铜绿假单胞菌LexA蛋白的纯化及免疫活性分析

目的：对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的LexA蛋白进行表达、纯化,并检测其免疫活性。方法： lexA基因片段插入表达载体pET32a(+),在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解,镍离子亲合柱层析为第一步纯化,Superdex 75凝胶过滤层析作为第二步精细纯化,HPLC测定蛋白的浓度,将纯化的LexA蛋白经注射途径免疫家兔,制备兔抗LexA血清,采用免疫双扩、ELISA及Western Blot分析LexA的免疫活性。结果：LexA以包涵体形式表达,经镍离子亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98.97%,表达及纯化的LexA具有良好的免疫活性。     

铜绿假单胞菌SOS反应阻遏蛋白LexA的复性及活性研究

目的:对LexA蛋白复性方法进行优化,对复性后的LexA蛋白的生物学活性进行分析。方法:采用含有GSH/GSSG的缓冲液,一步稀释法对变性LexA蛋白进行复性,用镍离子亲合柱及阳离子柱层析法对复性后的LexA蛋白进行纯化,再以Sephadex G-25凝胶柱脱盐,采用非变性聚丙烯酰胺凝胶电泳和RP-HPLC法检测复性效果,Western blot法分析复性前后及经DTT处理后的LexA蛋白的免疫反应性,凝胶滞留电泳试验检测复性LexA蛋白与DNA的特异性结合能力。结果:复性后的LexA蛋白出现单体和多聚体的形式,多聚体是由单条肽链聚合而成。LexA单体和多聚体与兔抗LexA多克隆抗体均有较好的反应性。复性后的LexA蛋白能与SOS盒序列发生特异性结合。     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Heterogeneity in expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> colicin K activity gene <Emphasis Type="Italic">cka</Emphasis> is controlled by the SOS system and stochastic factors

Phenotypic diversity provides populations of prokaryotic and eukaryotic organisms with the flexibility required to adapt to and/or survive environmental perturbations. Consequently, there is much interest in unraveling the molecular mechanisms of heterogeneity. A classical example of heterogeneity in Escherichia coli is the subset (3%) of the population that expresses the colicin K activity gene (cka) upon nutrient starvation. Here, we report on the mechanism underlying this variable response. As colicin synthesis is regulated by the LexA protein, the central regulator of the SOS response, we focused on the role of LexA and the SOS system in the variable cka expression. Real-time RT-PCR showed that the SOS system, without exogenous DNA damage, induces moderate levels of cka expression. The use of cka-gfp fusions demonstrated that modification of the conserved LexA boxes in the cka promoter region affected LexA binding affinity and the percentage of cka-gfp expressing cells in the population. A lexA-gfp fusion showed that the lexA gene is highly expressed in a subset of bacteria. Furthermore, cka-gfp fusions cloned into higher copy plasmid vectors increased the percentage of cka-gfp positive bacteria. Together, these results indicate that the bistability in cka expression in the bacterial population is determined by (1) basal SOS activity, (2) stochastic factors and possibly (3) the interplay of LexA dimers at cka operator. Other LexA regulated processes could exhibit similar regulation.     

Intradomain LexA rotation is a prerequisite for DNA binding specificity

In the absence of DNA damage the LexA protein represses the bacterial SOS system. We performed molecular dynamic simulations of two LexA dimers bound to operators. Our model predicted that rotation of the LexA DNA binding domain, with respect to the dimerised C-terminal domain, is required for selective DNA binding. To confirm the model, double and quadruple cysteine LexA mutants were engineered. Electrophoretic mobility-shift assay and surface plasmon resonance showed that disulfide bond formation between the introduced cysteine residues precluded LexA specific DNA binding due to blocked domain reorientation. Our model could provide the basis for novel drug design.     

Interaction of the CRP-cAMP complex with the cea regulatory region

Summary Analysis of the induction of expression of cea-lacZ fusions in cya and crp mutants showed that catabolite repression affects the kinetics of induction and the rate of induced synthesis. In a cya mutant, addition of cAMP reduced the induction lag and increased the amount of -galactosidase produced. The CRP-cAMP complex was found to bind to two sites 5 to the cea promoter, but deletion analysis showed that only one of these was involved in the control of cea. Deletion of this site resulted in a loss of the stimulatory effects of cAMP in a cya mutant.     

Two overlapping SOS-boxes in ColE operons are responsible for the viability of cells harboring the Col plasmid

In this study, oligonucleotide-directed site-specific mutagenesis was used to change the consensus sequences of the LexA binding motifs in either one of the two SOS-boxes of the ColE7 operon. The results indicated that both mutants produced larger amounts of colicin than cells harboring the wild-type ColE7 plasmid. This finding would imply that two biologically functional SOS boxes exist in the ColE7 operon. In the non-induced state, no lysis of cells harboring wild-type plasmids occurred at 37°C, whereas, cells harboring recombinant plasmids containing either one of the mutated SOS boxes underwent lysis within 100 min under the same conditions. This result indicated that adaptation of two SOS boxes of the ColE operon would obviously tightly control the expression of ColE operons. In such a way that it may prevent excessive expression of the lysis (cel) gene, thus safeguard the host cells from being lysed in ordinary living conditions.     

大肠杆菌（Escherichia coli）LexA蛋白的结构与功能

LexA蛋白首先在大肠杆菌（Escherichia coli）中作为SOS反应的重要调节因子之一被发现. LexA蛋白含有202个氨基酸,由N端DNA结合结构域和C端催化核心结构域构成. 细胞中LexA蛋白大都以二聚体形式存在,并且有可切割和不可切割两种构象. 在正常生理条件下,LexA特异性结合16 bp的保守序列5′-CTGTN8ACAG-3′,即SOS盒,抑制约50个基因的表达. 当发生DNA损伤时,活化的RecA蛋白通过稳定LexA蛋白可切割构象,促进LexA蛋白Ala84-Gly85间肽键的切割,产生的C端LexA85 202和N端LexA1 84被蛋白酶ClpXP和Lon快速降解. LexA蛋白切割后,SOS基因以一定的顺序开始表达,并且完成DNA损伤修复. 本文回顾和总结了LexA蛋白分子结构,自我切割分子机制和影响因素,以及在SOS反应中的作用等方面的研究进展. 同时,也讨论了LexA蛋白在原核细胞中的进化保守性.     

LexA protein of cyanobacterium Anabaena sp. strain PCC7120 exhibits in vitro pH-dependent and RecA-independent autoproteolytic activity

The LexA protein of the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 exhibits a RecA-independent and alkaline pH-dependent autoproteolytic cleavage. The autoproteolytic cleavage of Anabaena LexA occurs at pH 8.5 and above, stimulated by the addition of Ca²⁺ and in the temperature range of 30–57 °C. Mutational analysis of Anabaena LexA protein indicated that the cleavage occurred at the peptide bond between Ala-84 and Gly-85, and optimal cleavage required the presence of Ser-118 and Lys-159, as also observed for LexA protein of Escherichia coli. Cleavage of Anabaena LexA was affected upon deletion of three amino acids, ⁸⁶GLI. These three amino acids are unique to all cyanobacterial LexA proteins predicted to be cleavable. The absence of RecA-dependent cleavage at physiological pH, which has not been reported for other bacterial LexA proteins, is possibly due to the absence of RecA interacting sites on Anabaena LexA protein, corresponding to the residues identified in E. coli LexA, and low cellular levels of RecA in Anabaena. Exposure to SOS-response inducing stresses, such as UV-B and mitomycin C neither affected the expression of LexA in Anabaena nor induced cleavage of LexA in either Anabaena 7120 or E. coli overexpressing Anabaena LexA protein. Though the LexA may be acting as a repressor by binding to the LexA box in the vicinity of the promoter region of specific gene, their derepression may not be via proteolytic cleavage during SOS-inducing stresses, unless the stress induces increase in cytoplasmic pH. This could account for the regulation of several carbon metabolism genes rather than DNA-repair genes under the regulation of LexA in cyanobacteria especially during high light induced oxidative stress.     

Genetic analysis of the LexA repressor: Isolation and characterization of LexA(Def) mutant proteins

Summary We report the isolation of LexA mutant proteins with impaired repressor function. These mutant proteins were obtained by transforming a LexA-deficient recA-lacZ indicator strain with a randomly mutagenized plasmid harbouring the lexA gene and subsequent selection on MacConkey-lactose indicator plates. A total of 24 different lexA(Def) missense mutations were identified. All except three mutant proteins are produced in near-normal amounts suggesting that they are fairly resistant to intracellular proteases. All lexA(Def) missense mutations are situated within the first 67 amino acids of the amino-terminal DNA binding domain. The properties of an intragenic deletion mutant suggest that the part of the amino-terminal domain important for DNA recognition or domain folding should extent at least to amino acids 69 or 70. A recent 2D-NMR study (Lamerichs et al. 1989) has identified three a helices in the DNA binding domain of LexA. The relative orientation of two of them (helices 2 and 3) is reminiscent of, but not identical to, the canonical helix-turn-helix motif suggesting nevertheless that helix 3 might be involved in DNA recognition. The distribution of the lexA(Def) missense mutations along the first 67 amino-terminal amino acids indeed shows some clustering within helix 3, since 8 out of the 24 different missense mutations are found in this helix. However one mutation in front of helix 1 and five mutations between amino acids 61 and 67 suggest that elements other than helices 2 and 3 may be important for DNA binding.     

Two modes of binding of DinI to RecA filament provide a new insight into the regulation of SOS response by DinI protein

RecA protein plays a principal role in bacterial SOS response to DNA damage. The induction of the SOS response is well understood and involves the cleavage of the LexA repressor catalyzed by the RecA nucleoprotein filament. In contrast, our understanding of the regulation and termination of the SOS response is much more limited. RecX and DinI are two major regulators of RecA's ability to promote LexA cleavage and strand exchange reaction, and are believed to modulate its activity in ongoing SOS events. DinI's function in the SOS response remains controversial, since its interaction with the RecA filament is concentration dependent and may result in either stabilization or depolymerization of the filament. The 17 C-terminal residues of RecA modulate the interaction between DinI and RecA. We demonstrate that DinI binds to the active RecA filament in two distinct structural modes. In the first mode, DinI binds to the C-terminus of a RecA protomer. In the second mode, DinI resides deeply in the groove of the RecA filament, with its negatively charged C-terminal helix proximal to the L2 loop of RecA. The deletion of the 17 C-terminal residues of RecA favors the second mode of binding. We suggest that the negatively charged C-terminus of RecA prevents DinI from entering the groove and protects the RecA filament from depolymerization. Polymorphic binding of DinI to RecA filaments implies an even more complex role of DinI in the bacterial SOS response.     

Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind^–) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations o ^c in the umuDC and umuD'C operons and also used the o ₉₈ ^c -recA mutation. The o ₁ ^c -umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutgenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.