Ribose Utilization by Veillonella alcalescens

The utilization of ribose by Veillonella alcalescens has been further investigated. Nonfermentation of ribose is not a result of a phosphorylation lesion since ribose-phosphorylating activity was measured in cell extracts. Resting cells accumulated ribose-5-phosphate and nucleotides when ¹⁴C-ribose was provided; no other sugar phosphates were detectable. Resting cells that were shifted to growth conditions polymerized rather than degraded the accumulated ribose compounds. Cell extracts contained a fructose diphosphate phosphatase. Ribose-5-phosphate, glucose-6-phosphate, and fructose-6-phosphate were not hydrolyzed. It is postulated that the nonfermentation of ribose is not due to any metabolic lesions, but is a consequence of metabolic control at the fructose diphosphate level of glycolysis.     

d-Ribose Formation by Pseudomonas reptilivora

During the course of some works on sugar metabolism in bacteria, we could find out bacteria having producibility of free d-ribose. Among 1395 strains isolated from soil, only nine strains were found to be able to produce aldopentose which was identified chromato-graphically as ribose. From cultured broth of Pseudomonas reptilivora S-1104, a representative strain among these nine strains, d-ribose was isolated in crystalline form as aldopentose. It was also found that ribose was formed not only from glucose but also from d-fructose, d-arabitol, gluconic acid, etc., and that d-fructose and a glucoside (remained unknown) were also accumulated at the same time in the culture broth of Pseudomonas reptilivora S-1104.     

Lactate Metabolism by Veillonella parvula

A strain of Veillonella parvula M4, which grows readily in lactate broth without a requirement for carbon dioxide, has been isolated from the oral cavity. Anaerobic, washed cells of this organism fermented sodium lactate to the following products (moles/100 moles of lactate): propionate, 66; acetate, 40; carbon dioxide, 40; and hydrogen, 14. Cells grew readily in tryptone-yeast extract broth with pyruvate, oxaloacetate, malate, and fumarate, but poorly with succinate. The fermentation of pyruvate, oxaloacetate, or lactate plus oxaloacetate by washed cells resulted in the formation of propionate and acetate in ratios significantly lower than those observed with lactate as the sole carbon source. This was primarily due to increased acetate production. Cell-free extracts were unable to degrade lactate but metabolized lactate in the presence of oxaloacetate, indicating the presence of malic-lactic transhydrogenase in this organism. Lactic dehydrogenase activity was not observed. Evidence is presented for oxaloacetate decarboxylase and malic dehydrogenase activities in extracts.     

d-Ribose contributes to the glycation of serum protein

d-Ribose is active in glycation and rapidly produces advanced glycation end products, leading to cell death and to cognitive impairment in mice. Glycated serum protein (GSP) is a relatively short-term biomarker for glycemic control in diabetes mellitus. However, whether d-ribose is related to GSP is unclear. The aim of this work was to identify the contribution of d-ribose to GSP compared to d-glucose. Here, we showed that the yield of glycated human serum albumin with d-ribose was at least two-fold higher than that with d-glucose in a 2-week incubation. The glycation of human serum albumin (HSA) with d-ribose was much faster than that with d-glucose, as determined by monitoring changes in the fluorescent intensity of glycation products with time. Liquid chromatography-mass spectrometry/mass spectrometry revealed that 17 and 7 lysine residues on HSA were glycated in the presence of d-ribose and d-glucose, respectively, even when the concentration ratio [d-ribose]/[d-glucose] was 1/50. The intraperitoneal injection of d-ribose significantly increased the GSP levels in Sprague Dawley rats, but the injection of d-glucose did not. The level of d-ribose was more positively associated with GSP than the level of d-glucose in streptozotocin-treated rats. In diabetic patients, the levels of both d-ribose and d-glucose were closely related to the level of GSP. Together, these in vitro and in vivo findings indicated that d-ribose is an important contributor to the glycation of serum protein, compared to d-glucose. To assess GSP levels in diabetes mellitus, we should consider the contribution from d-ribose, which plays a nonnegligible role.     

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.     

Energy conservation by succinate decarboxylation in Veillonella parvula.

Veillonella parvula cannot grow with succinate as sole energy source. However, succinate decarboxylation simultaneous with malate or lactate fermentation increased growth yields by 2.4-3.5 g (mol succinate)-1. Malate was fermented stoichiometrically to acetate and propionate whereas lactate fermentation produced more acetate and considerable amounts of H2. Aspartate was utilized only in the presence of succinate as co-substrate. Methylmalonyl-CoA decarboxylase and ATP-dependent pyruvate carboxylase, but not methylmalonyl-CoA:pyruvate transcarboxylase, were detected in cell-free extracts of malate- or lactate-grown cells. The energetic aspects of these fermentation patterns are discussed.     

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.     

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.     

Polysaccharide-lipid complexes from Veillonella parvula

Mergenhagen, Stephan E. (National Institutes of Health, Bethesda, Md.). Polysaccharide-lipid complexes from Veillonella parvula. J. Bacteriol. 90:1730-1734. 1965.-A strain of Veillonella parvula (V2) elaborates an extracellular slime when grown in a nutrient medium containing only dialyzable components. Deproteinization with chloroform-butanol of ethyl alcohol-precipitated material from the supernatant culture fluid leads to the isolation of a water-soluble lipopolysaccharide (LPS1). Another component (LPS2), showing similarity in biological and immunological properties to the endotoxic antigen (LPC) isolated from whole cells, was extracted with phenol from the insoluble emulsion remaining after chloroform-butanol extraction of slime. Analysis of polysaccharides by thin-layer chromatography demonstrated the presence of glucose and galactose in LPS1 and glucose, glucosamine, galactosamine, and a methyl pentose in LPC. LPS1 failed to give a positive epinephrine skin test after intravenous injection in rabbits and failed to kill pertussis-sensitized mice, whereas LPS2 and LPC were active in both of these bioassays. Both lipopolysaccharides (LPS1 and LPC) exhibited type-specific haptenic activity in hemagglutination tests with numerous anti-Veillonella rabbit sera. LPS1 was found in these tests to be unrelated to a heterologous strain of Veillonella possessing a related somatic antigen. These experiments reveal the presence of two chemically and immunologically distinguishable polysaccharide-lipid complexes in this strain of V. parvula.     

Degradation of α-Ketoglutarate by Veillonella alcalescens

Veillonella alcalescens degrades alpha-ketoglutarate to CO(2), H(2), and propionate by a thioclastic mechanism.     

Inhibition of Salmonella typhimurium by the products of tartrate metabolism by a Veillonella species

The inhibition of the growth of Salmonella typhimurium by a Veillonella species grown on media supplemented with tartrate was examined. Growth of Salmonella typhimurium was not inhibited by the concentrations of products metabolized by Veillonella cultures on media supplemented with 0 or 50 mmol 1^-1 of tartrate, but was inhibited on media supplemented with 100 or 150 mmol 1^-1 of tartrate. Inhibition of Salm. typhimurium was correlated with the increased production of acetate and propionate from tartrate by the Veillonella species.     

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.     

Nitrite reduction in Veillonella alcalescens.

Nitrite reduction was examined in Veillonella alcalescens C-1, and obligate anaerobe with an ATP-yielding nitrate-reducing system. Hydrogen donors for nitrite reduction included hydrosulfite, hydrogen gas, and pyruvate, but not pyridine nucleotides, in the presnece or absence of flavins. Pyruvate-linked nitrite reduction was not inhibited by 4,4,4-trifluoro-1-(2-thienyl) 1,3-butanedione, dicoumarol, or 2-heptyl-4-hydroxy-quinoline-N-oxide. The noninvolvement of membrane-bound factors was supported by the fact that 100% of pyruvate-linked activity remained in the soluble fraction after fractionation of crude extracts by ultracentrifugation. Using DEAE-cellulose column chromatography, however, the participation of ferredoxin in nitrite reduction was demonstrated. The product of nitrite reduction appeared to be ammonia, as determined from H2-to-NO2- ratios. Nitrite reductase was induced by nitrate or nitrite and was repressed by increased levels of reduced nitrogenous compounds.     

Multiple Impairment of Glycolysis in Veillonella alcalescens

The property of glucose nonfermentation, characteristic of the genus Veillonella, was investigated in V. alcalescens C1, a strain of sheep rumen origin. Cell-free extracts as well as intact cells were incapable of glucose fermentation, thereby eliminating the possibility of nonpermeation. Assimilation of (14)C-glucose was not detectable. Of the 10 glycolytic enzymes, hexokinase, phosphoglyceromutase, and pyruvate kinase were not detectable. The other glycolytic enzymes were present.     

Regulatory Properties of Acetokinase from Veillonella alcalescens

Ghosts of T4 bacteriophage inhibit the uptake of thiomethyl-beta-galactoside (TMG), alpha-methylglucoside, glucose-6-phosphate, and glycerol in Escherichia coli B. The transport of orthonitrophenyl-beta-galactoside (ONPG) is also inhibited to a lesser degree and without alteration of the apparent K(m) of transport. These effects of ghosts parallel those of energy poisons on these systems. However, no one energy poison can produce such pronounced inhibitory effects in all these systems. The effect of the intact phage in these systems was either absent or very slight relative to the ghost. The effect of ghosts on the uptake of TMG was not immediate; at 10 C, no effect of the ghosts was apparent for at least 2 min. This suggests that a step, more temperature dependent than the attachment of the ghost, is necessary for the inhibitory action. The intracellular level of adenosine triphosphate (ATP) in the ghost-infected cells fell to less than 25% of the control value, and the ATP lost from the cell appeared in extracellular medium. Phage, on the other hand, caused no decrease in the intracellular ATP level. This loss of ATP from the cells after ghost infection suggests an alteration of the barrier properties of the membrane so that ATP can leave the cell; however, the accessibility of extracellular ONPG to intracellular beta-galactosidase does not increase. The dissimilarity of the actions of phage and ghosts on all properties examined does not support the model that the initial events in their infections are identical but that the intact phage, unlike the ghost, can provide information for the repair of its effects.     

Acetate kinase from Veillonella alcalescens. Regulation by succinate and substrates.

The kinetic properties of acetate kinase from Veillonella alcalescens were investigated. In the presence of high concentrations of nucleotide both forward and reverse reactions were observed. In the presence of succinate the degree of cooperativity between subunits of the homodimer decreased, i.e. the Hill coefficient, n, decreased from 2.5 to 1.4 for acetyl phosphate in the presence of succinate. At low substrate concentrations hyperbolic kinetic data were observed with succinate. We have proposed a modified version of the concerted symmetry model to describe the kinetics observed with this enzyme. The primary differentiating feature of the proposed model is the requirement for activator ligand binding for catalysis. In the absence of succinate, the substrate (acetate or acetyl phosphate) also functions as an activating ligand.     

Truncated Glycolytic System in Veillonella.

Rogosa, M., (National Institutes of Health, Bethesda, Md.), M. I. Krichevsky, and F. S. Bishop. Truncated glycolytic system in Veillonella. J. Bacteriol. 90:164-171. 1965.-Intact Veillonella cells do not utilize carbohydrates for growth, nor are carbohydrates fermented. In cell extracts, there is no detectable glucokinase or fructokinase. Cell extracts do not degrade glucose or fructose unless supplemented with yeast hexokinase. Under these conditions, triose phosphates are formed in the presence of a hydrazine trap. When glucose-C(14) plus added hexokinase or fructose-1,6-diphosphate-C(14) was incubated with cell extracts, the production of CO(2), acetate, pyruvate, propionate, and lactate was detected. It is concluded that, except for a hexokinase, all the activities required for a glycolytic system are present.