Article Figures & Data
Figures
- 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.
(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.
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
- Table 1.
Strains and plasmids used
Strain or plasmid Relevant phenotype(s) Source or reference E. coli strains SM10λpir thi recA thr leu tonA lacY supE Laboratory strain RP4-2-Tc::Mu λ::pir strain AC-E184 SM10λpir(pAC160) 5 AC-E189 SM10λpir(pAC214) 5 DSM-E713 SM10λpir(pDSM375) 17 V. cholerae strains C6709-1 El Tor biotype, Smr Roberts et al.a AC-V168 C6709-1 lacZ::pGP704, LacZ− 5 DSM-V382 C6709-1rpoS::pGP704 17 DSM-V490 C6709-1 rpoS::pCVD442 This work DSM-V491 C6709-1 ΔrpoS 17 DSM-V506 C6709-1 ΔrpoS (pFY7) This work DSM-V583 C6709-1(pMMB67EH), LacZ− This work DSM-V714 C6709-1(pFY7) This work DSM-V717 DSM-V506 cured of pFY7 This work DSM-V719 DSM-V491 reverted to wild type This work DSM-V720 DSM-V717 reverted to wild type This work Plasmids pAC160 pGP704::′iviVI′ 5 pAC214 pGP704::′orf3′ 5 pFY7 pMMB67EH::′nlpD-rpoS-mutS′ 22 pDSM375 pGP704::′rpoS′ 17 pDSM747 pCVD442::′nlpD-rpoS-mutS′ This work pMMB67EH IncQ broad-host-range cloning vector 22 ↵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 straina Relevant genotype Competition indexb nc In vitro In vivo DSM-V382 rpoS::pGP704 2.0 0.20 4 DSM-V490 rpoS::pCVD442 1.0 0.25 8 DSM-V491 ΔrpoS 1.2 0.21 8 DSM-V506 ΔrpoS (pFY7) 32 1.2 8 DSM-V506d ΔrpoS(pFY7) 2.0 1.7 5 DSM-V717 DSM-V506 cured of pFY7 4.2 0.12 5 DSM-V719 DSM-V491 reverted 1.1 1.1 8 DSM-V720 DSM-V717 reverted 2.9 0.88 5 ↵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 insertion Transferred plasmid (insert size [bp]) Insertion frequency (transconjugants/recipient)a rpoS pDSM375 (287) 1.4 × 10−6 ± 1.1 × 10−6 iviVI pAC160 (249) 3.1 × 10−7 ± 1.0 × 10−7 orf3 pAC214 (295) 9.5 × 10−7 ± 3.4 × 10−7 ↵a Average ± standard deviation of three separate matings.
