A decrease in S-adenosylmethionine synthetase activity increases the probability of spontaneous sporulation

Abstract

Starting with a relaxed (relA) strain, mutants with reduced activity of adenosine triphosphate:L-methionine S-adenosyl transferase (EC 2.5.1.6; SAM synthetase) were isolated in Bacillus subtilis. One such mutant (gene symbol metE1) had only 3% of the normal SAM synthetase activity but grew almost as well as the parent strain. Another mutant was isolated (gene symbol spdC1) as being able to sporulate continually at a high frequency; it had one-half the normal SAM synthetase activity at 33 degrees C. Both mutants continually and spontaneously entered spore development at a higher frequency than the parent strain in a medium containing excess glucose, ammonium ions, and phosphate. Sporulation was prevented by a high concentration of SAM (1 mM or more) or by the combination of adenosine and methionine (0.5 mM or more each), both of which are precursors of SAM. In contrast to this continual increase in the spore titer, addition of decoyinine, an inhibitor of GMP synthetase, rapidly initiated massive sporulation. Various amino acid analogs also induced sporulation in the relA strain, the methionine analogs ethionine and selenomethionine being most effective.

A Novel Approach for Gene Expression Optimization through Native Promoter and 5′ UTR Combinations Based on RNA-seq, Ribo-seq, and TSS-seq of Streptomyces coelicolor

“…the sporulation abilities of the two MetK overexpressing mutants were also compared because the effect of MetK on delaying sporulation is also known in Streptomyces strains. , When R5 – complex agar media was used, the MetK mutant using P 4808 R 3083 combination started to sporulate on day two just as in WT, whereas the P 4505 R 5755 - metK mutant began its sporulation around day three (Figure S3). The strongly expressed MetK mutant with P 4505 R 5755 resulted in incomplete sporulation, similar to the result from previous reports …”

“…The absorbance at 640 nm of the supernatant was measured with the use of a Multiskan spectrum (Thermo scientific, USA). 40 ■ ASSOCIATED CONTENT * S Supporting Information…”

“…The supernatant was acquired by centrifugation at 13500 rpm for 5 min at room temperature. The absorbance at 640 nm of the supernatant was measured with the use of a Multiskan spectrum (Thermo scientific, USA) …”