Regulation of ribosomal protein synthesis in Vibrio cholerae

Abstract

We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria.

FIG. 1.

(A) Maps of the V. cholerae S10 operon and plasmids used for targets of L4-mediated regulation. The reporter gene (S10′/lacZ′ or lacZ) is expressed from the IPTG-inducible trc promoter (P_trc). RBS-S10 and RBS-lacZ refer to the Shine-Dalgarno regions of the S10 and lacZ genes, respectively. (B and C) Promoter and leader regions of the V. cholerae and E. coli S10 operons. The secondary structures of the leaders from E. coli and V. cholerae were described previously (1). The DNA sequences upstream of the transcription start site are shown in lowercase letters, with the presumptive −35 and −10 sequences indicated. The transcribed regions are shown in uppercase letters. The boxed sequences indicate the Shine-Dalgarno (SD) sequence and the AUG initiation codon of the S10 structural gene.

FIG. 2.

Growth of cells carrying P_ara-L4 plasmids. Either E. coli (Ec) or V. cholerae (Vc) host cells carrying an arabinose-inducible L4 gene from the indicated source were streaked on Luria-Bertani plates with or without arabinose and incubated at 37°C for about 24 h. E. coli L4-AraR is a regulatory-defective mutant of the E. coli L4 protein (25).

FIG. 3.

Regulation of the E. coli (Ec) and V. cholerae (Vc) S10 operons mediated by L4. E. coli or V. cholerae strains with the indicated reporter plasmids and L4 sources were pulse-labeled with [³⁵S]methionine before (−) or 10 min after (+) induction of L4 by addition of arabinose. Extracts were fractionated by sodium dodecyl sulfate gel electrophoresis. The radioactivity in each S10′/lacZ′ or lacZ fusion protein band was normalized to the total radioactivity in the same lane. L4-mediated regulation (+L4/−L4) was expressed as the normalized radioactivity in the fusion band after L4 induction divided by the normalized radioactivity in the fusion band before L4 induction. Each experiment was performed at least four times. The standard deviations are shown in parentheses. (A) E. coli or V. cholerae cells carrying the V. cholerae leader-S10′/lacZ′ plasmid and a P_ara plasmid harboring the V. cholerae L4 gene. (B) V. cholerae cells carrying an S10′/lacZ′ plasmid with the wild-type (wt) V. cholerae leader or the V. cholerae leader containing a deletion of the indicated hairpins. (C) V. cholerae cells carrying an S10′/lacZ′ plasmid with either the wild-type V. cholerae leader or a V. cholerae leader with a deletion of the HD hairpin. The L4 source was V. cholerae. (D) V. cholerae cells carrying an operon fusion plasmid with the wild-type V. cholerae leader.

FIG. 4.

(A) Maps of the V. cholerae spc operon and the plasmid used for the target of S8-mediated regulation. The map of the spc operon is based on the genome sequence reported by Heidelberg et al. (7). (B) Secondary structures of the S8 targets in 16S rRNA and spc mRNA. The core binding regions of S8 on E. coli 16S (4, 9, 15) and on E. coli spc mRNA (5) are indicated by filled boxes. The same region is also required for S8-mediated regulation of the spc operon in E. coli (2, 26). As an analogy, the same region is indicated on the V. cholerae mRNA. The Shine-Dalgarno sequence and the initiation codon of the L5 gene are indicated by open boxes. (C) Regulation of the E. coli (Ec) and V. cholerae (Vc) spc operons mediated by S8. E. coli or V. cholerae strains with the indicated target reporter plasmid and S8 source were pulse-labeled with [³⁵S]methionine before (−) or 10 min after (+) induction of S8 by addition of arabinose. The radioactivity in each L5′/lacZ′ band was normalized to the total radioactivity in the same lane. S8-mediated regulation (+S8/−S8) was expressed as the normalized radioactivity in the fusion band after S8 induction divided by the normalized radioactivity in the fusion band before S8 induction. Each experiment was performed at least two times. The standard deviations are shown in parentheses.