparaginase II secreted by O157:H7 inhibits T-lymphocyte proliferation, but the role of EF-Tu at the surface of this bacterium remains to be elucidated. In the case of OmpT, we show its participation in the adhesion of O157:H7 to human epithelial cells. Thus, this study extends the knowledge of the pathogenic mechanisms of STEC.

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Secretin of the Enteropathogenic Escherichia coli Type III Secretion System Requires Components of the Type III Apparatus for Assembly and Localization

Infection and Immunity ◽

10.1128/iai.71.6.3310-3319.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3310-3319 ◽

Cited By ~ 114

Author(s):

Annick Gauthier ◽

Jose Luis Puente ◽

B. Brett Finlay

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Biochemical Analysis ◽

Host Cells ◽

Type Iii Effectors ◽

Type Iii ◽

Secretion Pathway ◽

Detergent Extraction ◽

Inner Membrane Protein ◽

And Function

ABSTRACT At least 16 proteins are thought to be involved in forming the enteropathogenic Escherichia coli (EPEC) type III translocation apparatus which delivers virulence factors into host cells, yet their function and location have not been determined. A biochemical analysis was performed on three components: EscN, a predicted cytoplasmic ATPase; EscV, a predicted inner membrane protein; and EscC, a predicted outer membrane secretin. Wild-type EPEC and mutants constructed in these genes were fractionated by lysozyme treatment, ultracentrifugation, and selective detergent extraction. Fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC, suggesting a continuous channel. Epitope-tagged EscC, EscV, and EscN were localized by fractionation, confirming computer modeling predictions for their location. Transcomplementation experiments revealed that localization of EscV and EscN was unaffected by mutations in other examined type III components. Remarkably, localization of EscC was altered in escV or escN mutants, where EscC accumulated in the periplasm. EscC was correctly localized in the escF needle component mutant, indicating that secretin localization is independent of needle formation. These data indicate that, contrary to previous indications, correct insertion and function of EscC secretin in the outer membrane depends not only on the sec-dependent secretion pathway but also on other type III apparatus components.

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Effects of bfp Mutations on Biogenesis of Functional Enteropathogenic Escherichia coli Type IV Pili

Journal of Bacteriology ◽

10.1128/jb.182.9.2498-2506.2000 ◽

2000 ◽

Vol 182 (9) ◽

pp. 2498-2506 ◽

Cited By ~ 50

Author(s):

Ravi P. Anantha ◽

Kelly D. Stone ◽

Michael S. Donnenberg

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Signal Sequence ◽

Outer Membrane Proteins ◽

Laboratory Strain ◽

Type Iv Pili ◽

Host Cells ◽

Wild Type ◽

Type Iv ◽

The Individual

ABSTRACT Enteropathogenic Escherichia coli expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells. A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of E. coli. We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA. Here we report the construction and analysis of nonpolar mutations in six genes of thebfp cluster, bfpG, bfpB,bfpC, bfpD, bfpP, andbfpH, as well as the further analysis of a previously described bfpA mutant strain that is unable to express bundlin, the pilin protein. We found that mutations inbfpB, which encodes an outer membrane protein;bfpD, which encodes a putative nucleotide-binding protein; and bfpG and bfpC, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA. The mutation in bfpP, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA. The mutation in bfpH, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA. For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes. We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins. These studies indicate that many bfp gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly.

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Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

Journal of Bacteriology ◽

10.1128/jb.01493-06 ◽

2006 ◽

Vol 189 (2) ◽

pp. 522-530 ◽

Cited By ~ 22

Author(s):

Bum-Yeol Hwang ◽

Navin Varadarajan ◽

Haixin Li ◽

Sarah Rodriguez ◽

Brent L. Iverson ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Outer Membrane ◽

Amino Acid Sequence Identity ◽

Catalytic Efficiency ◽

Wild Type ◽

Peptide Substrate ◽

Peptide Substrates ◽

E Coli ◽

Sequence Identity

ABSTRACT Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k cat/Km = 3.0 × 106 M−1s−1). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1′ sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3′. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1′, site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F′ episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.

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Chlamydia trachomatis OmcB protein is a surface-exposed glycosaminoglycan-dependent adhesin

Journal of Medical Microbiology ◽

10.1099/jmm.0.46801-0 ◽

2007 ◽

Vol 56 (1) ◽

pp. 15-22 ◽

Cited By ~ 42

Author(s):

Sanaa Fadel ◽

Adrian Eley

Keyword(s):

Escherichia Coli ◽

Chlamydia Trachomatis ◽

Outer Membrane ◽

Hela Cells ◽

Polyclonal Antibodies ◽

Full Length ◽

Host Cells ◽

Gene Encoding ◽

Antigenic Properties ◽

Entire Gene

The OmcB protein of Chlamydia trachomatis is a cysteine-rich outer membrane polypeptide with important functional, structural and antigenic properties. The entire gene encoding the OmcB protein from C. trachomatis serovar LGV1 was cloned and expressed in Escherichia coli and the full-length protein used to raise polyclonal antibodies. Recombinant OmcB was used to show that OmcB is a surface-exposed protein that functions as a chlamydial adhesin. Infectivity inhibition assays carried out using HeLa cells with serovar LGV1 in the presence of purified anti-OmcB serum showed inhibition of infectivity, suggesting that some of the OmcB was surface exposed. Moreover, using recombinant OmcB in infectivity inhibition assays resulted in 70 % inhibition of infectivity, confirming that OmcB plays a role as an adhesin in C. trachomatis. Furthermore, recombinant OmcB protein bound to the surface of HeLa and Hec1B cells, but binding to glycosaminoglycan (GAG)-deficient cells (pgsA-745 and pgsD-677) was markedly reduced, indicating that OmcB binds to GAG-like receptors on host cells.

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