Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products

Using linker insertion mutations, we determined the most stable region of the parE gene which encodes a toxic protein (ParE) that inhibits growth of Escherichia coli. The toxicity of ParE was sustained until a 144bp linker was inserted into this region. We have developed a 3 T-overhang vector based on these characteristics of the GST-ParE toxin, and named pTOC-T. Because pTOC-T uses a post-segregational killing system, all transformants grown up on the plates can be considered as recombinants containing foreign DNA. pTOC-T not require X-Gal, IPTG or other substrates for selection. This T-vector using a positive selection system can be applied to various E. coli strains such as XL1-Blue, BL21, DH5, JM109, and JM110.Revisions requested 28 July 2004; Revisions received 7 September 2004     

ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase

Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication-proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase-DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.     

Function inferences from a molecular structural model of bacterial ParE toxin

Toxin-antitoxin (TA) systems contribute to plasmid stability by a mechanism that relies on the differential stabilities of the toxin and antitoxin proteins and leads to the killing of daughter bacteria that did not receive a plasmid copy at the cell division. ParE is the toxic component of a TA system that constitutes along with RelE an important class of bacterial toxin called RelE/ParE superfamily. For ParE toxin, no crystallographic structure is available so far and rare in vitro studies demonstrated that the target of toxin activity is E. coli DNA gyrase. Here, a 3D Model for E. coli ParE toxin by molecular homology modeling was built using MODELLER, a program for comparative modeling. The Model was energy minimized by CHARMM and validated using PROCHECK and VERIFY3D programs. Resulting Ramachandran plot analysis it was found that the portion residues failing into the most favored and allowed regions was 96.8%. Structural similarity search employing DALI server showed as the best matches RelE and YoeB families. The Model also showed similarities with other microbial ribonucleases but in a small score. A possible homologous deep cleft active site was identified in the Model using CASTp program. Additional studies to investigate the nuclease activity in members of ParE family as well as to confirm the inhibitory replication activity are needed. The predicted Model allows initial inferences about the unexplored 3D structure of the ParE toxin and may be further used in rational design of molecules for structure-function studies.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

The phage infection process: a functional role for the distal linker region of bacteriophage protein 3

The filamentous bacteriophage infects Escherichia coli by interaction with the F pilus and the TolQRA complex. The virus-encoded protein initiating this process is the gene 3 protein (g3p). The g3p molecule can be divided into three different domains separated by two glycine-rich linker regions. Though there has been extensive evaluation of the importance of the diverse domains of g3p, no proper function has so far been assigned to these linker regions. Through the design of mutated variants of g3p that were displayed on the surface of bacteriophage, we were able to elucidate a possible role for the distal glycine-rich linker region. A phage that displayed a g3p comprised of only the N1 domain, the first linker region, and the C-terminal domain was able to infect cells at almost the same frequency as the wild-type phage. This infection was proven to be dependent on the motif between amino acid residues 68 and 86 (i.e., the first glycine-rich linker region of g3p) and on F-pilus expression.     

Crystal structure of the bacterial cell division inhibitor MinC

Bacterial cell division requires accurate selection of the middle of the cell, where the bacterial tubulin homologue FtsZ polymerizes into a ring structure. In Escherichia coli, site selection is dependent on MinC, MinD and MINE: MinC acts, with MinD, to inhibit division at sites other than the midcell by directly interacting with FTSZ: Here we report the crystal structure to 2.2 A of MinC from Thermotoga maritima. MinC consists of two domains separated by a short linker. The C-terminal domain is a right-handed beta-helix and is involved in dimer formation. The crystals contain two different MinC dimers, demonstrating flexibility in the linker region. The two-domain architecture and dimerization of MinC can be rationalized with a model of cell division inhibition. MinC does not act like SulA, which affects the GTPase activity of FtsZ, and the model can explain how MinC would select for the FtsZ polymer rather than the monomer.     

Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2.

A 3.2-kb region of the broad-host-range plasmid RK2 has been shown to encode a highly efficient plasmid maintenance system that functions in a vector-independent manner. This region, designated par, consists of two divergently arranged operons: parCBA and parDE. The 0.7-kb parDE operon promotes plasmid stability by a postsegregational killing mechanism that ensures that plasmid-free daughter cells do not survive after cell division. The 2.3-kb parCBA operon encodes a site-specific resolvase protein (ParA) and its multimer resolution site (res) and two proteins (ParB and ParC) whose functions are as yet unknown. It has been proposed that the parCBA operon encodes a plasmid partitioning system (M. Gerlitz, O. Hrabak, and H. Schwabb, J. Bacteriol. 172:6194-6203, 1990; R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). To further define the role of this region in promoting the stable maintenance of plasmid RK2, the parCBA and parDE operons separately and the intact (parCBA/DE) par region (3.2 kb) were reintroduced into an RK2 plasmid deleted for par and assayed for plasmid stability in two Escherichia coli strains (MC1061K and MV10delta lac). The intact 3.2-kb region provided the highest degree of stability in the two strains tested. The ability of the parCBA or parDE region alone to promote stable maintenance in the E. coli strains was dependent on the particular strain and the growth temperature. Furthermore, the insertion of the ColE1 cer site into the RK2 plasmid deleted for the par region failed to stabilize the plasmid in the MC1061K strain, indicating that the multimer resolution activity encoded by parCBA is not by itself responsible for the stabilization activity observed for this operon. To examine the relative contributions of postsegregational cell killing and a possible partitioning function encoded by the intact 3.2-kb par region, stability assays were carried out with ParD provided in trans by a compatible (R6K) minireplicon to prevent postsegregational killing. In E. coli MV10delta lac, postsegregational killing appeared to be the predominant mechanism for stabilization since the presence of ParD substantially reduced the stability of plasmids carrying either the 3.2- or 0.7-kb region. However, in the case of E. coli MC1061K, the presence of ParD in trans did not result in a significant loss of stabilization by the 3.2-kb region, indicating that the putative partitioning function was largely responsible for RK2 maintenance. To examine the basis for the apparent differences in postsegregational killing between the two E. coli strains, transformation assays were carried out to determine the relative sensitivities of the strains to the ParE toxin protein. Consistent with the relatively small contribution of the postsegregational killing to plasmid stabilization in MC1061K, we found that this strain was substantially more resistant to killing by ParE in comparison to E. coli MV10delta lac. A transfer-deficient mutant of thepar-deleted plasmid was constructed for the stable maintenance studies. This plasmid was found to be lost from E. coli MV10delta lac at a rate three times greater than the rate for the transfer-proficient plasmid, suggesting that conjugation can also play a significant role in the maintenance of plasmid RK2.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli.

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Barnase fusion as a tool to determine the crystal structure of the small disulfide-rich protein McoEeTI

We present a fusion system suited to determine the crystal structure of small disulfide-rich proteins. McoEeTI, a hybrid inhibitor cystine knot microprotein, was produced as a soluble fusion to a catalytically inactive variant of the RNAse barnase in Escherichia coli. Functioning as a versatile tag, barnase facilitated purification, crystallization and high-resolution structure determination. Flexibility of the linker region allows for different relative orientations of barnase and the fusion partner in two crystallographically independent molecules and may thereby facilitate crystal packing. Nevertheless, the linker region is well ordered in both molecules. This system may prove more generally useful to determine the crystal structure of peptides and small proteins.     

一个高效表达、快速纯化大肠杆菌精氨酰-tRNA合成酶的新系统(英文)

编码大肠杆菌精氨酰ｔ Ｒ Ｎ Ａ 合成酶（ Ａｒｇ Ｒ Ｓ） 的基因ａｒｇ Ｓ 被克隆到ｐ Ｍ Ｆ Ｔ７５ 载体上。将此质粒转化的大肠杆菌 Ｊ Ｍ１０９（ Ｄ Ｅ３） 中, 该转化子粗抽液的比活是宿主菌的２ ５００ 倍。通过 Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ６ Ｂ Ｆａｓｔ Ｆｌｏｗ 和 Ｂｌｕｅ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ６ Ｂ两步柱层析在一天内即可将精氨酰ｔ Ｒ Ｎ Ａ 合成酶纯化至电泳一条带, 比活为３６ ０００ ｕ／ｍｇ , 总收率可达６９ ％ 。与以前报道的 Ａｒｇ Ｒ Ｓ的高表达质粒相比, 使用该重组质粒可以很方便地将昂贵的标记氨基酸高效地参入酶分子内。目前的研究结果表明,该新系统能够很方便地提供大量的更高比活的大肠杆菌精氨酰ｔ Ｒ Ｎ Ａ 合成酶以进行该酶的 Ｎ Ｍ Ｒ 和结晶学研究     

Purification and characterization of DNA topoisomerase IV in Escherichia coli.

The subunits of topoisomerase IV (topo IV), the ParC and ParE proteins in Escherichia coli, were purified to near homogeneity from the respective overproducers. They revealed type II topoisomerase activity only when they were combined with each other. In the presence of Mg2+ and ATP, topo IV was capable of relaxing a negatively or positively supercoiled plasmid DNA or converting the knotted P4 phage DNA, whether nicked or ligated, to a simple ring. However, supercoiling activity was not detected. The topoisomerase activity was not detectable when the purified ParC and ParE proteins were combined with the purified GyrB and GyrA proteins, respectively. This is consistent with the result that neither a parC nor a parE mutation was compensated by transformation with a plasmid carrying either the gyrA or the gyrB gene. Simultaneous introduction of both the gyrA and gyrB plasmids corrected the phenotypic defect of parC and parE mutants. The results suggest that DNA gyrase can substitute for topo IV at least in some part of the function for chromosome partitioning. Antisera were prepared against the purified ParC, ParE, GyrA, and GyrB proteins and used to investigate cellular localization of these gene products. ParC protein was found to be specifically associated with inner membranes only in the presence of DNA. This result suggests that one of the functions of topo IV might be to anchor chromosomes on membranes as previously proposed for eukaryotic topoisomerase II.     

大肠杆菌甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)的敲除及其对甘油产量的影响

利用Red重组系统构建了大肠杆菌JM109甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)缺失的双突变菌株JM109B,然后将表达酿酒酵母3-磷酸甘油脱氢酶基因(GPD1)和3-磷酸甘油酯酶基因(HOR2)的质粒pSE-gpd1-hor2转化到JM109B突变菌株中,在含1%葡萄糖的摇瓶发酵培养基中37℃发酵24 h,甘油的最高产量为5.61 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍;在30 L发酵罐中发酵28 h,甘油的最高产量为103.12 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍,是原始菌株BL21/pSE-gpd1-hor2甘油产量的1.41倍,葡萄糖转化率为50.39%。     

大肠杆菌ptsG基因敲除及其缺陷株生长特性研究

在大肠杆菌磷酸转移酶系统中,葡萄糖主要由ptsG基因编码的酶ⅡCB^Glc转运入细胞。利用代谢工程技术构建ptsG基因缺陷株,有望降低葡萄糖的摄取速率,减少乙酸累积,促进菌体生长。运用PCR技术,扩增出两翼与ptsG基因上下游序列同源,中间为氯霉素抗性基因的DNA片段。经电转化,将外源DNA片段分别转入Escherichia coli DH5a、JM109中。在Red重组酶的作用下,外源DNA片段与染色体上同源区域重组,将基因ptsG敲除,构建ptsG基因缺陷株：DH5αP,JM109P。在LB培养基中,ptsG基因缺陷株的生长状况与亲株无明显差异。在含有葡萄糖的LB培养基中,DH5αP、JM109P的最高菌密度分别是对照菌株DH5α,JM109的3．47倍和4．25倍,ptsG基因缺陷株对葡萄糖的摄入量也明显高于对照菌株。重组蛋白肿瘤坏死因子(TNF)在DH5αP、JM109P中的表达量分别占全菌蛋白的24．3％、20．8％,A600分别为8．28、7．62,TNF在缺陷株中单位体积的表达量明显高于对照菌株。以上结果说明,大肠杆菌ptsG基因缺陷株具有良好的生长能力和表达外源蛋白的能力,在大肠杆菌高密度发酵研究方面具有良好的应用前景。     

苏云金芽孢杆菌鲇泽变种7—29杀虫蛋白质结构基因的...

The regulative region (181bp) and the fifth toxic active domain (217bp) were removed from the insecticidal protein gene of Bacillus thuringiensis var. aizawai 7-29. After the synthesis of the adaptor (15bp) that contains initiation codon (ATG) and the PCR synthesis of the fifth toxic active domain (229bp) that contains stop codon (TAA), were inserted into on 5' truncated and 3' truncated of the coding fod N-terminal peptid's DNA fragment, that to become a modified structural gene. The modified structural gene can be play initiatic translation-function and stop translation-function during translation of insecticidal protein. The insecticidal protein was determined by western blotting, showed the expression of modified structural gene in Escherichia coli JM 103. The bioassay of insecticidal proteins showed the 3' truncated and 5' truncated of insecticidal gene was higher toxic active than the 3' truncated of insecticidal gene in Escherichia coli JM 103.     

High-pressure infrared spectroscopic study of human proinsulin gene expression in live Escherichia coli cells

Infrared spectra of E. coli strain JM103 and transformants which overproduced recombinant proinsulin have been measured as a function of pressure up to 38 kbar. It is the first time that high-pressure infrared spectra of live bacteria have been successfully measured. In ambient conditions, spectra of the host strain JM103 and the transformants are generally identical. However, under pressure, distinct shifting pattern can be observed in specific spectral parameters of transformants, presumably due to accumulation of proinsulin in form of cytoplasmic inclusion bodies. In particular, the pressure-induced frequency shift of the amide III band (1235 cm-1) in the proinsulin-producing transformants is much smaller than in the host JM103. This pressure effect can potentially be an efficient approach to monitor maximum gene expression in microorganisms. Contrary to predictions based on model system, the pressure-induced denaturation and the sharp transition from disordered liquid crystalline state to the ordered gel state commonly observed in the aqueous solution of protein and aqueous bilayer dispersion of lipids, respectively, do not occur in the bacterial proteins and cell membrane of E. coli.     

Characterization of the type 3 fimbriae with different MrkD adhesins: possible role of the MrkD containing an RGD motif

Four novel mrkD alleles namely mrkD(V1), mrkD(V2), mrkD(V3), and mrkD(V4) were identified in seventeen Klebsiella pneumoniae meningitis strains using PCR-RFLP and sequence determination. Comparative analysis revealed a most variable region containing an RGD motif in the receptor domain of MrkD(V3). In order to determine if the sequence confers the K. pneumoniae mrkD(V3) the highest level of the fimbrial activity, a type 3 fimbriae display system was constructed in Escherichia coli. The E. coli JM109[pmrkABCD(V3)F] displaying meshwork-like fimbriae also had the most fimbrial activity, supporting a possible role of the varied sequences. In a dose-dependent manner, the GRGDSP hexapeptide appeared to inhibit the adhesion of the E. coli JM109[pmrkABCD(V3)F] to HCT-8, an ileocecal epithelial cell line. In addition, the adhesion activity was reduced by the addition of anti-alpha5beta1 integrin monoclonal antibody, indicating that the RGD containing region in MrkD(V3) is responsible for the binding of type 3 fimbriae to integrin.