TheVibrio harveyiGTPase CgtA_VIs Essential and Is Associated with the 50S Ribosomal Subunit

Abstract

It was previously reported that unlike the other obg/cgtA GTPases, the Vibrio harveyi cgtA V is not essential. Here we show that cgtA V was not disrupted in these studies and is, in fact, essential for viability. Depletion of CgtA V did not result in cell elongation. CgtA V is associated with the large ribosomal particle. In light of our results, we predict that the V. harveyi CgtA V protein plays a similar essential role to that seen for Obg/CgtA proteins in other bacteria.All living organisms express one or more Obg/CgtA GTPase. Elucidating the function of these GTPases has been complicated by somewhat contradictory phenotypes associated with specific mutants in different model systems. Some of the confusion stems from the dual function of these proteins (ribosome assembly and stress response) in at least some organisms, as well as from potential species-specific differences that may result in different phenotypes observed for orthologous obg/cgtA mutants. Perhaps the most surprising of these inconsistent reports is that of the nonessential nature of the Vibrio harveyi cgtA V gene (5) and its pleiotropic phenotypes (32,34,42), observations at odds with what has been reported for other obg/cgtA mutants. Here we show that, in contrast to these studies, the V. harveyi cgtA V gene is essential. Furthermore, we demonstrate that the trimethoprim resistance associated with a cgtA V clone was conferred by the linked folA gene. We also show that CgtA V is associated with the 50S ribosomal particle. From these studies, we predict that the role of CgtA V is similar to that of other bacterial Obg/CgtA proteins.Bacterial strains, plasmids, and growth conditions. The strains and plasmids used in the present study are listed in Table 1. Escherichia coli cells were grown at 37°C (unless otherwise indicated) in Luria-Bertani media (LB; 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl/liter) or LB agar (1.5% agar) containing antibiotics, as required (gentamicin, 30 g/ml; kanamycin, 30 g/ml; chloramphenicol, 10 g/ml; trimethoprim, 100 g/ml; ampicillin, 100 g/ml). V. harveyi strains were derived from BB7 (2) and maintained at 30°C on BOSS medium (10 g of Bacto Peptone, 3 g of beef extract, 30 g of NaCl, and 1 ml of 100% glycerol/liter) or BOSS agar (1.5% agar) supplemented with antibiotics (gentamicin, 30 g/ml; kanamycin, 50 g/ml).The V. harveyi cgtA V gene is essential. We were perplexed by the reported viability of the V. harveyi cgtA V transposon insertion mutant BB7X (5) for several reasons. First, in all other organisms examined, obg/cgtA is an essential gene (1,16,22,25,37,40). Second, given the sequence similarity among the Obg/CgtA proteins, we would predict that CgtA proteins play similar roles in all bacteria, and yet the viable nature and reported phenotypes of the V. harveyi cgtA V insertional mutant (5, 32, 34, 42) were different from those reported for other obg/cgtA mutant strains (7,16,17,22,26,38). Therefore, we reinvestigated the consequences of CgtA V depletion in V. harveyi. Plasmids used in the presen...

Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae

“…It has also been reported that the E. coli Obg protein is involved in monitoring DNA damage during DNA replication (22). In contrast, CgtA in V. harveyi does not affect does not affect chromosome segregation (17). In V. cholerae, CgtA-depletion does not cause cell elongation, and no chromosomal defects were observed when nucleoids were visualized with DAPI staining (data not shown).…”

“…This is probably a conserved function because in other organisms, such as V. harveyi, B. subtilis, C. crescentus, and E. coli, CgtA interacts with the 50S ribosomal subunit and has been shown to be involved in assembly or stability of the 50S subunit (17,23,27,28). Interestingly, a yeast mitochondrial CgtA homolog is essential for translation, likely through an involvement in ribosome biogenesis (29).…”

“…It is often annotated as obg/GTP1 or yhbZ and is essential in every organism in which it has been studied, including Escherichia coli, Vibrio cholerae, Vibrio harveyi, Caulobacter crescentus, and Bacillus subtilis (13)(14)(15)(16)(17). CgtA/Obg is involved in sporulation in B. subtilis and morphological development in S. coelicolor (18)(19)(20).…”

Chromosome segregation control by Escherichia coli ObgE GTPase

“…Previous studies of Obg orthologues have examined different temperature-sensitive alleles and point mutations to deplete functional Obg in Gram-negative bacteria (Kobayashi et al, 2001;Datta et al, 2004;Sato et al, 2005;Sikora et al, 2006). The results of these studies have suggested various and diverse cellular roles, including ribosome biogenesis, stress response and chromosome segregation.…”

The Growth-Promoting and Stress Response Activities of the Bacillus subtilis GTP Binding Protein Obg Are Separable by Mutation

“…Genetics studies have also revealed a growth-essential role for the Obg orthologs of Escherichia coli, Caulobacter crescentus, and Vibrio and Streptomyces species (25,34,35,39,43). In vitro, purified B. subtilis and C. crescentus Obgs display high GTP/GDP dissociation exchange rates (K d , ϳ1.5 s Ϫ1 ) but relatively low GTP hydrolysis rates (half-life, ϳ23 min) (32,52).…”