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A bacterium, designated strain lac18(T), was isolated in pure culture from the mucosal jejunum of a healthy pig, using a medium selective for anaerobic lactic acid bacteria and containing porcine gastric mucin as the main carbon and nitrogen source. Cells of this strain were coccus-shaped, arranged singly or in pairs and were Gram-stain-negative, oxidase-negative, non-spore-forming, anaerobic and microaerotolerant. An analysis based on 16S rRNA gene sequences indicated that strain lac18(T) should be assigned to the genus Veillonella, class Clostridia, phylum Firmicutes. 16S rRNA and dnaK gene sequence-based phylogenetic analyses both indicated that the most closely related species were Veillonella ratti ATCC 17746(T) (similarities of 96.6 and 84.5 %, respectively) and Veillonella criceti ATCC 17747(T) (similarities of 96.6 and 83.4 %, respectively). The results of DNA-DNA hybridizations between strain lac18(T) and these Veillonella species and the type species of the genus, Veillonella parvula DSM 2008(T), confirmed the genotypic distinctness of the novel isolate. Data from phenotypic studies also served to differentiate strain lac18(T) from related strains. Therefore strain lac18(T) represents a novel species of the genus Veillonella, for which the name Veillonella magna sp. nov. is proposed. The type strain is lac18(T) (=CCUG 55454(T)=CIP 109767(T)=DSM 19857(T)=JCM 15053(T)).

The cell wall of Veillonella alcalescens was shown to have a typically Gram-negative appearance and composition. The wall contains 24% lipid, 0.8% phosphorus, and 6.8% hexosamine. It is estimated to contain about 5% murein, unlike the 24% reported by other for Veillonella parvula. The amounts of 19 amino acids, including diaminopimelic acid, were determined. Though Veillonella sp. cannot metabolize sugars for energy, V. alcalescens incorporates ribose and fructose by separate, specific mechanisms and uses most of the incorporated sugar in nucleic acid synthesis. Large excesses of either sugar in the medium do not repress gluconeogenesis from the pyruvate level. We have been unable to detect phosphoglyceromutase (EC 2.7.5.3) by several assay methods but have no indication of a gluconeogenic pathway other than reverse glycolysis.

As a basis for physicochemical studies on the membranes of the strictly anaerobic bacteria Veillonella parvula, Anaerovibrio lipolytica, and Megasphaera elsdenii, the fatty acyl and alk-1-enyl moieties on the phosphoglycerides of these organism were characterized. Uncommon is the high proportion of a heptadecenoic acyl and alk-1-enyl moiety in these three lactate-fermenting bacteria. In contrast to V. parvula and A. lipolytica, M. elsdenii contains high amounts of branched-chain acyl and alk-1-enyl moieties. Freeze-etching electron microscopy showed that the lipids of the plasma membranes of V. parvula and A. lipolytica go from the liquid crystalline to the gel state upon lowering of the temperature, indicating that the membrane lipids are predominantly in the fluid state. No lipid-protein segregation could be detected in the plasma membrane of M. elsdenii. This can be explained by the abundance of branched-chain fatty acyl and alk-1-enyl residues in the membranes of this organism which may prevent lipid-protein segregation during the lipid-phase transition.

A cocci-filament association was discovered in bacterial cultural studies of a subgingival plaque sample. Components were isolated and identified as Veillonella parvula and Eubacterium saburreum. A E. saburreum cell-associated material consisting of approximately 25% glucose and 70% protein was found which may play a role in the adherence of V. parvula to this filament. Acid end product analysis via gas liquid chromatography showed an uptake of lactic and succinic acids by V. parvula resulting in increased levels of acetic, propionic and n-butyric acids in mixed culture of the E. saburreum and the V. parvula strains. The ability of E. saburreum to adhere to nichrome wire and to glass surfaces as well as the secondary plaque forming ability of V. parvula indicates that these organisms may play a role in the maturation of human dental plaque.

Veillonella alcalescens subsp. dispar was grown in a synthetic medium containing either radiolabeled thymidine or uridine to monitor cell lysis by assay of the release of deoxyribonucleic acid or ribonucleic acid (RNA), respectively. Biochemical analyses demonstrated that, although human or hen egg white lysozymes alone did not release deoxyribonucleic acid or RNA, the nucleic acids were liberated in equal amounts from lysozyme-treated cells by the addition of low concentrations of the sodium salts of HCO-3, SCN-, Cl-, and F-, RNA release was dependent on enzyme and anion concentration. Human lysozyme was more potent than hen egg white lysozyme, and bicarbonate was the most effective anion in promoting bacteriolysis. Surprisingly, ultrastructural analyses differed from biochemical results. Lysozyme alone caused lysis in approximately 40% of the cell population. Detailed ultrastructural examination revealed aggregated cytoplasmic components which appeared as small clumps, explaining why nucleic acids were not measurable in the biochemical assays. In reaction mixtures containing lysozyme plus inorganic salts, electron microscopy results were compatible with biochemical data. Ultrastructural studies demonstrated that the addition of inorganic salts to lysozyme-treated cells resulted in the solubilization of the protoplasmic aggregates of lysed cells, presumably freeing the complexed RNA, and in the rapid lysis of the remaining cells (approximately 60%). These data suggest that electron microscopy must be used in conjunction with biochemical assays to assess lytic damage of bacterial cells.

Veillonella alcalescens ATCC 17745, a strictly anaerobic, gram-negative small coccus, requires putrescine or cadaverine for growth (M. B. Ritchey, and E. A. Delwiche, J. Bacteriol. 124:1213-1219, 1975). Both putrescine and cadaverine were demonstrated to be incorporated exclusively into the peptidoglycan layer of V. alcalescens ATCC 17745. V. parvula GAI 0574 also proved to contain putrescine as a component of peptidoglycan. The primary chemical structure of the peptidoglycan common to the two Veillonella species is N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acid gamma-meso-diaminopimelic acid-D-alanine. Putrescine or cadaverine links covalently to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan is necessary for normal cell growth. In V. alcalescens ATCC 17745, above 40% saturation at cadaverine linked to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan is necessary for normal growth.