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.

The walls of Bacillus subtilis var. niger WM, grown in a Mg2+-limited chemostat culture (carbon source glucose, dilution rate = 0.2 h-', 37 "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 yielded I-U-b-glucosylglycerol phosphate (together with small amounts of glycerol phosphate) and 13C nuclear magnetic resonance spectra of this hydrolysis product, and its derivative after alkaline phosphatase treatment, confirmed that the monomeric unit was l-O-~-glucosylglycerol-3-phosphate. Assignment of the resonances in the spectrum of undegraded teichoic acid revealed that the polymer was a poly [(2,3)glycerol phosphate], glucosidically substituted on C-1 of glycerol with /?-glucose.The walls of Bacillus subtilis var. niger contain teichoic acid under conditions of chemostat culture where phosphorus is not the growth-limiting nutrient [l, 21. However, the precise structure of this polymer is not known with certainty and therefore, in order to study its biosynthesis, it is first necessary to determine its exact composition. Previous investigations have shown that B. subtilis var. niger produces a teichoic acid built up from glycerol, phosphate and glucose [l], but no conclusive evidence has been presented about the sequence of the monomcrs in this polymer [3]; because of the complexity of the chemical techniques generally used, we looked for a more direct and unequivocal method for the sequence determination.13C nuclear magnetic resonance (I3C NMR) has scarcely been used for structural analysis of bacterial wall polymers, although Bundle et al. [4] have shown the value of this technique in elucidating the structure of 2-acetamino-2-deoxy-~-glucose phosphate polymers from Neisseriu meningitidis and Staphylococcus lactis. However, no reports have been published, to the best of our knowledge, on the application of 13C NMR techniques to teichoic acid analysis.In this paper we describe the analysis of the structure of the monomeric units of teichoic acid from Ahhrr~iarion. NMR, nuclear magnetic resonance.B. suhtilis var. niger WM by an alkaline hydrolysis procedure [5,6], and the use of 13C NMR as an analytical tool for the determination of the sequence of the monomers in the undegraded teichoic acid. MATERIALS AND METHODS Cultivation of BacteriaBacillus subtilis var. niger WM (a spontaneously occurring mutant from B. suhtilis var. niger which forms white instead of reddish colonies on peptone agar + 1 %glucose) was cultured in a 1-1 (LH-Engineering) chemostat with the dilution rate set to a value of 0.2 h-', the pH value controlled at 7.0 and the temperature regulated at 37 "C. The MgZ+-limited medium was made up essentially as described by Evans et al. [7], except that the overall concentration of each nutrient was three-quarters of that specified. Glucose was provided as the carbon source and was added to a final concentration of 22.5 g/l. The outflow tube of the chemostat was connec...

Cell wall polymers were measured both in the cells and in the cell-free medium of samples from steady-state chemostat cultures of Bacillus subtilis, growing at various rates under magnesium or phosphate limitation. The presence of both peptidoglycan and anionic wall polymers in the culture supernatant showed the occurrence of wall turnover in these cultures. Variable proportions of the total peptidoglycan present in the culture samples were found outside the cells in duplicate cultures, indicating that the rate of peptidoglycan turnover is variable in B. subtilis. Besides peptidoglycan, anionic wall polymers were detected in the culture supernatant: teichoic acid in magnesium-limited cultures and teichuronic acid in phosphate-limited cultures. In several samples, the ratio between the peptidoglycan and the anionic polymer concentrations was significantly lower in the extracellular fluid than in the walls. This divergency was attributed to the occurrence of direct secretion of anionic polymers after their synthesis.

A study was made to determine whether factors other than the availability of phosphorus were involved in the regulation ofsynthesis ofteichoic 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-'. 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.

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.