Leuconostoc Strains Research Articles

Transposons Tn916 and Tn925 can transfer from Enterococcus faecalis to Leuconostoc oenos

The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oenos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Lactococcus lactis LM2301. Further analysis of L. lactis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no efficient conjugal or transformation systems previously described for this microorganism.

Identification and sequence analysis of IS1297, an ISS1-like insertion sequence in a Leuconostoc strain

The insertion sequence (IS) ISS1 from Lactococcus lactis was amplified from lactococcal genomic DNA using a primer to the 18-bp inverted repeat sequence. The amplified product hybridized to a single EcoRI fragment in a total genomic DNA digest of Leuconostoc mesenteroides ssp. dextranicum NZDRI 2218. The DNA sequence of this ISS1-like element (IS1297) and the Le. mesenteroides sequences flanking the IS were determined and compared with other iso-ISS1 elements. No direct repeats were found immediately flanking IS1297; however, direct repeats were present approximately 60 bp on either side of the insertion site. IS1297 contained a major open reading frame (ORF) of 681 bp, encoding a putative 226-amino-acid protein with 96.5% homology to the presumed transposase of ISS1. An overlapping ORF of 174 bp in the same orientation was also present. A putative ORF in the opposite orientation to the transposase ORF, which has been shown in some iso-ISS1 elements, was not present in IS1297. IS1297 was shown to hybridize with other dairy Leuconostoc strains. This is the first sequence of an ISS1-like element from a genus other than Lactococcus; however, ISI297 has close similarity to the lactococcal iso-ISS1 elements, especially the iso-ISS1 element from the lactose plasmid, pTDI.

Differentiation of dextran-producing Leuconostoc strains from fermented rice cake ( puto) using pulsed-field gel electrophoresis

Lactic acid bacteria were isolated from puto, a fermented rice cake consumed as a breakfast and snack food in the Philippines. The microflora was dominated by dextran-producing leuconostocs, and these were differentiated into four groups using pulsed-field gel electrophoresis of restriction enzyme digested chromosomal DNA, in conjunction with taxonomic tests. The four groups corresponded to the species Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc citreum and Leuconostoc fallax. Several strains showed an unusual clumping phenotype, and two of these were capable of inhibiting other strains of lactic acid bacteria.

Identification of Leuconostoc oenos from South African fortified wines by numerical analysis of total soluble cell protein patterns and DNA‐DNA hybridizations

Eight ethanol‐tolerant (17% v/v) strains of Leuconostoc were isolated from South African fortified wines which underwent spoilage in the bottle. The levels of genotypic relatedness of these strains to Leuconostoc oenos, Leuconostoc lactis, Leuconostoc argentinum, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc mesenteroides subsp. dextranicum and Leuconostoc paramesenteroides (recently reclassified as Weissella paramesenteroides) were determined by performing numerical analysis of total soluble cell protein patterns and DNA‐DNA hybridizations. The fortified wine strains grouped at r≥ 0.90 into one cluster with the reference strains of Leuc. oenos and shared a DNA homology of 82–95% with the type strains of Leuc. oenos (NCFB 1674T). The fortified wine strains were genotypically unrelated to the non‐acidophilic Leuconostoc spp.

Characterisation of four Leuconostoc bacteriophages isolated from dairy fermentations

Four bacteriophages (phages) growing on the same Leuconostoc strain were characterised. Electron micrographs showed these phages to be similar in morphology to the commonly isolated lactococcal phages with head diameters ranging from 49–55 nm and tail lengths of 117–131 nm. A distinctive base plate and collar were also present. From restriction enzyme analysis of purified phage DNA, the genome sizes were 23–29 kb. All four phages showed one major structural protein (of approximately 24 kDa) on SDS polyacrylamide gels. Hybridization experiments confirmed that the phages belonged to the same homology group. There was no homology between DNA from these phages and DNA from a prolate or small isometric lactococcal phage.

Characterisation of fourLeuconostocbacteriophages isolated from dairy fermentations

Four bacteriophages (phages) growing on the same Leuconostoc strain were characterised. Electron micrographs showed these phages to be similar in morphology to the commonly isolated lactococcal phages with head diameters ranging from 49–55 nm and tail lengths of 117–131 nm. A distinctive base plate and collar were also present. From restriction enzyme analysis of purified phage DNA, the genome sizes were 23–29 kb. All four phages showed one major structural protein (of approximately 24 kDa) on SDS polyacrylamide gels. Hybridization experiments confirmed that the phages belonged to the same homology group. There was no homology between DNA from these phages and DNA from a prolate or small isometric lactococcal phage.

Evaluation of three disk tests for identification of enterococci, leuconostocs, and pediococci

Simple rapid tests for presumptive identification of catalase-negative non-beta-hemolytic cocci (i.e., enterococci, leuconostocs, and pediococci) have not previously been available. Seven hundred thirty-four strains of aerobic and facultatively anaerobic, catalase-negative, non-beta-hemolytic gram-positive cocci were tested for susceptibility to vancomycin (Vans) by a screening procedure and production of leucine aminopeptidase (LAPase) and pyrrolidonylarylamidase (PYRase) in disk tests. Three unique patterns of activity in response to the three disks (30 micrograms of vancomycin, PYRase, and LAPase) can be used to presumptively identify the vancomycin-resistant (Vanr) enterococci (Vanr and PYRase and LAPase positive), leuconostocs (Vanr and PYRase and LAPase negative), and pediococci (Vanr, PYRase negative, and LAPase positive). The results indicate that, together with Gram stain characteristics and the catalase test, the vancomycin, LAPase, and PYRase disk tests can be used to presumptively identify Vanr strains of enterococci as well as Leuconostoc and Pediococcus strains from human infections.

Sequence analysis, distribution and expression of an aminopeptidase N-encoding gene from lactobacillus helveticus CNRZ32

Lactobacillus (Lb.) helveticus CNRZ32 possesses a 97-kDa metalloenzyme with aminopeptidase activity (PepN; EC 3.4.11.2). A 3.8-kb fragment encoding PepN was cloned into pIL253 and designated pSUW34. Transformation of Lactococcus (Lc.) lactis LM0230 with pSUW34 resulted in > 180-fold increase in general aminopeptidase (AP) activity using L-lysine-p-nitroanilide. Southern hybridization was conducted to determine the distribution of homology to the CNRZ32 pepN gene among lactic-acid bacteria (LAB). Hybridization was observed with strains of lactobacilli, pediococci, leuconostoc, streptococci and lactococci. The pepN gene was sequenced and found to encode a protein containing 844 amino acid (aa) residues. A comparison of Lb. helveticus CNRZ32 pepN to Lb. delbrueckii ssp. lactis DSM7290 pepN indicated 69.5% nucleotide (nt) identity and 71.8% aa identity, while comparison to pepN from Lc. lactis ssp. cremoris MG1363 indicated 61.1% nt identity and 49.2% aa identity. Alignment of peptidase aa sequences of LAB, Escherichia coli, yeast and mammalian origin display homology in the zinc-binding domain, as well as a conserved region upstream from the putative active site.

Principal characteristics of β-galactosidase from Leuconostoc spp.

Abstract β-Galactosidase (β-gal) and phospho-β-galactosidase (P-β-gal) from 33 strains belonging to different species of Leuconostoc have been investigated. P-β-gal was not detected, suggesting that the lactose transport system by translocation (PEPP-TS) is not functional in Leuconostoc. A great number of strains possess a high level of β-gal in the presence of glucose. Characterization of β-gal from several strains of Leuconostoc, in comparison with an industrial strain of S. thermophilus which was used as a reference, showed that the β-gal of Leuconostoc strains has a pH optimum at 7.2. The temperature optimum varied with the strain, ranging from 43 to 58 °C. The Km values of the enzymes on substrate o-nitrophenyl-β- d -galactopyranoside (ONPG) varied from 0.68 to 4.72 m m . The Leuconostoc β-gal had a lower affinity for lactose than that of S. thermophilus. Comparison of SDS-PAGE protein profiles for a Lac− mutant and its wild Lac+ parental strain allowed the apparent molecular weight of β-gal to be estimated at approximately 80 000 for Leuc. mesenteroides subsp. mesenteroides, which is different from that of the β-gal of Leuconostoc lactis.

Bacteriocins of Leuconostocs isolated from meat

Several bacteriocin-producing Leuconostoc strains have been isolated from meat and identified as Leuconostoc gelidum UAL 187, Leuconostoc paramesenteroides-La7a, Leuconostoc carnosum-Ta11a and Leuconostoc carnosum-La54a. All strains produce bacteriocins that are active against Listeria monocytogenes and other lactic acid bacteria of concern in meat spoilage. All of the bacteriocins studied are heat stable in acidic environments and are inactivated by a range of proteolytic enzymes but not by catalase or lysozyme. Most are detected early in the growth cycle and are produced at refrigeration temperatures and in a pH range of 4.0–7.0. Leucocin A-UAL187, produced by Leuconostoc gelidium UAL 187, is a small peptide (MW 3930) translated as a 61 amino acid prepeptide consisting of a 24 amino acid leader region and 37 amino acid active bacteriocin that is secreted. A probe designed from a region of the leucocin gene has been used to locate the bacteriocin genes in the other strains (La7a, La54a and Ta11a). Strong hybridization signals were detected from 8.9 MDa, 32 MDa and 8.9 MDa plasmids in strains La7a, La54a and Ta11a, respectively. The bacteriocin structural gene from Leuconostoc carnosum-Ta11a (leucocin B-Ta11a) has been cloned and sequenced and the bacteriocin shows 100% homology to leucocin A-UAL187; however, the prepeptide differs in six residues. The mature extracellular bacteriocin from strain UAL 187 was purified and characterized by precipitation, gel filtration, hydrophobic interaction chromatography followed by RP-HPLC and amino-terminal sequencing, whilst those of the other strains are in the process of being purified and characterized using similar techniques. Preliminary results indicate that they are all small peptides in the range of 3.5–4.0 kDa.

Effect of pH and the bacteriocin carnocin 54 on growth and cell morphology of two Leuconostoc strains

Leuconostoc carnosum LA54A produces carnocin 54, a bacteriocin inhibitory to Listeria and closely related lactic acid bacteria. The effects of the pH of cell-free LA54 culture supernatants on the antibacterial activity of carnocin 54 was assessed using Leuc. mesenteroides DSM 20343 and TA10C as indicator strains. Carnocin 54 showed greatest activity against both strains at pH 4.5. At pH 6.5, activity was reduced, especially against Leuc. mesenteroides TA10C. Scanning electron microscopy showed irregular and rough surfaces on bacteriocin-treated cells at both pH values.

Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non‐growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1‐1 and decreased at higher concentrations. The main degradation products resulting from the co‐metabolism of citrate (10 mmol 1‐1) and glucose (2 mmol 1‐1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.

INHIBITION OF STAPHYLOCOCCUS AUREUS IN BUFFER, CULTURE MEDIA AND FOODS BY LACIDIN A, A BACTERIOCIN PRODUCED BY LACTOBACILLUS ACIDOPHILUS OSU133

ABSTRACTThirty seven strains of lactobacilli and leuconostocs were screened for bacteriocin production. A bacteriocin‐like substance (designated lacidin A) was detected in culture filtrate of Lactobacillus acidophilus OSU133. Lacidin A inhibited Enterococcus faecalis, L. acidophilus, Lactobacillus delbrueckii subsp. lactis, and Staphylococcus aureus. The bacteriocinogenic nature of lacidin A was confirmed by eliminating the inhibitory effects of acid, hydrogen peroxide and phage and by loss of activity upon hydrolysis with proteolytic enzymes. The bacteriocin is relatively heat‐stable and has considerable bactericidal action against S. aureus in phosphate buffer. Lacidin A also demonstrated bacteriostatic action against S. aureus in brain heart infusion and heat‐treated milk and liquid whole egg.

Properties of two bacteriocins synthesized byLeuconostoc strains

Mesenterocin 52 (Mes-52), was produced byLeuconostoc mesenteroides ssp.mesenteroides strain FR52 and dextranicin 24 (Dex-24) byLeuconostoc mesenteroides ssp.dextranicum strain J24. Dex-24 had a very narrow spectrum of antibacterial activity: it inhibited only some other strains ofLeuconostoc, contrary to Mes-52, which antagonized alsoListeria andEnterococcus sp. Mes-52 was partially purified by ammonium sulfate precipitation, gel filtration, cation exchange, and hydrophobic interaction chromatography. Mes-52 was biosynthesized and excreted into the medium during growth phase. At different temperatures media, there was an inverse relationship between the final bacteriocin-specific productivity and the mean growth rate. After novobiocin treatment, different mutants of strain FR 52 were obtained: nonproducing-sensitive (Bac−Imm−) and nonproducingimmune (Bac−Imm+) strains. Many changes in the plasmid profiles of several mutants were detected by electrophoresis.

Numerical taxonomy and identification of lactic acid bacteria from spoiled, vacuum-packaged vienna sausages.

Sixty-one lactic acid bacteria from spoiled vacuum-packaged vienna sausages and 15 reference strains were tested for 72 phenotypic characteristics. An identification key and a computer data base, both specific for lactic acid bacteria from meat sources, were used for identification and the results were compared. There was a high correlation (86.9%) between the two procedures in the identification of strains to genus level. However, only a 54.8% correlation was obtained in identifying strains to the species level. With numerical taxonomy (Ssm matching coefficient with average linkage clustering) 60 strains were recovered in six clusters at the 89% similarity level. While most Leuconostoc strains clustered separately from the Lactobacillus strains, the identity of many leuconostocs was not clarified. The presence of a heterogeneous cluster containing typical and 'atypical' strains of the Lactobacillus saké/curvatus group and a separate homogeneous Lact. curvatus cluster was noted. Closer examination of the data suggested that the 'atypical' lactobacilli were all strains of Lact. saké.

Antibacterial activity of three Leuconostoc strains isolated from vacuum‐packaged processed meats

One hundred and fifty lactic acid bacteria (LAB) isolated from vacuum-packaged processed meats were screened for antagonistic activity against various food spoilage microorganisms and foodborne pathogens. Nineteen strains produced bacteriocins active against closely related LAB and Listeria strains. Leuconostoc carnosum (LA54a and TA26b) and Leuconostoc mesenteroides subspecies dextranicum (TA33a) produced bacteriocins that were susceptible to proteolytic enzymes, but not to catalase, lysozyme or chloroform. They were heat stable up to 100 degrees C for thirty minutes at pH 2 to 7, and exerted a bacteriolytic effect. Bacteriocin production by all Leuconostoc strains was growth associated, occurring at incubation temperatures of 0 degrees C to 30 degrees C and initial medium pH 4.5 to 7.5. Probing of plasmid DNA from the three Leuconostoc strains with an oligonucleotide probe homologous to the nucleotide sequence of leucocin A-UAL 187 indicated plasmid-mediated bacteriocin production. Homology of the three Leuconostoc bacteriocin-coding genes to the amino-terminal end of the leucocin A-UAL 187 gene from Leuconostoc gelidum UAL 187 is therefore suggested. This evidence implies that all three Leuconostoc strains produce type 2, Listeria active bacteriocins.

Selective enumeration of Leuconostoc on Vancomycin agar media

Two selective agar media for Leuconostoc enumeration and/or isolation were obtained by adding Vancomycin to MRS or NL media. Under the conditions described, Leuconostoc strains were able to grow on these media, while Lactococcus strains do not. Comparison of colony counts showed no difference with the MRS medium. Enumeration of Leuconostoc mesenteroides subsp. cremoris in mixed culture with Lactococcus strains was performed successfully using these media. In a milk medium, mixed cultures with lac+ prt+ strains of Lactococcus· showed a rapid decrease of the proportion of Leuconostoc strain in the total bacterial population.

Comparison of Leuconostoc oenos Strains by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis of chromosomal DNA digested with NotI or SfiI was used to differentiate individual strains of Leuconostoc oenos. L. oenos isolates with 13 different restriction digest patterns were detected in New Zealand wines undergoing malolactic fermentation. The average genome size was estimated to be 1,800 kb.

Leuconostoc argentinum sp. nov., Isolated from Argentine Raw Milk

The levels of genotypic relatedness among seven strains of Leuconostoc spp. isolated from Argentine raw milk, Leuconostoc lactis DSM 20202T (T = type strain) and DSM 20198, Leuconostoc mesenteroides subsp. mesenteroides NCDO 523T, Leuconostoc mesenteroides subsp. dextranicum NCDO 529T, and Leuconostoc paramesenteroides DSM 20288T were determined by performing a numerical analysis of total soluble cell protein patterns and DNA-DNA hybridization data. The Argentine raw milk strains formed a tight genotypic cluster at an r value of = 0.85 and exhibited low levels of DNA homology with the other species included in this study. These strains represent a new species within the group of leuconostocs isolated from milk or dairy products. The name Leuconostoc argentinum sp. nov. is proposed; the type strain is strain LL76.

Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides FR 52.

One hundred and sixty-five isolates of Leuconostoc spp. were tested for bacteriocin production. Only one strain, Leuc. mesenteroides ssp. mesenteroides FR 52, isolated from a raw milk, produced a bacteriocin which was named Mesenterocin 52. This bacteriocin inhibited other Leuconostoc strains and several strains of Enterococcus and Listeria spp. No activity was found against lactococci and lactobacilli. The antibacterial spectrum differed from that of previously described Leuconostoc bacteriocins. Mesenterocin 52 was secreted into the medium during the growth phase. It was inactivated with protease treatments. At pH 7.0 it had a relative stability after heating at 100 degrees C (15 min), but it had a greater stability at pH 4.5 than at pH 7.0 after 6 h at 80 degrees C. The apparent molecular mass was estimated to be less than 10 kDa by ultrafiltration. Mesenterocin 52 showed a bactericidal effect on Leuconostoc paramesenteroides DSM 20288.