Article Figures & Data
Figures
- FIG. 1.
Comparison of the growth and colony-forming ability of wild-type (LS2) and ΔrpoH (LS1) V. cholerae at 22°C and 42°C. LS1 (black lines) and LS2 (gray lines) cells were grown in LB with chloramphenicol and arabinose at 22°C to an OD600 of 0.03. The cells were then harvested, washed, and resuspended in fresh medium supplemented with chloramphenicol and either arabinose (solid lines) or glucose (dotted lines) and grown in a shaker incubator at 42°C (A and C) or 22°C (B and D). Each graph is representative of at least two independent experiments; the data in panels A and B and those in panels C and D come from two different experiments. Time zero indicates the time at which the cells were placed in the incubator at either 22°C or 42°C. Note that the y axis is a logarithmic scale.
- FIG. 2.
Maintenance of a plasmid-borne copy of rpoH in wild-type versus ΔrpoH V. cholerae at various temperatures. Retention of the Cmr plasmid pRpoH in LS1 (Δ rpoH) and LS2 (wild type) after 32 h at 15°C, or after 8 h at 30°C, 37°C and 42°C, is shown as the normalized ratio of Smr Cmr to Smr CFU. The black bars represent LS1 cells, the light gray bars represent LS2 cells, and the dark gray bars represent wild-type cells carrying the empty vector pBAD18-Cm. Normalization was done by dividing the ratios of Smr Cmr/Smr cells after incubation at the various temperatures by the ratio of Smr Cmr/Smr cells at the time of inoculation (t = 0). Note that the y axis is a logarithmic scale. The results are the mean values of two experiments, and the error bars represent standard deviations.
- FIG. 3.
Temperature influences the requirement of an rpoH deletion mutant for RpoH in the presence of kanamycin. In panel A, 10 μl of serial 10-fold dilutions from a freshly thawed frozen stock of LS1 were spotted on LB and LB-kanamycin (LB Kn) plates. The numbers in the upper panel indicate the dilution factor. Plates on the left contain arabinose (0.1%), and plates on the right are supplemented with glucose (0.2%). The CFU/ml of the freshly thawed frozen stocks plated on LB supplemented with chloramphenicol are shown in the bar graph (B). Gray bars indicate plates supplemented with arabinose, and black bars indicate plates supplemented with glucose. Note that the y axis is a logarithmic scale. The results are the mean values of at least two experiments, and the error bars represent standard deviations.
- FIG. 4.
Comparison of the growth of LS1 and LS2 in LB at 37°C and in suckling mice. Prior to inoculation, the strains were grown at 22°C in either glucose or arabinose. Then, the two strains were mixed 1:1 and inoculated into suckling mice or LB broth. The ratio of LS1 to LS2 CFU in intestinal homogenates or in the overnight LB broth cultures was divided by the ratio of these strains in the inocula to yield the competitive indices. Open triangles and diamonds represent mice that had no detectable LS1 CFU.
- FIG. 5.
Relative levels of transcripts of four RpoH-regulated genes in ΔrpoH versus wild-type V. cholerae. RNA was isolated from LS1 and LS2 20 min after a temperature up-shift from 15°C to 37°C. The names of the transcripts quantified by real-time RT-PCR are indicated below the x axis. The transcript levels were normalized to the amount of rpoB transcripts in the cells. The results presented are the mean values of two experiments. The error bars represent the standard deviations.
- FIG. 6.
Bioinformatics approach for determination of a putative V. cholerae σ32 binding site consensus sequence. (A) Protocol used to determine a consensus sequence for the V. cholerae RpoH binding site shown in panel B. Sequence databases are shown as blue ovals, computer programs are shown in black ovals, and outputs are shown as magenta ovals. The consensus sequence was used to search upstream of all V. cholerae ORFs to identify putative RpoH-dependent promoters. The graph at the bottom of panel A shows the score distribution of these putative RpoH-dependent promoters. The pink lines correspond to putative promoters identified upstream of RpoH-regulated genes in Table 2. (B) Consensus sequence of the RpoH binding site displayed with WebLogo (http://weblogo.berkeley.edu ) (7, 40). The height of each column indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position. The number of times each nucleotide is found at each position in the consensus is shown below the sequence. N(13-16) indicates the gap between the two blocks.
- FIG. 7.
dnaK and vca0446 transcription start sites. (A) Two dnaK transcription start sites were identified by 5′ RACE. P1 and P2 are positioned 6 and 7 bp downstream from the −10 motif of the two predicted σ32 binding sites, respectively. (B) One transcription start site upstream from vca0446 was identified by 5′ RACE. P1 is located 9 bp downstream from the −10 motifs of the predicted σ32 binding site. The translation start codon for each gene is boxed, and the likely Shine-Dalgarno (SD) sequence is indicated by a dashed line. The beginning of each ORF is shown with uppercase letters.
- FIG. 8.
Features of the rpoH promoter region. Three rpoH transcription start sites, P1, P2, and P3, were identified by 5′ RACE. The likely −10 and −35 elements for P2 and P3 suggest that these promoters depend on σ70. Potential σ24 −10 and −35 sequences were identified upstream of the P1 transcription start site. The RpoH translation start site is boxed, and the likely Shine-Dalgarno (SD) sequence is indicated by a dashed line. The beginning of the rpoH ORF is shown with uppercase letters. The asterisks denote the ftsX translation stop codon. The dashed box indicates a putative DnaA binding sequence.
Tables
- TABLE 1.
Primers used in this study
Name Sequence 0177Fd TATTCGATTCCCTGAACTGGA 0177Rv TTGACCAGAGAGCACCTTCA 0706Fd GTGGCTTCTGCAATTCACG 0706Rv TTCGCTCAGTTCCGTTTTAT 2675Fd TGAAAGGCAATGCACGTAAA 2675Rv CCTTGGTGCATCTGCAACTT DSP CGACGACCGATCAGACGCTTG DSP1 CAATTTTGTAAGGCATGAT DSP2 GATATCGCGCTGAACTTCTTCG GroES2Fd AAAATCAAACCGTGGCAAAG GroES2Rv TCCGTTTTCACACCGTAACC HSP CCTCGACATTCGCGACAATATG HSP1 GGCATGATGATTACATTTAG HSP2 GGATTACTAGCTTCACAATGCAAG kan3 CGGAATTCCCCGCGCTGGAGGATCATC kan5 CGGGATCCAGCTACTGGGCTATCTGGAC KSP GTTGGCTACCCGTGATATTGC KSP1 GGAAAATGGCCGCTTTTC KSP2 GGGTGTGGCGGACCGCTATC lacZ1 CCCAAGCTTAACAATTTCACACAGGAAACAG lacZ2 ATAGTTTAGCGGCCGCGAGAACAGAGAAATAGCGGC PgrpE1 GAAGATCTTGAACGGCTTTTTCATAAGG PgrpE2 CCCAAGCTTCACTTCATACTGGTCAGG RpB2Fd AACCTGTCTCAAGCCGGTTA RpB2Rv TTTCTACCAGTGCAGAGATGC rpoH3 GCTCTAGATTGGTTTGCCCGTTTAGAT rpoH4 CGGAATTCTCAATTCCTCATCATTCTCTGCTC rpoH5 CGGGATCCATGCGTAAGCTGAAAGAAGC rpoH6 ACATGCATGCCGGTATTCTATACCATCCC rpoH8 TGCTCTAGACCTTTTCTTATCAAGCTG rpoHH1 GCTCTAGAAATTCATGAATATGTGCTACG rpoHH2 TCAGTGATGGTGATGGTGATGGAACTCGCCCACCGCTTC rpoHH3 CATCACCATCACCATCACTGATTTCACATTGCGTAACAGC rpoHH4 TCATGCATGCCGGTATCTATACCATCCCTGAG RSP CTCTTGCACTAAGTCCGCCATT RSP1 CTCAGGGTTAAAGCGTTT RSP2 CCGCTTTCATCAGACCGATATT RSP3 CCAGATCAGAGAATGATGAGGAA - TABLE 2.
V. cholerae RpoH-regulated genes
Gene category Ratioa E. coli regulonb Designationc Name Productc υ λ τ Degradation of proteins, peptides, and glycopeptides 0.27 υ λ τ vc0188 prlC Oligopeptidase A 0.05 υ λ τ vc0711 clpB-1 ClpB 0.45 vc1343 Peptidase, M20A family 0.33 υ λ τ vc1920 lon ATP-dependent protease LA 0.25 υ τ vc2674 hslU Protease HslVU, ATPase subunit HslU 0.25 υ λ τ vc2675 * hslV Protease HslVU, subunit HslV Protein folding and stabilization 0.33 υ λ τ vc0018 ibpA 16-kDa heat shock protein A 0.40 υ λ τ vc0854 grpE Heatshock protein GrpE 0.05 υ λ τ vc0855 dnaK DnaK 0.08 υ τ vc0856 dnaJ DnaJ 0.14 υ λ τ vc0985 htpG Heat shock protein HtpG 0.06 vc2664 groEL-1 Chaperonin, 60-kDa subunit 0.05 vc2665 groES-1 Chaperonin, 10-kDa subunit 0.05 υ τ vca0819 * groES-2 Chaperonin, 10-kDa subunit 0.06 υ λ τ vca0820† groEL-2 Chaperonin, 60-kDa subunit Regulatory functions 0.35 λ τ vc0706* σ54 modulation protein, putative 0.26 vc2485 Transcriptional regulator, LysR family Transcription 0.14 vc0150 rpoH RNA polymerase σ32 factor Transport and binding proteins 0.37 vc0280 Cadaverine/lysine antiporter CadB, putative 0.47 vca1028 ompS Maltoporin 0.13 vca0910 tonB1 TonB1 0.29 vc0589 ABC transporter, ATP-binding protein 0.37 vc0590 Permease, putative Biosynthesis of cofactors, prosthetic groups, and carriers 0.48 vc0468 gshB Glutathione synthetase 0.38 vc1950 bizZ Biotin sulfoxide reductase 0.42 υ vc2673 menA 1,4-Dihydroxy-2-naphthoate octaprenyltransferase Pathogenesis 0.36 vca0446 Hemagglutinin 0.29 vca0447 Hemagglutinin-associated protein Metabolism 0.49 vc1589 aldC α-Acetolactate decarboxylase 0.46 vc1951 yecK Cytochrome c-type protein YecK 0.11 τ vca0752 trxC Thioredoxin 2 Conserved hypothetical proteins 0.20 vc0466 0.41 vc0467 0.44 vc0469 0.40 vc0490 0.18 λ τ vc0977 0.48 vc1055 0.41 vc1358 0.45 vc1871 0.46 υ τ vc2735 Hypothetical proteins 0.04 vc0177* 0.44 vc0189 0.36 vc0886 0.49 vc1080 0.20 vc2486 0.17 vc2663 0.15 vca0065 0.46 vca0396 0.16 vca0821 ↵ a To be included here, the ratio of mutant/wild-type transcript must be ≤0.5. Values are averages from three independent experiments.
↵ b Genes that are also found in the E. coli regulon, defined as follows: τ, Zhao et al. (54); λ, Wade et al. (47); υ, Nonaka et al. (33).
↵ c As annotated by The Institute for Genomic Research. The genes that possess a potential σ32 binding sequence in their promoter region are in boldface type. Symbols: *, gene chosen for real time RT-PCR; †, data not available for one of the three experiments for vca0820 (groEL-2) (the ratio reported for this gene is the average from two of three experiments).
