An RNA Polymerase Mutation Causing Temperature-Sensitive Sporulation in Bacillus subtilis

Abstract

A single-site mutant of Bacillus subtilis with a rifampin-resistant RNA polymerase has been isolated; this mutation causes temperature-sensitive sporulation. The temperature-sensitive mutation was only expressed during a limited time period, covering the middle third of the sporulation sequence. Mutant cells grown at the nonpermissive temperature exhibited the normal change in RNA polymerase template specificity, accumulated extracellular proteolytic activity and antibiotic activity, but failed to accumulate alkaline phosphatase and, hence, were blocked at or near stage III in the sporulation sequence. Pulse-labeled RNA synthesis was seriously deranged during postexponential growth phase in mutant cells incubated at the nonpermissive temperature.Bacterial sporulation represents an ideal system in which to study the differential control of gene expression during a simple process of cell differentiation. Bacillus subtilis is particularly suitable for investigations of this type, since a great deal is known about the genetics (1, 2), physiology (3, 4), and biochemistry (3-6) of the developmental process. Previous studies have indicated that some form of transcriptional control must be operative during spore formation, since unique mRNA species are found in sporulating cells (6). Furthermore, it is known that a proteolytic cleavage of the 3 subunit of RNA polymerase is an absolute requirement for sporulation to proceed past state 0 (7-9). Mutants with an altered serine protease (7) or an altered RNA polymerase (8, 9) block sporulation at stage 0 by interfering with the clippage of 3 subunit. An important and unanswered question is whether sporulation-specific alterations in RNA polymerase molecular structure are necessary at other stages in the sporulation process. This communication establishes that a mutation of RNA polymerase can specifically affect development over a limited time during midsporulation. MATERIALS AND METHODSBacillus. s-sbtilis W168 was grown (7, 10) in a modified Schaeffer's Medium. Bacillus subtilis cys and bacteriophage PBS1 were from L. R.'Brown, and Bacillus subtilis trp-, guawas from J. A. Hoch. Procedures for the estimation of refractile bodies (10), protease accumulation (7), antibiotic activity (7), alkaline phosphatase accumulation (10), RNA synthesis (7), and RNA polymerase template activity (7) were described.Temperature-sensitive sporulation (spot") mutants with defects in RNA polymerase were isolated by screening for a subclass of rifampin-resistant (rifr) mutants with the expected properties. Bacillus subtilis W168 spores were muta-1179 genized with ethane methyl sulfonate to 80% survival (11).Survivors were plated onto tryptose-blood agar-base plates at 35°. Incubation was continued for 4 hr at 35°. The plates were then spread with 0.2 ml of a 700 ,g/ml solution of rifampin. Incubation was continued until resistant colonies appeared. All colonies were replicated onto similar plates at 350 and 480. Asporogenous mutants lyse on such plates after 24-48 hr of incubation (7). Th...

Use of temperature-sensitive mutants to study gene expression during sporulation in Bacillus subtilis

“…These problems may be studied by the use of temperature-sensitive mutants (19,20,31,32) that are shifted from permissive to nonpermissive conditions, and vice versa, during sporulation. Esposito et al (12), working with temperature-sensitive mutants ofSaccharomyces cerevisiae, showed that it is possible to make allowances for both (i) different rates of development at permissive and nonpermissive temperatures and (ii) different rates of development of different strains under identical conditions, so that the position in the sporulation sequence of the temperature-sensitive period of a mutant may be determined.…”

Genetic Control of Sporulation

“…kinases), or other regulatory macromolecules that alter its specificity and allow it to transcribe sporulation-specific genes. A streptovaricin resistant RNA polymerase mutant which has a mutation close to the rifampicin locus also has a phenotype similar to that exhibited by the Class IV mutants (87); it was of interest that no streptolydigin-resistant RNA polymerase mutants were conditionally temperature sensitive during sporulation. Since streptolydigin only inhibits elongation and not initiation, it is possible that this function is identical in both vegetative and sporulation RNA polymerase molecules.…”

Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T

“…Leighton and his co-workers have used a more direct approach. They have isolated various ts mutants-of B. subtilis affecting specific enzymes which they believed were involved in sporulation (Leighton 1973;Leighton et nl. 1972Leighton et nl.…”

“…, 1973. One series of ts mutants isolated by Leighton (1973) include rifampin-resistant RNA polymerase mutants which are affected at uniquely different time periods during sporulation. One of them, ts-14, produces several of the products involved in the early stages of sporulation but is affected in forespore membrane synthesis and engulfment and synthesis of alkaline phosphatase (Leighton 1973;Santo et 01.…”