Identification of a positive regulatory protein in Escherichia coli: the product of the cysB gene

Summary From the specialized transducing bacteriophage cysB, recombinant phages cysB242 and cysB257 have been obtained, each of which carries an amber mutation in the cysB cistron. A comparison of polyacrylamide gel electrophoretic profiles of labelled extracts from uv-irradiated bacteria that had been infected with cysB ⁺ or with cysB-amber phages, led to the identification of a 39,000-dalton polypeptide as the product of the cysB gene. The native protein was purified to near radiochemical purity and was found to be an oligomer with an isoelectric point close to pH 7.     

Fertility functions of resistance factors

Summary The isolation of transducing phages carrying the tolPAB cluster is described. These genes map between gltA and gal in Escherichia coli, and thus are relatively close to att. To isolate these transducing phages, it was necessary to use a strain deleted of most of the intervening genes (nadA to chlD) between tolPAB and att. Using a lysogen of such a deletion strain, several defective dtol phages were isolated that carry different amounts of the tolPAB cluster.All of these dtolPAB phages were defective in both lysogenization and vegetative growth, and in this respect were similar to dgal transducing phages.The usefulness of such specialized transducing phages in studying the cell surface is discussed.Research Fellow of the National Cancer Institute of Canada.     

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.     

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.     

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.     

Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis.

Summary Hybrid ColE1 plasmids called ColE1-cos-guaA or ColE1-cos-gal can be efficiently transduced into various E. coli K-12 cells through packaging into phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-cos-guaA and ColE1-cos-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-cos-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos-guaA about 7-fold. (5) The same ColE1-cos-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA ⁺ gen function(s) and suggest that ColE1 plasmid itself provides no recA ⁺-like functions.     

lambdaimm P22dis: a hybrid of coliphage lambda with both immunity regions of Salmonella phage P22.

Summary Genetically marked and P22 phages were recombined in Escherichia coli-Salmonella typhimurium hybrid WR4028, a host sensitive to infection by both of these phages. Hybrid phages that acquired the immC region of P22, but retained the genes for the protein coat were selected on WR4027 (), a -immune, P22-resistant derivative of WR4028. In these immP22 hybrids, at least the c through P genes of were replaced with functionally related P22 genes. Phage recombinants with more extensive regions of the P22 genome were selected on the double lysogen WR4027 (, immP22). One such hybrid, immP22dis, was determined by heteroduplex analysis to contain approximately 40% of the P22 genome. Genetic studies established that immP22dis possesses the two widely separated immunity control regions of P22 (immC and immI) and that these loci are expressed in E. coli K-12 lysogenic for immP22dis. In addition, immP22dis contains the P22 a1 locus responsible for somatic 0–1 antigen conversion in Salmonella. Although the immP22dis phage particle has the head and tail, the phage genome also carries P22 tail gene 9 as evidenced by the production of free P22 tails. It also has the P22 att site as indicated by the integration of the immP22dis prophage near the proA locus on the bacterial chromosome.     

Characterization of dnaA gene carried by lambda transducing phage

Summary Specialized transducing phages dnaA were obtained by inducing lysogens in which tna was integrated at the tnaA region of the Escherichia coli chromosome; the tnA region is located in the vicinity of the dnaA gene. The dnaA ^- deletion derivatives of dnaA were isolated from the lysate of dnaA grown on bacteria carrying a transposon Tn3.The structures of various transducing phages thus obtained were determined by heteroduplex DNA mapping. From these results, the transducing fragment of 13.8-kb-long was divided into nine domains. Upon infection of UV-irradiated cells with the phage, production of polypeptides of 49 kD and 42 kD was specifically associated with infections by the dnaA and recF transducing phages. Polypeptides of 49 kD and 42 kD appeared to be coded for by dnaA and recF genes, respectively. The dnaA gene was assigned to the region of 2.8-kb-long which extends by 2.4 kb in the counterclockwise direction on the E. coli genetic map and 0.4 kb in the opposite direction, as measured from the nearest HindIII site close to the tnaA gene. The recF gene was also discovered to lie very close to dnaA in the order of tnaA-dnaA-recF.Merogenotes heterozygous for the dnaA gene were constructed by introducing F100-12 carrying dnaA into the recipients with different mutations at or near dnaA. For combinations, F(dnaA ⁺)/dnaA46 and F(dna ⁺)/dna-83, dnaA ⁺ was trans-dominant, whereas the dnaA ⁺ was recessive for F(dnaA ⁺)/dna-5. For F(dnaA ⁺)/dna-167, the result of the transdominance test was affected by the growth media employed; dnaA ⁺ was dominant on a -broth plate, and dna-167 was dominant on an M9-minimal plate. Thus, transdominance of dnaA ⁺ in heterozygotes is affected by difference in mutations and growth media.     

Lambda transducing phages for the nalA gene of Escherichia coli and conditional lethal nalA mutations.

Summary Defective transducing phages for the nalA region of the Escherichia coli chromosome were isolated from a lysogen in which is inserted in the nearby glpT gene. The three classes of transducing phages designated nrdA, dubiG, and dnalA contained bacterial DNA extending from glpT through nrdA, ubiG, and nalA, respectively. The bacterial genes are in the left arm of the chromosome. Of the eleven polypeptides coded by dnalA that were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate only one was not also specified by dubiG This 105,000 dalton polypeptide is the nalA gene product. The electrophoretic mobility and isoelectric point of this protein were unaffected by a nalA mutation (nalA48) that confers nalidixic acid resistance. Temperature-sensitive and amber mutations in the nalA gene were isolated using a dnalA48 lysogen which is heterodiploid for nalA. The conditional lethality of these mutations proves that nalA is an essential locus.     

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function

Summary Escherichia coli mutants, called groNB, which block the growth of bacteriophage at the level of action of the gene N product, have been isolated as survivors at 42°C of bacteria carrying a) the defective prophage bio1 1 i cI857 H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage cI857. In addition, groNB bacterial mutants have been isolated at 37° C, as large colony formers in the presence of i cI h ⁴³⁴, i cI h , and i cI h ⁸⁰ phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42° C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro⁺ for growth. Lambda transducing phages carrying the groNB ⁺ bacterial gene have been isolated. GroNB mutant bacterial lysogenized by the transducing phage acquire the Gro⁺ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.     

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.     

Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome

Summary Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting dilv phage were selectively labelled with radioactive amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from dilv phage into pBR322 and shown to be dominant to a rho mutation on the host cromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.     

The origin of Q-independent derivatives of phage lambda

Summary qsr (Q-independent) phages are characterised by the replacement of the region of the genome that contains Q, S, R, and the late gene promoter, P_R, with host-derived DNA that codes for functions analogous to those deleted. Restriction endonuclease analysis and DNA/DNA hybridisation methods have been used to show that p4 and qin A 3, two such Q-independent phages, are the product of recombination between and a defective lambdoid prophage (the qsr prophage) located at an as yet unidentified site in the E. coli K 12 chromosome. The qsr prophage is distinct from the defective lambdoid prophage Rac (Kaiser and Murray 1979). In the E. coli K 12 strain AB1157 from which qsr phages cannot be generated, the qsr prophage has suffered an internal deletion. That the qsr prophage appears not to carry a full complement of essential late genes suggests one explanation for its apparently defective nature.     

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.     

Analysis of a temperature sensitive mutation in gene cII of bacteriophage lambda

Summary The mutation cIIts612 was found to map outside the immunity region of phage imm21 hybrid. As expected of a cII mutation, cIIts612 is unable to stimulate either cI repressor or Int synthesis during the establishment of lysogeny. These results indicate that part of the cII gene of is homologous to that of imm21 phage.     

Tandem duplication induced by an unusual ampA1-, ampC-transducing lambda phage: A probe to initiate gene amplification

Summary Secondary attachment site -lysogens were isolated in an Escherichia coli strain carrying multiple tandem 9.8 kb repeats. The repeat carried the structural gene for chromosomal -lactamase, ampC. One lysogen produced lysates with amp-transducing activity. Three types of phages with different densities were obtained from this lysogen. The one with the lowest density was found to be a helper cI857S7 phage. The other two phages showed identical restriction endonuclease fragmentation patterns. The difference in density was due to the presence or absence of phage tail. In damp the right cohesive end segment was deleted in a random fashion with the majority ending between 81.0% and 82.4% of . The chromosomal segment of damp was most likely located at the attachment site. The damp DNA was compared to that of a ColE1 hybrid carrying the chromosomal amp segment and a ColE1 hybrid carrying the same 9.8 kb amp repeat as the lysogen from which damp was isolated. It was found that the chromosomal part of damp constituted 9.8 kb, i.e. the size of one repeat. Moreover, the novel joint between adjacent repeats was present. In a attB-deleted E. coli K-12 strain, lysogenic for damp, highly ampicillin-resistant mutants occurred at an exceedingly high frequency. They were found to contain in the chromosome an amplified 9.8 kb repeat. This suggested that integration of the novel joint from damp into the amp region gives rise to an amplifiable duplication. In E. coli lysogenized for damp at attB highly ampicillin-resistant clones were also found at a high frequency. These clones carried multiple tandem repeats of damp DNA, each with an intact right end segment.     

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.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of thebio specialized transducing phage inEscherichia coli. Because mostbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^– phenotype), we selectedbio transducing phage by their Spi^– phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio and DNAs, except for a hotspot which accounts for 57% of UV-inducedbio transducing phages and 77% of spontaneously inducedbio transducing phages. The hotspot sites onE. coli and DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio and hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.     

Normal expression of the viral gene N interferes with growth of bacteriophage lambda in Escherichia coli 15T-.

Summary Growth of and of some lambdoid phages is considerably inhibited on strain 3057 derived from E. coli 15T^-. Mutants of which overcome this inhibition map in gene N. Some of these hty mutants are temperature sensitive for growth on E. coli K12. Thus plating of on strain 3057 allows one to isolate temperature sensitive N mutants. The hty mutants produce less than normal N activity as judged by their low efficiency of plating on a nus ^- host and by the extended latent period of some of them on normal hosts. The inability of strain 3057 to propagate can be at least partially reversed by addition of thymidine to the medium and the growth difference between hty and in 3057 increases with decreasing thymidine concentration. The amount of DNA produced by in 3057 at low thymidine concentration is lower than that produced by hty under the same conditions. Only a small percentage of the DNA produced by in 3057 is packaged into viable phage particles. This suggests that not only produces less DNA in 3057 than hty but that an important part of the DNA in 3057 is in a form which can not be packaged or which is noninfective for other reasons. A hypothesis is discussed that hty mutations enable to grow on E. coli 15T^- at low thymidine concentration because they lead to reduction in the number of single strand nicks in the DNA by reducing the intracellular endonuclease activity. Under permissive conditions conditional lethal N mutants are favored for growth on 3057 over N ⁺ which confirms the idea that N activity or the activity of a gene under N control interferes with growth in 3057 at low thymidine concentration.     

Human λ light chain locus: Organization and DNA sequences of three genomicJ regions

Evidence for the genomic organization of human lambda light chain joining (J) region gene segments is presented. A mouse J probe was used in Southern hybridizations to localize joining region sequences in a cosmid clone containing the genomic cluster of six human lambda constant (C) region gene segments. The results of these hybridizations suggest the presence of at least one J gene segment upstream from each constant region gene segment. The DNA sequences indicate that the human JI, J2, and J3 gene segments have consensus nonamer and heptamer sequences, proposed to be involved in V-J joining, are capable of encoding the known amino acid sequences for the respective J peptides, and have a sequence which could give functional RNA splice site at the end of their coding regions. Our data show that a single functional J is located 1.3 or 1.6 kb upstream of each of the C gene segments known to encode the Mcg, Kern^– Oz^–, and Kern^–Oz⁺ isotypes. Therefore, the gene organization of this region of the human lambda locus is J1 CI -J2C2-J3C3. The DNA sequences ofJ 1,J 2, andJ 3 presented in this paper establish that a singleJ gene segment precedes each expressed C gene segment, and support a model for the evolution of the human JC clusters where JICI andJ2C2-J3C3. arose from different ancestral JC units.