The vacB Gene Required for Virulence inShigella flexneri and Escherichia coli Encodes the Exoribonuclease RNase R

Abstract

vacB, a gene previously shown to be required for expression of virulence in Shigella and enteroinvasive Escherichia coli, has been found to encode the 3-5 exoribonuclease, RNase R. Thus, cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy of vacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacB gene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacB be renamed rnr.Exoribonucleases play an important role in RNA maturation, turnover, and degradation (for reviews, see Refs. 1 and 2). In Escherichia coli eight distinct exoribonucleases have been characterized. Most of them display a degree of overlap in their function. For example, six of the eight, including RNases II, D, BN, T, PH, and polynucleotide phosphorylase (PNPase), 1 participate in the 3Ј-maturation of tRNA precursors (3). Recently, the maturation of the small stable RNAs, M1 RNA, 10Sa RNA/tmRNA, 6S RNA and 4.5S RNA, was examined and found to involve many of the same exoribonucleases (4). It is also known that strains lacking RNases II, D, BN, T, and PH in combination are inviable, but the presence of any one of the five enzymes is sufficient to confer viability, although with varying degrees of effectiveness (5).RNase R is one of the eight exoribonucleases. It acts nonspecifically on poly(A), poly(U), and ribosomal RNAs (rRNA) in vitro (1, 6 -8). The enzyme was initially identified 20 years ago in an E. coli strain deficient in RNase II (6). Whereas RNase II accounts for more than 95% of the activity against poly(A) and poly(U) in crude cell extracts, the residual activity against these substrates and rRNA is due primarily to RNase R (1, 5, 7). Based on its gel filtration properties, RNase R is apparently a protein of ϳ85 kDa (8). However, despite all of this biochemical information, essentially nothing was known about the gene encoding RNase R other than that it mapped to the last quarter of the E. coli chromosome. 2In this paper we report the identification and characterization of the gene that encodes RNase R and show that it is the E. coli vacB gene. vacB was originally described in Shigella flexneri as a chromosomal gene required for expression of the virulence genes carried on the large plasmid of this organism (9). We were led to consider vacB as a candidate for the gene encoding RNase R because (a) sequence analysis revealed that it is homologous to the rnb gene that encodes another exoribonuclease with similar properties, RNase II (10); (b) the deduced size of the VacB protein (ϳ92 kDa...

Modulation of Yersinia Type Three Secretion System by the S1 Domain of Polynucleotide Phosphorylase

“…In a transposon mutagenesis screen, a Shigella flexneri was isolated that displayed reduced host cell invasion. The disruption was mapped to a locus that was later identified as the gene (vacB/rnr) encoding RNaseR (27,28). More pertinent to this study and as mentioned in the introduction, a pnp mutant of Salmonella enterica was found to have a disregulated TTSS (12).…”

Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R

“…RNase R has been purified to homogeneity and been shown to be a 3Ј to 5Ј exoribonuclease (8). The rnr gene encoding RNase R was identified and shown to be identical to the vacB gene required for virulence in Shigella and enteroinvasive E. coli (9). Efforts to construct mutant strains deficient in RNase R, alone or in combination with other exoribonucleases, showed that RNase R Ϫ single mutant strains are essentially normal, as are multiple mutant strains lacking RNases R and T, RNases R and PH, or RNases R, II, D, and BN (9).…”

“…Efforts to construct mutant strains deficient in RNase R, alone or in combination with other exoribonucleases, showed that RNase R Ϫ single mutant strains are essentially normal, as are multiple mutant strains lacking RNases R and T, RNases R and PH, or RNases R, II, D, and BN (9). However, a mutant strain lacking both RNase R and PNPase activities could not be made (9), suggesting that such cells are inviable and that the two exoribonucleases might overlap in some important function that had not yet been identified.…”

Efficient gene transfer and targeted mutagenesis in Fusobacterium nucleatum

“…The rnr gene, which encodes a 5′-3′ exoribonuclease, was originally identified as the vacB gene by Tn5 mutagenesis in Shigella flexneri based on reduced invasion (8,37). In E. coli RNase R is believed to function along with a polynucleotide phosphorylase in the maintenance of ribosomal RNA quality control through the removal of defective RNAs (7).…”

“…RNase R is not essential to viability, although inactivation of both the rnr and polynucleotide phosphorylase genes results in bacterial cell death (7). Further, the loss of RNase R activity in E. coli is not evident in the presence of RNase II activity (8). To our knowledge, exoribonuclease activity has not been studied in fusobacteria, and we have not detected any alteration in exoribonuclease activity in the F. nucleatum rnr mutant in comparison to the parental wild type strain (data not shown).…”