A comparison of the adhesion, coaggregation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius

Fibrillar and fimbriate strains of Streptococcus salivarius were compared for their ability to adhere to buccal epithelial cells and saliva-coated hydroxyapatite beads, and for their ability to coaggregate with Veillonella strains. The fibrillar Lancefield group K strains adhered statistically significantly better to both buccal epithelial cells and saliva-coated hydroxyapatite beads than the fimbriate strains, which lacked the Lancefield group K antigen. After 1 h the fibrillar strains coaggregated statistically significantly better than the fimbriate strains with V. parvula strain V1, but after 24 h, coaggregation both of fibrillar and of fimbriate strains reached approximately 90%. Freshly isolated Veillonella strains all coaggregated with the S. salivarius strains, but the percentage coaggregation varied considerably after 1 h depending on the Veillonella strain. Coaggregation was independent of the presence of Ca2+. S. salivarius strain HB-V5, a mutant of strain HB that had lost the Veillonella-binding protein, coaggregated weakly with V. parvula strain V1, but coaggregated very well with other wild-type veillonellae, suggesting the presence of an alternative mechanism for Veillonella-binding for strain HB. Fibrillar strains were, therefore, more adhesive to oral surfaces and coaggregated with veillonellae after 1 h better than the fimbriate S. salivarius strains. Both fibrillar and fimbriate strains were highly hydrophobic in the hexadecane-buffer partition assay.     

Acetate kinase in the genus Veillonella: effect of succinate, serological cross-reactivity, and separation by electrophoresis

Acetate kinases from the genus Veillonella were divided into two types: a succinate-stimulated enzyme and a succinate-independent enzyme. Three strains, V. parvula ATCC 17743 (antigenic group II), V. parvula ATCC 17744 (V), and V. parvula ATCC 10790 (VI), contained the succinate-stimulated enzyme. Among four types strains of V. alcalescens, three strains, ATCC 17747 (I), ATCC 17746 (III), and ATCC 17748 (VII), contained the succinate-independent enzyme, whereas only one strain, ATCC 17745 (IV), contained the succinate-stimulated enzyme. Small amounts of antiserum to the purified acetate kinase from V. alcalescens ATCC 17748 completely inhibited the purified and crude enzyme activity from the strain. Classification of the enzymes on the basis of stimulation by succinate was consistent with classification based on serological reactions using the antiserum as an independent parameter. The succinate-stimulated enzyme could be separated into two classes according to the degree of sensitivity to succinate: (i) enzymes from V. parvula ATCC 17744 and V. alcalescens ATCC 17745, which could be demonstrated on gel after electrophoresis by a histochemical method to be highly stimulated by the presence of succinate in the reaction mixture, and (ii) enzymes from V. parvula ATCC 10790 and V. parvula ATCC 17743, which could be easily demonstrated without succinate. Four groups of acetate kinases from the genus Veillonella were separated by gel electrophoretic mobility. The results showed that almost all enzymes from the seven type strains were heterogeneous at the molecular level.     

Adjuvant activity of Klebsiella O3 lipopolysaccharide: no contribution of proteins to the expression of adjuvant activity

Previously we found that Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant of strain Kasuya (O3: K1) or its decapsulated mutant strain LEN-1 (O3: K1-) exhibited very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice. The preparation of KO3 LPS after deproteinization by four cycles of treatment with chloroform-butanol (5: 1) usually contained a small percentage of proteins and a definite amount of another antigen which was destroyed by heating at 100 C for 1 hr. This antigen proved to be derived from type 1 fimbriae which are responsible for mannose-sensitive hemagglutination of guinea pig erythrocytes. The preparation of KO3 LPS isolated from culture supernatant of the strains which did not produce type 1 fimbriae exhibited strong adjuvant activity similar to that of the preparation from those which produced them. The preparation of KO3 LPS treated with hot phenol water which is known to remove lipid A-associated proteins exhibited a similar strong adjuvant activity. The preparation of KO3 LPS after extensive deproteinizing, two cycles of pronase treatment followed by ten cycles of treatment with chloroform-butanol, no longer contained detectable amounts of proteins and the fimbrial antigen, but this preparation also exhibited similar strong adjuvant activity. Moreover, there was no difference in strength of the adjuvant activity between the preparation of KO3 LPS isolated from culture supernatant and that isolated by the phenol method from bacterial cells. The present study demonstrates that the strong adjuvant activity of the preparation of KO3 LPS does not depend in any way on proteins contaminating the preparation.     

Genetic transformation of Veillonella parvula

Veillonellae are one of the most prevalent and predominant microorganisms in both the supra- and subgingival plaques of the human oral cavity. Veillonellae's mutualistic relationships with the early, middle, and late colonizers of the oral cavity make them an important component of oral biofilm ecology. Unlike other ubiquitous early colonizers in the oral cavity, surprisingly little is known about Veillonella biology due to our lack of ability to genetically transform this group of bacteria. The objective of this study was to test the transformability of veillonellae. Using Veillonella parvula strain PK1910, we first obtained spontaneous mutations conferring streptomycin resistance. These mutations all carry a K43N substitution in the RpsL protein. Using the mutated rpsL gene as a selection marker, a variety of conditions were tested and optimized for electroporation. With the optimized protocol, we were able to introduce the first targeted mutation into the chromosome of V. parvula PK1910. Although more studies are needed to develop a robust genetic manipulation system in veillonellae, our results demonstrated, for the first time, that V. parvula is transformable, at least for strain PK1910.     

Production of Slime Polysaccharides by Shigella dysenteriae Type 1

Electron microscopy of ruthenium red-stained ultrathin section of strains of Shigella dysenteriae type 1 grown in the Casamino Acids-yeast extract broth medium showed the presence of an extracellular slime layer. The slime appeared as a dense sheath covering bacteria. The presence of slime promoted hemagglutinating activity of the bacteria. The slime polysaccharide (SPS) isolated from the cell-free culture supernatant or the bacterial surface was less than 162,000 daltons in size and immunochemically similar. The SPS showed cross-reaction with lipopolysaccharide (LPS) antigen in immunological tests; however, it also appeared to be different from LPS since it did not contain 2-keto-3-deoxyoctonate, a core sugar of LPS. A different pattern of separation from LPS was also observed by silver staining of SDS-polyacrylamide gels. From these data it appeared that either LPS and SPS are contaminated with each other or that SPS is the polysaccharide portion of LPS.     

Complete genome sequence of Acetohalobium arabaticum type strain (Z-7288)

Veillonella parvula (Veillon and Zuber 1898) Prévot 1933 is the type species of the genus Veillonella in the family Veillonellaceae within the order Clostridiales. The species V. parvula is of interest because it is frequently isolated from dental plaque in the human oral cavity and can cause opportunistic infections. The species is strictly anaerobic and grows as small cocci which usually occur in pairs. Veillonellae are characterized by their unusual metabolism which is centered on the activity of the enzyme methylmalonyl-CoA decarboxylase. Strain Te3(T), the type strain of the species, was isolated from the human intestinal tract. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the large clostridial family Veillonellaceae, and the 2,132,142 bp long single replicon genome with its 1,859 protein-coding and 61 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Lipid-phase transitions of the strictly anaerobic bacteria Veillonella parvula and Anaerovibrio lipolytica.

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.     

Phospholipid biosynthesis in some anaerobic bacteria.

We have identified and characterized enzymes of phospholipid synthesis in two plasmalogen-rich anaerobes. Megasphaera elsdenii and Veillonella parvula, and one anaerobe lacking plasmalogens. Desulfovibrio vulgaris. All three species contained phosphatidate cytidylyltransferase and phosphatidylserine synthase. Phosphatidylglycerophosphate synthesis was detected only D. vulgaris extracts. Phosphatidylserine (diacyl form) was the major product of the phosphatidylserine synthase assay with particles from M. elsdenii or V. parvula. The amounts of phosphatidylethanolamine formed were very low. Only D. vulgaris particles had an active phosphatidylserine decarboxylase.     

Chemically defined media for growing anaerobic bacteria of the genus Veillonella

A chemically defined medium for Veillonella parvula and V. alcalescens is described. Some nutritional aspects of the two strains used were examined: the optimum concentration of reducing agents, the requirement for amino acids, diamines, vitamins and other growth factors, and the conditions needed for well balanced nutrition.No specific requirements for single amino acids were observed. A combination of l-cysteine, dl-aspartic acid, l-glutamic acid, l-serine and l-tyrosine, promoted growth. In V. alcalescens, serine could substitute both arginine and tryptophan (or histidine). No growth was obtained with ammonium salts as the sole N source.Decarboxylation of l-ornithine, l-lysine and l-arginine was not demonstrated in the Veillonella parvula strain, which required putrescine or cadaverine for growth. Spermine, spermidine, l-lysine, l-ornithine and l-arginine, could not substitute putrescine in Veillonella parvula. Veillonella alcalescens, which does not require putrescine in the medium, was able to decarboxylate l-ornithine while forming putrescine.     

Chemotypes of Veillonella lipopolysaccharides

Lipopolysaccharides (LPS) were isolated by phenol-water extraction from 34 strains of Veillonella, and examined by paper chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-deoxy-octonate (KDO). Several preparations were also examined for neutral sugars by gas liquid chromatography. The LPS had in common glucosamine, galactosamine, L-glycero-D-manno-heptose glucose and KDO. Most LPS contained galactose, and a few rhamnose. D-glycero-D-manno-heptose was found in LPS from one of the strains. Based on the sugar composition of the LPS, the Veillonella strains could be classified into four chemotypes.     

Studies on the Bacteriophage 2 Receptors of Pseudomonas aeruginosa

The lysogenization of Pseudomonas aeruginosa strain BI with phage 2 resulted in the loss of the capacity to adsorb the same phage. The absence of phage 2 receptors on the surface of the lysogenized strain BI(2)(8) was confirmed by the failure of purified slime polysaccharide (SPB) or lipopolysaccharide (LPS) to inactivate phage 2. SPB and LPS from a phage 2-resistant strain also failed to inactivate phage 2 in contrast to the phage inactivation exhibited by the SPB and LPS obtained from the wild-type strain BI. Chemically, quantitative differences were apparent when the SPB and LPS of strains BI(2)(8) and BI/2S(2) were compared with those of the wild-type strain BI. The most striking difference noted was the absence of amino sugars in the SPB of strain BI/2S(2). The SPB of strain BI(2)(8) also contained a lower percentage of amino sugars compared with the SPB of the wild-type strain BI.     

Chemical Characterization of the Lipopolysaccharide of Pseudomonas solanacearum

The carbohydrates present in lipopolysaccharide (LPS) from Pseudomonas solanacearum are rhamnose, xylose, 2-amino-2-deoxyglucose, glucose, heptose, and 2-keto-3-deoxyoctonate. LPS extracted from cultures grown on either glycerol or glucose (as the major source of carbon) and extracted after various incubation periods had similar compositions. The LPS from several strains of the bacterium contained the same component sugars, but the amounts of each sugar varied considerably. It was observed, however, that xylose and 2-amino-2-deoxyglucose increased proportionately with rhamnose, the major component. Phenol-water-extracted LPS contained measurable amounts of nucleic acid, protein, and arabinan, but none of these polymers were detected in LPS extracted with phenol-chloroform-petroleum ether. Polysaccharides liberated from LPS by mild acid hydrolysis were purified by gel filtration. Carbohydrate analysis of the LPS from a virulent, fluidal strain (K60) showed that the O-specific antigen consisted of rhamnose, xylose, and 2-amino-2-deoxyglucose in the proportions 4:1:1. The LPS of an avirulent, afluidal strain (B1) lacked the O-specific antigen; the R-core region consisted of rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate. Methylation analysis indicated that the K60 O-specific antigen was composed of a hexasaccharide repeating unit containing 3-, 2-, and 3,4-substituted rhamnopyranosyl residues, 3-substituted 2-amino-2-deoxyglucose, and terminal xylopyranose in the molar ratios 2:1:1:1:1.     

Protective cross activity of the extracellular mucus of Pseudomonas aeruginosa

Extracellular slime was isolated from 15 P. aeruginosa typing strains of different O-serotypes (immunotypes). The isolated slime, partially purified by ethanol precipitation, was later referred to as crude slime. Glycolipoprotein was obtained from crude slime and lipopolysaccharide (LPS) was obtained from acetone-dried microbial cells by the method of aqueous-phenol extraction. All these antigenic preparations were studied in the active mouse cross-protection tests: immunized mice were challenged with 7 strains of different immunotypes, strain No. 170 019 or toxigenic strain PA-103. In experiments on mice the slime of different P. aeruginosa serotypes (immunotypes) was found to stimulate immunity to intraperitoneal infection with P. aeruginosa, both homologous or heterologous in respect to their immunotype, including toxigenic strains. Slime glycoprotein also stimulated active cross-immunity in mice, but the level of this immunity was higher than that of immunity stimulated by crude slime. LPS showed mostly weak protective activity in experiments on mice.     

Cell wall composition and incorporation of radio-labelled compounds by Veillonella alcalescens.

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.     

A Comparative Study of the Sugar Composition of O-antigenic Lipopolysaccharides Isolated from Vibrio alginolyticus and Vibrio parahaemolyticus

A comparative study of the sugar composition of O-antigenic lipopolysaccharides (LPS) isolated from Vibrio alginolyticus and those from V. parahaemolyticus was carried out. 3-Deoxy-d-mannooctulosonic acid, 2-keto-3-deoxy octonate (KDO), a regular sugar constituent of gram-negative bacterial LPS, was totally absent from LPS of all V. alginolyticus strains examined as it was from those of V. parahaemolyticus. Furthermore, a KDO-like thiobarbituric acid test-positive substance, identical with that of either V. parahaemolyticus 07 or 012, was also found in LPS from three strains, 505–78, 905–78, and 1013–79 (designated tentatively as group I), out of the five strains of V. alginolyticus tested. LPS from the members of group I contained, as component sugars, glucose, galactose, l-glycero-d-manno-heptose, glucosamine, galactosamine, the KDO-like substance, and an unidentified amino sugar P1. Thus, LPS of the members of group I possessed a similar sugar composition which is similar to that of LPS from either V. parahaemolyticus 07 or 012. LPS of strain 1027–79, one of the other two strains (designated tentatively as gorup II), contained as component sugars, glucose, l-glycero-d-mannoheptose, glucosamine, galactosamine, and the other unidentified amino sugar P2, while LPS of strain 53–79, the other member of group II, contained galactose as an additional component. The results indicate that LPS of strain 1027–79 has a sugar composition similar to that of V. parahaemolyticus 09 LPS.     

Expression of the surface properties of the fibrillar Streptococcus salivarius HB and its adhesion deficient mutants grown in continuous culture under glucose limitation

Streptococcus salivarius HB and four adhesion deficient mutants, HB-7, HB-V5, HB-V51 and HB-B, were grown in continuous culture in a defined medium under glucose limitation over a range of growth rates from 0.1 to 1.1 h-1. The ability to coaggregate with Veillonella parvula V1 cells and the ability to adhere to buccal epithelial cells did not alter with increasing growth rate. Cell surface hydrophobicity decreased markedly with increasing growth rate for the non-fibrillar non-adhesive mutant HB-B but not for the other four strains which all carry different combinations of fibril classes. The thickness of the ruthenium red staining layer (RRL) also varied with growth rate for strain HB-B, ranging from 19.5 +/- 3.8 nm at high growth rate to a minimum of 12.3 +/- 4.8 nm at low growth rate. Low cell surface hydrophobicity correlated with a thicker RRL for strain HB-B. Strains HB-V5 and HB-7 also showed a significant increase in RRL thickness at high growth rates although to a lesser degree than HB-B. SDS-PAGE revealed a large number of protein bands common to all strains at all growth rates, with the major common protein occurring at 15.6 kDa. Protein bands at 70, 56, 40.5 and 39 kDa appeared stronger at high growth rates than at low. A protein band at 82 kDa showed strongly only at low growth rates. Therefore, adhesion and coaggregation are not phenotypically variable with increasing growth rate but RRL thickness, hydrophobicity and cell surface proteins may be phenotypically variable depending on the strain.     

宋内Ⅰ相O抗原及霍乱毒素B亚基在福氏2a痢疾菌中的表达及其免疫保护效果的观察

本文利用基因重组的方法,将宋内Ｉ相Ｏ抗原基因以及霍乱毒素Ｂ亚单位基因（ｃｔｘ－Ｂ）克隆至带链球菌的ａｓｄ基因的表达载体,然后转化至ａｓｄ－痢疾菌苗株福氏２ａＴ３２。脂多糖银染以及Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ实验证实以上两基因都能在宿主菌中稳定表达。动物（小白鼠）保护实验表明,该重组菌对福氏２ａ、宋内氏痢疾菌的保护效率达１００％,对霍乱弧菌的保护效率也达７０％。该菌具有稳定、无抗生素标记、多价的特点。     

Putrescine and cadaverine are constituents of peptidoglycan in Veillonella alcalescens and Veillonella parvula.

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.     

Induction of an outer membrane protein of 78 kDa in Vibrio vulnificus cultured in the presence of desferrioxamine B under iron-limiting conditions

Fermentation balances and growth yields were determined with various bacteria fermenting lactate to acetate plus propionate either via methylmalonyl-CoA or via acrylyl-CoA. All strains fermented lactate to acetate plus propionate at approximately a 1:2 ratio. Growth yields of Propionibacterium freudenreichii were more than twice as high as those of Clostridium homopropionicum or Veillonella parvula. Hydrogen was formed as a side product to a significant extent only by V. parvula and Pelobacter propionicus; the latter formed hydrogen preferentially when using ethanol as substrate. Acrylyl-CoA reductase of C. homopropionicum and Clostridium neopropionicum was found nearly exclusively in the cytoplasm thus confirming that this reduction step is unlikely to be involved in energy conservation. C. homopropionicum exhibited higher K(S) and higher micro(max) values, as well as higher specific substrate turnover rates than P. freudenreichii. The results allow us to conclude that C. homopropionicum using the acrylyl-CoA pathway with low growth yield obtains its specific competitive advantage compared to P. freudenreichii not through higher substrate affinity or metabolic shift toward enhanced acetate-plus-hydrogen formation but through faster specific substrate turnover.