Study of tyrosine transfer ribonucleic acid modification in relation to sporulation in Bacillus subtilis

Abstract

MATERIALS AND METHODS Strains. B. subtilis W168, B. subtilis W23 D obtained from R. H. Doi, and W23 S obtained from P. Schaeffer were stored as spore suspensions. Growth conditions. Cells were cultivated at 37 C with vigorous agitation on different media: nutrient broth medium (28); Penassay broth (Difco); SCM medium (11) (synthetic medium supplemented with Casamino Acids lDifco]); MS medium (29) (synthetic medium with 0.2% NH4Cl as a nitrogen source and 0.5% malate as a carbon source). MSS medium (8), synthetic sporulation medium supplemented with or without iron, contains (in grams per liter): sodium citrate, 2.0; sodium acetate, 2.0; glutamic acid, 2.0; NH4C1, 0.5; glycerol, 0.5. Iron was extracted from a 5 x-concentrated stock solution with chloroform-8-hydroxyquinoline by the method of Waring and Werkman (35). Before use, MnCl2 and (NO3)2Ca, filter sterilized, were added from stock solutions to give final concentrations of 10 ,uM and 0.5 mM, respectively. This medium was supplemented with or without 1 ml of 1 mM SO4Fe per liter, filter sterilized. Experimental cultures were inoculated from growing precultures in the same medium, themselves already having been inoculated with cells from overnight cultures at 30C in agar-nutrient broth medium.

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

“…The 2-D gel patterns of tRNA samples labeled during vegetative growth, sporulation, and outgrowth are stikingly simila. This result and reports that the alterations observed in at least four tRNA's (tRNALYO, tRNAT', tRNAPhe, and tRNATh") during sporulation are due to posttranscriptional modifications of vegetative tRNA's (2,15,21,37) strongly suggest that the same tRNA genes are transcribed at all stages of the B. subtilis life cycle.…”

“…U in the anticodon [23]), the effect of tRNA modifications on gel migration has not been analyzed in any rigorous manner and can only be inferred from other data (9,11,27,31). We feel that it is likely that a tRNA base modification such as the thiomethylation reported to occur for some tRNA species during sporulation (2,15,21,37) would not produce an altered tRNA migration pattern in the 2-D gel system that we used.…”

“…The extent of labeling was about one radiolabeled methyl group per tRNA molecule (14). Considering that tRNA's undergo modification during sporulation (15,21,37), the fact that there were so few radiolabeled groups added per tRNA molecule makes it possible that in vivo tRNA modification could block or expose the site(s) available for in vitro methylation. Nonuniform labeling of different tRNA species could make the estimates of the number of tRNA species transcribed at different growth stages invalid.…”

“…Investigations of mechanisms controlling bacterial spore development have involved an intense examination of the regulatory role that tRNA may play in this system (18,20,21,35). Sporulation in Bacillus subtilis is accompanied by significant qualitative and quantitative changes in at least eight iso-accepting tRNA species (37).…”

Thiomethylation of tyrosine transfer ribonucleic acid is associated with initiation of sporulation in Bacillus subtilis: effect of phosphate concentration

“…Bacillus subtilis tRNATcan be separated by chromatography into two major species. One of these (tRNATyI) is predominant in vegetative cells, whereas the other (tRNATYIII) is the major component in stationary-phase cells and spores (1,10,12,18). tRNAT-" modification begins during the last division of exponential growth phase and is over by 2 h after the end of this phase.…”