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The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.

Streptococcus faecalis strain DS-5 has been shown previously to contain three plasmids, pAMalpha1, pAMbeta1, and pAMgamma1. Mixed incubation of DS-5 with strain JH2-2, a plasmid-free S. faecalis recipient, results in the transfer of pAMalpha1 (which determines resistance to tetracycline) and/or pAMgamma1. Analyses of recipients carrying various combinations of these plasmids have revealed the pAMgamma1 codes for toxin (hemolysin) production and two bacteriocin activities. JH2-2 strains carrying both pAMalpha1 and pAMgamma1, or pAMgamma1 only, can donate their plasmids to other recipients, whereas strains carrying only pAMalpha1 cannot serve as donors. This indicates that pAMgamma1 actually mobilizes the otherwise nontransferable pAMalpha1.

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.

The non-ionic detergent octyl glucoside solubilizes a substantial amount of Streptococcus faecalis membrane protein without loss of the monitored enzyme activities. A secondary detergent, dioctanoyl phophatidycholine, appears to increase the yield of solubilized material. In addition, the effect of ionic strength indicates that it may be possible to selectively extract groups of membrane proteins by their characteristic solubility at different ionic strengths. The solubilized membrane-associated enzymes, ATPase and NADH dehydrogenase, enter polyacrylamide gels as distict species. Electrophoretic studies suggest that there are two membrane-associated ATPase in the Streptococcus faecalis, one which dissociates from the membrane in the absence of Mg-2+ ions and the other which remains particulate until solubilized by detergents. Octyl glucoside can be easily removed from a solution containing solubilized proteins and lipid by dialysis.

1. The occurrence of a novel series of triterpenoid carotenes in Streptococcus faecium UNH 564P is reported. 2. This series, which comprises the C(30) analogues of phytoene, phytofluene, zeta-carotene, 7,8,11,12-tetrahydrolycopene and neurosporene, appears to be analogous to the C(40) Porter-Lincoln series and a pathway of triterpenoid carotene dehydrogenation is proposed to account for the formation of these compounds. 3. Two cis isomers of the C(30) analogue of neurosporene are described. 4. An appropriate system of nomenclature for these novel compounds is proposed.

Strains of Streptococcus faecalis var. zymogenes, designated JH1 and JH3, produced a hemolysin and a bacteriocin. Hemolytic activity was lost from a low percentage of cells grown in broth at either 37 or 45 C. All nonhemolytic (Hly-) variants had lost bacteriocin activity (Ben-), and those from strain JH3 had also lost resistance to the bacteriocin (Bnr-). The majority of Hly-, Ben- variants from JH1 retained bacteriocin resistance (Bnrplus). Strains JH1 and JH3 contained a plasmid deoxyribonucleic acid species of molecular weight 38 times 10-6 (plasmids pJH2 and pJH3, respectively), and strain JH1 also contained a 50 times 10-6 molecular weight plasmid (pJH1) which has previously been shown to carry the genes determining resistance to the antibiotics kanamycin, neomycin, streptomycin, erythromycin, and tetracycline. Hly-, Bcn-, Bnr- variants of strain JH3 had completely lost plasmid pJH3. Hly-, Bcn-, Bnr- variants of strain JH1 had completely lost plasmid pJH2 and retained plasmid pJH1, but Hly-, Bcn-, Bnrplus variants had retained both plasmids pJH2 and pJH1. The Hlyplus, Bcnplus, Bnrplus traits from both parental strains were transferable to nonhemolytic S. faecalis strains during mixed incubation in broth at 37 C, and hemolytic recipient strains were found to have received plasmid pJH2 from strain JH1 and pJH3 from JH3. We conclude that the Hlyplus, Bnrplus traits are borne on plasmid pJH2 in strain JH1 and pJH3 in strain JH3 and that, in Hly-, Bcn-, Bnrplus variants of strain JH1, plasmic pJH2 has suffered a mutation affecting hemolysin and bacteriocin expression. We infer that the plasmids transfer by conjugation. Beta-hemolytic activity is the only property distinguishing the zymogenes variety from S. faecalis. Since we have shown that this activity is plasmid borne in strains JH1 and JH3, we endorse the view that the varietal status of zymogenes should be dropped.

Protoplasts of Streptococcus faecalis ATCC 9790 were produced with the aid of lysozyme, and the ability of these bodies to synthesize soluble, peptide cross-linked peptidoglycan (PG) fragments was examined. Lysozyme digests of PG isolated using gel filtration from the supernatant medium of protoplasts grown in the presence of [14C]acetate and L-[3H]lysine contained small amounts of PG having KD expected for peptide cross-linked dimers and trimers. Addition of benzyl penicillin (300 mug/ml) to growing protoplast cultures did not affect the net amount of PG fragments synthesized but resulted in inhibition of synthesis of dimer and trimer fractions by 27 and 59%, respectively. Failure of penicillin to completely inhibit the accumulation of the dimer fraction was attributed to the presence of atypical forms of dimer. In fact, the supernatant medium of penicillin-treated cultures did not contain detectable amounts of typical peptide cross-linked dimer. The degree of peptide cross-linkage of protoplast PG was at most only 13% of that found in walls isolated from intact streptococci. The relative amounts of monomers, dimers, and trimers synthesized during early and late stages of protoplast growth was approximately the same. Protoplasts synthesized soluble PG fragments in amounts which were of the same order of magnitude as that expected for insoluble PG produced by an equivalent amount of intact streptococci.

1. From Streptococcus faecalis a third novel phosphoglucolipid was isolated. It contained D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 2 : 3 : 4 : 2. 2. The structure was shown to be 3(1)-O-[6 inches-(sn-glycerol-1-phosphoryl), 6 foot-(1,2 diacyl-sn-glycerol-3-phosphoryl)-2 foot-O-(alpha-D-glucopyranosyl)-alpha-D-glucopyranosyl]-1(3),2-diacylglycerol. The novel lipid is related to the earlier described phosphatidyl and glycerophosphoryl kojibiosyl diacylglycerol from Streptococcus faecalis by combininb their substituents in its structure. 3. The structure of the deacylated core was accomplished by analyses of the breakdown products obtained on Smith degradation and strong alkaline hydrolysis. The location of the acyl groups was achieved by degradation of the intact lipid with 60% HF (w/v) which resulted in the formation of inorganic phosphate, glycerol, diacylglycerol and diglucosyl diacylglycerol. The sn-glycerol-1-phosphoryl and 3-sn-phosphatidyl substituents were located by hydrolysis of the lipid with 98% acetic acid (v/v) and subsequent analysis of the resultant phosphatidyl diglucosyl diacylglycerol. 4. The composition and positional distribution of the constituent fatty acids at the two diacylglycerol portions was studied by combination of chemical and enzymatic degradations. The fatty acids are the same as with the other polar lipids of S. faecalis, they are present in nearly the same proportions and display the same non-random distribution pattern. 5. A possible relationship between sn-glycerol 1-phosphate containing phosphoglucolipids and lipoteichoic acid will be discussed.

1. The characterization of two novel triterpenoid xanthophylls occurring in Streptococcus faecium UNH 564P is described. 2. Both are derived formally, and probably biosynthetically, from the C(30) analogue of neurosporene and have been identified as 4-hydroxy-4,4'-diaponeurosporene and its glucoside, 4-d-glucopyranosyloxy-4,4'-diaponeurosporene. 3. Problems associated with the use of specific glucosidases in defining the anomerism of carotenoid glucosides are discussed.

1. A lipid-teichoic acid complex was isolated from Streptococcus faecalis N.C.I.B. 8191. The covalent nature of the linkage between teichoic acid and lipid was established. 2. The complex exhibits macromolecular properties in solution, and ultracentrifugation studies show that these are due to micelle formation. 3. From chemical studies it is concluded that the teichoic acid is a poly(glycerol phosphate) in which some of the glycerol hydroxyl groups possess kojibiosyl [2-O-alpha-d-glucopyranosyl-(1-->2)-alpha-d- glucopyranosyl] substituents, together with d-alanine ester residues. 4. The lipid is 1-kojibiosyl diglyceride, already known as a membrane component of this organism, with probably a phosphatidyl substituent. The phosphatidyl kojibiosyl diglyceride is attached to the teichoic acid through a phosphodiester linkage, and the chain of the teichoic acid contains 28-35 units. 5. Although the complex represents the whole of the membrane teichoic acid in this organism, only about 12% of the membrane glycolipid is associated with teichoic acid. 6. Two phosphatidyl glycolipids, closely resembling that bearing the teichoic acid, were isolated from the lipids of the organism and were partly characterized.

Deoxyribonucleic acid base compositions were determined for 25 Mycoplasma strains and 6 L-phase variant strains. Values obtained correlated well with the results of other investigators.

Biochemical capabilities of bacterial and L-phase organisms of 15 bacterial strains were examined by using a variety of modified routine diagnostic biochemical tests. The results demonstrated that 13 of the tests examined were suitable for use with L-phase variants, that L-phase variants and revertant bacterial phase organisms retained diagnostically significant capabilities of the respective bacterial or L-phase organisms from which they were derived, and that the 13 biochemical tests could be usefully employed to relate a given L-phase variant to a given bacterial phase organism, to distinguish L-phase variants of different species, and to aid in the identification of nonreverting L-phase variants.

Properties relating to the recovery of three heat-injured strains of Streptococcus faecalis were studied. All strains were cultured in all purpose plus Tween broth (APT) at 30 C for 24 hr before being subjected to heat in fresh APT broth. APT recovery medium containing various added amounts of NaCl, KCl, MgCl(2), or KCl and MgCl(2) was used to assess the effect of salts on the recovery of thermally injured S. faecalis. It was evident that, upon exposure to heat, S. faecalis cells became sensitive to increased salt concentrations. Analyses to determine the ribonucleic acid (RNA) content of heated cells showed a reduction of cellular RNA, but the per cent reduction was not directly proportional to the per cent reduction of the viable cells.

Relative fatty acid compositions of 37 enterococci were examined by gas chromatography. Streptococcus faecalis, S. faecium, and S. faecium var. durans yielded similar fatty acid patterns. Strains of S. faecium var. casseliflavus, and a motile yellow-pigmented streptococcus, contained very low levels of C(19:0) cyclopropane fatty acid and four unidentified components, compared to the other strains of enterococci examined. There were no significant differences in the fatty acid patterns of enterococci grouped according to plant, animal, or human source.

A stable L-form of Streptococcus faecalis (T(53)) was transferred in media containing decreasing concentrations of sucrose until it grew in medium without added osmotic stabilizer. This L-form (designated T(53I)) was compared with T(53) from which it was derived. The survival of these two L-forms suspended at different osmolalities showed that maximal survival for T(53I) was 350 to 400 milliosmolal and for T(53) was 900 to 1,000 milliosmolal. Both peaks were at the osmolality of their growth media. Measurement of intracellular potassium and sodium showed that the concentration of these ions was reduced in T(53I).

The amino acid composition of the murein (peptidoglycan) of 75 strains of enterococci was investigated. All strains designated Streptococcus faecalis according to their physiological properties contained the lysine-alanine type of murein; all strains classified as S. faecium contained the lysine-aspartic acid type of murein.

The fatty acid composition of the membranes of three different penicillin-produced L-forms of Streptococcus faecalis was determined: (i) a stable (nonreverting) L-form (T(53)) cultured in brain heart infusion (BHI) with 0.5 M sucrose; (ii) a stable L-form (T(531)) cultured in BHI without sucrose; and (iii) an unstable L-form (T(9)) cultured in BHI with 0.5 M sucrose and 1,000 U of penicillin per ml. L-forms were obtained by centrifugation and lysed by washing in 1 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer. The parent S. faecalis was also cultured in BHI and BHI containing 0.5 M sucrose, and washed with buffer. The fatty acid composition of L-forms of S. faecalis cultured in BHI without sucrose (370 mosmol) had higher C(18:1) and lower C(18) than L-forms cultured in the same media with added 0.5 M sucrose (950 mosmol) in both exponential and stationary cultures. In the stationary phase of growth, C(19) was reduced in the L-forms cultured without sucrose. Similar changes were seen in the parent S. faecalis cultured in the two types of media. These changes in membrane fatty acids may relate to osmo-regulation of the L-forms.

A statistical method for quantifying the relatedness among proteins was used to perform 2926 paired comparisons of amino-acid composition among 77 contractile and membrane-associated proteins from diverse species and sources. Relatedness of amino-acid compositions correlates with homology of amino-acid sequence. A high degree of relatedness was detected among K(+)-dependent membrane ATPase of Streptococcus faecalis, coupling factors F(1) and CF(1) from mitochondria and chloroplasts, outer fiber protein of cilia, ciliary dynein, tubulin, various actins, and myosin subfragment S-1. Heavy meromyosin and tropomyosin were related to each other but not to the first group of proteins. Differences in the degree of methylation may account for some differences in physiological function. Because of their diverse sources, the high degree of relatedness among these proteins is more compatible with evolution from common ancestral genes than with convergent evolution. Squid axon filarin, molluscan paramyosin, and bacterial flagellins appear to be unrelated either to each other or to any of the other proteins studied. Existence of persistent homologies among so many diverse proteins implies conservation of genetic information during evolution by utilization of codons for preferred amino-acid sequences in various proteins.

C(55)-isoprenyl alcohol and its derivatives have been isolated from Streptococcus faecalis and characterized. The relative amounts present as free alcohol, neutral lipid esters, and phosphate ester derivatives were determined. The chain lengths, mass spectra, and cis to trans ratio of double bonds are reported.

Autoradiograms of total lipid extracts from Streptococcus faecalis ATCC 9790, harvested in the stationary phase from a medium containing (32)P-orthophosphate, showed six major spots. The corresponding compounds were identified as diphosphatidylglycerol (possibly with a penta acyl structure); phosphatidylglycerol; a provisionally identified mixture of alanylphosphatidylglycerol and of the 2'-lysyl-derivative of phosphatidylglycerol; the 3'-lysyl-derivative of phosphatidylglycerol, probably together with some arginylphosphatidylglycerol; a diglucosyl derivative of phosphatidylglycerol; and a compound which was tentatively identified as the 2',3'-dilysyl derivative of phosphatidylglycerol.

Intact cells of Streptococcus faecalis and Micrococcus lysodeikticus were found to have high-frequency electric conductivities of 0.90 and 0.68 mho/m, respectively. These measured values, which reflect movements of ions both within the cytoplasm and within the cell wall space, were only about one-third of those calculated on the basis of determinations of the amounts and types of small ions within the cells. Concentrated suspensions of bacteria with damaged membranes showed similarly large disparities between measured and predicted conductivities, whereas the conductivities of diluted suspensions were about equal to predicted values. Thus, the low mobilities of intracellular ions appeared to be interpretable in terms of the physicochemical behavior of electrolytes in concentrated mixtures of small ions and cell polymers. In contrast to the low measured values for conductivity of intact bacteria, values for intracellular osmolality measured by means of a quantitative plasmolysis technique were higher than expected. For example, the plasmolysis threshold for S. faecalis cells indicated an internal osmolality of about 1.0 osmol/kg, compared with a value of only 0.81 osmol/liter of cell water calculated from a knowledge of the cell content and the distribution of small solutes. In all, our results indicate that most of the small ions within vegetative bacterial cells are free to move in an electric field and that they contribute to cytoplasmic osmolality.

The isolation and some properties of two mutants of Streptococcus faecalis ATCC 9790 (S. faecium) which autolyze at a much slower rate than the wild type are described. Compared with the wild type, mutant E71 autolyzed more slowly, contained less active but more latent autolysin in the isolated wall fraction, and possessed a wall of very similar chemical composition and degree of cross-bridging. Ultrastructural studies of exponential phase cells showed that cells of E71 were on the average slightly longer and had slightly thickened walls compared to the wild type. Mutant E81 autolyzed much more slowly, grew exponentially in long chains (8 to 40 cells compared with mainly diplococci), contained much less active and latent autolysin in the wall, and possessed a wall of very similar chemical composition but with about twice the content of N-terminal groups. Mutant E81 walls were more susceptible to isolated autolysin but possessed an autolysin of the same specificity as the wild type. Ultrastructurally E81 cells were, on the average, significantly longer and had thicker walls than the wild type. Mutant E71 may be partially blocked at either transport of autolysin to the wall or in conversion of latent to active autolysin. The pleitropic effects noted in mutant E81 have been taken to suggest a possible membrane defect and to support the role of the autolysin in cell separation.