Molecular characterization of Vibrio cholerae ΔrelA ΔspoT double mutants

Abstract

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae DeltarelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA (+ )background, but unlike E. coli, several V. cholerae DeltarelA DeltaspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli DeltarelA DeltaspoT mutant (ppGpp(0) strain), the V. cholerae DeltarelA DeltaspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of DeltarelA DeltaspoT mutants could be reverted back to DeltarelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae.

ppGpp Conjures Bacterial Virulence

“…As predicted by data for E. coli reported in the literature, relA mutant cells do not accumulate ppGpp under conditions of amino acid starvation, but the V. cholerae mutants still respond to carbon source starvation. However, a relA spoT double mutant still accumulates ppGpp during glucose starvation (56). Furthermore, the relA spoT double mutant grows in M9 minimal medium; is resistant to 3-amino-1,2,4-triazole, a reagent that induces histidine limitation; and exerts a stringent control of stable RNA synthesis when glucose is limiting.…”

Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae

“…Bacterial cells were also grown in M9 minimal (M9M) solution (SigmaAldrich) or agar (1.5 %; Difco) containing 0.4 % glucose as a carbon source (Das & Bhadra, 2008;Das et al, 2009) and growth of bacterial cells was usually checked after 24-30 h after incubating the plates at 37 uC. Bacterial strains were preserved at 270 uC in LB containing 20 % sterile glycerol (Haralalka et al, 2003).…”

“…Restriction and nucleic acid-modifying enzymes were purchased from New England Biolabs, and were used essentially as directed by the manufacturer. Electrocompetent V. cholerae cells were prepared essentially as reported earlier (Das & Bhadra, 2008) and transformants were selected on LA plates containing appropriate antibiotics.…”

Functional Analysis of the Essential GTP-Binding-Protein-Coding GenecgtAofVibrio cholerae

“…To show this, initially chromosomal DNA flanking the cgtA gene (The Institute for Genome Research annotation no. VC0437) was PCR amplified using the Cgta-F/Cgta-R primer pair (Table 2), which was followed by cloning of the amplicon in pCR4TOPO (28) to obtain the desired deletion in the cgtA gene of V. cholerae using a method essentially described previously (5). Despite several attempts we were unable to obtain a ⌬cgtA V. cholerae strain.…”

“…Raskin et al (24) reported that V. cholerae ⌬cgtA cells are viable if the parent strain is a relA null mutant, which apparently indicates critical involvement of the cgtA gene function with the stringent response of the organism. Furthermore, we know that spoT is essential in a relA ϩ background in both E. coli and V. cholerae (5,24,33). It has been shown that CgtA of E. coli copurifies with SpoT, indicating that these two proteins interact with each other (32).…”