Regulation of aspartokinase in Lactobacillus plantarum

As a rational approach to the genetic development of a stable lysine overproducing strain of Lactobacillus plantarum for the fermentation of 'ogi', a Nigerian fermented cereal porridge, regulation of lysine biosynthesis in this species was investigated. Spontaneous lysine overproducing mutants of Lact. plantarum were obtained and their aspartokinase activities compared with those of wild-type strains under different conditions. Results showed that aspartokinase activity of Lact. plantarum cell extracts was not inhibited by either lysine, threonine, methionine or combinations of lysine and threonine. Instead, methionine enhanced aspartokinase activity in vitro. Results indicated that lysine biosynthesis in Lact. plantarum could be regulated by lysine via the control of aspartokinase production in a way different to that described for other bacteria.     

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.     

Plantaricins S and T, Two New Bacteriocins Produced by Lactobacillus plantarum LPCO10 Isolated from a Green Olive Fermentation

Twenty-six strains of Lactobacillus plantarum isolated from green olive fermentations were tested for cross-antagonistic activities in an agar drop diffusion test. Cell-free supernatants from four of these strains were shown to inhibit the growth of at least one of the L. plantarum indicator strains. L. plantarum LPCO10 provided the broadest spectrum of activity and was selected for further studies. The inhibitory compound from this strain was active against some gram-positive bacteria, including clostridia and propionibacteria as well as natural competitors of L. plantarum in olive fermentation brines. In contrast, no activity against gram-negative bacteria was detected. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. Since the inhibitory activity of the active supernatant was lost after treatment with various proteolytic enzymes, this substance could be classified as a bacteriocin, designated plantaricin S. Plantaricin S was also sensitive to glycolytic and lipolytic enzymes, suggesting that it was a glycolipoprotein. It exhibited a bactericidal and nonbacteriolytic mode of action against indicator cells. This bacteriocin was heat stable (60 min at 100 degrees C), active in a pH range of 3.0 to 7.0, and also stable in crude culture supernatants during storage. Ultrafiltration studies indicated that plantaricin S occurred as multimolecular aggregates and that the size of the smallest active form is between 3 and 10 kDa. In sodium dodecyl sulfate-polyacrylamide gels, plantaricin S migrated as a peptide of ca. 2.5 kDa. Maximum production of plantaricin S was obtained in a fermentor system in unregulated pH and log-phase cultures of L. plantarum LPCO10 in MRS broth plus 4% NaCl. In these culture conditions, a second bacteriocin (designated plantaricin T) was produced in late-stationary-phase cultures of L. plantarum LPCO10. On the basis of its biological activity, its sensitivity to various enzymes, and its molecular weight (lower than that of plantaricin S) as assessed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, plantaricin T appeared different from plantaricin S. Curing experiments with L. plantarum LPCO10 resulted in the appearance of variants that no longer produced either of the two bacteriocins but that were still immune to both of them.     

Utilization of high lysine-producing strains of Lactobacillus plantarum as starter culture for nutritional improvement of ogi

A high lysine-producing mutant of Lactobacillus plantarum (OG 261-5) derived from a wild type strain (OG 261) previously isolated from fermenting ogi was evaluated for nutritional improvement of ogi in a modified fermentation process. Results indicate that ogi obtained by fermentation with the pure culture of the mutant compared to traditional ogi increased in concentration of available lysine from 228.5 ± 12.0 mg/100 g to 525.1 ± 25.8 mg/100 g, tryptophan from 58.6 ± 8.0 mg/100 g to 114.3 ± 11.0 mg/100 g and tyrosine from 408.5 ± 13.7 mg/100 g to 4636.5 ± 11.3 mg/100 g. However, the contents of valine, leucine, isoleucine and phenylalanine were substantially reduced which may affect the protein quality of the modified ogi. The modified process shortened the period of traditional ogi production from five days to one by reducing the two-stage fermentation (i.e. soaking and souring stages) to a one-stage process and the total protein recovery was better compared to traditional ogi processing. There was no significant difference (P > 0.05) in organoleptic quality attributes of colour, flavour, and overall acceptability of ogi produced by the modified process and the traditional ogi.     

Genome sequence of Lactobacillus pentosus IG1, a strain isolated from Spanish-style green olive fermentations

Lactobacillus pentosus is the most prevalent lactic acid bacterium in Spanish-style green olive fermentations. Here we present the draft genome sequence of L. pentosus IG1, a bacteriocin-producing strain with biotechnological and probiotic properties isolated from this food fermentations.     

Molecular analysis of the 21-kb bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a

The complete 21,344-bp DNA sequence of the bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a was determined. Thirty-four putative open reading frames which could code for proteins longer than 42 amino acids were found. Those included the structural genes encoding for the previously described bacteriocins enterocin I and J (also named as enterocins L50A and L50B). After comparison to sequences in public databases, analysis of the gene organization of pEF1 suggests a modular structure with three different functional domains: the replication region, the bacteriocin region and the mobilization plus UV-resistance region. This genetic mosaic structure most probably evolved through recombination events promoted by transposable elements. The hypothesis that the bacteriocin cluster on pEF1 could act as a functional plasmid stabilization module in E. faecium 6T1a is discussed.     

Optimization of Bacteriocin Production by Batch Fermentation of Lactobacillus plantarum LPCO10

Optimization of bacteriocin production by Lactobacillus plantarum LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 2³_5-2 fractional factorial two-level design and tested for their influence on maximal bacteriocin production by L. plantarum LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 3²_3-1 fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27°C, salt concentration from 2.3 to 2.5%, and L. plantarum LPCO10 inoculum size ranging from 10^7.3 to 10^7.4 CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 × 10⁴ times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from L. plantarum LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from L. plantarum LPCO10 so that bacteriocins can be used as preservatives in canned foods.     

Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of gram-positive bacteria

Lactobacillus plantarum NC8 was shown to produce plantaricin NC8 (PLNC8), a recently purified and genetically characterized inducible class IIb bacteriocin, only when it was co-cultured with other gram-positive bacteria. Among 82 strains belonging to the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Staphylococcus, and Streptococcus, 41 were shown to induce PLNC8 production in L. plantarum NC8. There was apparently no relationship between the sensitivity of the strains and their ability to induce the bacteriocin, indicating that the inducer and sensitive phenotypes may not be linked. In some instances, induction was promoted by both living and heat-killed cells of the inducing bacteria. However, no PLNC8-inducing activity was found in the respective cell-free, pure culture supernatants. Inducer strains also promoted the production of a PLNC8-autoinducing activity by L. plantarum NC8, which was found only in the cell-free co-culture supernatants showing inhibitory activity. This PLNC8-autoinducing activity was diffusible, heat resistant, and of a proteinaceous nature, and was different from the bacteriocin itself. Taken together, the results suggest that the presence of specific gram-positive bacteria acts as an environmental stimulus activating both PLNC8 production by L. plantarum NC8 and a PLNC8-autoinducing activity, which in turn triggers or maintains bacteriocin production in the absence of inducing cells.     

Use of Lactobacillus plantarum LPCO10, a Bacteriocin Producer, as a Starter Culture in Spanish-Style Green Olive Fermentations

Bacteriocin-producing Lactobacillus plantarum LPCO10 and its non-bacteriocin-producing, bacteriocinimmune derivative, L. plantarum 55-1, were evaluated separately for growth and persistence in natural Spanish-style green olive fermentations. Both strains were genetically marked and selectively enumerated using antibiotic-containing media. Plasmid profile and bacteriocin production (bac⁺) were used as additional markers. When olive brines were inoculated at 10⁵ CFU/ml, the parent strain, LPCO10, proliferated to dominate the epiphytic microflora, sharing high population levels with other spontaneously occurring lactobacilli and persisting throughout the fermentation (12 weeks). In contrast, the derivative strain could not be isolated after 7 weeks. Stability of both plasmid profile and bac⁺ (LPCO10 strain) or bac^- (55-1 strain) phenotype was shown by L. plantarum LPCO10 and L. plantarum 55-1 isolated throughout the fermentation. Bacteriocin activity could be found in the L. plantarum LPCO10-inoculated brines only after ammonium sulfate precipitation and concentration (20 times) of the final brine. Spontaneously occurring lactobacilli and lactic coccus populations, which were isolated from each of the fermenting brines studied during this investigation, were shown to be sensitive to the bacteriocins produced by L. plantarum LPCO10 when tested by the drop diffusion test. The declines in both pH and glucose levels throughout the fermentative process were similar in L. plantarum LPCO10- and in L. plantarum 55-1-inoculated brines and were comparable to the declines in the uninoculated brines. However, the final concentration of lactic acid in L. plantarum LPCO10-inoculated brines was higher than in the L. plantarum 55-1-inoculated brines and uninoculated brines. These results indicated that L. plantarum LPCO10 may be useful as a starter culture to control the lactic acid fermentation of Spanish-style green olives.     

Vitamin and amino acid requirements of Lactobacillus plantarum strains isolated from green olive fermentations

The requirement for essential amino acids and vitamins was determined in wild-type Lactobacillus plantarum strains isolated from green olive fermentation brines. All the strains were found to be auxotrophic with respect to the amino acids but some of them were prototrophic for pyridoxal, p -aminobenzoic acid and/or nicotinic acid. Their growth response to these nutrients was also studied and found to be quite heterogeneous. Nutritional requirement pattern as a criteria for selecting starter cultures is discussed.