tmRNA-dependent trans-translation is required for sporulation in Bacillus subtilis.

SUMMARY: Spore formation in Bacillus subtilis is significantly impaired by the deletion of the gene for tmRNA (ssrA), which facilitates the trans-translation reaction that rescues stalled ribosomes and degrades incompletely synthesized peptides. Microscopic analysis revealed that the sporulation of most DeltassrA cells is blocked after forespore formation. Expression analysis of lacZ-fused genes directed by several RNA polymerase sigma factors showed that the synthesis of active sigma(K), encoded by the sigK gene, is predominantly inhibited in DeltassrA cells. The defect in sigma(K) synthesis is attributable to a defect in the skin element excision, which generates the sigK gene, caused in turn by reduced expression of SpoIVCA (recombinase) in DeltassrA cells.

Participation of 3'-to-5' exoribonucleases in the turnover of Bacillus subtilis mRNA.

Four 3'-to-5' exoribonucleases have been identified in Bacillus subtilis: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Mutant strains were constructed that were lacking PNPase and one or more of the other three ribonucleases or that had PNPase alone. Analysis of the decay of mRNA encoded by seven small, monocistronic genes showed that PNPase was the major enzyme involved in mRNA turnover. Significant levels of decay intermediates, whose 5' ends were at the transcriptional start site and whose 3' ends were at various positions in the coding sequence, were detected only when PNPase was absent. A detailed analysis of rpsO mRNA decay showed that decay intermediates accumulated as the result of a block to 3'-to-5' processivity at the base of stem-loop structures. When RNase R alone was present, it was also capable of degrading mRNA, showing the involvement of this exonuclease in mRNA turnover. The degradative activity of RNase R was impaired when RNase PH or YhaM was also present. Extrapolation from the seven genes examined suggested that a large number of mRNA fragments was present in the PNPase-deficient mutant. Maintenance of the free ribosome pool in this strain would require a high level of activity on the part of the tmRNA trans translation system. A threefold increase in the level of peptide tagging was observed in the PNPase-deficient strain, and selective pressure for increased tmRNA activity was indicated by the emergence of mutant strains with elevated tmRNA transcription.