The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coliα-hemolysin family

Abstract Most enterohemorrhagic Escherichia coli O157:H7 strains harbor a large-sized (90 kb) plasmid designated pO157 and show an enterohemolytic phenotype. In this study the hemolytic activity of E. coli O157:H7 strain EDL933 was investigated. Curing of strain EDL933 from pO157 resulted in loss of its hemolytic activity. By transformation with Tn801-tagged pO157 (pSK3), the hemolysin-negative E. coli K-12 strains C600 and DH5 _α became positive for hemolysin production. By transformation of recombinant plasmids carrying a 11.9 kb Bam HI fragment and a 5.3 kb Sal I fragment of pSK3 hemolytic activity is revealed when tranformed in E. coli C600 or DH5α DNA-hybridization of pO157 and subclones with the α-hemolysin specific DNA probe was only found under conditions of low stringency. No hybridization was found with enterohemolysin I (EHly1) and enterohemolysin II (EHly2) probes. Our results indicate that a hitherto not described hemolysin belonging to the α-hemolysin family is encoded by the 90 kb plasmid of E. coli O157 strains.     

The construction of Streptomyces cyaneus genomic libraries in Escherichia coli is dependent upon the use of Mcr-deficient strains.

Streptomyces cyaneus genomic DNA ligated into either lambda phage or plasmid vectors was very inefficiently cloned into standard Escherichia coli host strains. However, the same material could be efficiently cloned using Mcr-deficient E. coli strains. These results suggest that the S. cyaneus genome contains 5-methylcytosine residues, some of which occur within the recognition sequences of the E. coli Mcr restriction system.     

Mapping of three plasmids from Lactobacillus helveticus ATCC 15009

A 1.4 kb DNA fragment from the chromosomal DNA of Penicillium nalgiovense was isolated which confers proteolytic activity to E. coli DH5α cells when cloned under the control of the E. coli lacZ promoter. The protein was excreted by the cells as was shown by the formation of a clearing zone in skim milk medium. A retransformation of the plasmid carrying the protease gene into P. nalgiovense leads to transformants with both increased and with nearly no proteolytic activity under neutral conditions. Southern blotting experiments revealed that the transforming plasmid had apparently integrated into the homologous locus and thereby inactivated the residual gene.     

Characterization of the α-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding α-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire α- hly CABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited α- hly determinants. The α- hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli α-hly plasmid pHly152, in particular, the structural α- hly CABD genes (99.2% identity) and the regulatory hly R regions (98.8% identity). pEO5 and α-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural α- hly CABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS 2 element upstream of the hly C gene in pHly152. The presence of transposon-like structures at both ends of the α- hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.     

DNA from Serratia marcescens confers a hydrophobic character in Escherichia coli

Abstract In order to determine whether hydrophobic surface properties of Serratia marcescens can be transferred to Escherichia coli , E. coli DH5α cells were transformed by DNA fragments from S. marcescens RZ. Fifteen-hundred E. coli transformants were screened for adhesion to hexadecane and polystyrene. One transformant exhibited increased adhesion to hexadecane droplets, as well as altered kinetics of aggregation in the presence of ammonium sulfate. Western colony blotting revealed that antibodies raised against S. marcescens RZ recognized components) on the transformant outer surface.     

Comparative proteomic and genetic analyses reveal unidentified mutations in Escherichia coli XL1-Blue and DH5α

Escherichia coli has been used widely in laboratory and the biotech industry. However, the genetic and metabolic characteristics remain inadequately studied, particularly for those strains with extensive genetic manipulations that might have resulted in unknown mutations. Here, we demonstrate a comparative proteomics and genetics approach to identify unknown mutations in E. coli K-12 derivatives. The comparative proteomic and genetic analyses revealed an IS5 disruption of the kdgR gene in two commonly used derivative strains of E. coli K-12, XL1-Blue and DH5α, compared with K-12 wild-type strain W3110. In addition, a controversial deoR mutation was clarified as a wild type in E. coli DH5α using the same approach. This approach should be useful in characterizing the unknown mutations in various mutant strains developed. At the same time, comparative proteomic analysis also revealed the distinct metabolic characteristic of the two derivatives: higher biosynthetic flux to purine nucleotides. This is potentially beneficial for the synthesis of plasmid DNA.     

The class A macrophage scavenger receptor type I (SR-AI) recognizes complement iC3b and mediates NF-κB activation

The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis. In addition, SR-AI modulates macrophage activation through cell signaling. However, investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors. Therefore, we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling. On 293T cells, SR-AI could respond to E. coli DH5α, leading to NF-κB activation and IL-8 production. However, this requires E. coli DH5α to be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion. Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5α and blocks DH5α stimulation of SR-AI signaling. Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b. By mutagenesis, The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α. These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.     

Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5alpha)

Plasmid DNA (pDNA) is an emerging experimental vaccine, produced in E. coli, initially targeted for viral diseases. Unlike traditional protein vaccines whose average dose is micrograms, the average dose of pDNA is on the scale of milligrams. Production yields are, therefore, important for the future development of this vaccine. The E. coli strains currently used for pDNA production, JM109 and DH5alpha, are both suitable for production of stable pDNA due to the deletion of recA and endA, however, these two E. coli K strains are sensitive to growth conditions such as high glucose concentration. On the other hand E. coli BL21 is less sensitive to growth conditions than E. coli JM109 or DH5alpha, this strain grows to higher densities and due to its active glyoxylate shunt and anaplerotic pathways is not sensitive to high glucose concentration. This strain is used for recombinant protein production but not for pDNA production because of its inability to produce stable pDNA. To adapt E. coli BL21 for stable pDNA production, the strain was mutated by deleting both recA and endA, and a proper growth and production strategy was developed. Production values, reaching 2 g/L were obtained using glucose as a carbon source. The produced plasmid, which was constructed for HIV clinical study, was found to have identical properties to the plasmid currently produced by E. coli DH5alpha.     

ompC突变造成大肠杆菌在碱性条件下对渗透压敏感

大肠杆菌DH42突变株碱性条件下对高渗透压敏感。采用mini-Tn5转座突变质粒,同源重组构建突变菌株和DNA片段亚克隆等技术确定了造成大肠杆菌DH42在碱性条件下,对高渗透压敏感的原因是ompC基因突变。通过P1转导,构建了大肠杆菌D9（W3110 ompC：：kan）菌株。比较D9菌株和DH42菌株在不同pH和不同盐浓度条件下的生长,发现大肠杆菌ompC基因是大肠杆菌在碱性条件下应对高渗透压环境胁迫的必须基因。     

大肠杆菌holo-ACP的过表达、分离纯化及长链脂酰ACP的合成

[目的]获得高纯度大肠杆菌holo-ACP和多种长链脂酰ACP,为研究细菌脂肪酸、类脂A和N-酯酰高丝氨酸内脂等物质的合成提供底物.[方法和结果]采用PCR方法扩增得到大肠杆菌酰基载体蛋白基因(acpP)和holo-ACP合成酶基因(acpS).使用载体pBAD24、pBAD34和pET28b分别克隆了acpP和acpS,得到pBAD-ACP、pET-ACP和pET-ACP-ACPS 3个ACP表达质粒和一个AcpS表达质粒pBAD-ACPS.分别用3个ACP表达质粒转化大肠杆菌DH5a和BL21(DE3),构建了DH5αpBAD-ACP、BL21(DE3)/pET-ACP和BL21(DE3)/pET-ACP-ACPS 3种ACP生产菌株.与holo-ACP纯化常用菌株DK574相比,虽然三菌株在诱导时均能过量表达ACP,但是holo-ACP所占比例偏低.为了提高ACP生产菌株holo-ACP的产量,用质粒pBAD-ACPS分别转化上述3种ACP生产菌株,获得了3种携带双质粒的ACP生产菌株.表达结果显示携带pBAD-ACP和pBAD-ACPS双质粒的DH5a菌株比DK574菌株能产生更多的holo-ACP,且纯度也得到提高(纯度达99%).同时使用UNOsphere Q阴离子交换层析从这一菌株培养物中分离纯化到了高纯度的holo-ACP,并以纯化到的holo-ACP和多种长链脂肪酸为底物在哈氏弧菌脂酰ACP合成酶的催化下,合成了多种长链脂酰ACP.[结论]通过研究获得一株holo-ACP高产菌株,并证明在大肠杆菌菌株中,同时表达acpP基因和acpS基因,有利于holo-ACP的产生.     

Mosquito larvicidal binary toxin localization in recombinant bacteria: identification using an immunogold labelling technique

An immunogold labelling technique has been developed and used to characterize the localization of mosquito larvicidal binary toxin in Bacillus sphaericus 2362, asporogenous mutants of Bacillus sphaericus 2362, Bacillus megaterium pWH1520 TK and E. coli DH5 pAR5. Different patterns of binary toxin concentration occur between the wild type and recombinant strains which may be related to their effective toxicities. © Rapid Science Ltd. 1998     

棒杆菌宿主中质粒不稳定性的研究

重组质粒pNAR4是由钝齿棒杆菌质粒pNAT65和大肠杆菌质粒pACYCl84构建的穿梭质粒。质粒pNAR4转化不同棒杆菌,在钝齿棒杆菌T6—13和答氨酸棒杆菌l0l47中为结构型不稳定,在钝齿棒杆菌B9中为分离型不稳定。而pNAT65转化谷氨酸棒杆菌10147后,转化于中的质粒分子大小及主要酶切位点与pxz10145相同。DNA杂交实验结果表明．在10147菌中有一种与z10145高度同源的超螺旋DNA组分,而这一组分与pxz10145的来源宿主中的另一小质粒具有相同的分子量。提出了质粒结构不稳定与宿主中存在的pxz10145高度同源的小质粒(超螺旋组分)有关,并提出产生质粒分子结构反复变化原因的假设。     

嗜盐古菌启动子DNA片段的功能检测

将来源于嗜盐古菌染色体DNA的启动子片段RM07或RM13插入到启动子探针载体pYLZ_2的报告基因lacZ之前,通过β_半乳糖苷酶酶活性的检测,进一步确证RM07和RM13片段在大肠杆菌(Escherichia coli)中的启动功能。同时用微量热技术检测了大肠杆菌DH5α及其重组菌株在LB培养基中37℃生长过程的热输出功率。T2(pYLZ_2)、TE07(pYL726)、TE07_2(pYL702)、TE131(pYL131)和TE132(pYL132)菌株的生长速率分别比大肠杆菌DH5α降低了6.5%、11%4、1.1%4、7.5%和42.7%。当启动子启动了基因表达时,菌株的生长速率显著降低,热力学参数与酶活性检测结果有较好的一致性。微量热结果表明基因的表达比质粒DNA的复制过程需要消耗更多的能量,对细菌的生理代谢有较大改变。微量热技术为检测基因的表达和转录调控提供了新的方法和思路。     

利用大肠杆菌工程菌廉价高效生产聚羟基丁酸酯

利用大肠杆菌生产聚羟基脂肪酸酯是近来国际上生物可降解塑料的研究热点,本研究通过对适宜于聚羟基脂肪酸酯生产的大肠杆菌菌株的选择和碳源利用试验,初步确立了大肠杆菌代谢工程改造生产聚羟基脂肪酸酯的基础。并在此基础上,通过对大肠杆菌磷酸烯醇式丙酮酸葡萄糖转移酶系统的改造和工程菌环境诱导系统的应用,解决了大肠杆菌工程菌无法同时利用多种碳源合成聚羟基脂肪酸酯的难题。发酵试验证明,工程化改造的大肠杆菌利用廉价底物在5L发酵罐中分批培养32h后,菌体终浓度能够达到8.24g/L,聚羟基脂肪酸酯占细胞干重的84.6%。     

An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1

AIMS: To examine Escherichia coli strains EQ1, DH5alpha, BLR and BL21 for known pathogenic mechanisms. METHODS AND RESULTS: Using specific DNA probes, the strains were shown not to carry the genes encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins. The strains were unable to express long-chain lipopolysaccharide and were susceptible to the effects of serum complement. Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut. Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut. In Merino sheep, only strain EQ1 was detected 6 d post-infection. CONCLUSIONS: Escherichia coli strains EQ1, DH5alpha, BLR and BL21 did not carry the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli strains EQ1, DH5alpha, BLR and BL21 were considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.     

Immunogenicity and protective efficacy of GroEL (hsp60) of Streptococcus pneumoniae against lethal infection in mice

The present study was carried out to evaluate the immunogenicity and protective efficacy of GroEL (hsp60) of Streptococcus pneumoniae , by expressing full length GroEL in heterologous host Escherichia coli BL21(DE3). PCR-amplified groEL was ligated in pQE 30 expression vector and subsequently transformed in E. coli DH5α strains. Cloning of groEL was confirmed by double digestion, followed by DNA sequencing. The His-tag containing recombinant GroEL was purified by Ni-NTA affinity chromatography. To determine the immunogenicity of GroEL, the mice were immunized by injecting 40 μg GroEL protein per mouse intraperitoneally. The results showed a significant increase in antibody titre and lymphocyte proliferation in animals immunized with GroEL as compared with control. Further, there was an appreciable increase in interleukin-2 (IL-2) and IL-4 production in lymphocytes isolated from immunized mice as compared with control. To determine the efficacy of GroEL in eliciting protection, the mice were challenged with the lethal dose of S. pneumoniae A66 type 3 capsular strain intranasally after the seventh day of the last immunization. In the GroEL-immunized mice the onset of death was insignificantly delayed and all the mice died by the seventh day postinfection.     

构建重组系统分析回文结构对基因表达的影响

回文结构序列是最常见的一种DNA序列,它是许多基因表达调控因子的顺式作用元件。本文设计了不同长度的回文结构序列,构建了Cre\|loxP重组系统,通过共转化含有cre基因的质粒和含有loxp位点的质粒,观察共转化菌株质粒图谱的变化,研究了重组系统在细菌中对基因表达的作用,同时研究不同长度回文结构对基因表达的影响,结果发现长度在20bp以下回文序列对下游基因的表达没有影响,而长于34bp的回文结构序列则会抑制下游基因的表达。     

Molecular cloning and nucleotide sequence of the Corynebacterium glutamicum pheA gene.

The pheA gene of Corynebacterium glutamicum encoding prephenate dehydratase was isolated from a gene bank constructed in C. glutamicum. The specific activity of prephenate dehydratase was increased six-fold in strains harboring the cloned gene. Genetic and structural evidence is presented which indicates that prephenate dehydratase and chorismate mutase were catalyzed by separate enzymes in this species. The C. glutamicum pheA gene, subcloned in both orientations with respect to the Escherichia coli vector pUC8, was able to complement an E. coli pheA auxotroph. The nucleotide sequence of the C. glutamicum pheA gene predicts a 315-residue protein product with a molecular weight of 33,740. The deduced protein product demonstrated sequence homology to the C-terminal two-thirds of the bifunctional E. coli enzyme chorismate mutase-P-prephenate dehydratase.