Exposure of exponentially growing cultures of Streptococcus mutans strains FA-1 and GS-5 to various concentrations of benzylpenicillin (Pen G) resulted in inhibition of turbidity increases at low concentrations (0.02 to 0.04 ,tg/ml). In many bacterial species, addition of a sufficient concentration of benzylpenicillin (Pen G), or other antibiotic inhibitors of cell wall peptidoglycan (PG) assembly, to prevent further growth of cultures is rapidly followed by cell death, accompanied in many cases by cellular lysis (1,33,34). The availability of bacterial species that are tolerant to both the lethal and the lytic consequences of inhibition of cell wall PG assembly permits studies of the processes that lead to growth inhibition of intact cells without the complications of rapid losses of viability. Furthermore, comparisons of the events occurring in such tolerant species with those occurring in species that are killed by treatment with these drugs will help provide insights into the lytic and other possible mechanisms of lethality. Currently (34), it is clear that the lethal t Present address:

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A clinical isolate of Staphylococcus aureus was found to be tolerant (MBC >> MIC) to a number of beta-lactam antibiotics, including oxacillin. Biophotometric analysis showed that a number of concentrations of oxacillin were capable of stimulating rapid cellular lysis in this organism, but the extent of lysis was antibiotic concentration dependent and limited. Cell cultures treated with an antibiotic concentration yielding the maximum rate and extent of lysis were analyzed for protein and RNA synthesis by pulse-labeling techniques.RNA synthesis was initially stimulated and then severely inhibited. Protein synthesis was not inhibited initially; however, the increase in the rate of synthesis expected as the result of logarithmic growth was not observed. Instead, the antibiotic-treated culture maintained for approximately 50 min the rate of protein synthesis ongoing at the time of antibiotic addition. The rate of protein synthesis declined thereafter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein samples taken 1 and 3 h after antibiotic addition showed that the shutdown of protein synthesis was not coordinate but rather was suggestive of the operation of a stress regulon perhaps similar to those responsible for heat shock, SOS, and oxidation stress.Certain bacteria are so constituted that they resist the killing but not the growth inhibitory action of beta-lactam antibiotics. Standard clinical testing of these organisms shows that the MBC is much greater than the MIC. The biochemical mechanism of this so-called tolerant (12) response of certain strains to normally bactericidal antibiotics is not known; however, one possible explanation suggests the presence, in tolerant strains, of a special form of regulatory "cross talk" such that the almost immediate consequence after inhibition of peptidoglycan synthesis by a beta-lactam antibiotic is the inhibition of protein synthesis (9, 10). This inhibition of protein synthesis was postulated to antagonize whatever lytic signal was sent to the cell wall autolytic system of the tolerant bacterium by the beta-lactam antibiotic in the same way that inhibitors of protein synthesis are known to antagonize. the bactericidal activity of penicillins and cephalosporins (5).In the present study, we looked more closely at the protein synthesis inhibition caused by beta-lactam treatment and asked whether all of the proteins were inhibited in a manner consistent with the operation of a coordinated control mechanism or whether the effect on protein synthesis was specific, such that certain proteins were either enriched or repressed while others were unaffected. MATERIALS AND METHODSOrganism and culture.

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Turnover of the cell wall peptidoglycan fraction of six different strains of Streptococcus mutans and eight different strains of Streptococcus sanguis was examined. Cells were grown in the presence of [3H]lysine and ['4C]leucine for at least eight generations and then chased in growth medium lacking the two labels. At intervals during the chase, samples of cultures were removed, and the amounts of the two labeled precursors remaining in the peptidoglycan and protein fractions were quantitated. Similar experiments were done in which the pulse-labeling technique was used. In addition, cells were labeled in the presence of tetracycline or penicillin, chased with growth medium containing no inhibitor, and assayed at intervals during the chase for the amount of [3H]lysine present in peptidoglycan fractions. Studies of cultures of S. mutans strains FA-1, OMZ-61, OMZ-176, 6715, GS-5, and Ingbritt and of S. sanguis strains 10558, M-5, Wicky, DL-101, DL-1, 71X26, and 71X48 maintained in the exponential phase of growth in a chemically defined medium failed to show evidence of loss of insoluble peptidoglycan via turnover. Similarly, for the strains of S. mutans, insoluble peptidoglycan assembled during 2 h of benzylpenicillin or tetracycline treatment was also conserved during recovery from growth inhibition.

Strains of Streptococcus mutans are very susceptible to growth inhibition by benzylpenicillin, but are tolerant to lysis when exposed to even high concentrations of this drug. These properties enabled this study of S. mutans GS-5 surface growth and peptidoglycan, ribonucleic acid, protein, and deoxyribonucleic acid syntheses in the absence of osmotic stabilization. Inhibition of syntheses of peptidoglycan, ribonucleic acid, and protein was dose dependent. Synthesis of peptidoglycan was most susceptible. Substantial but less severe inhibitions of ribonucleic acid and protein syntheses rapidly followed decreased peptidoglycan synthesis, whereas inhibition of deoxyribonucleic acid synthesis was delayed and minimal. Computer-assisted reconstructions of surface growth zones and poles observed in electron micrographs of replicas were performed and indicated that at low concentrations of benzylpenicillin (0.03 micrograms/ml), growth sites reached abnormally large sizes and surface/volume ratios. The observed shifts in surface/volume ratio were attributed to an inhibition of the normal constrictive division mechanism. The poles of these cells also increased in size over those of the controls, but the relatively smaller change in surface/volume ratio confirmed the visual impression that the shape of the poles was much less altered than the shape of the growth sites. As the concentration of benzylpenicillin used was raised from 0.03 to 2 micrograms/ml, the ability of growth sites and poles to enlarge was restricted in a manner that most closely agreed with the extent of inhibition of peptidoglycan (rather than deoxyribonucleic acid, ribonucleic acid, or protein) synthesis. This correlation suggested that increases in cell size may be regulated by the supply of peptidoglycan precursors.

Bacillus megaterium cells have been examined during outgrowth for their macromolecular content, ability to undergo microcycle sporulation, the time of their growth division, the time of deoxyribonucleic acid (DNA) replication initiation, and their ability to synthesize DNA after transfer to sporulation medium. The increase in total DNA content of the cells increased discontinuously beginning at 90 min. Thymidine incorporation became insensitive to chloramphenicol between 90 and 105 min of outgrowth. At 90 min the cells acquired the ability to undergo microcycle sporulation and the degree of sporulation depended on the time spent in outgrowth, with maximal sporulation occurring at 180 min. During outgrowth, cells underwent one synchronous growth division beginning at 225 min and ending at 270 min. Outgrowing cells were not able to continue DNA synthesis after transfer to sporulation medium. The data suggest that DNA replication starts before cells are able to undergo microcycle sporulation; however, the initiation of replication may not be the only requirement for microcycle sporulation.

Rates of protein and peptidoglycan synthesis were determined by pulse-labeling techniques before and after treatment of exponentially growing cultures of Streptococcus mutans FA-1 with a number of concentrations of penicillin G (0.05, 0.1, 0.3, and 0.4 pg/ml). These penicillin concentrations were all less than that required to saturate the specific penicillin-binding sites present on the surface of this organism (0.5 ug/ml), but were all greater than and, in fact, were multiples of the minimum inhibitory concentration (0.02 ug/ml). Low concentrations of penicillin G (2.5x the minimum inhibitory concentration) immediately halted the exponential increase in the rate of peptidoglycan synthesis normally expected as the result of cell multiplication, but allowed the rate of peptidoglycan synthesis occurring at the time of penicillin addition to be maintained for almost 1 h. An increased penicillin concentration (5x the minimum inhibitory concentration) allowed the rate of peptidoglycan synthesis occurring at the time of penicillin addition to be maintained for a shorter length of time (-0.67 h). Still greater penicillin concentrations caused an immediate inhibition of the peptidoglycan synthetic rate. The effect of penicillin on the rate of protein synthesis was similar, although less pronounced. Samples were taken for scanning electron microscopy immediately before and after 3 h of treatment with a low (2.5x the minimum inhibitory concentration) concentration of penicillin. The surface areas and volumes of the cells in these samples were calculated from the electron micrographs by using computer reconstruction techniques. From the frequency distributions of surface area, the plots of surface area to volume ratio as a function of surface area, and the pulse-labeling data mentioned previously, low, growth-inhibitory concentrations (2.5x the minimum inhibitory concentration) of penicillin are proposed (i) to inhibit the constriction of the division septum, (ii) to prevent the establishment or maturation of new envelope growth sites, and (iii) to have no immediate effects on the synthesis of cell wall peptidoglycan already in progress at the time of penicillin addition.Streptococus mutans FA-1 has recently (18) been described as an organism which is tolerant to the lethal and lytic effects that often follow the inhibition of cell wall peptidoglycan (PG) assembly (1,21,22), although it is highly susceptible to growth inhibition by several antibiotics that inhibit PG synthesis. Penicillin G (Pen G) is a structural analog of D-alanyl-D-alanine (26) and is for this reason thought to inhibit the transpeptidation step of PG assembly, although examples are known oforganisms in which transpeptidation is not susceptible to penicillin inhibition (3, 9).The standard methods used to measure the efficacy with which an antibiotic interferes with bacterial metabolisn have been quantitation of the antibiotic concentration necesary to inhibit growth (minimum inhibitory concentration [MIC]) and the concentration of that antibiotic necess...