Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis

Summary A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A phage library was first constructed by ligating a Sau3A (GATC) partial DNA digest of the entire E. coli chromosome into the BamHI (G GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRB1) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: 3, 4, 5, 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of 4 using different restriction fragments. Plasmids pGL444 and pBS105 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement a dnaGts chromosomal marker at nonpermissive 40° C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.     

The glucose kinase gene of Streptomyces coelicolor and its use in selecting spontaneous deletions for desired regions of the genome

Summary A number of deletions in the glucose kinase (glk) region of the Streptomyces coelicolor chromosome were found among spontaneous glk mutants. The deletions were identified by probing Southern blots of chromosomal DNA from glk mutants with cloned glk DNA. The deletions ranged in size from 0.3 kb to greater than 2.9 kb. When cloned glk DNA was introduced on a C31 phage vector into a glk mutant that contained a deletion of the entire homolgous chromosomal glk region, glucose kinase activity was detected in extracts of these cells. The entire coding information for at least a subunit of glucose kinase is there-fore present on the cloned glk DNA. The 0.3 kb glk chromosomal deletion was used to demonstrate that transfer of chromosomal glk mutations on the the C31::glk phage could occur by recombination in vivo. Since glk mutations frequently arise from deletion events, a method was devised for inserting the cloned glk DNA at sites in the chromosome for which cloned DNA is available, and thus facilitating the isolation of deletions in those DNA regions. C31::glk vectors containing a deletion of the phage att site cannot lysogenize S. coelicolor recipients containing a deletion of the glk chromosomal gene unless these phages contain S. coelicolor chromosomal DNA. In such lysogens, the glk gene becomes integrated into the chromosome by homologous recombination directed by the chromosomal insert on the phage DNA. In appropriate selective conditions, mutants which contain deletions of the glk gene that extend into the adjacent host DNA can be easily isolated. This method was used to insert glk into the methylenomycin biosynthetic genes, and isolate derivatives with deletions of host DNA from within the prophage into the adjacent host DNA. Phenotypic and Southern blot analysis of the deletions showed that there are no genes essential for methylenomycin biosynthesis for at least 13 kb to the left of a region concerned with negative regulation of methylenomycin biosynthesis. Many of the deletions also removed part of the C31 prophage.     

Transposition of a DNA sequence determining kanamycin resistance into the single-stranded genome of bacteriophage fd

Summary Derivatives of bacteriophages fd which transduce kanamycin resistance were selected after growth of the phage in an E. coli strain that carried transposon 5 (Tn5). Different clones of transducing phage and their DNAs were characterized by gel electrophoresis, electron microscopy, and by their ability to multiply in the absence of helper phage. Integration of the intact transposon into the full size phage genome was correlated with an increase in size of the phage particle from 0.95 to 1.7 , and with the appearance in the phage DNA of the stem loop structure characteristic for single-stranded Tn5 DNA. In nondefective phages the site of insertion was mapped by heteroduplex analysis within the intergenic region of the phage genome. Defective transducing phages were characterized as an insertion of Tn5 into a phage gene, and/or as a partial deletion or duplication of phage and transposon DNA. The size of the transducting phage from different defective clones varied from 0.6 to 3.0 and was directly proportional to the DNA content. These results demonstrate that filamentous bacteriophage are highly capable to replicate and package very different amounts of foreign DNA.This work was presented at the EMBO Workshop on single-stranded DNA viruses, October 1976, Harpert, The Netherlands     

Integration specificity of an artificial kanamycin transposon constructed by the in vitro insertion of an internal Tn5 fragment into IS2

Summary IS2 has been marked genetically by the in vitro insertion into its HindIII site of a 3.3 Kb HindIII fragment of Tn5 conferring resistance to kanamycin. The transposition of the IS2::Km, thus obtained, to has been found and insertion sites were characterised. Each of ten independent IS2::Km insertions were found at the same site at 61.2% of the map, always in the same orientation (orientation II relative to the xis gene). The integration sites of IS2::Km in five of the kanamycin-transducing phages were determined by DNA sequence analysis, and were found to be identical at the nucleotide level. Further transposition of IS2::Km from to the bacterial chromosome was demonstrated.     

Origin of replication, oriC, or the Escherichia coli chromosome on specialized transducing phages lambda asn.

Summary Specialized transducing phages asn harboring chromosomal DNA and genetic markers on either side of the asn gene were isolated. Phages carrying chromosomal DNA counterclockwise of the asn gene can upon infection establish themselves as self-replicating plasmids in asn, recA hosts lysogenic for lambda. It is concluded that this bypassing of normal lambda immunity is due to the presence of the chromosomal replication origin, oriC, in this class of phages. Genetic analysis and the determination of restriction endonuclease cleavage patterns of the different asn lead to the allocation of oriC within 1.5 megadaltons of the asn gene towards the uncA, uncB genes at 82 min on the genetic map of E. coli, The clockwise order of genes on the chromosome is found to be: bglB, (pst, glmS), (uncA, uncB), criC, asn, trkD, rbs, rrnC, ilv.Abbreviations CCC covalently closed circular - MD 10⁶ Daltons; mw, molecular weight - R. restriction endonuclease - LFT, HFT low (high) frequency transducing - a.p.i. average phage input - m.o.i. multiplicity of infection     

Coordinate expression of Escherichia coli dnaA and dnaN genes

Summary The defects of temperature-sensitive dnaA and dnaN mutants of Escherichia coli are complemented by a recombinant lambda phage, which carries the bacterial DNA segment composed of two EcoRI segments of 1.0 and 3.3 kilobases. Derivatives of the phage, which have an insertion segment of Tn3 in the dnaA gene, are much less active in expressing the dnaN gene function than the parent phage. The dnaN gene activity was determined as the efficiency of superinfecting phage to suppress loss of the viability of lysogenic dnaN59 cells at the nonpermissive temperature. Deletions that include the end of the dnaA gene distal to the dnaN gene also reduce the expression of the dnaN gene fuction. Deletion and insertion in the dnaN gene do not affect the expression of the dnaA gene function. The expression of the dnaN gene function by the dnaA ^- dnaN ⁺ phages remains weak upon simultaneous infection with dnaA ⁺ dnaN ^- phages. Thus the insertion and deletion in the dnaA gene influence in cis the expression of the dnaN gene. We propose that the dnaA and dnaN genes constitute an operon, where the former is upstream to the latter.     

Molecular cloning of the fnr gene of Escherichia coli K12

Summary Mutations in the fnr gene of Escherichia coli have pleiotropic effects leading to deficiencies in the reduction of fumarate and nitrate, hydrogen production and the ability to grow anaerobically with fumarate or nitrate as terminal electron acceptors. Transducing phages (fnr) carrying the wild-type fnr gene were isolated from populations of artificially-constructed recombinant lambda phages by their ability to complement the lesions of fnr mutants. The fnr phages restored anaerobic growth with fumarate and nitrate as electron acceptors and, as prophages, they promoted normal synthesis of fumarate reductase, nitrate reductase and hydrogenase in fnr mutants. Five independently-isolated fnr phages each contained a R.HindIII fragment (11.5 kilobases) that possessed three internal R.EcoRI targets and had inserted with the same orientation relative to the phage. A physical map of the fnr region was constructed by restriction analysis and flanking fragments were identified by DNA:DNA hybridization.     

Recombinant P4 bacteriophages propagate as viable lytic phages or as autonomous plasmids in Klebsiella pneumoniae

Summary We demonstrate the use of bacteriophage P4 as a molecular cloning vector in Klebsiella pneumoniae. A hybrid P4 phage, constructed in vitro, that contains a K. pneumoniae hisDG DNA fragment can be propagated either as a lytic viable specialized transducing phage or as an autonomous, self-replicating plasmid. Hybrid P4 genomes existing as plasmids can be readily converted into non-defective P4-hybrid phage particles by superinfection with helper phage P2. Infection of a K. pneumoniae hisD non-P2 lysogen with P4-hisD hybrid phage results in approximately 90% of the infected cells becoming stably transduced to HisD⁺. Because P4 interferes with P2 growth, high titre stocks of P4 hybrid phages are relatively free (10^-6) of P2 contamination. The hisG gene product was detected in ultraviolet light irradiated host cells infected by the P4-hisDG hybrid phage. A mutant of P4 (P4sidl) that directs the packaging of P4 DNA into P2 sized capsids should permit the construction of hybrid phages carrying 26 kilobase inserts.     

Lambda transducing phages derived from a FinO- R100::lambda cointegrate plasmid: proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant.

Summary Three transducing phages have been isolated from pEDR20, an R100:: cointegrate plasmid in which the insertion inactivated the R100 finO gene. Physical analysis of the three phages showed that the is inserted at kilobase coordinate 81.3 of R100. All three phages carry different amounts of R100 DNA in the left arm of . Each phage contains IS1b, the mer genes and the region between coordinate 81.3 and 88.6; thus, all contain the genes necessary for R100 replication. One phage, VA73, contains the entire r-determinant of R100 in addition to the above DNA. Five proteins coded by the region between 81.3 and 88.6 were detected. These had subunit molecular weights of 10,400; 12,200; 16,200; 19,600; and 38,300. The first was made constitutively and the other four only from a promoter. Other constitutive proteins were one from the cml fus region with a molecular weight of 22,400 (cml) and two from the str sul region with molecular weights of 31,500 (str?) and 30,100 (sul?). Mercuric ion induced synthesis of at least 10 proteins. Six of these were known from earlier work. The total size of the proteins which appear to derive from the mer genes exceeds by a factor of 1.5, the coding capacity of this region without overlapping genes. Some, or all of these extra proteins may be chromosomal in origin, possibly derepressed in response to mercury gene products.     

Isolation of a transducing phage carrying rpsT, the structural gene for ribosomal protein S20

Summary transducing phages carrying segments of the Escherichia coli chromosome in the dapB region have been isolated and their in vivo gene products analyzed by two-dimensional gel electrophoresis. One of these phages, ddapB-2, carries the structural genes for ribosomal protein S20 (rpsT) and isoleucyltransfer RNA synthetase (ileS). The most likely gene order is thr-rpsT-ileS-dapB-pyrA.     

A physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome

A combined physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleasesSwaI (5-ATTTAAAT-3),PacI (5-TTAATTAA-3), andPmeI (5-GTTTAAAC-3) yielding 26, 27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacentSwaI fragments, a genomic cosmid library ofC. glutamicum was screened for chromosomal inserts containingSwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect theSwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blottedSwaI digests using as probesPacI andPmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partialSwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes toSwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.     

Site-directed insertion mutagenesis with cloned fragments in Escherichia coli by P1 phage transduction

Summary A cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage gt11. P1 phage grown on cells lysogenized with the recombinant phage could transduce the mutant gene into the original site on the Escherichia coli chromosome.     

A technique for targeted mutagenesis of the EF-Tu chromosomal gene by M13-mediated gene replacement

A generally applicable system for targeted mutagenesis of a chromosomal sequence is described. The Escherichia coli tufA gene was mutated using a recombinant M13mp9 phage vector carrying a tuf gene. Integration via crossing over with the chromosomal tufA target gene produced an M13 lysogen. These lysogens were screened for resistance to kirromycin. The M13 phage carrying tufA mutations were efficiently retrieved by a genetic procedure. Genetic mapping was performed with the M13 vectors. The same recombinant M13 phage was used for mutagenesis, lysogen formation, gene replacement, retrieval, mapping and sequencing of kirromycin mutants. Three different mutations yielding resistance to kirromycin were found: two of these have previously been found and characterised, while the third mutation, Gly316 Asp, is a new mutant. We also report the identification of a fourth kirromycin-resistant mutant, Gln124 Lys.     

Mapping of insertion element IS30 in the Escherichia coli K12 chromosome

Summary We identified seven phage clones containing the insertion element IS30 in a phage library mini-set, which includes 476 clones carrying chromosomal segments that cover almost the entire chromosome ofEscherichia coli K12 W3110 (Kohara et al. 1987). We could assign locations and orientations to four copies of IS30 (namedis30A tois30D) on the W3110 chromosome by restriction analysis of phage DNAs containing them. These IS30s were present at the same locations in chromosomes of both W3110 and anotherE. coli K12 strain JE5519, and thus are assumed to be present in otherE. coli K12 derivatives, including early isolates. Among the IS30 copies found, one (is30B) contained a large deletion and possessed only a 181 by stretch of the right terminal region of IS30.EMBL Accession Number: The EMBL accession number of the sequence reported in this paper is X17345     

Genetic Analysis of Chromosomal Regions of Lactococcus lactis Acquired by Recombinant Lytic Phages

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi⁺) with phage 31. HindIII restriction maps of the variants (31.1, 31.2, 31.7, and 31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage 31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, 31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the 31.1 DNA, which replaced the 31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of 31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.     

Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.     

Genetic and physical mapping of recF in Escherichia coli K-12

Summary Two factor transductional crosses place recF at approximately 82 min on the E. coli chromosome; recF is highly cotransducible with dnaA and gyrB (cou). Transductional analysis with a series of tna specialized transducing phages carrying chromosomal DNA from the tnaA region place recF between dnaA and gyrB. This analysis also indicates that a gene lying in the same region and producing an easily detectable protein (estimated MW of 45 kD) is dnaN and not recF.     

The isolation of evolutionarily conserved Eag I end-clones from mouse chromosome 17 using cloned DNA.

To isolate DNA markers from mouse chromosome 17, a genomic phage library was constructed from the mouse-hamster CMGT cell hybrid RcE-B52. This hybrid contains a chromosomal fragment from the distal end/flanking region of the t complex on mouse chromosome 17. Recombinants of mouse origin were identified by using a panel of mouse-specific repetitive sequences as a probe. A total of 1,500 mouse phage recombinants were isolated. These were found to represent 250-300 individual recombinants, comprising about 4 Mbp of cloned mouse DNA. The pooled mouse recombinant phages were used to construct an Eag I end-library. This was achieved by the specific insertion of a marker plasmid in Eag I recognition sites when present in the mouse inserts of the recombinant phages. The Eag I end-fragments were subsequently subcloned using a simple procedure taking advantage of the inserted plasmid. A total of 56 individual Eag I end-fragments were identified. These were found to contain recognition sites for rare cutting enzymes at high frequency. A large proportion (73%) were found to be evolutionarily conserved in human DNA. Furthermore, a significant fraction of these fragments, two of six tested, appears to detect specific cDNAs in a 8.5-day mouse embryo cDNA library.     

Identification of the E. coli dnaJ gene product

Summary We have previously shown that a mutation (groPC259) in the E. coli dnaJ gene renders the cell inviable at high temperatures and arrests bacteriophage DNA replication at all temperatures (Sunshine et al., 1977). We have isolated dnaJ ⁺⁺ transducing phages both by in vitro cloning and by abnormal excision of a dnaK transducing phage integrated near the dnaJ locus. The dnaJ gene product has been identified on SDS polacrylamide gels after infection of UV-irradiated E. coli cells by dnaJ ⁺⁺ derivative phages. It is a polypeptide chain with an apparent molecular weight of 37,000-daltons. This has been verified by the fact that a transducing phage carrying an amber mutation in the dnaJ gene fails to induce the synthesis of the 37,000-dalton polypeptide chain upon infection of sup ⁺⁺ bacteria, but does so upon infection of supF or supD bacteria.