Construction of recombinant lambda phages that carry the E. coli recB and recC genes

Summary A fragment of the E. coli chromosome including the recC gene has been cloned by in vitro recombinant DNA techniques into a phage vector to give the recombinant phage drecC. This was used to derive the phage drecBC by in vivo recombination. On lysogenisation of recB and recC mutants with drecBC wild type levels of UV-resistance and RecBC DNase activity were restored. Infection of E. coli with drecBC led to the synthesis of phage-coded proteins of 125 kilodaltons (kd) and 135 kd that were not synthesised on infection with the original vector, whereas a 125 kd protein but not a 135 kd protein was synthesised in similar experiments with drecC. The recombinant phages, which are unable to form plaques, presumably due to the deletion of essential phage genes during their construction, provide useful starting points from which to subclone the recB, recC, and the neighbouring thyA and argA genes individually into multiple copy plasmid vectors.     

Genetic recombination in Escherichia coli. IV. Isolation and characterization of recombination-deficiency mutants of Escherichia coli K12

19 independent recombination-deficient mutants were isolated. 7 carried mutations that mapped near or in the recB and recC genes between thyA and argA. 10 mutants carried mutations cotransducible with pheA and exhibited no complementation with recA in temporary zygotic diploids.     

The Evolution of RecD Outside of the RecBCD Complex

The common understanding of the function of RecD, as derived predominantly from studies in Escherichia coli, is that RecD is one of three enzymes in the RecBCD double-stranded break repair DNA recombination complex. However, comparative genomics has revealed that many organisms possess a recD gene even though the other members of the complex, recB and recC, are not present. Further, bioinformatic analyses have shown that there is substantial sequence dissimilarity between recD genes associated with recB and recC (recD1), and those that are not associated with recBC (recD2). Deinococcus radiodurans, known for its extraordinary DNA repair capability, is one such organism that does not possess either recB or recC, and yet does possess a recD gene. The recD of D. radiodurans was deleted and this mutant was shown to have a capacity to repair double-stranded DNA breaks equivalent to wild-type. The phylogenetic history of recD was studied using a dataset of 120 recD genes from 91 fully sequenced species. The analysis focused upon the role of gene duplication and functional genomic context in the evolution of recD2, which appears to have undergone numerous independent events resulting in duplicate recD2 genes. The role of RecD as part of the RecBCD complex appears to have a divergence from an earlier ancestral RecD function still preserved in many species including D. radiodurans.     

An Rts1-derivative plasmid conferring UV sensitivity on Escherichia coli host

A deletion mutant was isolated from a kanamycin resistance R plasmid Rtsl. This mutant plasmid, pTW20, was found to enhance the lethal effect of UV irradiation on $\text{Escherichia}\text{coli}$ host, especially at 42°C. A cloning experiment with pTW20 DNA demonstrated that the gene, $\text{puv}$, being responsible for the UV sensitivity was located on the kanamycin resistance gene containing $\text{Bam}$H1 fragment of pTW20. This fragment conferred a sensitivity to methyl methane sulfonate on its host along with the sensitivity to UV, suggesting that a reapir process of the host chromosome is impaired by the presence of $\text{puv}$.     

Derivation of the absorvance by circularly symmetrical assembly of chromophores viewed through limited apertures

The recombination of two plasmids, pRDK101 and pACYC184, was examined in wild-type Escherichia coli and in various E. coli strains containing different recombination-deficient mutations. Recombination-induced oligomer formation from monomeric plasmids was found to be dependent on the functions of the recA, recB, recC and recFgenes. Intramolecular recombination of tetrameric plasmids to form lower-order oligomers and monomers also required the functions of the recA and recF genes but did not require the function of the recB and recC genes. In all cases where recA, recB, recC and recF mutations appeared to block the formation or reduction of plasmid circular oligomers, these effects could be reversed by the presence of an sbcA mutation.Further studies on recombination in vivo were carried out utilizing two tetracycline-sensitive derivatives of the compatible plasmids pACYC184 and pBR322. Recombination events between these two plasmids could be quantitated by measuring the production of tetracycline-resistant cells during the growth of transformants. Plasmid recombination was found to be reduced by recA and recF mutations, and the effect of these two mutations was reversed by the presence of an sbcA mutation. Plasmid recombination measured by this genetic assay was stimulated by recBrecC mutations, and electrophoretic analysis of the recombination products demonstrated that they were primarily circular monomers. A role for the recBrecC gene product, exonuclease V, in the resolution of recombination intermediates is discussed.     

Genetic analysis of the recF pathway to genetic recombination in Escherichia coli k12: Isolation and characterization of mutants

A recombination proficient strain ofEscherichia coli which is recB^? recC^? sbcB^? has been subjected to mutagenesis by nitrosoguanidine. Among the recombination deficient mutants isolated one was sbcB⁺, three were recA^～ and 11 were mutants in at least four newrec genes: recF, recJ, recK and recL. recF143 and recL152 are cotransducible with ilv but they lie on opposite sides of the ilv operons as determined by F$\text{\$}\text{?}$studies. recF, recL and recK are not involved in the RecBC pathway of recombination since a recB⁺recC⁺sbcB^? strain carrying a mutation in one of these genes is recombination proficient. Hence the hypothesis that a RecF pathway of recombination can operate as a partially independent substitute for the RecBC pathway of recombination is supported. recF^?recB⁺ and recF⁺recB^? single mutants are sensitive to u.v. irradiation while the recF^?recB^? double mutant is more sensitive than either single mutant. The sensitivity of the recB^?recC^?sbcB^?recF^? strain approaches the sensitivity of a recA^? single mutant. This is interpreted to mean that there are partially independent RecF and RecBC pathways for the repair of u.v. damage. recJ and mutations were not mapped precisely; hence the mutant properties they confer can not be stated conclusively.     

β-Lactamase genes of Streptomyces badius,Streptomyces cacaoi and Streptomyces fradiae: Cloning and expression in Streptomyces lividans

Genes encoding extracellular β-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The β-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi β-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The β-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the β-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three β-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi β-lactamases exhibited a 10–100-times lower activity in S. lividans, whereas the S. fradiae β-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.     

Inhibition of exonuclease V after infection of E. coli by bacteriophage T7

Exonuclease V ($\text{recBC}$ DNase) is inactivated in $\text{E. coli}$ between 4 and 7 min after infection by T7. This process requires protein sythesis. The inactivation does not occur when T7 is deficient for its RNA polymerase and thus does not express the genes involved in DNA replication and phage maturation. Some implications of this new function of T7 are discussed with respect to the processes of infection and DNA replication.     

Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/molBam HI pSa fragment carrying determinants of resistance to four antibiotics in the uniqueBam HI site of pNH602. The resultingin vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 perE. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in theBam HI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymesBam HI andEco RI and its physical and genetic map was constructed.     

Molecular cloning and amplification of the gene for thymidylate synthetase of E. coli

The thyA gene of Escherichia coli, which directs the synthesis of the enzyme thymidylate synthetase, has been subcloned from a recombinant λ phage (Hickson et al., 1982) into the multicopy plasmid pBR325 to give the plasmid pPE245. To identify the thyA gene product, the transposon Tn1000 was inserted into pPE245 and derivative plasmids isolated that were no longer able to complement thyA mutations. When proteins synthesised by these plasmids and by pPE245 were labelled and analysed on SDS-polyacrylamide gels a protein of 33000 M_r, presumably the thyA⁺ gene product was absent whenever the thyA gene was inactivated. On assaying cell extracts prepared from cells harbouring pPE245 for thymidylate synthetase, the level of this enzyme was found to be elevated by a factor of at least 25.     

DNA sequence of a plasmid-encoded dihydrofolate reductase

Summary The sequence of the methotrexate-resistant dihydrofolate reductase (DHFR) gene borne by the plasmid R-388 was determined. The gene was subcloned and mapped by an in vitro mutagenesis method involving insertion of synthetic oligonucleotide decamers encoding the BamHI recognition site. Sites of insertion that destroyed the methotrexate resistance fell in two regions separated by 300 bp within a 1.2 kb fragment. One of these regions encodes a 78 amino acid polypeptide homologous to another drug-resistant DHFR. The second region essential for DHFR expression appears to be the promoter of the DHFR gene.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12. IV. Analysis of recombinants formed by a recombination deficient (recB21 recC22) strain

Summary Residual genetic recombination is carried out by recB ^- recC ^- mutants of E. coli. Recombinants (for one gene) formed by a recB ^- recC ^- parent were shown to be as recombination deficient as their parent, when recombination of a second gene is measured. Therefore the resididual recombination cannot be attributed to a genetically recombination proficient fraction of the parent recB ^- recC ^- culture. I conclude that each recB ^- recC ^- parent cell is capable of carrying out genetic recombination. This conclusion is consistent with the existence of an alternate (and minor) recombination mechanism in E. coli K12, independent of the recB ⁺ recC ⁺ mediated steps.The previous paper in this series was Capaldo-Kimball and Barbour, Involvement of Recombination Genes in Growth and Viability, J. Bact. April (1971).     

Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome

Summary Mutations at the cpxA locus of Escherichia coli K-12 affect cellular processes that are not otherwise related. We have now determined the physical and genetic structure of the E. coli chromosome in the region of cpxA (87.5 min). Our results indicate that cpxA is a single gene. Previous studies showed cpxA to be linked to tpiA. We therefore isolated two tpiA ⁺ recombinant plasmids, pRA200 and pRA300, from EcoRI and BamHI digests of F133, respectively. By genetic complementation or enzyme overproduction, the 9.5 kb EcoRI fragment in pRA200 was shown to include glpK, tpiA and cdh. The 13.6 kb BamHI fragment of pRA300 lacks glpK, but includes tpiA, pfkA and cpxA. Neither fragment complemented a deletion of the rha operon. These data indicate the chromosomal gene order: 87 min-rha-cpxA-pfkA-cdh-tpiA-glpK-88 min. The EcoRI and BamHI fragments overlap in an interval corresponding to about 8.2 kb of DNA. The total region of the E. coli K12 chromosome covered by the two fragments is about 15 kb. A terminal 2 kb EcoRI-BamHI fragment from pRA300 complemented the chromosomal cpxA2[Ts] allele with respect to isoleucine and valine synthesis, RNA bacteriophage sensitivity and surface exclusion in Hfr strains, and envelope protein composition. Complementation occurred when the fragment was subcloned in pBR325 but not when it was subcloned in pBR322, suggesting that the 2 kb fragment lacks expression sequences that are supplied by cat (chloramphenicol acetyltransferase gene) expression sequences of pBR325. The cpxA locus on the E. coli chromosome was established with respect to two chromosomal Tn10 insertions by a combination of genetic and physical analyses. The locus established by those analyses was consistent with the location of the 2 kb EcoRI-BamHI fragment in the physical map of the region. Physical analyses of (rha-pfkA) and (rha-tpiA) deletion strains showed that they lack cpxA and surrounding genes. Since these strains were viable, cpxA is not essential under all growth conditions.     

Molecular cloning vectors derived from the CoLE1 type plasmid pMB1.

$\text{In}$$\text{vitro}$ recombination via restriction endonucleases and $\text{in}$$\text{vivo}$ genetic translocation of an impicillin resistance gene, have been utilized for the construction of a series of cloning vehicles. These vectors have been derived by combining the essential replication properties of plasmid pMBl, a CoLEl type plasmid, with one, two or three resistance markers (tetracycline, ampicillin, chloramphenicol and colicin El production). During the construction of these vectors, the position of restriction sites used for cloning DNA fragments, relative to the antibiotic resistance genes and the origin of DNA replication, have been determined. The use of the cloning vectors pMB9, pBR313, pBR322, pBR324, pBR325 permits the molecular cloning and easy selection of $\text{Eco}$ RI, $\text{Bam}$ HI, $\text{Bgl}$ II, $\text{Dpn}$ II, $\text{Hind}$ III, $\text{Sal}$ I, $\text{Xho}$ I, $\text{Xam}$ I, $\text{Taq}$ I, $\text{Pst}$ I, $\text{Hinc}$ II, $\text{Pvu}$ I, $\text{Sma}$ I, $\text{Xma}$ I, $\text{Ba}$l I, $\text{Hpa}$ I, $\text{Ava}$ I, $\text{Pvu}$ II, $\text{Hae}$ III, $\text{Alu}$ I, $\text{Hpa}$ II, $\text{Eco}$ RII and virtually any blunt-ended generated DNA fragment.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^? strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^– strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.Deceased, December 15, 1995     

Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12

Summary pTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull^* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II^* from the plasmid ompA ⁺ gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH₂-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH₂-terminal fragment. However, the NH₂-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII^*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II^*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO₂H-terminal residues of wildtype protein II^* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.     

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.