Structural studies of the capsular polysaccharide from Aerococcus viridans var. homari.

The capsular polysaccharide from Aerococcus viridans var. homari has been investigated, using n.m.r. spectroscopy, methylation analysis, and specific degradations as the main methods. The polysaccharide is composed of tetrasaccharide repeating-units having the following structure. (Formula; see text) In this structure, D-QuiN stands for 2-amino-2,6-dideoxy-D-glucose (quinovosamine). Two of the three acidic sugars found, namely, L-altruronic acid and 4-O-[(S)-1-carboxyethyl]-D-glucose, have not been found in any other natural source. As evident from the n.m.r. spectra, the L-altruronic acid is not present in the 1C4 conformation, but flips to a conformation close to this on carboxyl reduction.

Phenotypic characterization, cellular fatty acid composition, and DNA relatedness of aerococci and comparison to related genera.

Aerococci can be misidentified as streptococci, enterococci, pediococci, lactococci, or leuconostocs. To distinguish the genus and determine if another species is needed in the present taxon, we analyzed 37 aerococci for cellular fatty acids and compared them with 377 strains of gram-positive cocci, including the species type strains from each of the related genera. The cellular fatty acid profile of aerococci was distinguishable from other genera. Two relatively novel fatty acids found in the aerococci were identified as C16:1 omega 9c and C16:1 omega 9t. Eleven strains of aerococci (including a strain originally identified as "Gaffkya" species) were chosen for DNA-DNA reassociation studies with the type strain Aerococcus viridans ATCC 11563; DNAs from eight of these strains were more than 75% related to the type strain and had 1 to 4% divergence in related sequences. The remaining three strains were 60 to 70% related to the type strain, had 7 to 11.5% divergence, and may represent a second species, Aerococcus genospecies 2. beta-Glucuronidase, alpha-galactosidase, and beta-galactosidase were useful in characterizing the aerococci.

Identification of monounsaturated fatty acids of Aerococcus viridans with dimethyl disulfide derivatives and combined gas chromatography-mass spectrometry.

Location of the double-bond position of monounsaturated fatty acids of Aerococcus viridans was accomplished by combined gas chromatography (GC)-mass spectrometry analysis of dimethyl disulfide (DMDS) derivatives. The monoenoic fatty acids from whole bacterial cells were converted to methyl esters and then to DMDS adducts and analyzed by capillary GC-mass spectrometry. The mass spectra of DMDS adducts gave an easily recognizable molecular ion (M+) and two major diagnostic ions attributable to fragmentation between the two CH3S groups located at the original site of unsaturation. Two relatively novel acids that distinguish aerococci from bacteria of closely related genera were identified as C16:1 omega 9c and C16:1 omega 9t from their mass spectrometry fragmentation patterns and retention characteristics on nonpolar capillary GC columns.

Porphyrin test as an alternative to benzidine test for detecting cytochromes in catalase-negative gram-positive cocci.

A total of 66 strains of gram-positive cocci, including 21 catalase-negative members of the family Streptococcaceae and strains of Stomatococcus mucilaginosus, were investigated for the ability to produce porphobilinogen and porphyrin from delta-aminolevulinic acid as an alternative to the benzidine test for detecting the presence of cytochromes. Production of porphobilinogen correlated 100% with membership in the family Micrococcaceae.

Characterization of peptidoglycan stem lengths by solid-state 13C and 15N NMR.

Lyophilized whole cells of Aerococcus viridans (Gaffkya homari) grown on a synthetic medium containing D-[2-13C, 15N]Ala, or containing both L-[1-13C]Lys and D-[15N]Ala, have been examined by double cross-polarization magic-angle spinning 13C and 15N nuclear magnetic resonance. Results from the double-labeled alanine experiment confirm the absence of metabolic scrambling of alanine by A. viridans. Results from the combined single-label experiment can be used to count directly the number of adjacent L-Lys and D-Ala units in peptide chains of cell-wall peptidoglycan. This count leads to the conclusion that there are no terminal D-Ala or D-Ala-D-Ala units in uncross-linked chains of the peptidoglycan of A. viridans.

Lipoquinones of some spore-forming rods, lactic-acid bacteria and actinomycetes.

The respiratory quinones of 73 strains of Gram-positive bacteria including spore-forming rods, lactic-acid bacteria and actinomyctes were examined. Menaquinones with seven isoprenoid units (MK-7) were the main quinone type found in representatives of the genus Bacillus and in Sporolactobacillus inulinus. However, a strain of B. thuringiensis produced MK-8 in addition to MK-7, and strains of B. lentus and B. pantothenticus appeared to produce MK-9 and MK-8, respectively, with no MK-7. In the clostridia and lactic-acid bacteria, no quinones were found, except in Pediococcus cerevisiae NCTC 8066 and Lactobacillus casei subsp. rhamnosus ATCC 7469, which contained menaquinones, and Streptococcus faecalis NCTC 775 and HIM 478-1, which contained demethylmenaquinones, in relatively low concentrations. Menaquinones were also found in the actinomycetes (except Actinomyces odontolyticus and Bifidobacterium bifidum which did not produce any quinones) and in Protaminobacter alboflavus ATCC 8458, the so-called Actinobacillus actinoides ATCC 15900 and Noguchia granulosis NCTC 10559.

Lipoquinones of some bacteria and mycoplasmas, with considerations on their functional significance.

In a comparative study the lipoquinones of some chemoorganotrophic, facultatively aerobic bacteria, and representative Acholeplasma, Mycoplasma, Spiroplasma, and Thermoplasma strains were investigated. The quinones were partly purified by preparative thin layer chromatography of lipid extracts, and characterized by their difference spectra (reduced minus oxidized) and Rf values. Respiring bacteria expectedly contained benzoquinones and/or naphthoquinones in micromolar concentrations whereas some aerotolerant, cytochrome-less, gram-positive bacteria were found to contain menaquinones in nanomolar concentrations, or even no quinones; only Streptococcus faecalis, an organism supposed to use a rudimentary, flavin-terminated respiratory chain system produced desmethyl menaquinone in amounts ranging between "high" and "low" quinone contents. Among the mycoplasmas investigated, only Thermoplasma acidophilum was found to be capable of synthesizing quinones (MK-7) in the micromolar order of magnitude indicating a respiratory electron transport system. The presence of energetically useful respiratory chain systems in Acholeplasma, Mycoplasma, and Spiroplasma is questioned since these organisms contain quinones (MK-4) in nanomolar concentrations, or no quinones, depending on the presence of exogeneous MK-6 in the growth medium. The possible metabolite role of menaquinones present in "low" amounts, as well as the role of NADH oxidase systems more or less tightly bound to the cytoplasmic membrane with the mycoplasmas deserves further investigation.

Modified biochemical tests for characterization of L-phase variants of bacteria.

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.