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.     

Regulation of ilvEDA expression occurs upstream of ilvG in Escherichia coli: additional evidence for an ilvGEDA operon.

A low-copy-number plasmid was prepared that contained the entire ilv gene cluster of Escherichia coli. The introduction of an ilvO mutation allowed the ilvG gene of the plasmid to be expressed and imparted valine resistance to strains carrying it. Insertion of Tn10 into the ilvG gene of the plasmid resulted in a strong polar effect on ilv genes E, D, and A. Replacement of a region of ilv deoxyribonucleic acid between two KpnI sites on the high-copy-number plasmid carrying the entire ilv gene cluster with a KpnI fragment carrying an ilv-lac fusion but not extending into the ilv-specific control region resulted in a plasmid expressing the lacZ gene under ilv control when the fusion had been inserted in its normal orientation but not when it had been inserted in the opposite orientation. These experiments indicate that ilv-specific control over ilvE, ilvD, and ilvA expression is dependent on these genes being continguous with deoxyribonucleic acid that lies upstream of ilvG. The results also add further support to the concept of an ilvGEDA operon in E. coli.     

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.     

Physical analysis of deletion mutations in the ilvGEDA operon of Escherichia coli K-12.

DNA-DNA hybridization of cloned segments of the Escherichia coli K-12 ilvGEDA operon to genomic blots was used to determine the physical dimensions of a series of deletion mutations of the ilvGEDA operon. The smallest mutation resulted from the deletion of approximately 200 base pairs from within ilvD, whereas the largest mutation resulted from the deletion of 17 kilobases including the rep gene. The structure of three of these mutants indicates that formation of the deletions was mediated by Tn5 (or Tn5-131) that is retained in the chromosome. This is the first observation of this type of Tn5-mediated event. Our analysis of the total acetohydroxy acid synthase activity of strains containing deletions of ilvG indicates that the truncated ilvG polypeptide of wild-type E. coli K-12 lacks enzyme activity. The small 200-base-pair deletion of ilvD confirms the presence of a strong polar site 5' to ilvA. The detailed structure of these deletions should prove useful for the investigation of other genes in this region. This genomic analysis demonstrates that the ilv restriction site map that was established previously by the analysis of recombinant bacteriophage and plasmids is identical to that on the genome.     

Molecular cloning and expression of the ilvGEDAY genes from Salmonella typhimurium

The ilvGEDAY genes of Salmonella typhimurium were cloned in Escherichia coli K-12 by in vitro recombination techniques. A single species of recombinant plasmid, designated pDU1, was obtained by selecting for Valr Ampr transformants of strain SK1592. pDU1 was shown to contain a 14-kilobase EcoRI partial digestion product of the S. typhimurium chromosome inserted into the EcoRI site of the pVH2124 cloning vector. The ilvGEDAY genes were found to occupy a maximum length of 7.5 kilobases. Restriction endonuclease analysis of the S. typhimurium ilv gene cluster provided another demonstration of the gene order as well as established the location of ilv Y between ilvA and ilvC. The presence of a ribosomal ribonucleic acid operon on the pDU1 insert, about 3 kilobases from the 5' end of ilvG, was shown by Southern hybridization. The expression of the ilvGEDA operon from pDU1 was found to be elevated, reflecting the increased gene dosage of the multicopy plasmid. A polarity was observed with respect to ilvEDA expression which is discussed in terms of the possible translational effects of the two internal promoter sequences, one located proximal to ilvE and the other located proximal to ilvD.     

Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-). Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead. After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region. Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting. When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage. All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim. Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction.     

Isolation and analysis of aroFo mutants by using an aroF-lac operon fusion

The lac structural genes were fused to the regulatory region of the aroF-tyrA operon so that the expression of beta-galactosidase was regulated by the tyrR+ gene product. Transducing phage carrying the aroF-lac fusion were isolated, and a lambda aroF-lac lysogen was used to select for aroFo mutants. A plasmid vector was constructed onto which the aroFo mutations were transferred by recombination in vivo.     

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.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

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.     

Functioning of amino acid operons in Escherichia coli strains with an altered transcription and translation apparatus. II. The effect of mutations in genes coding ribosomal protein S5 and translation elongation factor G on the functioning of the ilv operon

It has been shown in our previous study that mutations in genes relA, relC and rpsL result in the delay in Escherichia coli ilv operon derepression; the complete inhibition of derepression of the ilv operon is observed in the double mutants having alterations in rpsL and relA or relC genes. At present, some mutations occurring in the fus gene and altering the structure of the translational elongation G factor have been also found to delay derepression of E. coli ilv operon and complete inhibition in fusr and rel double mutants. Phenotypical ile and val auxotrophy is also detected in the double E. coli mutants with spectinomycin resistance mutation in rpsE gene coding for the structure of ribosomal S5 protein and mutations in relA or relC genes. The suggestion of participation of the ilv operon in regulation of other E. coli amino acid operons expression is discussed.     

Cloning and characterization of the cysAMK region of Salmonella typhimurium.

A total of 30 kilobases of DNA comprising the cysAMK region of S. typhimurium was cloned as a series of fragments in phage lambda 1059. The genetic organization of this region was established through studies of gene expression from fragments subcloned in pBR322 and from blot hydridization analyses of restriction sites in chromosomal DNA from multisite deletion strains. The results give a gene order of cysA-cysM-crr-ptsl-ptsH-cysK over a distance of approximately 12 kilobases. cysM and cysA have been cloned and expressed in pBR322; attempts to obtain stable pBR322 derivatives carrying cysK were unsuccessful.     

Synthesis and activities of branched-chain aminoacyl-tRNA synthetases in threonine deaminase mutants of Escherichia coli.

Valyl-, isoleucyl-, and leucyl-tRNA synthetase activities were examined in an Escherichia coli K-12 strain that possessed a deletion of three genes of the ilv gene cluster, ilvD, A, and C, and in a strain with the same deletion that also carried the lambdadilvCB bacteriophage. It was observed that the branched-chain tRNA synthetase activities of both strains were considerably less than those of the normal strain during growth in unrestricted medium. Furthermore, during an isoleucine limitation, there was a further reduction in isoleucyl-tRNA synthetase activity and an absence of the isoleucine-mediated derepression of valyl-tRNA synthetase formation in both of these mutants, as compared with the normal strain. In addition, it was observed that these branched-chain synthetase activities were reduced in steady-state cultures of several ilvA point mutants. However, upon the introduction of the ilv operon to these ilvA mutants by use of lambda bacteriophage, there was a specific increase in the branched-chain synthetase activities to levels comparable to those of the normal strain. These results support our previous findings that the stability and repression control of synthesis of these synthetases require some product(s) missing in the ilvDAC deletion strain and strongly suggest this component is some form of the ilvA gene product, threonine deaminase.