单核细胞增多性李斯特菌氨基肽酶Lmo1711体外表达及其酶活分析

对单核细胞增多性李斯特菌(简称单增李斯特菌)lmo1711基因编码的氨基肽酶进行克隆表达与纯化,并研究该重组蛋白的体外酶学特性。首先通过生物信息学分析预测Lmo1711与氨基肽酶家族成员的亲缘关系及关键活性位点的保守性。利用SWISS-MODEL模拟预测该蛋白的空间结构;构建Lmo1711原核表达载体并转化入E.coli Rosetta中,诱导表达重组目的蛋白,并利用镍离子亲和层析方法纯化目的蛋白;以氨基酸-对硝基苯胺偶联物为底物,Lmo1711通过水解底物N端氨基酸残基产生游离对硝基苯胺单体,405 nm处检测吸光值对该产物进行检测从而分析Lmo1711的酶学特性。在此基础上系统研究Lmo1711对不同氨基酸残基底物的催化特异性,及不同金属离子对该酶活性的影响。经原核表达纯化获得49.3 kDa的重组Lmo1711蛋白,与预测分子量一致;生物信息学分析推测Lmo1711属于M29氨基肽酶家族,且存在保守关键氨基酸活性位点(Glu250、Glu316、His345、Tyr352、His378、Asp380);酶活分析显示,Lmo1711具有较强的氨基肽酶活性,针对不同底物的结合和催化能力差异较大,对亮氨酸残基的亲和程度最高;Lmo1711氨基肽酶活性具有金属离子依赖性,Co~(2+)、Cd~(2+)、Zn~(2+)等多种金属离子均能显著增强其活性,其中Co~(2+)的激活效应最显著。本试验首次发现并证实,单增李斯特菌Lmo1711属于M29氨基肽酶家族成员,具有较强的催化活性,且对金属离子具有不同程度的依赖性。     

N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.     

硫氧还蛋白Lmo1903介导单增李斯特菌抵抗氧化应激的生物学功能研究

【目的】以单增李斯特菌(Listeria monocytogenes, LM)硫氧还蛋白Lmo1903为研究对象,研究其在细菌环境适应过程中的抗氧化应激生物学作用。【方法】使用生物信息学方法分析Lmo1903的进化关系和关键活性位点,使用酶切连接的方法构建Lmo1903蛋白表达载体,获得纯化的重组蛋白,以胰岛素为底物分析其氧化还原酶学活性;同时制备鼠源多克隆抗体,分析其在细胞内的定位;采用核苷酸定点突变技术构建CX1X2C基序中的半胱氨酸点突变蛋白,分析关键位点半胱氨酸对Lmo1903酶活的影响;采用同源重组原理构建lmo1903基因缺失株Δlmo1903和回补株CΔlmo1903,研究lmo1903在单增李斯特菌生长、运动和抗氧化应激方面发挥的功能。【结果】生物信息学分析显示,Lmo1903含有CX1X2C基序,与枯草芽孢杆菌(Bacillussubtilis)的TrxA的亲缘关系较近,属于硫氧还蛋白家族成员,主要定位在细菌细胞质中,具有较强的还原酶学活性,突变CX1X2C基序中的半胱氨酸残基会显著降低Lmo1903的还原酶活能力。缺失lmo1903不影响单增李斯特菌的生长能力,但显...     

Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria and often consists a polyglycerolphosphate backbone chain that is linked to the membrane by a glycolipid. In Listeria monocytogenes this glycolipid is Gal-Glc-DAG or Gal-Ptd-6Glc-DAG. Using a bioinformatics approach, we have identified L. monocytogenes genes predicted to be involved in glycolipid ( lmo2555 and lmo2554 ) and LTA backbone ( lmo0644 and lmo0927 ) synthesis. LTA and glycolipid analysis of wild-type and mutant strains confirmed the function of Lmo2555 and Lmo2554 as glycosyltransferases required for the formation of Glc-DAG and Gal-Glc-DAG. Deletion of a third gene, lmo2553 , located in the same operon resulted in the production of LTA with an altered structure. lmo0927 and lmo0644 encode proteins with high similarity to the staphylococcal LTA synthase LtaS, which is responsible for polyglycerolphosphate backbone synthesis. We show that both proteins are involved in LTA synthesis. Our data support a model whereby Lmo0644 acts as an LTA primase LtaP and transfers the initial glycerolphosphate onto the glycolipid anchor, and Lmo0927 functions as LTA synthase LtaS, which extends the glycerolphosphate backbone chain. Inactivation of LtaS leads to severe growth and cell division defects, underscoring the pivotal role of LTA in this Gram-positive pathogen.     

Listeria monocytogenes EGD lacking penicillin-binding protein 5 (PBP5) produces a thicker cell wall

We report on the cloning of the structural gene for penicillin-binding protein 5 (PBP5), lmo2754. We also describe the enzymatic activity of PBP5 and characterize a mutant lacking this activity. Purified PBP5 has dd-carboxypeptidase activity, removing the terminal D-alanine residue from murein pentapeptide side chains. It shows higher activity against low molecular weight monomeric pentapeptide substrates compared to dimeric pentapeptide compound. Similarly, PBP5 preferentially cleaves monomeric pentapeptides present in high-molecular weight murein sacculi. A Listeria monocytogenes mutant lacking functional PBP5 was constructed. Cells of the mutant are viable, showing that the protein is dispensable for growth, but grow slower and have thickened cell walls.     

Identification of an essential gene of Listeria monocytogenes involved in teichoic acid biogenesis

Listeria monocytogenes is a facultative intracellular gram-positive bacterium responsible for severe opportunistic infections in humans and animals. We had previously identified a gene encoding a putative UDP-N-acetylglucosamine 2-epimerase, a precursor of the teichoic acid linkage unit, in the genome of L monocytogenes strain EGD-e. This gene, now designated lmo2537, encodes a protein that shares 62% identity with the cognate epimerase MnaA of Bacillus subtilis and 55% identity with Cap5P of Staphylococcus aureus. Here, we addressed the role of lmo2537 in L. monocytogenes pathogenesis by constructing a conditional knockout mutant. The data presented here demonstrate that lmo2537 is an essential gene of L. monocytogenes that is involved in teichoic acid biogenesis. In vivo, the conditional mutant is very rapidly eliminated from the target organs of infected mice and thus is totally avirulent.     

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.     

A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.