Oxacillin-induced inhibition of protein and RNA synthesis in a tolerant Staphylococcus aureus isolate.

A clinical isolate of Staphylococcus aureus was found to be tolerant (MBC much greater than 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.

Effects of low penicillin concentrations on cell morphology and on peptidoglycan and protein synthesis in a tolerant Streptococcus strain.

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 mug/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 mug/ml), but were all greater than and, in fact, were multiples of the minimum inhibitory concentration (0.02 mug/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.

Effects of penicillin on macromolecular synthesis and surface growth of a tolerant streptococcus as studied by computer reconstruction methods.

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.

Conservation of cell wall peptidoglycan by strains of Streptococcus mutans and Streptococcus sanguis.

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 [14C]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.

Inhibition of peptidoglycan, ribonucleic acid, and protein synthesis in tolerant strains of Streptococcus mutans.

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 mug/ml). However, in contrast to some other streptococcal species, growth inhibition was not accompanied by cellular lysis or by a rapid loss of viability. In both strains, synthesis of insoluble cell wall peptidoglycan was very sensitive to Pen G inhibition and responded in a dose-dependent manner to concentrations of about 0.2 and 0.5 mug/ml for strains GS-5 and FA-1, respectively. Higher Pen G concentrations failed to inhibit further either growth or insoluble peptidoglycan assembly. Somewhat surprisingly, Pen G also inhibited both ribonucleic acid (RNA) and protein syntheses, each in a dose-dependent manner. Compared with inhibition of peptidoglycan synthesis, inhibition of RNA and protein syntheses by Pen G was less rapid and less extensive. Maximum amounts of radiolabeled Pen G were specifically bound to intact cells upon exposure to about 0.2 and 0.5 mug/ml of Pen G for strains GS-5 and FA-1, respectively, concentrations consistent with those that resulted in maximum or near-maximum inhibitions of the synthesis of cellular peptidoglycan, RNA, and protein. Five polypeptide bands that had a very high affinity for [(14)C]Pen G were detected in a crude cell envelope preparation of strain FA-1. After exposure of cultures of strain FA-1 to the effects of saturating concentrations of the drug for up to 3 h, addition of penicillinase was followed by recovery of growth after a lag. The length of the lag before regrowth depended on both Pen G concentration and time of exposure. On the basis of these and other observations, it is proposed that the secondary inhibitions of cellular RNA or protein synthesis, or both, are involved in the tolerance of these organisms to lysis and killing by Pen G and other inhibitors of insoluble peptidoglycan assembly.

Does penicillin kill bacteria?.

The thesis is presented that the bactericidal action of penicillin and of other inhibitors of cell wall peptidoglycan synthesis, such as vancomycin and cycloserine, is secondary or tertiary to their ability inhibit specific reactions in the assembly of an osmotically protective cell wall. Examples are given of the inhibition of these reactions, which results in inhibition of cell growth (bacteriostatic action) in the absence of either cellular lysis or rapid loss of viability. Thus, in some instances, inhibitory concentrations of these drugs are, in effect, sublethal; this is true, for example, for Streptococcus mutans, a species of bacteria that is part of the normal flora of the oropharynx and that can cause subacute bacterial endocarditis. On the other hand, the damaging effects of the subminimal inhibitory concentrations of penicillin G on Streptococcus faecalis, a species with an active autolytic enzyme system, can be uncovered and converted to a lytic (and lethal) response by partial inhibition of fatty acid synthesis with low concentrations of cerulenin. Some theoretical and practical implications of the occurrence and inhibition of these secondary lethal consequences are discussed.

Requirement of deoxyribonucleic acid synthesis for microcycle sporulation in Bacillus megaterium.

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.