乳酸菌高效膜蛋白表达系统的构建及其自溶性质的研究
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摘要
乳酸菌是一类能够利用碳水化合物产生乳酸的革兰氏阳性细菌的统称,是一群由多个属组成的庞杂细菌群体,主要包括乳球菌属(Lactococcus),乳杆菌属(Lactobacillus),片球菌属(Pediococcus),链球菌属(Streptococcus),肠球菌属(Enterococcus),双歧杆菌属(Bifidobacterium)等。大多数乳酸菌对人体有益,它们通过降解糖类、蛋白和脂肪产生多种生物活性物质促进人体健康。此外,大部分乳酸菌本身就是益生菌(probiotics),其益生性质体现在维持人体肠道菌群平衡、调节人体免疫以及抑制有害病原微生物的生长等多个方面。
乳酸菌有些益生功能与细菌表面性质有关,比如絮凝(aggregation)、黏附(adhesion)、自溶(autolysis)等。自溶(自裂解),是指细菌在生长繁殖的某个阶段因肽聚糖水解酶(自溶酶)的作用而引起的自身裂解的现象。自溶酶除了裂解细胞发生自溶外,还参与细菌生长代谢的多个生理过程,如新合成肽聚糖的延伸、细胞增大以及子代细胞的产生等,是一类极其重要的细胞壁水解酶类。研究表明,有些抗生素、细菌素的抗菌作用也是通过肽聚糖水解酶的活性而完成的,因此对肽聚糖水解酶的深入认识对于揭示乳酸菌的益生作用以及充分发挥抗生素的杀菌作用尤为重要。
除了作为益生菌剂,乳酸菌还是重要的工业微生物菌株,在食品加工、乳制品发酵等领域具有悠久的应用历史,是公认的安全级(Generally Regarded as Safe, GRAS)微生物。有些乳酸乳球菌和乳杆菌作为发酵剂广泛应用于奶酪的生产。其中,奶酪的成熟过程是一个缓慢而昂贵的过程,而乳酸菌发酵剂的自溶释放出的脂肪酶、蛋白酶有利于风味物质的形成和奶酪的成熟,因此实时控制和深入认识自裂解机制是加速奶酪成熟的有效手段。截至目前,很多研究都着重于通过基因工程的方法构建可控裂解的乳酸乳球菌发酵剂菌株从而达到控制与缩短奶酪成熟时间的目的。而遗憾的是,发酵剂菌株的过度裂解会导致正常细胞数量减少,从而引起乳糖的过量剩余和奶酪性质的不稳定。所以,在非发酵剂菌株(主要指乳酸杆菌)中构建可控裂解系统可有效避免上述问题的发生。
乳酸菌基因组小、代谢简单、蛋白酶活力弱、易于进行人工改造等特征使其成为微生物学及分子生物学研究中的重要模式菌株。其中乳酸乳球菌已建立起较完善的遗传操作系统,利用严谨型启动子NisA可控表达系统(the nisin-controlled expression, NICE)已实现了多种异源蛋白的表达。由于乳酸菌是单分子膜,遗传操作系统可控性强,它还是膜蛋白表达的良好宿主
本论文主要以乳酸乳球菌与干酪乳杆菌(Lactobacillus casei)为实验材料,以自溶性质以及膜蛋白表达为研究重点,构建了一个新的高效乳酸菌膜蛋白表达载体,实现了绿色巴夫藻Δ4去饱和酶和嗜酸乳杆菌亚油酸异构酶在乳球菌中的过量表达；并且对乳酸菌的自溶性质以及相关肽聚糖水解酶的鉴别与生物特性进行了研究和探讨。具体的工作内容和取得的结果如下：
1.多不饱和脂肪酸合成相关功能基因的克隆与表达
多不饱和脂肪酸是人体所必需却不能自然合成的重要营养物质,其相关合成基因的克隆和表达是研究者关心的热点。本文以绿色巴夫藻(Pavlova viridis)为材料,通过反转录、SEFA-PCR (self-formed adaptor PCR)、SOE (splicing by overlap extension)等方法分别扩增得到了在EPA(二十碳五烯酸)与DHA(二十二碳六烯酸)合成中起重要作用的△5去饱和酶和A4去饱和酶的基因全长及表达序列。与之前实验室克隆和特征化的C20延伸酶(△5延伸酶)一起,完成了对绿色巴夫藻中EPA和DHA合成所涉关键基因的全部克隆工作。其中,Δ4去饱和酶与其同源蛋白有75%的同源序列。为了研究该酶的性质,在尝试以大肠杆菌和毕赤酵母为宿主利用多种载体表达A4去饱和酶时均没有成功,主要原因是膜蛋白的过量表达对宿主细胞有毒害作用。随后,将来源于枯草芽孢杆菌(Bacillus subtilis)、能够自动折叠于细胞膜的整合型膜蛋白短肽Mi stic与Δ4去饱和酶进行融合连接,结果实现了A4去饱和酶在大肠杆菌的过量表达。目的蛋白条带在SDS-PAGE中显著可见,此研究成为真核微藻多不饱和脂肪酸去饱和酶基因在原核生物中高效表达的首个成功例证。
2.乳酸乳球菌高效膜蛋白表达载体的构建与应用
膜蛋白对真核与原核细胞的生命活动具有重要作用,比如信号传导、能量转换、营养物质转运等。在已测序的基因组中,膜蛋白占据所有开放阅读框(ORF)的20%到25%。可是相比于可溶蛋白的研究,已明确结构特征和生化基础的膜蛋白数量很少,而通过异源表达制备具有高分辨率结构的膜蛋白则更是屈指可数。阻碍膜蛋白研究的一个重要瓶颈就是膜蛋白过量表达。在Mi stic介导Δ4去饱和酶在大肠杆菌表达工作的基础上,进一步以乳酸乳球菌为宿主,以pNZ8148为骨架,结合Mi stic结构元件构建了一个膜蛋白表达载体pNM110,探讨和改进膜蛋白在乳酸乳球菌中的表达。通过Mi stic与GFP的融合表达和GFP荧光的检测,证实了短肽Mi stic可以成功插入乳酸乳球菌的细胞膜,并且具有促进膜蛋白过量表达的潜力。为了验证该载体在乳球菌中表达膜蛋白的有效性,将克隆自绿色巴夫藻的A4去饱和酶基因pkjDes4和嗜酸乳杆菌的亚油酸异构酶基因pkjLi分别与Mistic融合连接,构建了重组载体pNMDes4和pNMLi,并成功实现了上述两种膜蛋白在乳酸乳球菌中的过量表达,其表达水平分别达到了膜蛋白总量的4.4%与45.2%,这个表达水平是目前以乳酸乳球菌为宿主表达膜蛋白产量最高的。为了进一步验证所表达亚油酸异构酶的功能,将重组的乳酸乳球菌菌株与底物亚油酸在一定条件下培养,结果发现重组乳酸菌具有转化亚油酸生成共轭亚油酸的能力,产量可达0.852mg/ml,转化率为28.4%,该结果首次实现了乳杆菌亚油酸异构酶的异源表达和功能鉴定。
3.乳酸菌自溶性质的研究
细菌编码的自溶酶除了用于细胞分裂产生子代细胞外,对于乳酸菌来说,还有一个重要的作用是在乳酪成熟中释放出胞内水解酶类而有助于风味物质的形成和缩短成熟时间;同时自溶酶具有对病原微生物的杀菌效果而可作为杀菌剂用作菌种保藏。因此对自溶酶和自溶表型的研究为乳酸茵的研究热点之一。本文采用GM17和MRS选择培养基,结合不同培养条件,先后从发酵乳、鸡肠道和人粪便等不同生境中分离得到了多株乳酸菌并对其自溶能力进行了测量和筛选。结果发现,同种细菌不同菌株之间的自溶能力有显著差异,这说明自溶具有高度菌株特异性。此外,粪肠球菌的自溶能力明显强于乳酸乳球菌。以抗生素为代表的生长抑制剂的使用是诱发细菌自溶现象的一个重要途径。本文检测了多种生长抑制剂在不同生长时期对乳酸乳球菌自溶性质的影响。结果表明,某些抗生素能够在碳源严格限制性培养基中对处于特定生长时期的乳酸乳球菌产生显著裂解的现象,而且抑制细胞壁合成类抗生素的促自溶效果更为明显,这些结果对于研究抗生素与乳酸菌自溶的关系以及乳酸菌自溶的调控机制提供了实验依据。
4.干酪乳杆菌肽聚糖水解酶AclB的鉴别与生物特性研究
相比于乳酸乳球菌,除了植物乳杆菌(Lactobacillus plantarum)的Acm2外,尚无乳杆菌肽聚糖水解酶鉴别与生物特性研究的报道。本文以干酪乳杆菌为研究对象,鉴别并克隆到5种推定的肽聚糖水解酶(自溶酶)基因,并对非内肽酶的基因在指数生长时期的不同阶段的表达情况做了测定,发现它们在指数期的转录量有着相似的变化趋势。随后,AclA与AclB分别在大肠杆菌中进行了大量表达和纯化。酶活性质测定表明,AclB在pH5.0的酸性条件下具有最大的水解活性,而最适温度是37℃,此结果与乳酸乳球菌肽聚糖水解酶AcmB、AcmC的酶活性质相似。为了研究AclB在细胞分离中的作用,使用自杀性整合载体对干酪乳杆菌S1的AclB基因进行了敲除失活。结果发现敲除后的乳杆菌除了在自溶能力以及自絮凝能力上有变化外,最显著的特征是部分突变菌体拉长或者扭曲,表明AclB在细胞分裂后的分离中起着重要的作用。本文首次对乳杆菌的肽聚糖水解酶做了全面的鉴别和分析,并首次对干酪乳杆菌中的AclB酶进行了生物特性研究,为该菌株的后续应用研究奠定了基础。
5.干酪乳杆菌可控裂解系统的建立及在奶酪发酵中的潜在应用
干酪乳杆菌作为发酵剂或非发酵剂乳酸菌株(non-starter lactic acid bacteria,NSLAB)应用于切达奶酪等多种奶酪的发酵生产中。建立干酪乳杆菌可控裂解系统可实现胞内酶的快速释放,同时可保持发酵剂一定的活细胞数,是加快奶酪成熟的有效途径。本文选用了来自于乳酸乳球菌的AcmA与来自于粪肠球菌的AtlA两种自溶酶,以NICE系统为基础,以干酪乳杆菌为宿主分别构建了两个可控裂解体系。在nisin的诱导下,AcmA与AtlA均能够成功表达并水解细胞壁,从而裂解干酪乳杆菌细胞并释放出胞内蛋白。为了进一步验证该体系在奶酪生产中的应用潜力,我们进行了实验室规模的模拟奶酪实验,在使用AtlA裂解体系的奶酪凝乳中检测到了5倍于对照组的乳酸脱氢酶活性,这标志着更多的胞内酶在生产过程中被释放到凝乳中。此外,在整个模拟奶酪实验中,所使用的发酵菌株乳酸乳球菌的数量以及整个凝乳的pH值较对照组没有显著改变,证明奶酪性质保持了相对的稳定。该研究证实了对非发酵剂乳杆菌进行可控裂解的可行性和优势,该思路和方法应具有应用于奶酪发酵生产的潜力。
Lactic acid bacteria (LAB) are a group of Gram positive bacteria utilizing carbohydrates to produce lactic acid. It is a mixing population constituted by a variety of genera, mainly including Lactococcus, Lactobacillus, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, etc. Except for a small population, most lactic acid bacteria play essential roles in many physiological functions involved in the interests of human-beings. The bioactive compounds, obtained by degrading the carbohydrates, proteins and fats, essentially contribute to the human health. The significance of LAB towards human-beings was also shown by its status of probiotics, namely living microorganisms to exert beneficial effects on the hosts when administered in the human gut. The probiotic effects include maintaining the microbiota balance of human intestinal tract, immunomodulating, inhibiting the growth of pathogens, and so on.
The probiotic properties of lactic acid bacteria were regarded to be associated with their surface characteristics, such as aggregation, adhesion and autolysis. Aggregation was the coherence and clumping mediated by the surface interaction between the same and different species of bacteria. Like adhesion, aggregation was considered to be an integral process of biofilm formation. They remove the pathogens and protect the epithelial cells through the competitive exclusion and precipitating pathogens. Autolysis can be observed under conditions which result in cessation of peptidoglycan synthesis, such as stationary phase or exposure to antibiotics, possibly from an uncontrolled action of PGHs (peptidoglycan hydrolases). Autolysins participate in many physiological processes of bacterial growth and metabolization, such as cell wall extension, peptidoglycan turnover and recycling, daughter cell separation and cell division, and thus are a family of enzymes functioning on the cell surface. The relevant studies revealed that the autolysis induced by antibiotics and bacteriocins had to be mediated by the autolysins. Thus the research on autolysins is important for the bacteria having tight commensal relationships with human beings. In addition, the inactivation of autolysin genes of LAB usually leads to the alteration of aggregation characteristics, demonstrating that the surface properties of LAB are tightly correlated.
In addition to the probiotic properties, lactic acid bacteria are industrially important dairy microorganisms, too. Due to its GRAS (Generally Regarded as Safe) status, LAB were traditionally applied in food and dairy fermentation, among which, the cheese manufacture using lactococcal and lactobacillus strains as starters attracted extensive attention. Cheese ripening was a slow and costly process, and considered to be tightly associated with the lysis of the starters. Then autolysis of the relevant LAB has become a critical subject. To date, many researchers focus on establishing controllable lysis system in the starter LAB by means of genetic engineering, so as to control and reduce cheese ripening time. However, to our disappointment, the excessive lysis of the starters could usually lead to very few starter cells remaining in the gel, causing excessive residual lactose and instability of cheese properties. Therefore, constructing controllable lysis system in non-starter lactic acid bacteria was expected to be of critical application values.
Lactic acid bacteria are also important objects and tools for the microbiology and molecular biology research. Because they had a series of advantageous characteristics, such as small genome size, high cell densities, multiple amino acid auxotrophic strains and mild proteolytic capabilities, LAB are supposed to be the beneficial hosts for heterologous protein expression. Particularly, since a strong and tightly regulated promoter system:NICE (the nisin-controlled expression) system was established and extensively used, lactic acid bacteria (mainly referring to Lactococcus lactis) have been given more attention in this respect. As for the membrane protein study, which has been lagged far behind due to the difficulties in heterologous expression, LAB hosts also have special values.
Using L. lactis and Lactobacillus casei as the studying objects, concentrating on the bacterial autolysis and membrane protein expression, this dissertation mainly introduced a novel and highly efficient membrane protein expression vector in L. lactis, investigated the autolysis of LAB, as well as identified and characterized the associated autolysins. The detailed contents and achievements are listed on the following list:
1. Identification and overexpression of functional genes involved in PUFA synthesis
Polyunsaturated fatty acids (PUFAs) are among the nutrient that human-beings need essentially but could not synthesize naturally. So the studies on the identification and characterization of the relevant functional genes are of economic use. This dissertation used Pavlova viridis as the raw material to obtain the genomic and cDNA sequences of△5and△4desaturase, responsible for the synthesis of EPA (Eicosapentaenoic acid) and DHA (Docosahexaenoic acid), respectively. The involved techniques included reverse transcription, SEFA-PCR (self-formed adaptor PCR), SOE (splicing by overlap extension), and so on. Along with the C20elongase (△5elongase), cloned and characterized previously in our lab, the work concerning the key enzymes involved in the EPA and DHA synthesis of P. viridis were finished. Among the results,△4desaturase obtained shared75%sequence identity with the homologous proteins. As a eukaryotic transmembrane protein, difficulties were met when attempting to heterologously express△4desaturase in a range of vectors from E. coli and Pichia pastor is. The phenominon that no obvious expression was observed might be ascribed to the severe toxicity towards host cells. Subsequently, after being fused with Mistic, a short peptide from the Bacillus subtilis and with unique properties,△4desaturase (pkjDes4) was successfully overexpressed in the cytoplasmic membrane of E. coli. The objective protein band was clearly visible on SDS-PAGE gel, and thus became the first example of any PUFA desaturase genes from eukaryotic microalgae overexpressed in prokaryotic organisms.
2. Construction and application of a high-yield membrane protein expression system in Lactococcus lactis
Membrane proteins occupy20%to25%of all the open reading frames (ORFs), and fulfill many biological functions both in the eukaryotes and prokaryotes, such as signal transduction, energy conversion, nutrient transportation. But compared to the studies on soluble proteins, very few high-resolution structures of membrane proteins were obtained, and those acquired from the heterogenous overexpression were even fewer. One of the bottlenecks to hamper the membrane proteins'research was that most of them could hardly be heterologously overexpressed. In order to overcome the difficulty, this dissertation used Lactococcus lactis as the host and used Mistic as the featuring element to establish a membrane protein expression vector pNM110. The potency of this system was demonstrated in the expression of a eukaryotic membrane protein (pkjDes4) and a prokaryotic membrane protein (pkjLi). The expression level could reach up to4.4%and45.2%, respectively. The latter is a newly isolated linoleate isomerase from Lactobacillus acidophilus, the catalyzing nature of which was first characterized in this species via the activity determination after heterologous overexpression in L. lactis. The system established here not only proved the Mistic chaperoning strategy could be applied to L. lactis hosts efficiently, but also exhibited its extraordinary capacity to facilitate the overproduction of some intractable membrane proteins.
Conjugated linoleic acid (CLA) was also an essential polyunsaturated fatty acid. It attracted much attention due to their numerous health-promoting properties, such as anti-obesity, anti-diabetes, immunomodulating and lowering cholesterol content. Some LAB (mainly referring to Lactobacillus) strains are able to transform linoleic acid (LA) to CLA, and thus were widely investigated. The CLA synthesis was the result of the enzymatic activity of linoleate isomerase (LI). As a membrane protein, LIs of the lactobacillus origin have never been overexpresseed and functionally characterized. In this dissertation, the pkjLI, a newly-cloned LI from Lactobacillus acidophilus, was functionally overexpressed in L. lactis, based on pNM110established above. The recombinant L. lactis strain obtained the capacity to synthesize CLA after the addition of substrate LA. The production yield could reach up to0.852mg/mL, and the conversion rate was28.4%. Because of its probiotic property, the recombinant strain was anticipated to have applications in anti-proliferative activity and fatty acid modulation in host adipose tissue.
3. Screening and autolysis assays of lactic acid bacteria
Lactic acid bacteria are the bacterial resources with economic significance. The widespread genera have decided the diversity of their habitats. In this dissertation, we combined the use of selective medium and different culturing conditions to successively screen and isolate a range of lactic acid bacteria from commercial yogurt, milk, chicken intestine and human faeces. The isolated strains were then identified by the16S rDNA sequencing. Subsequently, the fundamental bacterial properties, such as growth curve, aggregation mediated by flocculants and autolysis were determined. It was revealed from autolysis that, different strains from the same species could exhibit significantly distinct autolyzing abilities, indicating that autolysis was a strain-specific characteristic. Besides, the autolyzing ability of Enterococcus strains was regarded to be better than that of L. lactis strains. Subsequently, the influence of growth inhibitors on the autolysis of LAB was investigated in this dissertation. The results revealed that the rapid autolysis in the diauxic growth point under some circumstances was observed. In addition, although most growth inhibitors exhibited obvious bactericidal effects towards lactococcal cells under the used concentrations, their influence on the autolysis of L. lactis was variable. The results provided experimental proofs for the studies on the relationship between antibiotic agents and autolysis of LAB, as well as on the investigation of the regulation mechanisms underlying the autolysis phenotype.
4. The peptidoglycan hydrolase complement of Lactobacillus casei: identification and characterization of AcIB
In comparison with the research of L. lactis, no peptidoglycan hydrolases (autolysins) were reported to be identified and characterized in Lactobacillus, except for an Acm2from Lb. plantarum and an S-protein from Lb. acidophilus. In this dissertation, five putative autolysins were identified and cloned in Lb. casei. The expression profiles of the enzymes, except for endopeptidases, were determined in the different growth stages, indicating that the amount of the transcripts of different genes had the similar dynamics. Then AclA and AcIB were overexpressed and purified in E. coli. The enzymatic activity assay showed that the optimal condition for AcIB activity was pH4.0and37℃, which was a similar behavior with the PGHs form L. lactis: AcmB, AcmC and YjgB. Subsequently, with the aids of suicidal integration vector, the AcIB gene was inactivated. And in addition to the alterations in autolysis and autoaggregation, the most obvious phenomenon of the knock-out strain was that a proportion of bacteria were elongated and twisted, a typical characteristic for the PGH-inactivation strain, indicating AcIB plays key roles in cell separation. In summary, the peptidoglycan hydrolases in the Lactobacillus were for the first time extensively investigated and analysed, and this was also a novel instance to characterize a peptidoglycan hydrolase in Lb. casei. The study could not only be considered as the novel basis research on the PGH complement in the species of bacteria, but also be expected to be widely used in the autolysis-involving applications.
5. Construction of the controlled autolysis systems in Lactobacillus casei and their potential applications in cheese manufacture
Lactobacillus casei was used in the production of many types of cheese as starter LAB or nonstarter LAB. It was a novel pathway to use nonstarter LAB to establish the controlled autolysis system to accelerate the cheese ripening, because the system could not only achieve the rapid release of intracellular peptidases, but was favorable for maintaining the cell number of the starters. In order to remove the potential problems caused by prophage-sourced lysin, two autolysins, AcmA from L. lactis and AtlA from Enter-ococcus faecalis, were adopted to establish the autolysis system in L. casei, which was based on the principles of NICE application. The induction experiments in broth showed that upon nisin induction, both AcmA and AtlA could be successfully expressed and positioned at the cell wall to function, causing the lysis of the cells and the release of the intracellular enzymes. Subsequently, to examine the application potential of this system in the cheese production, the laboratory-scale model cheese experiment was carried out. The results indicated that approximately5-fold of lactate dehydrogenase (LDH), compared to the control group, were detected in the cheese utilizing AtlA-autolyzing system, indicating that more intracellular enzymes were released into the curd during the process. Besides, in this experiment, the cell population of L. lactis cells used as starters, as well as the pH of the curd, were not changed significantly, demonstrating that the fundamental properties of cheese were maintained. This work demonstrated the feasibility and advantages of the controlled autolysis system to be established in non-starter LAB, and was readily available for the future cheese manufacture.
乳酸菌有些益生功能与细菌表面性质有关,比如絮凝(aggregation)、黏附(adhesion)、自溶(autolysis)等。自溶(自裂解),是指细菌在生长繁殖的某个阶段因肽聚糖水解酶(自溶酶)的作用而引起的自身裂解的现象。自溶酶除了裂解细胞发生自溶外,还参与细菌生长代谢的多个生理过程,如新合成肽聚糖的延伸、细胞增大以及子代细胞的产生等,是一类极其重要的细胞壁水解酶类。研究表明,有些抗生素、细菌素的抗菌作用也是通过肽聚糖水解酶的活性而完成的,因此对肽聚糖水解酶的深入认识对于揭示乳酸菌的益生作用以及充分发挥抗生素的杀菌作用尤为重要。
除了作为益生菌剂,乳酸菌还是重要的工业微生物菌株,在食品加工、乳制品发酵等领域具有悠久的应用历史,是公认的安全级(Generally Regarded as Safe, GRAS)微生物。有些乳酸乳球菌和乳杆菌作为发酵剂广泛应用于奶酪的生产。其中,奶酪的成熟过程是一个缓慢而昂贵的过程,而乳酸菌发酵剂的自溶释放出的脂肪酶、蛋白酶有利于风味物质的形成和奶酪的成熟,因此实时控制和深入认识自裂解机制是加速奶酪成熟的有效手段。截至目前,很多研究都着重于通过基因工程的方法构建可控裂解的乳酸乳球菌发酵剂菌株从而达到控制与缩短奶酪成熟时间的目的。而遗憾的是,发酵剂菌株的过度裂解会导致正常细胞数量减少,从而引起乳糖的过量剩余和奶酪性质的不稳定。所以,在非发酵剂菌株(主要指乳酸杆菌)中构建可控裂解系统可有效避免上述问题的发生。
乳酸菌基因组小、代谢简单、蛋白酶活力弱、易于进行人工改造等特征使其成为微生物学及分子生物学研究中的重要模式菌株。其中乳酸乳球菌已建立起较完善的遗传操作系统,利用严谨型启动子NisA可控表达系统(the nisin-controlled expression, NICE)已实现了多种异源蛋白的表达。由于乳酸菌是单分子膜,遗传操作系统可控性强,它还是膜蛋白表达的良好宿主
本论文主要以乳酸乳球菌与干酪乳杆菌(Lactobacillus casei)为实验材料,以自溶性质以及膜蛋白表达为研究重点,构建了一个新的高效乳酸菌膜蛋白表达载体,实现了绿色巴夫藻Δ4去饱和酶和嗜酸乳杆菌亚油酸异构酶在乳球菌中的过量表达；并且对乳酸菌的自溶性质以及相关肽聚糖水解酶的鉴别与生物特性进行了研究和探讨。具体的工作内容和取得的结果如下：
1.多不饱和脂肪酸合成相关功能基因的克隆与表达
多不饱和脂肪酸是人体所必需却不能自然合成的重要营养物质,其相关合成基因的克隆和表达是研究者关心的热点。本文以绿色巴夫藻(Pavlova viridis)为材料,通过反转录、SEFA-PCR (self-formed adaptor PCR)、SOE (splicing by overlap extension)等方法分别扩增得到了在EPA(二十碳五烯酸)与DHA(二十二碳六烯酸)合成中起重要作用的△5去饱和酶和A4去饱和酶的基因全长及表达序列。与之前实验室克隆和特征化的C20延伸酶(△5延伸酶)一起,完成了对绿色巴夫藻中EPA和DHA合成所涉关键基因的全部克隆工作。其中,Δ4去饱和酶与其同源蛋白有75%的同源序列。为了研究该酶的性质,在尝试以大肠杆菌和毕赤酵母为宿主利用多种载体表达A4去饱和酶时均没有成功,主要原因是膜蛋白的过量表达对宿主细胞有毒害作用。随后,将来源于枯草芽孢杆菌(Bacillus subtilis)、能够自动折叠于细胞膜的整合型膜蛋白短肽Mi stic与Δ4去饱和酶进行融合连接,结果实现了A4去饱和酶在大肠杆菌的过量表达。目的蛋白条带在SDS-PAGE中显著可见,此研究成为真核微藻多不饱和脂肪酸去饱和酶基因在原核生物中高效表达的首个成功例证。
2.乳酸乳球菌高效膜蛋白表达载体的构建与应用
膜蛋白对真核与原核细胞的生命活动具有重要作用,比如信号传导、能量转换、营养物质转运等。在已测序的基因组中,膜蛋白占据所有开放阅读框(ORF)的20%到25%。可是相比于可溶蛋白的研究,已明确结构特征和生化基础的膜蛋白数量很少,而通过异源表达制备具有高分辨率结构的膜蛋白则更是屈指可数。阻碍膜蛋白研究的一个重要瓶颈就是膜蛋白过量表达。在Mi stic介导Δ4去饱和酶在大肠杆菌表达工作的基础上,进一步以乳酸乳球菌为宿主,以pNZ8148为骨架,结合Mi stic结构元件构建了一个膜蛋白表达载体pNM110,探讨和改进膜蛋白在乳酸乳球菌中的表达。通过Mi stic与GFP的融合表达和GFP荧光的检测,证实了短肽Mi stic可以成功插入乳酸乳球菌的细胞膜,并且具有促进膜蛋白过量表达的潜力。为了验证该载体在乳球菌中表达膜蛋白的有效性,将克隆自绿色巴夫藻的A4去饱和酶基因pkjDes4和嗜酸乳杆菌的亚油酸异构酶基因pkjLi分别与Mistic融合连接,构建了重组载体pNMDes4和pNMLi,并成功实现了上述两种膜蛋白在乳酸乳球菌中的过量表达,其表达水平分别达到了膜蛋白总量的4.4%与45.2%,这个表达水平是目前以乳酸乳球菌为宿主表达膜蛋白产量最高的。为了进一步验证所表达亚油酸异构酶的功能,将重组的乳酸乳球菌菌株与底物亚油酸在一定条件下培养,结果发现重组乳酸菌具有转化亚油酸生成共轭亚油酸的能力,产量可达0.852mg/ml,转化率为28.4%,该结果首次实现了乳杆菌亚油酸异构酶的异源表达和功能鉴定。
3.乳酸菌自溶性质的研究
细菌编码的自溶酶除了用于细胞分裂产生子代细胞外,对于乳酸菌来说,还有一个重要的作用是在乳酪成熟中释放出胞内水解酶类而有助于风味物质的形成和缩短成熟时间;同时自溶酶具有对病原微生物的杀菌效果而可作为杀菌剂用作菌种保藏。因此对自溶酶和自溶表型的研究为乳酸茵的研究热点之一。本文采用GM17和MRS选择培养基,结合不同培养条件,先后从发酵乳、鸡肠道和人粪便等不同生境中分离得到了多株乳酸菌并对其自溶能力进行了测量和筛选。结果发现,同种细菌不同菌株之间的自溶能力有显著差异,这说明自溶具有高度菌株特异性。此外,粪肠球菌的自溶能力明显强于乳酸乳球菌。以抗生素为代表的生长抑制剂的使用是诱发细菌自溶现象的一个重要途径。本文检测了多种生长抑制剂在不同生长时期对乳酸乳球菌自溶性质的影响。结果表明,某些抗生素能够在碳源严格限制性培养基中对处于特定生长时期的乳酸乳球菌产生显著裂解的现象,而且抑制细胞壁合成类抗生素的促自溶效果更为明显,这些结果对于研究抗生素与乳酸菌自溶的关系以及乳酸菌自溶的调控机制提供了实验依据。
4.干酪乳杆菌肽聚糖水解酶AclB的鉴别与生物特性研究
相比于乳酸乳球菌,除了植物乳杆菌(Lactobacillus plantarum)的Acm2外,尚无乳杆菌肽聚糖水解酶鉴别与生物特性研究的报道。本文以干酪乳杆菌为研究对象,鉴别并克隆到5种推定的肽聚糖水解酶(自溶酶)基因,并对非内肽酶的基因在指数生长时期的不同阶段的表达情况做了测定,发现它们在指数期的转录量有着相似的变化趋势。随后,AclA与AclB分别在大肠杆菌中进行了大量表达和纯化。酶活性质测定表明,AclB在pH5.0的酸性条件下具有最大的水解活性,而最适温度是37℃,此结果与乳酸乳球菌肽聚糖水解酶AcmB、AcmC的酶活性质相似。为了研究AclB在细胞分离中的作用,使用自杀性整合载体对干酪乳杆菌S1的AclB基因进行了敲除失活。结果发现敲除后的乳杆菌除了在自溶能力以及自絮凝能力上有变化外,最显著的特征是部分突变菌体拉长或者扭曲,表明AclB在细胞分裂后的分离中起着重要的作用。本文首次对乳杆菌的肽聚糖水解酶做了全面的鉴别和分析,并首次对干酪乳杆菌中的AclB酶进行了生物特性研究,为该菌株的后续应用研究奠定了基础。
5.干酪乳杆菌可控裂解系统的建立及在奶酪发酵中的潜在应用
干酪乳杆菌作为发酵剂或非发酵剂乳酸菌株(non-starter lactic acid bacteria,NSLAB)应用于切达奶酪等多种奶酪的发酵生产中。建立干酪乳杆菌可控裂解系统可实现胞内酶的快速释放,同时可保持发酵剂一定的活细胞数,是加快奶酪成熟的有效途径。本文选用了来自于乳酸乳球菌的AcmA与来自于粪肠球菌的AtlA两种自溶酶,以NICE系统为基础,以干酪乳杆菌为宿主分别构建了两个可控裂解体系。在nisin的诱导下,AcmA与AtlA均能够成功表达并水解细胞壁,从而裂解干酪乳杆菌细胞并释放出胞内蛋白。为了进一步验证该体系在奶酪生产中的应用潜力,我们进行了实验室规模的模拟奶酪实验,在使用AtlA裂解体系的奶酪凝乳中检测到了5倍于对照组的乳酸脱氢酶活性,这标志着更多的胞内酶在生产过程中被释放到凝乳中。此外,在整个模拟奶酪实验中,所使用的发酵菌株乳酸乳球菌的数量以及整个凝乳的pH值较对照组没有显著改变,证明奶酪性质保持了相对的稳定。该研究证实了对非发酵剂乳杆菌进行可控裂解的可行性和优势,该思路和方法应具有应用于奶酪发酵生产的潜力。
Lactic acid bacteria (LAB) are a group of Gram positive bacteria utilizing carbohydrates to produce lactic acid. It is a mixing population constituted by a variety of genera, mainly including Lactococcus, Lactobacillus, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, etc. Except for a small population, most lactic acid bacteria play essential roles in many physiological functions involved in the interests of human-beings. The bioactive compounds, obtained by degrading the carbohydrates, proteins and fats, essentially contribute to the human health. The significance of LAB towards human-beings was also shown by its status of probiotics, namely living microorganisms to exert beneficial effects on the hosts when administered in the human gut. The probiotic effects include maintaining the microbiota balance of human intestinal tract, immunomodulating, inhibiting the growth of pathogens, and so on.
The probiotic properties of lactic acid bacteria were regarded to be associated with their surface characteristics, such as aggregation, adhesion and autolysis. Aggregation was the coherence and clumping mediated by the surface interaction between the same and different species of bacteria. Like adhesion, aggregation was considered to be an integral process of biofilm formation. They remove the pathogens and protect the epithelial cells through the competitive exclusion and precipitating pathogens. Autolysis can be observed under conditions which result in cessation of peptidoglycan synthesis, such as stationary phase or exposure to antibiotics, possibly from an uncontrolled action of PGHs (peptidoglycan hydrolases). Autolysins participate in many physiological processes of bacterial growth and metabolization, such as cell wall extension, peptidoglycan turnover and recycling, daughter cell separation and cell division, and thus are a family of enzymes functioning on the cell surface. The relevant studies revealed that the autolysis induced by antibiotics and bacteriocins had to be mediated by the autolysins. Thus the research on autolysins is important for the bacteria having tight commensal relationships with human beings. In addition, the inactivation of autolysin genes of LAB usually leads to the alteration of aggregation characteristics, demonstrating that the surface properties of LAB are tightly correlated.
In addition to the probiotic properties, lactic acid bacteria are industrially important dairy microorganisms, too. Due to its GRAS (Generally Regarded as Safe) status, LAB were traditionally applied in food and dairy fermentation, among which, the cheese manufacture using lactococcal and lactobacillus strains as starters attracted extensive attention. Cheese ripening was a slow and costly process, and considered to be tightly associated with the lysis of the starters. Then autolysis of the relevant LAB has become a critical subject. To date, many researchers focus on establishing controllable lysis system in the starter LAB by means of genetic engineering, so as to control and reduce cheese ripening time. However, to our disappointment, the excessive lysis of the starters could usually lead to very few starter cells remaining in the gel, causing excessive residual lactose and instability of cheese properties. Therefore, constructing controllable lysis system in non-starter lactic acid bacteria was expected to be of critical application values.
Lactic acid bacteria are also important objects and tools for the microbiology and molecular biology research. Because they had a series of advantageous characteristics, such as small genome size, high cell densities, multiple amino acid auxotrophic strains and mild proteolytic capabilities, LAB are supposed to be the beneficial hosts for heterologous protein expression. Particularly, since a strong and tightly regulated promoter system:NICE (the nisin-controlled expression) system was established and extensively used, lactic acid bacteria (mainly referring to Lactococcus lactis) have been given more attention in this respect. As for the membrane protein study, which has been lagged far behind due to the difficulties in heterologous expression, LAB hosts also have special values.
Using L. lactis and Lactobacillus casei as the studying objects, concentrating on the bacterial autolysis and membrane protein expression, this dissertation mainly introduced a novel and highly efficient membrane protein expression vector in L. lactis, investigated the autolysis of LAB, as well as identified and characterized the associated autolysins. The detailed contents and achievements are listed on the following list:
1. Identification and overexpression of functional genes involved in PUFA synthesis
Polyunsaturated fatty acids (PUFAs) are among the nutrient that human-beings need essentially but could not synthesize naturally. So the studies on the identification and characterization of the relevant functional genes are of economic use. This dissertation used Pavlova viridis as the raw material to obtain the genomic and cDNA sequences of△5and△4desaturase, responsible for the synthesis of EPA (Eicosapentaenoic acid) and DHA (Docosahexaenoic acid), respectively. The involved techniques included reverse transcription, SEFA-PCR (self-formed adaptor PCR), SOE (splicing by overlap extension), and so on. Along with the C20elongase (△5elongase), cloned and characterized previously in our lab, the work concerning the key enzymes involved in the EPA and DHA synthesis of P. viridis were finished. Among the results,△4desaturase obtained shared75%sequence identity with the homologous proteins. As a eukaryotic transmembrane protein, difficulties were met when attempting to heterologously express△4desaturase in a range of vectors from E. coli and Pichia pastor is. The phenominon that no obvious expression was observed might be ascribed to the severe toxicity towards host cells. Subsequently, after being fused with Mistic, a short peptide from the Bacillus subtilis and with unique properties,△4desaturase (pkjDes4) was successfully overexpressed in the cytoplasmic membrane of E. coli. The objective protein band was clearly visible on SDS-PAGE gel, and thus became the first example of any PUFA desaturase genes from eukaryotic microalgae overexpressed in prokaryotic organisms.
2. Construction and application of a high-yield membrane protein expression system in Lactococcus lactis
Membrane proteins occupy20%to25%of all the open reading frames (ORFs), and fulfill many biological functions both in the eukaryotes and prokaryotes, such as signal transduction, energy conversion, nutrient transportation. But compared to the studies on soluble proteins, very few high-resolution structures of membrane proteins were obtained, and those acquired from the heterogenous overexpression were even fewer. One of the bottlenecks to hamper the membrane proteins'research was that most of them could hardly be heterologously overexpressed. In order to overcome the difficulty, this dissertation used Lactococcus lactis as the host and used Mistic as the featuring element to establish a membrane protein expression vector pNM110. The potency of this system was demonstrated in the expression of a eukaryotic membrane protein (pkjDes4) and a prokaryotic membrane protein (pkjLi). The expression level could reach up to4.4%and45.2%, respectively. The latter is a newly isolated linoleate isomerase from Lactobacillus acidophilus, the catalyzing nature of which was first characterized in this species via the activity determination after heterologous overexpression in L. lactis. The system established here not only proved the Mistic chaperoning strategy could be applied to L. lactis hosts efficiently, but also exhibited its extraordinary capacity to facilitate the overproduction of some intractable membrane proteins.
Conjugated linoleic acid (CLA) was also an essential polyunsaturated fatty acid. It attracted much attention due to their numerous health-promoting properties, such as anti-obesity, anti-diabetes, immunomodulating and lowering cholesterol content. Some LAB (mainly referring to Lactobacillus) strains are able to transform linoleic acid (LA) to CLA, and thus were widely investigated. The CLA synthesis was the result of the enzymatic activity of linoleate isomerase (LI). As a membrane protein, LIs of the lactobacillus origin have never been overexpresseed and functionally characterized. In this dissertation, the pkjLI, a newly-cloned LI from Lactobacillus acidophilus, was functionally overexpressed in L. lactis, based on pNM110established above. The recombinant L. lactis strain obtained the capacity to synthesize CLA after the addition of substrate LA. The production yield could reach up to0.852mg/mL, and the conversion rate was28.4%. Because of its probiotic property, the recombinant strain was anticipated to have applications in anti-proliferative activity and fatty acid modulation in host adipose tissue.
3. Screening and autolysis assays of lactic acid bacteria
Lactic acid bacteria are the bacterial resources with economic significance. The widespread genera have decided the diversity of their habitats. In this dissertation, we combined the use of selective medium and different culturing conditions to successively screen and isolate a range of lactic acid bacteria from commercial yogurt, milk, chicken intestine and human faeces. The isolated strains were then identified by the16S rDNA sequencing. Subsequently, the fundamental bacterial properties, such as growth curve, aggregation mediated by flocculants and autolysis were determined. It was revealed from autolysis that, different strains from the same species could exhibit significantly distinct autolyzing abilities, indicating that autolysis was a strain-specific characteristic. Besides, the autolyzing ability of Enterococcus strains was regarded to be better than that of L. lactis strains. Subsequently, the influence of growth inhibitors on the autolysis of LAB was investigated in this dissertation. The results revealed that the rapid autolysis in the diauxic growth point under some circumstances was observed. In addition, although most growth inhibitors exhibited obvious bactericidal effects towards lactococcal cells under the used concentrations, their influence on the autolysis of L. lactis was variable. The results provided experimental proofs for the studies on the relationship between antibiotic agents and autolysis of LAB, as well as on the investigation of the regulation mechanisms underlying the autolysis phenotype.
4. The peptidoglycan hydrolase complement of Lactobacillus casei: identification and characterization of AcIB
In comparison with the research of L. lactis, no peptidoglycan hydrolases (autolysins) were reported to be identified and characterized in Lactobacillus, except for an Acm2from Lb. plantarum and an S-protein from Lb. acidophilus. In this dissertation, five putative autolysins were identified and cloned in Lb. casei. The expression profiles of the enzymes, except for endopeptidases, were determined in the different growth stages, indicating that the amount of the transcripts of different genes had the similar dynamics. Then AclA and AcIB were overexpressed and purified in E. coli. The enzymatic activity assay showed that the optimal condition for AcIB activity was pH4.0and37℃, which was a similar behavior with the PGHs form L. lactis: AcmB, AcmC and YjgB. Subsequently, with the aids of suicidal integration vector, the AcIB gene was inactivated. And in addition to the alterations in autolysis and autoaggregation, the most obvious phenomenon of the knock-out strain was that a proportion of bacteria were elongated and twisted, a typical characteristic for the PGH-inactivation strain, indicating AcIB plays key roles in cell separation. In summary, the peptidoglycan hydrolases in the Lactobacillus were for the first time extensively investigated and analysed, and this was also a novel instance to characterize a peptidoglycan hydrolase in Lb. casei. The study could not only be considered as the novel basis research on the PGH complement in the species of bacteria, but also be expected to be widely used in the autolysis-involving applications.
5. Construction of the controlled autolysis systems in Lactobacillus casei and their potential applications in cheese manufacture
Lactobacillus casei was used in the production of many types of cheese as starter LAB or nonstarter LAB. It was a novel pathway to use nonstarter LAB to establish the controlled autolysis system to accelerate the cheese ripening, because the system could not only achieve the rapid release of intracellular peptidases, but was favorable for maintaining the cell number of the starters. In order to remove the potential problems caused by prophage-sourced lysin, two autolysins, AcmA from L. lactis and AtlA from Enter-ococcus faecalis, were adopted to establish the autolysis system in L. casei, which was based on the principles of NICE application. The induction experiments in broth showed that upon nisin induction, both AcmA and AtlA could be successfully expressed and positioned at the cell wall to function, causing the lysis of the cells and the release of the intracellular enzymes. Subsequently, to examine the application potential of this system in the cheese production, the laboratory-scale model cheese experiment was carried out. The results indicated that approximately5-fold of lactate dehydrogenase (LDH), compared to the control group, were detected in the cheese utilizing AtlA-autolyzing system, indicating that more intracellular enzymes were released into the curd during the process. Besides, in this experiment, the cell population of L. lactis cells used as starters, as well as the pH of the curd, were not changed significantly, demonstrating that the fundamental properties of cheese were maintained. This work demonstrated the feasibility and advantages of the controlled autolysis system to be established in non-starter LAB, and was readily available for the future cheese manufacture.
引文
Acosta MP. Polomino MM. Allievi MC. Rivas CS. Ruzal SM. Murein hydrolase activity in the surface layer of Lactobacillus acidophilus ATCC 4356. Applied and Environmental Microbiology 2008; 74 (24):7824-7827
Antignac A. Sieradzki K. Tomasz A. Perturbation of cell wall synthesis suppressed autolysis in Staphylococcus aureus. evidence for coregulation of cell wall synthetic and hydrolytic enzymes. Journal of Bacteriology,2007; 189 (21):7573-7580
Altieri C. Bevilacqua A. D'Amato D. Nobile A. Sinigaglia M. Modelling the survival of starter lactic acid bacteria and Bifidobacterium bifidum in single and simultaneous cultures. Food Microbiology 2008; 25:729-734
Apiradee H. Kalyanee P. Pongsathon P. Patcharaporn D. Matura S. Sanjukta S. Supapon C. Morakot T. The expression of three desaturase genes of Spirulina platensis in Escherichia coli DH5α. Molecular Biology Reports 2004; 31:177-189
Beliveau C. Potvin C. Trudel J. Asselin A. Bellemare G. Cloning sequencing and expression in Escherichia coli of a Streptococcus faecal is autolysin. Journal of Bacteriology 1991; 173:5619-5623
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Bravetti AL. Mesnage S. Lefort A. Chau F. Eckert C. Garry L. Arthur M. Fantin B. Contribution of the autolysin AtlA to the bactericidal activity of amoxicillin against Enterococcus faecalis JH2-2. Antimicrobial Agents and Chemotherapy 2009; 53 (4): 1667-1669
Brunskill EW. Bayles KW. Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus. Journal of Bacteriology 1996; 178 (3):611-618
Buist G. Kok J. Leenhouts KJ. Dabrowska M. Venema G. Haandrikman AI. Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. Journal of Bacteriology 1995; 177(6):1554-1563
Buist G. Karsens H. Nauta A. van Sinderen D. Venema G. Kok J. Autolysis of Lactococcus lactis caused by induced overproduction of its major autolysin, AcmA. Applied and Environmental Microbiology 1997; 63 (7):2711-2718
Chi X. Zhang X. Guan X. Ding L. Li Y. Wang M. Lin H. Qin S. Fatty acid biosynthesis in eukaryotic photosynthetic microalgae:identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii. The Journal of Microbiology 2008; 46(2):189-201
Chung J. Yoon HE. Shin HC. Choi E. Byeon W. Induction of growth phase-specific autolysis in Bacillus subtilis 168 by growth inhibitors. The Journal of Microbiology 2009; 47(1):50-59
Cibik R Chapot-Chartier MP. Characterization of autolytic enzymes in Laclobacillus pentosus. Letters in Applied Microbiology 2004; 38:459-463
Drew D. Froderberg L. Baars L. de Gier JL. Assembly and overexpression of membrane proteins in Escherichia coli. Biochimica et Biophysica Acta 2003; 1610: 3-10
Drew D. Slotboom D. Friso G. Reda T. Genevaux P. Rapp M. Meindl-Beinker NM. Lambert W. Lerch M. Daley DO. Van Wijk K. Hirst J. Kunji E. De Gier J. A scalable, GFP-based pipeline for membrane protein overexpression screening and purification Protein Science 2005; 14:2011-2017
Drew, D. Lerch M. Kunji E. Slotboom DJ. de Gier JW. Optimization of membrane protein overexpression and purification using GFP fusions. Nature Methods 2006; 3: 303-313
Dvir H. Matthew E. Lundberg E. Maji SK. Riek R. Choe S. Mistic:cellular localization, solution behavior, polymerization, and fibril formation. Protein Science 2009; 18:1564-1570.
Eckert C. Lecerf M. Dubost L. Arthur M. Mesnage S. Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis. Journal of Bacteriology 2006; 188 (24):8513-8519
Emirian A. Fromentin S. Eckert C. Chau F. Dubost L. Delepierre M. Gutmann L Arthur M. Mesnage S. Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB. FEBS Letters 2009; 583:3033-3038
Feirtag JM. McKay CC. Isolation of Streptococcus lactis C2 mutants selected for temperature sensitivity and potential use in cheese manufacture. Journal of Dairy Science 1987; 70:1773-1778
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Hannon JA. Kilcawley KN. Wilkinson MG. Delahunty CM. Beresford TP. Production of ingredient-type cheddar cheese with accelerated flavor development by addition of enzyme-modified cheese powder. Journal of Dairy Science 2006; 89 (10):3749-3762
Hickey RM. Ross RP. Hill C. Controlled autolysis and enzyme release in a recombinant lactococcal strain expressing the metalloendopeptidase enterolysin A. Applied and Environmental Microbiology 2004,70 (3):1744-1748
Huard C. Miranda G. Wessner F. Bolotin A. Hansen J. Foster SJ. Chapot-Chartier M. Characterization of AcmB, an N-acetylglucosaminidase autolysin from Lactococcus lactis. Microbiology 2003; 149:695-705
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Jebava I. Plockova M. Lortal S. Valence F. The nine peptidoglycan hydrolases genes in Lactobacillus helveticus are ubiquitous and early transcribed. International Journal of Food Microbiology 2011; 148(1):1-7
Jenkins JK. Courtney PD. Lactobacillus growth and membrane composition in the presence of linoleic or conjugated linoleic acid. Canadian Journal of Microbiology 2003; 49:51-57
Kleerebezem M. Hols P. Bernard E. Rolain T. Zhou M. Siezen RJ. Bron PA. The extracellular biology of the lactobacilli. FEMS Microbiology Reviews 2010; 34: 199-230
Krogh A. Larsson B. von Heijne G. Sonnhammer ELL. Predicting transmembrane protein topology with a Hidden Markov Model:application to complete genomes. Journal of Microbiology and Biotechnology 2001; 305:567-580
Kunji ER. Slotboom DJ. Poolman B. Lactococcus lactis as host for overproduction of functional membrane proteins. Biochimica et Biophysica Acta 2003; 1610:97-108
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Ledala N. Wilkinson BJ. Jayaswal RK. Effect of oxacillin and tetracycline on autolysis, autolysin processing and atl transcription in Staphylococcus aureus. International Journal of Antimicrobial Agents 2006; 27:518-524
Leonard AE. Pereira SL. Sprecher H. Huang YS. Elongation of long-chain fatty acids. Progress in Lipid Research 2004; 43 (1):36-54
Leroy F. De Vuyst L. Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends in Food Science & Technology 2004; 15:67-78
Li X. Huang X. Shao X. Li L. Functional cell surface display of endo-beta-1,3-1,4-glucanase in Lactococcus lactis using N-acetylmuraminidase as the anchoring motif. Sheng Wit Gong Cheng Xue Bao 2009; 25(1):89-94
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Altieri C. Bevilacqua A. D'Amato D. Nobile A. Sinigaglia M. Modelling the survival of starter lactic acid bacteria and Bifidobacterium bifidum in single and simultaneous cultures. Food Microbiology 2008; 25:729-734
Apiradee H. Kalyanee P. Pongsathon P. Patcharaporn D. Matura S. Sanjukta S. Supapon C. Morakot T. The expression of three desaturase genes of Spirulina platensis in Escherichia coli DH5α. Molecular Biology Reports 2004; 31:177-189
Beliveau C. Potvin C. Trudel J. Asselin A. Bellemare G. Cloning sequencing and expression in Escherichia coli of a Streptococcus faecal is autolysin. Journal of Bacteriology 1991; 173:5619-5623
Bernaudat F. Frelet-Barrand A. Pochon N. Dementin S. Hivin P. Boutigny S. Rioux J. Salvi D. Seigneurin-Berny D. Richaud P. Joyard J. Pignol D. Sabaty M. Desnos T. Pebay-Peyroula E. Darrouzet E. Vernet T. Rolland N. Heterologous expression of membrane proteins:choosing the appropriate host. PLoS One 2011; 6 (12):e29191
Bravetti AL. Mesnage S. Lefort A. Chau F. Eckert C. Garry L. Arthur M. Fantin B. Contribution of the autolysin AtlA to the bactericidal activity of amoxicillin against Enterococcus faecalis JH2-2. Antimicrobial Agents and Chemotherapy 2009; 53 (4): 1667-1669
Brunskill EW. Bayles KW. Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus. Journal of Bacteriology 1996; 178 (3):611-618
Buist G. Kok J. Leenhouts KJ. Dabrowska M. Venema G. Haandrikman AI. Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. Journal of Bacteriology 1995; 177(6):1554-1563
Buist G. Karsens H. Nauta A. van Sinderen D. Venema G. Kok J. Autolysis of Lactococcus lactis caused by induced overproduction of its major autolysin, AcmA. Applied and Environmental Microbiology 1997; 63 (7):2711-2718
Chi X. Zhang X. Guan X. Ding L. Li Y. Wang M. Lin H. Qin S. Fatty acid biosynthesis in eukaryotic photosynthetic microalgae:identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii. The Journal of Microbiology 2008; 46(2):189-201
Chung J. Yoon HE. Shin HC. Choi E. Byeon W. Induction of growth phase-specific autolysis in Bacillus subtilis 168 by growth inhibitors. The Journal of Microbiology 2009; 47(1):50-59
Cibik R Chapot-Chartier MP. Characterization of autolytic enzymes in Laclobacillus pentosus. Letters in Applied Microbiology 2004; 38:459-463
Drew D. Froderberg L. Baars L. de Gier JL. Assembly and overexpression of membrane proteins in Escherichia coli. Biochimica et Biophysica Acta 2003; 1610: 3-10
Drew D. Slotboom D. Friso G. Reda T. Genevaux P. Rapp M. Meindl-Beinker NM. Lambert W. Lerch M. Daley DO. Van Wijk K. Hirst J. Kunji E. De Gier J. A scalable, GFP-based pipeline for membrane protein overexpression screening and purification Protein Science 2005; 14:2011-2017
Drew, D. Lerch M. Kunji E. Slotboom DJ. de Gier JW. Optimization of membrane protein overexpression and purification using GFP fusions. Nature Methods 2006; 3: 303-313
Dvir H. Matthew E. Lundberg E. Maji SK. Riek R. Choe S. Mistic:cellular localization, solution behavior, polymerization, and fibril formation. Protein Science 2009; 18:1564-1570.
Eckert C. Lecerf M. Dubost L. Arthur M. Mesnage S. Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis. Journal of Bacteriology 2006; 188 (24):8513-8519
Emirian A. Fromentin S. Eckert C. Chau F. Dubost L. Delepierre M. Gutmann L Arthur M. Mesnage S. Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB. FEBS Letters 2009; 583:3033-3038
Feirtag JM. McKay CC. Isolation of Streptococcus lactis C2 mutants selected for temperature sensitivity and potential use in cheese manufacture. Journal of Dairy Science 1987; 70:1773-1778
Fredriksen L. Mathiesen G. Moen A. Bron PA. Kleerebezem M. Eijsink VGH. Egge-Jacobsen W. The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation. Journal of Bacteriology 2012; 194 (2):325
Fuglsang A. Lactic acid bacteria as prime candidates for codon optimization. Biochemical and Biophysical Research Communications 2003; 312:285-291
Gullard RR. Ryther JH. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Candadian Journal of Microbiology 1962; 8:229-239
Hannon JA. Kilcawley KN. Wilkinson MG. Delahunty CM. Beresford TP. Production of ingredient-type cheddar cheese with accelerated flavor development by addition of enzyme-modified cheese powder. Journal of Dairy Science 2006; 89 (10):3749-3762
Hickey RM. Ross RP. Hill C. Controlled autolysis and enzyme release in a recombinant lactococcal strain expressing the metalloendopeptidase enterolysin A. Applied and Environmental Microbiology 2004,70 (3):1744-1748
Huard C. Miranda G. Wessner F. Bolotin A. Hansen J. Foster SJ. Chapot-Chartier M. Characterization of AcmB, an N-acetylglucosaminidase autolysin from Lactococcus lactis. Microbiology 2003; 149:695-705
Huard C. Miranda G. Redko Y. Wessner F. Foster SJ. Chapot-Chartier MP. Analysis of the peptidoglycan hydrolase complement of Lactococcus lactis:identification of a third N-acetylglucosaminidase, AcmC. Applied and Environmental Microbiology 2004; 70 (6):3493-3499
Jebava I. Plockova M. Lortal S. Valence F. The nine peptidoglycan hydrolases genes in Lactobacillus helveticus are ubiquitous and early transcribed. International Journal of Food Microbiology 2011; 148(1):1-7
Jenkins JK. Courtney PD. Lactobacillus growth and membrane composition in the presence of linoleic or conjugated linoleic acid. Canadian Journal of Microbiology 2003; 49:51-57
Kleerebezem M. Hols P. Bernard E. Rolain T. Zhou M. Siezen RJ. Bron PA. The extracellular biology of the lactobacilli. FEMS Microbiology Reviews 2010; 34: 199-230
Krogh A. Larsson B. von Heijne G. Sonnhammer ELL. Predicting transmembrane protein topology with a Hidden Markov Model:application to complete genomes. Journal of Microbiology and Biotechnology 2001; 305:567-580
Kunji ER. Slotboom DJ. Poolman B. Lactococcus lactis as host for overproduction of functional membrane proteins. Biochimica et Biophysica Acta 2003; 1610:97-108
Kunji ER. Chan KW. Slotboom DJ. Floyd S. O'Connor R. Monne M. Eukaryotic membrane protein overproduction in Lactococcus lactis. Current Opinion in Biotechnology 2005; 16:546-551.
Ledala N. Wilkinson BJ. Jayaswal RK. Effect of oxacillin and tetracycline on autolysis, autolysin processing and atl transcription in Staphylococcus aureus. International Journal of Antimicrobial Agents 2006; 27:518-524
Leonard AE. Pereira SL. Sprecher H. Huang YS. Elongation of long-chain fatty acids. Progress in Lipid Research 2004; 43 (1):36-54
Leroy F. De Vuyst L. Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends in Food Science & Technology 2004; 15:67-78
Li X. Huang X. Shao X. Li L. Functional cell surface display of endo-beta-1,3-1,4-glucanase in Lactococcus lactis using N-acetylmuraminidase as the anchoring motif. Sheng Wit Gong Cheng Xue Bao 2009; 25(1):89-94
Loir YL. Gruss A. Ehrlich SD. Langella P. A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. Journal of Bacteriology 1998; 180(7):1895-1903
Lortal S. Valence F. Bizet C. Maubois JL. Electrophoretic pattern of peptidoglycan
hydrolases, a new tool for bacterial species identification:application to 10 Lactobacillus species. Research in Microbiology 1997; 148 (6):461-474
Macouzet M. Robert N. Lee BH. Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus. Applied Microbiology and Biotechnology 2010; 87:1737-1742
Margot P. Pagni M. Karamata D. Bacillus subtilis 168 gene lytF encodes a γ-D-glutamate-meso-diaminopimelate muropeptidase expressed by the alternative vegetative sigma factor, σD. Microbiology 1999; 145:57-65
Martinez-Cuesta MC. Pelaez C. Juarez M. Requena T. Autolysis of Lactococcus lactis ssp. lactis and Lactobacillus casei ssp. casei. Cell lysis induced by a crude bacteriocin. International Journal of Food Microbiology.1997; 38:125-131
Martinez-Cuesta MC. Kok J. Herranz E. Pelaez C. Requena T. Buist G. Requirement of autolytic activity for bacteriocin-induced lysis. Applied and Environmental Microbiology 2000; 66 (8):3174-3179
Maslennikov I. Kefala G. Johnson C. Riek R. Choe S. Kwiatkowski W. NMR spectroscopic an analytical ultracentrifuge analysis of membrane protein detergent complexes BMC Structural Biology 2007; 7:74
Mercier C. Durrieu C. Briandet R. Domakova E. Tremblay J. Buist G. Kulakauskas S. Positive role of peptidoglycan breaks in lactococcal biofilm formation. Molecular Microbiology 2002; 46 (1):235-243
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