Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.     

The PecM protein is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators of virulence-factor synthesis in Erwinia chrysanthemi

The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS. Purification and characterization of PecS, expressed either from E. coli, from the wild-type E. chrysanthemi strain or from a pecM mutant, showed that the PecS protein produced in these three genetic backgrounds displays the same biochemical properties. Band-shift assay analysis with the three PecS isoforms confirmed the involvement of the PecM protein in modulating the PecS DNA-binding capacity. Moreover, determination of the Kd_app for operator regions of the PecS protein, produced either by the wild-type E. chrysanthemi or by E. coli, reveals similar affinities. Thus, in E. coli, there is likely to be at least one other PecM-like protein able to cross-react with the E. chrysanthemi PecS protein.     

Cloning of a galacturonic acid uptake gene from Erwinia chrysanthemi EC16

Abstract A 3.4 kb fragment of Erwinia chrysanthemi EC16 DNA capable of complementing galacturonic acid uptake mutants ( exuT ) was identified and cloned into a multicopy vector. In E. chrysanthemi B374 exuT mutants, the cloned DNA provided for growth of the mutant strains on galacturonic acid by complementing the galacturonic acid uptake defect. Alkaline phosphatase ( phoA ) gene fusions with the cloned DNA suggested that most of the cloned DNA was necessary for complementation of exuT mutant strains. Using anti-alkaline phosphatase antibody, a hybrid ExuT-PhoA protein was localized to the membrane fraction of the bacterium.     

Export of Erwinia chrysanthemi (EC16) protease by Escherichia coli

Abstract During exponential growth, Erwinia chrysanthemi (EC16) exports 99% of the protease (PRT) into the growth medium. By screening an EC16 genomic library in Escherichia coli HB101, several Prt⁺ clones were identified. A 16-kb Eco RI fragment, carrying the prt gene, was subcloned into pBR322 (pAKC326). E. coli HB101[pAKC326] cells exported PRT into the growth medium during exponential growth. PRT export was not accompanied by periplasmic leakage. E. coli HB101 carrying EC16 prt and pel genes (encoding pectate lyase) exported PRT but retained PEL in the periplasm. These findings indicate the occurrence of a PRT-specific export system in EC16, which is also functional in an E. coli strain carrying the prt ⁺ DNA segment.     

High‐throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays

Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray‐based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.     

不同调控元件驱动下菊欧氏杆菌纤维素酶celY基因在大肠杆菌中表达的研究

目的:以菊欧氏杆菌(Erwinia chrysanthemi)基因组DNA为模板,通过PCR方法找到了该菌的β-1,4-内切葡聚糖酶celY基因及其调控元件并克隆至pUC19载体。为提高纤维素酶celY基因在原核细胞中的分泌表达量,比较了由不同启动子和信号肽调控的celY基因在大肠杆菌中的表达水平。方法:分别构建了由脂蛋白启动子、T7启动子、果胶酶信号肽调控的多种表达载体与由纤维素酶celY基因自身的启动子和信号肽调控的表达载体相比较。结果:由脂蛋白启动子、T7启动子、果胶酶信号肽调控的表达载体都不同程度提高了celY基因的分泌表达量。结论:脂蛋白启动子、T7启动子、果胶酶信号肽都不失为构建强分泌表达载体的可选元件。     

水稻基腐病细菌毒素的遗传特性和产毒相关的分子标记

[目的]水稻基腐病(Erwinia chrysanthemi pv.zeae)是水稻上重要的细菌病害之一,本论文对该病菌的毒素遗传特性和产毒相关的分子标记进行了研究.[方法]通过化学诱变方法,筛选基腐细菌去质粒的突变体Ech7-mu1;应用RAPD技术,筛选产毒素相关的分子标记.[结果]毒素活性测定结果表明,野生菌Ech7和去质粒菌株Ech7-mu1都能产生毒素.从260条随机引物中,筛选出引物K10,该引物能从不产生毒素的突变株Ech7-4中扩增出大小为2139bp的DNA特异片段,但不能扩增野生菌Ech7,将该片段克隆,测序分析,设计特异引物,在突变体Ech7-4中获得了与毒素产生相关的SCAR分子标记(标记符合率为100%).该基因片段有5个ORFs,其中2个ORFs分别编码NADH-黄素还原酶和N-乙酰转移酶,另外2个不完整的ORFs编码的蛋白分别与Pseudomonas aerginosa(ZP00136947)和Yersinia Pestis(ZP01177873)的抗菌素代谢转运蛋白通透酶(DMT)具有66%和46%的同源率.[结论]水稻基腐细菌毒素的生物合成是由染色体基因编码,与质粒无关.不产生毒素的突变菌株基因突变的位点位于SCAR标记DNA的3'末端.     

1.88 A crystal structure of the C domain of hCyP33: a novel domain of peptidyl-prolyl cis-trans isomerase

Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis-trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a beta-barrel covered by two alpha-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.     

Cyclosporin A binding to Mycobacterium tuberculosis peptidyl-prolyl cis-trans isomerase A--investigation by CD, FTIR and fluorescence spectroscopy

Circular dichroism and resolution-enhanced Fourier transform infrared reveal induction of secondary structural elements on peptidyl-prolyl cis–trans isomerase A (PpiA) from Mycobacterium tuberculosis upon binding cyclosporin A (CsA). Thermal denaturation shows aggregation of PpiA at higher temperatures (>70 °C) and CsA fails to impart stabilization in protein structure. However, CsA stabilizes PpiA structure in urea denaturation. In presence/absence of CsA, urea-induced reversible unfolding of secondary and tertiary structures follows two-state and three-state transition, respectively. The chemical unfolding results also demonstrate that loss in the tertiary structure precedes the loss in secondary structure both in presence and absence of CsA at the initial stages. Fluorescence quenching suggests presence of a positive barrier around tryptophan microenvironment of PpiA.     

大黄欧文氏菌蔗糖异构酶基因克隆表达及其酶学特性研究

通过PCR克隆得到了不包括信号肽序列的109～1803bp大黄欧文氏菌蔗糖异构酶基因,以pQE30为表达载体可以在大肠杆菌中高效表达SI基因,但容易形成包涵体。通过降低诱导剂IPTG至10μmol／L,28℃低温诱导表达可以改进表达产物的可溶性。重组SI的最适pH为6．0,最适反应温度为30℃。测定的Km为46．8mmol／L,Vm甜为152．2u／mg。重组SI对麦芽糖、海藻糖、棉子糖、三氯蔗糖及廿甲基葡萄糖苷等均不起作用,只利用蔗糖为底物转糖苷,也可以水解对硝基苯酚-α-葡萄糖苷。同时发现重组SI具有转化异麦芽酮糖生成海藻酮糖的活性。     

Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity.

We identified and characterized an Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation that prevents disulfide bond formation in periplasmic proteins. This gene, dsbC, codes for a 24 kDa periplasmic protein that contains a characteristic active site sequence of disulfide isomerases, Phe-X-X-X-X-Cys-X-X-Cys. Besides the active site, DsbC has no homology with DsbA, thioredoxin or eukaryotic protein disulfide isomerase and it could define a new subfamily of disulfide isomerases. Purified DsbC protein is able to catalyse insulin oxidation in a dithiothreitol dependent manner. The E.coli gene xprA codes for a protein functionally equivalent to DsbC. The in vivo function of DsbC seems to be the formation of disulfide bonds in proteins. The presence of XprA could explain the residual disulfide isomerase activity existing in dsbA mutants. Re-oxidation of XprA does not seem to occur through DsbB, the protein that probably re-oxidizes DsbA.     

Erwinia chrysanthemi strains cause death of human gastrointestinal cells in culture and express an intimin-like protein

The bacterium Erwinia chrysanthemi is a model plant pathogen, responsible for causing cell death in plant tissue. Cell-wall depolymerizing enzymes and avirulence proteins essential for parasitism by this bacterium utilize dedicated type II and type III secretion systems, respectively. Although E. chrysanthemi is not recognized as a mammalian pathogen, we have observed that the bacterium can adhere to, cause an oxidative stress response in and kill cultured human adenocarcinoma cells. These bacteria express a surface protein that bears immunological identity to intimin, a protein required for full virulence of enterohemorrhagic and enteropathogenic Escherichia coli. A type III secretion mutant of E. chrysanthemi was observed to have a significantly lower capability of causing death than the wild-type strain in parallel cultures of human colon adenocarcinoma cells. These observations suggest that E. chrysanthemi has the potential to parasitize mammalian hosts as well as plants.     

Comparative evaluation of pectolytic and proteolytic enzyme production by free and immobilized cells of some strains of the phytopathogenic Erwinia chrysanthemi

Whole cells of the phytopathogenic Erwinia chrysanthemi strains were immobilized in k-carrageenan and grown in high-calcium Xanthomonas campestris medium containing sodium polypectate as carbon source. All the strains used survived immobilization into k-carrageenan beads. Immobilized E. chrysanthemi strains displayed higher pectolytic and proteolytic enzyme activities than free cells in liquid suspension. Carrageenan immobilization techniques could provide a system to mimic the conditions of E. chrysanthemi cells in the infected plant tissue. This could prompt a thorough study of the factors governing the biosynthesis of virulence factors by this bacterium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 215–219. Received 04 April 2001/ Accepted in revised form 12 June 2001     

Flavohaemoglobin HmpX from Erwinia chrysanthemi confers nitrosative stress tolerance and affects the plant hypersensitive reaction by intercepting nitric oxide produced by the host

Host cells respond to infection by generating nitric oxide (NO) as a cytotoxic weapon to facilitate killing of invading microbes. Bacterial flavohaemoglobins are well-known scavengers of NO and play a crucial role in protecting animal pathogens from nitrosative stress during infection. Erwinia chrysanthemi, which causes macerating diseases in a wide variety of plants, possesses a flavohaemoglobin (HmpX) whose function in plant pathogens has remained unclear. Here we show that HmpX consumes NO and prevents inhibition by NO of cell respiration, indicating a role in protection from nitrosative stress. Furthermore, infection of Saintpaulia ionantha plants with an HmpX-deficient mutant of E. chrysanthemi revealed that the lack of NO scavenging activity causes the accumulation of unusually high levels of NO in host tissue and triggers hypersensitive cell death. Introduction of the wild-type hmpX gene in an incompatible strain of Pseudomonas syringae had a dramatic effect on the hypersensitive cell death in soya bean cell suspensions, and markedly reduced the development of macroscopic symptoms in Arabidopsis thaliana plants. These observations indicate that HmpX not only protects against nitrosative stress but also attenuates host hypersensitive reaction during infection by intercepting NO produced by the plant for the execution of the hypersensitive cell death programme.