Cell lysis was efficiently induced in Staphylococcus aureus by the addition of 0.3 M NaCl to exponentially growing cultures at 30 degrees C. When cells harvested at the exponential phase were incubated in buffer with NaCl, autolysis occurred. Treatment with chloramphenicol failed to induce cell lysis by NaCl. The effects of NaCl on growing cells and harvested cells were inhibited by the addition of sodium polyanethole sulfonate, subtilisin, cardiolipin, and lipoteichoic acid. These agents diminished the activity of a cell wall-lytic enzyme liberated from the cells in the presence of NaCl. Lysis induced by salt appears to be catalyzed by a similar lytic enzyme in growing and harvested cells.
d-Serine inhibited the growth of Mycobacterium smegmatis and induced the morphological alteration of the bacilli. The growth inhibitory action of d-serine was partially reduced by an equimolecular concentration of d-alanine. The combination of glycine with d-alanine reversed the growth inhibition produced by d-serine more than did d-alanine alone. In cells cultured in the presence of d-serine, the amounts of alanine, diaminopimelic acid, and glycine inserted into the cell wall mucopeptide were reduced, and serine was increased. The intracellular accumulation of a precursor of cell wall mucopeptide was increased by d-serine, and this accumulation was reduced by d-alanine. d-Serine competed with glycine for incorporation into the cell wall mucopeptide. The incorporation of l-aspartic acid into diaminopimelic acid residues in the cell wall mucopeptide was markedly inhibited by d-serine. Three mutants resistant to d-serine were isolated by nitrosoguanidine treatment. In these mutants the effects of d-serine on the sites of cell wall mucopeptide synthesis were all reduced. Thus, d-serine inhibition of the growth is due to replacement of glycine residues of the cell wall mucopeptide with d-serine and inhibition of the cell wall synthesis by blocking the formation of d-alanine and diaminopimelic acid.
The transport of D-alanine, D-glutamic acid, and D-valine in Mycobacterium smegmatis was compared quantitatively with that of their L-isomers. It appeared that the uptake of D-alanine was mediated by an active process displaying saturation kinetics characteristic of enzyme function, whereas the uptake of D-glutamic acid was accomplished by a passive process showing diffusion kinetics. Both processes were involved in the uptake of L-alanine, L-glutamic acid, D-valine, and L-valine. D-Valine competed with L-valine for entry into the cell through a single active process. D-Alanine and L-alanine also utilized the same active process, but the D-isomer could not enter the cell through the passive process. The passive process exhibited characteristics of diffusion, but was sensitive to sulfhydryl-blocking reagents and showed competition among structurally related amino acids. These last findings suggested that the passive process is a facilitated diffusion.
L-Aspartic acid was taken up by Mycobacterium smegmatis cells at a greater rate than D-aspartic acid. Kinetic studies of L-aspartic acid uptake revealed a curvilinear Lineweaver-Burk plot. The observed kinetics could be accounted for by the existence oftwo different processes, a saturable and a nonsaturable. In contrast, the latter process was solely active for D-aspartic acid uptake. Competition studies indicated that aspartic acid and glutamic acid were similarly affected by the operation of 2 different processes. The existence of 2 similar catalytic transport processes, specific for dicarboxylic amino acids, was discussed.In the previous paper [17], the transport of D-alanine, D-glutamic acid, and D-valine in Mycobacteriuin smegmatis was compared quantitatively with that of their L-isomers. It was found that for these amino acids the transport of the L-isomers exceeded that of the D-isomers. In addition, evidence for the existence of 2 kinetically different transport processes for these amino acids was presented. One of these 2 processes displayed saturation kinetics characteristic of an enzyme reaction that is assumed to reflect the involvement of permease [6], and the other exhibited linear, nonsaturation kinetics which was sensitive to sulfhydrylblocking agents and showed competition among several amino acids. The present paper concerns the transport of D-and Laspartic acid in M. smegmatis ATCC 607 and the characterization of the nonsaturation process involved in the transport of Dand L-aspartic acid.
MATERIALS AND METHODS
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