Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.     

pMB9 plasmids bearing the Salmonella typhimurium his operon and gnd gene

A plasmid containing the entire Salmonella typhimurium his operon was constructed from plasmid pM89 and an EcoRI fragment of phi 80 his imm lambda DNA. The recombinant pST41 also includes the glucose 6-phosphate dehydrogenase (gnd) gene and has one EcoRI endonuclease cleavage site in the integrated fragment. This plasmid served as a source for the construction of two additional plasmids, one carrying the OGDC-region of the his operon and the other a CBHAFIE segment of the his gene along with the gnd gene. The presence of the his operon in the constructed plasmids was confirmed by hybridization to S. typhimurium his RNA. The location of the gnd gene in the CBHAFIE fragment of the his gene was confirmed genetically: after transfection with the plasmid bearing the gnd gene, a gnd recipient gained the capacity to utilize gluconate as a sole carbon source. The DNAs of the three hybrid plasmids were analyzed by gel electrophoresis. By comparing the EcoRI endonuclease cleavage pattern of these three hybrid plasmids with the DNA cleavage pattern of phi 80 his imm lambda, phi 80 imm lambda and lambda phages, the EcoRI cleavage map of phi 80 his imm lambda was obtained.     

Plasmid vehicles for direct cloning of Escherichia coli promoters.

A multicopy plasmid cloning vehicle, pGA22, which carries genes for ampicillin resistance (Apr), tetracycline resistance (Tcr), chloramphenicol resistance (Cmr), and kanamycin resistance (Kmr) has been constructed. This plasmid has five unique sites for restriction endonucleases EcoRI, PstI, XhoI, SmaI, and SalI within antibiotic resistance genes. pGA22, which is 5.1 megadaltons in size, has a low copy number (probably fewer than 10 per genome), is capable of relaxed replication, and is mobilized by F-factor at a frequency of 10(-5). A series of promoter-cloning vehicles, pGA24, pGA39, and pGA46, has been developed from pGA22. In these plasmids the natural promoter for Tcr has been removed and has been replaced by small deoxyribonucleic acid fragments carrying unique sites for several restriction endonucleases. Cells carrying these vectors are sensitive to tetracycline unless insertional activation of the Tcr occurs by cloning a promoter-carrying deoxyribonucleic acid fragment in one of the unique sites adjacent to the 5' end of Tcr. In this way, promoters carried on a HindIII-generated deoxyribonucleic acid fragment can be inserted at the HindIII site of plasmid pGA24, pGA39, or pGA46. A promoter in fragments generated by digestion with restriction endonuclease XmaI or PstI or by any restriction endonucleases which generate flush ends, such as SmaI, PvuII, HpaI, HincII, or HaeIII, can be clones in plasmid pGA39. Plasmid pGA46 can be used to detect a promoter fragment carried on a BglII, BamHI, MboI, or PstI fragment. We also describe a plasmid, pGA44, with a unique KpnI site in the rifampin resistance gene rpoB.     

Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac.

Bacteriophage Mu d1 (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate-dehydratase. A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions. A restriction enzyme cleavage map of lambda ppheA-lac for the enzymes HindIII and PstI is presented.     

DNA probes with different specificities from a cloned 23S rRNA gene of Micrococcus luteus

A 7500 bp PstI restriction fragment of chromosomal DNA from Micrococcus luteus containing a 23S rRNA gene was cloned in vector pHE3 in E. coli RR 28 (the recombinant plasmid was designated pAR1). A recombinant phage (pAR5) hybridizing to all eubacteria tested was constructed by shotgun subcloning of the PstI fragment in phage M13mp8. Further subcloning of the fragments of the 23S rRNA gene in the vectors pTZ18R and pTZ19R using selected restriction sites of the gene enabled us to select cloned fragments of the 23S rRNA gene representing different specificities. Probes specific for Micrococcus luteus-Micrococcus lylae (pAR28), for the Arthrobacter-Micrococcus group (pAR27), for eubacteria (pAR5), and for the detection of eu- and archaebacteria (the so-called universal probe pAR17) were constructed. The specificity of each probe was analysed by dot hybridization to the chromosomal DNAs of representatives of most of the main phyla of eu- and archaebacteria.     

Identification of deoxyribonucleic acid restriction fragments of beta-converting corynebacteriophages that carry the gene for diphtheria toxin.

Deoxyribonucleic acid fragments bearing the gene for diphtheria toxin have been identified in restriction enzyme digests of deoxyribonucleic acids from beta-converting and gamma-nonconverting corynebacteriophages. A combination of physical and genetic evidence has established that the Bam HI band C fragment of beta phage deoxyribonucleic acid, which carries the specific phage attachment site (Buck and Groman, J. Bacteriol. 148:131-142, 1981), also carries most, and probably all, of the gene for diphtheria toxin. A detailed restriction map of this tox-bearing Bam HI fragment has been developed, and the locations and orientation of the tox gene and the attP site within this fragment have been established.     

The identification and partial characterization of a plasmid containing the gene for the membrane-associated hydrogenase from E. coli

Escherichia coli DNA was digested with restriction endonuclease PstI and ligated into the PstI site of plasmid pBR322. Recombinant plasmids that were constructed in this manner were used to transform E. coli H61, a mutant with a decreased level of hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for the membrane-associated E. coli hydrogenase in plasmid pBL101. In E. coli minicells, the pBL101 DNA directed the synthesis of a protein of a size corresponding to that of the precursor of the E. coli membrane-associated hydrogenase, which appears to contain an uncleaved leader peptide. A restriction map of the cloned DNA was determined for 14 endonucleases.     

Cloning of sporulation gene spoOB of Bacillus subtilis and its genetic and biochemical analysis.

A specialized transducing phage carrying a sporulation gene (spoOB) was constructed from Bacillus subtilis temperate phage rho 11 by in vitro and in vivo recombinations. Transformation experiments showed that the spoOB gene resides on a 1.4-megadalton fragment generated by EcoRI endonuclease treatment of the phage deoxyribonucleic acid (DNA). Mutants of this phage which lost transducing activity were isolated and used for genetic complementation tests and the analysis of protein(s) coded by the 1.4-megadalton fragment. The spoOB locus was shown to be composed of one cistron. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins synthesized in ultraviolet-irradiated cells infected with these phages showed that the 1.4-megadalton fragment codes at least one protein, of molecular weight 39,000, which is synthesized in both vegetative and sporulating cells. A cleavage map of the phage DNA was constructed by use of restriction endonucleases, EcoRI, BamHI, and SalI, and the site of integration of the 1.4-megadalton fragment was determined. Expression and function of the spoOB gene are discussed.     

Cloning and expression of the gene(s) for cephalosporinase production of Citrobacter freundii

The chromosomal gene(s) for cephalosporinase production of Citrobacter freundii GN346 has been cloned into vector plasmid pMK1, initially as a 7.3 kb EcoRI fragment. From the substrate profile and the response to anti-GN346 CSase serum of the enzyme produced, it was confirmed that the hybrid plasmid (pTY71) carries the relevant chromosomal cephalosporinase gene from C. freundii GN346. A restriction endonuclease cleavage map of cloned EcoRI fragments was constructed, and the structural gene of the cephalosporinase could be limited in the 1.5 kb BamHI fragment. The cloned gene(s) was expressed at an extremely low level in Escherichia coli. Furthermore, its expression was constitutive in E. coli, although inducible in its own cytoplasm.     

Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12.

A hybrid plasmid from the Clarke and Carbon collection has been isolated. This plasmid carries the trmA gene of E. coli, which is necessary for the formation of 5-methyluridine (m5U,ribothymidine) present in all transfer ribonucleic acid (tRNA) chains of the organism so far sequenced. A restriction map of the argCBH-trmA regions is presented. By using cloning in vitro, the trmA gene was located on a 2.9-kilobase pair deoxyribonucleic acid (DNA) fragment. These results and comparison with lambda dargECBH transducing phages established the gene order: argECBH trmA bfe in the 88-min region of the E. coli chromosomal map. Plasmids carrying this 2.9-kilobase pair DNA fragment overproduce the enzyme tRNA(m5U)methyltransferase (EC 2.1.1.35) 20 to 40 times. When this 2.9-kilobase pair chromosomal DNA fragment was expressed in a minicell system, a polypeptide of a molecular weight of 42,000 was synthesized. This polypeptide was tentatively identified as the tRNA(m5U)methyltransferase. These results support the earlier suggestion that the trmA gene is the structural gene for the tRNA(m5U)methyltransferase.     

The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the Xts+d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied. The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.     

Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance.

The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kilobases) were determined for the BglII, PstI, and SmaI restriction enzymes, and the determinants for tetracycline and ampicillin resistance were localized. The cleavage sites were clustered at or near the drug resistance genes. To localize regions required for plasmid replication and maintenance in Escherichia coli, we deleted nonessential regions of RK2 by partial digestion with the restriction endonuclease HaeII to produce small derivatives. The smallest stable replicon obtained contained five HaeII fragments of RK2 which total 5.4 kilobases. These fragments were derived from three regions of RK2 that are separated from each other by antibiotic resistance genes. One of these HaeII fragments (0.75 kilobases) has the properties expected of the origin of replication. The outer four fragments, located in two separate regions of RK2, were found to provide, in trans, functions that permit the replication of the HaeII fragment carrying the origin of the replication. These results indicate that at least two plasmid-encoded genes, capable of acting in trans, and a replication origin are required for RK2 replication and maintenance.     

Transfer of the chromosomal bla gene from Enterobacter cloacae to Escherichia coli by RP4::mini-Mu

The resistance gene for beta-lactamase-stable cephalosporins from Enterobacter cloacae was transferred to Escherichia coli by the aid of RP4::mini-Mu. The R-prime plasmids generated carried 60 to 80 kilobases (kb) of E. cloacae DNA and coded for the chromosomal E. cloacae beta-lactamase. The gene was fully expressed in the recipient. Restriction endonuclease EcoRI fragments of the R-prime plasmid pBP100 were cloned into the vector pBP328, yielding the plasmid pBP102 with a size of 14 kb. A restriction map of this plasmid was constructed. By digesting pBP102 into seven PstI fragments, ligating the fragments, and looking for the smallest plasmid generated, pBP103 was isolated. It consisted of three PstI fragments, two of them (together 4.2 kb) necessary for resistance. During the experiment (performed in a recA+ background) the largest PstI fragment had undergone a substitution of a 0.3-kb segment of pBP102 by a 0.7-kb segment in pBP103 (as deduced by heteroduplex analysis). The bla gene of resistant E. cloacae strains was dominant over the gene of susceptible organisms.     

Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro

The structural gene for alkaline phosphatase (phoA) of Escherichia coli was cloned into the PstI site of pBR322, from a transducing bacteriophage, lambda p(phoA-proC). The restriction map of the plasmid was established. Based upon this information, several phoA deletion plasmids as well as a smaller phoA+ plasmid were constructed. The genetic map and restriction map were correlated by recombination analysis. Cells carrying one of the phoA+ plasmids overproduce alkaline phosphatase 10-fold upon phosphate limitation. However, both regulation and processing of the enzyme were found to be normal.     

Isolation of Specialized Transducing Bacteriophages for Gluconate 6-Phosphate Dehydrogenase (gnd) of Escherichia coli

Specialized transducing phages for gluconate 6-phosphate dehydrogenase (gnd), a constitutive enzyme in Escherichia coli, have been isolated using a method previously described for other genes. The gnd-his region, carried on an F' episome, was first transposed to tonB. Rare phages carrying gnd were selected, by transduction, from phi80 lysogens of these strains; one phage also carried his (phi80gndhis). From the transductants, high-frequency transducing lysates were obtained; low multiplicity of infection then yielded defective lysogens. tonB deletion analysis of the phi80dgndhis lysogen shows the order of genes in the prophage to be imm80...hisOGD...gnd; according to a marker rescue experiment most phage late genes have been replaced by bacterial deoxyribonucleic acid. A heat-inducible, lysis-defective lambda-phi80 hybrid derivative of phi80dgndhis has been prepared.     

Cloning of bacteriophage T5 DNA fragments in the plasmid pBR322. Analysis of recombinant plasmids by the method of bonding with RNA-polymerase from Escherichia coli on nitrocellulose filters

DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.