Transcription After Bacteriophage SPP1 Infection in Bacillus subtilis

The role of the host polymerase in Bacillus subtilis infected with phage SPP1 was studied in vivo with regard to production of phage-specific and host-specific ribonucleic acid (RNA) and to phage yield. Evidence is presented that the subunit(s) of B. subtilis RNA polymerase which is sensitive to rifampin and streptolydigin is necessary at all times during infection for phage production. The synthesis of phage RNA and the phage yield in strains resistant to either antibiotic were unaffected by the drug. Host RNA synthesis continued throughout infection; phage-specific RNA never accounted for more than 20% of pulse-labeled RNA at any time during infection.     

Initiation of Bacillus subtilis Ribonucleic Acid Polymerase on Deoxyribonucleic Acid from Bacteriophages 2C, φ29, T4, and Lambda

Ribonucleic acid (RNA) synthesis primed by bacteriophage T4 or lambda deoxyribonucleic acid (DNA) with Bacillus subtilis RNA polymerase is severely inhibited by high ionic strength. In contrast, RNA synthesis on B. subtilis bacteriophage 2C, SPO1, or phi29 DNA is only moderately affected under similar conditions. The basis of this inhibition lies in the inability of the enzyme to initiate RNA chains with adenosine triphosphate or guanosine triphosphate (ATP, GTP). Binding to templates and the rate of catalysis in high salt after initiation do not seem to be affected. Incorporation of gamma-(32)P-ATP and GTP under a variety of conditions suggests that the specificity of B. subtilis RNA polymerase is different from that of the Escherichia coli enzyme and that it recognizes few promoters on T4 and lambda DNA. Although B. subtilis RNA polymerase initiates RNA chains primarily with ATP or GTP, initiations with pyrimidines can occur on DNA molecules in which hydroxymethyluracil replaces thymine. RNA synthesis on denatured DNA does not seem to be inhibited by high ionic strength, and on native T4 or lambda DNA the inhibition of initiation at constant ionic strength is inversely but not linearly proportional to the ionic radii of cations used to stabilize bihelical DNA to denaturation.     

New types of RNA polymerase mutations causing temperature-sensitive sporulation in bacillus subtilis.

A single site mutant of Bacillus subtilis with a streptovaricin-resistant RNA polymerase has been isolated; this mutation caused temperature-sensitive sporulation, but had no effect on vegetative growth. The mutant (ts710) temperature-sensitive period irreversibly affected the middle and late stages of sporulation. Mutant cells grown at the nonpermissive temperature exhibited abnormal serine protease accumulation, serine esterase accumulation, alkaline phosphatase accumulation, RNA polymerase template specificity changes, and pulse-labeled RNA synthesis profiles. The accumulation of metal protease was not affected at the nonpermissive temperature. Attempts to isolate single site mutants which were streptolydigin-resistant, and temperature-sensitive for sporulation, were unsuccessful.     

Sigma factor is not released during transcription in Bacillus subtilis

Molecular Genetics and Genomics - The relationship between sigma (σ) and delta (δ) factors of Bacillus subtilis RNA polymerase has been analyzed during initiation of RNA synthesis. When...     

Medium-dependent control of the bacterial growth rate

By combining results from previous studies of nutritional up-shifts we here re-investigate how bacteria adapt to different nutritional environments by adjusting their macromolecular composition for optimal growth. We demonstrate that, in contrast to a commonly held view the macromolecular composition of bacteria does not depend on the growth rate as an independent variable, but on three factors: (i) the genetic background (i.e. the strain used), (ii) the physiological history of the bacteria used for inoculation of a given growth medium, and (iii) the kind of nutrients in the growth medium. These factors determine the ribosome concentration and the average rate of protein synthesis per ribosome, and thus the growth rate. Immediately after a nutritional up-shift, the average number of ribosomes in the bacterial population increases exponentially with time at a rate which eventually is attained as the final post-shift growth rate of all cell components. After a nutritional up-shift from one minimal medium to another minimal medium of higher nutritional quality, ribosome and RNA polymerase syntheses are co-regulated and immediately increase by the same factor equal to the increase in the final growth rate. However, after an up-shift from a minimal medium to a medium containing all 20 amino acids, RNA polymerase and ribosome syntheses are no longer coregulated; a smaller rate of synthesis of RNA polymerase is compensated by a gradual increase in the fraction of free RNA polymerase, possibly due to a gradual saturation of mRNA promoters. We have also analyzed data from a recent publication, in which it was concluded that the macromolecular composition in terms of RNA/protein and RNA/DNA ratios is solely determined by the effector molecule ppGpp. Our analysis indicates that this is true only in special cases and that, in general, medium adaptation also depends on factors other than ppGpp.     

Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. II. Fraction of RNA polymerase engaged in the synthesis of stable RNA at different steady-state growth rates

The relative rates of stable RNA synthesis (rate of stable synthesis/rate of total RNA synthesis) were determined for Escherichia coli$\text{B}\text{r}$ growing in succinate (μ = 0.69 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) media. The relative rates were 0.29, 0.50 and 0.66 at these growth rates. From the relative rates, the fraction of RNA polymerase engaged in the synthesis of stable RNA, ψ_s, was calculated to be 0.22, 0.36 and 0.48, respectively, by taking into account the difference between the RNA chain growth rate of stable and that of unstable RNA. The relationship between these ψ_s values and μ and our previously determined chain growth rate of stable RNA has two implications for the control of RNA synthesis during a nutritional shift-up: (1) the increase in the net rate of RNA synthesis after a shift-up results from a transfer of RNA polymerase molecules from unstable to stable RNA genes, and a concomitant increase in the stable RNA chain growth rate, but does not require an activation of RNA polymerase; (2) the synthesis of functioning RNA polymerase enzymes is subject to a growth rate-dependent control.     

Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis

6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.