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Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第5期65卷 2078-2083页

作者： Sleator, RD Gahan, CGM Abee, T Hill, C Natl Univ Ireland Univ Coll Cork Dept Microbiol Cork Ireland Natl Univ Ireland Univ Coll Cork Natl Food Biotechnol Ctr Cork Ireland Wageningen Univ Agr Dept Food Sci Wageningen Netherlands

The trimethylammonium compound glycine betaine (N,N,N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13, The betL gene is preceded by a consensus sigma(B)-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions, BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group, The observed K-m value of 7.9 mu M for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL(+) parent, signifying the role of the transporter in Listeria osmotolerance.

关键词：氨基酸序列细菌蛋白质类/遗传学细菌蛋白质类/代谢碱基序列甜菜碱/代谢生物转运主动/遗传学载体蛋白质类/遗传学载体蛋白质类/代谢克隆分子 DNA引物/遗传学 DNA 细菌/遗传学大肠杆菌/遗传学基因细菌遗传互补测验动力学利斯特菌单核细胞增生/遗传学利斯特菌单核细胞增生/代谢分子序列数据突变氯化钠水电解质平衡/遗传学

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Mutational analysis of the role of HPr in Listeria monocytogenes

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第5期65卷 2112-2115页

作者： Christensen, DP Benson, AK Hutkins, RW Univ Nebraska Dept Food Sci & Technol Lincoln NE 68583 USA Univ Nebraska Sch Biol Sci Lincoln NE 68583 USA

The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes, By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we,were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.

关键词：腺苷三磷酸/代谢细菌蛋白质类/遗传学细菌蛋白质类/代谢碱基序列结合部位/遗传学 DNA引物/遗传学果糖二磷酸盐类/药理学基因表达基因细菌利斯特菌单核细胞增生/遗传学利斯特菌单核细胞增生/代谢诱变定点磷酸烯醇丙酮酸糖磷酸转移酶系统/遗传学磷酸烯醇丙酮酸糖磷酸转移酶系统/代谢磷酰化点突变丝氨酸/代谢

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Detection of Salmonella spp. and Listeria monocytogenes in suspended organic waste by nucleic acid extraction and PCR

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第5期65卷 2235-2237页

作者： Burtscher, C Fall, PA Wilderer, FA Wuertz, S Tech Univ Munich Inst Water Qual Control & Waste Management D-85748 Garching Germany

A nucleic acid-based method for the detection of the bacterial pathogens Salmonella spp. and Listeria monocytogenes in biological waste was developed. The detection limits were less than 10 cells per mi of biological ... 详细信息

关键词：碱基序列 DNA引物/遗传学 DNA 细菌/遗传学 DNA 细菌/分离和提纯电泳琼脂凝胶利斯特菌单核细胞增生/遗传学利斯特菌单核细胞增生/分离和提纯聚合酶链反应垃圾处理沙门菌属/遗传学沙门菌属/分离和提纯

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Division of Listeria monocytogenes serovar 1/2a strains into two groups by PCR and restriction enzyme analysis

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第5期65卷 2054-2056页

作者： Unnerstad, H Nilsson, I Ericsson, H Danielsson-Tham, ML Bille, J Bannerman, E Tham, W Swedish Univ Agr Sci Fac Vet Med Dept Food Hyg S-75007 Uppsala Sweden Univ Lausanne Hosp Med Bacteriol Lab Lausanne Switzerland

Altogether, 100 strains of listeria;ria monocytogenes serovar 1/2a isolated from humans, animals, food, and the environment were typed by a combination of PCR and restriction enzyme analysis (REA), A PCR product of 2,916 bp, containing the downstream end of the gene inlA (955 bp), the space between inlA and inlB (85 bp), and 1,876 bp of the gene inlB, was cleaved with the enzyme AluI, and the fragments generated were separated by gel electrophoresis. By this method two different cleavage patterns were obtained, Seventy of the 100 strains shared one restriction profile, and the remaining 30 strains shared the second one, No relation was found between the types differentiated by PCR-REA and the origins of the strains.

关键词：细菌蛋白质类/遗传学细菌分型技术碱基序列 DNA引物/遗传学 DNA限制酶类环境微生物学食品微生物学基因细菌利斯特菌单核细胞增生/分类利斯特菌单核细胞增生/遗传学利斯特菌单核细胞增生/分离和提纯膜蛋白质类/遗传学聚合酶链反应血清分型动物人类

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Cloning, sequencing, and transcriptional analysis of the dnaK heat shock operon of Listeria monocytogenes

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CELL STRESS & CHAPERONES 2000年第1期5卷 21-29页

作者： Hanawa, T Kai, M Kamiya, S Yamamoto, T Kyorin Univ Sch Med Dept Microbiol Tokyo 1818611 Japan Natl Inst Infect Dis Leprosy Res Ctr Dept Microbiol Tokyo 1890002 Japan

The complete dnaK operon of Listeria monocytogenes was isolated by chromosome walking using the previously cloned dnaK gene as a probe. Molecular analysis of the locus identified 6 genes in the order hrcA, grpE, dnaK, dnaJ, orf35, and orf29. Primer extension analysis revealed 3 transcription start sites-S1, S2, and S3-upstream of the hrcA, grpE, and dnaJ, respectively. The transcription from S1 was heat inducible. Analysis of the sequences revealed the consensus promoter sequences of gram-positive bacteria, P1 and P2 upstream of the hrcA and dnaJ, respectively. The hrcA gene and a regulatory sequence, designated CIRCE (controlling inverted repeat of chaperone expression), play a role in the regulation of expression of the dnaK focus in response to heat shock in several grampositive bacteria. Their presence upstream of the dnaK locus in L monocytogenes suggested a similar regulatory mechanism for the transcription initiated at the promoter, P1. Northern blot analysis led to the detection of 4 mRNA species of 4.9 kb, 3.6 kb, 3.6 kb, and 1.2 kb;the first 2 species were heat inducible. The current results indicate that 4 distinct transcripts directed by 3 promoters are involved in the expression of the dnaK operon of L. monocytogenes.

关键词：氨基酸序列细菌/遗传学细菌蛋白质类/遗传学克隆分子 DNA结合蛋白质类大肠杆菌蛋白质类基因表达调控细菌基因细菌 HSP40热休克蛋白质类 HSP70热休克蛋白质类/遗传学热休克蛋白质类/遗传学利斯特菌单核细胞增生/遗传学分子序列数据分子量操纵子 RNA 信使/遗传学调控序列核酸阻遏蛋白质类/遗传学序列分析种特异性转录遗传

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Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: Analysis of plasmid DNA encoding a CTL epitope derived from microorganisms

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第2期261卷 445-451页

作者： Nagata, T Uchijima, M Yoshida, A Kawashima, M Koide, Y Hamamatsu Univ Sch Med Dept Microbiol & Immunol Hamamatsu Shizuoka 4313192 Japan

Interspecific difference of codon usage is one of the major obstacles for effective induction of specific immune responses against bacteria and protozoa by DNA immunization. Using genes encoding major histocompatibility complex class I-restricted cytotoxic T-lymphocyte (CTL) epitopes, derived from an intracellular bacterium, Listeria monocytogenes and a mouse malaria parasite, Plasmodium yoelii, we report here that the codon optimization level of the genes is not precisely proportional to, but does correlate well with the translational efficiency in mammalian cells, which is concomitantly associated with the induction level of specific CTL response in the mouse. These results suggest that DNA immunization using the gene codon-optimized to mammals through the entire region is very effective. (C) 1999 Academic Press.

关键词：抗原细菌/遗传学抗原原虫/遗传学人工基因融合密码子/遗传学表位/遗传学免疫法利斯特菌单核细胞增生/遗传学利斯特菌单核细胞增生/免疫学萤光素酶类/遗传学小鼠近交BALBC 质粒/遗传学疟原虫约氏/遗传学疟原虫约氏/免疫学蛋白质生物合成 T淋巴细胞细胞毒性/免疫学疫苗 DNA/遗传学动物小鼠

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