乳杆菌噬菌体的分离、功能基因表达及抗噬菌体菌株的选育
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摘要
乳杆菌为乳制品发酵中的重要菌株,而乳杆菌噬菌体的普遍存在给乳制品行业带来了巨大危害。为有效地对乳杆菌噬菌体进行防治及应用,本文以德氏乳杆菌烈性噬菌体phiLdb和发酵乳杆菌温和噬菌体φPYB5为中心展开研究,具体结果如下：
1.德氏乳杆菌烈性噬菌体的分离及性质研究
德氏乳杆菌保加利亚亚种Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus)为酸奶生产中的重要发酵剂,在奶制品发酵过程中主要负责乳酸的快速积累,产品风味物质的形成,质地的改善等。当L. bulgaricus受噬菌体侵染时,其发酵活性将受到破坏,整个发酵过程迅速减慢或完全终止,从而严重影响产品质量。然而,与乳球菌噬菌体相比,关于乳杆菌噬菌体的研究信息非常有限。本论文从发酵异常的酸奶样品中分离获得一个L. bulgaricus的烈性噬菌体,并命名为phiLdb。该噬菌体具有一个直径大小为47.7±0.9 nm的头部和一条长129.8±2 nm的尾,因此被归为Siphoviridae科。噬菌体phiLdb基因组大小约41 kbp,而且不含粘性末端。该噬菌体的一步生长曲线显示,其潜伏期和裂解期分别为45 min和75min,裂解量为56±2 PFU。噬菌体phiLdb对L. bulgaricus的侵染具高度专一性。二价阳离子Ca2+或Mg2+对促进宿主细胞裂解和提高噬菌斑的形成是必需的。噬菌体phiLdb在pH 2-10的范围内均维持高的存活率,并表现出对乙醇和异丙醇的高耐受性。然而,90℃高温处理40 min可将噬菌体phiLdb彻底灭活。Ca2+,pH及温度对噬菌体phiLdb的吸附影响较弱。另外,噬菌体phiLdb对正常宿主细胞和经灭活处理的细胞的吸附曲线相似,说明宿主细胞的生理状态对该噬菌体吸附影响也不明显。本论文对新分离到的噬菌体phiLdb的各方面性质的研究为生产环境中采用最有效的噬菌体防治策略奠定了理论基础。
2.噬菌体保存方法研究
噬菌体是专性活细菌细胞内寄生的病毒,离开宿主易于死亡。为长期保存噬菌体,本论文以大肠杆菌λ噬菌体和德氏乳杆菌噬菌体phiLdb为实验材料,分别在不同稳定剂和不同温度下保存6个月,检测期间噬菌体的存活性,探索最有效的保存方法。结果显示,保存效果最好的稳定剂为甘油,其次为二甲基亚砜,效果最差的稳定剂为脱脂奶粉；效果最好的温度为4℃,其次是-80℃。因此,以甘油或二甲基亚砜为稳定剂,置于4℃,-20℃或-80℃保存均为比较简便有效的噬菌体保存方法。
3.德氏乳杆菌抗噬菌体菌株的选育及性质研究
噬菌体侵染造成的巨大损失促使许多噬菌体防治策略的提出及应用。然而,噬菌体抗性菌株通常是通过DNA重组或携带噬菌体抗性基因的质粒的转移获得的,因而赋予所得菌株潜在的安全隐患。而自发突变的方法,由于不涉及基因操作,已被认为是一种简便有效且“天然”的获得抗噬菌体菌株的策略。60Co-γ射线是最常用的物理诱变剂之一,并且60Co-γ射线诱变已成功用于微生物菌种的改造。本论文通过自发突变及60Co-γ射线辐照诱变的方式分别获得30株抗噬菌体的L. bulgaricus突变株。所有60株突变株均表现出对噬菌体phiLdb的完全抗性(EOP<10-9)。溶源性测定结果表明,除2株自发突变获得的抗性菌株外,其他58株突变菌株的噬菌体抗性均与溶源性无关。所有抗性菌株对噬菌体phiLdb的吸附率明显下降,说明突变菌株可干扰噬菌体的吸附。实验证明,噬菌体phiLdb的吸附受体与肽聚糖-多糖的附属聚合物有关。自发突变获得的抗性菌株间的蛋白酶活性差别较显著,但是大多数抗性菌株的蛋白酶活性及pH值与初始菌株L.bulgaricus ATCC11842的类似。根据各抗性菌株的蛋白酶活性与pH值,我们以BIM 10和γ19为代表,进行发酵乳试验。两株抗性菌株不管噬菌体phiLdb的存在与否,均表现出良好的发酵特性。因此,除自发突变方法外,60Co-γ射线辐照诱变也被证明是一种获得发酵性能优良的Lactobacillus delbrueckii抗噬菌体菌株的简便有效方法。此外,本研究为工业生产中提供了大量抗噬菌体德氏乳杆菌,可作为替换受噬菌体感染发酵剂的后备资源。
4.可控性自裂解乳球菌的构建
在复制周期的最后一步,噬菌体将产生一系列酶类来裂解宿主细胞,从而释放子代颗粒。所有双链DNA噬菌体编码的裂解相关酶类均涉及两个蛋白,即穿孔素(holin)和裂解素(lysin),组成了“两组分裂解盒”。对发酵乳杆菌温和噬菌体φPYB5的基因组进行测序过程中获得了其“两组分裂解盒”的基因：holin (hyb5)和lysin(lyb5)。为阐明噬菌体φPYB5编码的由穿孔素(Hyb5)和裂解素(Lyb5)组成的裂解盒在乳酸菌中的作用特征,并有效利用该裂解盒构建可控性自裂解的乳酸菌株,我们将基因hyb5-lyb5克隆到E. coli/L. lactis穿梭质粒pSEC的nisin诱导型启动子下游,构建了重组质粒pSEC-hyb5-lyb5。将pSEC-hyb5-lyb5电转入Lactococcus lactis NZ9000,获得重组菌株NZphl。经nisin诱导后,Hyb5-Lyb5的表达表现出高的裂解活性,使NZphl菌液的浊度(OD600)急剧下降,并释放出大量胞内蛋白,为利用自裂解菌株生产优质乳制品奠定了基础。5.发酵乳杆菌温和噬菌体裂解盒在大肠杆菌中的克隆与表达
与其他裂解盒不同,Hyb5经预测只含有一个跨膜区,且Lyb5具有N-端信号肽。为研究该组合奇特的裂解盒的作用特征,并阐明Lyb5作为诊疗剂的潜在应用,本论文中,将hyb5, lyb5及hyb5-lyb5分别插入大肠杆菌表达载体,实现了Hyb5,Lyb5在大肠杆菌中的单独表达和共表达。SDS-PAGE结果显示,Hyb5的分子量大小约19 kDa, Lyb5大小为45 kDa。Hyb5或Lyb5的单独表达均可引起宿主菌的裂解,导致胞内β-galactosidase的释放。然而,Hyb5-Lyb5裂解盒的共表达则使宿主菌裂解地更迅速,更彻底。双层平板观测法及zymogram复性胶检测确定了Lyb5对宿主菌的胞外裂解活性。经zymogram复性胶分析,Lyb5展现出非常广泛的裂解谱,可裂解包括致病菌金黄色葡萄球菌(Staphylococcus aureus)在内的所有测试革兰氏阳性菌及部分革兰氏阴性菌如沙门氏菌(Salmonella typhi)等,显示出Lyb5作为诊断工具及治疗剂的巨大潜力。6. Hyb5-Lyb5裂解机制初步研究
基于大肠杆菌噬菌体的研究结果,holin-lysin裂解盒的裂解作用模型及holin控制裂解时间的机制已有报道。但对侵染其他菌的噬菌体的裂解机制研究比较缺乏。本论文试图通过对Lyb5蛋白N-端信号肽的功能分析及对hyb5不同基因片段的表达和活性分析来阐明Hyb5-Lyb5裂解作用模型及单次跨膜Hyb5控制裂解时间的作用机制。我们将lyb5或△SARlyb5(除掉N端SAR序列的lyb5)插入载体pETDuet-1的多克隆位点1,将hyb5, hyb5p(hyb5 5'端324 bp基因片段)或hyb5N30(编码Hyb5 N-端跨膜区30个氨基酸的序列)插入载体pETDuet-1的多克隆位点2,构建了一系列重组载体。并实现了各基因的单独表达及不同基因间的共表达,观察宿主菌的裂解情况。结果显示,△SARlyb5与Hyb5p的共表达使宿主菌的OD值明显下降,而△SARlyb5的单独表达反而使宿主菌生长更旺盛,说明SAR序列可作为信号肽引导Lyb5穿膜,但Lyb5的裂解活性不依赖于SAR序列。Hyb5的N-端跨膜区单独表达时抑制了宿主菌的生长,表明Hyb5的N-端30个氨基酸可以完成跨膜功能,表现出对细胞的毒性。而N30hyb5与Lyb5或△SARlyb5的共表达并未引起宿主菌的大量裂解,暴露了N30hyb5辅助Lyb5或△SARlyb5穿膜功能的缺陷性。比较有趣的是,缺少C-端46个氨基酸的Hyb5对Lyb5穿膜的促进作用明显高于Hyb5,暗示了Hyb5 C-端序列对其行使运输活性的调节作用。至于SAR在裂解过程中是否被切割以及Hyb5作用时本身及其他调节因素的阐明,以致最后总结出Hyb5-Lyb5奇特组合的裂解机制模型和像Hyb5这样不具备其他Holin具有的典型的双翻译起始位点且仅具一个跨膜区的蛋白控制裂解时间机制模型的提出均需要大量的进一步的实验摸索,推测及证实。
Lactobacilli are one of the most important strains in dairy fermentation. However, the infection of Lactobacillus phages would result in large damage to dairy industries. In order to control and apply Lactobacillus phages effectively, we studied mainly on characterizations of the Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) virulent phage phiLdb and the Lactobacillus fermentum temperate bacteriophageφPYB5, the results were described as follows:
1. Isolation and characterization of a novel virulent phage (phiLdb) of Lactobacillus delbrueckii
L. bulgaricus is an essential starter contributing to fast lactic acid development, and also to flavor and texture modifications in fermented milks. However, this starter activity of L. bulgaricus can be seriously affected when phage infection occurs. Consequently, the fermentation process may be slowed or completely stopped, thereby reducing the quality of the final products. Nevertheless, in comparison with phages of lactococi, the available knowledge about lactobacilli phages is limited and only a few of them have been studied in detail. In this study, A new virulent phage (phiLdb) of L. bulgaricus was isolated from a Chinese yogurt sample showing slow acidification. It belonged to the Siphoviridae family with an icosahedral capsid of 47.7±0.9 nm in diameter and a long tail of 129.8±2 nm. The genome of phage phiLdb was estimated to be approximately 41 kbp, and did not contain cohesive ends. One-step growth kinetics of its lytic development revealed latent and burst periods of 45 min and 75 min, respectively, with a burst size of 56±2 phage particles per infected cell. Phage phiLdb was highly specific for L. bulgaricus. The presence of calcium or magnesium ions was necessary to accelerate cell lysis and improve plaque formation. Phage phiLdb was able to survive in a pH range between 2 and 10, and resist ethanol and isopropanol. However, a treatment of 90℃for 40 min was observed to inactive phage phiLdb thoroughly. Calcium ions, pH as well as temperature did not show significant influence on phage adsorption, and the adsorption kinetics were similar on viable and nonviable cells. The characterization of this novel phage was helpful to establish a basis for adopting the most effective phage control strategies in industrial plants.
2. Research on storage methods of phages
Phages are obligate intracellular parasite of bacteria and will easily lose activity without host cells. In order to store phages for a long period, Escherichia coliλphage and Lactobacillus delbrueckii phage phiLdb were kept with a variety of stablizers and temperature for 6 months. And at one month intervals, the phage titers were enumerated and compared to optimize the storage conditions. The results showed that the best storage temperature was 4℃and secondly-80℃; The best stablizer was glycerine, and secondly dimethyl sulfoxide. Therefore, phage storages with glycerine or dimethyl sulfoxide at 4℃,-20℃or-80℃are simple, efficient and recommendable methods.
3. Isolation and characterization of phage-resistant derivatives of Lactobacillus delbruekii.
The phage problem has promoted the adoption of various strategies. However, phage-resistant starter cultures were traditionally obtained by recombinant DNA technology or conjugal transfer of plasmids confering phage resistance, which holded health potential dangers. Fortunately, the isolation of spontaneous phage-resistant mutants has been considered as a convenient, simple and "natural" strategy to replace phage-sensitive strains.60Co-γray is one of the most common used physical mutagens. And recently,60Co-γray mutagenesis was also used to improve microbe strains. In this study, a total of 60 phage-resistant mutants were isolated from L. bulgaricus ATCC11842 by spontaneous mutaion (30 mutants) and 60Co-γray irradiation (30 mutants). All mutants appeared to be completely refractory to infection with the efficiency of plaquing lower than 10-9, and with high phage-resistant stability. Except for 2 spontaneous mutants, the phage-resistant mechanisms of the last 58 mutants were irrelevant with lysogeny. The obvious decrease of the adsorption rates exhibited by all mutants indicated the presence of adsorption interference during phage infection. And the accessory polysaccharide-peptidoglycan complex was proved to be involved in the phage receptor sites. Proteolytic activities were heterogeneous distributed among the spontaneous mutants. Nevertheless, most of the mutants revealed proteolytic activities and pH values similar to those observed for the parent strain. Two mutants, BIM 10 and y 19 were chosen as model starters for pilot fermentations. Both of them showed favorable technological characteristics regardless of the absence or presence of phage. Therefore, besides spontaneous mutation, the 60Co-γray irradiation was also proved to be a simple and effective method for obtaining phage-resistant mutants from Lactobacillus delbrueckii strains with high technological performance. Moreover, some of the phage-resistant mutants obtained in this work might be used as potential starters when commercial strains become phage-sensitive.
4. Construction of a controlled autolysis Lactococcus strain
In the last stage of reproduction cycles, bacteriophages produce a set of enzymes that lyse the host cells to release their progeny particles. The lysis enzymes encoded by all double-stranded DNA phages involve two proteins:a holin and a lysin (also termed endolysin or lysozyme), which constitute a "two-component cell lysis cassette". The temperate bacteriophageφPYB5 was isolated from Lactobacillus fermentum YB5 strain. A putative holin (hyb5) and lysin (lyb5), obtained by sequencing of the genome library ofφPYB5 in E. coli, constituted a two-component cell lysis cassette. To elucidate the characterization of the lysis cassette (Hyb5-Lyb5) encoded by temperate bacteriophageφPYB5 in lactic acid bacteria, we cloned the hyb5 and lyb5 gene into the plasmid pSEC under the control of the nisin-inducible promoter, generating the recombinant plasmid pSEC-hyb5-lyb5. Then, pSEC-hyb5-lyb5 was induced into Lactococcus lactis NZ9000 by electroporation, yielding NZphl. Hyb5 and Lyb5 were expressed successfully under the induction of nisin and induced the lysis of NZphl effectively, resulting in the rapid reduction of OD600 and leakage of abundant intracellular proteins. These results will facilitate the application of autolysis lactic acid bacteria in fermentation to improve the quality of products.
5. Clone and expression of the two-component cell lysis cassette encoded by temperate bacteriophageφPYB5 of Lactobacillus fermentum in E. coli.
Different to others, Hyb5 was predicted to have a single transmembrane domain and Lyb5 possess an N-terminal signal transmembrane domain. In order to characterize this novel two-component cell lysis cassette encoded byφPYB5, and illustrate the potential application of Lyb5 as therapeutic agents, the hyb5, lyb5 and hyb5-lyb5 cassette were cloned and expressed in E. coli in this paper. The molecular weight of Hyb5 indicated by SDS-PAGE was 19 kDa, and Lyb5 was 45 kDa. Both Hyb5 and Lyb5 protein could induce cell lysis alone, resulting in the leakage ofβ-galactosidase. However, the Hyb5-Lyb5 cassette lysed the host cells more rapidly and extensively. By zymogram analysis, the Lyb5 produced in E. coli exhibited a broad lytic spectrum against Gram positive strains including Staphylococcus aureus as well as Gram negative strains such as Salmonella typhi, suggesting that Lyb5 provides a potential alternative of diagnostic tools and therapeutic agents.
6. Lysis mechanism studies on "Hyb5-Lyb5".
The lysis mechanism of holin-lysin cassette and the role of holin in the regulation of lysis time were elucidated based on the studies on E. coli phages. In this study, we tried to illuminate the lysis model of Hyb5-Lyb5 cassette and the mechanism of the regulation of Hyb5 in lysis through analyzing the function of N-trminal signal sequence in Lyb5 and determining the activities of different segments in Hyb5. We cloned lyb5 or△SARlyb5 (lyb5 lack of the SAR sequence) into the MCS1 of the pETDuet-1 vector, and inserted hyb5, hyb5p, or hyb5N30 (N-terminal sequence encoding the TMD with 30 amino acids) into the MCS2 of the pETDuet-1 vector, generating a series of recombinant vectors. By determining the lysis of different recombinants, we revealed that the co-expression of△SARlyb5 and Hyb5p resulted in obvious decrease of the culture turbidity in OD600, while the expression of△SARlyb5 accelerated the growth of the host strain, indicating that the SAR was unnecessary to the lysis activity of Lyb5, but was essential to the transmemberane of Lyb5. When hyb5N30 was expressed, it restrained the growth of E. coli, suggesting that the 30 AA could achieve transmemberane alone. However, the co-action of N30hyb5 and Lyb5 or△SARlyb5 did not induce large lysis of E. coli, exhibiting the limination of N30hyb5 in assisting Lyb5 or△SARlyb5 to the periplasmic space. Interestingly, Hyb5 lacking the C-terminal 30 AA could assist the transfer of Lyb5 more effectively than Hyb5, implying that the C-termina might inhibit the activity of Hyb5. However, the cleavability of SAR in the lysis process, the regulation of Hyb5 itself or other factors to the activity of Hyb5 and finally elucidation of the lysis mechanism of the novel Hyb5-Lyb5 cassette as well as the regulation of Hyb5 in the lysis process, all of which require abundant and further investigations.
1.德氏乳杆菌烈性噬菌体的分离及性质研究
德氏乳杆菌保加利亚亚种Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus)为酸奶生产中的重要发酵剂,在奶制品发酵过程中主要负责乳酸的快速积累,产品风味物质的形成,质地的改善等。当L. bulgaricus受噬菌体侵染时,其发酵活性将受到破坏,整个发酵过程迅速减慢或完全终止,从而严重影响产品质量。然而,与乳球菌噬菌体相比,关于乳杆菌噬菌体的研究信息非常有限。本论文从发酵异常的酸奶样品中分离获得一个L. bulgaricus的烈性噬菌体,并命名为phiLdb。该噬菌体具有一个直径大小为47.7±0.9 nm的头部和一条长129.8±2 nm的尾,因此被归为Siphoviridae科。噬菌体phiLdb基因组大小约41 kbp,而且不含粘性末端。该噬菌体的一步生长曲线显示,其潜伏期和裂解期分别为45 min和75min,裂解量为56±2 PFU。噬菌体phiLdb对L. bulgaricus的侵染具高度专一性。二价阳离子Ca2+或Mg2+对促进宿主细胞裂解和提高噬菌斑的形成是必需的。噬菌体phiLdb在pH 2-10的范围内均维持高的存活率,并表现出对乙醇和异丙醇的高耐受性。然而,90℃高温处理40 min可将噬菌体phiLdb彻底灭活。Ca2+,pH及温度对噬菌体phiLdb的吸附影响较弱。另外,噬菌体phiLdb对正常宿主细胞和经灭活处理的细胞的吸附曲线相似,说明宿主细胞的生理状态对该噬菌体吸附影响也不明显。本论文对新分离到的噬菌体phiLdb的各方面性质的研究为生产环境中采用最有效的噬菌体防治策略奠定了理论基础。
2.噬菌体保存方法研究
噬菌体是专性活细菌细胞内寄生的病毒,离开宿主易于死亡。为长期保存噬菌体,本论文以大肠杆菌λ噬菌体和德氏乳杆菌噬菌体phiLdb为实验材料,分别在不同稳定剂和不同温度下保存6个月,检测期间噬菌体的存活性,探索最有效的保存方法。结果显示,保存效果最好的稳定剂为甘油,其次为二甲基亚砜,效果最差的稳定剂为脱脂奶粉；效果最好的温度为4℃,其次是-80℃。因此,以甘油或二甲基亚砜为稳定剂,置于4℃,-20℃或-80℃保存均为比较简便有效的噬菌体保存方法。
3.德氏乳杆菌抗噬菌体菌株的选育及性质研究
噬菌体侵染造成的巨大损失促使许多噬菌体防治策略的提出及应用。然而,噬菌体抗性菌株通常是通过DNA重组或携带噬菌体抗性基因的质粒的转移获得的,因而赋予所得菌株潜在的安全隐患。而自发突变的方法,由于不涉及基因操作,已被认为是一种简便有效且“天然”的获得抗噬菌体菌株的策略。60Co-γ射线是最常用的物理诱变剂之一,并且60Co-γ射线诱变已成功用于微生物菌种的改造。本论文通过自发突变及60Co-γ射线辐照诱变的方式分别获得30株抗噬菌体的L. bulgaricus突变株。所有60株突变株均表现出对噬菌体phiLdb的完全抗性(EOP<10-9)。溶源性测定结果表明,除2株自发突变获得的抗性菌株外,其他58株突变菌株的噬菌体抗性均与溶源性无关。所有抗性菌株对噬菌体phiLdb的吸附率明显下降,说明突变菌株可干扰噬菌体的吸附。实验证明,噬菌体phiLdb的吸附受体与肽聚糖-多糖的附属聚合物有关。自发突变获得的抗性菌株间的蛋白酶活性差别较显著,但是大多数抗性菌株的蛋白酶活性及pH值与初始菌株L.bulgaricus ATCC11842的类似。根据各抗性菌株的蛋白酶活性与pH值,我们以BIM 10和γ19为代表,进行发酵乳试验。两株抗性菌株不管噬菌体phiLdb的存在与否,均表现出良好的发酵特性。因此,除自发突变方法外,60Co-γ射线辐照诱变也被证明是一种获得发酵性能优良的Lactobacillus delbrueckii抗噬菌体菌株的简便有效方法。此外,本研究为工业生产中提供了大量抗噬菌体德氏乳杆菌,可作为替换受噬菌体感染发酵剂的后备资源。
4.可控性自裂解乳球菌的构建
在复制周期的最后一步,噬菌体将产生一系列酶类来裂解宿主细胞,从而释放子代颗粒。所有双链DNA噬菌体编码的裂解相关酶类均涉及两个蛋白,即穿孔素(holin)和裂解素(lysin),组成了“两组分裂解盒”。对发酵乳杆菌温和噬菌体φPYB5的基因组进行测序过程中获得了其“两组分裂解盒”的基因：holin (hyb5)和lysin(lyb5)。为阐明噬菌体φPYB5编码的由穿孔素(Hyb5)和裂解素(Lyb5)组成的裂解盒在乳酸菌中的作用特征,并有效利用该裂解盒构建可控性自裂解的乳酸菌株,我们将基因hyb5-lyb5克隆到E. coli/L. lactis穿梭质粒pSEC的nisin诱导型启动子下游,构建了重组质粒pSEC-hyb5-lyb5。将pSEC-hyb5-lyb5电转入Lactococcus lactis NZ9000,获得重组菌株NZphl。经nisin诱导后,Hyb5-Lyb5的表达表现出高的裂解活性,使NZphl菌液的浊度(OD600)急剧下降,并释放出大量胞内蛋白,为利用自裂解菌株生产优质乳制品奠定了基础。5.发酵乳杆菌温和噬菌体裂解盒在大肠杆菌中的克隆与表达
与其他裂解盒不同,Hyb5经预测只含有一个跨膜区,且Lyb5具有N-端信号肽。为研究该组合奇特的裂解盒的作用特征,并阐明Lyb5作为诊疗剂的潜在应用,本论文中,将hyb5, lyb5及hyb5-lyb5分别插入大肠杆菌表达载体,实现了Hyb5,Lyb5在大肠杆菌中的单独表达和共表达。SDS-PAGE结果显示,Hyb5的分子量大小约19 kDa, Lyb5大小为45 kDa。Hyb5或Lyb5的单独表达均可引起宿主菌的裂解,导致胞内β-galactosidase的释放。然而,Hyb5-Lyb5裂解盒的共表达则使宿主菌裂解地更迅速,更彻底。双层平板观测法及zymogram复性胶检测确定了Lyb5对宿主菌的胞外裂解活性。经zymogram复性胶分析,Lyb5展现出非常广泛的裂解谱,可裂解包括致病菌金黄色葡萄球菌(Staphylococcus aureus)在内的所有测试革兰氏阳性菌及部分革兰氏阴性菌如沙门氏菌(Salmonella typhi)等,显示出Lyb5作为诊断工具及治疗剂的巨大潜力。6. Hyb5-Lyb5裂解机制初步研究
基于大肠杆菌噬菌体的研究结果,holin-lysin裂解盒的裂解作用模型及holin控制裂解时间的机制已有报道。但对侵染其他菌的噬菌体的裂解机制研究比较缺乏。本论文试图通过对Lyb5蛋白N-端信号肽的功能分析及对hyb5不同基因片段的表达和活性分析来阐明Hyb5-Lyb5裂解作用模型及单次跨膜Hyb5控制裂解时间的作用机制。我们将lyb5或△SARlyb5(除掉N端SAR序列的lyb5)插入载体pETDuet-1的多克隆位点1,将hyb5, hyb5p(hyb5 5'端324 bp基因片段)或hyb5N30(编码Hyb5 N-端跨膜区30个氨基酸的序列)插入载体pETDuet-1的多克隆位点2,构建了一系列重组载体。并实现了各基因的单独表达及不同基因间的共表达,观察宿主菌的裂解情况。结果显示,△SARlyb5与Hyb5p的共表达使宿主菌的OD值明显下降,而△SARlyb5的单独表达反而使宿主菌生长更旺盛,说明SAR序列可作为信号肽引导Lyb5穿膜,但Lyb5的裂解活性不依赖于SAR序列。Hyb5的N-端跨膜区单独表达时抑制了宿主菌的生长,表明Hyb5的N-端30个氨基酸可以完成跨膜功能,表现出对细胞的毒性。而N30hyb5与Lyb5或△SARlyb5的共表达并未引起宿主菌的大量裂解,暴露了N30hyb5辅助Lyb5或△SARlyb5穿膜功能的缺陷性。比较有趣的是,缺少C-端46个氨基酸的Hyb5对Lyb5穿膜的促进作用明显高于Hyb5,暗示了Hyb5 C-端序列对其行使运输活性的调节作用。至于SAR在裂解过程中是否被切割以及Hyb5作用时本身及其他调节因素的阐明,以致最后总结出Hyb5-Lyb5奇特组合的裂解机制模型和像Hyb5这样不具备其他Holin具有的典型的双翻译起始位点且仅具一个跨膜区的蛋白控制裂解时间机制模型的提出均需要大量的进一步的实验摸索,推测及证实。
Lactobacilli are one of the most important strains in dairy fermentation. However, the infection of Lactobacillus phages would result in large damage to dairy industries. In order to control and apply Lactobacillus phages effectively, we studied mainly on characterizations of the Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) virulent phage phiLdb and the Lactobacillus fermentum temperate bacteriophageφPYB5, the results were described as follows:
1. Isolation and characterization of a novel virulent phage (phiLdb) of Lactobacillus delbrueckii
L. bulgaricus is an essential starter contributing to fast lactic acid development, and also to flavor and texture modifications in fermented milks. However, this starter activity of L. bulgaricus can be seriously affected when phage infection occurs. Consequently, the fermentation process may be slowed or completely stopped, thereby reducing the quality of the final products. Nevertheless, in comparison with phages of lactococi, the available knowledge about lactobacilli phages is limited and only a few of them have been studied in detail. In this study, A new virulent phage (phiLdb) of L. bulgaricus was isolated from a Chinese yogurt sample showing slow acidification. It belonged to the Siphoviridae family with an icosahedral capsid of 47.7±0.9 nm in diameter and a long tail of 129.8±2 nm. The genome of phage phiLdb was estimated to be approximately 41 kbp, and did not contain cohesive ends. One-step growth kinetics of its lytic development revealed latent and burst periods of 45 min and 75 min, respectively, with a burst size of 56±2 phage particles per infected cell. Phage phiLdb was highly specific for L. bulgaricus. The presence of calcium or magnesium ions was necessary to accelerate cell lysis and improve plaque formation. Phage phiLdb was able to survive in a pH range between 2 and 10, and resist ethanol and isopropanol. However, a treatment of 90℃for 40 min was observed to inactive phage phiLdb thoroughly. Calcium ions, pH as well as temperature did not show significant influence on phage adsorption, and the adsorption kinetics were similar on viable and nonviable cells. The characterization of this novel phage was helpful to establish a basis for adopting the most effective phage control strategies in industrial plants.
2. Research on storage methods of phages
Phages are obligate intracellular parasite of bacteria and will easily lose activity without host cells. In order to store phages for a long period, Escherichia coliλphage and Lactobacillus delbrueckii phage phiLdb were kept with a variety of stablizers and temperature for 6 months. And at one month intervals, the phage titers were enumerated and compared to optimize the storage conditions. The results showed that the best storage temperature was 4℃and secondly-80℃; The best stablizer was glycerine, and secondly dimethyl sulfoxide. Therefore, phage storages with glycerine or dimethyl sulfoxide at 4℃,-20℃or-80℃are simple, efficient and recommendable methods.
3. Isolation and characterization of phage-resistant derivatives of Lactobacillus delbruekii.
The phage problem has promoted the adoption of various strategies. However, phage-resistant starter cultures were traditionally obtained by recombinant DNA technology or conjugal transfer of plasmids confering phage resistance, which holded health potential dangers. Fortunately, the isolation of spontaneous phage-resistant mutants has been considered as a convenient, simple and "natural" strategy to replace phage-sensitive strains.60Co-γray is one of the most common used physical mutagens. And recently,60Co-γray mutagenesis was also used to improve microbe strains. In this study, a total of 60 phage-resistant mutants were isolated from L. bulgaricus ATCC11842 by spontaneous mutaion (30 mutants) and 60Co-γray irradiation (30 mutants). All mutants appeared to be completely refractory to infection with the efficiency of plaquing lower than 10-9, and with high phage-resistant stability. Except for 2 spontaneous mutants, the phage-resistant mechanisms of the last 58 mutants were irrelevant with lysogeny. The obvious decrease of the adsorption rates exhibited by all mutants indicated the presence of adsorption interference during phage infection. And the accessory polysaccharide-peptidoglycan complex was proved to be involved in the phage receptor sites. Proteolytic activities were heterogeneous distributed among the spontaneous mutants. Nevertheless, most of the mutants revealed proteolytic activities and pH values similar to those observed for the parent strain. Two mutants, BIM 10 and y 19 were chosen as model starters for pilot fermentations. Both of them showed favorable technological characteristics regardless of the absence or presence of phage. Therefore, besides spontaneous mutation, the 60Co-γray irradiation was also proved to be a simple and effective method for obtaining phage-resistant mutants from Lactobacillus delbrueckii strains with high technological performance. Moreover, some of the phage-resistant mutants obtained in this work might be used as potential starters when commercial strains become phage-sensitive.
4. Construction of a controlled autolysis Lactococcus strain
In the last stage of reproduction cycles, bacteriophages produce a set of enzymes that lyse the host cells to release their progeny particles. The lysis enzymes encoded by all double-stranded DNA phages involve two proteins:a holin and a lysin (also termed endolysin or lysozyme), which constitute a "two-component cell lysis cassette". The temperate bacteriophageφPYB5 was isolated from Lactobacillus fermentum YB5 strain. A putative holin (hyb5) and lysin (lyb5), obtained by sequencing of the genome library ofφPYB5 in E. coli, constituted a two-component cell lysis cassette. To elucidate the characterization of the lysis cassette (Hyb5-Lyb5) encoded by temperate bacteriophageφPYB5 in lactic acid bacteria, we cloned the hyb5 and lyb5 gene into the plasmid pSEC under the control of the nisin-inducible promoter, generating the recombinant plasmid pSEC-hyb5-lyb5. Then, pSEC-hyb5-lyb5 was induced into Lactococcus lactis NZ9000 by electroporation, yielding NZphl. Hyb5 and Lyb5 were expressed successfully under the induction of nisin and induced the lysis of NZphl effectively, resulting in the rapid reduction of OD600 and leakage of abundant intracellular proteins. These results will facilitate the application of autolysis lactic acid bacteria in fermentation to improve the quality of products.
5. Clone and expression of the two-component cell lysis cassette encoded by temperate bacteriophageφPYB5 of Lactobacillus fermentum in E. coli.
Different to others, Hyb5 was predicted to have a single transmembrane domain and Lyb5 possess an N-terminal signal transmembrane domain. In order to characterize this novel two-component cell lysis cassette encoded byφPYB5, and illustrate the potential application of Lyb5 as therapeutic agents, the hyb5, lyb5 and hyb5-lyb5 cassette were cloned and expressed in E. coli in this paper. The molecular weight of Hyb5 indicated by SDS-PAGE was 19 kDa, and Lyb5 was 45 kDa. Both Hyb5 and Lyb5 protein could induce cell lysis alone, resulting in the leakage ofβ-galactosidase. However, the Hyb5-Lyb5 cassette lysed the host cells more rapidly and extensively. By zymogram analysis, the Lyb5 produced in E. coli exhibited a broad lytic spectrum against Gram positive strains including Staphylococcus aureus as well as Gram negative strains such as Salmonella typhi, suggesting that Lyb5 provides a potential alternative of diagnostic tools and therapeutic agents.
6. Lysis mechanism studies on "Hyb5-Lyb5".
The lysis mechanism of holin-lysin cassette and the role of holin in the regulation of lysis time were elucidated based on the studies on E. coli phages. In this study, we tried to illuminate the lysis model of Hyb5-Lyb5 cassette and the mechanism of the regulation of Hyb5 in lysis through analyzing the function of N-trminal signal sequence in Lyb5 and determining the activities of different segments in Hyb5. We cloned lyb5 or△SARlyb5 (lyb5 lack of the SAR sequence) into the MCS1 of the pETDuet-1 vector, and inserted hyb5, hyb5p, or hyb5N30 (N-terminal sequence encoding the TMD with 30 amino acids) into the MCS2 of the pETDuet-1 vector, generating a series of recombinant vectors. By determining the lysis of different recombinants, we revealed that the co-expression of△SARlyb5 and Hyb5p resulted in obvious decrease of the culture turbidity in OD600, while the expression of△SARlyb5 accelerated the growth of the host strain, indicating that the SAR was unnecessary to the lysis activity of Lyb5, but was essential to the transmemberane of Lyb5. When hyb5N30 was expressed, it restrained the growth of E. coli, suggesting that the 30 AA could achieve transmemberane alone. However, the co-action of N30hyb5 and Lyb5 or△SARlyb5 did not induce large lysis of E. coli, exhibiting the limination of N30hyb5 in assisting Lyb5 or△SARlyb5 to the periplasmic space. Interestingly, Hyb5 lacking the C-terminal 30 AA could assist the transfer of Lyb5 more effectively than Hyb5, implying that the C-termina might inhibit the activity of Hyb5. However, the cleavability of SAR in the lysis process, the regulation of Hyb5 itself or other factors to the activity of Hyb5 and finally elucidation of the lysis mechanism of the novel Hyb5-Lyb5 cassette as well as the regulation of Hyb5 in the lysis process, all of which require abundant and further investigations.
引文
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