Chromosome and cell wall segregation in Streptococcus faecium ATCC 9790.

Segregation was studied by measuring the positions of autoradiographic grain clusters in chains formed from single cells containing on average less than one radiolabeled chromosome strand. The degree to which chromosomal and cell wall material cosegregated was quantified by using the methods of S. Cooper and M. Weinberger, dividing the number of chains labeled at the middle. This analysis indicated that in contrast to chromosomal segregation in Escherichia coli and, in some studies, to that in gram-positive rods, chromosomal segregation in Streptococcus faecium was slightly nonrandom and did not vary with growth rate. Results were not significantly affected by strand exchange. In contrast, labeled cell wall segregated predominantly nonrandomly.     

Interactions between beta-lactam antibiotics and isolated membranes of Streptococcus faecalis ATCC 9790.

The DD-carboxypeptidase-exchange membrane-bound enzyme in Streptococcus faecalis ATCC 9790 reacts with beta-lactam antibiotics to form complexes with rather long half-lives. Depending upon the antibiotic, the second-order rate constants for complex formation range from 0.75-560 M-1 S-1 (at 37 degrees C and in water) and the first-order rate constants for complex breakdown range from 1.3 to 26 x 10(-5) s-1 (at 37 degrees C and in 5 mM phosphate buffer pH 7.5). There are about 30 pmol of DD-carboxypeptidase-exchange enzyme per mg of membrane protein. The degradation products arising from benzylpenicillin are phenylacetylglycine and probably N-formyl-D-penicillamine. Isolated membranes also contain other penicillin binding sites (about 70 pmol/mg membrane protein). That part of benzylpenicillin which reacts with at least some of these latter sites is slowly degraded into penicilloic acid. Normal functioning of the DD-carboxypeptidase-exchange membrane-bound enzyme is important, if not essential, for cell growth. With the beta-lactam antibiotics tested inhibition of cell growth is mainly related to the rates of formation of the inactive enzyme-antibiotic complexes. The relationship, however, is not a direct one probably due to the competitive effect exerted by the other penicillin binding sites.     

Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790.

Cultures of Enterococcus faecium growing at various rates were examined for timing of cell division cycle events by using the method of residual divisions and a morphological analysis. Both methods gave essentially the same timing for the onset of D1 (completion of chromosome replication) and of D2 (completion of septation). Frequencies of cells exhibiting a phase-reversed center in bovine serum albumin at various growth rates were determined. The data fit a model in which rapidly growing cells increase in refractive index (which is assumed to represent central density) at completion of the chromosome replication cycle involved in the ongoing division, whereas slowly growing cultures increase in central density at the time of completion of septation. There was no correlation between the timing of increase in central density and the timing of initiation of new sites of surface growth.     

Variation between penicillin-tolerant and penicillin-sensitive states in Streptococcus faecium ATCC9790

Abstract Lineage studies of single-colony isolates from the ATCC9790 strain of Streptococcus faecium are consistent with a reversible high-frequency variation between a penicillin-tolerant and a penicillin-sensitive state. Tolerant and sensitive derivatives have been partially characterized.     

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin.

A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a beta-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a [14C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.     

Division blocks in temperature-sensitive mutants of Streptococcus faecium (S. faecalis ATCC 9790).

Two hundred nine temperature-sensitive growth or division (or both) mutants of Streptococcus faecium ATCC 9790 were isolated. These strains were examined for timing of the division block in the cell division cycle. About 42% of the isolates were blocked at terminal stages of cell division. A second large group appeared to be blocked at various stages of septation. Only five of the temperature-sensitive isolates were blocked at a stage before the completion of chromosome replication. Thirty temperature-sensitive isolates lysed after one or more doublings at the nonpermissive temperature.     

Properties of cell wall-associated DD-carboxypeptidase of Enterococcus hirae (Streptococcus faecium) ATCC 9790 extracted with alkali.

DD-Carboxypeptidase (DD-CPase) activity of Enterococcus hirae (Streptococcus faecium) ATCC 9790 was extracted from intact bacteria and from the insoluble residue (crude cell wall fraction) of mechanically disrupted bacteria by a brief treatment at pH 10.0 (10 mM glycine-NaOH) at 0 degrees C or by extraction with any of several detergents. Extractions with high salt concentrations failed to remove DD-CPase activity from the crude wall fraction. In contrast to N-acetylmuramoylhydrolase (both muramidase 2 and muramidase 1) activities, DD-CPase activity failed to bind to insoluble cell walls or peptidoglycan matrices. Thus, whereas muramidase 1 and muramidase 2 activities can be considered to be cell wall proteins, the bulk of the data are consistent with the interpretation that the DD-CPase of this species is a membrane protein that is sometimes found in the cell wall fraction, presumably because of hydrophobic interactions with other proteins and cell wall polymers. The binding of [14C]penicillin to penicillin-binding protein 6 (43 kilodaltons) was proportional to DD-CPase activity. Kinetic parameters were also consistent with the presence of only one DD-CPase (penicillin-binding protein 6) in E. hirae.     

Variation in buoyant density of whole cells and isolated cell walls of Streptococcus faecium (ATCC 9790).

The buoyant density of whole cells of Streptococcus faecium varies with growth rate and during the cell cycle. Two possible explanations for this were explored: (i) the density of cell walls may vary, and (ii) the proportions of wall and cytoplasm may vary. We tested the first possibility by isolating walls from chilled, unfixed populations of S. faecium cells and fractionating them on Percoll density gradients. Mean cell wall density averaged 4% less than whole-cell density and did not vary significantly with growth rate. In addition, walls isolated from heavy and light fractions of a population of cells did not differ significantly in density. Thus, variation in the density of isolated cell walls could not account for the observed variation in whole-cell density within or between populations. Using previously published measurements of the physical dimensions of S. faecium cells, we also found that the relative proportions of wall and cytoplasm (see the second possibility above) could not account for the observed changes in whole-cell buoyant density.     

Enzymatic deacylation of lipoteichoic acid by protoplasts of Streptococcus faecium (Streptococcus faecalis ATCC 9790).

High-molecular-weight, micellar lipoteichoic acid (LTA) was converted to a lower-molecular-weight, apparently deacylated polymer when the former was incubated in the presence of growing protoplasts of Streptococcus faecium (S. faecalis ATCC 9790), but not when incubated in fresh or spent protoplast medium. The mobility of the low-molecular-weight polymer upon agarose gel electrophoresis was indistinguishable from that of native extracellular lipoteichoic acid LTA(X) from this organism or from chemically deacylated LTA. Native LTA(X) was shown to contain less than one fatty acid equivalent per 18 LTA(X) molecules, in contrast to the 4:1 ratio of fatty acids to polyglycerolphosphate chains in micellar LTA.     

Dodecylglycerol. A new type of antibacterial agent which stimulates autolysin activity in Streptococcus faecium ATCC 9790

Dodecylglycerol has a minimum inhibitory concentration of 4 micrograms/ml compared to 9 micrograms/ml for monolaurin (dodecanoylglycerol) with Streptococcus faecium ATCC 9790 as the test organism. The greater potency of dodecylglycerol can be correlated to its greater retention by the cell. Gram-positive bacteria were more susceptible than Gram-negative bacteria to dodecylglycerol. The antibacterial action of dodecylglycerol is not through the physical dissolution of cell walls, but rather as an enzymatic effector. The autolysin activity of whole cells of S. faecium was greatly stimulated by dodecylglycerol. The stimulation of autolytic activity and inhibition of growth respond in parallel to different concentrations of dodecylglycerol, to dodecylglycerol versus some poorer effector such as monolaurin or a glycerol alkyl ether with a longer or shorter fatty alkyl side chain than dodecanol, and to the antagonistic effects of diphosphatidlyglycerol. This close relationship implies that the stimulation of autolysin activity could be a primary, but not necessarily the only, mechanism by which dodecylglycerol and related compounds exert their antibacterial activity. However, the autolysin activity is not stimulated by a direct interaction between the enzyme and dodecylglycerol. A non-wall entity, such as a proteinase, has been implicated as an intermediary (Ved, H. S., Gustow, E., and Pieringer, R. A. (1984) J. Biol. Chem. 259, 8122-8124).     

Mode of elongation of the glycerol phosphate polymer of membrane lipoteichoic acid of Streptococcus faecium ATCC 9790.

Specific degradation of membrane lipoteichoic acid of Streptococcus faecium ATCC 9790 by a phosphodiesterase from Aspergillus niger and by periodate oxidation has demonstrated that the enzymatic synthesis of the glycerol phosphate polymer of the molecule occurs by an external elongation system. Evidence of this type of mechanism was obtained with lipoteichoic acid synthesized in vivo or in vitro by differential radioisotope labeling techniques. The glycerol phosphate repeating units were transferred from phosphatidylglycerol and became linked through a phosphodiester bond to the glycerol phosphate unit of the chain farthest from or most external to the lipid end of the polymer.     

The involvement of the proteinase of Streptococcus faecium ATCC 9790 in the stimulation of its autolysin activity by dodecylglycerol

Treatment of Streptococcus faecium ATCC 9790 with 3.5 micrograms/ml of dodecylglycerol produces a nonwall entity found in the 25,000 X g supernatant cell fraction which activates the autolysin activity of S. faecium. The stimulation of the autolysin activity by dodecylglycerol mimics the activation of the autolysin from a latent to an active form by trypsin and other proteolytic enzymes. This stimulation of autolytic activity by dodecylglycerol can be reversed by specific proteinase inhibitors. Dodecylglycerol also markedly stimulates the proteinase activity endogenous to S. faecium, and this stimulation can be reversed by several proteinase inhibitors. It is concluded that one primary antibacterial mode of action of dodecylglycerol is to stimulate the proteinase of S. faecium which activates the cell's autolysin and thereby prevents bacterial growth.     

Inhibition of peptidoglycan synthesis ofStreptococcus faecium ATCC 9790 andStreptococcus mutans BHT by the antibacterial agent dodecyl glycerol

Dodecyl glycerol inhibits the synthesis of the peptidoglycans ofStreptococcus faecium ATCC 9790 andStreptococcus mutans BHT. This metabolic regulation represents the second known mode by which dodecyl glycerol expresses antibacterial activity. The first mode of action of dodecyl glycerol was shown to stimulate autolysin activity which degrades cell-wall peptidoglycan (Ved HS, Gustow E, Mahadevan V and Pieringer RA, 1984, J. Biol. Chem.259, 8115–8121).     

Autoradiographic studies of chromosome replication during the cell cycle of Streptococcus faecium.

Analysis of the distribution of autoradiographic grains around cells of Streptococcus faecium which had been either continuously or pulse-labeled with tritiated thymidine (mass doubling time, 90 min) showed a non-Poisson distribution even when the distribution of cell sizes in the populations studied was taken into account. These non-Poisson distributions of grains were assumed to reflect the discontinuous nature of chromosome replication. To study this discontinuous process further, we fitted an equation to the grain distribution observed for the pulse-labeled cells that assumed that in any population of cells there were subpopulations in which there were zero, one, or two replicating chromosomes. This analysis predicted an average time for chromosome replication and for the period between completion of rounds of chromosome replication and division of 55 and 43 min, respectively, which were in excellent agreement with estimates made by other techniques. The present investigation extended past studies in indicating that the initiation and completion of rounds of chromosome replication are poorly phased with increases in cell volume and that the amount of chromosome replication may be different in different cell halves.     

Buoyant density, growth rate, and the cell cycle in Streptococcus faecium.

The buoyant density in rapidly growing Streptococcus faecium 9790 cells varies over the cell cycle, in contrast to the density in Escherichia coli. Buoyant density in S. faecium was measured by using Percoll (Pharmacia Fine Chemicals, Piscataway, N.J.) density gradients. We found that the mean and coefficient of variation of the population density increased with growth rate; and within a population, the mean cell volume, which was measured electronically, increased with density. These results were compared with electron microscopic measurements of the size distributions of cell wall growth sites within each fraction of the density gradient. As the density increased within a population, the frequency of large cells increased and the frequency of newly initiated cell wall growth sites increased. These effects were more marked as the growth rate increased. Next, these data were regrouped by cell size by using the size of the central growth site as an index of cell cycle stage. Each frequency value was weighted by the proportion of the population represented by that density fraction. Then, the average buoyant density was calculated for each value of cell size. In all cell populations, the density decreased and then increased as the central site enlarged. Peripheral growth sites were initiated as density reached a maximum. At faster growth rates, density increased more steeply, and new peripheral growth sites opened up at a higher frequency. We suggest that the rate at which density increases during the cell cycle correlates with the initiation of new cell wall growth sites.     

Relationship of shape to initiation of new sites of envelope growth in Streptococcus faecium cells treated with beta-lactam antibiotics.

Exponential-phase cells of Streptococcus faecium were treated with concentrations of ampicillin and cephalothin which, over 60 min, had little effect on increase in culture mass but resulted in about a 65% inhibition of increase in cell numbers. The resulting drug-treated cells underwent about a doubling in cell mass and volume above that of the untreated cells. The newly divided cells produced in the presence of drugs were shown to be due to the division of central or primary sites of envelope growth present at the time of treatment. Sites that were newly initiated (secondary sites) at the time of treatment or sites initiated after treatment did not divide but enlarged in length and girth to give abnormally large cells. Although the increase in average total volume was the same after each interval of treatment with ampicillin and cephalothin, the primary growth sites of the cephalothin-treated cells grew somewhat more slowly, and their secondary sites grew somewhat more quickly, than did those of the ampicillin-treated cells. Cephalothin-treated cells initiated secondary sites at a rate similar to that of the untreated cells, whereas the ampicillin-treated cells exhibited reduced rates of secondary site initiation. Two models are presented that account for these results.     

Solubilization and isolation of the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC9790. Properties of the purified enzyme.

Streptococcus faecalis ATCC 9790 possesses six membrane-bound, penicillin-binding proteins. That numbered 6 (Mr 43000) is the most abundant one and is the DD-carboxypeptidase studied previously. The enzyme has been solubilized and purified to the stage where one single protein band can be detected by gel electrophoresis. The purification procedure does not alter the properties that the enzyme exhibits when it is membrane-bound. The DD-carboxypeptidase itself may be a killing target for penicillin in S. faecalis.     

Inhibition of peptidoglycan biosynthesis at a postcytoplasmic reaction in a stable L-phase variant of Streptococcus faecium.

Cultures of a stable L-phase variant of Streptococcus faecium F24 produced and retained peptidoglycan precursors intracellularly over the entire growth cycle in a chemically defined medium. The identity of the most abundant precursor, UDP N-acetylmuramyl-L-alanyl-D-glutamyl-L-lysyl-D-alanyl-D-alanine (UDP-MurNAc-pentapeptide), was confirmed by demonstrating in vitro the presence of enzymes required for the cytoplasmic stage of peptidoglycan biosynthesis. The initial membrane-bound reaction in peptidoglycan biosynthesis involving phospho-MurNAc-pentapeptide translocase and undecaprenyl-phosphate membrane carrier was catalyzed by protoplast membrane preparations but not by L-phase membrane preparations. However, both protoplast and L-phase membranes incorporated radioactivity from dTDP-L-[14C]rhamnose, the presumed precursor to a non-peptidoglycan cell surface component, into high-molecular-weight material. dTDP-L-rhamnose did not accumulate in growing cultures but was synthesized from D-glucose-1-phosphate and dTTP by cell-free extracts of the streptococcus and L-phase variant. Neither rhamnose- nor muramic acid-containing compounds were detected in culture fluids. It is suggested that continued inhibition of cell wall biosynthesis in this stable L-phase variant is the result of a defect expressed at the membrane stage of peptidoglycan biosynthesis specifically involving the translocation step.     

Cell cycle changes in the buoyant density of exponential-phase cells of Streptococcus faecium.

Cell buoyant densities were determined by centrifugation in Percoll gradients containing exponential-phase cells of Streptococcus faecium ATCC 9790 grown at a mass doubling time of about 33 min. This bacterium showed the highest average density values (1.13 g/ml) measured to date for any eucaryotic or procaryotic organism. Fractions having the highest densities were enriched with cells that were in the process of dividing or had just divided. These high-density fractions were also enriched with cells that had newly initiated sites of cell wall growth. It appears that S. faecium shows minimum cell densities in the midportion of its cycle.     

Streptococcus faecium ATCC 9790 penicillin-binding proteins and penicillin sensitivity are heavily influenced by growth conditions: proposal for an indirect mechanism of growth inhibition by beta-lactams

The effects of variations in growth conditions on the penicillin response of Streptococcus faecium ATCC 9790 were studied. Changes in the growth temperature and medium composition were found to cause striking changes in the bacterial generation time, cellular penicillin sensitivity (minimum inhibitory concentration), sensitivity of peptidoglycan synthesis to inhibition by penicillin, rate of autolysis, and labeling pattern of penicillin-binding proteins. However, no constant relationship between these parameters and the minimum inhibitory concentration could be observed. Similar electrophoretic patterns for penicillin-binding proteins were observed in cells grown in different media at the optimal growth temperature. Inhibition of cell division by penicillin in cells grown at this temperature (but not at higher or lower temperatures) caused filamentation of the bacteria. In cells grown in a chemically defined medium at the optimal temperature (but not at temperatures above or below), complete inhibition of cell division was associated with only partial inhibition (34% after 150 min) of peptidoglycan synthesis. It is suggested that the status and physiological importance of individual penicillin-binding proteins in S. faecium are heavily influenced by growth conditions. Depending on the growth conditions, different penicillin-binding proteins may perform the cellular function, indispensible for bacterial growth.