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.     

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.     

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.     

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.     

Lipoteichoic acid from Listeria monocytogenes.

A lipoteichoic acid (LTA) was extracted from Listeria monocytogenes (serotype 1) by phenol-water partition and isolated by gel-filtration chromatography. The LTA exhibited amphiphilic properties by changes in gel-filtration mobility in the presence of detergent buffers and after mild base hydrolysis. In a hemagglutination assay, Listeria LTA bound antibody prepared against a known LTA from Streptococcus spp. Listeria LTA inhibited the binding of anti-LTA antibody to a Lactobacillus LTA in a hemagglutination inhibition assay. The Listeria LTA contained glucose, galactose, fatty acids, glycerol, and phosphate with molar ratios of 0.05, 0.07, 0.21, 0.94, and 1.0 to phosphate, respectively. Adjacent glycerols were linked between the C-1 and C-3 positions by phosphodiesters (structural type 1). The average chain length was 19 +/- 2 (standard deviation) glycerol-phosphate repeating units. Approximately one glycosyl side chain was present per LTA molecule. The side chain was a galactose-containing disaccharide. The lipid portion of the LTA was a galactose- and glucose-containing glycolipid which may have been a phosphoglycolipid, but the structure was not confirmed. Major fatty acids of LTA and the glycolipid were 17:anteiso, 15:anteiso, 16:iso, 16:n, and 18:n. L. monocytogenes contained cell wall products typical of gram-positive bacteria which is in contrast to the reports by others of the presence of lipopolysaccharides from L. monocytogenes.     

Cloning and expression of the Listeria monocytogenes haemolysin in Escherichia coli

Abstract Listeriolysin, an SH-activated haemolysin probably involved in Listeria pathogenicity, has been cloned into the cosmid vector pHC79 and was expressed in Escherichia coli HB101 cells. Chromosomal DNA of Listeria monocytogenes serovar 1/2 was partially digested with Mbo I and ligated to the Bam HI cleaved cosmid. From 2000 recombinant clones examined, 12 (0.6%) produced haemolysin in solid and liquid media. All of them contained chromosome fragments of Listeria of about 40 kb. The cloning of the listeriolysin determinant will lead to a better understanding of the basis of Listeria pathogenicity.     

Acetoin production as an indicator of growth and metabolic inhibition of Listeria monocytogenes

It has been shown that Listeria monocytogenes produces acetoin from glucose under aerobic conditions. A defined medium with glucose as the sole carbon source was used in an aerobic shake flask culture to reliably produce acetoin. Acetoin, the reactive compound in the Voges–Proskauer test, was assayable in the medium and was used to quantify the metabolic response when inhibitors were added to the medium. Inhibitors such as lactic, acetic, propionic and benzoic acids were used to demonstrate the utility of acetoin production as an indicator of metabolic disruption. With increasing levels of inhibitor, the metabolic and growth responses were measured by acetoin production and optical density change, respectively. Both measurements decreased in a similar manner with increasing inhibitor concentrations. The data also showed the apparent mode of action of the inhibitors. A bacteriostatic effect was observed for the protonated organic acids, acetic (4 mmol l⁻¹) and propionic (4 mmol l⁻¹), whereas protonated lactic (4 mmol l⁻¹) and benzoic (0·16 mmol l⁻¹) acids gave an irreversible (apparent bacteriocidal) effect. Lactic, acetic, and propionic acids showed stimulation of metabolic activity at low concentrations, but benzoic did not. Acetoin production is a novel method for quantifying and assessing the mode of action of inhibitors against L. monocytogenes . This system can be used to screen inhibitors for applications in food safety.