Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro

Inversion of the G segment in bacteriophage Mu DNA occurs by a site-specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible λ pL promoter and a synthetic Shine-Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to ˜10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg²⁺, a super-coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(−) orientation is as efficient as the switch from G(−) to G(+).     

Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli

In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap^? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup⁺ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap⁺ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.     

The relationship of two invertible segments in bacteriophage Mu and Salmonella typhimurium DNA

Summary A Mu gin ^- mutant which lacks the function required for inversion of the G segment can be complemented by hybrid plasmids and phages carrying the invertible segment that controls flagellar phase variation in Salmonella typhimurium. This suggests that the same kind of site-specific recombination mechanism is responsible for these inversions. Based on the different features of the two invertible segments we propose a model for their evolutionary relationship.     

Molecular cloning of the SUF2 frameshift suppressor gene from Saccharomyces cerevisiae

A genetic approach to the molecular cloning of frameshift suppressor genes from yeast is described. These suppressors act by suppressing +1 G:C base-pair insertion mutations in glycine or proline codons. The cloning regimen involves an indirect screen for yeast transformants which harbor a functional suppressor gene inserted into the autonomously replicating “shuttle” vector YEp13, followed by transfer of the hybrid plasmid from yeast into Escherichia coli. Using this procedure a 10.7-kb DNA fragment carrying the SUF2 frameshift suppressor gene has been isolated. This suppressor acts specifically on +1 G:C insertions in proline codons. When inserted into an integrative vehicle and reintroduced into yeast by transformation, this fragment integrates by homologous recombination in the region of the SUF2 locus on chromosome III. A large proportion of the fragment overlaps with another cloned DNA segment which carries the closely linked CDC10 gene. The SUF2 fragment carries at least two tRNA genes. The SUF2 gene and one of the tRNA genes are located on a 0.85-kb restriction fragment within the 10.7-kb segment. A method is also described for the isolation of DNA fragments carrying alternative alleles of the SUF2 locus. Using this procedure, the wild-type suf2⁺ allele has been cloned.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

Cloning and characterization of the gene for Salmonella typhimurium serine hydroxymethyltransferase

A plasmid containing the glyA gene of Salmonella typhimurium LT2 was constructed in vitro using plasmid pACYC184 as the cloning vector and a λgt7-glyA transducing phage as the source of glyA DNA. The recombinant plasmid (pGS30) contains a 10-kb EcoRI insert fragment. Genetic and biochemical experiments established that the fragment contains a functional glyA gene. From plasmid pGS30 we subcloned a 4.4-kb SalI-EcoRI fragment containing the glyA gene and its neighboring regions (plasmid pGS38). The location and orientation of the glyA gene within the 4.4-kb insert fragment was determined in four ways: (1) comparison of the physical map of the 4.4-kb SalI-EcoRI fragment with the physical map of a 2.6-kb SalI-PvuII fragment that carries the Escherichia coli glyA gene; (2) deletion analysis; (3) transposon Tn5 insertional inactivation experiments; (4) deoxyribonucleic acid sequencing and comparison of the S. typhimurium DNA sequence with the E. coli DNA sequence. A presumptive glyA-encoded polypeptide of M_r 47000 was detected using plasmid pGS38 as template in a minicell system, but not when the glyA gene was inactivated by insertion of a Tn5 element.     

In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions

Summary The construction of a plasmid carrying the right end PstI·B fragment of bacteriophage Mu DNA and of plasmids containing in addition the left end EcoRI·C fragment of Mu DNA into the vector pBR322 is described. Inversion of the G segment still occurs in all these plasmids. By marker rescue and complementation experiments the right PstI cleavage site was located to the left of gene Q. The composite plasmids inheriting also the left end EcoRI fragment of Mu DNA express both the immunity and killing functions of Mu and direct the in vitro synthesis of presumably Mu-specific polypeptides. These results demonstrate that Mu-specific functions can be analyzed from cloned fragments.     

Cloning of a gene required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli

A clone bank, consisting of approx. 8100 colonies, has been created for the spirochete Leptospira biflexa serovar patoc in Escherichia coli using pBR322 as the vector. One of these clones contains the genetic information needed to complement a defect in the trpE gene of E. coli. The information resides on a 20.5-kb plasmid designated pYCl, which carries a 16-kb insert consisting of three HindIII fragments. It does not complement defects in other genes needed for the biosynthesis of tryptophan in E. coli.     

Cloning and expression of an extracellular-agarase gene from Streptomyces coelicolor A3(2) in Streptomyces lividans 66

An extracellular agarase gene was cloned from Streptomyces coelicolor A3(2) strain M130 into S. lividans 66 using the multicopy plasmid vector pIJ702. Various deletion derivatives of the initial clone (pMT605) were obtained by in vitro and in vivo methods. This allowed the gene to be localised to a 1.9-kb segment of DNA. The agarase enzyme was overproduced (up to 500 times) and exported efficiently into the medium. The agarase protein was identified as a 28-kDal band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE); in the case of one derivative, pMT608, this band accounted for nearly 50% of the total extracellular protein. Differences in agarase production between the deletion derivatives correlated well with plasmid stability.     

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects

This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion.In vitro reconstitution of infectious phage particles has shown that four genes (E, D, I, J) control the formation of phage heads and that a cluster of eight genes (K, L, M, N, P, Q, R, S) controls the formation of phage tails.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10³M_r); H (64 × 10³M_r); F (54 × 10³M_r); G (16 × 10³M_r); L (55 × 10³M_r); N (60 × 10³M_r); P (43 × 10³M_r) and S (56 × 10³M_r). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V, W or R (41.5 × 10³M_r, and 45 × 10³M_r); U (20.5 × 10³M_r) and of genes located in the β region (24 × 10³M_r (gpgin) and 37 × 10³M_r (possibly gpmom)).Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10³, a second head polypeptide of 54 × 10³ (gpF) and two major tail polypeptides with molecular weights of 55 × 10³ and 12.5 × 10³ (gpL and gpY, respectively). In addition, there are five minor components of the tail (including gpN, gpS and gpU) and approximately seven minor components of the head structure of the virion (including gpH).     

Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

A Bacillus subtilis gene coding for an endo-β-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage λ and plasmid vectors. The gene is contained within a 1.6-kb EcoRI-PvuI DNA fragment and directs the synthesis in E. coli of a β-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates.     

Analysis of foldback sequences and repetitive sequences in cloned segments of nuclear DNA from Physarum polycephalum

The properties of DNA segments containing foldback elements were studied after their selection from a ‘random’ recombinant library of Physarum polycephalum nuclear DNA sequences, cloned using the plasmid vector pBR322. Hybridisation of in vitro labelled recombinant plasmids to Southern blots of genomic restriction fragments demonstrated that each cloned segment contained repetitive elements located at several hundred sites in the genome. Two of the DNA clones generated hybridisation patterns which suggested that they contain repetitive elements with internal cleavage sites for the restriction endonuclease HaeIII. Cross-hybridisation of all combinations of the cloned sequences showed that most contain different arrangements of repetitive elements derived from different sequence families. The results are consistent with a model proposed previously on the basis of studies on total nuclear DNA, for the organisation of sequences closely associated with foldback elements in the Physarum genome     

Cloning and biological characterization of the immunity region of Escherichia coli phage Mu.

The construction of three hybrid plasmids containing different parts of the left or immunity and end of phage Mu DNA is described. The recombinant plasmids pKN05 and pKN54 carry the HindIII.C and PstI.C fragments of Mu DNA, respectively. Neither of these plasmids expresses the killing function. Moreover, they do not allow plating of superinfecting Mu phages. Plasmid pKN62 harbors the fragment located in between the left PstI and EcoRI cleavage sites on Mu DNA, allows plating of superinfecting Mu phages, but does not express the killing function. These data suggest that the gene coding for the killing function is either positively regulated by a product from the EcoRI.C fragment, or the killing function requires a second product not coded for by pKN62. Mu Vir A- or Mu Vir B- phages are able to grow on bacteria harboring the recombinant plasmid pKN001 which carries the left and EcoRI-C fragment of Mu DNA. This indicates that the superinfecting phages can induce the corresponding gene functions from pKN001. No such induction could be detected in cells harboring the hybrid plasmids pKN05, pKN54 or pKN62.     

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene,and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H. K. Kuramitsu, Plasmid, 1995, 34, 85–95). This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb BglII fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3′-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3′-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.