乳链菌肽Nisin的生物合成及表达调控机制

乳链菌肽Nisin是乳酸乳球菌（Lactococcus lactis）产生的一种多肽类抗菌物质,是一种由34个氨基酸组成的羊毛硫细菌素。与Nisin合成有关的基因有11个,构成一个基因簇nisA（Z）BTCIPRKFEG。这些相关基因组成三个操纵子进行转录,分别是nisA（Z）BTCIP、nisRK和nisFEG。Nisin通过NisRK双组分调节系统诱导自身合成,而NisI和NisFEG赋予了Nisin产生菌对Nisin的免疫性。对于Nisin的生物合成机制人们展开了非常广泛的研究。本文对Nisin的结构、Nisin合成相关的基因簇、Nisin的生物合成及表达调控机制以及Nisin产生菌对Nisin的免疫性进行了综述。     

Nisin生物合成相关基因分析

Nisin是乳酸乳球菌某些菌株产生的一种对细菌芽孢和多种革兰氏阳性菌有较好杀伤作用的小肽,广泛用于食品加工、医疗保健等诸多领域。合成Nisin的基因位于染色体上-可接合型蔗糖转座子,由三个启动nisA、nisR、nisF控制的nisA/ZBTCIPRKFEG构成,其中nisA因具有更强的启动强度,且表达诱导物和宿主菌均为食品级,方便、经济、安全,应用最为广泛。其他基因分别在Nisin合成、调控过程中起不同作用：自身保护性基因ninI、nisF、nisE、nisG的表达,使菌体获得对Nisin较好的耐受性;NisB和NisC与翻译后修饰有关;NisT可促进乳链菌肽前体转移至胞外;NisP则与nisin前体信号肽的切除有关。     

Nisin A前体基因nisA的过量表达对Nisin A产量的影响

Nisin是一种广泛应用于食品工业的抗菌素。通过基因工程手段分别构建了Nisin A前体结构基因nisA的穿梭表达载体pMG36ek-nisA和整合型载体pDG780-nisA,并转入Nisin A产生菌乳酸乳球菌Lactococcus lactis ATCC 11454中,得到两株基因工程菌FMM1和FMM2。对比工程菌和原始产生菌的生长状态及Nisin A产量,结果表明FMM1的生长速度、生物量以及发酵液的酸碱度没有显著变化,而Nisin A产量提高了31%;相反,FMM2的生物量较原始菌株显著降低,但Nisin A产量也有一定程度的提高。通过RT-PCR检测工程菌与原始产生菌Nisin A生物合成基因簇中11个基因的转录水平,结果显示FMM1和FMM2的11个基因的转录水平均有提高,其中FMM1提高更为显著。因此推测,nisA是Nisin A高产的关键因素之一,其游离型过量表达能显著提高Nisin A的产量。该研究为采用基因工程手段提高Nisin A的产量提供了新的思路,并对NisinA的大规模工业生产有指导意义,同时,也为其他抗菌肽产生菌的基因工程改造提供了参考。     

大肠杆菌重组乳链菌肽抗性蛋白(NSR)的表达纯化及其功能分析

乳链菌肽(Nisin)是由某些乳酸菌产生的一种阳离子抗菌肽, 而Nisin抗性蛋白(Nisin resistance protein, NSR)的表达则使一些非 Nisin产生菌获得Nisin抗性。为深入探索NSR的作用机制, 本研究在大肠杆菌中表达了去除N末端38个氨基酸残基的NSR(NSRΔ38)与GST的融合蛋白GST-NSRΔ38。通过谷胱甘肽(GSH)亲和层析和GST标签的切除后, 得到纯化的NSRΔ38, 并测定了该蛋白可能的nisin降解活性。反应产物的抑菌活性测定结果表明被NSRΔ38作用     

VirAB在单核细胞增生李斯特菌耐药性及生物被膜形成中的作用

【背景】单核细胞增生李斯特菌(Listeria monocytogenes,Lm)对一些临床常用抗生素、乳酸链球菌素(Nisin)等抗菌药物的敏感性下降,然而其背后的机制仍未完全阐明。【目的】调查转运蛋白VirAB在Lm对抗菌药物的耐药性及生物被膜形成中的作用。【方法】利用同源重组技术构建Lm基因缺失突变株,比较野生株和缺失株对抗菌药物的耐药性;利用微孔板法观测突变株生物被膜形成能力的变化;利用平板泳动法研究菌株的泳动能力。【结果】与野生株相比,virAB缺失突变株对头孢类抗生素、Nisin和溴化乙锭的敏感性增加;当培养基中分别添加亚致死浓度的苯扎氯铵、卡那霉素和四环素时,突变株均表现出不同程度的生长缺陷。缺失virAB后菌株形成生物被膜的能力下降。【结论】VirAB在Lm对头孢类等抗菌药物的耐药及生物被膜形成方面具有重要作用。     

Nisin对幽门螺杆菌生物学作用的实验研究

目的：探讨乳链菌肽（Nisin)在柠檬酸的协同作用下对幽门螺杆菌（Helicobacter pylori,Hp)的生物学作用,寻求一种新的防治Hp微生态制剂,为临床治疗Hp提供理论和实践指导。方法：运用国际通用的药敏试验方法纸片法（Kirby-Bauer)和倾注培养法（Pour Culture)对96例从胃病患者分离出的临床株Nisin和柠檬酸协同作用的生物学实验,然后电镜观察被Nisin作用后的Hp菌株细胞结构并进行分析处理。结果：Nisin在柠檬酸的协同作用下对Hp具有明显的抑杀作用,电镜观察被作用后的Hp菌株细胞质膜破碎和细胞发生球形样变。结论：Nisin作用机制主要表现在对Hp菌株的细胞质膜上。     

乳链菌肽（Nisin）的研究进展

Nisin亦称乳链菌肽或乳酸链球菌素 ,源于 N群链球菌抑菌物质 (Group N Inhibitory Substance.Mattick,Hirsch1947) ,它是由一些乳酸链球菌产生的一种小分子多肽抗菌物质或细菌素。Nisin的作用范围相对较窄 ,它仅对大多数革兰阳性菌起抑制作用 ,如葡萄球菌、链球菌、乳球菌、微球菌、单核细胞增生利斯特菌、分枝杆菌、棒状杆菌等 ,并能抑制芽胞杆菌属或梭状芽胞杆菌属胞子的形成 ,但对真菌或革兰阴性菌没有作用 [1～ 4] 。最近研究表明 Nisin与某种物质连接 ,如EDTA二钠以及一些因素的协同下如降低作用时的温度、增加作用时的压力等 [5…     

环状RNA:一种基因调控RNA

环状RNA是一种通过共价结合线性RNA分子的3′端和其上游5′形成的闭合环状结构RNA,广泛存在于真核生物系统内,其结构具有稳定性,序列具有保守性,表达具有时空特异性。目前研究发现环状RNA的形成是受多个因素调控的,并可通过吸附miRNA分子,在pre-mRNA的剪切过程中与线性RNA形成竞争或促进其来源的基因表达,对基因的表达具有调控作用,同时在组织的分化发育和肿瘤、心肌肥厚、动脉粥样硬化、神经系统等多种疾病的发生发展过程中具有重要作用。     

植物抗病基因结构、功能及其进化机制研究进展

植物与病原菌在长期的共进化和相互选择的过程中,逐渐形成了组织障碍、非寄主抗性和小种专化抗性等有效的防御机制。小种专化抗性(基因对基因抗性)主要是由植物抗病基因识别相应的病原菌无毒基因并激活植物体内抗病信号进而抵御病原菌的侵染。从目前已克隆的 70 多个抗病基因来看,它们在结构上具有高度保守性,主要包括核苷酸结合位点(NBS),亮氨酸重复结构(LRR), 蛋白激酶结构域(PK), 果蝇蛋白 Toll 和哺乳动物蛋白质白细胞介素 1 受体[interleukin(IL)-1 receptor]类似结构域(TIR), 双螺旋结构(CC)或亮氨酸拉链(LZ)和跨膜结构域(TM)等,其在抗病基因与病原菌无毒(效应)蛋白互作以及植物内部免疫信号传导中起着重要的作用。同时,抗病基因又通过基因复制、遗传重组等进化机制形成多基因家族,为植物抗病的专化性和多样性提供了重要的遗传基础。本文主要讨论了近来已克隆抗病基因的结构特征、功能以及抗病基因进化机制研究的进展。     

具有抗菌效应的聚羟基脂肪酸酯生物塑料的制备与功能表征

聚羟基脂肪酸酯(Polyhydroxyalkanoates,PHAs)作为一类新型的生物高分子材料,因其多样的材料性质与高度的生物可降解性日益受到关注。使用乳酸链球菌素(Nisin),一种被公认为安全的天然食品防腐剂,制备了具有高效、持久抗菌效应的PHA塑料。首先采用溶剂浇铸的方法将Nisin整合到正3-羟基丁酸-3羟基己酸共聚酯(PHBHHx),一种具备高度生物相容性的PHA中,从而获得了具有抗菌效应的PHBHHx薄膜。激光共聚焦显微镜观察表明Nisin在PHBHHx中呈颗粒状均匀分布。随后以条件致病菌藤黄微球菌Micrococcus luteus为测试菌株,通过琼脂扩散法,测定PHBHHx薄膜抗菌效应对Nisin含量的依赖关系;在液体培养条件下测量PHBHHx薄膜的Nisin释放效果与抗菌效应。结果表明Nisin可从PHBHHx薄膜顺利释放且Nisin的含量高于25μg/g时即表现出显著的抑菌效果且可长时间维持。该研究为工业化生产具有抗菌效应的PHA奠定了重要的技术基础,拓展了PHA在医学和食品领域的应用潜力。     

乳链菌肽(nisin)抗性机制的研究进展

乳链菌肽(nisin)是某些乳酸乳球菌产生的一种羊毛硫细菌素。其对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,是目前世界上唯一被允许用作食品添加剂的细菌素。nisin的广泛使用虽未引发大范围的抗性,但在自然界或实验室的选择压力下,某些非nisin产生菌也获得了抵御nisin攻击的抗性机制。nisin抗性机制通常涉及两种方式,即非特异性的生理适应机制和特异性蛋白酶介导的主动防御机制。本文综述了近年来nisin抗性机制的研究进展。     

Genetics of subtilin and nisin biosyntheses

Several peptide antibiotics have been described as potent inhibitors of bacterial growth. With respect to their biosynthesis, they can be devided into two classes: (i) those that are synthesized by a non-ribosomal mechanism and (ii) those that are ribosomally synthesized. Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics. They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyl-lanthionine. They are derived from prepeptides which are post-translationally modiffied and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al. 1988). Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984). It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al. 1987). Nisin occurs as a partially amphiphilic molecule (Van de Ven et al. 1991). Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al. 1980). In several countries nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al. 1988; Kaletta & Entian 1989). Nisin has two natural variants, nisin A and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al. 1991; De Vos et al. 1993). Subtilin is produced by Bacillus subtilis ATCC 6633. Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988). Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig. 1), and both lantibiotics possess similar antibiotic activities. Due to its easy genetic analysis B. subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al. 1995b). The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig. 2). These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium. In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein.     

Genetics of lipopolysaccharide biosynthesis in enteric bacteria.

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.     

乳酸乳酸球菌AL2产生的乳链菌肽的提纯和性质

用NaCl饱和的乳酸乳酸球菌(Lactococcus lactis subsp. Lactis)AL2发酵液经正丙醇提取和CM-Sephadex C-25柱层析,得到聚丙烯酰胺凝胶电泳纯的乳链菌肽组分,比活力从24427IU/mg提高到39865IU/mg,活力回收为41.7％。Α—胰凝乳蛋白酶可使乳链菌肽丧失活性;在低pH条件下,乳链菌肽对热较稳定;对许多革兰氏阳性菌有强烈抑制作用,而对革兰氏阴性菌、酵母菌和霉菌没有作用。     

Quorum-sensing regulation of gene expression: Fundamental and applied aspects and the role in bacterial communication

Quorum sensing (QS) is a specific type of regulation of gene expression in bacteria; it is dependent on the population density. QS systems include two obligate components: a low-molecular-weight regulator (autoinducer), readily diffusible through the cytoplasmic membrane, and a regulatory receptor protein, which interacts with the regulator. As the bacterial population reaches a critical level of density, autoinducers accumulate to a necessary threshold value and abrupt activation (induction) of certain genes and operons occurs. By means of low-molecular-weight regulators, bacteria accomplish communication between cells belonging to the same or different species, genera, and even families. QS systems have been shown to play a key role in the regulation of various metabolic processes in bacteria and to function as global regulators of the expression of bacterial genes. Data are presented on different types of QS systems present in bacteria of various taxonomic groups, on the species specificity of these systems, and on communication of bacteria by means of QS systems. The possibility is considered of using QS regulation systems as targets while combating bacterial infections; other applied aspects of QS investigation are discussed.     

Quorum-sensing regulation of gene expression: fundamental and applied aspects and the role in bacterial communication

Quorum sensing (QS) is a specific type of regulation of gene expression in bacteria; it is dependent on the population density. QS systems include two obligate components: a low-molecular-weight regulator (autoinducer), readily diffusible through the cytoplasmic membrane, and a regulatory receptor protein, which interacts with the regulator. As the bacterial population reaches a critical level of density, autoinducers accumulate to a necessary threshold value and abrupt activation (induction) of certain genes and operons occurs. By means of low-molecular-weight regulators, bacteria accomplish communication between cells belonging to the same or different species, genera, and even families. QS systems have been shown to play a key role in the regulation of various metabolic processes in bacteria and to function as global regulators of the expression of bacterial genes. Data are presented on different types of QS systems present in bacteria of various taxonomic groups, on the species specificity of these systems, and on communication of bacteria by means of QS systems. The possibility is considered of using QS regulation systems as targets while combating bacterial infections; other applied aspects of QS investigation are discussed.     

Identification and characterization of some bacterial membrane sulfhydryl groups which are targets of bacteriostatic and antibiotic action

Covalent modification of sulfhydryl groups which become sensitive toward sulfhydryl agents during germination of Bacillus cereus spores exerts a profound bacteriostatic effect, resulting in outgrowth inhibition. The modified spore components are membrane species of 13,000, 28,000, and 29,000 daltons. Detergent disruption of the membrane inactivated the sulfhydryl groups. A highly sigmoid inhibition curve (n = 11.8) with diamide suggested the participation of closely neighboring sulfhydryl groups. Substate and substrate analogs of the lactose and dicarboxylic acid permeases protected the sulfhydryl groups against modification. Nisin, a 34-residue peptide antibiotic, inhibited spore outgrowth and sulfhydryl modification at a concentration of about 0.1 microM. Since these sulfhydryl groups have been implicated as involved with the bacteriostatic action of nitrite, substances directed toward them may be a useful new class of bacteriostatic agents and antibiotics.     

Molecular recognition of lipopolysaccharide by the lantibiotic nisin

Nisin is a lanthionine antimicrobial effective against diverse Gram-positive bacteria and is used as a food preservative worldwide. Its action is mediated by pyrophosphate recognition of the bacterial cell wall receptors lipid II and undecaprenyl pyrophosphate. Nisin/receptor complexes disrupt cytoplasmic membranes, inhibit cell wall synthesis and dysregulate bacterial cell division. Gram-negative bacteria are much more tolerant to antimicrobials including nisin. In contrast to Gram-positives, Gram-negative bacteria possess an outer membrane, the major constituent of which is lipopolysaccharide (LPS). This contains surface exposed phosphate and pyrophosphate groups and hence can be targeted by nisin. Here we describe the impact of LPS on membrane stability in response to nisin and the molecular interactions occurring between nisin and membrane-embedded LPS from different Gram-negative bacteria. Dye release from liposomes shows enhanced susceptibility to nisin in the presence of LPS, particularly rough LPS chemotypes that lack an O-antigen whereas LPS from microorganisms sharing similar ecological niches with antimicrobial producers provides only modest enhancement. Increased susceptibility was observed with LPS from pathogenic Klebsiella pneumoniae compared to LPS from enteropathogenic Salmonella enterica and gut commensal Escherichia coli. LPS from Brucella melitensis, an intra-cellular pathogen which is adapted to invade professional and non-professional phagocytes, appears to be refractory to nisin. Molecular complex formation between nisin and LPS was studied by solid state MAS NMR and revealed complex formation between nisin and LPS from most organisms investigated except B. melitensis. LPS/nisin complex formation was confirmed in outer membrane extracts from E. coli.     

Mechanism of copper surface toxicity in Escherichia coli O157:H7 and Salmonella involves immediate membrane depolarization followed by slower rate of DNA destruction which differs from that observed for Gram-positive bacteria

We have reported previously that copper I and II ionic species, and superoxide but not Fenton reaction generated hydroxyl radicals, are important in the killing mechanism of pathogenic enterococci on copper surfaces. In this new work we determined if the mechanism was the same in non-pathogenic ancestral (K12) and laboratory (DH5α) strains, and a pathogenic strain (O157), of Escherichia coli. The pathogenic strain exhibited prolonged survival on stainless steel surfaces compared with the other E.?coli strains but all died within 10?min on copper surfaces using a 'dry' inoculum protocol (with approximately 10(7) cfu?cm(-2) ) to mimic dry touch contamination. We observed immediate cytoplasmic membrane depolarization, not seen with enterococci or methicillin resistant Staphylococcus aureus, and loss of outer membrane integrity, inhibition of respiration and in situ generation of reactive oxygen species on copper and copper alloy surfaces that did not occur on stainless steel. Chelation of copper (I) and (II) ionic species still had the most significant impact on bacterial survival but protection by d-mannitol suggests hydroxyl radicals are involved in the killing mechanism. We also observed a much slower rate of DNA destruction on copper surfaces compared with previous results for enterococci. This may be due to protection of the nucleic acid by the periplasm and the extensive cell aggregation that we observed on copper surfaces. Similar results were obtained for Salmonella species but partial quenching by d-mannitol suggests radicals other than hydroxyl may be involved. The results indicate that copper biocidal surfaces are effective for Gram-positive and Gram-negative bacteria but bacterial morphology affects the mechanism of toxicity. These surfaces could not only help to prevent infection spread but also prevent horizontal gene transmission which is responsible for the evolution of virulent toxin producing and antibiotic resistant bacteria.