Proteomic analysis of Lactococcus lactis,a lactic acid bacterium

Lactococcus lactis is a Gram-positive bacteria, which belongs to the group of lactic acid bacteria among which several genera play an essential role in the manufacture of food products. Cytosolic proteins of L. lactis IL1403 cultivated in M17 broth have been resolved by two-dimensional gel electrophoresis using two pH gradients (pH 4-7, 4.5-5.5). More than 230 spots were identified by peptide mass fingerprints, corresponding to 25% of the predicted acid proteome. The present study made it possible to describe at the proteome level a significant number of cellular pathways (glycolysis, fermentation, nucleotide metabolism, proteolysis, fatty acid and peptidoglycan synthesis) related to important physiological processes and technological properties. It also indicated that the fermentative metabolism, which characterizes L. lactis is associated with a high expression of glycolytic enzymes. Thirty-four proteins were matched to open reading frames for which there is no assigned function. The comparison at the proteome level of two strains of L. lactis showed an important protein polymorphism. The comparison of the proteomes of glucose- and lactose-grown cells revealed an unexpected link between the nature of the carbon source and the metabolism of pyrimidine nucleotides.     

Lactic acid bacteria as a cell factory: rerouting of carbon metabolism in Lactococcus lactis by metabolic engineering

Lactic acid bacteria display a relatively simple metabolism wherein the sugar is converted mainly to lactic acid. The extensive knowledge of metabolic pathways and the increasing information of the genes involved allows for the rerouting of natural metabolic pathways by genetic and physiological engineering. We discuss several examples of metabolic engineering of Lactococcus lactis for the production of important compounds, including diacetyl, alanine and exopolysaccharides.     

Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.     

Urinary analysis of 16(5alpha)-androsten-3alpha-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis

We present a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6 per thousand, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual delta(13)C-values for 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9 per thousand. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1 per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Evolution of lactic acid bacteria in the order Lactobacillales as depicted by analysis of glycolysis and pentose phosphate pathways

Lactic acid bacteria (LAB) represent a functional group of bacteria that are fundamental in human nutrition because of their prominent role in fermented food production and their presence as commensals in the gut. LAB co-evolution and niche-adaptation have been analyzed in several phylogenomic studies due to the availability of complete genome sequences. The aim of this study was to provide novel insights into LAB evolution through the comparative analysis of the metabolic pathways related to carbohydrate metabolism. The analysis was based on 42 LAB genome sequences of representative strains belonging to Enterococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae. A reference phylogenetic tree was inferred from concatenation of 42 ribosomal proteins; then 42 genes belonging to the Embden–Meyerhof–Parnas (or glycolysis; EMPP) and pentose phosphate (PPP) pathways were analyzed in terms of their distribution and organization in the genomes. Phylogenetic analyses confirmed the paraphyly of the Lactobacillaceae family, while the distribution and organization of the EMPP and PPP genes revealed the occurrence of lineage-specific trends of gene loss/gain within the two metabolic pathways examined. In addition, the investigation of the two pathways as structures resulting from different evolutionary processes provided new information concerning the genetic bases of heterofermentative/homofermentative metabolism.     

Presence of sourdough lactic acid bacteria in commercial total mixed ration silage as revealed by denaturing gradient gel electrophoresis analysis

Aims: To characterize the bacterial communities in commercial total mixed ration (TMR) silage, which is known to have a long bunk life after silo opening. Methods and Results: Samples were collected from four factories that produce TMR silage according to their own recipes. Three factories were sampled three times at 1‐month intervals during the summer to characterize the differences between factories; one factory was sampled 12 times, three samples each during the summer, autumn, winter and spring, to determine seasonal changes. Bacterial communities were determined by culture‐independent denaturing gradient gel electrophoresis. All silages contained lactic acid as the predominant acid, and the contents appeared stable regardless of factories and product seasons. Acetic acid and 1‐propanol contents were different between factories and indicated seasonal changes, with increases in warm seasons compared to cool seasons. Both differences and similarities existed among the bacterial communities from each factory and product season. Lactobacillus parabuchneri was found in the products from three of four factories. Various sourdough lactic acid bacteria (LAB) were identified in commercial TMR silage; Lactobacillus panis, Lactobacillus hammesii, Lactobacillus mindensis, Lactobacillus pontis, Lactobacillus frumenti and Lactobacillus farciminis were detected in many products. Moreover, changes owing to product season were distinctive, and Lact. pontis and Lact. frumenti became detectable in summer products. Conclusion: Sourdough LAB are involved in the ensiling of commercial TMR silage. Silage bacterial communities vary more by season than by factory. The LAB species Lact. parabuchneri was detected in the TMR silage but may not be essential to the product’s long bunk life after silo opening. Significance and Impact of the Study: Commercial TMR silage resembles sourdough with respect to bacterial communities and long shelf life. The roles of sourdough LAB in the ensiling process and aerobic stability are worth examining.     

Applying stable carbon isotopic analysis at the natural abundance level to determine the origin of docosahexaenoic acid in the brain of the fat-1 mouse

A key factor limiting the study of the origin and metabolism of brain fatty acids is the lack of cost-efficient methods available to trace fatty acids. Here, through the application of compound-specific isotope analysis (CSIA), a novel, cost-efficient method, we successfully differentiated between brain DHA originating directly from dietary omega (n)?3 polyunsaturated fatty acids (PUFA), and brain DHA biochemically synthesized to determine the origin of brain DHA in fat-1 mice. Fat-1 mice and their wild-type littermates were either weaned onto n?6 PUFA rich, n?3 PUFA deficient diets or diets rich in both n?3 and n?6 PUFA. Isotopic analysis of fatty acid methyl esters from brain and liver tissue was conducted via gas chromatography- isotope ratio mass spectrometry. Our data demonstrates that in the presence of n?3 and n?6 PUFA, fat-1 mice obtain their brain DHA solely from n?3 PUFA sources. This study reflects the first application of CSIA to a complex multivariate model to determine the origin of brain fatty acids.     

UPLC/MS based method for quantitative determination of fatty acid composition in Gram-negative and Gram-positive bacteria

Quantitative fatty acid composition of microorganisms at various growth space points is required for understanding membrane associated processes of cells, but the majority of the relevant publications still restrict to the relative compositions. In the current study, a simple and reliable method for quantitative measurement of fatty acid content in bacterial biomass without prior derivatization using ultra performance liquid chromatography-electrospray ionization mass spectrometry was developed. The method was applied for investigating the influence of specific growth rate and pH on the fatty acid profiles of two biotechnologically important microorganisms — Gram-negative bacteria Escherichia coli and Gram-positive bacteria Lactococcus lactis grown in controlled physiological states. It was found that the membranes of slowly growing cells are more rigid and that the fatty acid fraction of the cells of L. lactis diminishes considerably with increasing growth rate.