Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12.

We previously characterized a set of lambda dilv phages by genetic, restriction enzyme, and heteroduplex analyses and tentatively correlated isoleucine-valine gene products with specific ilv DNA segments by using cloned ilv segments in maxicells and lambda dilv phage infection of UV-irradiated cells. In this work, the identity of the ilvC gene product, alpha-acetohydroxy acid isomeroreductase, was confirmed by demonstrating its induction by the physiological inducers alpha-acetolactate and alpha-acetohydroxybutyrate. The identity of the ilvE gene product, transaminase, B, was confirmed by antibody precipitation of the purified enzyme. Phage derivatives with ilv regulatory mutations were found to have the predicted effect upon the ilvGEDA and ilvC protein products. The distribution of the ilvGEDA and ilvC gene products in the soluble, periplasmic, inner membrane, and outer membrane fractions was examined, and no significant membrane association was observed. The expression of the ilv genes in the lambda dilv phage from ilv and phage lambda promoters was compared in order to determine the fractional contribution of each to ilv gene expression. An additional protein of 54,000 daltons that was not detected in the previous analysis was observed to be coded by a bacterial gene but was produced only by readthrough from phage promoters.     

Molecular characterization of ilvC specialized transducing phages of Escherichia coli K-12

A series of lambda defective ilvC specialized transducing phage has been isolated which carry regions of isoleucine and valine structural and regulatory genes derived from the ilv cluster at minute 83 on the linkage map of the chromosome of Escherichia coli K-12. The ilv genes carried by these phages and their order have been determined by transduction of auxotrophs. The ilvC+ lysogen of an ilvC- strain gave rise, after heat induction of the lysogen, to transducing particles which carried the wild-type allele of the cya-marker. Further experiments have shown that the lambda defective ilvC phages were able to cotransduce a rho-15ts mutation as well as a rep-5 mutation. Hence, the order of the clockwise excision of the ilv cluster was found to be ilvC-rho-rep-cya. Enzyme levels in strains carrying the lambda defective ilvC phages indicated the the ilvC gene was not altered by the insertion of lambda into the ilv cluster. The isolation and digestion of lambda defective ilvC DNA by EcoRI and HindIII restriction endonucleases demonstrated that the specialized transducing phages carried part of the genome from the E. coli K-12 chromosome.     

Isolation and analysis of two Escherichia coli K-12 ilv attenuator deletion mutants with high-level constitutive expression of an ilv-lac fusion operon.

A lysogenizing lambda phage, lambda dilv-lac11, was constructed to carry an ilvD-lac operon fusion. Expression from the phage of the ilvE and lacZ genes is controlled by an intact ilv control region also carried by this phage. Two spontaneous mutants of lambda dilv-lac11 that have high-level constitutive expression of the ilv-lac fusion operon were isolated by growth on a beta-chloroalanine selective medium. The mutants were shown by nucleotide sequence determination to contain large deletions (delta 2216, approximately 1.6 kilobases; delta 2219, approximately 1.9 kilobases), which in both cases remove the proposed ilv attenuator terminator. The rest of the ilv leader and promoter region DNA remains intact in these mutants. Deletion 2216 also removed part of the downstream ilvG gene, whereas delta 2219 extended through the entire ilvG gene into the ilvGE intercistronic region. A possible mechanism of deletion formation is discussed.     

Identification of a protein of 15,000 daltons related to isoleucine-valine biosynthesis in Escherichia coli K-12.

The effect of the ilvG671, ilvG468, and ilvG603 mutations (phenotype, IlvG+ Valr; formerly ilvO) upon proteins synthesized was determined by infection of irradiated Escherichia coli K-12 cells, using specifically constructed derivatives of lambda dilv phage. These ilvG alleles are similar to the previously studied ilvG2096(Valr) allele in that they activate the latent ilvG gene which is present in the wild-type strain, leading to the synthesis of a 62,000-dalton protein. In addition, all of these ilvG (Valr) alleles increase the synthesis of a 15,000-dalton protein. To localize the gene coding for the 15,000-dalton protein, the proteins produced in maxicells containing plasmids with specific deletions of ilv and rrnX DNA segments were analyzed. The gene coding for the 15,000-dalton protein was located within a region about 1,000 base pairs long between ilv and trpT. The function of the 15,000-dalton protein is not known.     

Location of the multivalent control site for the ilvEDA operon of Escherichia coli

A strain of Escherichia coli K-12 containing a deletion extending from early in the ilvE gene toward the ilvG gene was shown to exhibit a higher expression of the downstream genes, ilvD and ilvA, than did an ilv+ strain. The elevated expression was under apparently normal ilv-specific control, however. The deletion was transferred to the ilv region of lamba h80dilv and shown by restriction endonuclease and heteroduplex analysis to extend through the deoxyribonucleic acid (DNA) shown, in the preceding paper (C. S. Subrahmanyam, G. M. McCorkle, and H. E. Umbarget, J. Bacteriol 142:547--555, 1980), to contain the ilvO determinant. The deletion was also transferred to an ilv-lac fusion strain and shown to cause an increase in beta-galactosidase formation while allowing retention of ilv-specific control. Transducing phages excised from these fusion strains with and without the ilvO determinant were compared. The phage carrying the ilvO+ determinant contained ilv DNA extending only into but not through the ilvG gene. It did not exhibit an ilv-specific control of beta-galactosidase formation. The phage carrying the deletion of ilvO but containing ilv DNA extending beyond the ilvG gene exhibited ilv-specific control of beta-galactosidase formation. It was concluded that the multivalently controlled ilv-specific promoter affecting ilv operon expression lies upstream from ilvG and that the ilvO region in the wild-type K-12 strain is a region of polarity preventing ilvG expression and reducing ilvEDA expression.     

Relative map location of the rep and rho genes of Escherichia coli.

The rep gene of Escherichia coli was mapped between ilvC and rho by three-factor P1 transductional crosses and also by complementation with a set of lambda transducing phages that contain known amounts of bacterial DNA linked to ilvC. The physical distance between ilvC and rep and between rep and rho were calculated with an accuracy of +/- 0.4 kilobase to be 0 less than or equal to ilvC-rep less than or equal to 3.4 kilobases and 2.0 less than or equal to rep-rho less than or equal to 6.0 kilobases. It was shown that rho-15 is Gro+ for phage ST-1. An ilv::Tn10 mutation was located in ilvY.     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

The isolation of lambda phage carrying DNA from the histidine and isoleucine-valine regions of the Bacillus subtilis chromosome

From a lambda gtWES library of the chromosome of Bacillus subtilis, phages carrying DNA from the hisA and ilv-leu regions were isolated. They were identified by their ability to form complementing plaques on hisB, ilvC or leuB mutants of Escherichia coli K12 under selective conditions and in the presence of a helper phage. The his phages complemented E. coli his A, B or D mutations and could transform seven mutations in the hisA region of the B. subtilis chromosome; each carried a single EcoR1 insert of about 8.2 kb. Phages complementing E. coli ilvC or leuB mutations and carrying the equivalent B. subtilis genes ilvC and leuC transformed a range of mutations in the B. subtilis ilv-leu region. The distribution of genetic markers carried by the phages suggests that the entire ilv-leu cluster from az1A through leuD is covered in the collection of phages obtained and is carried in three EcoR1 restriction fragments of approximately 6.7, 4.7 and 2.85 kb.     

Physical characterization of ilv-lac fusions.

Electron microscopic heteroduplex analysis and comparative restriction digests have been used to characterize lambda p123(209), the complementary pair of phages used in the Casadaban technique of gene fusion. Derivatives of lambda 1(209) constructed to carry fusions of the lac genes to the control regions of the ilvC and ilvEDA operons were also analyzed. These physical maps have provided confirmation of the genetic models for these constructions and physical specifications important in interpreting the behavior of these ilv-lac fusions.     

Mapping of ilvO loci of Escherichia coli K-12 with bacteriophage lambda dilv.

A set of lambda dilv phage have been used in a deletion mapping procedure to determine the location of two previously characterized ilvO alleles. In contrast to earlier conclusions derived from three-factor crosses and episome-shortening techniques with phage P1, the order found is ilvG-ilvO-ilvEDA. A three-factor cross with phage P1 is described that is not consistent with this location for an ilvO allele. Further analysis of this particular three-factor cross revealed than an artifact attributable to a mutual syntrophism had skewed the apparent frequency of inheritance of the ilvO locus. The role of mutual syntrophism is discussed as a source of mapping errors for the ilvO locus. The value of this set of lambda dilv phage and this mapping procedure for obtaining comparatively unambiguous data on the locations of the ilv structural and regulatory genes is demonstrated.     

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.     

New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.     

Identification of the Escherichia coli cell division gene sep and organization of the cell division-cell envelope genes in the sep-mur-ftsA-envA cluster as determined with specialized transducing lambda bacteriophages.

From a lysogen with lambda integrated in the leu operon, specialized transducing phages that carry the cell division, murein biosynthesis, and envelope permeability genes located about 0.5 min to the right of leu were isolated. These phages were used to identify the previously undiscovered cell division gene sep. A genetic map proves that sep is located in the sequence leuA sep murE murF murC ddl ftsA envA. A physical map of this region was prepared by heteroduplex analysis of the phage DNAs. Overlapping segments of host DNA extended rightward for as much as 26.4 kilobase pairs from the prophage insertion point (thought to be in leuA) to include all the genes through envA.     

Isolation of specialized lambda transducing bacteriophages for flagellar genes (fla) of Escherichia coli K-12.

Specialized transducing lambda phages carrying the region III flagellar genes (fla) of Escherichia coli K-12 were isolated by a new method. A strain carrying both a cryptic lambda prophage near the his genes and a deletion of the attlambda gene was used as a starting strain. The lysogen of lambdacI857pga18-bio69 was isolated in which the prophage was integrated within the lambda cryptic genes by means of recombination with the residual lambda DNA. The strains with deletions starting within the prophage and ending in these fla genes were selected from among the heat-resistant survivors of the lysogen. They were then infected with heat-inducible and lysis-defective lambda phages and, thus, specialized transducing phage lines for hag and fla were obtained. High-frequency transfer lines of rare phages carrying the fla genes were isolated by inducing a strain carrying a heat-inducible lambda prophage near the his genes and selecting by transduction of a fla deletion strain. Preliminary characterization of these transducing phages is also reported.     

Isolation of Plaque-Forming, Galactose-Transducing Strains of Phage Lambda

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.—A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.     

Genetic mapping of the ilv7434 mutation providing threonine deaminase resistance to isoleucine inhibition in Escherichia coli

Location of previously isolated ilv7434 mutation was determined by use of transductional shortening of the F'14 episome. The ilv7434 mutation causes resistance of threonine deaminase (coded for by ilvA gene) to feed-back inhibition by isoleucine. Another phenotype characteristics of the ilv7434 mutant is the ability to feed a lawn of isoleucine auxotrophs in the cross-streak test. The F'14 strain AB1206 carrying ilv7434 mutation was used as a donor for making transductionally shortened episomes in recA recipient. These shortened F'14 episomes containing variable segments of the ilv cluster were then tested for their ability to transfer ilv7434 phenotype by complementation with ilv recA recipients. The data of complementation test suggest that ilv7434 is situated between ilvD and ilvC genes. One of 20 tested shortened episomes carrying, as shown by complementation test, incomplete ilvA gene was found to transfer ilv7434 phenotype by recombination with ilvA527 recA+ recipient. These data allow to conclude that ilv7434 mutation is located within the ilvA gene.     

Transposition of lambda placMu is mediated by the A protein altered at its carboxy-terminal end

Lambda placMu phages are derivatives of bacteriophage lambda that use the transposition machinery of phage Mu to insert into chromosomal and cloned genes. When inserted in the proper fashion, these phages yield stable fusions to the Escherichia coli lac operon in a single step. We have determined the amount of DNA from the c end of phage Mu present in one of these phages, lambda placMu3, and have shown that this phage carries a 3137-bp fragment of Mu DNA. This DNA segment carries the Mu c-end attachment site and encodes the Mu genes cts62, ner+, and gene A lacking 179 bp at its 3' end (A'). The product of this truncated gene A' retains transposase activity and is sufficient for the transposition of lambda placMu. This was demonstrated by showing that lambda placMu derivatives carrying the A am1093 mutation in the A' gene are unable to transpose by themselves in a Su- strain, but their transposition can be triggered by coinfection with lambda pMu507(A+ B+). We have constructed several new lambda placMu phages that carry the A' am1093 gene and the kan gene, which confers resistance to kanamycin. Chromosomal insertions of these new phages are even more stable than those of the previously reported lambda placMu phages, which makes them useful tools for genetic analysis.