Characterization of the small untranslated RNA RyhB and its regulon in Vibrio cholerae

Abstract

Numerous small untranslated RNAs (sRNAs) have been identified in Escherichia coli in recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved in Vibrio cholerae, and both identification and characterization of sRNAs in V. cholerae remain at a preliminary stage. We have characterized one of the few sRNAs conserved between E. coli and V. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB in V. cholerae is significantly larger than in E. coli. As in E. coli, V. cholerae RyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB in V. cholerae and E. coli appear to differ, although some overlap is evident. Analysis of gene expression in V. cholerae in the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host by V. cholerae.

FIG. 1.

Northern blot analyses of ryhB expression and RyhB interactions in V. cholerae. A. RNA was isolated from wild-type N16961 (wt) and from ryhB and hfq derivatives of this strain, which were grown in the media indicated with (+D) or without dipyridyl. RNA was electrophoresed on denaturing acrylamide gels and transferred to membranes by electroblotting before hybridization to a RyhB probe. B. Effect of addition of FeSO₄·7 H₂O to cells in M63 medium. RNA was electrophoresed on an agarose gel. Total concentration of FeSO₄·7 H₂O is shown. C. Role of Fur in iron-regulated expression of ryhB. RNA from wt and fur cells grown in LB with or without 100 μM dipyridyl (Dip.) was electrophoresed on an agarose gel. D. RyhB interaction with Hfq. HfqH6 and associated RNAs were affinity purified on Ni-NTA agarose from lysate of an hfq::H6 strain (HfqH6); a control purification was performed in parallel using lysate from wt cells (wt). Protein purification was assessed on Western blots probed with an anti His5 antibody (upper panel), and RyhB purification was assessed on Northern blots of agarose gels (lower panel). Protein and RNA were extracted from total lysates (lanes 1 and 5), “depleted lysates” (lanes 2 and 6), washes containing 250 mM imidazole (lanes 3 and 7), and Ni-NTA agarose pellets after 250 mM imidazole wash (lanes 4 and 8).

FIG. 2.

Boundaries, features, and potential base pairing for V. cholerae RyhB. A. Start and end sites of V. cholerae RyhB were determined by 5′ and 3′ RACE, respectively. Potential binding sites for RNA polymerase (−35 and −10) were identified using BPROM. The candidate Fur binding site (underlined) matches a consensus sequence at 12 of 19 positions. A ∼60-nt region is conserved in V. cholerae, V. vulnificus and V. parahaemolyticus. A ∼28-nt region (bold) is conserved among vibrios, Yersinia spp., E. coli, Salmonella spp., Photobacterium profundum, and other bacteria. B. Sequence complementarity between V. cholerae RyhB and the leader sequence of sodB. C. Sequence complementarity between V. cholerae RyhB and the 5′ end of sdhC. The first translated codon of sdhC is boxed.

FIG. 3.

Northern blot analyses of genes identified in microarray analyses as differentially expressed in wt and ryhB V. cholerae. RNA was isolated from wt, ryhB, and hfq V. cholerae grown in M63-dipyridyl. Blots were hybridized to riboprobes complementary to transcripts of VC2045 (A), VC1169 (B), VC2213 (C), VC1255 (D), and VC1451 (E).

FIG. 4.

Comparative growth analyses of wt and ryhB V. cholerae in vitro and in the suckling mouse. Growth curves for wt and ryhB V. cholerae grown in M63 (A) and M63-25 μM dipyridyl (B) are shown. Cultures for each strain were grown in duplicate; both measurements were plotted. C. Competitive indices for wt and ryhB V. cholerae grown in the suckling mouse (in vivo) and in LB medium (in vitro). The competitive index is defined as follows: (mutant_ouput/wt_output)/(mutant_input/wt_input). Thus, a competitive index of <1.0 indicates a strain with a competitive disadvantage.