Glutathione protects Lactococcus lactis against acid stress

Previously we showed that glutathione (GSH) can protect Lactococcus lactis against oxidative stress (Y. Li et al., Appl. Environ. Microbiol. 69:5739-5745, 2003). In the present study, we show that the GSH imported by L. lactis subsp. cremoris SK11 or produced by engineered L. lactis subsp. cremoris NZ9000 can protect both strains against a long-term mild acid challenge (pH 4.0) and a short-term severe acid challenge (pH 2.5). This shows for the first time that GSH can protect a gram-positive bacterium against acid stress. During acid challenge, strain SK11 containing imported GSH and strain NZ9000 containing self-produced GSH exhibited significantly higher intracellular pHs than the control. Furthermore, strain SK11 containing imported GSH had a significantly higher activity of glyceraldehyde-3-phosphate dehydrogenase than the control. These results suggest that the acid stress resistance of starter culture can be improved by selecting L. lactis strains capable of producing or importing GSH.     

Glutathione Protects Lactococcus lactis against Oxidative Stress

Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to ~60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H₂O₂ compared to the resistance of cells without intracellular glutathione. The resistance to H₂O₂ treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H₂O₂. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.     

Properties of the Cell Walls of Lactococcus lactis subsp. cremoris SK110 and SK112 and Their Relation to Bacteriophage Resistance

Resistance of Lactococcus lactis subsp. cremoris SK110 to bacteriophage sk11G, encoded on the plasmid pSK112, is due to poor phage adsorption. Its phage-sensitive variant SK112, cured of pSK112, adsorbs phages effectively. Incubation of SK112 with concanavalin A remarkably reduced phage adsorption to this strain. This treatment also caused agglutination of SK112 that was not found with SK110, indicating different concanavalin A adsorption characteristics of cell walls of both strains. The differences between the two strains were reduced by a mild alkali treatment of cells. This resulted in a positive agglutination with concanavalin A for both strains and in parallel adsorption of phage sk11G to both. Moreover, isolated cell walls of the two strains were investigated, and both bound phage sk11G. These observations suggest the presence of phage receptor material in SK112 as well as in SK110. SK110 contained a relatively high level of bound galactose when compared with the phage-sensitive SK112. After the mild alkali treatment, however, the galactose content of SK110 was diminished such that it became comparable with that of SK112. It is hypothesized that the alkali treatment liberates a galactose-containing component from the cell wall and causes phage sensitivity in L. lactis subsp. cremoris SK110.     

Cooperation between Lactococcus lactis and Nonstarter Lactobacilli in the Formation of Cheese Aroma from Amino Acids

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of α-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced α-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.     

Pulsed-Field Gel Electrophoresis of SmaI Digests of Lactococcal Genomic DNA, a Novel Method of Strain Identification

The pulsed-field gel electrophoresis (PFGE) pattern of SmaI digests of 29 strains of Lactococcus lactis subsp. lactis and subsp. cremoris were determined. Unrelated strains yielded markedly different patterns of digestion products. Bacteriophage-resistant derivatives of four strains, generated by a method analogous to that used regularly in some cheese factories, yielded patterns that were identical or almost identical to that of the parent strain. It is proposed that a 16-h PFGE run with a pulse time increasing linearly from 1 to 20 s, which separates fragments between 50 and 240 kilobase pairs (kbp) and produces a pattern containing around 15 bands, can be used as a reliable procedure for strain identification in the lactococci. SmaI digests of 24 of the strains were analyzed by PFGE at three different pulse times to determine accurately the sizes of fragments bigger than 8 kbp. The sum of the sizes of all of the fragments in the digest of a strain provided an estimate of the genome size of the strain. For all the strains analyzed, this estimate was within the range of 2.0 to 2.7 Mbp, with no apparent difference between L. lactis subsp. lactis, L. lactis subsp. lactis biovar diacetylactis and L. lactis subsp. cremoris strains.     

Diversity analysis of dairy and non-dairy strains of Lactococcus lactis ssp. lactis by multilocus sequence analysis (MLSA)

The genetic diversity of 31 identified strains of Lactococcus lactis ssp. lactis isolated from different dairy and non-dairy sources were investigated at gene level using multilocus sequence analysis (MLSA) and PCR-RFLP based on the differences in four selected partial protein coding gene sequences: araT, encoding aromatic amino acid-specific aminotransferase; dtpT, encoding di/tri peptide transporter; yueF, encoding non-proteolytic protein, peptidase, M16 family; and pdhA, encoding pyruvate dehydrogenase E1 component α-subunit. A set of seven test strains from different isolation sources and one reference strain, L. lactis ssp. lactis NCDC 094, were analyzed by MLSA. The strains showed distinct diversity among themselves and exhibited a greater percent similarity with reference strains L. lactis ssp. lactis CV56 (CP002365.1), IL1403 (AE005176.1), and KF147 (CP001834.1) in comparison with L. lactis ssp. cremoris NZ9000 (CP002094.1), MG1363 (AM406671.1), and SK11 (CP00425.1). The MLSA revealed one distinct genomic lineage within strains exclusively of L. lactis ssp. lactis. This analysis also revealed no source-wise genetic relationship in the test strains analyzed. Further, PCR-RFLP of araT, dtpT, yueF and pdhA also characterized the single genomic lineage exclusively of L. lactis ssp. lactis within a total of 24 test strains.     

Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis

Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.     

Improved acid-stress tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000 and <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 by overexpression of the anti-acid component <Emphasis Type="Italic">recT</Emphasis>

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.     

Detection of Specific Strains and Variants of Streptococcus cremoris in Mixed Cultures by Immunofluorescence

Antisera against four different strains of Streptococcus cremoris were raised by injecting rabbits with washed suspensions of whole cells. These antisera interacted specifically with the corresponding strain in a mixture of up to nine different S. cremoris strains. The antisera could be used for analyzing the composition of mixed cultures containing these strains by immunofluorescence. Competition experiments were performed in batch and continuous cultures under amino acid limitation. A bacteriophage-sensitive variant of S. cremoris SK11 (SK1128) could be distinguished from a bacteriophage-resistant variant (SK1143) by the same immunofluorescence technique. The competition between the two variants and the stability of both variants in pure cultures were followed with the specific antibodies. Antibodies against the purified proteolytic system of S. cremoris Wg2 were used to determine the presence of proteases by immunofluorescence in several S. cremoris strains under different culture conditions. The described immunofluorescence methods can be used to analyze complex mixed starter cultures common in the dairy industry as the strains and variants present in these mixtures can be recognized microscopically.     

异源表达德氏乳杆菌保加利亚亚种TCS1调控酸适应基因提高乳酸乳球菌NZ9000耐酸性

[目的] 在乳酸乳球菌NZ9000中异源表达德氏乳杆菌保加利亚亚种中由双组分系统TCS1（JN675228/JN675229）调控的与酸适应相关基因,进而探究德氏乳杆菌保加利亚亚种应对酸胁迫的机制。[方法] 通过逆转录聚合酶链式反应和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳验证由德氏乳杆菌保加利亚亚种TCS1调控的与酸适应相关基因中腺嘌呤磷酸核糖转移酶（aprt）、D-丙氨酸-D-丙氨酸连接酶（ddl）、寡肽ABC转运蛋白（oppDII）和延伸因子Ts（tsf）在乳酸乳球菌NZ9000中的表达情况。酸处理实验验证基因表达对宿主菌酸胁迫耐受能力的影响。并采用酵母双杂交验证双组分系统TCS1与表达的酸适应相关基因之间的互作关系及具体的互作部位。[结果] 结果表明,乳酸乳球菌NZ9000中成功表达了aprt、ddl、oppDII和tsf。aprt、ddl基因使重组菌对酸胁迫的抗性分别提高了75倍和114倍。oppDII和tsf基因的表达对重组菌株的耐酸能力没有明显影响。酵母双杂交实验表明TCS1中的组氨酸蛋白激酶HPK1与Ddl之间存在相互作用,且HPK1-C结构域是二者相互作用的关键区域。[结论] aprt和ddl过表达菌株酸刺激的适应能力显著高于对照菌株,该研究结果可为德氏乳杆菌保加利亚亚种及类似菌株耐酸性特性的获得策略提供参考。     

Receptor Binding Domain of Escherichia coli F18 Fimbrial Adhesin FedF Can Be both Efficiently Secreted and Surface Displayed in a Functional Form in Lactococcus lactis

Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae. In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF. In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated. Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L. lactis subsp. cremoris protein PrtP for evaluation of secretion efficiency. To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L. lactis AcmA anchor or the PrtP cell wall binding domain. An HtrA-defective L. lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins. Recombinant L. lactis clones secreting the receptor binding domain of F18 fimbriae as a fusion with the H domains of L. lactis protein PrtP were first constructed by using two different signal peptides. FedF-PrtP fusions, directed by the signal sequence of L. brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L. lactis Usp45. In the surface display systems tested, the L. lactis AcmA anchor performed significantly better, particularly in the L. lactis NZ9000ΔhtrA strain, compared to the L. lactis PrtP anchor region. Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L. lactis cells to porcine intestinal epithelial cells. These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L. lactis.     

Inactivation of the Glutamate Decarboxylase Gene in Lactococcus lactis subsp. cremoris

Lactococcus lactis subsp. lactis strains show glutamate decarboxylase activity, whereas L. lactis subsp. cremoris strains do not. The gadB gene encoding glutamate decarboxylase was detected in the L. lactis subsp. cremoris genome but was poorly expressed. Sequence analysis showed that the gene is inactivated by the frameshift mutation and encoded in a nonfunctional protein.     

Comparative Analyses of Prophage-Like Elements Present in Two Lactococcus lactis Strains

In this study, we describe the genetic organizations of six and five apparent prophage-like elements present in the genomes of the Lactococcus lactis subsp. cremoris strains MG1363 and SK11, respectively. Phylogenetic investigation as well bioinformatic analyses indicates that all 11 prophages belong to subdivisions of the lactococcal P335 group of temperate bacteriophages.     

Introducing glutathione biosynthetic capability into Lactococcus lactis subsp. cremoris NZ9000 improves the oxidative-stress resistance of the host

This study describes how a metabolic engineering approach can be used to improve bacterial stress resistance. Some Lactococcus lactis strains are capable of taking up glutathione, and the imported glutathione protects this organism against H(2)O(2)-induced oxidative stress. L. lactis subsp. cremoris NZ9000, a model organism of this species that is widely used in the study of metabolic engineering, can neither synthesize nor take up glutathione. The study described here aimed to improve the oxidative-stress resistance of strain NZ9000 by introducing a glutathione biosynthetic capability. We show that the glutathione produced by strain NZ9000 conferred stronger resistance on the host following exposure to H(2)O(2) (150 mM) and a superoxide generator, menadione (30 microM). To explore whether glutathione can complement the existing oxidative-stress defense systems, we constructed a superoxide dismutase deficient mutant of strain NZ9000, designated as NZ4504, which is more sensitive to oxidative stress, and introduced the glutathione biosynthetic capability into this strain. Glutathione produced by strain NZ4504(pNZ3203) significantly shortens the lag phase of the host when grown aerobically, especially in the presence of menadione. In addition, cells of NZ4504(pNZ3203) capable of producing glutathione restored the resistance of the host to H(2)O(2)-induced oxidative stress, back to the wild-type level. We conclude that the resistance of L. lactis subsp. cremoris NZ9000 to oxidative stress can be increased in engineered cells with glutathione producing capability.     

Cell Surface Characteristics of Bacteriophage-Resistant Lactococcus lactis subsp. cremoris SK110 and Its Bacteriophage-Sensitive Variant SK112

Several cell surface characteristics of bacteriophage-resistant Lactococcus lactis subsp. cremoris SK110 were compared with those of its phage-sensitive derivative SK112. After centrifugation, SK110 cells resisted suspension more strongly than SK112 cells. SK112 was more negatively charged and had a more hydrophobic cell surface than SK110. Furthermore, SK112 was agglutinated in the presence of concanavalin A, whereas SK110 was not. The opposite was observed upon incubation of cells of either strain with a lectin from Ricinus communis. A mild alkali treatment decreased the differences in the cell surface characteristics of the two strains remarkably.     

pSEUDO, a Genetic Integration Standard for Lactococcus lactis

Plasmid pSEUDO and derivatives were used to show that llmg_pseudo_10 in Lactococcus lactis MG1363 and its homologous locus in L. lactis IL1403 are suitable for chromosomal integrations. L. lactis MG1363 and IL1403 nisin-induced controlled expression (NICE) system derivatives (JP9000 and IL9000) and two general stress reporter strains (NZ9000::PhrcA-GFP and NZ9000::PgroES-GFP) enabling in vivo noninvasive monitoring of cellular fitness were constructed.     

Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.     

Characterization of starter lactic acid bacteria from the Finnish fermented milk product viili

Aims: Phenotypic and molecular methods were used to identify and compare the strain composition of three industrial dairy starters used for the manufacture of viili. Methods and Results: Preliminary differentiation was made by phenotypic methods. Genotypic differentiation was carried out using polymerase chain reaction (PCR) and further characterization at strain level by pulsed‐field gel electrophoresis (PFGE). The isolates could be assigned as acid‐producing Lactococcus lactis strains of both lactis and cremoris subspecies, and aroma producers, identified as L. lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides. PCR analysis discriminated between the lactococcal subspecies, and cluster analysis of the digestion patterns of PFGE analysis revealed different genotypes in each subspecies. Each Leuconostoc‐genotype seemed to be specific to only a single starter mix. Conclusions: The work proved that in addition to L. lactis subsp. lactis biovar diacetylactis and Leuc. mesenteroides subsp. cremoris, commercial viili starters of traditional origin may contain (i) only L. lactis subsp. cremoris, (ii) both L. lactis subsp. cremoris and L. lactis subsp. lactis as a minority, and – as a new discovery – (iii) only L. lactis subsp. lactis. Significance and Impact of the Study: The results obtained give an overview of the microbial population of viili starters and can be exploited in the development of optimized starter cultures for industrial‐scale manufacture of viili.