A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG1614 with 60 international units (IU) nisin/ml selection yielded approximately 10⁵ transformants/μg DNA. MG1614 carrying pLEB590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB590 was succesfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications. Electronic Publication     

The nisin-controlled gene expression system: construction, application and improvements

Lactic acid bacteria are widely used in industrial fermentation. The potential use of these bacteria as homologous and heterologous protein expression hosts has been investigated extensively. The NIsin-Controlled gene Expression system (the NICE system) is an efficient and promising gene expression system based on the autoregulation mechanism of nisin biosynthesis in the Lactococcus lactis. In the NICE system, the membrane-located histidine kinase NisK senses the inducing signal nisin and autophosphorylates, then transfers phosphorous group to intracellular response regulator protein NisR which activates nisA promoter to express the downstream gene(s). The NICE system allows regulated overproduction of a variety of interest proteins by several Gram-positive bacteria, especially L. lactis. The essential elements for system construction, its application for expression of some biotechnologically important proteins and further improvements of this system are discussed.     

乳链菌肽细胞分子传感器的构建与应用

【背景】乳链菌肽主要是由乳酸乳球菌生产的一类多肽,对革兰氏阳性菌有抑菌作用,是目前联合国粮食及农业组织/世界卫生组织唯一批准使用的天然食品防腐剂。但是其产量低、缺乏简便高效的检测方法,限制了其研究和应用。【目的】构建一种可输出肉眼可见红色荧光的细胞分子传感器,以期能简单方便地检测样品中的乳链菌肽,同时应用该传感器筛选乳链菌肽生产菌株。【方法】用Golden-Gate克隆方法构建含乳链菌肽诱导启动子和下游红色荧光蛋白基因(两种)的载体,转入Lactococcus lactis中。用细胞传感器筛选可能的乳链菌肽生产菌株。【结果】构建的两种乳链菌肽细胞分子传感器都能对2?200 ng/mL乳链菌肽有灵敏的响应,可用于定量测定。两种传感器的最大荧光强度和表型也有所不同。利用细胞传感器确定了Lactococcus lactis ATCC 11454乳链菌肽的产生,同时排除了一个能产其他抗菌化合物的菌株。【结论】构建的细胞分子传感器能特异性地响应乳链菌肽,并能简单快速地筛选乳链菌肽菌株。     

Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.     

乳酸链球菌肽发酵动力学的研究

提出了在恒定不同pH的发酵条件下,乳酸链球菌SM526的菌体生长、底物消耗、乳酸及Nisin产生的动力学模型。菌体生长、乳酸及Nisin产生用逻辑方程描述,而底物消耗是菌体生长和乳酸产生速率的函数。模型表明,乳酸链球菌SM526菌体生长和乳酸产生的最佳pH为7．0,而Nisin产生的最佳pH却为6．5。     

A lactococcal expression system for engineered nisins.

The nisin-producing Lactococcus lactis strain FI5876 has been modified and developed for use as an expression system for engineered nisin variants. Insertional inactivation of the resident nisA gene had a polar effect on downstream genes, including those involved in nisin immunity. However, subsequent chromosomal rearrangements in this region involving a newly discovered insertion element (IS905) generated a strain that was deficient in the nisA gene product but expressed those nisin determinants necessary for prenisin maturation, secretion, and immunity. Complementation of the lesion in the nisA gene by plasmid-encoded nisA genes containing site-specific mutations resulted in the exclusive production of altered nisins containing specific amino acid substitutions.     

Microplate bioassay for nisin in foods,based on nisin-induced green fluorescent protein fluorescence

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 microg of nisin in cheese, and 1 microg of nisin per ml in salad dressings.     

A lactococcal expression system for engineered nisins.

The nisin-producing Lactococcus lactis strain FI5876 has been modified and developed for use as an expression system for engineered nisin variants. Insertional inactivation of the resident nisA gene had a polar effect on downstream genes, including those involved in nisin immunity. However, subsequent chromosomal rearrangements in this region involving a newly discovered insertion element (IS905) generated a strain that was deficient in the nisA gene product but expressed those nisin determinants necessary for prenisin maturation, secretion, and immunity. Complementation of the lesion in the nisA gene by plasmid-encoded nisA genes containing site-specific mutations resulted in the exclusive production of altered nisins containing specific amino acid substitutions.     

Characterization of Lactic Acid Bacteria Coexisting with a Nisin Z Producer in <Emphasis Type="Italic">Tsuda</Emphasis>-Turnip Pickles

Bacteriocin-producing lactic acid bacteria (LAB) are believed to be associated with many types of fermented food. The present study reports the identification of lactic acid bacterium MS27 producing a bacteriocin isolated from the Tsuda-turnip pickle, which is a Japanese fermented food, and characterization of LAB coexisting with the bacteriocin producers in the Tsuda-turnip pickle. The strain MS27 was identified as Lactococcus lactis subsp. lactis based on a partial 16S rRNA gene sequence and sugar fermentation pattern analyses. Mass spectroscopy and genetic analysis revealed that it produces nisin Z. Microbial population analysis revealed that the LAB community in the Tsuda-turnip pickle comprises nisin Z-sensitive and nisin Z-insensitive LAB (nonbacteriocin producers) and nisin Z producers at population rates of 52.5%, 37.5%, and 10.0%, respectively. This revealed that Leuconostoc spp. (nisin Z insensitive) is the dominant species among LAB microflora and that nisin Z insensitivity of a bacterial strain is proportional to its ability to dominate the population in Tsuda-turnip pickles. Competitive growth assay revealed that Leuconostoc spp. considerably suppressed the bacteriocin production of L. lactis MS27. These results suggested that Leuconostoc spp. contributes to the formation of the LAB community with a wide variety of microorganisms in Tsuda-turnip pickles.     

Bioassay for nisin in milk, processed cheese, salad dressings, canned tomatoes, and liquid egg products

A sensitive nisin quantification bioassay was constructed, based on Lactococcus lactis chromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variant gfp(uv) gene under the control of the nisin-inducible nisA promoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.     

Synergistic antimicrobial effect of nisin whey permeate and lactic acid on microbes isolated from fish

The antimicrobial activity of a combination of lactic acid and whey permeate fermented by a nisin-producing Lactococcus lactis strain was tested by the agar diffusion method using bacteria isolated from fish as test organisms. Lactic acid inhibited all bacterial strains studied, but nisin whey permeate inhibited Gram-positive bacteria only. The combination was more effective than lactic acid alone against Pseudomonas fluorescens and Staphylococcus hominis isolated from fish, and Pseudomonas aeruginosa ATCC9721 and Micrococcus luteus ATCC9341.     

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。     

Identification and characteristics of nisin Z-producing Lactococcus lactis subsp. lactis isolated from Kimchi

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100 degrees C for 1 h and at 121 degrees C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or beta-glucosidase. There were some differences in characteristics from those of nisins described previously.     

Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin

Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.     

基于DNA扣除法的Real-time RT-PCR对工程乳酸菌外源基因表达的绝对定量分析

本研究旨在利用Real-time RT-PCR对外源基因在工程乳酸菌中的表达进行定量分析,建立一种新的Real-time RT-PCR分析方法。采用玻璃珠热酚法提取工程乳酸菌总RNA,对外源目的基因的反转录(含有cDNA和DNA)样品和非反转录(仅含DNA)样品进行Real-time PCR检测,根据经典绝对定量方法并结合DNA扣除法进行分析,将得到的Ct值通过标准曲线换算为样品拷贝数,通过从反转录样品中扣除DNA样品的拷贝数的量,去除了DNA对实验结果的影响,得出最终的定量结果。采用以上方法分析工程乳酸乳球菌NZ9000中外源纤维素酶基因CBHⅡ的表达情况,对表达量较低的目的基因进行转录水平的分析,避免了RNA的损失,得到了外源基因表达的量为(1.28±0.02)×10-1 copies/cfu。这种基于DNA扣除法的Real-time RT-PCR绝对定量方法可以有效地对外源基因在工程乳酸菌中的表达进行分析。     

Growth of nisin-producing lactococci in cooked rice supplemented with soybean extract and its application to inhibition of Bacillus subtilis in rice miso

Lactic acid fermentation of cooked rice and rice koji by supplementation with soybean extract (SBE) and its application to rice miso fermentation were investigated. By supplementing the cooked rice with SBE, lactic acid bacteria (LAB) grew well without any unfavorable effects on the rice such as off-flavor or coloration. Lactococcus lactis subsp. lactis IFO12007 (Lc. lactis, a producer of the bacteriocin nisin) proliferated at 10(8 to approximately 9) cells/g after 24 h of incubation and produced high activity of nisin. The fermented rice with Lc. lactis strongly inhibited not only Bacillus subtilis ATCC19659 but also the other Bacillus strains. While some strains of LAB markedly inhibited the growth of Asp. oryzae, resulting in failure of koji fermentation, Lc. lactis did not affect the growth of these molds. When Lc. lactis was used for rice miso fermentation as a lactic acid starter culture, Lc. lactis rapidly proliferated and produced high nisin activity of 6,400 IU/g, in the steamed rice, resulting in complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation. The rice miso after 12 weeks of aging had a suitable pH, and favorable taste and color. Furthermore, hyposalting of rice miso could be done without difficulty by lactic acid fermentation of both rice and soybeans.     

Isolation, screening and characterization of bacteriocin-producing lactic acid bacteria isolated from traditional fermented food

100 lactic acid bacterial strains isolated from traditional fermented foods (yoghurt, milk cream, sour dough and milk) were screened for bacteriocin production. Twenty six strains producing a nisin-like bacteriocin were selected. Most of these isolates gave only a narrow inhibitory spectrum, although one showed a broad inhibitory spectrum against the indicator strains tested, this strain was determined as Lactococcus lactis. The influence of several parameters on the fermentative production of nisin by Lactococcus lactis was studied. Production of nisin was optimal at 30 degrees C and in the pH range 5.5-6.3. The effect of different sulphur and nitrogen sources on Lactococcus lactis growth and nisin production was studied. Magnesium sulfate and manganese sulfate were found to be the best sulphur sources while triammonium citrate was the best inorganic nitrogen source and meat extract, peptone and yeast extract were the best organic nitrogen source for nisin production.     

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。