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Molecular Pathogenesis

Vibrio cholerae Requires rpoS for Efficient Intestinal Colonization

D. Scott Merrell, Anna D. Tischler, Sang Ho Lee, Andrew Camilli
D. Scott Merrell
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
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Anna D. Tischler
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
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Sang Ho Lee
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
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Andrew Camilli
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
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DOI: 10.1128/IAI.68.12.6691-6696.2000
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  • Fig. 1.
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    Fig. 1.

    (A) Southern blot analysis of the rpoSregion. Chromosomal DNA harvested from the indicated strains was digested with HindIII and hybridized with a probe specific for nlpD, which lies immediately upstream of therpoS coding sequence. Lane C6709-1 shows the presence of the wild-type rpoS HindIII fragment, while lane DSM-V491 shows a smaller band due to an in-frame deletion of the rpoS coding sequence. Three and two independent isolates of reverted DSM-V506 and DSM-V491, respectively, are shown. In each case, the ΔrpoSband is absent and the wild-type rpoS band is regained. (B) Hydrogen peroxide sensitivity, shown as percent survival after 30 min of exposure to hydrogen peroxide as described in Materials and Methods. In each of the three data sets, a C6709-1 wild-type control is shown, as the three experiments were performed on different days.

  • Fig. 2.
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    Fig. 2.

    (A) Growth kinetics of DSM-V506 and C6709-1. For each strain, overnight cultures were diluted 1:200 into fresh LB and grown with aeration at 37°C. At the indicated times, the OD600was read for each and plotted as a function of time. (B) In vitro competition growth kinetics of DSM-V506 and DSM-V583. A 1:1 mixture of overnight cultures of each strain was diluted 1:1,000 into fresh LB. The titer of each strain was determined at the indicated time points by plating on LB supplemented with X-Gal. In addition, the OD600 was read at each time point. The percentage of each colony type found at each time point was used to extrapolate the OD600 of each strain based on the total OD600.

  • Fig. 3.
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    Fig. 3.

    Genetic manipulations of V. cholerae C6709-1 presented in this report. Strains of equivalent genotype and phenotype are in boxes. The box with a solid line represents the presence of a wild-type chromosomal copy of rpoS and wild-type growth and virulence; the box with a dashed line represents loss ofrpoS function, sensitivity to H2O2in vitro, and reduced colonization in vivo; the box with alternating short and long dashes represents the presence of a plasmid expressingrpoS in trans, resistance to H2O2, increased competitive fitness in vitro, and wild-type colonization.

Tables

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  • Table 1.

    Strains and plasmids used

    Strain or plasmidRelevant phenotype(s)Source or reference
    E. coli strains
     SM10λpirthi recA thr leu tonA lacY supELaboratory strain
    RP4-2-Tc::Mu λ::pir strain
     AC-E184SM10λpir(pAC160)5
     AC-E189SM10λpir(pAC214)5
     DSM-E713SM10λpir(pDSM375)17
    V. cholerae strains
     C6709-1El Tor biotype, SmrRoberts et al.a
     AC-V168C6709-1 lacZ::pGP704, LacZ−5
     DSM-V382C6709-1rpoS::pGP70417
     DSM-V490C6709-1 rpoS::pCVD442This work
     DSM-V491C6709-1 ΔrpoS17
     DSM-V506C6709-1 ΔrpoS (pFY7)This work
     DSM-V583C6709-1(pMMB67EH), LacZ−This work
     DSM-V714C6709-1(pFY7)This work
     DSM-V717DSM-V506 cured of pFY7This work
     DSM-V719DSM-V491 reverted to wild typeThis work
     DSM-V720DSM-V717 reverted to wild typeThis work
    Plasmids
     pAC160pGP704::′iviVI′5
     pAC214pGP704::′orf3′5
     pFY7pMMB67EH::′nlpD-rpoS-mutS′22
     pDSM375pGP704::′rpoS′17
     pDSM747pCVD442::′nlpD-rpoS-mutS′This work
     pMMB67EHIncQ broad-host-range cloning vector22
    • ↵a A. Roberts, G. D. Pearson, and J. J. Mekalanos, Proc. 28th Joint Conf., U.S.-Japan Cooperative Med. Sci. Prog. Cholera Related Diarrheal Dis., p. 43–47, 1992.

  • Table 2.

    In vitro and in vivo competition assays

    Competing strainaRelevant genotypeCompetition indexbnc
    In vitroIn vivo
    DSM-V382rpoS::pGP7042.00.204
    DSM-V490rpoS::pCVD4421.00.258
    DSM-V491ΔrpoS1.20.218
    DSM-V506ΔrpoS (pFY7)321.28
    DSM-V506dΔrpoS(pFY7)2.01.75
    DSM-V717DSM-V506 cured of pFY74.20.125
    DSM-V719DSM-V491 reverted1.11.18
    DSM-V720DSM-V717 reverted2.90.885
    • ↵a All competition assays were between the indicated strain and the isogenic wild-type strain C6709-1 (LacZ+) or AC-V168 (LacZ−) unless otherwise indicated.

    • ↵b Calculated by dividing mutant output number by wild-type output number and correcting the quotient for input deviations from 1:1.

    • ↵c n, number of animals used per competition experiment. All competitions were conducted in duplicate. Results from a single representative experiment are shown.

    • ↵d Competed against DSM-V714 (wild-type carrying pFY7).

  • Table 3.

    Frequencies of replication-defective plasmid integration into rpoS and two other loci

    Site of insertionTransferred plasmid (insert size [bp])Insertion frequency (transconjugants/recipient)a
    rpoSpDSM375 (287)1.4 × 10−6 ± 1.1 × 10−6
    iviVIpAC160 (249)3.1 × 10−7 ± 1.0 × 10−7
    orf3pAC214 (295)9.5 × 10−7 ± 3.4 × 10−7
    • ↵a Average ± standard deviation of three separate matings.

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Vibrio cholerae Requires rpoS for Efficient Intestinal Colonization
D. Scott Merrell, Anna D. Tischler, Sang Ho Lee, Andrew Camilli
Infection and Immunity Dec 2000, 68 (12) 6691-6696; DOI: 10.1128/IAI.68.12.6691-6696.2000

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Vibrio cholerae Requires rpoS for Efficient Intestinal Colonization
D. Scott Merrell, Anna D. Tischler, Sang Ho Lee, Andrew Camilli
Infection and Immunity Dec 2000, 68 (12) 6691-6696; DOI: 10.1128/IAI.68.12.6691-6696.2000
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KEYWORDS

Bacterial Proteins
Intestines
sigma factor
Vibrio cholerae

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