Utilization Of L-lactate Research Articles

Staphylococcus aureus lactate- and malate-quinone oxidoreductases contribute to nitric oxide resistance and virulence.

Staphylococcus aureus is a Gram-positive pathogen that resists many facets of innate immunity including nitric oxide (NO·). Staphylococcus aureus NO-resistance stems from its ability to evoke a metabolic state that circumvents the negative effects of reactive nitrogen species. The combination of l-lactate and peptides promotes S. aureus growth at moderate NO-levels, however, neither nutrient alone suffices. Here, we investigate the staphylococcal malate-quinone and l-lactate-quinone oxidoreductases (Mqo and Lqo), both of which are critical during NO-stress for the combined utilization of peptides and l-lactate. We address the specific contributions of Lqo-mediated l-lactate utilization and Mqo-dependent amino acid consumption during NO-stress. We show that Lqo conversion of l-lactate to pyruvate is required for the formation of ATP, an essential energy source for peptide utilization. Thus, both Lqo and Mqo are essential for growth under these conditions making them attractive candidates for targeted therapeutics. Accordingly, we exploited a modelled Mqo/Lqo structure to define the catalytic and substrate-binding residues.We also compare the S. aureus Mqo/Lqo enzymes to their close relatives throughout the staphylococci and explore the substrate specificities of each enzyme. This study provides the initial characterization of the mechanism of action and the immunometabolic roles for a newly defined staphylococcal enzyme family.

Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli

BackgroundDue to its abundance and low-price, glycerol has become an attractive carbon source for the industrial production of value-added fuels and chemicals. This work reports the engineering of E. coli for the efficient conversion of glycerol into L-lactic acid (L-lactate).ResultsEscherichia coli strains have previously been metabolically engineered for the microaerobic production of D-lactic acid from glycerol in defined media by disrupting genes that minimize the synthesis of succinate, acetate, and ethanol, and also overexpressing the respiratory route of glycerol dissimilation (GlpK/GlpD). Here, further rounds of rationale design were performed on these strains for the homofermentative production of L-lactate, not normally produced in E. coli. Specifically, L-lactate production was enabled by: 1), replacing the native D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase (L-LDH), 2) blocking the methylglyoxal bypass pathways to avoid the synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of toxic intermediate, methylglyoxal, and 3) the native aerobic L-lactate dehydrogenase was blocked to prevent the undesired utilization of L-lactate. The engineered strain produced 50 g/L of L-lactate from 56 g/L of crude glycerol at a yield 93% of the theoretical maximum and with high optical (99.9%) and chemical (97%) purity.ConclusionsThis study demonstrates the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported in the literature prior to our work. The engineered biocatalysts produced L-lactate from crude glycerol in defined minimal salts medium at high chemical and optical purity.

D-Lactate altered mitochondrial energy production in rat brain and heart but not liver

BackgroundSubstantially elevated blood D-lactate (DLA) concentrations are associated with neurocardiac toxicity in humans and animals. The neurological symptoms are similar to inherited or acquired abnormalities of pyruvate metabolism. We hypothesized that DLA interferes with mitochondrial utilization of L-lactate and pyruvate in brain and heart.MethodsRespiration rates in rat brain, heart and liver mitochondria were measured using DLA, LLA and pyruvate independently and in combination.ResultsIn brain mitochondria, state 3 respiration was 53% and 75% lower with DLA as substrate when compared with LLA and pyruvate, respectively (p < 0.05). Similarly in heart mitochondria, state 3 respiration was 39% and 86% lower with DLA as substrate when compared with LLA or pyruvate, respectively (p < 0.05). However, state 3 respiration rates were similar between DLA, LLA and pyruvate in liver mitochondria. Combined incubation of DLA with LLA or pyruvate markedly impaired state 3 respiration rates in brain and heart mitochondria (p < 0.05) but not in liver mitochondria. DLA dehydrogenase activities were 61% and 51% lower in brain and heart mitochondria compared to liver, respectively, whereas LLA dehydrogenase activities were similar across all three tissues. An LDH inhibitor blocked state 3 respiration with LLA as substrate in all three tissues. A monocarboxylate transporter inhibitor blocked respiration with all three substrates.ConclusionsDLA was a poor respiratory substrate in brain and heart mitochondria and inhibited LLA and pyruvate usage in these tissues. Further studies are warranted to evaluate whether these findings support, in part, the possible neurological and cardiac toxicity caused by high DLA levels.

Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

BackgroundCorynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032.ResultsHere, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer.ConclusionsCg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer.

Proposal to assign Aeromonas diversa sp. nov. as a novel species designation for Aeromonas group 501

The Aeromonas group 501, also named Aeromonas sp. HG13, is taxonomically close to A. schubertii. Results obtained in previous studies, including DNA–DNA hybridization and DNA fingerprinting, suggest that Aeromonas group 501 could constitute a different Aeromonas species. In this work we have performed a polyphasic study with the two strains comprising the Aeromonas sp. HG13 in order to propose a formal species name. They could be differentiated from A. schubertii by the indole and lysine decarboxylase tests and the utilization of l-lactate. Phenotypically, both strains were also easily separated from the other Aeromonas species. Sequence analysis of the 16S rRNA gene showed high sequence similarities (>97%) between Aeromonas group 501 and all Aeromonas species. Nevertheless, sequence divergences of cpn60, dnaJ, gyrB and rpoD genes were higher than the intraspecific threshold values established for each gene (3.5%, 3.3%, 2.3% and 2.6%, respectively), while sequence divergences between strains CDC 2478-85 T and CDC 2555-87 were low (0.6–1.1%). The DNA G+C content of the type strain was 62.2 mol%. Phenotypic and genotypic evidence strongly suggests that the Aeromonas group 501 is a novel species of the genus Aeromonas, for which the name Aeromonas diversa sp. nov. is proposed. The type strain is CDC 2478-85 T (=CECT 4254 T=ATCC 43946 T=LMG 17321 T).

Aeromonas bivalvium sp. nov., isolated from bivalve molluscs

A polyphasic study was performed to determine the taxonomic position of two Aeromonas strains, 665N and 868E(T), isolated from bivalve molluscs, that could not be identified at the species level in a previous numerical taxonomy study. The DNA G+C content of these isolates was 62.3 and 62.6 mol%, respectively. Sequence analysis of the 16S rRNA gene showed that the two new strains were closely related to members of the genus Aeromonas. Fluorescence amplified fragment length polymorphism fingerprinting revealed that strains 665N and 868E(T) clustered together with a similarity of 78 % but did not cluster with any of the Aeromonas genomospecies. DNA-DNA hybridization experiments revealed a high level of relatedness between the two new isolates (76 %) but low levels of relatedness between these and phylogenetically most closely related Aeromonas genomospecies (30-44 %). Useful tests for the phenotypic differentiation of strains 665N and 868E(T) from other mesophilic Aeromonas species included those for gas from glucose, lysine decarboxylase, Voges-Proskauer reaction, acid from L-arabinose, hydrolysis of aesculin and utilization of L-lactate. On the basis of genotypic and phenotypic evidence, strains 665N and 868E(T) are considered to represent a novel species of the genus Aeromonas, for which the name Aeromonas bivalvium sp. nov. is proposed. The type strain is 868E(T) (=CECT 7113(T)=LMG 23376(T)).

Influence of oocyte-secreted factors and culture duration on the metabolic activity of bovine cumulus cell complexes

Intracellular communication between the cumulus cell complex and the oocyte is essential for numerous processes during oocyte maturation. The aim of this study was to determine the interaction between oocyte-secreted factors and the metabolic activity of bovine cumulus cell complexes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were aspirated from ovaries derived from an abattoir and divided into four treatment groups: (i) intact COCs, (ii) oocytectomized complexes (OOX), in which the ooplasm was microsurgically removed, (iii) OOX co-cultured with denuded oocytes (OOX+DO) and (iv) DO. The complexes were cultured individually in IVM media. After 0-4, 10-14 and 20-24 h of culture, the utilization of oxygen, glucose, pyruvate and L-lactate by the complexes was measured. The metabolic activity of the DO was undetectable. There were no significant differences in metabolic measurement among any of the treatment groups, indicating that the metabolism of the cumulus complex is not affected by the presence of the oocyte. When metabolic activity for the complexes was analysed relative to time in culture, there was an approximate twofold increase in the consumption of oxygen, glucose and pyruvate over the 24 h period (P<0.05), although production of L-lactate remained constant. The relationship between total glucose uptake and L-lactate production indicated that the majority of glucose consumed at the start of culture was being utilized via glycolysis, but by the cessation of the maturation period, there was significant utilization of glucose elsewhere, possibly for the formation of cumulus extracellular matrix. These results indicate that metabolism of COC does not reflect biochemical activity of the oocyte. Nevertheless, the metabolic requirements of the COC increase throughout maturation.

Bacterial Leaf Spot of Cilantro in Florida.

In the 1995 to 1996 winter vegetable season, a leaf spot disease of cilantro (fresh coriander) (Coriandrum sativum L.) was widespread throughout commercial plantings in the Everglades Agricultural Area south and east of Lake Okeechobee. Symptoms first appeared as water-soaked spots ≤1 mm in diameter. These spots became dark brown to black and enlarged up to 2 mm in diameter. No chlorotic haloes developed around the lesions. The disorder was observed in numerous plantings of the cultivar Longstanding at four separate locations. Symptoms were apparent throughout each planting, with disease severity ranging from a few individual spots to numerous lesions covering nearly entire leaflets. Severe outbreaks were correlated with heavy precipitation events. In several instances, disease levels were great enough to render entire plantings totally unmarketable. A non-fluorescent bacterium was consistently isolated on King's medium B when a cooled inoculation needle was pushed through lesions. Six representative strains were chosen for further characterization. All strains were aerobic, gram-negative rods, and were oxidase and arginine dihydrolase negative. Levan was produced, but potato slices were not rotted. Tests for utilization of l-tartrate, l-lactate, and erythritol were negative. Biolog analysis identified all strains of the bacterium as Pseudomonas syringae. Highest similarity indices (0.52 to 0.81) were with P. syringae pv. pisi for four of the six strains. Suspensions of each strain were swab inoculated onto leaves of 4-week-old Longstanding cilantro seedlings in the greenhouse. Control plants were swabbed with sterile water only. Plants were covered with clear polyethylene bags for 72 h. Watersoaked spots were evident on test plants when bags were removed. Typical brown, greasy-looking leaf spots were seen by 6 days after inoculation. Control plants were symp-tomless. In a host-range study, cilantro and the following plants were mist-inoculated with a 107 CFU/ml suspension of each of the six test strains: carrot (Daucus carota L. 'Fancy Pack'), celery (Apium graveolens L. var. dulce (Mill.) Pers., 'June Belle'), garden pea (Pisum sativum L. 'Melting Sugar'), snap bean (Phaseolus vulgaris L. 'Pod Squad'), and onion (Allium cepa L. 'Evergreen Bunching'). Six days after inoculation, characteristic symptoms were evident on the cilantro. Four of the six strains produced a few (less than 10 per plant) bacterial leaf spot symptoms in carrot from which P. syringae was readily recovered. Some necrosis was observed on young, emerging leaves of snap bean. No symptoms were recorded for the other host species or the controls. This disease is similar to one reported on cilantro in California (1) and Germany (2).

Membrane Permeability and Utilization of L-Lactate and Pyruvate in Carp Red Blood Cells

ABSTRACT L-Lactate and pyruvate permeability and utilization in carp (Cyprinus carpio) red blood cells was studied in vitro with tracer methods. Transport of lactate and pyruvate across the carp red blood cell membrane is rapid. At low plasma concentrations, lactate and pyruvate are transported into carp red blood cells mainly via a specific monocarboxylate carrier. This is shown by a study of the saturation kinetics and by inhibition using α-cyano-4-hydroxycinnamic acid and, more powerfully, p-chloromercuriphenylsulphonic acid. At higher plasma concentrations both simple diffusion and, apparently, the band 3 anion exchange system become increasingly important transport pathways. Carbon dioxide production rates from lactate and pyruvate as a function of their extracellular concentrations showed saturation kinetics. The transport rates of lactate and pyruvate are considerably higher than those required for their maximal rate of oxidation. The rapid transport of lactate and pyruvate into carp red blood cells thus guarantees that substrate availability is not the rate-limiting factor for the oxidation of these substrates.

Transport of lactate and other monocarboxylates across mammalian plasma membranes

Transport of L-lactate across the plasma membrane is of considerable importance to almost all mammalian cells. In most cells a specific H(+)-monocarboxylate cotransporter is largely responsible for this process; the capacity of this carrier is usually very high, to support the high rates of production or utilization of L-lactate. The best characterized H(+)-monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates, including pyruvate and the ketone bodies acetoacetate and beta-hydroxybutyrate. This carrier is inhibited by alpha-cyanocinnamate derivatives and some stilbene disulfonates and has been identified as a protein of 35-50 kDa on the basis of purification and specific labeling experiments. Other cells possess similar alpha-cyanocinnamate-sensitive H(+)-linked monocarboxylate transporters, but in some cases there are significant differences in the properties of these systems, sufficient to suggest the existence of a family of such carriers. In particular, cardiac muscle and tumor cells have transporters that differ in their Km values for certain substrates (including stereoselectivity for L- over D-lactate) and in their sensitivity to inhibitors. Mitochondria, bacteria, and yeast also possess H(+)-monocarboxylate transporters that share some properties in common with those in the mammalian plasma membrane but are adapted to their specific roles. However, there are distinct Na(+)-monocarboxylate cotransporters on the luminal surface of intestinal and kidney epithelia, which enable active uptake of lactate, pyruvate, and ketone bodies in these tissues. This article reviews the properties of these transport systems and their role in mammalian metabolism.

Effect of molybdenum on the growth and metabolism of Veillonella parvula and Streptococcus mutans.

Molybdenum inhibited production of active nitrate reductase by Veillonella parvula and increased utilization of L-lactate, but had little effect on cell yield. KNO3spared con sumption of L -lactate. Cell yield of Streptococcus mutans was increased in the presence of molybdenum, but there was no change in L -lactate production or terminal pH.

Metabolism of Pyruvate and L-Lactate by Rat Adipose Tissue

Abstract The metabolism of l-lactate and pyruvate, labeled uniformly with 14C, by epididymal fat pad tissue of fasted and fasted-refed rats was studied. In tissue of fasted-refed rats, pyruvate and l-lactate were utilized at equal rates. l-Lactate was nearly all converted into CO2 and fatty acids, and pyruvate into CO2, fatty acids, and l-lactate. The incorporation of 14C into glycerol was very low. l-Lactate was a better precursor of fatty acids than pyruvate. In tissue of fasted rats, pyruvate utilization was depressed, but still proceeded at an appreciable rate, and pyruvate carbon was incorporated into fatty acids and glycerol. The utilization of l-lactate by tissue of fasted rats was very low, and there was insignificant incorporation into lipids. Addition of unlabeled pyruvate or glucose to tissue of fasted rats metabolizing uniformly labeled l-lactate-14C increased 14C incorporation into CO2 and very markedly into lipid, but there was no net uptake of l-lactate. The 14C yields reflect exchange between labeled l-lactate and unlabeled pyruvate. Addition of malate, aspartate, α-ketoglutarate, and glutamate did not increase the utilization of uniformly labeled l-lactate-14C by tissue of fasted rats, but aspartate and α-ketoglutarate when added together stimulated l-lactate uptake, and incorporation of 14C into CO2 and lipids. Addition of oxalacetate and propionate increased to some extent 14C incorporation into CO2 and lipid, without appreciably stimulating l-lactate uptake. Addition of α-ketobutyrate or acetoacetate stimulated incorporation of l-lactate carbon into CO2 and lipids and net utilization of l-lactate by tissue of fasted rats. There was accumulation of α-hydroxybutyrate but not of β-hydroxybutyrate. Addition of acetate or β-hydroxybutyric acid was without effect. We conclude that in adipose tissue of rats there is no transfer of hydrogen equivalents from cytoplasm to mitochondria. When lipogenesis is pronounced, cytoplasmic hydrogen equivalents are all used in reductive biosynthesis; in the fasted state DPNH accumulates in the absence of lipogenesis, hydrogen equivalents from l-lactate oxidation cannot be utilized, and no l-lactate oxidation to pyruvate can occur.