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MOLECULAR BIOLOGY OF PATHOGENS

The ompU Paralogue vca1008 Is Required for Virulence of Vibrio cholerae

Carlos G. Osorio, Hector Martinez-Wilson, Andrew Camilli
Carlos G. Osorio
1Programa de Microbiología y Micología, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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Hector Martinez-Wilson
2Tufts University Medical School, Tufts University, Boston, Massachusetts 02111
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Andrew Camilli
2Tufts University Medical School, Tufts University, Boston, Massachusetts 02111
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  • For correspondence: andrew.camilli@tufts.edu
DOI: 10.1128/JB.186.15.5167-5171.2004
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  • FIG. 1.
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    FIG. 1.

    Phylogenetic tree for select known and putative porins of V. cholerae and E. coli constructed by the neighbor-joining algorithm (CLUSTAL W, v1.81). Protein names (when available) and locus names obtained from The Institute for Genomic Research (http://www.tigr.org/tigr-scripts/CMR2/CMRHomePage.spl ) are listed on the right. The V. cholerae OmpU and paralogue VCA1008 cluster with the E. coli classical nonspecific porins OmpF, OmpC, and PhoE. V. cholerae OmpS and OmpW cluster with E. coli LamB and OmpW, respectively. The V. cholerae OmpT and VC0972 are distantly related to the other proteins.

  • FIG. 2.
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    FIG. 2.

    SDS-PAGE analysis of outer membrane proteins of V. cholerae strains. The molecular masses of markers in lane 1 are indicated to the left of the gel. Lane 2, wild-type; lane 3, Δvca1008; lane 4, ΔompU; lane 5, Δvca1008 ΔompU; lane 6, ΔompT Δvc0972; lane 7, ΔompU ΔompT Δvc0972. The most intense band in lanes 2, 3 and 6, which runs between the 32.5- and 47.5-kDa markers (marked by arrow), corresponds to OmpU (see the text for discussion).

Tables

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  • TABLE 1.

    Strains and plasmids used in this study

    Strain or plasmidRelevant genotype and phenotypeaSource or reference
    Strains
        V. cholerae
            CVD110E7946 El Tor biotype Δ(cep-ctxB) hlyA::mer::ctxB Hgr 8
            GOA1264Spontaneous Rifr of CVD110; Rifr This work
            GOA1713GOA1264 Δvca1008 This work
            GOA1714GOA1264 ΔompU This work
            GOA1715GOA1264 Δvca1008 ΔompU This work
            GOA1716GOA1264 ΔompT Δvc0972 This work
            GOA1717GOA1264 ΔompT Δvc0972 ΔompU This work
            GOA6WGOA1264 lacZ::pGP704; LacZ− This work
            GOA1245GOA1264 lacZ::res-neo-sacB-res Rifr Kmr Sucs C. Osorio, J. Crawford, J. Michalsky, J. Kaper, and A. Camilli, unpublished data
            GOA1705GOA1245 vca1008::tnpR135 lacZ::res-neo-sacB-res Rifr Kmr Sucs C. Osorio, J. Crawford, J. Michalsky, J. Kaper and A. Camilli, unpublished data
        E. coli
            SM10αλpir thi recA thr leu tonA lacY supE RP4-2-Tc::Mu λ::pir Kmr Laboratory strain
            DH5αλpir F− φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR1 supE44 thi-1 gyrA96 relA1::pir 5
    Plasmids
        pCVD442 oriR6K mobRP4 sacB Apr Sucs 4
        pGP704 oriR6K mobRP4 Apr 9
        pMMB67EH oriV mobRP4 Apr 10
        pMMB67EH-neopMMB67EH Δbla::neo; Kmr This work
        pVCA1008-FpMMB67EH-neo::vca1008 forwardThis work
        pVCA1008-RpMMB67EH-neo::vca1008 reverseThis work
    • ↵ a Rifr, Rif resistant; Kmr, kanamycin resistant; Sucr, sucrose resistant; Apr, ampicillin resistant.

  • TABLE 2.

    Oligonucleotide primers

    Primerasequence (5′ - 3′)bUsec
    vca1008F1CAGGACGCAATGGAGTAGTCSOE PCR
    vca1008F2GGTAGAAAAATGTAATCACACTGCCTTAAACSOE PCR
    vca1008R1AGTGTGATTACATTTTTCTACCCTATTAGSOE PCR
    vca1008R2TGGCGTGATCAAACGGCTGGACSOE PCR
    ompUF1CAGCATGGTATTCCGCATTCSOE PCR
    ompUF2ATGGACAATACTTCAGGTCACACGCCAAACSOE PCR
    ompUR1GACCTGAAGTATTGTCCATAAATTTGSOE PCR
    ompUR2GATCAGGTTGTCGGACTCTTGSOE PCR
    ompTF1TGATCACTGATCCTGCGASOE PCR
    ompTR1GATCTTACAACTCTTTGTTTGGTCACCACSOE PCR
    ompTF2CAAAGAGTTGTAAGATCTCGAACACGTTTASOE PCR
    ompTR2CATCCCTCTTGCCAAGCCAGSOE PCR
    vc0972F1GGTAGACTCTGCTCTAGCSOE PCR
    vc0972R1CGTCTGATTACATGGATAACTCCTAAAAATGSOE PCR
    vc0972F2GTTATCCATGTAATCAGACGAACCCTGCSOE PCR
    vc0972R2AAGAGTGAGCATGGCTTTAGSOE PCR
    vca1008F0CAAGCAATTAAATTGCACACDeletion confirmation
    ompUF0TAGCTTGTATTCGATATCACDeletion confirmation
    ompUR0AGTAAAAACGGTGTCCCAAGGDeletion confirmation
    ompTF0ACGTTCGCTACAACAATAACDeletion confirmation
    vc0972F0ACCGTAACCAACAAGTGATCDeletion confirmation
    GOA3 TCTAATGAATTCTGCGTGGCAACATTGATGTG vca1008 cloning
    GOA4 TCTAATTCTAGAGGTGATGTTTAATGCTCATG vca1008 cloning
    GOA5 TCTAATTCTAGATGCGTGGCAACATTGATGTG vca1008 cloning
    GOA6 TCTAATGAATTCGGTGATGTTTAATGCTCATG vca1008 cloning
    • ↵ a The beginning of each primer name corresponds to the V. cholerae N16961 gene being deleted, and the F and R indicate forward and reverse primers, respectively.

    • ↵ b Underlined bases indicate a noncomplementary 5′ tail and restriction site.

    • ↵ c SOE PCR refers to splicing-by-overlap-extension PCR used to construct gene deletions.

  • TABLE 3.

    Competition assays in vitro and in infant mouse

    CompetitionCIa for test strain genotype:
    Δvca1008Δvca1008 ΔompUΔvc0972 ΔompTΔvc0972 ΔompT ΔompUΔvca1008 (pVCA1008-F)Δvca1008 (pVCA1008-R)
    In vitro0.920.5*2.32.81.9*1
    In vivo0.025*0.013*1.613*4.1*0.8
    • ↵ a The CI were calculated as described in Materials and Methods. Each in vitro competition was done by using two independent LB broth cultures, and each in vivo competition used 10 infant mice. Asterisks indicate significant differences from the parent strain (P < 0.05) as determined by the Student's two-tailed t test with, as control group, competitions between GOA1264 and GOA6W.

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The ompU Paralogue vca1008 Is Required for Virulence of Vibrio cholerae
Carlos G. Osorio, Hector Martinez-Wilson, Andrew Camilli
Journal of Bacteriology Jul 2004, 186 (15) 5167-5171; DOI: 10.1128/JB.186.15.5167-5171.2004

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The ompU Paralogue vca1008 Is Required for Virulence of Vibrio cholerae
Carlos G. Osorio, Hector Martinez-Wilson, Andrew Camilli
Journal of Bacteriology Jul 2004, 186 (15) 5167-5171; DOI: 10.1128/JB.186.15.5167-5171.2004
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KEYWORDS

Adhesins, Bacterial
Bacterial Outer Membrane Proteins
Gene Expression Regulation, Bacterial
Intestine, Small
Vibrio cholerae

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