Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Dissolution of xylose metabolism in Lactococcus lactis

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl(-). Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl(+) strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.     

Identification of Lactococcus lactis genes required for bacteriophage adsorption

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.     

Molecular-genetic differentiation of the dairy yeast Kluyveromyces lactis and its closest wild relatives

The currently accepted formal division of the species Kluyveromyces lactis into two taxonomic varieties, Kl. lactis var. lactis and Kl. lactis var. drosophilarum, is based arbitrarily on phenotypic and ecological characters. On the other hand, the genetic hybridisation analysis and molecular karyotyping of its synonyms allowed us [FEMS Yeast Res. 2 (2002) 39] to reinstate them in the genus Zygofabospora Kudriavzev emend G. Naumov (=Kluyveromyces Kurtzman et al., 2001) as the varieties Zf. lactis var. lactis, Zf. lactis var. krassilnikovii, Zf. lactis var. drosophilarum, Zf. lactis var. phaseolospora and Zf. lactis var. vanudenii. In the present work, we studied forty Kl. lactis strains of different geographic and ecological origins by means of restriction analysis of the PCR-amplified non-coding nrDNA regions encompassing the intergenic spacer 2 (IGS2) and the internal transcribed spacers (ITS1 and ITS2). The results confirmed the complex structure of Kl. lactis. Moreover, four additional genetic populations were identified: three in North America ('aquatic', 'pseudovanudenii' and 'new') and one in Far-East Asia ('oriental'). Comparative sequence analysis of the 5.8S-rRNA gene and the two internal transcribed spacers revealed that the populations 'aquatic' and 'oriental' formed distinct taxa which are phylogenetically separate from the five known populations. However, some discrepancies were observed between the restriction and sequencing data. Genetic hybridisation analysis needs to be done to further elucidate the genetic relationships between the populations of Kl. lactis.     

Cloning, expression, and nucleotide sequence of genes involved in production of lactococcin DR, a bacteriocin from lactococcus lactis subsp. lactis.

The partial nucleotide sequence of a Lactococcus lactis subsp. lactis ADRIA 85LO30 bacteriocin-producing operon was determined. The first two open reading frames of the operon are necessary to get bacteriocin expression in L. lactis IL1403R.     

Relationship between the presence of the citrate permease plasmid and high electron-donor surface properties of Lactococcus lactis ssp. lactis biovar. diacetylactis

Some strains of Lactococcus lactis subspecies possess a citrate permease that enables them to utilize citrate and to produce diacetyl. Such strains are classified as diacetylactis biovariants (L. lactis ssp. lactis biovar. diacetylactis). We investigated the electron-donor surface properties of L. lactis strains and observed that the diacetylactis biovariants presented increased adhesion to electron-acceptor solvents (microbial adhesion to solvents electron-donor characteristics of cells of <27% for L. lactis and about 50% for L. lactis ssp. lactis biovar diacetylactis). We investigated the properties of a pCitP- derivative and observed for a diacetylactis biovariant strain a loss of the electron-donor characteristics falling from 47% for a pCitP+ strain to 8% for its pCitP- derivative. This suggests that the presence of high electron-donor characteristics on the surface of L. lactis results to a large extent from the presence of the citrate permease plasmid.     

Preliminary attempts at bacteriocin typing of lactic streptococci

Preliminary attempts at typing Streptococcus lactis, S. lactis subsp. diacetylactis and Streptococcus cremoris strains by bacteriocins (lactostrepcins) are presented. Among 106 strains used about 85% were sensitive to lactostreptocins. The highest proportion of bacteriocin-typing strains was observed in S. lactis species. Lactostrepcin-sensitive strains could be divided into 6 types. The results confirm some individual features of S. diacetylactis compared with S. lactis.     

Molecular markers for differentiation between the closely related dairy yeast Kluyveromyces lactis var. lactis and wild Kluyveromyces lactis strains from the European "krassilnikovii" population

A comparative molecular genetic study of 37 Kluyveromyces strains of different origin has made it possible to find molecular markers that can differentiate between the dairy yeast Kluyveromyces lactis var. lactis and the genetically close wild Kl. lactis strains from the European "krassilnikovii" population, which are unable to ferment lactose. A restriction fragment length polymorphism analysis of the IGS2 region of the strains' rDNA reveals two different AluI profiles, one of which corresponds to Kl. lactis var. lactis while the other corresponds to yeasts from the "krassilnikovii" population. The AluI restriction profile of the IGS2 region of the rDNA also makes it possible to differentiate between the physiologically similar species Kl. marxianus and Kl. lactis. The origin of clinical Kl. lactis var. lactis isolates is discussed.     

Plasmid transfer via transduction from Streptococcus thermophilus to Lactococcus lactis

Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.     

乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

Molecular description and industrial potential of Tn6098 conjugative transfer conferring alpha-galactoside metabolism in Lactococcus lactis

A novel 51-kb conjugative transposon of Lactococcus lactis, designated Tn6098, encoding the capacity to utilize α-galactosides such as raffinose and stachyose, was identified and characterized. Alpha-galactosides are a dominant carbon source in many plant-derived foods. Most dairy lactococcus strains are unable to use α-galactosides as a growth substrate, yet many of these strains are known to have beneficial industrial traits. Conjugal transfer of Tn6098 was demonstrated from the plant-derived donor strain L. lactis KF147 to the recipient L. lactis NZ4501, a derivative of the dairy model strain L. lactis MG1363. The integration of Tn6098 into the genome of the recipient strain was confirmed by Illumina sequencing of the transconjugant L. lactis NIZO3921. The molecular structure of the integration site was confirmed by a PCR product spanning the insertion site. A 15-bp direct repeat sequence (TTATACCATAATTAC) is present on either side of Tn6098 in the chromosome of L. lactis KF147. One copy of this sequence is also present in the L. lactis MG1363 chromosome and represents the sole integration site. Phenotypic characterization of all strains showed that the transconjugant has not only acquired the ability to grow well in soy milk, a substrate rich in α-galactosides, but also has retained the flavor-forming capabilities of the recipient strain L. lactis MG1363. This study demonstrates how (induced) conjugation can be used to exploit the beneficial industrial traits of industrial dairy lactic acid bacteria in fermentation of plant-derived substrates.     

乳酸乳球菌载体疫苗

乳酸乳球菌是一种在食品工业中广泛应用的安全级微生物,应用基因工程手段能使乳酸乳球菌表达多种病毒、细菌、寄生虫的外源蛋白。乳酸乳球菌可经粘膜途径免疫,能有效递呈抗原,诱导外源蛋白的特异性免疫应答,并能同时诱导粘膜免疫与全身免疫,因此可作为潜在的疫苗载体。本文对乳酸乳球菌载体疫苗的优势、应用以及疫苗设计时需要考虑的问题进行了概述。     

A host factor absent from Lactococcus lactis subspecies lactis MG1363 is required for conjugative transposition

In matings between Lactococcus lactis strains, the conjugative transposons Tn916 and Tn919 are found in the chromosome of the transconjugants in the same place as in the chromosome of the donor, indicating that no transposition has occurred. In agreement with this, the frequency of L. lactis transconjugants from intraspecies matings is the same whether the donor contains the wild-type form of the transposon or the mutant Tn916-int1, which has an insertion in the transposon's integrase gene. However, in intergeneric crosses with Bacillus subtilis or Enterococcus faecalis donors, Tn916 and Tn919 transpose to different locations on the chromosome of the L. lactis transconjugants. Moreover, Tn916 and Tn919 could not be transferred by conjugation from L. lactis and B. subtilis, E. faecalis or Streptococcus pyogenes. This suggests that excision of these elements does not occur in L. lactis. When cloned into E. coli with adjacent chromosomal DNA from L. lactis, the conjugative transposons were able to excise, transpose and promote conjugation. Therefore, the inability of these elements to excise in L. lactis is not caused by a permanent structural alteration in the transposon. We conclude that L. lactis lacks a factor required for excision of conjugative transposons.     

Genetics and molecular physiology of the yeast Kluyveromyces lactis

With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology. In particular, comparative yeast research has been providing insights into the strikingly different physiological strategies that are reflected by dominance of respiration over fermentation in K. lactis versus Saccharomyces cerevisiae. Other than S. cerevisiae, whose physiology is exceptionally affected by the so-called glucose effect, K. lactis is adapted to aerobiosis and its respiratory system does not underlie glucose repression. As a consequence, K. lactis has been successfully established in biomass-directed industrial applications and large-scale expression of biotechnically relevant gene products. In addition, K. lactis maintains species-specific phenomena such as the "DNA-killer system, " analyses of which are promising to extend our knowledge about microbial competition and the fundamentals of plasmid biology.     

Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.

The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by Tn5 mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. lactis subsp. lactis ME2) and abi416 (Abi+, from L. lactis subsp. lactis IL416). Unlike abiA, the action of abiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.     

Cholate resistance in Lactococcus lactis is mediated by an ATP-dependent multispecific organic anion transporter

The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363. The cholate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artificial pH probe 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in L. lactis C41-2. Further experiments with L. lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.     

Selection of lactic yeast producing glucosylceramide from cheese whey

From 2150 isolates from raw milk and milk products, yeast strains were surveyed to produce glucosylceramide from cheese whey. Most of the 54 strains that had accumulated a detectable amount of glucosylceramide were identified as Kluyveromyces lactis var. lactis. The cells of K. lactis var. lactis strain M-11 derived from domestic raw milk accumulated glucosylceramide 2.5-fold higher than K. lactis var. lactis NBRC 1267, the reference strain selected from the culture collections. Strain M-16 of K. lactis var. lactis derived from the same origin was found to synthesize a considerable amount of steryl glucoside in addition to glucosylceramide. Sequence analysis of ribosomal DNA intergenic spacer two regions revealed that strains M-11 and M-16 were diverged from a type strain of K. lactis var. lactis in the same species.     

Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis

Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters - ethylacetate (EA) being the major one - and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA - a common precursor of MTA and EA - but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.