Functional analysis of the essential GTP-binding-protein-coding gene cgtA of Vibrio cholerae

Abstract

The cgtA gene, coding for the conserved G protein CgtA, is essential in bacteria. In contrast to a previous report, here we show by using genetic analysis that cgtA is essential in Vibrio cholerae even in a Delta relA background. Depletion of CgtA affected the growth of V. cholerae and rendered the cells highly sensitive to the replication inhibitor hydroxyurea. Overexpression of V. cholerae CgtA caused distinct elongation of Escherichia coli cells. Deletion analysis indicated that the C-terminal end of CgtA plays a critical role in its proper function.

FIG. 1.

Effect of CgtA depletion on growth of V. cholerae. Experimental and control strains were grown in LB in the absence (− ara) or presence (+ ara) of arabinose (0.01%), as indicated on the left. After 5 h of growth, 10-fold serial dilutions (from left to right) were spotted onto LA plates without or with arabinose (0.01%) or with glucose (0.4%), as indicated on the right.

FIG. 2.

Effect of HU on CgtA-depleted V. cholerae cells. (A) Phase-contrast micrographs of cells grown in LB without (− ara) or with (+ ara) arabinose (0.01%) and HU (1 mM) as indicated. (B) Cells were grown in LB with or without arabinose for 5 h, and 10-fold serial dilutions were spotted on plates containing only HU (1 mM) or HU (1 mM) plus arabinose (0.01%) as indicated.

FIG. 3.

SEM analysis of morphology of bacterial cells overexpressing CgtA. The top and bottom panels show SEM of E. coli and V. cholerae cells, respectively. V. cholerae (N16961 wild-type) or E. coli (CF1648 wild-type) cells carrying the cgtA expression plasmid pSBORF or an empty vector (pBAD24) were used as controls. Cells were grown in LB with arabinose (0.2%) for 5 h, and samples were processed for SEM and visualized using a VEGA II LSU microscope (Tescan, Czech Republic) at 10 kV. The arrows indicate distinct bulging of the surface of E. coli cells, which is not present in V. cholerae. Bar, 1 μm.

FIG. 4.

Overexpression of whole and C-terminally truncated CgtA proteins and effects of these proteins on cell morphology. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of arabinose (0.2%)-induced and uninduced total cellular proteins of V. cholerae cells. Arabinose-induced or uninduced cells were grown for 5 h at 37°C with shaking, and whole-cell lysates were prepared, which was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Lane 1, N16961(pBAD24) without arabinose; lane 2, N16961(pBAD24) induced with arabinose; lane 3, N16961(pSBORF) grown without arabinose; lane 4, N16961(pSBORF) induced with arabinose; lane 5, N16961(pSBΔ87) induced with arabinose; lane 6, N16961(pSBΔ189) grown with arabinose; lane 7, protein molecular weight markers (sizes are indicated on the right). The arrows indicate overexpressed full-length CgtA protein (∼44 kDa [lane 4]) and its C-terminally truncated forms (∼41 and ∼38 kDa [lanes 5 and 6, respectively]). (B) Phase-contrast micrographs of E. coli strain CF1648 (top panels) and V. cholerae strain N16961 (bottom panels) overexpressing full-length CgtA using plasmid pSBORF or overexpressing C-terminally truncated CgtA proteins using plasmids pSBΔ87 and pSBΔ189. Cells were grown in LB with arabinose (+ ara) or without arabinose (− ara) for 5 h at 37°C with shaking.