Regulation of the Bacterial Cell Wall: Analysis of a Mutant of Bacillus subtilis Defective in Biosynthesis of Teichoic Acid

Abstract

Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (Nacyl muramyl-L-alanine amidase). Genetic analysis of the mutant by PBS1mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-i) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the midportion of the chromosome of B. subtilis.

The Potential for Genetic Exchange by Transformation within a Natural Population of Bacillus subtilis

“…These DNA preparations were used to transform an auxotrophic derivative of strain 168 (strain 1A40) toward tryptophan and lysine prototrophy, using a protocol modified from Boylan et al (1972). Briefly, the recipient strain was inoculated from a refrigerated spore culture (on AK agar [BBL] with 5 mM MgS0 4 ) onto a TBAB plate, and incubated overnight at 30°C.…”

“…Cultures were grown in a gyratory incubator (120 rpm, 37°C) until 90 min after the end of logarithmic phase (as determined colorimetrically). The culture was then diluted into a stepdown liquid medium (GM2 of Boylan et al [1972]) and cultured for 60 min, at which time DNA was added to the culture at a final concentration of 6~g/ml. Cultures were incubated with DNA for 30 min, and then DNase was added at a final concentration of 20~g/ml.…”

Cell wall assembly in Bacillus subtilis: how spirals and spaces challenge paradigms

“…Assembly of this minor teichoic acid polymer is not well characterized but is known to be catalysed by enzymes encoded in the gga locus, presumably using nucleotide-activated sugar precursors (Estrela et al ., 1991). The synthesis of this polymer is impaired at elevated temperature although the cause of this phenomenon has not been identified (Boylan et al ., 1972).…”

Identification of the protein encoded by rodC, a cell division gene from Bacillus subtilis

“…Thus, conditional mutants must be used. This has restricted previous studies to genetic mapping (Karamata etal., 1972(Karamata etal., ,1987Van Alstyne and Simon, 1971), morphological appearance (Rogers et al, 1970;Van Alstyne and Simon, 1971), or to biochemical analysis (Boylan et al, 1972;Pooley et al, 1987). Although much is known in these areas, the pleiotropic effect of many of these conditional mutants has complicated investigations and has impaired the understanding of the molecular aspects of cell division.…”