Transfer ribonucleic acid synthesis during sporulation and spore outgrowth in Bacillus subtilis studied by two-dimensional polyacrylamide gel electrophoresis

Abstract

The synthesis of transfer ribonucleic acid (tRNA) was examined during spore formation and spore outgrowth in Bacillus subtilis by two-dimensional polyacrylamide gel electrophoresis of in vivo 32P-labeled RNA. The two-dimensional gel system separated the B. subtilis tRNA's into 32 well-resolved spots, with the relative abundances ranging from 0.9 to 17% of the total. There were several spots (five to six) resolved which were not quantitated due to their low abundance. All of the tRNA species resolved by this gel system were synthesized at every stage examined, including vegetative growth, different stages of sporulation, and different stages of outgrowth. Quantitation of the separated tRNA's showed that in general the tRNA species were present in approximately the same relative abundances at the different developmental periods. tRNA turnover and compartmentation occurring during sporulation were examined by labeling during vegetative growth followed by the addition of excess phosphate to block further 32P incorporation. The two-dimensional gels of these samples showed the same tRNA's seen during vegetative growth, and they were in approximately the same relative abundances, indicating minimal differences in the rates of turnover of individual tRNA's. Vegetatively labeled samples, chased with excess phosphate into mature spores, also showed all of the tRNA species seen during vegetative growth, but an additional five to six minor spots were also observed. These are hypothesized to arise from the loss of 3'-terminal residues from preexisting tRNA's.