Plasmid transformation by electroporation of Leuconostoc paramesenteroides and its use in molecular cloning.

In this report, we demonstrate the utility of electroporation as an efficient method for genetic transformation of Leuconostoc paramesenteroides. We optimized several factors which determine the transformation frequency, resulting in transformation efficiencies of up to 4 x 10(3) transformants per micrograms of pNZ12 DNA, which contains the promiscuous Lactococcus lactis pSH71 replicon. Slightly lower efficiencies were obtained with a deletion derivative of the broad-host-range plasmid pAM beta 1. These plasmids could be stably maintained in L. paramesenteroides NZ6009 for more than 100 generations, even in the absence of selective pressure. In order to show the use of the developed host-vector system, we cloned the Lactococcus lactis gene encoding phospho-beta-galactosidase in L. paramesenteroides. Expression of this heterologous gene in L. paramesenteroides under control of Lactococcus lactis expression signals was evident from the presence, in transformants, of phospho-beta-galactosidase activity and a specific phospho-beta-galactosidase protein band on Western blots (immunoblots). In addition, we transformed a lactose-deficient derivative of L. paramesenteroides with a plasmid carrying a Lactococcus lactis-Escherichia coli lacZ gene fusion. The resulting transformants synthesized high levels of beta-galactosidase, indicating the efficiency of heterologous gene expression signals in L. paramesenteroides.     

Structure and expression of the Lactococcus lactis gene for phospho-beta-galactosidase (lacG) in Escherichia coli and L. lactis

The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).     

Expression of a beta-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis

A beta-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high beta-galactosidase activity but utilized lactose only slightly faster than the recipient. beta-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the beta-galactosidase gene. Growth rates of Lac+ H1 and 7962 derivatives were not affected after introduction of the clostridial beta-galactosidase, even though beta-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-beta-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-beta-galactosidase activity. We suggest that beta-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-beta-galactosidase genes.     

Lactobacillus plantarum and Lactobacillus buchneri as Expression Systems: Evaluation of Different Origins of Replication for the Design of Suitable Shuttle Vectors

The objectives of this study were to establish transformation protocols for Lactobacillus plantarum CD033 and Lactobacillus buchneri CD034, two industrial silage strains and to test the influence of selected origins of replication on plasmid copy number, plasmid stability, and plasmid incompatibility in these strains. Electro-transformation protocols were optimized by examination of the influence of different electroporation solutions and cell wall weakening agents on transformation efficiency. Using Lithium acetate as cell wall weakening agent, we could achieve transformation efficiencies of 8?×?10(4) transformants per 1?μg DNA for L. buchneri CD034 which is to our knowledge the highest described for this species up to now. In order to test feasibility of previously described origins of replication derived from Bacillus subtilis, L. plantarum, Lactococcus lactis, and two novel L. buchneri CD034 plasmids to drive replication in our two selected Lactobacillus strains, six shuttle vectors were constructed. Results indicate that, in terms of stable propagation and high gene copy numbers (up to 238 copies/chromosome), the most suitable origins of replication for the construction of expression vectors for the selected silage strains were the ones derived from the novel L. buchneri CD034 plasmids.     

IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis.

The lactococcal insertion sequence IS946 was used to construct suicide vectors for insertion of heterologous DNA into chromosomal and plasmid sequences of Lactococcus lactis subsp. lactis. Electroporation of L. lactis strains, including the recombination-deficient strain MMS362, with the suicide vector pTRK145 yielded 10(1) to 10(3) transformants per micrograms of DNA. pTRK145 insertions occurred primarily in the chromosome, with one insertion detected in a resident plasmid. Vector-specific probes identified junction fragments that varied among transformants, indicating random insertions of pTRK145.     

IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis.

The lactococcal insertion sequence IS946 was used to construct suicide vectors for insertion of heterologous DNA into chromosomal and plasmid sequences of Lactococcus lactis subsp. lactis. Electroporation of L. lactis strains, including the recombination-deficient strain MMS362, with the suicide vector pTRK145 yielded 10(1) to 10(3) transformants per micrograms of DNA. pTRK145 insertions occurred primarily in the chromosome, with one insertion detected in a resident plasmid. Vector-specific probes identified junction fragments that varied among transformants, indicating random insertions of pTRK145.     

Food-grade host/vector expression system for <Emphasis Type="Italic">Lactobacillus casei</Emphasis> based on complementation of plasmid-associated phospho-β-galactosidase gene <Emphasis Type="Italic">lacG</Emphasis>

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.     

利用乳酸乳球菌AcmA表面展示b-1, 3-1, 4-葡聚糖酶

采用PCR扩增乳酸乳球菌(Lactococcus lactis)MB191菌株的全长肽聚糖水解酶基因acmA, 通过C-末端融合构建了与绿色荧光基因gfp的融合基因acmA-gfp, 再连接于表达载体pMG36k上后得到可组成型表达AcmA-GFP融合蛋白的重组质粒pMB137, 然后将该质粒电转化导入到乳酸乳球菌AS1.2829中获得重组菌MB137。经SDS-PAGE检测, 重组菌MB137可表达预期的分子量约74 kD的蛋白质。Western blotting、细胞分级分离组分的荧光活性测定和特异GFP二抗标记的流式细胞仪检测证实GFP被成功锚定在重组菌细胞表面, 被锚定蛋白约占总表达融合蛋白的35%。进一步通过从枯草芽胞杆菌BF7658基因组中扩增去信号肽序列的b-1, 3-1, 4葡聚糖酶基因gls, 来取代pMB137中的gfp, 得到携带融合基因acmA-gls的重组质粒pMB138, 经导入到乳酸乳球菌AS1.2829后得到重组菌MB138, 其全细胞b-1, 3-1, 4-葡聚糖水解酶的活性约为12 U/mL菌液, 明显高于对照菌株。     

Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.     

Expression of Lactobacillus casei ATCC 393 β-galactosidase encoded by plasmid pLZ15 in Lactococcus lactis CNRZ 1123

Lactococcus lactis subsp. lactis CNRZ 1123, a Lac^- derivative of CNRZ 1122 was transformed by electroporation with the Lactobacillus casei ATCC 393 plasmid pLZ15, which bears a β-galactosidase gene. The transformants expressed a constitutive β-galactosidase activity at a higher level than in Lact. casei , and in the cell-free extract two additional protein bands were detected by SDS-PAGE which could correspond to lactose metabolism enzymes. Both plasmid and β-gal activity were stable in Lactococcus after 100 generations in glucose-containing medium.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA.

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Alternative lactose catabolic pathway in Lactococcus lactis IL1403

In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 beta-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside), and we identify several genes essential for lactose assimilation. Among these are ccpA (encoding catabolite control protein A), bglS (encoding phospho-beta-glucosidase), and several genes from the Leloir pathway gene cluster encoding proteins presumably essential for lactose metabolism. The functions of these genes were demonstrated by their disruption and testing of the growth of resultant mutants in lactose-containing media. By examining the ccpA and bglS mutants for phospho-beta-galactosidase activity, we showed that expression of bglS is not under strong control of CcpA. Moreover, this analysis revealed that although BglS is homologous to a putative phospho-beta-glucosidase, it also exhibits phospho-beta-galactosidase activity and is the major enzyme in L. lactis IL1403 involved in lactose hydrolysis.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes.

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.     

Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-beta-galactosidase and beta-galactosidase genes and their expression products

The mesophilic (S. lactis and S. cremoris) and thermophilic (S. thermophilus) dairy lactic streptococci, which are used in industrial dairy fermentations, contain two different lactose hydrolysing enzymes, a phospho-beta-galactosidase and a beta-galactosidase. The central role of these enzymes in the pathways used for lactose transport and degradation is discussed along with their properties and distributions in lactic streptococci. In addition, recent results on the cloning, expression and sequence organization of the genes for the mesophilic phospho-beta-galactosidase and thermophilic beta-galactosidase are reviewed. Original data are presented concerning heterologous gene expression in the study of lactose hydrolysis in lactic streptococci. These include 1) the purification of the S. lactis phospho-beta-galactosidase from an overproducing Escherichia coli, and 2) the expression of the E. coli beta-galactosidase (lacZ) gene in S. lactis employing a lactic streptococcal expression vector.     

Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media

The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.     

Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media.

The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.     

乳酸菌载体pMG36e的应用现状

乳酸乳球菌通用表达质粒pMG36e是一个经典的人工构建的组成型表达载体,是以乳酸乳球菌乳脂亚种蛋白酶基因的转录和翻译信号为基础构建而成的。它包含一个强启动子,能够在多种细菌中表达外源蛋白。已用于研究细菌素作用机制,乳酸菌基因工程菌株的改造以及口服疫苗的开发等,应用领域十分广泛,已成为乳酸菌基因工程研究的重要工具质粒之一。本文主要从载体构成、基因表达与食品级载体改造等三方面的应用对其进行综述,旨在为该质粒今后研究提供资料。     

Transformation of, and heterologous protein expression in, Lactobacillus agilis and Lactobacillus vaginalis isolates from the chicken gastrointestinal tract

Lactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derived Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 10(8) and 10(6) transformants per microgram of plasmid DNA, respectively. The third strain tested, L. crispatus Lc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter gene gfp was analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3 ldhL promoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression in L. agilis and L. vaginalis. The ability of these strains to express heterologous proteins in vitro indicates their potential utility as in vivo delivery vectors for therapeutic peptides to the chicken gastrointestinal tract.