Cloning and expression in Escherichia coli of a recA-like gene from Bacteroides fragilis

The recombinant plasmid pHG100, containing a 5.2-kb DNA fragment from Bacteroides fragilis, complemented defects in homologous recombination, DNA repair and prophage induction to various levels in an Escherichia coli recA mutant strain. There was no DNA homology between the cloned B. fragilis recA-like gene and E. coli chromosomal DNA. pHG100 produced two proteins with Mr of approx. 39,000 and 37,000 which cross-reacted with antibodies raised against E. coli RecA protein. The production of these proteins was not increased after UV induction. The cloned B. fragilis recA-like gene product did not enhance the production of native but defective E. coli RecA protein after UV irradiation.     

Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli

A cosmid gene bank of Vibrio cholerae 395, classical Ogawa, was screened in Escherichia coli HB101 for expression of the vibrio neuraminidase (NANase) gene nanH (N-acylneuraminate glycohydrolase). Positive clones were identified by their ability to cleave the fluorogenic NANase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Seven NANase-positive clones were detected after screening 683 cosmid isolates with a rapid, qualitative plate assay method. The nanH gene was subcloned from one of the cosmids and was located within a 4.8-kilobase-pair BglII restriction endonuclease fragment. Evidence that nanH was the NANase structural gene was obtained by transposon mutagenesis and by purification and comparison of the cloned gene product with the secreted NANase purified from the parent V. cholerae strain. The sequence of the first 20 amino-terminal amino acids of the secreted NANase purified from V. cholerae was determined by automated Edman degradation and matched perfectly with the amino acid sequence predicted from nucleotide sequencing of nanH. The sequence data also revealed the existence of a potential signal peptide that was apparently processed from NANase in both V. cholerae and E. coli. In contrast to V. cholerae, E. coli nanH+ clones did not secrete NANase into the growth medium, retaining most of the enzyme in the periplasmic compartment. Kinetic studies in V. cholerae showed that nanH expression and NANase secretion were temporally correlated as cells in batch culture entered late-exponential-phase growth. Similar kinetics were observed in at least one of the E. coli nanH+ clones, suggesting that nanH expression in E. coli might be controlled by some of the same signals as in the parent V. cholerae strain.     

Cloning and expression in Escherichia coli of a recA-like gene from the acidophilic autotroph Thiobacillus ferrooxidans

A recombinant plasmid, pRSR100, containing the functional analogue of the Escherichia coli recA gene was isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. The plasmid complemented defects in DNA repair and homologous recombination in E. coli recA mutant strains. Antiserum raised against E. coli RecA protein reacted with the native but defective E. coli HB101 RecA protein; it did not react with protein extracts from the recA deletion mutant E. coli JK696, but it reacted with two protein bands in extracts of E. coli JK696(pRSR100). A single band with an apparent Mr equal to the higher-Mr band in E. coli JK696(pRSR100) was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum.     

Cloning of Clostridium acetobutylicum genes and their expression in Escherichia coli and Bacillus subtilis

Summary DNA from Clostridium acetobutylicum ABKn8 was partially digested with Sau3A and the fragments obtained were inserted into the unique BamHI site of the cloning vector pHV33. The recombinant plasmids were used to transform Escherichia coli HB101 with selection for ampicillin resistance. A collection of ampicillin-resistant, tetracycline-sensitive clones representative of the Clostridium acetobutylicum genome was made. The clones were shown to carry recombinant plasmids each containing an insert of 2 to 16 kb in size. Several of them complemented the HB101 proA2 or leuB6 auxotrophic mutations. The cloned sequences were shown by Southern blot hybridization to be homologous to the corresponding ABKn8 DNA fragments.     

Interplay of SOS induction,recombinant gene expression,and multimerization of plasmid vectors in Escherichia coli

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.     

Cloning and expression in Escherichia coli of a species-specific Chlamydia trachomatis outer membrane antigen

Abstract A gene library of Chlamydia trachomatis serovar L₂ (strain 434) was constructed in Escherichia coli using plasmid pBR322. Amongst 200 recombinants we have identified and characterized a recombinant E. coli that expresses a protein antigen of M _r 74 000 similar in size to an outer membrane antigen produced by elementary bodies of C. trachomatis . Immunologically, the molecule synthesised by E. coli has the same specificity as the protein encoded by serovar L₂. A 1.8 kb DNA fragment from the recombinant insert, used as a hybridization probe, confirmed the species specificity of this clone at the gene level.     

Molecular cloning and expression of an extracellular nuclease of Serratia marcescens in Escherichia coli

Abstract The gene encoding an extracellular nuclease of Serratia marcescens was cloned in Escherichia coli using the vector pBR322. Transformants were selected by their ability to grow in the presence of ampicillin, and nuclease-positive clones were detected on a commercially available DNase test agar. The production of a nuclease could be detected in recombinant strains and enzyme activity was found in culture supernatants of such strains. Deletion derivatives of the parental recombinant plasmid were constructed to define the region of DNA encoding the expression of the nuclease. The smallest DNA fragment found to produce the nuclease was determined to be 2.2 kb in length, although a somewhat smaller fragment appeared to be partially active.     

Cloning and expression in Escherichia coli of dextranase genes from Bacteroides thetaiotaomicron

A genomic bank was constructed in Escherichia coli HB101, consisting of DNA fragments from Bacteroides thetaiotaomicron strain 489 inserted within the vector pBR322. By screening on complex medium containing blue dextran, 10 stable dextranase-positive (Dex+) clones were isolated. Seven groups of Dex+ inserts were identified on the basis of their restriction maps and hybridization responses. Dextanase activity of the recombinant clones was weak, and was revealed on the selection medium after 15 days. Subcloning of a Sau3AI partially digested 3.2-kb insert in the expression vector pDR720 greatly enhanced dextranse activity on blue dextran plates in one clone, but the delay remained unaltered. This suggested that the enzyme was released by cell lysis. Expression of this 0.7-kb subcloned insert was dependent on the promoter region of tryptophan operon carried by pDR720.     

Taq DNA耐热聚合酶在大肠杆菌中的克隆和高表达

从水生栖热菌(Thermus aquaticus)YT-1中分离得到的Taq DNA 聚合酶是一种广泛应用于PCR的耐热DNA聚合酶。由于天然菌株酶产量较低,培养条件要求严格,酶的纯化过程极为繁琐而使产品成本较高,因而促使人们构建适合于大规模生产的基因工程菌株。已有人分别通过不同的途径,使用不同的载体,成功地在大肠杆菌菌株中表达了Taq 耐热DNA聚合酶基因。我们通过与前人不同的途径,把这一基因克隆到大肠杆菌的载体质粒pJLA503上并使其得以高表达,为降低生产成本和进一步研究该酶的各种特性提供了有利条件。     

Cloning and study of the function of recA-like gene of Yersinia pestis in Escherichia coli cells

A 2 kb fragment of Yersinia pestis genome cloned in Escherichia coli cells of the strain HB101 contains a gene able to complement the recA-dependent deficiency of E. coli cells in UV-resistance, resistance to alkylating agents, UV- and MNNG-induced mutability. Cellular capability for homologous recombination in crosses with HfrH donor, derepressed synthesis of bacteriocins (colicin E1 and pesticin 1) is also complemented by the fragment in E. coli recA-strains. The obtained data suggest the functional homology of the cloned recA-like gene product with the product of E. coli recA-gene.     

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.     

Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli

Summary To measure the degree of phr gene induction by DNA-damaging agents, the promoter region was fused to the coding region of the lacZ gene in plasmid pMC1403. The new plasmids were introduced into Escherichia coli cells having different repair capabilities. More efficient induction of phr gene expression was detected in a uvrA ^– strain as compared with the wild-type strain. In addition, obvious induction was detected in uvrA ^– cells treated by 4-nitroquinoline 1-oxide and mitomycin C. Nalidixic acid, an inhibitor of DNA gyrase, also induced phr gene expression. In contrast, little induced gene expression was noted in UV-irradiated lexA ^– and recA ^– strains. It is suggested from these results that induction of the phr gene is one of the SOS responses. Possible nucleotide sequences which could be considered to constitute an SOS box were found at the regulator region of the phr gene.Abbreviations phr photoreactivation - UV ultraviolet light - 4NQO 4-nitroquinoline 1-oxide - MMC mitomycin C - PRE photoreactivating enzyme - E. coli Escherichia coli     

Comparative sequence analysis of recA gene among Vibrio cholerae isolates from Iran with globally reported sequences

Aims: To study the genetic relatedness between V. cholerae isolates from Iran and other countries based on housekeeping gene recA sequence analysis. Methods and Results: A 995‐bp region of the recA gene from 24 V. cholerae isolates obtained from human and surface water origins in Iran over a 5‐year period was sequenced and compared with the sequence data from the isolates belonging to other places. Cluster analysis of the constructed dendrogram based on recA sequence divergence for our clinical isolates showed one sequence type (ST), whereas environmental isolates revealed eight STs. Interestingly, one of our environmental isolates was intermixed with clinical isolates in the largest cluster containing the epidemic strains. Our 24 isolates plus 198 global isolates available in the GenBank showed 77 sequence types (STs) with at least one nucleotide difference. Conclusions: Our result suggested that recA sequencing is a reliable analysis method for understanding the relatedness of the local isolates with the isolates obtained elsewhere. Significance and Impact of the Study: Understanding the genetic relatedness between V. cholerae isolates could give insights into the health care system for better control and prevention of the cholera.     

Hemolysin from Vibrio cholerae eltor: cloning and expression of genes in Escherichia coli]

A 6.56-kb V. cholerae eltor DNA fragment encoding hemolysin synthesis was cloned in pUC18. The resultant recombinant plasmid pES4H (9.25 kb) was mapped by restriction analysis and shown to express in different E. coli strains as well as in nonhemolytic V. cholerae strains. Application of the cloned fragment as a molecular probe revealed homologous sequences in all V. cholerae strains tested independently on their biotypes, hemolytic activity and presence of vct-genes in their genomes while none of other Vibrio species and related microorganisms contained such sequences. A recombinant E. coli strain, a V. cholerae eltor hemolysin producer, was constructed. The simultaneous expression of hemolytic and toxinogenic properties by the same V. cholerae strains is discussed.     

外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.