Range Of Lactic Acid Bacteria Research Articles

A novel bacterium-like particles platform displaying antigens by new anchoring proteins induces efficacious immune responses.

Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of Escherichia coli outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria Levilactobacillus brevis 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system's effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of Clostridium perfringens as the model antigen. The vaccine can induce more than 50% protection rate against C. perfringens type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.

Characterization of probiotic potential of lactic acid bacteria isolated from aquaculture objects of Uzbekistan

Lactic acid bacteria have a high potency to combat infections in the body. The range of lactic acid bacteria isolated from aquaculture in Uzbekistan has been described in this article. We found that Lactobacillus delbrueckii, L. plantarum, L. sakei, L. brevis, Lactococcus lactis, Pediococcus acidilactici, P. pentosaceus, Enterococcus faecium, E. hirae, E. mundii, E.faecalis, Leuconostoc citreum, and Weisella sibaria strains are represented in hydrobionts. Among them isolates Lactobacillus delbrueckii R1, Lactobacillus plantarum Kr5, Pediococcus acidilactici B, Enterococcus faecium R2, Lactobacillus plantarum R3, Pediococcus pentosaceus R1 showed high antagonistic activity against aquaculture pathogens. Four strains: Lactobacillus plantarum Kr5, Pediococcus acidilactici B, Enterococcus faecium R2, and Pediococcus pentosaceus R1, meet all the criteria for probiotics and can be recommended as part of probiotic feed additives.

Исследование бактериофагов, лизирующих молочнокислые бактерии

Bacteriophages harm food production, disrupt fermenting, spoil dairy products, and cause financial loss. The article describes the biodiversity and properties of bacteriophages capable of lysing lactic acid bacteria used in fermented dairy and meat products.
 The research featured bacteriophages obtained from fermented meat and dairy products. The methods included microbiological analyses, sensory evaluation, physico-chemical tests, genetic studies, electron microscopy, optical reassociation, and mathematical data processing.
 Violation of the fermentation process always resulted in bacteriophages in the finished products, e.g., fermented dairy products, curd whey, raw smoked and dry-cured sausages, etc. The list of bacteriophages of lactic acid bacteria included 20 bacteriophages of Lactococcus ssp., 11 – of Streptococcus salivarius subsp. thermophilus, and 5 – of Lactobacillus delbrueckii subsp. bulgaricus. The study revealed the diversity of the isolated bacteriophages, their molecular and biological profile, the titer and range of their new hosts, and the differences from standard ones. The bacteriophages proved to be able to infect a wider range of lactic acid bacteria.
 The article describes the change in the biodiversity and evolution of bacteriophages depending on their lytic action and virulence. The improved collection of bacteriophages and their detection cultures contribute to an earlier and more effective identification of bacteriophages in fermented products.

Comparative genomics analysis of genus Leuconostoc resolves its taxonomy and elucidates its biotechnological importance

Genus Leuconostoc consists of a diverse range of lactic acid bacteria (LAB) from dairy, food and environmental ecology. Even though the species of Leuconostoc are commercially significant, their taxonomy is largely based on old, low-resolution classical methods. Several taxonomic reclassifications in the past were inadequate for microbiologist and food industry professionals to demarcate any new strain of genus Leuconostoc. The current taxonomy of the genus is largely based on classical approaches, which are in utmost need of reinvestigation by whole genome-based approaches. In the present study, the taxono-phylogenomic analysis depicted sixteen species, including three novel genomospecies and several reshufflings across the species, namely, L. mesenteroides, L. pseudomesenteroides, L. gelidum and L. lactis. Genus-wide T3PKS, CAZymes, and vector plasmids supports its biotechnological potential. However, detection of the antibiotic resistance genes in such an important LAB genus raises concern over their utility in industry. Present, large-scale in-depth genome-based study can shed light on the genome dynamics of the member species, help to obtain a more robust taxonomy and elucidate its biotechnology importance.

Genomic characterisation of Lacticaseibacillus paracasei phage ΦT25 and preliminary analysis of its derived endolysin

Bacteriophage infection is a serious problem in milk fermentation where it causes a delay in the fermentation process and reduces product quality. This study provides the first molecular characterisation of a lactic acid bacteriophage from spoiled fermented milk tanks in the dairy industry in Thailand. Bacteriophage ΦT25 is virulent, and shows strong lytic activity against Lacticaseibacillus paracasei LPC. Genome analysis demonstrated that phage ΦT25 possesses a linear, double-stranded DNA genome of 38,421 bp. To assay endolysin activity of phage ΦT25 (ORF35), Lysin vector pET21a was constructed by cloning the LysinT25 gene into plasmid pET21a. The size of LysinT25 endolysin was predicted to be ∼41 kDa LysinT25 was able to lyse a wide range of lactic acid bacteria and other Gram-positive bacteria, such as Staphylococcus aureus and Listeria monocytogenes. In addition, this endolysin has a synergistic inhibitory effect against Escherichia coli when combined with chloroform as an outer-membrane permeabiliser.

Antibacterial activity of lactic acid bacteria isolated from milk and traditional fermented dairy products of south Algeria against multidrug resistance pathogenic bacteria

The alarming situation of the increasing problem of the antibiotics resistance in the whole world requires an immediate need of the exploration of better alternatives for combating resistance. The main aim of the present work was the isolation and the screening of large range of lactic acid bacteria with important potential of antibacterial activity against multidrug resistance pathogenic strains and the characterization of the molecules responsible for anti-bacterial activity. The primary screening indicated the isolation of total of 200 lactic acid bacteria (LAB) from harvested milk (camel and goat) and tradi-tional fermented dairy products (Jben and kamaria) of south Algeria and in-vestigated for their antibacterial activity against clinical pathogens bacteria. Furthermore, Nineteen representative isolates (19) were selected and char-acterized with regard to their functional properties. The obtained results indicated that produced bacteriocin by both bacterial strains L. raffinolactis LAB9 and Leuconostoc mesenteroides subsp. cremoris LAB13 has manifested an excellent antagonistic activity against P.aeruginosa (PA) and Staphylococcus aureus (MRSA), which was Methicillin resistant Staphylococcus aureus (MRSA). A Further investigation indicated that the produced bacteriocin by LAB9 and LAB 13 were heat stable, where their inactivation was observed when treated with pepsin and trypsin. Additionally, the purification of the produced bacteriocin by L.raffinolactis LAB9 and Leuconostoc mesenteroides subsp. cremoris LAB13 has been achieved by the using of the partial ammonium sulfate precipitation. The technological properties and the stability of bacteriocins of LAB may lead for bioprospection of antibacterial components in the current struggle against increasing pandrug resistance and slowing down the expansion of multi drug resistance.

Antibacterial Activity of Different Kefir Types Against Various Plant Pathogenic Bacteria

Kefir is a probiotic, dairy product produced by the fermentative activity of a diverse range of lactic acid bacteria, acetic acid bacteria, and yeast. In this study, we revealed the antimicrobial spectra of five types of kefir supernatants (EG, AN, KF, KY and SD) from different regions of Turkey fermented for 24 and 48 h against seven plant pathogenic bacteria and one bacterial biocontrol agent in vitro and in vivo for the first time. In vitro, antibacterial activity was investigated by the disk diffusion agar method. Their antibacterial potencies varied according to the type of kefir and the fermentation time. Also, we showed that the antimicrobial activity of kefir could be attributable to antimicrobial substances in supernatants rather than the low pH. In vivo, studies using the most potent kefir type on cucumber and common bean with their pathogenic bacteria in the climate chamber showed no remarkable decrease in diseases but revealed an increase in some plant growth parameters. The application resulted in an increase of 22% in shoot fresh weight, 20% in shoot dry weight, 79% in root fresh weight and 113% in root dry weight in common bean, on the other hand, 25% in shoot fresh weight, 34% in root fresh weight and 30% in shoot dry weight in cucumber.

Susceptibility of dairy associated molds towards microbial metabolites with focus on the response to diacetyl

Food spoilage may cause food waste, economic losses and even brand image deterioration. In fermented dairy products, spoilage by molds is frequently occurring and the molds are commonly being introduced at the consumer stage after opening. Diverse molds may be involved with the main genera being Penicillium, Mucor, and Cladosporium. Fungal growth may be inhibited by bioprotective cultures by competition and/or production of inhibitive compounds. Several compounds produced by a range of lactic acid bacteria have been reported as having an antifungal activity against certain fungi. Susceptibility tests of a panel of dairy associated molds towards twelve of these pure compounds were performed using microtiter plate well method. Diacetyl was the most potent antifungal among the compounds followed by octanoic acid. Large variations were seen within the Penicillium strains tested with P. commune being more sensitive than P. roquefortii, and two Mucor strains generally exhibiting high tolerance. The growth of two susceptible Penicillium strains in yoghurt serum with pH 4.6 with increasing amounts of diacetyl added were followed. Susceptibility varied but at 64 μg/mL, both strains were completely inhibited on yoghurt regardless of temperature. At higher pH (5.6) in malt extract agar (MEA), growth was faster but inhibition was still observed. Upon exposure to diacetyl, the level of cytoplasmic reactive oxygen species (ROS) increased and impairment of membrane integrity and leakage of cellular materials were observed. These findings provide new knowledge of the activity of diacetyl against molds.

Diversity of the metabolic profiles of a broad range of lactic acid bacteria in soy juice fermentation

This study explores the ability of lactic acid bacteria (LAB) to ferment soy juice. The ability of 276 LAB strains from 25 species to ferment the principal soy carbohydrates, sucrose, raffinose or stachyose was tested in synthetic media and a soy juice. Fermented soy juices (FSJs) were characterized for their odor. Selected FSJs were characterized by targeted metabolomics. All Streptococcus, 83% of Leuconostoc and Lactobacillus and 41% of Lactococcus strains were sucrose-positive, while only 36% of all the LAB strains tested were raffinose-positive and 6% stachyose-positive. Nearly all (97%) the sucrose-positive strains fermented soy juice, indicating that an ability to use sucrose is a good criterion to select strains for soy juice fermentation. Among the most efficient acidifying strains, 46 FSJs had an odor deemed to be acceptable. FSJ composition was dependent on both species and strains: 17/46 strains deglycosylated soy juice isoflavones, the 27 S. thermophilus strains converted a mean 4.4 ± 0.1 g/L of sucrose into 3.0 ± 0.1 g/L of lactic acid versus 5.2 ± 0.1 g/L into 2.2 ± 0.1 g/L for the 18 Lactobacillus and one Lactococcus strains. This study highlights the diversity of the metabolic profiles of LAB strains in soy juice fermentation.

Occurrence of Multidrug Resistant Salmonella spp. in Poultry and Approach for Its Indigenous Bio-control

A significant limitation to flourish poultry industry in Bangladesh is the emergence of multidrug resistance pathogenic bacteria like Salmonella spp. due to uncontrolled use of antibiotics for disease treatment. An alternative to antibiotics could be the application of probiotics. About 120 cloacal-swabs from poultry birds were investigated, seventy two (72) isolates of Salmonella spp. and twenty two (22) isolates of lactic acid bacteria (out of 50)were identified respectively by cultural, morphological and biochemical tests presumptively. Antibiotic sensitivity test of Salmonella spp. was performed, followed by Kirby-Bauer disc-diffusion assay with six antibiotic groups. All those isolates of Salmonella spp. were found to be highly resistant to â-lactam, cephalosporin, tetracycline and macrolide, highly sensitive to carbapenem and moderately sensitive to aminoglycosides. Then Salmonella spp. were used as a target for the prospective probiotic bacteria which were screened based on antimicrobial activity against those multidrug resistance Salmonella spp. In antagonism assay such as disc diffusion and one-streak method, it was revealed that five lactic acid bacteria showed antimicrobial activity against Salmonella spp. Further, lactic acid bacteria were characterized based on their tolerance ability to pH and NaCl, antibiotic susceptibility test. The tolerance range of Lactic acid bacteria was about pH (3.5-9.5), NaCl (4-8) % and also resistant to antibiotics groups like B lactam, aminoglycosides, and quinolone. Then 16S rDNA gene sequence analysis was performed for molecular identification of potential probiotic bacteria. One representative isolate was identified as phylogenetically closed relative to Pediococcus acidilactici. This study was able to demonstrate that Pediococcus acidilactici as anindigenous probiotic candidate to inhibit the growth of isolated multidrug resistant Salmonella spp. in poultry. The potent probiotic candidate Pediococcus sp. could be used to counter bacterial diseases in poultry, thereby it could ensure food safety in the poultry industries of Bangladesh.
 Bangladesh J Microbiol, Volume 34 Number 2 December 2017, pp 83-90

Probiotic potency of Lactobacillus plantarum KX519413 and KX519414 isolated from honey bee gut.

The Indian honey bee Apis cerana indica, which harbors an abundant and diverse range of lactic acid bacteria (LAB) in their gut with beneficial effects, was used as the source for the isolation of LAB. In the present study, two LAB isolates from honey bee gut were selected primarily based on their phenotypic and selective biochemical characterization, followed by PCR and identified using 16S rRNA sequencing as Lactobacillus plantarum and were registered in National Centre for Biotechnology Information under accession number KX519413 and KX519414. The probiotic potency of test strains indicated their survivability at acidic pH, bile salts and viability in simulated gastric juice enabling them to withstand gastrointestinal tract conditions. Evaluation of cell surface properties suggested that they possess an important defense mechanism against the pathogen since they are hydrophobic, auto-aggregative and have co-aggregative ability. Further, efficient exopolysaccharide production by them indicates not only their ability to enrich biofilm formation and auto-aggregation, but also enhances bacterial adhesion and colonization on the host intestinal tract. The present study concluded that L. plantarum from the gut of Apis cerana indica possesses probiotic potency, and potential candidates for use as food besides application in nutraceutical and pharmaceutical industries.

Exopolysaccharides produced by lactic acid bacteria: from health-promoting benefits to stress tolerance mechanisms.

A wide range of lactic acid bacteria (LAB) is able to produce capsular or extracellular polysaccharides, with various chemical compositions and properties. Polysaccharides produced by LAB alter the rheological properties of the matrix in which they are dispersed, leading to typically viscous and "ropy" products. Polysaccharides are involved in several mechanisms such as prebiosis and probiosis, tolerance to stress associated to food process, and technological properties of food. In this paper, we summarize the beneficial properties of exopolysaccharides (EPS) produced by LAB with particular attention to prebiotic properties and to the effect of exopolysaccharides on the LAB-host interaction mechanisms, such as bacterial tolerance to gastrointestinal tract conditions, ability of ESP-producing probiotics to adhere to intestinal epithelium, their immune-modulatory activity, and their role in biofilm formation. The pro-technological aspect of exopolysaccharides is discussed, focusing on advantageous applications of EPS in the food industry, i.e., yogurt and gluten-free bakery products, since it was found that these microbial biopolymers positively affect the texture of foods. Finally, the involvement of EPS in tolerance to stress conditions that are commonly encountered in fermented beverages such as wine is discussed.

Short communication: 3-Phenyllactic acid production in milk by Pediococcus pentosaceus SK25 during laboratory fermentation process

3-Phenyllactic acid (PLA) is a broad-spectrum antimicrobial compound, produced by a wide range of lactic acid bacteria. A novel lactic acid bacteria strain with high PLA-producing ability, Pediococcus pentosaceus SK25, was isolated from traditional Chinese pickles. When grown in de Man, Rogosa, Sharpe broth at 30°C for 36h, this strain produced 135.6mg/L of PLA. Using this strain as starter for milk fermentation, 47.2mg/L of PLA was produced after fermentation for 12h. The PLA production was significantly improved by phenylalanine supplement, but was completely inhibited by tyrosine supplement.

A Screening of Class IIa Bacteriocins Produced by Lactic Acid Bacteria Isolated from Fermented Mare Milk

AbstractTwelve fermented mare milk samples were screened for class IIa bacteriocins based on a polymerase chain reaction (PCR) method. The positive PCR products were cloned and sequenced. The sequences shared 99% identity to enterocin A‐coding gene. The lactic acid bacteria, which showed positive PCR results, were detected with antimicrobial activity by means of traditional well‐diffusion method. The strains C71 and C102 showed antimicrobial activity to Listeria sp. These strains showed a potential application in food preservation. The results also indicated the PCR screening method proposed in this study can be used for the screening of antimicrobial peptides of class IIa.Practical ApplicationsIn the present study, 44 class IIa bacteriocins from a wide range of lactic acid bacteria were included in a cluster analysis, and primers were designed on basis of the cluster analysis. We screened class IIa bacteriocin‐producing lactic acid bacteria that were isolated from traditional fermented mare milk by means of a PCR‐based assay. Two strains showed potential application in food preservation. The results indicated the PCR screening method proposed in this study can be used for the screening of antimicrobial peptides of class IIa. This method is still in an exploratory stage. It needs to be improved in the future.

Anti‐listerial and Anti‐biofilm Activities of Potential Probiotic Lactobacillus Strains Isolated from Tunisian Traditional Fermented Food

AbstractThree Lactobacilli were isolated from traditional Tunisian fermented food and then characterized for its ability to inhibit Listeria monocytogenes growth. Antagonistic effect of Lactobacillus plantarum on four L. monocytogenes strains was tested in soft artisanal cheese. Anti‐biofilm activity of Lactobacillus extracts was also tested.Our results demonstrate that the selected lactic acid bacteria (LAB) extract exhibited a good antibacterial effect against L. monocytogenes ATCC 19115 with low MICs values: 8.33 ± 2.1, 20 ± 1.2 and 23.33 ± 1, respectively. L. plantarum also exhibited a stronger inhibitory effect against L. monocytogenes when grown in cheese. Moreover, a potential anti‐biofilm effect of the three LAB extracts with BIC50 values ranging from 5% to 15% for L. monocytogenes ATCC 19115 was demonstrated. Although LAB extracts were able to eradicate significantly a preformed L. monocytogenes biofilm (P < 0.05). Growth inhibition of preformed biofilm was more difficult to achieve. LAB could be used as a bioprotective culture in cheese ripening to prevent Listeria growth.Practical ApplicationsA wide range of lactic acid bacteria (LAB) produce bacteriocins, which were essentially active against the food‐borne pathogen Listeria monocytogenes. 2, 3‐bis (2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐5‐[(phenylamino) carbonyl]‐2H‐tetrazolium hydroxide (XTT) reduction method was used to assess the anti‐biofilm and anti‐listerial activity of LAB extract. XTT reduction assay is dependent on the microbial respiratory activity.Since XTT is used for quantification of the microbial respiratory activity, it was used to quantify viable mature biofilm and to compare LAB activity against planktonic and biofilm cells.

Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food

Ting is a spontaneously fermented sorghum food that is popular for its sour taste and unique flavour. Insight of the microbial diversity and population dynamics during sorghum fermentations is an essential component of the development of starter cultures for commercial production of ting. In this study, bacterial populations associated with spontaneous sorghum fermentations were examined using a culture-independent strategy based on denaturing gradient gel electrophoresis and sequence analysis of V3-16S rRNA gene amplicons, and a culture-dependent strategy using conventional isolation based on culturing followed by 16S rRNA and/or pheS gene sequence analysis. The entire fermentation process was monitored over a 54 h period and two phases were observed with respect to pH evolution and microbial succession. The first phase of the process (0–6 h) was characterized by relatively high pH conditions and the presence of Enterococcus mundtii, albeit that this species was only detected with the culture-dependent approach. The second phase of the fermentation process (12–54 h) was characterized by increased acidity and the predominance of a broader range of lactic acid bacteria, including Lactococcus lactis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Weissella cibaria, Enterococcus faecalis, and a close relative of Lactobacillus curvatus, as well as some members of the Enterobacteriaceae family. The Lb. curvatus-like species was only detected with PCR-DGGE, while the majority of the other species was only detected using the culture-dependent approach. These findings highlighted the fact that a combination of both approaches was essential in revealing the microbial diversity and dynamics during spontaneous sorghum fermentations.

The effect of bacteriocin-producing Lactobacillus plantarum strains on the intracellular pH of sessile and planktonic Listeria monocytogenes single cells

A wide range of lactic acid bacteria (LAB) produce bacteriocins mainly active against other closely related LAB, but some bacteriocins are also active against the food-borne pathogen Listeria monocytogenes. With the aim of increasing food safety it has thus been considered to utilise bacteriocins and/or bacteriocin-producing LAB as “natural” food preservatives in foods such as cheese, meat and ready-to-eat products. Some strains of Lactobacillus plantarum produce bacteriocins termed plantaricins. Using a single-cell based approach, the effect on the intracellular pH as a measure of the physiological state of sessile and planktonic L. monocytogenes (strains EGDe and N53-1) during co-culturing with plantaricin-producing L . plantarum (strains BFE 5092 and PCS 20) was investigated using fluorescence ratio imaging microscopy (FRIM). Mono-cultures of L. monocytogenes were used as control. Expression levels of plantaricin-encoding genes by sessile and planktonic L . plantarum were determined using qRT-PCR. L. plantarum BFE 5092 possesses the genes for plantaricin EF, JK and N, while L . plantarum PCS 20 contains the genes for plantaricin EF, although determination of the nucleotide sequence of the PCS 20 plantaricin E gene showed that this peptide is probably non-functional. When cultured as mono-culture, both L. monocytogenes strains maintained pH i at a constant level around 7.2–7.6 throughout the experiment, independently of the matrix. On a solid surface, L . plantarum BFE 5092 strongly affected pH i of L. monocytogenes N53-1 with only 20% of the cells being able to maintain pH i in the physiological optimal range with pH > 7 and 52% of the cells with pH i ~ pH ex, showing that the cells had no proton gradient towards the environment. The effect on L. monocytogenes EGDe was less pronounced, but still notable. L. plantarum PCS 20 left both strains of L. monocytogenes virtually unaffected when co-cultured on a solid surface. In liquid, both L . plantarum strains strongly affected the physiological state of L. monocytogenes EGDe as judged by pH i, whereas L. monocytogenes N53-1 was left virtually unaffected after 5 h of co-culturing and after 8 h 50% of the cells still maintained pH i ≥ 7. Higher concentrations of lactic acid were produced in liquid compared to a solid surface, and the different response of EGDe and N53-1 to the activities of the two L . plantarum strains probably reflect higher susceptibility of L. monocytogenes EGDe to organic acids compared to L. monocytogenes N53-1. Taken together, our results may be explained by the difference in the range of plantaricins produced by the two L . plantarum strains and matrix- and strain-related differences in the susceptibility of L. monocytogenes to plantaricins and organic acids. In conclusion, the present study represents the first demonstration of the ability of a bacteriocin-producing LAB to dissipate the proton gradient of sessile and planktonic L. monocytogenes.

Comparative study of the pln locus of the quorum-sensing regulated bacteriocin-producing L. plantarum J51 strain

Lactobacillus plantarum J51 strain was isolated from a Rioja red wine and it showed bacteriocin activity against a wide range of lactic acid bacteria of oenological importance. These characteristics conferred L. plantarum J51 a high interest both in wine microbiology and in the study of bacteriocin production. In this work the bacteriocin production regulated under the “quorum-sensing” mechanism is observed and the pln locus of the bacteriocin-producing L. plantarum J51 is fully characterized. A 20,667 bp fragment was completely sequenced (GenBank accession number DQ340868), and showed five operons ( plNC8βαc, plnLR-like, plnABCD, plnEFI, plnGHSTUVW) and a new region containing a putative operon with three new orfs that could encode a putative two-peptide bacteriocin.

Improvement of a nisin-inducible expression vector for use in lactic acid bacteria

The plasmid pMSP3535 is a popular vector for nisin-inducible expression of heterologous genes in lactic acid bacteria. However, the maximum protein expression level achievable with pMSP3535 is relatively low. In an effort to increase expression we modified pMSP3535 to create a high expression variant termed pMSP3535H2. Modifications included removal of a small NisA peptide fragment from the P nisA promoter and addition of a bidirectional transcription terminator. In addition the plasmid copy number was increased by replacing the pMSP3535 copy number control region with that of a high copy variant of the same replicon. As a result of these modifications, expression of two target proteins, the green fluorescent protein and the Escherichia coli antigen intimin, increased 5.0- and 7.5-fold, respectively. The increased range of inducible expression achieved with pMSP3535H2 will facilitate molecular studies in a range of lactic acid bacteria.

Population dynamics and antagonistic potential of enterococci colonizing the phyllosphere of grasses.

To investigate the spatial and temporal dynamics of enterococci colonizing forage grass and their ability to produce bacteriocins. Enterococci could be detected on above-ground plant parts throughout the growing season, with high continuity but low cell numbers (2.60 x 101-6.16 x 104 cfu g-1 fresh matter). A total of 750 strains were isolated and identified by their whole-cell protein patterns as Enterococcus faecalis (7.9%), Ent. mundtii (7.9%), Ent. casseliflavus (5.5%), Ent. faecium (5.2%) and Ent. sulfureus (0.1%). The vast majority of the strains (69.7%) formed a homogeneous 16S rDNA genotype that differed from those of known enterococci. A screening for antagonistic activity using an agar spot test revealed that 18.4% of all isolates were potential antagonists. Partially-purified proteins extracted from cell-free culture supernatant fluids of various species were characterized as pH- and heat-stable bacteriocins active against a wide range of lactic acid bacteria, clostridia and Listeria. The producing strains were antagonistically active even on 'phylloplane agar' at temperatures between 4 and 37 degrees C. Enterococci are a common part of the epiphytic microflora of grasses, displaying probably some antagonistic activity. The results provide new information on the distribution, species diversity and antagonistic potential of enterococci in the phyllosphere.