Cell wall turnover in growing and nongrowing cultures of Bacillus subtilis.

Cell wall turnover was studied in cultures of Bacillus subtilis in which growth was inhibited by nutrient starvation or by the addition of antibiotics. Concomitantly, the synthesis of wall, as measured by the incorporation of radioactively labeled N-acetylglucosamine, was followed in some of these cultures. In potassium- or phosphate-starved cultures, growth stopped, but wall turnover continued at a rate slightly lower than that in the control cultures. Lysis of cells did not occur. In glucose-starved cultures, continued wall turnover caused lysis of cells, since wall synthesis apparently was inhibited. The same phenomenon was observed after growth arrest by the addition of wall synthesis inhibitors such as fosfomycin, cycloserine, penicillin G, and vancomycin. Growth arrest by the addition of chloramphenicol allowed the continuation of wall synthesis; therefore, the observed turnover generally did not cause cell lysis.

Cell wall metabolism in Bacillus subtilis subsp. niger: effects of changes in phosphate supply to the culture.

Chemostat cultures of Bacillus subtilis subsp. niger WM were exposed to changes in the availability of phosphorus by means of a resuspension technique. Responses in wall metabolism were recorded by measuring the amounts of peptidoglycan and anionic polymers (teichoic or teichuronic acid) in the wall and extracellular fluid fractions. With respect to the wall composition, the effect of a change in orthophosphate supply was a complete shift in the nature of the anionic polymer fraction, the polymer originally present in the walls ("old" polymer) being replaced by the alternative ("new") anionic polymer. The peptidoglycan content of the walls remained constant. It was concluded that the incorporation of old polymer was completely blocked from the moment the orthophosphate supply was changed. However, from a measurement of the total amount of polymer in the whole culture during the course of the experiments, it was evident that synthesis of old polymer continued, but it was secreted. Synthesis of the new polymer started immediately, and it was incorporated exclusively into the wall. During adaption of the cells to the new environment, wall turnover continued in an identical fashion to that extant in steady-state cultures. It was concluded that the primary adaptive response to a change in orthophosphate supply occurred through a mechanism interacting with polymer incorporation and thus at the level of wall assembly at the membrane.

Cell wall turnover in batch and chemostat cultures of Bacillus subtilis.

Wall turnover was studied in Bacillus subtilis. The loss of radioactively labeled wall polymers was followed during exponential growth in batch and chemostat cultures. Turnover kinetics were identical under all growth conditions; pulse-labeled wall material was lost with first-order kinetics, but only after exponential growth for 1 generation time after its incorporation. Similarly, continuously labeled cells showed an accelerating decrease in wall-bound radioactivity starting immediately after removal of the labeled precursor and also reached first-order kinetics after 1 generation time. A mathematical description was derived for these turnover kinetics, which embraced the concept of "spreading" of old wall chains (H. M. Pooley, J. Bacteriol. 125:1127-1138, 1976). Using this description, we were able to calculate from our experimental data the rate of loss of wall polymers from cells and the fraction of the wall which was sensitive to turnover. We found that about 20% of the wall was lost per generation time and that this loss was affected by turnover activity located in the outer 20 to 45% of the wall; rather large variations were found with both quantities and also between duplicate cultures. These parameters were quite independent of the growth rate (the specific growth rate varied from 1.3 h-1 in broth cultures to 0.2 to 0.3 h-1 in chemostat cultures) and of the nature of the anionic polymer in the wall (which was teichoic acid in cultures with an excess of phosphate and teichuronic acid in phosphate-limited chemostat cultures). Some implications of the observed wall turnover kinetics for models of wall growth in B. subtilis are discussed.

Effects of carbon source and growth rate on cell wall composition of Bacillus subtilis subsp. niger.

A study was made to determine whether factors other than the availability of phosphorus were involved in the regulation of synthesis of teichoic and teichuronic acids in Bacillus subtilis subsp. niger WM. First, the nature of the carbon source was varied while the dilution rate was maintained at about 0.3 h-1. Irrespective of whether the carbon source was glucose, glycerol, galactose, or malate, teichoic acid was the main anionic wall polymer whenever phosphorus was present in excess of the growth requirement, and teichuronic acid predominated in the walls of phosphate-limited cells. The effect of growth rate was studied by varying the dilution rate. However, only under phosphate limitation did the wall composition change with the growth rate: walls prepared from cells grown at dilution rates above 0.5 h-1 contained teichoic as well as teichuronic acid, despite the culture still being phosphate limited. The wall content of the cells did not vary with the nature of the growth limitation, but a correlation was observed between the growth rate and wall content. No indications were obtained that the composition of the peptidoglycan of B. subtilis subsp. niger WM was phenotypically variable.

Further evidence for the structure of the teichoic acids from Bacillus stearothermophilus B65 and Bacillus subtilis var. niger WM.

Bacillus stearothermophilus B65 and Bacillus subtilis var. niger WM both contain teichoic acids in their walls composed of glycerol, phosphate and glucose. The 13C nuclear magnetic resonance spectrum of B. stearothermophilus teichoic acid showed 13C-31P coupling on the signals from the C-5 and C-6 carbon atoms of the glucose molecule and an alpha-glucosidic linkage between glucose and the C-1 atom of the glycerol moiety. These data are consistent with a poly[glucosylglycerol phosphate] as the cell-wall teichoic acid in this organism. B. subtilis var. niger WM teichoic acid was oxidized by periodate and incubated in glycine buffer at pH 10.5. This treatment did not significantly increase the phosphomonoester content (by beta-elimination of the phosphate groups) of the teichoic acid molecule (7.1 to 9.5%), which is in accordance with earlier data derived from 13C nuclear magnetic resonance spectroscopy [De Boer et al. (1976) Eur. J. Biochem. 62, 1-6], that in this organism the glucose is not an integral part of the polymer chain. Similar treatment of B. stearothermophilus B65 teichoic acid increased the phosphomonoester content of the preparation from 0.15 to 68.1%.

The structure of teichoic acid from Bacillus subtilis var, niger WM as determined by C nuclear-magnetic-resonance spectroscopy.

The walls of Bacillus subtilis var. niger WM, grown in a Mg2+ -limited chemostat culture (carbon source glucose, dilution rate = 0.2 h(-1), 37 degrees C, pH 7) contained 45% (w/w) teichoic acid, a polymer composed of glycerol, phosphate[ and glucose in the molar ratio 1.00:1.00:0.88, respectively. Alkaline hydrolysis of this teichoic acid yeilded 1-O-beta-glucosylglycerol phosphate (together with small amounts of glycerol phosphate0 and 13C nuclear magnetic resonance spectra of this hydrolysis product, and its derivative after alkaline phosphate treatment, confirmed that the monomeric unit was 1-O-beta-glucosylglycerol-3-phosphate. Assignment of the resonances in the spectrum of undergraded teichoic acid revealed that the polymer was a poly [(2,3) glycerol phosphate 1, glucosidically substituted on C-1 of glycerol with beta-glucose.