Initiation of wall assembly sites in Streptococcus faecium.

In electron micrographs of replicas of Streptococcus faecium, sites of wall growth are located between pairs of raised equatorial bands. Analysis of cells taken from cultures with mass doubling times between 30 and 125 min indicates that rounds of wall synthesis are initiated at a time close to division, which is temporally unrelated to the initiation or termination of chromosome replication. Growth sites are initiated at a relatively constant volume independent of growth rate when the volume contained within the two segments of wall adjoining an equatorial band marker approaches ca. 0.26 micrometer 3.     

Induction of autolysis in Streptococcus faecium

Autolysis of exponential-phase Streptococcus faecium cells was promoted by pretreating the bacteria (freezing-thawing; -70 degrees C) in Tris buffer, followed by incubation at 37 degrees C in the same buffer. The effect was dependent on Tris concentration. The pretreatment provoked ultrastructurally visible damage with extensive loss of K+ and leakage of UV-absorbing components. No autolysis was observed when the bacteria frozen-thawed in Tris were incubated in the presence of the autolysin inhibitor N-bromosuccinimide nor when they had been grown in the presence of chloramphenicol or tetracycline. Furthermore, two autolytic-defective mutants, EC31 and EC78, isolated from S. faecium, did not autolyse when frozen-thawed and incubated in Tris. Freezing-thawing in Tris, however, imparted extensive cell damage to the mutants and to the antibiotic-treated bacteria as well as considerable leakage of K+ and UV-absorbing materials. These observations indicate that the lysis of S. faecium reported above is due to the activity of the endogenous bacterial autolysin. Induction of autolysis of S. faecium by freezing-thawing was also observed, although to a lesser extent, when Tris was replaced by imidazole.     

Choline-containing bacteriophage receptors in Streptococcus pneumoniae.

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.     

Streptococcus faecium var. casselifavus, nov. var

Streptococcus faecium var. casseliflavus is a gram-positive, spherical cell. The cells occur chiefly as pairs within chains and elongate to ogive-shaped cells during growth. Growth is good on 5% bile salts-agar and in broth at 10 C, and in broth adjusted to pH 9.6 or containing 6.5% NaCl, but many strains fail to grow at 45 C. Litmus is reduced rapidly prior to formation of an acid curd. Few strains release ammonia from arginine or serine. The organism is not proteolytic and does not produce H(2)S or acetylmethylcarbinol, reduce nitrate, decarboxylate tyrosine, or produce slime on sucrose-agar. Most strains survive heating to 60 C for 30 min. It produces gray colonies on potassium tellurite agar, reduces 2,3,5-triphenyltetrazolium-HCl to a pink color, and ferments cellobiose, dextrin, maltose, mannose, and sorbitol, thus resembling S. faecalis. Like S. faecium, it produces peroxidase but not catalase on heated blood media, dissimilates malate, and ferments arabinose, melibiose, and salicin, but not melezitose. Like both species, it ferments dextrose, galactose, lactose, mannitol, sucrose, trehalose, and citrate. Properties peculiar to the variant include the high pH limiting initiation and termination of growth; the fermentation of alpha-methyl-d-glucoside, raffinose, and xylose; motility; and growth without blue button formation in ethyl violet broth. The water-soluble, pale lemon-yellow pigment is released into the aqueous phase only after the cell envelope is altered by fat solvents. The bacterium thrives as an epiphyte on plants.     

Cellular autolytic activity in synchronized populations of Streptococcus faecium.

The autolytic capacity of Streptococcus faecium (S. faecalis ATCC 9790) varied during synchronous cell division. This phenomenon was initially observed in rapidly dividing populations (TD=30 to 33 min) synchronized by a combination of induction and size selection techniques. To minimize the problems inherent in studies of cells containing overlapping chromosome cycles and possible artifacts generated by induction techniques, the autolytic capacities of slowly dividing populations (TD=60 to 110 min) synchronized by selection only were examined. Although the overall level of cellular autolytic capacity was observed to decline with decreasing growth rate, sharp, periodic fluctuations in cellular autolytic capacity were seen during synchronous growth at all growth rates examined. On the basis of similar patterns of cyclic fluctuations in autolytic capacity of cultures synchronized by (i) selection, (ii) amino acid starvation followed by size selection, and (iii) amino acid starvation followed by inhibition of DNA synthesis, a link of such fluctuations with the cell division cycle has been postulated.     

The ineffectiveness of tobramycin combination therapy in Streptococcus faecium endocarditis.

A patient required mitral valve replacement following ineffective antibiotic treatment of enterococcal endocarditis caused by Streptococcus faecium. Endocarditis had relapsed despite therapy with ampicillin and tobramycin for six weeks. A second relapse had occurred following treatment with penicillin and gentamicin. Initial failure of antibiotic therapy may be related to the known lack of in vitro and in vivo synergy between penicillin and tobramycin against S. faecium. Effective therapy of enterococcal endocarditis requires considerations of bacterial speciation, determination of high-level aminoglycoside resistance, and preferably adequate antibiotic synergy studies to assure effective therapy.     

Alpha-glycerophosphate oxidase in Streptococcus faecium F 24

alpha-Glycerophosphate oxidase, in a strain of Streptococcus faecium, was found to be adaptive to aerated conditions of growth. The enzyme was purified and found to mediate electron transfer from alpha-glycerophosphate to O(2), with the production of stoichiometric concentrations of H(2)O(2) and dihydroxyacetone phosphate. The enzyme is an anionic flavoprotein, with flavine adenine dinucleotide as the apparent prosthetic group. By manometric methods, a K(m) of 6 x 10(-3)m, with reference to substrate concentration, was obtained. An active reduced nicotinamide adenine dinucleotide diaphorase was closely associated with this enzyme in chromatographic mobility on ECTEOLA-cellulose. The purified alpha-glycerophosphate oxidase was not inhibited by KCN, azide, or sulfhydryl reagents, nor was it stimulated by alpha-lipoate, yeast extract, or other supplements.     

Morphological and physiological study of autolytic-defective Streptococcus faecium strains.

Three autolytic-defective mutants of Streptococcus faecium (S. faecalis ATCC 9790) were isolated. All three autolytic-defective mutants exhibited the following properties relative to the parental strain: (i) slower growth rates, especially in chemically defined medium; (ii) decreased rates of cellular autolysis and increased survival after exposure to antibiotics which block cell wall biosynthesis; (iii) decreased rates of cellular autolysis when treated with detergents, suspended in autolysis buffers, or grown in medium lacking essential cell wall precursors; (iv) a reduction in the total level of cellular autolytic enzyme (active plus latent forms of the enzyme); (v) an increased ratio of latent to active forms of autolysin; and (vi) increased levels of both cellular lipoteichoic acid and lipids.     

Lipids and lipoteichoic acid of autolysis-defective Streptococcus faecium strains.

Two of four previously isolated autolysis-defective mutants of Streptococcus faecium (Streptococcus faecalis ATCC 9790) incorporated substantially more [14C]glycerol into lipids and lipoteichoic acid than did the parent strain. Consistent with increased accumulation of lipids and lipoteichoic acid, significantly higher levels of phosphorus were found in the corresponding fractions of the two mutant strains than in the wild type. Although the autolysis-defective mutant strains contained the same assortment of lipids as the wild type, the relative amount of [14C]glycerol incorporated into diphosphatidylglycerol increased, accompanied by a decreased fraction of phosphatidylglycerol. These results suggested that increased cellular content of two types of substances, acylated lipoteichoic acid and lipids (notably diphosphatidylglycerol), which previously had been shown to be potent inhibitors of the N-acetylmuramoylhydrolase of this species, contributed to the autolysis-defective phenotype of these mutants. Consistent with this interpretation are observations that (i) cerulenin inhibition of fatty acid synthesis increased the rates of benzylpenicillin-induced cellular lysis and that (ii) Triton X-100 or Zwittergent 3-14 treatment could reveal the presence of otherwise cryptic but substantial levels of the active form of the autolysin in cells of three of four mutants and of the proteinase-activable latent form in all four mutants.     

Transfection of Streptococcus pneumoniae with bacteriophage DNA.

It was possible to transfect Streptococcus pneumoniae with DNA obtained from a newly isolated bacteriophage, diplophage-4 (Dp-4). Optimal frequency of transfection (0.9%) required the use of a nuclease-defective mutant; with wild-type bacteria, the transfection frequency was about 100-fold lower. Transfection requires physiological conditions that appear to be similar to the competent state needed for genetic transformation (A. Tomasz, J. Bacteriol. 91:1050--1061, 1966).     

Purification of Streptococcus group C bacteriophage lysin.

A simple procedure for the purification of Streptococcus group C phage lysin to apparent homogeneity is described. The electrophoretically pure, enzymatically stable polypeptide of 98,000 molecular weight converted Streptococcus (groups A, F, and H) cells into spheroplasts within 5 min at 0 degrees C or within less than a minute at 37 degrees C.     

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.     

Approximation of the cell cycle in synchronized populations of Streptococcus faecium.

Slowly growing populations (TD = 70 to 80 min) of Streptococcus faecium (S. faecalis ATCC 9790) were synchronized by selection after sucrose gradient fractionation. The cell cycle was approximated by correlating the patterns of DNA accumulation and cell division. More specifically, the beginning of cell cycle was equated with the beginning of a rapid linear increase in DNA accumulation. The DNA content of the culture approximately doubled during the period of accumulation, which lasted about 51 min. The period of rapid DNA accumulation, was followed by a period of reduced accumulation that lasted about 24 min. During synchronized growth, cell numbers increased rapidly in coordination with the period of rapid DNA accumulation and exhibited a plateau during the period of reduced DNA accumulation. In contrast, RNA and protein appeared to accumulate exponentially throughout the cell cycle at the same rate as culture mass.     

Activities of Arginine Dihydrolase and Phosphatase in Streptococcus faecalis and Streptococcus faecium

Strains of Streptococcus faecalis and S. faecium are known to produce ammonia from arginine, but only S. faecalis couples the adenosine triphosphate (ATP) produced through the arginine dihydrolase pathway to growth processes. The specific activities of the arginine dihydrolase enzymes were found to be much lower in S. faecium (0.01 to 0.10) than in S. faecalis (0.24 to 1.60). Phosphatase activities in both strains were similar (up to 0.11), but equaled or exceeded the activities of the arginine dihydrolase enzymes in S. faecium. The failure of S. faecium to show increased growth in arginine media is explained on the basis of low activities of the arginine dihydrolase enzymes coupled with sufficient phosphatase activity to negate any benefit from ATP formed.     

Purification and properties of the pyrrolidonecarboxylate peptidase of Streptococcus faecium.

Pyrrolidonecarboxylate peptidase (EC 3.4.11.8) from Streptococcus faecium was purified by fractionation with streptomycin sulphate and ammonium sulphate, by chromatography on Sephadex G200 and DEAE-cellulose, and by preparative electrophoresis on Sephadex G25. The purified enzyme on acrylamide gel showed a strong protein band which contained enzyme activity and a very faint band which had no activity. The subunit molecular weight of the purified enzyme was estimated by acrylamide gel electrophoresis in sodium dodecyl sulphate to be 42,000 +/- 1,000. The enzyme showed optimum activity at pH 7.6 and was unstable in the absence of 2-mercaptoethanol. The sensitivity of the enzyme to alkylating agents (N-ethylmaleimide and iodoacetamide) suggested that free sulphydryl groups were essential for enzyme activity. The enzyme was rapidly inactivated above 45 degrees C. The values of the Michaelis constants (Km) obtained with various L-pyrrolidonecarboxylyl dipeptides were similar although there was a 10-fold range in the maximal rates of hydrolysis of these substrates. Inhibition studies showed that the substrate analogues 2-pyrrolidone and pyrrolidonecarboxylate are competitive inhibitors of the enzyme. The binding of substrates and inhibitors to the active site of the enzyme is discussed.     

Regulation and function of lactate oxidation in Streptococcus faecium

Regulation of the synthesis and function of an l(+)-specific lactate-oxidizing enzyme system found in a homofermentative Streptococcus was investigated. With the exception of fructose, aerobic growth at the expense of a variety of substrates resulted in the formation of a lactate oxidation system; anaerobic growth resulted in a marked reduction or complete loss of lactate-oxidizing activity. Growth on fructose, under aerobic and anaerobic conditions, invariably produced a decrease in the activity of the lactate oxidation system. A negative control, activated by an early intermediate product of glycolysis, appeared to be responsible for repression of the lactate-oxidizing enzyme(s). The enzyme system confers upon the organism the ability to grow aerobically at the expense of l(+)-lactic acid.