Cloning of rel from Listeria monocytogenes as an Osmotolerance Involvement Gene

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.     

单增李斯特菌lmo2192基因克隆与表达

[目的]对单增李斯特菌新疆绵羊脑炎临床分离株LM90SB2的lmo2192基因进行克隆及原核表达。[方法]利用PCR方法扩增lmo2192基因,连接p MD19-T载体进行克隆,筛选阳性菌进行测序比对。构建重组表达质粒p ET32a-2192,将其转入大肠杆菌感受态细胞,经诱导表达,利用SDS-PAGE与Western Blotting鉴定重组蛋白。[结果]扩增lmo2192基因序列长度为969 bp,与预期一致。该基因在大肠杆菌中大量表达,经SDS-PAGE和Western Blotting鉴定分析该产物为56 k Da左右的融合重组蛋白,与预期大小一致。[结论]成功克隆lmo2192基因,并获得大量表达,为进一步研究该基因功能奠定理论基础。     

Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes.

Listeria monocytogenes is a gram-positive food-borne pathogen that is notably resistant to osmotic stress and can grow at refrigerator temperatures. These two characteristics make it an insidious threat to public health. Like several other organisms, L. monocytogenes accumulates glycine betaine, a ubiquitous and effective osmolyte, intracellularly when grown under osmotic stress. However, it also accumulates glycine betaine when grown under chill stress at refrigerator temperatures. Exogenously added glycine betaine enhances the growth rate of stressed but not unstressed cells, i.e., it confers both osmotolerance and cryotolerance. Both salt-stimulated and cold-stimulated accumulation of glycine betaine occur by transport from the medium rather than by biosynthesis. Direct measurement of glycine betaine uptake shows that cells transport betaine 200-fold faster at high salt concentration (4% NaCl) than without added salt and 15-fold faster at 7 than at 30 degrees C. The kinetics of glycine betaine transport suggest that the two transport systems are indistinguishable in terms of affinity for betaine and may be the same. Hyperosmotic shock and cold shock experiments suggest the transport system(s) to be constitutive; activation was not blocked by chloramphenicol. A cold-activated transport system is a novel observation and has intriguing implications concerning the physical state of the cell membrane at low temperature.     

单增李斯特菌新疆分离株lmo0160基因克隆及序列分析

【目的】单增李斯特菌是一种重要的食源性致病菌,常引起人和动物致病。其细胞壁表面LPXTG基序蛋白在单增李斯特菌致病过程中发挥重要作用,根据参考株全序列预测的41个LPXTG基序蛋白中仍有部分蛋白功能未知。对单增李斯特菌新疆绵羊脑分离株LM90SB2的LPXTG基序蛋白Lmo0160的基因进行克隆及生物信息学分析,为功能验证提供基础。【方法】根据GenBank中收录的lmo0160序列设计特异性引物,利用PCR方法对新疆分离株的lmo0160基因进行扩增,将扩增产物克隆到pMD 19-T载体,进行PCR、双酶切鉴定及序列测定,并对基因核苷酸序列和蛋白序列进行分析。【结果】分离株LM90SB2的lmo0160序列全长为1 708 bp,包含1 428 bp的开放阅读框,共编码475个氨基酸;LM90SB2株lmo0160核苷酸序列与CFSAN008100株(4b型,美国)、CFSAN023463株(4b型,美国)、J2-064株(4b型,美国)、F2365株(4b型,奶酪,美国)和NTSN株(4b型,绵羊脑,中国扬州)相似性为99.0%-99.1%;与M7株(4a型,牛奶,中国浙江)相似性为97.2%,与Finland1998株(1/2a型,美国)、N53-1株(1/2a型,熟火腿,瑞士)、N1546株(1/2a型,鱼,丹麦)和EGD-e株(1/2a型,美国)相似性为87.2%-91.1%;其推导的氨基酸序列与上述菌株相似性为91.8%-99.4%。系统进化树显示,LM90SB2菌株的lmo0160基因与CFSAN023463、F2365和NSTN菌株亲缘关系较近,处于同一分支上,而与标准株EGD-e菌株亲缘关系较远。蛋白质二级结构预测表明,LM90SB2的Lmo0160蛋白为亲水性蛋白,无信号肽,不形成跨膜结构。蛋白结构域预测表明Lmo0160蛋白含有胶原蛋白结合域和Cna B结构域。【结论】克隆了LM90SB2的lmo0160基因,为进一步研究LM90SB2的lmo0160基因功能奠定了基础。     

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.     

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes.

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.     

Characterization by molecular cloning and sequencing of the gene encoding an aminopeptidase from Listeria monocytogenes

The pepC gene of Listeria monocytogenes encodes aminopeptidase C that is predicted to share 72% amino acid sequence similarity and 53% sequence identity with the cysteine aminopeptidase PepC from Lactococcus lactis. The gene product also shows strong similarity to aminopeptidase C from Streptococcus thermophilus and Lactobacillus helveticus, and to a cysteine proteinase/bleomycin hydrolase from Saccharomyces cerevisiae. The enzyme from L. monocytogenes displayed broad N-terminal hydrolytic activity, with a similar substrate specificity to its lactic acid bacterial counterpart. The inhibition spectrum shows a great deal of similarity with enzymes from the family of lactic acid bacteria. In addition, one of the clones studied contained DNA sequences that could encode a regulatory protein of the deoR helix-turn-helix DNA binding protein family. The organization of the locus, designated pep, is presented along with the characterization of the gene products of the pep locus.     

Cloning, Sequencing, and Characterization of Genomic Subtracted Sequences from Listeria monocytogenes

Individual sequences of a genomic subtracted, PCR-amplified, mixed-sequence probe (GS probe) were cloned and sequenced. The GS probe differentiated restriction fragment length polymorphism patterns for Listeria monocytogenes but did not hybridize with members of other bacterial genera. Sequence analysis identified several L. monocytogenes sequences already present in the GenBank database; the putative identities of other sequences were inferred from homology data, and still other sequences did not exhibit significant levels of homology with any GenBank sequences.     

Cloning, sequencing, and characterization of genomic subtracted sequences from Listeria monocytogenes

Individual sequences of a genomic subtracted, PCR-amplified, mixed-sequence probe (GS probe) were cloned and sequenced. The GS probe differentiated restriction fragment length polymorphism patterns for Listeria monocytogenes but did not hybridize with members of other bacterial genera. Sequence analysis identified several L. monocytogenes sequences already present in the GenBank database; the putative identities of other sequences were inferred from homology data, and still other sequences did not exhibit significant levels of homology with any GenBank sequences.     

Identification and characterization of an essential gene in Listeria monocytogenes using an inducible gene expression system

The Listeria monocytogenes gene lmo1594 is a homolog of the Bacillus subtilis cell division gene ezrA. EzrA is a negative regulator of FtsZ ring formation, which is required for efficient cell division as it regulates the frequency and position of Z-rings in the cell and prevents aberrant polar cell division. Previously identified as a putative high pressure (HP) resistance mechanism; conferring enhanced barotolerance when heterologously expressed against an Escherichia coli background; the aim of the current study was to investigate whether lmo1594 plays a role in listerial barotolerance. When the creation of a deletion mutant proved unsuccessful, the role of lmo1594 was addressed by creating a conditional knockout mutant which demonstrated that the gene is in fact essential for cell survival and growth in L. monocytogenes. In order to investigate the effect of lmo1594 on barotolerance, the gene was over-expressed. The over-expression of lmo1594 increased survival levels in L. monocytogenes treated at 300 MPa, but survival levels similar to those of the wild-type strain were observed when treated at a higher pressure (≥400 MPa). In conclusion, this study reveals for the first time that lmo1594 is absolutely essential for listerial cell growth and survival, and also plays an important role in listerial barotolerance.     

Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe

A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridization conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed.     

Isolation of Listeria monocytogenes from Vegetation

A rural area in Virginia, in which clinical listeriosis of man and animals has been rare, was selected for this study. Vegetatino, which had died and remained in the fields over the winter, was collected and examined for Listeria monocytogenes by Gray's cold-holding procedure. Twelve fields were sampled and cultured, and eight strains of L. monocytogenes were isolated. Only two of the strains were pathogenic for the mouse. Compared with strains of L. monocytogenes isolated from listeric humans and animals in Virginia and the United States as a whole, these represented serotypes of low frequency.     

电化学方法在李斯特氏菌毒力基因检测中的应用

单核李斯特氏菌(Listeria monocytogenes, LM)是食源性李斯特氏病的病源菌, 该病可引起败血病、脑膜炎、流产等。李斯特氏菌的毒力因子listeriolysin O (LLO)是引发李斯特氏病的主要原因。文章使用一种特殊的电化学方法从样品中检测编码LLO的hlyA基因。该方法以化合物Nhydroxysulfosuccinimide (NHS) 和 N-(3-dimethylamion) propyl- N'-ethyl carbodiimidehydrochloride (EDC) 作为激活剂, 使单链DNA探针结合到金电极表面组成工作电极, 以[Co(phen)₃](ClO₄)₃ 作为指示剂来检测循环伏安曲线(Cyclic voltammetry , CV), 通过CV峰值的变化来估算hlyA基因的含量, 从而确定LM的污染情况。这种新颖的电化学方法用于免标记的目标DNA的杂交检测, 具有快速和方便的特点。