Leuconostoc mesenteroides SJRP55: A Bacteriocinogenic Strain Isolated from Brazilian Water Buffalo Mozzarella Cheese

The production of bacteriocins by Leuconostoc mesenteroides represents an important opportunity for exploration of their potential use for industrial purpose. The antimicrobial compounds produced by L. mesenteroides subsp. mesenteroides SJRP55 strain were characterized and purified. Cell-free supernatant of Leuc. mesenteroides subsp. mesenteroides SJRP55 produced antibacterial compounds against Listeria spp. strains and not inhibiting against Lactobacillus spp. The antimicrobial substances were stable at high temperatures (100 °C for 2 h and 121 °C for 20 min) and low pH (pH 2–4) values, but sensitive to proteolytic enzymes and resistant to α-amylase, lipase and catalase enzymes. The optimal temperature for active peptides production was 25 °C. The antimicrobial compounds were purified by ammonium sulfate precipitation, affinity column and reverse-phase chromatography. Mass spectrometry and amino acids analyses showed that the bacteriocins were identical to mesentericin Y105 and B105. The producer strain’s DNA analysis revealed presence of open reading frames possibly coding for virulence factors, such as enterococcal surface protein (esp), collagen adhesion (ace) and intrinsic vancomycin resistance (vanA); however, biogenic amines encoding genes were not observed. Leuc. mesenteroides subsp. mesenteroides SJRP55 is a promising biopreservative culture in fermented milk, and the purified bacteriocins can also be applied in food preservation.     

Instability of lactose and citrate metabolism of Leuconostoc strains

Summary The instability of Lac⁺ and Cit⁺ phenotypes was investigated inLeuconostoc mesenteroides subsp.cremoris ATCC 19245 and in four strains ofLeuconostoc mesenteroides subsp.dextranicum. The two phenotypes were linked respectively to a 14 Mdal and a 34 Mdal plasmid in Leuconostoc mesenteroides subsp.cremoris ATCC 19245. InLeuconostoc mesenteroides subsp.dextranicum the character Lac⁺ was linked to a 28 Mdal plasmid, while the Cit⁺ phenotype was stable.     

Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

Aims: To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent‐cell preparation and electroporation conditions were optimized. Methods and Results: Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pLeuCM was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (100‐fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency (1 × 10⁴ or more transformants per μg DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0·25 mol l^?1 sucrose and 0·8 μg ml^?1 penicillin G, followed by treatment with 600 U ml^?1 lysozyme and electroporation at a field strength of 10 kV cm^?1. When this protocol was used to transform pLeuCM into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. Conclusions: The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. Significance and Impact of the Study: These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria.     

Behavior of Psychrotrophic Lactic Acid Bacteria Isolated from Spoiling Cooked Meat Products

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10°C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10°C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4°C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10⁸ CFU/g at 10°C after 7 to 12 days.     

Antibiotic Susceptibility Profiles of Dairy Leuconostoc,Analysis of the Genetic Basis of Atypical Resistances and Transfer of Genes In Vitro and in a Food Matrix

In spite of a global concern on the transfer of antibiotic resistances (AR) via the food chain, limited information exists on this issue in species of Leuconostoc and Weissella, adjunct cultures used as aroma producers in fermented foods. In this work, the minimum inhibitory concentration was determined for 16 antibiotics in 34 strains of dairy origin, belonging to Leuconostoc mesenteroides (18), Leuconostoc citreum (11), Leuconostoc lactis (2), Weissella hellenica (2), and Leuconostoc carnosum (1). Atypical resistances were found for kanamycin (17 strains), tetracycline and chloramphenicol (two strains each), and erythromycin, clindamycin, virginiamycin, ciprofloxacin, and rifampicin (one strain each). Surprisingly, L. mesenteroides subsp. mesenteroides LbE16, showed resistance to four antibiotics, kanamycin, streptomycin, tetracycline and virginiamycin. PCR analysis identified tet(S) as responsible for tetracycline resistance in LbE16, but no gene was detected in a second tetracycline-resistant strain, L. mesenteroides subsp. cremoris LbT16. In Leuconostoc mesenteroides subsp. dextranicum LbE15, erythromycin and clindamycin resistant, an erm(B) gene was amplified. Hybridization experiments proved erm(B) and tet(S) to be associated to a plasmid of ≈35 kbp and to the chromosome of LbE15 and LbE16, respectively. The complete genome sequence of LbE15 and LbE16 was used to get further insights on the makeup and genetic organization of AR genes. Genome analysis confirmed the presence and location of erm(B) and tet(S), but genes providing tetracycline resistance in LbT16 were again not identified. In the genome of the multi-resistant strain LbE16, genes that might be involved in aminoglycoside (aadE, aphA-3, sat4) and virginiamycin [vat(E)] resistance were further found. The erm(B) gene but not tet(S) was transferred from Leuconostoc to Enterococcus faecalis both under laboratory conditions and in cheese. This study contributes to the characterization of AR in the Leuconostoc-Weissella group, provides evidence of the genetic basis of atypical resistances, and demonstrates the inter-species transfer of erythromycin resistance.     

Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains

A set of 83 lytic dairy bacteriophages (phages) infecting flavor-producing mesophilic starter strains of the Leuconostoc genus was characterized, and the first in-depth taxonomic scheme was established for this phage group. Phages were obtained from different sources, i.e., from dairy samples originating from 11 German dairies (50 Leuconostoc pseudomesenteroides [Ln. pseudomesenteroides] phages, 4 Ln. mesenteroides phages) and from 3 external phage collections (17 Ln. pseudomesenteroides phages, 12 Ln. mesenteroides phages). All phages belonged to the Siphoviridae family of phages with isometric heads (diameter, 55 nm) and noncontractile tails (length, 140 nm). With the exception of one phage (i.e., phage ΦLN25), all Ln. mesenteroides phages lysed the same host strains and revealed characteristic globular baseplate appendages. Phage ΦLN25, with different Y-shaped appendages, had a unique host range. Apart from two phages (i.e., phages P792 and P793), all Ln. pseudomesenteroides phages shared the same host range and had plain baseplates without distinguishable appendages. They were further characterized by the presence or absence of a collar below the phage head or by unique tails with straight striations. Phages P792 and P793 with characteristic fluffy baseplate appendages could propagate only on other specific hosts. All Ln. mesenteroides and all Ln. pseudomesenteroides phages were members of two (host species-specific) distinct genotypes but shared a limited conserved DNA region specifying their structural genes. A PCR detection system was established and was shown to be reliable for the detection of all Leuconostoc phage types.     

Differentiation of Dextran-Producing Leuconostoc Strains by a Modified Randomly Amplified Polymorphic DNA Protocol

Seven dextran-producing Leuconostoc strains were differentiated by using a modified randomly amplified polymorphic DNA (RAPD) protocol that incorporated specific primers designed from conserved regions of dextransucrase genes. RAPD profiles showed intraspecies differences among the Leuconostoc mesenteroides strains tested. This modified RAPD protocol will aid in the differentiation of polymer-producing leuconostocs, which are currently distinguished by time-consuming analyses of the dextrans they synthesize.     

Comparative genome analysis provides shreds of molecular evidence for reclassification of Leuconostoc mesenteroides MTCC 10508 as a strain of Leu. suionicum

This study presents the whole-genome comparative analysis of a Leuconostoc sp. strain, previously documented as Leu. mesenteroides MTCC 10508. The ANI, dDDH, dot plot, and MAUVE analyses suggested its reclassification as a strain of Leu. suionicum. Functional annotation identified a total of 1971 genes, out of which, 265 genes were mapped to CAZymes, evincing its carbohydrate transforming capability. The genome comparison with 59 Leu. mesenteroides and Leu. suionicum strains generated the core and pan-genome profiles, divulging the unique genes in Leuconostoc sp. MTCC 10508. For the first time, this study reports the genes encoding alpha-xylosidase and copper oxidase in a strain of Leu. suionicum. The genetic information for any possible allergenic molecule could not be detected in the genome, advocating the safety of the strain. The present investigation provides the genomic evidence for reclassification of the Leuconostoc sp. strain and also promulgates the molecular insights into its metabolic potential.     

Specific detection of Leuconostoc mesenteroides subsp. mesenteroides with DNA primers identified by randomly amplified polymorphic DNA analysis

Randomly amplified polymorphic DNA analysis using primer 239 (5' CTGAAGCGGA 3') was performed to characterize Leuconostoc sp. strains. All the strains of Leuconostoc mesenteroides subsp. mesenteroides (with the exception of two strains), two strains formerly identified as L. gelidum, and one strain of Leuconostoc showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L. mesenteroides subsp. mesenteroides strains.     

pH Homeostasis and Citric Acid Utilization: Differences Between Leuconostoc mesenteroides and Lactococcus lactis

This study presents the effects of citric acid and extracellular pH (pH_e) on the intracellular pH (pH_i) of wild-type and citrate negative variants (cit⁻) Leuconostoc mesenteroides subsp. mesenteroides (Ln. mesenteroides M) and Lactococcus lactis subsp. lactis bv. diacetylactis (L. lactis LD). A recent method using a pH-sensitive fluorescent indicator carboxyfluorescein succinimidyl ester (cFSE) was adapted to measure the pH_i of these two lactic acid bacteria in resting cells. Energized cells with 10 mM lactose of Ln. mesenteroides M and L. lactis LD modified their pH gradient (ΔpH) in the same manner; when the pH_e was decreased from 7 to 4, the pH_i decreased from 7 to about 5. The adjunction of 10 mM citric acid had no effect on the pH_i of wild-type and cit(−) variant of L. lactis LD, nor on the pH_i of Ln. mesenteroides cit(−) variant. Nevertheless, in Ln. mesenteroides M wild-type, citric acid utilization increased the pH_i, which was maintained at about 6.5–7.0 when the pH_e was decreased from 7 to 4. It could be concluded that citric acid allows the maintenance of pH homeostasis in Leuconostoc mesenteroides. Received: 7 March 1997 / Accepted: 14 April 1997     

Purification and N-Terminal Amino Acid Sequence of Dextranicin 24, a Bacteriocin of Leuconostoc sp.

Leuconostoc mesenteroides subsp. dextranicum strain J24 synthesized a bacteriocin named Dextranicin 24 (Dex-24), which inhibited only other Leuconostoc sp. strains. It was purified by a two-step procedure from the fraction of the bacteriocin bound to the producer cells at the end of the growth: desorption form the cells at acidic pH, followed by reserve phase HPLC. The N-terminal sequence of Dex-24 was the following: NH₂₋ K G V L G W L S M A S S A L T G P Q Q . . . Received: 26 January 1996 / Accepted: 28 March 1996     

Identification and Characterization of Leuconostoc carnosum, Associated with Production and Spoilage of Vacuum-Packaged, Sliced, Cooked Ham

Leuconostoc carnosum was shown to be the specific spoilage organism in vacuum-packaged, sliced, cooked ham showing spoilage during 3 weeks of shelf life. Identification of the specific spoilage organism was done by use of phenotypic data and ClaI, EcoRI, and HindIII reference strain ribopatterns. One hundred L. carnosum isolates associated with the production and spoilage of the ham were further characterized by pulsed-field gel electrophoresis (PFGE), together with some meat-associated Leuconostoc species: L. citreum, L. gelidum, L. mesenteroides subsp. dextranicum, and L. mesenteroides subsp. mesenteroides. ApaI and SmaI digests divided the industrial L. carnosum strains into 25 different PFGE types, ApaI and SmaI types being consistent. Only one specific PFGE type was associated with the spoiled packages. This type also was detected in air and raw-meat mass samples. The spoilage strain did not produce bacteriocins. Only seven isolates belonging to three different PFGE types produced bacteriocins. Similarity analysis of the industrial L. carnosum strains revealed a homogeneous cluster which could be divided into eight subclusters consisting of strains having at most three-fragment differences. The L. carnosum cluster was clearly distinguished from the other meat-associated leuconostoc clusters, with the exception of the L. carnosum type strain. Ribotyping can be very helpful in the identification of L. carnosum, but its discriminatory power is too weak for strain characterization. PFGE provides good discrimination for studies dealing with the properties of homogeneous L. carnosum strains.     

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese.     

DNA Probe for Lactobacillus delbrueckii

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-³²P-labeled DNA probe.     

Isolation and Characterization of Nisin-Resistant Leuconostoc mesenteroides for Use in Cabbage Fermentations

Leuconostoc mesenteroides strains that are resistant to high levels of nisin (up to 25,000 IU/ml in broth) were isolated. These nisin-resistant mutants were evaluated to determine their potential use as starter culture strains for cabbage fermentations. We found that some L. mesenteroides strains could be adapted to high levels of nisin resistance, while others could not. The nisin resistance trait was found to be stable for at least 35 generations, in the absence of nisin selection, for all mutants tested. The effects of nisin and salt, separately and in combination, on growth kinetics of the nisin-resistant strains were determined. Salt was the most influential factor on the specific growth rates of the mutants, and no synergistic effect between nisin and salt on specific growth rates was observed. The nisin-resistant strains were unimpaired in their ability to rapidly produce normal heterolactic fermentation end products. The use of these L. mesenteroides mutants as starter cultures in combination with nisin may extend the heterolactic phase of cabbage fermentations.     

Use of a Novel Escherichia coli-Leuconostoc Shuttle Vector for Metabolic Engineering of Leuconostoc citreum To Overproduce d-Lactate

Determination of the complete nucleotide sequence of a cryptic plasmid, pMBLT00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC13302 revealed that it contains 20,721 bp, a G+C content of 38.7%, and 18 open reading frames. Comparative sequence and mung been nuclease analyses of pMBLT00 showed that pMBLT00 replicates via the theta replication mechanism. A new, stable Escherichia coli-Leuconostoc shuttle vector, pMBLT02, which was constructed from a theta-replicating pMBLT00 replicon and an erythromycin resistance gene of pE194, was successfully introduced into Leuconostoc, Lactococcus lactis, and Pediococcus. This shuttle vector was used to engineer Leuconostoc citreum 95 to overproduce d-lactate. The L. citreum 95 strain engineered using plasmid pMBLT02, which overexpresses d-lactate dehydrogenase, exhibited enhanced production of optically pure d-lactate (61 g/liter, which is 6 times greater than the amount produced by the control strain) when cultured in a reactor supplemented with 140 g/liter glucose. Therefore, the shuttle vector pMBLT02 can serve as a useful and stable plasmid vector for further development of a d-lactate overproduction system in other Leuconostoc strains and Lactococcus lactis.     

The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.     

Purification of Leuconostoc mesenteroides Citrate Lyase and Cloning and Characterization of the citCDEFG Gene Cluster

A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the β subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the α subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD, citE, and citF, encoding, respectively, the three citrate lyase subunits γ (acyl carrier protein [ACP]), β (citryl-S-ACP lyase; EC 4.1.3.34), and α (citrate:acetyl-ACP transferase; EC 2.8.3.10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.Lactic acid bacteria of the genus Leuconostoc play important roles in the dairy industry because of their ability to produce carbon dioxide and C₄ aroma compounds through lactose heterofermentation and citrate utilization. The carbon dioxide produced is responsible for eye formation in certain types of cheese. Citrate utilization by these bacteria leads to the production of diacetyl, which is considered a main flavor compound of a range of fermented dairy products such as cultured butter, buttermilk, and cottage cheese.The citrate utilization by lactic acid bacteria requires specifically three enzymes involved in the conversion of citrate to pyruvate: a citrate permease, a citrate lyase, and an oxaloacetate decarboxylase. The energetic role of citrate metabolism in Leuconostoc mesenteroides has been recently described (24, 25). The citrate permease catalyzes an electrogenic exchange of divalent anionic citrate and monovalent lactate, resulting in the generation of a membrane potential (Fig. ​(Fig.1,1, reaction 1) (24, 25). The intracellular citrate is cleaved by a citrate lyase (EC 4.1.3.6), yielding acetate and oxaloacetate (Fig. ​(Fig.1,1, reactions 2 and 3). The oxaloacetate is decarboxylated into carbon dioxide and pyruvate in a reaction catalyzed by the enzyme oxaloacetate decarboxylase (Fig. ​(Fig.1,1, reaction 4). Open in a separate windowFIG. 1Citrate fermentation pathway in L. mesenteroides and role of the different subunits in the reaction catalyzed by citrate lyase (EC 4.1.3.6). The proteins involved are citrate permease (1), citrate lyase α subunit citrate:acetyl-ACP transferase (EC 2.8.3.10) (2), citrate lyase β subunit citryl–S-ACP lyase (EC 4.1.3.34) (3) oxaloacetate decarboxylase (4), acetate:SH-CL ligase (EC 6.2.1.22) (5), and lactate dehydrogenase (6). ACP, γ subunit of ACP; R, prosthetic group. Acetic anhydride is used for chemical specific acetylation of the prosthetic group. Acetic anhydride is an analog of the mixed anhydride of citric and acetic acids which corresponds probably to an intermediate analog in the acyl-exchange reaction (7a, 14a).Understanding of the molecular genetics of these lactic acid bacteria is not far advanced, and the genes encoding the enzymes citrate lyase and oxaloacetate decarboxylase are unknown.On the basis of previous studies (22, 33), the citrate lyase of Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc can be considered a functional complex (M_r, 585,000) composed of three proteins: α, β, and γ subunits in a stoichiometric relationship of 6:6:6. The structure and the mechanism of action are similar to those of the citrate lyase of Klebsiella pneumoniae, which has been extensively studied (1, 15, 16, 34, 36). The citrate lyase is active only if the thioester residue of the prosthetic group linked to its acyl carrier protein (ACP) (γ subunit) is acetylated. This activation is catalyzed by an acetate:SH-citrate lyase ligase (CL ligase) (EC 6.2.1.22), which converts HS-ACP with ATP and acetate into the acetyl-S-ACP (Fig. ​(Fig.1,1, reaction 5) (32). The α subunit replaces the acyl group with a citryl group to form the citryl-S-ACP (Fig. ​(Fig.1,1, reaction 2) (16). At last, the β subunit cleaves citryl-S-ACP into oxaloacetate and regenerates the acyl-S-ACP (Fig. ​(Fig.1,1, reaction 3) (16).Different mechanisms of regulation of citrate lyase have been reported, such as configurational changes, reversible covalent modification by acetylation-deacetylation, and phosphorylation-dephosphorylation (1, 2). In microorganisms like Klebsiella, in which the reactions of the tricarboxylic acid cycle are operative and therefore contain citrate synthase, a strict regulation of citrate lyase activity is necessary to avoid a futile cycle between citrate fermentation and the l-glutamate biosynthetic pathway. After citrate depletion from the growth medium or upon transfer from an anaerobic citrate medium to an aerobic glucose medium, the synthesis of l-glutamate from oxaloacetate and acetyl coenzyme A (CoA) via citrate can be ensured only if the citrate fermentation pathway is turned off. The intracellular l-glutamate concentration controls these pathways by modulating the activity of the citrate lyase complex (1, 2).An induction of citrate lyase activity has been observed in Leuconostoc but never in all Lactococcus strains tested (21, 26). In L. mesenteroides, the citrate lyase activity is induced by citrate and rapidly repressed after the citrate consumption in the medium. However, the regulation mechanisms remain unknown. In this paper, we report the purification of L. mesenteroides citrate lyase and an approach based on reverse genetics that yielded the full-length sequence of CL ligase and citrate lyase genes encoding the α, β, and γ subunits. The citrate lyase and CL ligase genes were sequenced and expressed in Escherichia coli.     

Influence of growth conditions on production and activity of mesenterocin 5 by a strain of Leuconostoc mesenteroides

The effects of various parameters on production and activity of mesenterocin 5, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides UL5, were investigated. Titres of bacteriocin and minimum inhibitory concentration values were determined by a critical dilution micromethod, using a sensitive strain of Listeria ivanovii as an indicator. Production of the antimicrobial compound was optimal at 37 and 40°C after 9 h of incubation, and was maximized in an aerobic fermentor maintained at pH 5.0. Tween 80 was a major factor in increasing mesenteroxin 5 production and specific production. Large quantities of bacteriocin could be obtained in whey and in whey permeate supplemented with yeast extract in the presence of the surfactant (0.1%). Most of the Listeria strains tested including L. monocytogenes were highly sensitive to the bacteriocin in the pH range 5.5 to 6.0 and at a temperature of 20 to 25°C.     

2-Hydroxy-dATP is incorporated opposite G by Escherichia coli DNA polymerase III resulting in high mutagenicity

Four kinds of oxidatively damaged DNA precursors, 8-hydroxydeoxyguanosine 5′-triphosphate (8-OH-dGTP), 2-hydroxydeoxyadenosine 5′-triphosphate (2-OH-dATP), 5-hydroxydeoxycytidine 5′-triphosphate (5-OH-dCTP) and 5-formyldeoxyuridine 5′-triphosphate (5-CHO-dUTP), were employed in in vitro gap-filling reactions of the supF gene conducted by the Escherichia coli DNA polymerase III holoenzyme, and these treated DNAs were transfected into various E.coli strains. When the manipulated DNAs were transfected into the repair-proficient strain, supF mutants were obtained much more frequently by the purine nucleotides than by the pyrimidine nucleotides (2-OH-dATP > 8-OH-dGTP >> 5-OH-dCTP ~ 5-CHO-dUTP). This result is in contrast to our previous observation that these four oxidatively damaged nucleotides induce chromosomal gene mutations with similar frequencies when incorporated directly into E.coli. 2-OH-dATP elicited G→T transversions, indicating the formation of G•2-OH-dATP pairs. These results demonstrate that 2-OH-dATP was highly mutagenic in this assay system containing the in vitro DNA synthesis by the E.coli replicative DNA polymerase, in addition to in the in vivo assay system reported previously. Slight increases in the mutant frequencies were observed when alkA (for 8-OH-dGTP and 2-OH-dATP) and mutY (for 2-OH-dATP) strains were used as hosts. This is the first report that clearly shows the formation of G•2-OH-dATP pairs.