Relevance of NADH Dehydrogenase and Alternative Two-Enzyme Systems for Growth of Corynebacterium glutamicum With Glucose, Lactate, and Acetate

The oxidation of NADH with the concomitant reduction of a quinone is a crucial step in the metabolism of respiring cells. In this study, we analyzed the relevance of three different NADH oxidation systems in the actinobacterial model organism Corynebacterium glutamicum by characterizing defined mutants lacking the non-proton-pumping NADH dehydrogenase Ndh (Δndh) and/or one of the alternative NADH-oxidizing enzymes, L-lactate dehydrogenase LdhA (ΔldhA) and malate dehydrogenase Mdh (Δmdh). Together with the menaquinone-dependent L-lactate dehydrogenase LldD and malate:quinone oxidoreductase Mqo, the LdhA-LldD and Mdh-Mqo couples can functionally replace Ndh activity. In glucose minimal medium the Δndh mutant, but not the ΔldhA and Δmdh strains, showed reduced growth and a lowered NAD+/NADH ratio, in line with Ndh being the major enzyme for NADH oxidation. Growth of the double mutants ΔndhΔmdh and ΔndhΔldhA, but not of strain ΔmdhΔldhA, in glucose medium was stronger impaired than that of the Δndh mutant, supporting an active role of the alternative Mdh-Mqo and LdhA-LldD systems in NADH oxidation and menaquinone reduction. In L-lactate minimal medium the Δndh mutant grew better than the wild type, probably due to a higher activity of the menaquinone-dependent L-lactate dehydrogenase LldD. The ΔndhΔmdh mutant failed to grow in L-lactate medium and acetate medium. Growth with L-lactate could be restored by additional deletion of sugR, suggesting that ldhA repression by the transcriptional regulator SugR prevented growth on L-lactate medium. Attempts to construct a ΔndhΔmdhΔldhA triple mutant were not successful, suggesting that Ndh, Mdh and LdhA cannot be replaced by other NADH-oxidizing enzymes in C. glutamicum.

222 EFFECT OF THE PRESENCE OR ABSENCE OF PERCOLL CENTRIFUGATION; PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE; AND HEPARIN ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll. Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte

Mercury Methylation by Interspecies Hydrogen and Acetate Transfer between Sulfidogens and Methanogens

ABSTRACT Cocultures of Desulfovibrio desulfuricans andMethanococcus maripaludis grew on sulfate-free lactate medium while vigorously methylating Hg2+. Individually, neither bacterium could grow or methylate mercury in this medium. Similar synergistic growth of sulfidogens and methanogens may create favorable conditions for Hg2+ methylation in low-sulfate anoxic freshwater sediments.

Role of Metabolic Intermediates in the Inhibition of Salmonella typhimurium and Salmonella enteritidis by Veillonella

A Veillonella sp. was isolated from the cecal contents of adult chickens. The Veillonella was grown on an agar medium supplemented with 200 mM of lactate, pyruvate, fumarate, or succinate and adjusted to a pH of 6.7, 6.5, 6.3, 6.1, 5.9, or 5.7. No metabolites were added to the control media, but it was adjusted to the same pH levels as the supplemented media. The agar medium on which the Veillonella was grown was overlaid with fresh agar medium. Cultures of Salmonella typhimurium or Salmonella enteritidis were spread on the surface of the agar overlay, and the plates were incubated at 37°C for 14–18 h. Veillonella did not inhibit the growth of either salmonellae on any of the control or pyruvate medium. Veillonella did inhibit the growth of both salmonellae on lactate medium that had been adjusted to pH 6.3, 6.1, or 5.9 and on succinate medium that had been adjusted to pH 5.7. Veillonella also inhibited the growth of S. typhimurium on fumarate medium that had been adjusted to pH 6.7, 6.5, 6.3, 6.1, or 5.9; and it inhibited the growth of S. enteritidis on fumarate medium that had been adjusted to pH 6.7, 6.5, 6.3, or 6.1. Inhibition on lactate agar was correlated with the production of acetate and propionate by Veillonella and residual lactate in the medium; inhibition on fumarate agar was correlated with the production of propionate and lactate by Veillonella; and inhibition on succinate agar was correlated to the production of propionate at low pH levels. The findings indicate that anaerobic bacteria that produce these metabolic intermediates and anaerobic bacteria that can convert the intermediates to volatile fatty acids may be important components of probiotic cultures that can be provided to chicks to reduce colonization by salmonellae.

Nutritional requirements for chloramphenicol biosynthesis in Streptomyces venezuelae

Cultures grown in a glycerol serine lactate medium were used to establish the inoculation procedure, aeration level, and trace-mineral nutrition optimizing chloramphenicol production in Streptomyces venezuelae. The stimulatory effect of lactate in this medium was concluded not to be an artifact of medium preparation but to reside in its influence on carbon-source utilization. In media with ammonium sulfate as a nonrestricting source of nitrogen, chloramphenicol production varied with the carbon source chosen. Production occurred during the growth phase and was highest on galactose, lactose, cellobiose, and starch. The rate of synthesis was related directed to the growth rate and decreased in the stationary phase. Variation of the nitrogen source with glucose as a nonrestricting source of carbon showed that the highest antibiotic titres were obtained with poorly utilized compounds such as isoleucine or phenylalanine. Proline gave yields comparable with those obtained in the more complex glycerol serine lactate medium in a shorter time. Although rate of growth is not the sole determining parameter, chloramphenicol synthesis is concluded to be a "growth-linked" process.

L-Methionine as an ethylene precursor in Saccharomyces cerevisiae

L-Methionine induced production of ethylene by Saccharomyces cerevisiae growing in lactate medium. The production induced by L-methionine was inhibited by pyruvate, and elevated by glucose. Labeled ethylene was produced when L-[U-14C]methionine, but not [U-14C]glucose, was fed to the yeast. The mutant S. cerevisiae G1332 (ade−, met−) did not produce significant amounts of ethylene unless L-methionine was added. Thus L-methionine acts as a precursor of ethylene in S. cerevisiae. The role of glucose appears to be other than as a precursor.

MUTANTS OF YEAST DEFECTIVE IN ISO-1-CYTOCHROME c

ABSTRACT A medium containing chlorolactate has been devised to enrich for mutants that are unable to utilize lactate for growth, and therefore that may be defective in cytochrome c. Complementation tests of 6,520 chlorolactate-resistant mutants that were obtained spontaneously or induced with UV, ICR-170, or nitrosoimidazolidone resulted in the identification of 195 mutations at the cycl locus, which controls the primary structure of iso-1-cytochrome c. These 195 mutants, with 16 cycl mutants previously isolated, were examined for total cytochrome c by spectroscopic methods, growth on lactate medium, suppressibility by defined nonsense suppressor, mutational sites by x-ray-induced recombination, ability to revert, and in 86 cases, whether intragenic revertants contain altered iso-1-cytochrome c. Except for the deletion mutant cycl-1, all of thb mutants appeared to contain single-site mutdons that could be assigned to at least 35 different sites within the gene. The cycl mutants either completely lacked iso-1-cytochrome c or contained iso-1-cytochromes c that were completely or partially nonfunctional. In spite of the fact that the cyd mutants obtained by the chlorolactate procedure were selected on the basis of defective function, 68% appeared to completely lack iso-1-cytochrome c. The remaining cycl mutants contained below normal amounts of iso-1-cytochromes c. Studies at several incubation temperatures indicated that these nonfunctional iso-1-cytochromes c were thermolabile. It is suggested that the predominant means for abolishing iso-1-cytochrome c by mutations are either through a complete loss, such as produced by chain terminating codons, or impairments through drastic changes of tertiary structure which lead to instability and thermolability.

Growth and macromolecular biosynthesis by Micrococcus sodonensis during the utilization of glucose and lactate

Micrococcus sodonensis can metabolize glucose to carbon dioxide and water via both the hexose monophosphate shunt and glycolysis but cannot use it as a sole source of carbon and energy. When lactate-grown cells of M. sodonensis are replaced on glucose there is a 50% increase in cell population. The synthesis of deoxyribonucleic acid, ribonucleic acid, and protein continues for about 1.5 to 2.0 h before it ceases. Cells replaced in lactate medium under the same conditions double in number in approximately 4 h. Cells replaced in glucose do not synthesize phosphorus-containing compounds as ascertained by inorganic phosphate uptake while lactate-utilizing cells take up phosphate at a significant and continuous rate. The results suggest that the inability to utilize glucose as a sole source of carbon and energy is related to this incapability of M. sodonensis to gain sufficient energy from glucose oxidation. Results with related strains of micrococci suggest this phenomenon may be widespread in this group of microorganisms.

Absorption and Screening in Phycomyces

In vivo absorption measurements were made through the photosensitive zones of Phycomyces sporangiophores and absorption spectra are presented for various growth media and for wavelengths between 400 and 580 mµ. As in mycelia, ß-carotene was the major pigment ordinarily found. The addition of diphenylamine to the growth media caused a decrease in ß-carotene and an increase in certain other carotenoids. Growth in the dark substantially reduced the amount of ß-carotene in the photosensitive zone; however, growth on a lactate medium failed to suppress ß-carotene in the growing zone although the mycelia appeared almost colorless. Also when diphenylamine was added to the medium the absorption in the growing zone at 460 mµ was not diminished although the colored carotenoids in the bulk of the sporangiophore were drastically reduced. Absorption which is characteristic of the action spectra was not found. Sporangiophores immersed in fluids with a critical refractive index show neither positive nor negative tropism. Measurements were made of the critical refractive indices for light at 495 and 510 mµ. The critical indices differed only slightly. Assuming primary photoreceptors at the cell wall, the change in screening due to absorption appears too large to be counterbalanced solely by a simple effect of the focusing change. The possibility is therefore advanced that the receptors are internal to most of the cytoplasm; i.e., near the vacuole.