Cell wall turnover in batch and chemostat cultures of Bacillus subtilis

Abstract

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.

Cell wall metabolism in Bacillus subtilis subsp. niger: accumulation of wall polymers in the supernatant of chemostat cultures

“…This would imply that, at the membrane, newly made peptidoglycan chains are heavily loaded with anionic polymers and that specific excision of many of the anionic polymers (linked to small peptidoglycan fragments) would occur in the turnover-sensitive layer of the wall (see reference 9). As was shown clearly by pulse-label experiments no peptidoglycan (and, most likely, no anionic polymers) is lost from the turnover-resistant inner wall layers (9). So all peptidoglycan-anionic polymer complexes released must originate from excision in the outer layers, at the rates indicated by k in Table 1 for peptidoglycan and the anionic polymer.…”

“…In a previous report (9), we showed that analysis of the way by which a specific radioactive peptidoglycan label was lost from B. subtilis by turnover provided some insight into the mechanisms of wall growth in this organism. Based on kinetic data, the conclusion (1,23) was reinforced that wall turnover in bacilli occurs from the outer wall layer and that wall surface enlargement takes place by spreading of peptidoglycan (9). The rate of wall turnover calculated from those previous experiments, however, referred merely to the peptidoglycan amino sugar t Present address: Gist-Brocades N. V., Microbiology Research Laboratory, Poatbus 1, 2600 MA Delft, The Netherlands.…”

“…The existence of a stabilized situation in a steady-state chemostat culture growing at a specified rate allows one to obtain the rate of loss of wall components simply from the measured concentrations. It has been suggested that the nature of the anionic wall polymer would exert a large influence on wall turnover (16); experiments with radioactive labeling as described above did not support such a hypothesis (9). In chemostat cultures, turnover can now be measured in exponentially growing cultures containing either TA or TUA as the anionic wall polymers by growing the cells under magnesium or phosphate limitation, respectively (19).…”

“…Such dissimilarity was not to be expected a priori. Cell wall turnover in B. subtilis is caused by autolytic enzymes (mainly N-acetylmuramyl-L-alanine amidase [3]) which act randomly on bonds within the peptidoglycan in the outer layer of the wall (9). Anionic polymers will appear in the supernatant because of their covalent coupling to the muramic acid moiety in peptidoglycan.…”

The growth strategy of the Gram-positive rod

“…Kinetic analysis of the cell wall turnover was initiated in Wouters' laboratory [17], but we have recently extended their treatment [15]. The Dutch kinetic model supposed that layers progressively moved outwardly as intact lamellae.…”