基于蛋白表达分析的干酪乳杆菌ATCC393交互胁迫保护机制

为考察干酪乳杆菌典型株ATCC393在交互胁迫环境下的生理应答机制,应用二维电泳和iTRAQ技术在蛋白水平上比较了交互胁迫前后干酪乳杆菌蛋白质组的变化情况。在对不同处理条件下细胞全蛋白的二维电泳分析中发现,干酪乳杆菌的主要蛋白分布在等电点pI4～7的范围,经酸预适应处理后细胞的蛋白表达产生了较大的变化。通过iTRAQ技术对细胞在酸适应前后以及相应致死条件下蛋白表达的定性及相对定量分析得知,酸诱导所产生的热胁迫应激蛋白（dnaK,dnaJ等）、氧胁迫应激蛋白（mutS,YeaO）以及与代谢相关的酶类上调可能是提高细胞对交互胁迫耐受能力的主要原因,而酸适应后GTP环化水解酶I和GMP合成酶的高表达可能与这一过程的诱导有关。上述研究结果为提高工业生产菌株在发酵生产及加工过程中对外界不利环境的抵御能力,进而通过调控与微生物生理应答机制密切相关的功能元器件实现生产菌株的性能强化提供了重要的生物信息和可借鉴的研究思路。     

Protective mechanism of Lactobacillus casei ATCC393 against multiple stresses by proteomics analysis

为考察干酪乳杆菌典型株ATCC 393在交互胁迫环境下的生理应答机制,应用二维电泳和iTRAQ技术在蛋白水平上比较了交互胁迫前后干酪乳杆菌蛋白质组的变化情况.在对不同处理条件下细胞全蛋白的二维电泳分析中发现,干酪乳杆菌的主要蛋白分布在等电点pI 4～7的范围,经酸预适应处理后细胞的蛋白表达产生了较大的变化.通过iTRAQ技术对细胞在酸适应前后以及相应致死条件下蛋白表达的定性及相对定量分析得知,酸诱导所产生的热胁迫应激蛋白( dna K,dna J等)、氧胁迫应激蛋白(mut S,rec O)以及与代谢相关的酶类上调可能是提高细胞对交互胁迫耐受能力的主要原因,而酸适应后GTP环化水解酶Ⅰ和GMP合成酶的高表达可能与这一过程的诱导有关.上述研究结果为提高工业生产菌株在发酵生产及加工过程中对外界不利环境的抵御能力,进而通过调控与微生物生理应答机制密切相关的功能元器件实现生产菌株的性能强化提供了重要的生物信息和可借鉴的研究思路.     

酸胁迫下琥珀酸放线杆菌的生理及转录应答

【目的】通过琥珀酸放线杆菌Actinobacillus succinogenes CGMCC1593对酸胁迫的生理应答和转录组学分析,探究琥珀酸放线杆菌酸胁迫的机制。【方法】测定不同pH对细胞生长、H+-ATPase、细胞内pH的影响;测定酸胁迫前后细胞膜和谷氨酸脱氢酶的变化、谷氨酸对琥珀酸放线杆菌生长的影响;通过RNA-seq测序分析酸胁迫条件下的差异表达基因。【结果】随pH值的降低,细胞生长受抑制,H+-ATPase的活性下降。pH 4.7酸胁迫后,细胞膜受到严重损伤,谷氨酸对酸胁迫后的细胞有保护作用,GDH酶活响应酸胁迫后略有增加。酸胁迫后,39个基因差异表达较为显著,其中49%基因属于应激蛋白、转运蛋白,小部分基因与代谢相关。【结论】本文探究了琥珀酸放线杆菌酸胁迫下的生理及转录应答,研究结果可为寻找增强琥珀酸放线杆菌耐酸性策略提供参考。     

应用双向荧光差异凝胶技术解析干酪乳杆菌Lactobacillus casei Zhang驯化菌株耐酸胁迫机制

以干酪乳杆菌Lactobacillus casei Zhang为出发菌株,通过适应性进化获得了干酪乳杆菌酸胁迫抗性驯化菌株.对细胞内微环境的检测发现,驯化菌株在酸胁迫过程中能够维持较高的磷酸烯醇式丙酮酸-糖转移酶系统活力,并具有较高的H+ -ATPase活性以及胞内ATP浓度.蛋白质组学分析结果表明,酸胁迫引发了细胞蛋白表达的变化,与原始菌株相比,驯化菌株保持了更高的代谢活性;同时,驯化菌株通过大量诱导应激蛋白如分子伴侣GroEL、GrpE,冷/热应激蛋白CspC、DnaK等维持了细胞的生理活性,有效提高了细胞对酸胁迫的抵御能力.本研究为进一步揭示酸胁迫下乳酸菌细胞的生理应答机制,探寻促进乳酸菌酸胁迫性能提升的最优策略,进而改善其在生产中的应用性能提供了可借鉴的思路.     

产气荚膜梭菌α毒素在干酪乳杆菌中的诱导表达及小鼠口服免疫保护效力的测定

摘要: 【目的】构建产气荚膜梭菌（Clostridium perfringens, C．perfringens）α 毒素基因的重组干酪乳杆菌口服疫苗,为产气荚膜梭菌毒素中毒的防治提供有效方法。【方法】将构建的重组产气荚膜梭菌α毒素基因细胞表面型载体pPG1及分泌表达载体pPG2电转化乳酸乳杆菌（Lactobacillus casei L.casei）,获得阳性重组菌pPG1-α/ L.casei 393 乳酸乳杆菌表面表达系统和pPG2-α/ L.casei393乳酸乳杆菌分泌表达系统。重组菌以1%乳糖为诱导物,在MRS培养基中进行诱导,通过Western-blot和间接免疫荧光方法鉴定,确定目的蛋白的表达。将重组菌口服免疫BALB/c小鼠,收集免疫小鼠粪便及眼冲洗液及外生殖道黏液样本测定小鼠产生抗α毒素的特异性sIgA 抗体水平,采集小鼠血液样本测定血清中抗α毒素的特异性IgG抗体水平。并对免疫小鼠进行α毒素的腹腔攻毒实验及对获得的抗血清进行α毒素中和试验测定。【结果】重组干酪乳杆菌pPG1-α/ L.casei 393及pPG2-/ L.casei 393免疫小鼠能够产生明显的抗α毒素的sIgA 和IgG 抗体水平,其对α毒素中和试验结果为完全保护。腹腔攻毒实验结果为能抵抗3倍最小致死剂量的α毒素攻击。【结论】表达产气荚膜梭菌α毒素免疫保护性抗原的重组乳酸乳杆菌口服免疫动物能够产生良好的局部和系统体液免疫应答和免疫中和效力。     

干酪乳杆菌12A碳源代谢的比较基因组学分析

【目的】在基因组学水平上,对干酪乳杆菌的碳源代谢特性及调控机制进行研究。【方法】基于BioCyc和MetaCyc数据库,利用Pathway Tools对菌株12A及7株已公布全基因序列的干酪乳杆菌进行全基因组水平的碳源代谢比较分析。【结果】全基因组比较分析结果显示,干酪乳杆菌12A可以将9种糖转运到细胞内代谢利用;可以将多种寡糖和多糖在细胞外水解成半乳糖和葡萄糖;干酪乳杆菌12A可经异型乳酸发酵或混合酸发酵途径生成乙醇及其副产物。【结论】干酪乳杆菌12A可以代谢多种类型碳源,底物选择范围宽泛,而且可以作为工业乙醇发酵的特定菌株;利用比较基因组学方法建立基因结构与细菌代谢能力的联系是可靠的。     

Rib类似蛋白在三种乳酸杆菌中的分布

【目的】阐明罗伊氏乳杆菌中Rib蛋白在细菌中的亚细胞定位,以及其在几种乳酸杆菌中的分布,探讨Rib蛋白潜在的生物学功能。【方法】以罗伊氏乳杆菌ATCC55730中Rib蛋白的基因为模板,设计特异性引物,通过PCR获得Rib蛋白N末端非重复区序列并在大肠杆菌中进行异源表达,经过亲和层析和分子筛获得纯化的Rib蛋白,制备Rib蛋白的小鼠多克隆抗体。通过蛋白质免疫印迹方法对11株乳酸杆菌中Rib类似蛋白进行检测。【结果】Western blot结果显示Rib蛋白主要分布于罗伊氏乳杆菌的细胞壁上。PCR和Western blot均表明Rib类似蛋白在4株罗伊氏乳杆菌、6株植物乳杆菌和1株干酪乳杆菌中均可被检测到。【结论】Rib类似蛋白可能在乳酸杆菌中分布较广泛,它们主要存在于细菌细胞壁上的现象表明其可能对这些乳杆菌在宿主肠道的定殖具有一定作用。     

短小芽孢杆菌β-1，4-甘露聚糖酶的酶学性质及其在干酪乳杆菌中的表达

摘要:【目的】干酪乳杆菌广泛的应用于食品加工和饲料行业,本研究拟构建表达甘露聚糖酶的重组干酪乳杆菌并进行相关评价。【方法】利用干酪乳杆菌表达载体pELX1和pELSH,将短小芽孢杆菌的β-1,4-甘露聚糖酶成熟肽的基因克隆到上述两个载体中,构建的重组质粒电转化到干酪乳杆菌宿主中,分别构建能够胞内表达和分泌表达甘露聚糖酶的重组干酪乳杆菌。【结果】重组干酪乳杆菌菌株经培养后,胞内表达的β-1,4-甘露聚糖酶在重组细胞总蛋白中最高可达23 U/mg,分泌表达培养基上清的β-1,4-甘露聚糖酶最高达到8.8 U/mL。【结论】本研究首次实现了甘露聚糖酶在干酪乳杆菌中的表达,结果表明该重组干酪乳杆菌具有较大的应用前景,值得进一步研究。     

重组粪肠球菌gshF基因对副干酪乳杆菌(Lactobacillus paracasei)L14抗逆性能的影响

乳酸菌作为重要的食品工业微生物,其在工业生产应用过程中会受到多种非生物胁迫。已有研究表明部分乳酸菌可以吸收培养基中或是自身合成谷胱甘肽(Glutathione,GSH),提高对各种胁迫的抵抗作用。克隆了粪肠球菌(Enterococcus faecalis)中的谷胱甘肽合成酶基因gshF,通过构建乳杆菌重组表达质粒,实现了gshF在副干酪乳杆菌(Lactobacillus paracasei)L14菌株中的异源表达。通过对重组gshF-副干酪乳杆菌阳性菌株的抗逆性性测定,结果表明在过氧化氢、酸、冻干脱水和渗透等胁迫条件下,gshF重组菌株的存活率与对照菌株相比均有显著提高。     

天冬氨酸提高乳酸乳球菌Lactococcus lactis NZ9000酸胁迫抗性的作用机制

【背景】乳酸菌作为重要的发酵微生物在应用过程中面临广泛存在的酸胁迫。【目的】确认天冬氨酸可有效提高乳酸乳球菌的酸胁迫抗性,通过解析天冬氨酸的作用机制,为进一步提高乳酸菌酸胁迫抗性提供可借鉴的思路。【方法】通过荧光定量PCR比较胁迫条件下天冬氨酸对L.lactisNZ9000产能和氨基酸代谢途径中关键基因转录水平的影响,并通过过量表达天冬酰胺酶增加胞内天冬氨酸的含量。【结果】天冬氨酸主要是在转氨酶的作用下生成草酰乙酸和谷氨酸。草酰乙酸参与三羧酸循环,为细胞提供更多的能量;谷氨酸经谷氨酸脱羧酶途径提高细胞的酸胁迫抗性。经pH4.0胁迫处理后,天冬氨酸使糖酵解和三羧酸循环产能途径中关键基因转录上调,胞内ATP含量为对照组的42倍;胞内谷氨酸含量为对照的1.99倍。通过过量表达天冬酰胺酶获得的重组菌株,在pH3.6条件下胁迫0.5h后,存活率约为对照组的11.11倍。【结论】在L. lactis NZ9000中探究了天冬氨酸提高酸胁迫抗性的作用机理,进一步完善了氨基酸代谢提高乳酸菌酸胁迫抗性的理论基础。     

Preparation and regeneration of bacteriophage PL-1 enzyme-induced Lactobacillus casei protoplasts.

Lactobacillus casei ATCC 27092 protoplasts were obtained by treatment with a bacteriolytic enzyme, which was produced in the PL-1 phage lysates of this bacterium, at 37 degrees C for 1.5 h in 50 mM Tris hydrochloride buffer (pH 7.2) containing 20% sucrose and 10 mM MgSO4. The protoplasts lacked the cell wall layer but retained the ability to transport L-[3H]glutamine. The frequency of regeneration was about 1%, whereas about 99% of the cells were osmotically sensitive.     

Preparation and regeneration of bacteriophage PL-1 enzyme-induced Lactobacillus casei protoplasts

Lactobacillus casei ATCC 27092 protoplasts were obtained by treatment with a bacteriolytic enzyme, which was produced in the PL-1 phage lysates of this bacterium, at 37 degrees C for 1.5 h in 50 mM Tris hydrochloride buffer (pH 7.2) containing 20% sucrose and 10 mM MgSO4. The protoplasts lacked the cell wall layer but retained the ability to transport L-[3H]glutamine. The frequency of regeneration was about 1%, whereas about 99% of the cells were osmotically sensitive.     

Detection of a Lactobacillus substance that inhibits Escherichia coli

Recent studies have shown that certain lactobacilli strains have the ability to interfere with the adherence and growth of uropathogenic bacteria. This interaction is believed to be important in the maintenance of a normal urogenital flora and in the prevention of infection in females. In the present study, Lactobacillus casei ssp. rhamnosus GR-1 and Lactobacillus acidophilus 76 were found to exert an inhibitory effect on pyelonephritogenic mutant Escherichia coli Hu 734 and E. coli ATCC 25922. The bioactivity of the inhibitor produced by strain GR-1 was retained under pH buffered conditions and was bactericidal. The bioactive substance was heat labile, not precipitated by up to 80% ammonium sulphate, and extractable in chloroform. The data indicated that the inhibitor is not lactic acid or hydrogen peroxide and has a molecular weight greater than 12,000-14,000. Human urine supported production of the inhibitor and reduced and delayed outgrowth of the E. coli. The ability of L. casei GR-1 and possibly other lactobacilli strains to produce inhibitors of uropathogenic bacteria may have clinical importance and significance in the microbial ecology of the urogenital tract.     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

Rapid PCR-based procedure to identify lactic acid bacteria: application to six common Lactobacillus species

The goal of this study was to develop a method allowing rapid identification of the lactic acid bacteria strains in use in the laboratory (Lactobacillus plantarum NCIMB8826; L. fermentum KLD; L. reuteri 100-23; L. salivarius UCC43321; L. paracasei LbTGS1.4; L. casei ATCC393), based on PCR amplification of 16S RNA coding sequences. First, specific forward oligonucleotides were designed in the variable regions of 16S RNA coding sequences of six Lactobacillus strains. The reverse oligonucleotide was designed in the region where the sequences were homologous for the six strains. The expected size of the amplification product was +/-1000 bp. The specificity of the method was tested on total chromosomal DNA. For five out of the six strains, the amplification of the fragment was strain-specific and the method was directly applicable to colonies. For the strain L. casei ATCC393, an additional argument to the classification of this bacteria in the paracasei group could be proposed. Validation of the developed method was performed by applying it to six Lactobacillus reference strains and to various species of bacteria.     

Effect of probiotic-fermented milk administration on gastrointestinal survival of Lactobacillus casei ATCC 393 and modulation of intestinal microbial flora

The aim of the present study was to assess the survival of free and immobilized Lactobacillus casei ATCC 393 on apple pieces, contained in probiotic-fermented milk, after gastrointestinal (GI) transit and to investigate the potential regulation of intestinal microbial flora in a rat model. In in vitro GI stress tolerance tests, immobilized L. casei ATCC 393 exhibited significantly higher survival rates compared to free cells. At a second stage, probiotic-fermented milk produced by either free or immobilized cells was administered orally at a single dose or daily for 9 days in Wistar rats. By 12 h after single-dose administration, both free and immobilized cells were detected by microbiological and molecular analysis at levels ≥6 logCFU/g of feces. Moreover, daily administration led to significant reduction of staphylococci, enterobacteria, coliforms and streptococci counts. In conclusion, L. casei ATCC 393 contained in fermented milk survived GI transit and modulated intestinal microbiota.     

Ribotyping ofLactobacillus casei group strains isolated from dairy products

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of L. casei and L. paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (= ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.     

Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.     

Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium.

The relationship of acid adaptation to tolerance of other environmental stresses was examined in Salmonella typhimurium. S. typhimurium was adapted to acid by exposing the cells to mildly acidic conditions (pH 5.8) for one to two cell doublings. Acid-adapted cells were found to have increased tolerance towards various stresses including heat, salt, an activated lactoperoxidase system, and the surface-active agents crystal violet and polymyxin B. Acid adaptation increased cell surface hydrophobicity. Specific outer membrane proteins were induced by acid adaptation, but the lipopolysaccharide component appeared to be unaltered. These results show that acid adaptation alters cellular resistance to a variety of environmental stresses. The mechanism of acid-induced cross-protection involved changes in cell surface properties in addition to the known enhancement of intracellular pH homeostasis.