Article Figures & Data
Figures
- Fig 1
Impaired use of alternative carbon sources by UTI89ΔcyaA and UTI89Δcrp. (A) MacConkey agar plate streaked with UTI89, UTI89ΔcyaA, and UTI89Δcrp. Growth of UTI89, UTI89ΔcyaA, UTI89Δcrp, and complemented mutants in modified M9 medium (B) and modified M9 medium lacking glucose (C). Mutant strains were complemented by uninduced expression of cyaA or crp from a Ptac promoter. Strains transformed with the empty vector pRR48 served as controls. Each curve reflects the means of results from a single experiment and is representative of at least three independent experiments performed in quadruplicate.
- Fig 2
UPEC requires functional cAMP-CRP for virulence in the murine urinary tract. Adult female CBA/J mice (A, B) or C3H/HeJ mice (C) were infected via catheterization with 1 × 107 CFU of wild-type UTI89 or isogenic mutants lacking cyaA or crp. Graphs show bacterial titers present in the bladder at 6 h (A) and 3 days (B, C) postinoculation. Bars indicate median values for each group; n ≥ 11 mice. P values were determined using Mann-Whitney U tests.
- Fig 3
UTI89ΔcyaA and UTI89Δcrp are sensitive to nitrosative stress and methyl viologen but resistant to H2O2. Growth of UTI89, UTI89ΔcyaA, and UTI89Δcrp in MES-LB broth (A), MES-LB broth + 1 mM ASN (B), MES-LB broth + 1 mM H2O2 (C), LB broth (D), and LB broth containing 1 mM MV (E). Growth curves show the means of results from a single experiment and are representative of at least three independent experiments carried out in quadruplicate.
- Fig 4
Complementation of UTI89ΔcyaA and UTI89Δcrp in the presence of nitrosative and oxidative stresses. Graphs show growth of UTI89 versus UTI89ΔcyaA (A to C) and UTI89Δcrp (D to F) in MES-LB (A, D), MES-LB ± 1 mM ASN (B, E), or MES-LB ± 1 mM H2O2 (C, F), all without added IPTG. Strains carried pcyaA, pcrp, or the control plasmid pRR48, as indicated. Growth curves show the means of results from a single experiment and are representative of at least three independent experiments carried out in quadruplicate.
- Fig 5
H2O2 resistance correlates with increased RpoS expression and catalase activity in UTI89ΔcyaA and UTI89Δcrp. (A) Western blot of RpoS (σS) in UTI89, UTI89ΔcyaA, and UTI89Δcrp after growth to mid-exponential phase in LB broth. Relative levels of RpoS normalized to loading control (Ctrl) are indicated. (B, C) Curves show growth of UTI89 and UTI89Δcrp carrying empty vector pRR48 or prpoS, as indicated, in MES-LB ± 1 mM H2O2. (D) Graph of catalase activity in UTI89, UTI89ΔcyaA, UTI89Δcrp, and UTI89/prpoS following growth to stationary phase in LB broth + 1 mM IPTG. Data are expressed relative to wild-type UTI89 as the means ± standard errors of three independent experiments carried out in triplicate. (E) Levels of trehalose present in UTI89ΔfimH, UTI89ΔotsBA, UTI89ΔcyaA, and UTI89Δcrp following growth to stationary phase (OD600 = 1.0). The ΔfimH mutant carries the same chloramphenicol resistance cassette as the ΔcyaA and Δcrp mutants and served as the control. (F, G) Graphs show growth of UTI89 and its mutant derivatives (ΔcyaA, Δcrp, and Δdps mutants) carrying pRR48 or pdps, as indicated, in MES-LB ± 1 mM H2O2. Each growth curve (B, C, F, and G) shows the means of results from a single experiment and is representative of at least three independent experiments carried out in quadruplicate. Dps and RpoS expression in these assays was induced by addition of 0.5 mM IPTG. (H) Survival of UTI89Δdps complemented with empty vector pRR48 or pdps following 15 min of exposure to 0.05 M CuSO4. Results were obtained without addition of IPTG and are presented relative to those of wild-type UTI89/pRR48. The indicated P values were determined using two-tailed unpaired t tests.
- Fig 6
Expression of RpoS or Dps enables wild-type UTI89 to survive low-pH stress at levels similar to those of the ΔcyaA and Δcrp mutants. After reaching mid-logarithmic growth phase in LB broth, UTI89 and the UTI89ΔcyaA, UTI89Δcrp, UTI89Δdps, UTI89ΔrpoS, UTI89ΔcrpΔdps, and UTI89ΔcrpΔrpoS mutants (± pRR48, pdps, and prpoS, as indicated) were exposed to acid (pH 3.5) stress for 30 min. Following washes in PBS, numbers of surviving bacteria were determined by dilution plating. Plasmid-containing strains were grown in the presence of 0.5 mM IPTG prior to challenge with pH stress. Data are expressed as the means ± standard deviations of three independent experiments. P values of <0.05 are indicated by one asterisk (*) for comparison with UTI89 and by two asterisks (**) for comparison with UTI89/pRR48, as determined by two-tailed unpaired t tests.
Tables
- Table 1
Bacterial strains and plasmids
Strain or plasmid Description Source Strains UTI89 UPEC strain (cystitis isolate, O18:K1:H7) 25 UTI89ΔcyaA UTI89cyaA::Clmr (pKD3) This study UTI89Δcrp UTI89crp::Clmr (pKD3) This study UTI89Δdps UTI89dps::Kanr (TT23691) This study UTI89ΔfimH UTI89fimH::Clmr (pKD3) 83 UTI89ΔotsBA UTI89otsBA::Kanr (pKD4) This study UTI89ΔrpoS UTI89rpoS::Kanr (TT23691) This study UTI89ΔcyaAΔotsBA UTI89cyaA::Clmr (pKD3) otsBA::Kanr (pDK4) This study UTI89ΔcrpΔrpoS UTI89crp::Clmr (pKD3) rpoS::Kanr (TT23691) This study UTI89ΔcrpΔdps UTI89crp::Clmr (pKD3) dps::Kanr (TT23691) This study TT23691 Strain with Kanr cassette flanked by universal primer sites 84 Plasmids pRR48 Ampr cloning plasmid containing an IPTG-inducible Ptac promoter upstream of the MCS 26 pKM208 Ampr plasmid; encodes IPTG-inducible lambda red recombinase 27 pKD3 Template plasmid for gene disruption; contains FRTa-flanked Clmr cassette 28 pKD4 Template plasmid for gene disruption; contains FRT-flanked Kanr cassette 28 pcrp Ampr plasmid; crp (from UTI89) cloned into PstI, KpnI sites of pRR48 This study pcyaA Ampr plasmid; cyaA (from UTI89) cloned into PstI, KpnI sites of pRR48 This study pdps Ampr plasmid; IPTG-inducible expression of E. coli Dps 55 prpoS Ampr plasmid; rpoS (from UTI89) cloned into PstI, HindIII sites of pRR48 This study ↵a FRT, FLP recombination target.
- Table 2
Primer sequences
Primer Sequence crp KOa Forward GCGCATGGTGCTTGGCAAACCGCAAACAGACCCGACTCTCTGTGTAGGCTGGAGCTGCTTCG Reverse CGCGCTACCAGGTAACGCGCCACTCTGACGGGATTAACGACATATGAATATCCTCCTTAG crp KO confirmation Forward GTATGCAAAGGACGCCACAT Reverse TTCGCCAAGCATTAACCCAA cyaA KO Forward GCGGAATCACAGTCATGACGGGTAGCAAATCAGGCGATACTGTGTAGGCTGGAGCTGCTTCG Reverse TACTGCTGCAACAGCGGCGCGTCATGCTCCTGATTGGCAGCATATGAATATCCTCCTTAG cyaA KO confirmation Forward AACCAGGCGCGAAAAGTGGT Reverse CTGAAAGGCGACGAGTGGAT otsBA KO Forward ATGTCTGTAAAGCGCGTTCTGCGCAACACAATAAGAAATGTGTAGGCTGGAGCTGCTTCG Reverse CTACGCAAGCTTAGGAAAGGTAGCAACTTTATCGCGCTGCCATATGAATATCCTCCTTAG otsBA KO confirmation Forward AGCGAAACGCACTGTCTGAT Reverse TTGCCTACGGTGAGTTAAGC dps KO Forward TTATTCGATGTTAGACTCGATAAACCACAGGAATTTATCCAGGTCGCGAGCACCAAACACCCCCCAAAACC Reverse GTGATAGGAACAGCCAGAATAGCGGAACACATAGCTGGTGCTATACTTAGCACACAACCACACCACACCAC dps KO confirmation Forward GATAGCAGATGGATGCACTA Reverse TGACAGTACGCAAAGAGAGC rpoS KO Forward CCAGCCTCGCTTGAGACTGGCCTTTCTGACAGATGCTTACCACCAAACACCCCCCAAAACC Reverse TGCCGCAGCGATAAATCGGCGGAACCAGGCTTTTGCTTGACACACAACCACACCACACCAC rpoS KO confirmation Forward AATGATGATTGCCGAATGTGACGCTG Reverse GCATTGTGTCGTTATGGGCGTAGG pcrp Forward CCCCC CTGCAG ATGGTGCTTGGCAAACCGCA Reverse CCCCC GGTACC TTAACGAGTGCCGTAAACGA pcyaA Forward CCCCC CTGCAG TTGTACCTCTATATTGAGAC Reverse CCCCC GGTACC TCACGAAAAATACTGCTGCA prpoS Forward CATTC CTGCAG ATGTTCCGTCAAGGGATCA Reverse AGTGC AAGCTT TTATTCGCGGAACAGCGCT pRR48 sequencing primer Forward CTGCTGAAGAGTACTTTGG Reverse CCAAAGCTGAAGACATCCAG ↵a KO, knockout.
