The Study of a Novel (p)ppGpp Synthase (YwaC) from Bacillus subtilis 168

Abstract

Adaptation to any undesirable change in the environment helps to ensure the survival of many microorganisms. During nutrient starvation, bacteria undergo a stringent response characterized by the accumulation of the alarmone (p)ppGpp. This results in the repression of stable RNA species and a change in colony morphology. In Gram-negative bacteria such as Escherichia coli, RelA and SpoT synthesize and hydrolyze these nucleotides, respectively, under conditions of nutrient starvation. In Gram-positive bacteria, the bifunctional enzyme Rei is responsible for the accumulation of (p)ppGpp. These enzymes catalyze the transfer of a pyrophosphate moiety from ATP to the 3' end of either GTP or GDP. The overproduction of (p)ppGpp has many diverse consequences on bacterial physiology such as sporulation, virulence, long term persistence of pathogenic bacteria, cell morphology, antibiotic synthesis and fatty acid metabolism. In Bacillus subtilis a novel (p)ppGpp synthase, YwaC, is also involved in the accumulation of (p)ppGpp but does not associate with the ribosome. Transcriptional analysis of ywaC has implicated it with cell wall stress especially associated with lesions in the teichoic acid biosynthetic pathway. The work described here includes a steady state kinetic analysis of the reaction catalyzed by YwaC. Recombinant YwaC was over-expressed in E. coli and purified to homogeneity. Steady-state kinetic experiments were performed utilizing a high-performance liquid chromatography assay. This examination yielded Km values for GDP and GTP of 5 J.1M and 6 J.1M respectively, while the kcat was measured to be 0.13 min"1 and 0.11 min"1 respectively. As is common with other (p)ppGpp synthases, the low activity ofYwaC may be increased in the presence of the appropriate effector molecule. To explore the functional phenotype associated with ywaC a deletion strain was made by replacing the gene on the chromosome of B. subtilis with a spectinomycin resistance cassette. A variety of antibiotics were used to probe the ywaC deletion strain in an attempt to detect antibiotic sensitivity in comparison to wildtype cells. In addition, the morphology of the ywaC deletion strain was investigated using phase contrast confocal microscopy. Length and shape remained the same in a ywaC knockout. Growth profiles performed over a 24-hour period showed that the knockout strain grew similarly to wildtype B. subtilis. Thus, the phenotype analysis described herein failed to further elucidate the function of YwaC. Nevertheless, rigorous biochemical analysis described here have established the enzymatic role of (p)ppGpp synthesis for YwaC, but there remains much room for further investigation.