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The properties and expression of polynucleotide phosphorylase (PNPase), capable of both RNA degradation and polymerization, have been studied for 60 years. In this issue of the Journal of Bacteriology, Park et al. (H. Park, H. Yakhnin, M. Connolly, T. Romeo, and P. Babitzke, J Bacteriol 197:3751-3759, 2015, http://dx.doi.org/10.1128/JB.00721-15) write the latest chapter on the complex regulation of pnp gene expression involving CsrA. I describe how this new piece of the puzzle fits into the global scheme of PNPase autoregulation and how this is influenced by central carbon metabolism at both the posttranscriptional level and that of enzyme activity., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The genes encoding the ribonucleases RNase J1 and RNase Y have long been considered essential for Bacillus subtilis cell viability, even before there was concrete knowledge of their function as two of the most important enzymes for RNA turnover in this organism. Here we show that this characterization is incorrect and that ΔrnjA and Δrny mutants are both viable. As expected, both strains grow relatively slowly, with doubling times in the hour range in rich medium. Knockout mutants have major defects in their sporulation and competence development programs. Both mutants are hypersensitive to a wide range of antibiotics and have dramatic alterations to their cell morphologies, suggestive of cell envelope defects. Indeed, RNase Y mutants are significantly smaller in diameter than wild-type strains and have a very disordered peptidoglycan layer. Strains lacking RNase J1 form long filaments in tight spirals, reminiscent of mutants of the actin-like proteins (Mre) involved in cell shape determination. Finally, we combined the rnjA and rny mutations with mutations in other components of the degradation machinery and show that many of these strains are also viable. The implications for the two known RNA degradation pathways of B. subtilis are discussed.

23S rRNA maturation in Bacillus subtilis is catalyzed by the recently characterized enzyme Mini-RNase-III. Mini-III is dispensable, however, and 23S rRNA is matured by other ribonucleases in strains lacking this enzyme. Here we show that these RNases are the 5'-to-3' exoribonuclease RNase J1 and the 3'-to-5' exoribonucleases, principally RNase PH and YhaM.

In Bacillus subtilis, maturation of 5S rRNA is catalyzed by an enzyme called RNase M5. We searched for potential mRNA substrates for RNase M5 by gene array technology, based on the premise that most endonucleolytic cleavages have an effect on the stability of RNA and hence on steady-state levels of expression. Only a handful of genes had significantly altered expression in rnmV mutants compared to wild-type strains that could subsequently be confirmed by Northern blotting. The effect of RNase M5 on the expression of the best candidates, the odhAB and sucCD operons, is indirect, by a mechanism we do not yet understand. We show that an effect of RNase M5 on the expression of the remaining candidate, ctsR, is due to the failure to process the 5S rRNA contained in the rrnW lying directly upstream. We thus conclude that RNase M5 has very few or possibly no mRNA substrates in B. subtilis.

The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrn deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Delta7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrn deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.