Utilization of fructose and ribose in lipopolysaccharide synthesis by Veillonella parvula.

Veillonella parvula, which cannot ferment or incorporate most sugars, incorporated radioactivity from [14C]ribose and [14C]fructose into cellular lipopolysaccharide (LPS) in the presence of lactate as an energy source. It was shown that virtually all of the fructose carbon which was assimilated into LPS material appeared in hydrophilic LPS components, and almost none was assimilated into fatty acid LPS components. The assimilation of lactate carbon into LPS in the presence of fructose was shifted from the hydrophilic toward the fatty acid components.

2-phosphoglycerate phosphatase and serine biosynthesis in Veillonella alcalescens.

The constituent enzymes for the phosphorylated and nonphosphorylated serine biosynthetic pathways in Veillonella alcalescens were identified and included phosphoserine phosphatase, 3-phosphoglycerate dehydrogenase, glycerate dehydrogenase, phosphoserine aminotransferase, and serine-pyruvate aminotransferase. Cell extracts of the organism were also found to cause the specific dephosphorylation of 2-phosphoglycerate. The phosphatase was purified 39-fold by manganese chloride precipitation, ammonium sulfate precipitation, and DEAE-cellulose chromatography. Sephadex G-200 gel filtration data established an apparent molecular weight of 50000 for the enzyme. The 2-phosphoglycerate phosphatase had a pH optimum of 5.5 and was distinct from phosphoglyceromutase. Assays conducted with the purified enzyme on a number of other phosphorylated intermediates indicated that the phosphatase was most specific for 2-phosphoglycerate. Glucerate, hydroxypyruvate, and serine inhibited the enzyme, whereas succinate stimulated activity. Veillonella 2-phosphoglycerate phosphatase is the first such enzyme to be described in a prokaryote and is probably involved in glycerate generation for the nonphosphorylated serine biosynthetic pathway.

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.

Nitrate-reductase electron-transport cofactors in Veillonella alcalescens.

Studies on the effects of inhibitors of the nitrate-reducing activity of Veillonella alcalescens extracts suggest the participation of a naphthoquinone, a b-type cytochrome, and non-heme iron in electron transport to nitrate. A nitrate-reductase-deficient mutant displayed a longer doubling time and a decreased molar growth yield on nitrate media. This mutant was phenotypically restored by the addition of molybdate to the growth medium, giving evidence for the functioning of molybdenum in the nitrate-reductase enzyme of V. alcalescens.

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.

Characterization of a naturally occurring diamine auxotroph of Veillonella alcalescens.

Veillonella alcalescens strain ATCC 17745 was shown to require putrescine or cadaverine for growth. None of the other compounds tried, including magnesium and spermidine, were able to substitute for the diamines. Studies with labeled diamines showed that spermidine was made from putrescine in this organism. A polyamine analogous to spermidine, but made from cadaverine, was not found. A combination of growth experiments and chemical assays suggested that protein synthesis was limited in diamine-starved cells. Protein synthesis occurred prior to nucleic acid synthesis when putrescine was added to starved cells.

Nitrate reduction and the growth of Veillonella alcalescens.

Veillonella alcalescens, a strict anaerobe, was found to possess a nitrate reductase system which has characteristics of both assimilatory and respiratory nitrate reduction. The nitrate reductase has been identified tentatively as a particulate enzyme which utilizes a variety of electron donors for the reduction of nitrate. By use of (15)N-labeled nitrate, it was shown that under appropriate conditions nitrate nitrogen is incorporated into cell material. V. alcalescens grown on pyruvate and nitrate has a greater growth rate than cells grown on pyruvate alone. Growth can occur in a medium with hydrogen and nitrate as the sole energy source. Ammonium chloride decreases the rate of nitrate reduction but does not completely inhibit reduction or incorporation. The results suggest that nitrate assimilation and respiration are not as distinct as in some other organisms.

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 (14)C-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.

Nonoxidative pentose phosphate pathway in Veillonella alcalescens.

Crude cell-free extracts of Veillonella alcalescens C1, an anaerobe unable to ferment glucose, were assayed for individual enzymes of the pentose phosphate pathway. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were not detectable. Constituent enzymes of the nonoxidative limb of the pentose phosphate pathway were demonstrable. The presence of transaldolase, transketolase, phosphoribose isomerase, and phosphoribulose epimerase in this organism suggests a primarily biosynthetic role for these enzymes. It is postulated that ribose is synthesized from lactate in V. alcalescens C1 via a modified reversal of glycolysis and the nonoxidative limb of the pentose phosphate pathway.

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.

Utilization of D-ribose by Veillonella.

Three strains of Veillonella, representing two species, were unable to utilize carbohydrates as energy sources for growth. Ribose, however, was utilized biosynthetically by all three strains. Exponentially growing cultures removed (14)C-ribose from the growth medium and retained radioactivity throughout the growth cycle. The kinetics of removal of ribose from the growth medium was found to depend on the initial ribose concentration. Uptake by resting cells was found to require active metabolism, was greatly stimulated by the presence of an energy source, and was insensitive to the presence of other pentoses. Fractionation of cells showed that the ribose was used for synthesis of acid-precipitable material, with as much as 92% of the radioactivity being found in the nucleic acid fraction. The intracellular distribution of ribose radioactivity did not change during growth after uptake was completed.

Degradiation of alpha-ketoglutarate by Veillonella alcalescens.

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