A new chemically defined medium for the growth of group A streptococci has been formulated. The advantages of this new medium over previously described defined media are: (i) rates of growth (i.e., doubling times) of 20 strains were comparable to the rates of growth in complex media; (ii) each strain grew to a higher culture density in the new defined medium than in complex media; (iii) transfer from complex media with small inocula was possible without any prior adaptation regimen; and (iv) the production of virulence factors (i.e., M protein and hyaluronic acid) and extracellular enzymes during growth in this new medium was comparable to that in complex media.

The penicillin-binding proteins (PBPs) are a set of enzymes responsible for the terminal stages of peptidoglycan biosynthesis, where they carry out transpeptidation, transglycosylation, endopeptidase, or carboxypeptidase functions. It is the transpeptidation, endopeptidase, and carboxypeptidase functions that are inhibited when these proteins are acylated by a ␤-lactam antibiotic (10). In all cases where this reaction has been investigated biochemically, the ␤-lactam ring opens and forms a covalent bond with an active-site serine residue on the PBP (10, 11). Among the most extensively investigated PBPs are those of the gram-negative bacillus Escherichia coli, and these have been the subject of numerous publications and reviews. It is for E. coli that we have perhaps the clearest understanding of the roles that the individual PBPs perform in peptidoglycan synthesis and cell morphogenesis (7,19,21). At the same time, however, much remains to be understood concerning the role of the PBPs in cell wall growth and their participation in cell division.More than 18 years ago, Spratt published an estimate of the numbers of individual PBPs in an average E. coli cell (25). In this seminal work, the PBPs of isolated cell membranes were labeled with [ 14 C]penicillin and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After electrophoresis, the radiolabeled PBPs were detected by fluorography. The numbers of PBPs per cell were calculated by scanning fluorographs, obtaining the areas under the curves and calculating a percentage distribution for each of the individual PBPs. This relative distribution was then combined with a literature value for the total amount of [ 14 C]cefoxitin bound per milligram (dry weight) and a literature value for cell number per milligram (dry weight) to arrive at absolute values of individual PBP numbers per cell (20,25). Although this was clearly meant as a rough estimate, this set of numbers has been cited repeatedly in numerous publications and several textbooks since that time (1,7,19,21,26).Because of the importance of these proteins in ␤-lactam antibiotic action, cell wall assembly, and cell division, we decided to measure the numbers of the individual PBPs of E. coli by a direct method. In the present study, the numbers of molecules per cell of the individual PBPs were calculated directly by measuring both the cell numbers present and the radioactivity in the individual PBPs from a precise quantity of cells. Care was taken to ensure that the individual PBPs were saturated with [ 3 H]penicillin, and a sufficiently large number of samples were taken in separate experiments to allow statistical assessment of the data sets. The results obtained in the present study are in some cases significantly different from the previously published values. In addition, measurements were performed with both rapidly growing cells in Luria broth (LB) medium as well as with cells growing in M9 minimal medium. The absolute numbers of PBPs were reduced in the cells growing at a lower ...

Streptococcus faecalis 39-5 is a haemolytic, bacteriocinogenic strain harbouring six plasmids. One of these plasmids, pPD1 (36.4 MDal) determines a bacteriocin and encodes a conjugative response to the sex pheromone cPD1 excreted by recipient (plasmid-free) strains. The pheromone response is characterized by the formation of mating aggregates of donors (responders) with recipients. Aggregation required the presence of phosphate and divalent cations and was inhibited by agents or conditions that destroy protein structure. Aggregation was postulated to be due to synthesis of a new proteinaceous molecule on the donor cell surface. Referred to as 'aggregation substance', such a material was identified and found to exhibit antigenic properties not associated with uninduced cells; it could be detected by immunoelectron microscopy. Aggregation substance could be extracted from induced cells but not uninduced cells as demonstrated by crossed immunoelectrophoresis. Antibody raised against the aggregation substance controlled by pPD1 cross-reacted with aggregation substance determined by other plasmid systems which respond to pheromones unrelated to cPD1.

Bacteria. The 853 isolates of gram-negative species and 296 isolates of gramn-positive species were predominantly of recent clinical origin from numerous sources of broad geographical distribution. The isolates were stored as described previously (2, 4). The 3-lactamase-producing strains used to generate the data in Table 3

The conjugative transfer of the Streptococcus faecalis plasmid pADl is characterized by a 10,000-fold increase in frequency following sex pheromone (cAD1) induction. Before the increase in plasmid transfer, donor cells synthesize a proteinaceous adhesin that facilitates the formation of mating aggregates. Four novel surface proteins appearing after exposure of pADl-containing cells to sex pheromone have been identified. Thirty minutes after induction, a 130-kilodalton (kDa) protein was detectable by Western blotting. A 74-kDa protein, the major species present, and a pair of bands at 153 and 157 kDa were evident 45 min after induction. Induced cells containing another conjugative S. faecalis plasmid, pPD1, gave rise to three high-molecularweight proteins of the same size (130, 153, and 157 kDa) as those synthesized by pADI-containing cells. These proteins cross-reacted with antisera raised against induced cells containing pAD1. However, the major protein species produced by pPD1-containing cells had a molecular weight of 78,000 and did not cross-react significantly with the corresponding band of the pADl system. Pheromone-induced transfer of the two plasmids, when both were present in the same cell, was independent; induction was limited to the pheromone-specified plasmid. The possibility that lipoteichoic acid might act as a receptor (binding substance) for the induced adhesin protein was also explored. Free lipoteichoic acid (isolated from S. faecalis) inhibited clumping of induced cells, apparently by acting as a competitive inhibitor of the cellular binding substance.Transfer of certain conjugative plasmids in Streptococcus faecalis is induced by a system of sex pheromones (8, 9). Potential recipient cells excrete small peptides which elicit a mating response in exposed donor cells. The pheromones are plasmid specific; when a cell acquires a certain plasmid, it shuts off the corresponding pheromone activity, but continues producing pheromones specific for different S. faecalis plasmids (7, 9). Exposure of donor cells to a pheromone for 90 min increases the frequency of transfer of resident plasmid DNA by about 4 orders of magnitude over that of the uninduced state (8,9). This increase in capacity to donate DNA, which is chloramphenicol and rifampin sensitive, can be separated into two phases: surface changes, allowing donors and recipients to aggregate into clumps, and changes which allow the plasmid DNA to be transferred between the two cells (4; for a review, see reference 6).The substance induced on the donor cell surface has been termed aggregation substance. This component, it is postulated, binds to a cell surface receptor, called binding substance. (Binding substance is presumed to be present on donors, as well as recipients, since self-clumping occurs upon induction by filtrates of recipients; the latter phenomenon is the basis of the pheromone assay.) The binding of the two surface determinants is dependent on the presence of divalent cations and phosphate (25). The formation of aggregates by induce...