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Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus.

Acetobacter pasteurianus, an obligately oxidative bacterium, is the first organism shown to utilize pyruvate decarboxylase (PDC) as a central enzyme for oxidative metabolism. In plants, yeast, and other bacteria, PDC functions solely as part of the fermentative ethanol pathway. During the growth of A. pasteurianus on lactic acid, the central intermediate pyruvate is cleaved to acetaldehyde and CO(2) by PDC. Acetaldehyde is subsequently oxidized to its final product, acetic acid. The presence of the PDC enzyme in A. pasteurianus was confirmed by zymograms stained for acetaldehyde production, enzyme assays using alcohol dehydrogenase as the coupling enzyme, and by cloning and characterization of the pdc operon. A. pasteurianus pdc was also expressed in recombinant Escherichia coli. The level of PDC activity was regulated in response to growth substrate, highest with lactic acid and absent with mannitol. The translated PDC sequence (548 amino acids) was most similar to that of Zymomonas mobilis, an obligately fermentative bacterium. A second operon ( aldA) was also found which is transcribed divergently from pdc. This operon encodes a putative aldehyde dehydrogenase (ALD2; 357 amino acids) related to class III alcohol dehydrogenases and most similar to glutathione-dependent formaldehyde dehydrogenases from alpha-Proteobacteria and Anabeana azollae.

Acetobacter↗

Lactosphaera gen. nov., a new genus of lactic acid bacteria, and transfer of Ruminococcus pasteurii Schink 1984 to Lactosphaera pasteurii comb. nov.

The phylogenetic position and physiology of strain KoTa2T (T = type strain), which was previously classified as a Ruminococcus pasteurii strain, were studied. A determination of the 16S ribosomal DNA sequence of this taxon revealed its position within the radiation of the gram-positive lactic acid bacteria having low DNA G+C contents and that it is closely related to the genus Carnobacterium. L-Lactic acid was produced from glucose by a fructose-1,6-bisphosphate-activated lactate dehydrogenase, and oxygen tolerance was observed, characteristics which are consistent with assignment to this group. On the basis of its phenotypic characteristics and unique signature nucleotides, we propose that strain KoTa2 (= DSM 2381 = ATCC 35945) should be transferred to a new genus, Lactosphaera gen. nov., as the type strain of the species Lactosphaera pasteurii comb. nov.

Anaerobiosis↗

Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation.

In the last decade, a variety of ribosomally synthesized antimicrobial peptides, or bacteriocins, produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into various fundamental aspects of biology and biochemistry such as bacteriocin processing and secretion, mechanisms of cell immunity, and structure-function relationships. In parallel, there has been a growing awareness that bacteriocins may be developed into useful antimicrobial food additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their significant biological activities and potential applications. The present review provides an overview of the knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances. The activity and potential food applications of class IIa bacteriocins are a major focus of this review.

Journal Article↗

Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine.

The potential to produce the biogenic amines tyramine, histamine and putrescine, was investigated for lactic acid bacteria (LAB) of various origin, including commercial malolactic starter cultures, type strains and 78 strains isolated from Spanish grape must and wine. The presence of biogenic amines in a decarboxylase synthetic broth was determined by reverse-phase high performance liquid chromatography (RP-HPLC). Tyramine was the main amine formed by the LAB strains investigated. Leuconostoc strains were the most intensive tyramine formers. No potential to form biogenic amines was observed in Oenococcus oeni strains. Two strains of Latobacillus buchneri were associated with putrescine formation. None of the lactic acid bacteria produced histamine. According to these in vitro results, the commercial starter bacteria analyzed did not produce histamine, tyramine and putrescine.

Biogenic Amines↗

Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3.

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40-60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2-3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

Animals↗

Carbohydrate, peptide and lipid metabolism of lactic acid bacteria in sourdough.

The metabolic pathways of lactic acid bacteria that influence bread quality are coupled to the central carbon flux by the availability of cofactors influencing the cellular and environmental redox potential. Homo- and heterofermentative metabolism differ fundamentally with respect to the requirement for regeneration of reduced cofactors, NADH or NADPH. The utilization of co-substrates such as oxygen or fructose as electron acceptors by obligate heterofermentative lactobacilli is coupled to an increased production of acetate in dough. Recently, several oxidoreductases involved in cofactor regeneration were characterized and glutathione and short-chain aldehydes derived from lipid oxidation were identified as substrates for cofactor regeneration by Lactobacillus sanfranciscensis. Based on the different metabolic requirements for cofactor regeneration, homo- and heterofermentative lactobacilli exert divergent effects on redox-reactions in sourdough that influence bread quality beyond the formation of acetate. Proteolysis, followed by peptide or amino acid metabolism by LAB is one of the key routes of flavour formation in bread flavour, and enables the strain-specific formation of antifungal metabolites. Peptide metabolism as well as the metabolism of cysteine, arginine, and phenylalanine in Lactobacillus plantarum, L. sanfranciscensis, and Lactobacillus pontis is increasingly understood and these insights provide new opportunities for the directed application of sourdough LAB for improved bread quality.

Bread↗

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria.

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.

Amino Acid Sequence↗

Absence of host specificity for in vitro adhesion of probiotic lactic acid bacteria to intestinal mucus.

Adhesion of probiotic lactic acid bacteria (LAB) has been reported to be host species specific. Host specificity is regarded as a desirable property for probiotic bacteria and therefore recommended as one of the selection criteria. However, previous studies have indicated that LAB originating from one host adhere well also to the mucus of other species. The aim of the study was to investigate the host specificity of LAB adhesion in human, canine, possum, bird and fish mucus in vitro. An in vitro mucus adhesion model was utilized in this study using immobilized mucus from faeces or intestinal material of these hosts. The results indicate that the adhesion trait was not host specific but rather was characteristic to LAB species. In conclusion, mucus adhesion properties are more dependent on the LAB strain than on the host. This suggests that animal models in probiotic adhesion assays may be more applicable to other species than thought earlier. Positive health effects facilitated by adherent probiotics in humans may also denote the possibility of similar outcome in other species and vice versa.

Animals↗

Lactic acid bacteria and proteomics: current knowledge and perspectives.

Lactic acid bacteria (LAB) are widely used in the agro-food industry. Some of the LAB also participate in the natural flora in humans and animals. We review here proteomic studies concerning LAB. Two methods of research can be distinguished. In the first one, a systematic mapping of proteins is attempted, which will be useful for taxonomy and to function assignment of proteins. The second one focuses particularly on proteins whose synthesis is induced by various environmental situations or stresses. However, both approaches are complementary and will give new insights for the use of bacteria in industry, in human health and in the struggle against bacterial pathogens. Interest in LAB is growing, showing thus an increasing concern of their rational use and one can foresee in the near future an increasing use of proteomics as well as genomics.

Bacterial Proteins↗

Influence of blood handling techniques on lactic acid concentrations.

Despite the popularity of measuring blood lactic acid concentrations, many of the common variations in technique have not been evaluated. The purposes of this study were to: 1) establish the relationship between plasma and blood lactate concentrations, 2) determine the inter-analyzer reliability, and 3) assess the stability of lactate concentration in blood stored for up to one week. Blood was sampled from 26 volunteers before exercise, at 80% of estimated maximum heart rate, and 5 minutes after a treadmill run to exhaustion. Inter-machine reliability was tested between two Yellow Springs Instruments analyzers with buffer treated with a lysing agent and between two without. Blood lactate levels at all three levels could be predicted from plasma with R2 greater than .95. Correlations between duplicates on the same machine were greater than .96 for blood and .97 for plasma. In the worst cases, between duplicate differences and between machine differences were 2%. Lactate in stored blood was in some cases significantly different after 24 hours of storage. Moderate and high lactate concentrations in plasma were not significantly altered after 2 days of storage.

Adult↗

Antibacterial activity of lactic acid bacteria isolated from vacuum-packaged meats.

Lactic acid bacteria isolated from vacuum-packaged fresh meat stored at 4 degrees C were shown to produce antagonistic substances active against closely related bacteria. Growth medium, pH and growth temperature all affected the production of the inhibitory substances. Ten strains including aciduric Lactobacillus-type organisms, Carnobacterium spp. and Leuconostoc spp. were selected that produced protein-aceous substances that caused inhibition of indicator strains. These were considered to be bacteriocins or bacteriocin-like compounds based on their inactivation with protease, generally narrow spectra of antibacterial activity and bactericidal or bacteriostatic modes of action. Activity was not lost from supernatant fluids as a result of heat treatment at 62 degrees C for 30 min, except for the Leuconostoc strains. Inhibitory spectra of some strains included Enterococcus spp. and Listeria monocytogenes. Some strains were of interest because their inhibitory substances were detected in the supernatant fluid early in the growth cycle. The inhibitory substances differed in characteristics between strains and there is evidence that more than one bacteriocin-like substance may be produced by some strains.

Animals↗

Antagonism of Lactic Acid Bacteria against Phytopathogenic Bacteria.

A variety of lactic acid bacteria, isolated from plant surfaces and plant-associated products, were found to be antagonistic to test strains of the phytopathogens Xanthomonas campestris, Erwinia carotovora, and Pseudomonas syringae. Effective "in vitro" inhibition was found both on agar plates and in broth cultures. In pot trials, treatment of bean plants with a Lactobacillus plantarum strain before inoculation with P. syringae caused a significant reduction of the disease incidence.

Journal Article↗

Differentiation of lactic acid bacteria strains by postelectrophoretic detection of esterases.

Lactic acid bacteria (LAB) comprise a diverse group of Gram-positive, non-spore-forming microorganisms. These bacteria are widely used in food technology. The species identification of LAB depends mainly on physiological and biochemical criteria. The esterolytic systems of LAB remain poorly characterized. Esterases (EC 3.1.1.3) represent a diverse group of hydrolases catalyzing the cleavage and formation of esters bonds Screening of esterases is usually performed either by employing chromophoric substances (e.g., alpha- or beta-naphthyl esters of short-chain fatty acids). The post-electrophoretic detection of esterases is a sensitive technique applied in bacterial systems, that mainly provides information on the similarity of strains within the same species or subspecies according to their esterase patterns. This technique is principally used to determine the number and substrate specificity of esterases and lipases, revealing the complexity of lipase and esterase systems. The present chapter describes the technique of polyacrylamide gel electrophoresis (PAGE; in the absence of sodium dodecyl sulfate [SDS]), in non-denaturing conditions, to find intracellular fractions for strain typing of LAB.

Electrophoresis↗

[Species and strain specific identification of lactic acid bacteria in complex microflora].

The identification of lactic acid bacteria in a complex microbiota using bacteriological culture in combination with phenotypic and genotypic identification techniques is laborious and time-consuming. New molecular methods permit a fast and culture-independent characterisation of such microbiota. Denaturing gradient gel electrophoresis (DGGE) of PCR fragments of the 16S rRNA gene has been proven to be a suitable tool. Here the use of PCR-DGGE with group specific primers is described to investigate the dynamic of sourdough microbiota from addition of the starter until the microbiota remained stable. Species were identified by applying an identification ladder obtained from reference strains or by sequence analysis of the PCR fragments. Furthermore, a method for detection of strains in complex microbiota is described. A strain specific chromosomal DNA fragment of Lactobacillus paracasei LTH 2579 was isolated applying the subtraction hybridisation. Based on the acquired target sequence a specific PCR system was established and combined with a PCR system specific for the species L. paracasei. Use of this detection system permitted to identify and quantitatively detect L. paracasei LTH 2579 in fermented sausages and upon consumption in faecal samples.

DNA Primers↗

Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli.

The gene encoding a poly(DL-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(epsilon-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.

Amino Acid Sequence↗

Effect of lactic acid fermentation on bacterial pathogens and indicator organisms in broiler processing waste.

Broiler processing offal (heads, viscera, and feet) was collected on 3 separate days from a commercial processing plant. Each sample was separately ground, supplemented with sucrose (6% initial concentration), inoculated with actively growing lactic acid bacteria (10(6) cfu/g of offal) from a commercial silage culture, and fermented at 37 C. Replicate samples were taken for standard microbiological analysis after 0, 48, and 120 h of fermentation. In fresh offal, heterotrophic plate count, total and fecal coliforms, fecal streptococci, and Aeromonas hydrophila concentrations were 7.4, 5.9, 5.9, 5.4, and 3.9 log10 cfu/g wet weight, respectively. After 48 h of fermentation, the bacterial concentrations were 7.6, 2.2, < 1.3, 5.5, and < 2.3 log10 cfu/g wet weight, respectively. After 120 h of fermentation, the bacterial concentrations were 6.9, < 1.1, < 1.1, < 1.1, and < 1.1 log10 cfu/g wet weight, respectively. Salmonella concentrations in fresh, 48-h fermented, and 120-h fermented offal samples were 3.7, < 1.5, and < 1.5 log10 most-probable-number/100 g wet weight, respectively. Lactic acid fermentation appears to be effective in reducing the number of bacterial pathogens and indicator organisms in poultry processing offal.

Animal Feed↗

Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA.

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an alpha-galactosidase gene, agaA, was followed by two genes in the same orientation, bgaB, encoding a 2-kb beta-galactosidase, and bgaC, encoding a structurally distinct 1.76-kb beta-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus lactis, for which the genome sequence is known. To determine if these sequences encoded enzymes with alpha- and beta-galactosidase activities, we subcloned the genes and examined the enzyme properties. The alpha-galactosidase, AgaA, hydrolyzes para-nitrophenyl-alpha-D-galactopyranoside and has optimal activity at 32 to 37 degrees C. The beta-galactosidase, BgaC, has an optimal activity at 40 degrees C and a half-life of 15 min at 45 degrees C. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both alpha- and beta-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated beta-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two beta-galactosidases may act synergistically with the alpha-galactosidase to degrade other polysaccharides available in the environment.

Bacterial Proteins↗

[The lactic acid enterococci Enterococcus faecium and Enterococcus durans: nucleotide sequence diversity in 16S rRNA genes].

Among the strains used as starters for making sour milk products on the territory of the CIS, the bacteria Enterococcus faecium and Enterococcus durans are frequently found. In this work, we studied a new collection of lactic acid enterococci and also obtained more complete data on the nucleotide sequences of 16S rRNA genes in some strains studied earlier and found that most strains had certain distinctions in their 16S rRNA genes as compared with the E. durans and E. faecium genes available in the NCBI database. Based on these data, it is suggested that the strains of lactic acid enterococci represent new, earlier unknown taxa of enterococci that use milk as an ecological niche.

Armenia↗