NikAB- or NikB-dependent intracellular recombination between tandemly repeated oriT sequences of plasmid R64 in plasmid or single-stranded phage vectors

The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.     

New recombination methods for Sinorhizobium meliloti genetics

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative alpha-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.     

xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase.

The heritable stability of ColE1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers. This plasmid stabilizing recombination system requires the presence in cis of the ColE1 cer region, plus at least two trans-acting factors encoded by the xerA and xerB genes of Escherichia coli. The xerB gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol. wt of 55.3 kd. The predicted amino acid sequence of this protein exhibits striking similarity to that of bovine lens leucine aminopeptidase (53 kd). The biological significance of this similarity is corroborated by genetic and biochemical evidence which suggests that xerB is identical to the E.coli and S.typhimurium pepA genes that encode aminopeptidase A.     

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.     

Transfer of mlt mutations into polyomavirus intronless genomes by intramolecular recombination in bacteria

We describe a modification of the procedure of Weber and Weissmann [Nucl. Acids Res. 11 (1983) 5661-5669] for the formation of hybrid genes by in vivo recombination to introduce two separate mutations into the same gene. The mutants of interest are inserted as head-to-tail tandems in a bacterial plasmid in such a way that the 5'-proximal mutation is located upstream from the mutant with the more distal mutation. Propagation of the plasmid in a rec+ strain of Escherichia coli allows recombination between homologous sequences in the insert. DNA with the size expected for the recombinant plasmid is isolated by agarose gel electrophoresis, cloned in a recA strain, and characterized by restriction endonuclease mapping. Using this procedure, we have transferred the deletion from polyomavirus mutant dl-8 into other mutant genomes lacking the intervening sequences for either middle T or large T.     

Construction of pBR322-ara hybrid plasmids by in vivo recombination

Summary In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analysis demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%–3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araCmutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.     

Gene engineering by selectable intraplasmid recombination: construction of novel dihydrofolate reductase minigenes

An intraplasmid recombination system in Escherichia coli has been designed to make possible the engineering of various genes using methods that greatly reduce dependence on appropriately placed restriction enzyme sites. This system has been used to manipulate intervening sequences in dihydrofolate reductase minigenes and to vary the number of 48-bp repeats in the promoter region. In this method, the two fragments to be recombined are cloned into a plasmid separated by a fragment of DNA containing an expressible galactokinase-encoding gene (galK). Selection for loss of the galK gene, but for retention of the plasmid in E. coli, results in a plasmid in which the two fragments have undergone homologous recombination. Several new plasmids are reported here which contain an expressible galK gene flanked by multiple restriction sites. These plasmids should be useful in recombination and as convenient sources of a gene for which both positive and negative selections are available in E. coli.     

Design of hybrid metalloproteinases from Bacillus]

Possibility of using recombination mechanisms for the construction of hybrids between relative proteins containing highly homologous regions has been demonstrated. In order to design hybrid neutral proteases (NPr) NPr B. amyloliquefaciens--NPr B. brevis genes encoding these enzymes have been cloned into the same plasmid in tandem orientation with subsequent recombination between them. With the help of sequential inactivation we have managed to ensure efficient selection at intermediate stages as well as at the completion stage of construction when the desirable hybrid forms exhibited proteolytic activity. Constructions containing expressed genes of the hybrid neutral proteases NPr B. amyloliquefaciens--NPr B. brevis were obtained. The presence of several regions of high homology between the genes of the two Bacilli neutral proteinases determines the possibility of obtaining of various variants of hybrid proteins with different properties.     

Self-cloning in the cyanobacterium Anacystis nidulans R2: Fate of a cloned gene after reintroduction

Functional analysis of cloned genes often makes use of complementation after introducing these genes into cells of a mutant strain. Problems with this self-cloning step in the cyanobacterium Anacystis nidulans R2 have been encountered, which were mainly due to recombinational instability of gene and vector after transformation. Therefore, conditions determining the exchange of material between chromosome, insert and plasmids were studied to achieve the necessary stability. The fate of plasmid pME1, containing a wild-type methionine gene from A. nidulans R2, was investigated after its introduction into a Tn901-induced methionine mutant strain as recipient, so that the mutant chromosomal gene could be distinguished from the plasmid-borne wild-type copy. Two different recipients were constructed, one containing and one lacking the resident plasmid pCH1, which is a derivative of the indigenous small plasmid pUH24. When using the pCH1-free strain and with combined selection for both wild-type gene and vector, the original configuration of the genes in chromosome and vector was retained in the majority of the transformed cells, while the remaining transformants were reciprocal recombinants; under conditions of single selection mainly nonreciprocal recombination or loss of the vector was observed. When the recipient strain contained pCH1 additional recombinational events took place. The results show that under appropriate conditions a chromosomal gene cloned on a plasmid vector can be stably maintained in a majority of the transformants, thus making self-cloning experiments feasible in A. nidulans R2. On the other hand, the introduction of foreign DNA into the chromosome can be achieved by deliberately exploiting recombination between chromosome and plasmid.     

In vivo generation of hybrids between two Bacillus thuringiensis insect-toxin-encoding genes

The parasporal crystal of Bacillus thuringiensis is composed of polypeptides highly toxic to a number of insect larvae. The structural genes (cryIA) encoding the Lepidoptera-specific toxin from different bacterial strains diverge primarily in a single hypervariable region, whereas the N-terminal and C-terminal parts of the proteins are highly conserved. In this report, we describe the generation of hybrid genes between two cryIA genes. Two truncated cryIA genes were cloned in a plasmid vector in such way as to have only the hypervariable region in common. The two truncated cryIA genes were separated by the tetracycline-resistance determinant (or part of it). In vivo recombination between the hypervariable regions of the cryIA genes reconstituted an entire hybrid cryIA gene. Direct sequence analysis of 17 recombinant plasmids identified eleven different crossover regions which did not alter the reading frame and allowed the production of eight different hybrid proteins. The recombination events were independent from the RecA function of Escherichia coli. Some of the hybrid gene products were more specific in their insecticidal action and one had acquired a new biological activity.     

New Recombination Methods for Sinorhizobium meliloti Genetics

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative α-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.     

Use of a bioluminescence gene reporter for the investigation of red-dependent and gam-dependent plasmid recombination in Escherichia coli K12

A plasmid recombination assay, which utilized mutated Vibrio fischeri luciferase genes, cloned in Escherichia coli plasmids was developed. Expression of the recombination product, a functional luxA gene, was assayed by measuring light intensity. This system was used to investigate the effect of E. coli gene functions on lambda Red- and Gam-dependent plasmid recombination. The genetic and physiological requirements for Red- and Gam-dependent plasmid recombination are similar to the conditions which allow synthesis of plasmid linear multimers. Both recombination and linear multimer synthesis are mediated by Red activity in recBrecC and in sbcB mutants and by Gam activity in sbcB and sbcA mutants, but neither recombination nor linear multimer synthesis is mediated by Red or Gam functions in RecBCD+ExoI+ cells. When mediated by Red in sbcB mutants, both recombination and linear multimer synthesis are RecA-independent, and when mediated by Gam, in the same genetic background, both are RecA-dependent. A role for replication in Red- and Gam-mediated plasmid recombination is suggested by the dependence of the recombination activity on DnaB. A model which hypothesizes mutual dependence of linear plasmid multimer synthesis and plasmid recombination by the RecE, RecF and Red pathways is presented. We propose that ends that are produced during this type of replication are recombinogenic in all three pathways and that new rounds of replication are primed by a recombination-dependent invasion of duplex DNA by 3' single strand ends.     

Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system.

The cry genes of Bacillus thuringiensis encode a diverse group of crystal-forming proteins that exhibit insecticidal activity, particularly against the larvae of lepidopteran, coleopteran, and dipteran insects. The efficacy of B. thuringiensis-based biopesticides may be improved through the genetic manipulation of these genes. A gene transfer system has been developed for the introduction and maintenance of cloned insecticidal cry genes on small plasmids in B. thuringiensis. This vector system combines a B. thuringiensis plasmid replicon and an indigenous site-specific recombination system that allows for the selective removal of ancillary or foreign DNA from the recombinant bacterium after introduction of the Cry-encoding plasmid. The site-specific recombination system is useful for engineering strains with unique combinations of cry genes, resulting in new active ingredients with improved insecticidal properties.     

Structure of a naturally occurring plasmid with genes for enterotoxin production and drug resistance.

A physical map of the 117-kilobase conjugative plasmid pCG86 was constructed using electron microscope heteroduplex analysis. This plasmid carries the genes elt, for heat-labile enterotoxin, and estA, for heat-stable enterotoxin, as well as the genes for resistance to tetracycline, streptomycin, sulfonamides, and mercury. These genes were mapped using deletions and Tn5 insertions as physical markers. Analysis of a heteroduplex between pCG86 and a previously described enterotoxin plasmid (EntP307) showed a 48-kilobase region of complete homology which included the genes elt and estA. An 8.8-kilobase BamHI fragment of EntP307 carrying elt, cloned by others, was also shown to be completely homologous with pCG86. The position of elt on the fragment was verified, and it was shown to carry estA as well. A 44-kilobase region of pCG86 showed partial homology with the region of EntP307 previously shown to contain conjugal transfer genes. The gene for tetracycline resistance is carried on a stem-loop structure with the dimensions of Tn10, and the genes for the other drug resistance markers are carried on a 14.6-kilobase segment that forms an insertion loop in heteroduplexes with EntP307. These studies suggest that pCG86 arose either by recombination between an enterotoxin plasmid of incompatibility group FI, like EntP307, and a multiple resistance factor of incompatibility group FII, or by transposition into EntP307 of two transposons.     

多变鱼腥藻ATCC 29413基因的一步插入失活

多变鱼腥藻ATCC 29413有潜力发展为异养生长产异形胞蓝藻的研究模式种, 但由于细胞内的限制-修饰系统等原因, 其基因转移效率极低。研究克隆了该藻株的两个甲基化酶(M. AvaⅠ和M. AvrⅡ)基因ava_3181和ava_4359, 构建了辅助质粒pHB6088, 对运载质粒进行甲基化保护以避免被细胞中的限制酶切割。以ava_1237和ava_4412基因为例, 研究证明利用该辅助质粒和接合转移系统可通过双交换对多变鱼腥藻ATCC 29413的基因实现一步插入失活。     

Hybrid plasmids containing the araBAD genes of Escherichia coli B/r.

The DNA fragments generated by restriction endonuclease BamI which contain the araCBAD genes from E.coli B/r have been cloned. The DNA fragments containing ara genes were idenified by a compairson of the BamI fragments of lambdah80dara phages containing different ara deletion mutations. The ara genes were cloned into the plasmid pBR317, a derivative of ColE1. The cloned DNA fragments were analyzed by digestion with pairs of restriction endonucleases to determine the molecular weight of the chimeras and to identify the cloned ara DNA fragments. The cloned ara fragments were also identified by genetic complementation and recombination tests.     

Phasmids: hybrids between ColE1 plasmids and E. coli bacteriophage lambda

Plasmids carrying cloned lambda att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of lambda. The cross, referred to as "lifting" the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed "lifting" lambda phages. One phage donates a short left arm and the other donates a short right arm. These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria. However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids. We call these recombinants phasmids. They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components. These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments. Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.     

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.     

Domain-Specific Rearrangement between the Two Arg-Gingipain-Encoding Genes in Porphyromonas gingivalis: Possible Involvement of Nonreciprocal Recombination

Porphyromonas gingivalis has two functional genes (rgpA and rgpB) encoding an arginine-specific cysteine proteinase (Arg-gingipain, RGP). Several RGP-encoding genes have been cloned and sequenced from various P. gingivalis strains, but all of the genes seem to be essentially equivalent to rgpA. In this study, we cloned and sequenced the second rgp gene (rgpB). A comparison of the rgpB gene and the rgp1 gene, one of the rgpA-equivalent genes, revealed that their gene structures were very similar to each other, except that the rgpB gene did not possess most of the hemagglutinin domain present in the C-terminal region of the rgpl gene, and provided strong evidence for homologous recombination between the proteinase domain regions of the two rgp genes. The presence of nonreciprocal recombination in P. gingivalis was experimentally proven using suicide/integration plasmid systems. The results provide one of the hypothetical scenarios of the generation of the two rgp genes; that is, they have been generated through the duplication of an ancestor rgp gene, insertion of the hemagglutinin domain region into one copy of the two resulting rgp genes (or deletion of the region from one rgp) and homologous recombination between the proteinase domain regions of the two rgp genes.