乳酸乳球菌Nisin抗性基因nisI的克隆及作为筛选标记的研究

摘要：【目的】为了优化LJ1菌株的培养条件使之产生高活性的胞外褐藻胶裂解酶。【方法】通过富集培养技术从海带筛选到一株褐藻胶裂解酶产生菌LJ1, 依据表型特征、脂肪酸组成分析及16S rRNA基因序列分析对该菌株进行鉴定。通过单因子和正交试验对LJ1 菌株产胞外褐藻胶裂解酶的培养条件进行了优化。【结果】LJ1菌株属于假交替单胞菌属（Pseudoalteromonas）。该菌株产酶的最佳培养基组成为：褐藻胶3 g/L、(NH4)2SO4 3 g/L、NaCl 20 g/L、KH2PO4 0.1 g/L、CaCl2 0.1 g/L;最佳培养条件为：250 mL三角烧瓶中装液量25 mL、接种量3%、摇瓶转速150 r/min、pH7.5、培养温度为28℃、培养时间为24 h。LJ1菌株所产褐藻胶裂解酶的最适温度为40℃,最适pH7.6,最适NaCl浓度为0.3 mol/L。1 mol/L金属离子Mg2+对酶活力有明显的促进作用,而Co2+ 和Zn2+对酶活力有较强的抑制作用。【结论】LJ1菌株是Pseudoalteromonas 新的胞外褐藻胶裂解酶产生菌,在最佳培养条件下,该菌株的酶活力提高了66%。     

南极假丝酵母脂肪酶B(CALB)基因在乳酸乳球菌MG1363中的整合及表达

根据南极假丝酵母脂肪酶B (CALB)的基因序列将CALB基因进行TA克隆、酶切鉴定及测序后,亚克隆至大肠杆菌-乳酸乳球菌穿梭表达栽体pMG36e-Nisl中,构建重组表达栽体pMG36e-Nisl-CALB.设计特异性引物P3和P4,对重组质粒pMG36e-NisI-CALB进行红霉素抗性基因的敲除,以构建食品级表达载体pMG36N-CALB,后再将两种重组质粒分别电转化入乳酸乳球菌MG1363,以Nisin为选择压力,考察CALB在MG1363中的表达情况.结果显示,成功构建了表达载体pMG36e-NisI-CALB及pMG36N-CALB,两株重组菌在含有20 IU Nisir/mL的培养基中均生长情况良好,遗传性能稳定,且经水解圈鉴定,CALB能够进行活性表达.进一步研究发现,CALB基因整合到乳酸乳球菌MG1363染色体中.     

幽门螺杆菌热休克蛋白A在乳酸乳球菌中的表达及免疫学活性分析

目的构建含幽门螺杆菌(H.pylori)热休克蛋白A编码基因的重组载体,并电转入乳酸乳球菌MG1363,表达目的蛋白并分析其免疫原性,为H.pylori基因工程口服疫苗的研究和开发奠定基础。方法以H.py-loriNCTC 11637株基因组DNA为模板,PCR扩增hspA基因,并克隆至乳酸乳球菌表达载体pMG36e中。将重组质粒转化E.coliDH5α,经鉴定的阳性重组质粒命名为pMG36e/hspA。以电穿孔法将pMG36e/hspA转化乳酸乳球菌MG1363并用Western blot检测HspA蛋白的表达。结果克隆重组后得到pMG36e/hspA。将pMG36e/hspA电转化MG1363后,收集菌体蛋白进行Western blot分析,在HspA的相对分子质量(Mr≈13 kDa)处出现特异性条带。结论首次成功构建了表达H.pyloriHspA的重组乳酸乳球菌MG1363,为进一步口服疫苗的相关研究奠定了基础。     

欧芹苯丙氨酸脱氨酶cDNA在乳酸乳球菌中的表达研究

将欧芹（Ｐｅｔｒｏｓｅｌｉｎｕｍｃｒｉｓｐｕｍ）苯丙氨酸脱氨酶（ＰＡＬ）ｃＤＮＡ亚克隆到组成型表达载体ｐＭＧ３６ｅ启动子Ｐ３２下游,电穿孔法转化乳酸乳球菌,获得有ＰＡＬ表达活性的乳酸乳球菌工程菌（ｐＭＧ３６ｅＰＡＬ／Ｌ．ｌａｃｔｉｓＭＧ１３６３）。通过递归ＰＣＲ合成了一段１２０ｂｐ的调控片段,用以将ｐＭＧ３６ｅ改造为分泌型表达载体ｐＸＨＳ,以翻译偶联的方式表达ＰＡＬ,可使ＰＡＬ的Ｎ末端带上ｕｓｐ４５信号肽,结果亦检测到ＰＡＬ酶活性。自行分离克隆了乳酸乳球菌热休克蛋白基因ｄｎａＪ的启动子区域,构建了热诱导表达载体ｐＸＨＪ,获得ＰＡＬ热诱导表达工程菌（ｐＸＨＪＰＡＬ／Ｌ．ｌａｃｔｉｓＩＬ１４０３）,经３０℃至３７℃热诱导,可使ＰＡＬ表达活性提高至２倍。本文还就乳酸乳球菌ＰＡＬ工程菌在经典型苯丙酮尿症防治中的应用进行了分析和讨论     

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.     

抗菌肽Bactenecin7重组质粒构建及其在乳酸菌的分泌表达和活性鉴定

构建抗菌肽Bactenecin7分泌表达载体,鉴定表达产物及检测其生物活性。采用重叠延伸PCR方法拼接合成Bactenecin7基因及其相关调控元件,将目的基因克隆到穿梭载体 pMG36e中,经鉴定后电击转化乳酸菌。通过RT PCR,Western blot检测目的基因表达情况,采用琼脂扩散法对表达产物的生物活性进行初步检测。结果显示成功构建了携Bactenecin7基因的重组质粒,将重组质粒转化至乳酸菌后,该乳酸菌能有效分泌表达Bactenecin7,经体外抑菌实验证实表达产物Bactenecin7具有抑菌活性。实验表明携Bactenecin7基因的乳酸菌能有效分泌表达具有生物活性的抗菌肽Bactenecin7,为进一步研究口服重组乳酸菌进行肠道细菌感染的治疗奠定了基础。     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Additive Effects of Two Different Plasmid-Linked Restriction and Modification Systems in Lactococcus

Two plasmids, pND801 and pND802, encoding different restriction and modification systems were isolated from Lactococcus lactis ssp. lactis LL42-1 and Lactococcus lactis ssp. cremoris LC14-1, respectively. pND802 contained one Sphl restriction enzyme site and the whole plasmid was cloned into the Sphl site of the streptococcal/ E. coli shuttle vector pSA3 generating the plasmid pND803. pND803 was stably maintained in L.lactis MG1363 harbouring pND801. The combination of the two R/M systems within L.lactis MG1363 resulted in an additive resistance towards both isometric phage and prolate phage.     

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.     

Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine beta-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.     

食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达

为构建乳酸乳球菌食品级分泌表达载体,通过PCR扩增质粒pMG36e的p32启动子片段及乳酸乳球菌MG1363未知分泌蛋白(Usp45)基因的核糖体结合位点、分泌信号肽和成熟肽前11个氨基酸的编码序列(SPusp45),克隆到食品级载体pSH91中,构建食品级分泌性表达载体pSQ;克隆报告基因金黄色葡萄球菌核酸酶(NucA)成熟肽的编码序列nucA到pSQ中分泌信号后,转化乳酸乳球菌MBP71,构建了乳酸乳球菌食品级分泌性表达系统L lactis/pSQ-nucA;通过TB-D法和酶谱法检测L lactis/pSQ-nucA的表达形式、表达量并与以前构建的L lactis/pSQZ-nucA系统表达能力进行比较,结果发现L lactis/pSQ-nucA能够分泌性表达NucA,分泌性表达的NucA量大约是胞内NucA的10倍;L lactis/pSQ-nucA的表达量高于lactis/pSQZ-nucA.为进一步目的蛋白的的分泌性表达及食品级疫苗的研制奠定了基础.     

大肠埃希菌Mn-SOD在乳酸乳球菌中的表达

本文根据GenBank中报道的大肠埃希菌MG1655全基因组DNA序列中SOD的编码基因序列设计引物,PCR扩增大肠埃希菌锰超氧化物歧化酶(Mn-SOD)基因,并将其克隆入原核高效表达质粒载体pBV220中构建重组质粒pBV220-sod,并将其电转入乳酸乳球菌MG1363中获得了成功表达,为SOD发酵奶的研制奠定了基础。     

Solution for promoting egl3 gene of Trichoderma reesei high-efficiency secretory expression in Escherichia coli and Lactococcus lactis

The objective of this study was to develop a solution for promoting egl3 gene of Trichoderma reesei (coding β-1,4-endoglucanase, EGIII) high-efficiency secretory expression in Escherichia coli and Lactococcus lactis and to investigate the effect of the best recombinant on degrading paper and wheat straw. The coding sequence of the egl3 gene fused with a gene fragment of Usp45 (usp45) of L. lactis was cloned to pMG36e and was expressed in E. coli DH 5α (DH 5α) and L. lactis subsp. lactis MG1363 (MG1363). The maximal productivity in recombinant DH 5α was 226 mU mL⁻¹ for extracellular EGIII and 535 mU mL⁻¹ for intracellular EGIII. The maximal productivity in recombinant MG1363 was 1118 mU mL⁻¹ for extracellular EGIII and 761 mU mL⁻¹ for intracellular EGIII. The plasmid stability in recombinant MG1363 was higher than 85% at 60 generations. Recombinant MG1363 vigorously degraded paper and wheat straw and produced sufficient acids. This study provided EGIII transgenic lactic acid bacteria for processing agricultural byproducts.     

Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion.

A physical and genetic map of the chromosome of the Lactococcus lactis subsp. cremoris reference strain MG1363 was established. The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments. The MG1363 chromosome appeared to be circular and 2,560 kb long. Seventy-seven chromosomal markers were located on the physical map by hybridization experiments. Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome. The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction. Comparison of the L. lactis subsp. cremoris MG1363 physical map with those of the two L. lactis subsp. lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism. At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.     

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.

The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during growth in milk in the absence of antibiotics. The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5. All three strains showed rapid growth in milk with concomitant rapid production of acid. The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.     

Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.     

Molecular characterization of plasmids pS7a and pS7b from Lactococcus lactis subsp. lactis bv. diacetylactis S50 as a base for the construction of mobilizable cloning vectors

Aims:  Strain Lactococcus lactis subsp. lactis bv. diacetylactis S50 harbours five theta-replicating plasmids (pS6, pS7a, pS7b, pS80 and pS140). The aim of this study was to characterize domains involved in the replication and conjugative mobilization of the small plasmids pS7a and pS7b, which are structurally very similar.
Methods and Results:  Complete nucleotide sequences of pS7a and pS7b were determined by cloning DNA fragments of different sizes into Escherichia coli vectors. Linearized plasmids and four Eco RI fragments of the pS7a and pS7b were cloned into an origin probe vector. Constructed plasmids (pSEV10, pSK10, pISE1a and pISE1b) were able to replicate in the strain L. lactis subsp. cremoris MG1363. In addition, experiments showed that plasmids pS7a and pS7b contained oriT sequences and their conjugative transfer directly depended on the presence of pS80 in donor cells.
Conclusions:  Plasmids pS7a and pS7b contained typical lactococcal theta replication origin and repB gene that enable them to replicate in the strain L. lactis subsp. cremoris MG1363. Plasmid pS80 plays a key role in the conjugative transfer of small plasmids.
Significance and Impact of the Study:  Plasmids pS7a and pS7b-based derivatives could be valuable tools for genetic manipulation, studying processes of plasmid maintenance and horizontal gene transfer in lactococci.