Cloning of specific regions of RK2, a broad host range incompatibility group P plasmid, has revealed three genes: kilA, kIB, and kiC. Each of these genes can cause loss of viability of an Escherichia col host. This effect on the host is normally prevented by the functions ofthree additional RK2 genes: korA, korB, and korC. Each kor gene is specific for a particular kil gene. The kil and kor genes are located in four distinct regions of the RK2 genome. The three kil genes are not clustered and, with the possible exception of kilA, they are also well separated from their corresponding kor genes. We have found that the korA and korB determinants are not peculiar to RK2 but instead are highly conserved throughout the incompatibility group P plasmids.

SUMMARY In Escherichia coli, RNA degradation often begins with conversion of the 5′-terminal triphosphate to a monophosphate, creating a better substrate for internal cleavage by RNase E. Remarkably, no homologue of this key endonuclease is present in many bacterial species, such as Bacillus subtilis and various pathogens. Here we report that the degradation of primary transcripts in B. subtilis can nevertheless be triggered by an analogous process to generate a short-lived, monophosphorylated intermediate. Like its E. coli counterpart, the B. subtilis RNA pyrophosphohydrolase that catalyzes this event is a Nudix protein that prefers unpaired 5′ ends. However, in B. subtilis this modification exposes transcripts to rapid 5′-exonucleolytic degradation by RNase J, which is absent in E. coli but present in most bacteria lacking RNase E. This pathway, which closely resembles the mechanism by which deadenylated mRNA is degraded in eukaryotic cells, explains the stabilizing influence of 5′-terminal stem-loops in such bacteria.

The pnpA gene of Bacillus subtilis, which codes for polynucleotide phosphorylase (PNPase), has been cloned and employed in the construction of pnpA deletion mutants. Growth defects of both B. subtilis and Escherichia coli PNPase-deficient strains were complemented with the cloned pnpA gene. RNA decay characteristics of the B. subtilis pnpA mutant were studied, including the in vivo decay of bulk mRNA and the in vitro decay of either poly(A) or total cellular RNA. The results showed that mRNA decay in the pnpA mutant is accomplished despite the absence of the major, P i -dependent RNA decay activity of PNPase. In vitro experiments suggested that a previously identified, Mn 2؉ -dependent hydrolytic activity was important for decay in the pnpA mutant. In addition to a cold-sensitive-growth phenotype, the pnpA deletion mutant was found to be sensitive to growth in the presence of tetracycline, and this was due to an increased intracellular accumulation of the drug. The pnpA deletion strain also exhibited multiseptate, filamentous growth. It is hypothesized that defective processing of specific RNAs in the pnpA mutant results in these phenotypes.Current models for the mechanism of mRNA decay in bacteria propose that decay occurs as a result of the combined action of endoribonucleases and exoribonucleases. An mRNA molecule is attacked initially by endonucleolytic cleavages at specific sites, and then the resultant RNA fragments are degraded exonucleolytically in the 3Ј-to-5Ј direction (4). 3Ј-to-5Ј exonucleases cannot attack mRNA without prior endonucleolytic cleavage, because the 3Ј terminal stem-loop structure of mRNA is bound by a protein complex called the exonuclease impeding factor, which is thought to block exonucleolytic activity (6). 5Ј-to-3Ј exonucleases capable of initiating decay at the 5Ј end have not been found in bacteria.In Escherichia coli, two 3Ј-to-5Ј exonucleases, RNase II and polynucleotide phosphorylase (PNPase), are thought to be involved in mRNA decay. The loss of both RNase II and PNPase activities in conditionally mutant E. coli strains results in the cessation of growth (11). RNase II degrades RNA by processive hydrolysis to 5Ј-monophosphate nucleosides, while PNPase degrades RNA by processive phosphorolysis to 5Ј-diphosphate nucleosides. Using 3 H-labeled poly(A) as a substrate for degradative activity, Deutscher and Reuven (10) showed that, in E. coli extracts with Mg 2ϩ present as the divalent cation, about 90% of the degradative activity was hydrolytic and was due to RNase II while 10% of the degradative activity was phosphorolytic and was due to PNPase. It is therefore likely that RNase II is the major mRNA-degradative enzyme in E. coli. By contrast, in Bacillus subtilis extracts, degradative activity was primarily phosphorolytic and was shown to be due to a B. subtilis PNPase-like activity. Thus, it was proposed that PNPase is the major mRNA-degradative enzyme in B. subtilis.Recently, the gene for B. subtilis PNPase was identified by the isolation of a transposon insertion mutant that ...

Ribonucleases (RNases) are mediators in most reactions of RNA metabolism. In recent years, there has been a surge of new information about RNases and the roles they play in cell physiology. In this review, a detailed description of bacterial RNases is presented, focusing primarily on those from Escherichia coli and Bacillus subtilis, the model Gram-negative and Gram-positive organisms, from which most of our current knowledge has been derived. Information from other organisms is also included, where relevant. In an extensive catalog of the known bacterial RNases, their structure, mechanism of action, physiological roles, genetics, and possible regulation are described. The RNase complement of E. coli and B. subtilis is compared, emphasizing the similarities, but especially the differences, between the two. Included are figures showing the three major RNA metabolic pathways in E. coli and B. subtilis and highlighting specific steps in each of the pathways catalyzed by the different RNases. This compilation of the currently available knowledge about bacterial RNases will be a useful tool for workers in the RNA field and for others interested in learning about this area.