Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.     

Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation. IV. Recombination characteristics of Gamr mutants

In the radiation-resistant Gamr444 mutant the inheritance frequency of long F' episomes ORF1 (purE+ tsx+ procC+ lac+) and F'14 (ilv+--argE+) is lower, and the frequencies of chromosome mobilization and integrative suppression of temperature-sensitive dnaA46 mutation by the sex factor F are much higher than those in the wild-type strain AB1157 and another radiation-resistant mutant Gamr445. In this respect, the mutant Gamr444 is very similar to the recRC sbcB mutant (RecF-pathway of recombination).     

Mutations in the purine nucleoside phosphorylase (pup) gene of Escherichia coli K-12 characterized by leaky damage to enzymatic activity and a pleiotropic effect]

The phenotype of earlier obtained mutants AIR38 and AIR6 is caused by leaky mutations of the structural gene for purine nucleoside phosphorylase (pup). These mutants are unable to grow on the medium with inosine as the only carbon source in the presence of thymidine. In contrast to ordinary leaky mutations, AIR38 and AIR6 are dominant in heterozygotes. When the strain F' with mutations AIR38 or AIR6 on episomes was used for conjugational matings with F- recA (pup+), recombinants with unexpected phenotype were observed: about 20% of recombinants F' became pup+ and thus the convertion of AIR38 and AIR6 alleles to pup+ took place. AIR38 mutation, unlike AIR6, is mapped in the proximal region of the pup gene and is characterized by pleyotropic effect on deo-genes: the inosine-induction of purine nucleoside phosphorylase in AIR38 mutant is absent and the induction of thymidine phosphorylase by exogenous thymidine is decreased. A speculation was made that the mutation AIR38 altered the structure of purine nucleoside phosphorylase in the site responsible for its interaction with membrane.     

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.     

Recombinant F′ Factors from Escherichia coli K-12 Strains Carrying recB or recC

The frequency of genetic exchanges between F' factors and the bacterial chromosome was studied in recombination-deficient Escherichia coli mutants under conditions in which the recombinant F' factors were immediately transferred to new hosts. In a series of double matings, F101-1 thr(+)leu(-) episomes were first transferred into each of four intermediate F(-)thr(-)leu(+) strains carrying various rec alleles. After the original F' donors were killed with phage T6, the F101-1 episomes were then transferred from the intermediate cells to F(-)thr(-)leu(-)Str(R)recA(-) females. Recipients of nonrecombinant episomes formed Thr(+) (Str(R)) colonies, and recipients of recombinant episomes formed Leu(+)(Str(R)) colonies. A comparison of the numbers of Leu(+)(Str(R)) and Thr(+)(Str(R)) colonies shows that recB(-) males formed 18 to 21% and recC(-) formed 47 to 60% of the wild-type level of recombinant episomes that could be detected after transfer. No recombinant episomes were detected using a recA(-) intermediate strain. If the intermediate strains harboring the F101 episomes were purified, allowed to grow for 50 generations, and then mated with the recA(-) recipient, recombinant episomes were transferred at 8% of the wild-type level for recB(-) and 13% for recC(-). In contrast, only 0.4 and 0.6% of the normal number of recombinants were obtained from crosses between Hfr Cavalli donors and the same recB(-) and recC(-) strains. Recombinant episomes were detected with greater frequency among newly formed rec(+), recB(-), and recC(-) partial diploids than in those which were 50 generations old.     

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.     

Positive control of ilvC expression in Escherichia coli K-12; identification and mapping of regulatory gene ilvY.

The construction of a plasmid carrying the ilvC::lacZ fusion is described. This plasmid provides a convenient source of template deoxyribonucleic acid for use in an in vitro protein-synthesizing system. We screened strains deleted in regions of the ilv cluster for their ability to support ilvC-dependent beta-galactosidase synthesis. The fact that two deletions prevented beta-galactosidase production indicated that ilv-C expression is under positive control. By use of plasmids carrying the positive-control factor structural gene ilvY, we were able to restore protein-synthesizing ability to these strains. These plasmids also enabled us to map ilvY between ilvA and ilvC.     

Gene relA function in the expression of amino acid operons. I. Effect of the allelic state of gene relA on phenotypic manifestations of auxotrophic threonine and isoleucine mutations in Escherichia coli K-12]

The substitution of the relA gene mutant allele with wild type allele of this gene in strictly auxotrophic strain of Escherichia coli K-12 GT25, carrying thr B1007 mitation, results in the appearance of the partial dependence of the bacterial growth upon threonine. On the other hand, the introduction of relA mutation into genome of incomplete threonine auxotroph, which was isolated as pseudorevertant from the strict threonine auxotroph CP78, recovered the strict dependency of the growth on the presence of threonine in the medium. The introduction of relA mutation into genome of partial isoleucine auxotroph, carrying a mutation in ilvA gene, reduces the residual activity of threonine deaminase under the conditions of derepression and results in the appearance of strict dependency of bacterial growth on the presence of isoleucine. These data indicate that operons, which control the biosynthesis of threonine and isoleucine, are positively regulated by the product of relA gene. The possibility of using leaky mutations, which lead to incomplete block of these amino acids synthesis, for testing allelic state of relA gene is discussed.     

Autogenous regulation of the synthesis of glutamine synthetase in Klebsiella aerogenes.

We isolated an F' episome of Escherichia coli carrying the glnA+ gene from K. aerogenes and an F' episome of E. coli carrying the glnA4 allele from K. aerogenes responsible for the constitutive synthesis of glutamine synthetase. Complementation tests with these episomes showed that the glnA4 mutation (leading to the constitutive synthesis of active glutamine synthetase) was in the gene identified by mutations glnA20, glnA51, and glnA5 as the structural gene for glutamine synthetase. By using these merodiploid strains we were able to show that the glnA51 mutation lead to the synthesis of a glutamine synthetase that lacked enzymatic activity but fully retained its regulatory properties. Finally, we discuss a model that explains the several phenotypes associated with mutations such as glnA4 located within the structural gene for glutamine synthetase leading to constitutive synthesis of active glutamine synthetase.     

Ordering of Mutant Sites in the Isoleucine-Valine Genes of Escherichia coli by Use of Merogenotes Derived from F14: a New Procedure for Fine-Structure Mapping

F-merogenotes derived from F(14) by transductional shortening have previously been found to consist of the sex factor plus one or more of the ilv genes. It is shown here that they carry one or more ilv genes and a variable portion of the adjacent proximal ilv gene. This observation was used to develop a method, analogous to deletion mapping, for ordering mutant sites within the ilv genes. This method requires the use of a series of merogenotes each carrying an increasingly longer segment of the gene being mapped. A simplified method of fine-structure mapping is also described which requires only one or two F' donor strains to map any one gene. This method is based on the large differences observed in recombination frequencies for mutant sites at various distances from the origin of the incomplete merogenote gene. The sequence of 25 mutant sites within three of the ilv genes was determined by use of the simplified procedure.     

cis-Dominant, transfer-deficient mutants of the Escherichia coli K-12 F sex factor.

Rare conjugational progeny formed by crossing each of five Hfr strains with a recA-F- strain have been characterized. Selection was made for a proximal Hfr marker, taking strict precautions to prevent transfer of recA+ to the zygotes. Most of the progeny were found to be F' strains containing deletion mutant plasmids. With two exceptions, these mutant plasmids have lost all of the tra genes, which are required to confer conjugational donor ability upon a host. In addition, all but the exceptional mutant plasmids were found to be very poorly transmissible from transient heterozygotes which also contain a wild-type F' plasmid. The poor transmissibility is a cis-dominant transfer-defective phenotype which may result from deletion of all or part of the origin of transfer replication (ori), or of a gene determining a cis-acting protein. The two exceptional mutant plasmids may carry short deletions of some of the tra genes or polar tra mutations. The remaining progeny were nonmutant F' strains and F- strains. The frequency with which the F- strains were recovered permits us to estimate that the maximum amount of recombination possible in a recA56 zygote is 10(-6) that of a recA+ zygote.     

Suppressors of a genetic regulatory mutation affecting isoleucine-valine biosynthesis in Escherichia coli K-12.

Escherichia coli K-12 mutant PS187 carries a mutation, ilvA538, in the structural gene for the biosynthetic L-threonine deaminase that leads to a leucine-sensitive growth phenotype, an isoleucine- and leucine-hypersensitive L-threonine deaminase, and pleiotropic effects resulting in abnormally low and invariant expression of some of the isoleucine-valine biosynthetic enzymes. Fifty-eight derivatives of strain PS187 were isolated as resistant to growth inhibition by leucine, by valine, or by valine plus glycly-valine and were biochemically, genetically, and physiologically characterized. All of these derivatives produced the feedback-hypersensitive L-threonine deaminase, and thus presumably possess the ilvA538 allele of the parent strain. Elevated synthesis of L-threonine deaminase was observed in 41 of the 58 isolates. Among 18 strains analyzed genetically, only those with mutations linked to the ilv gene clusters at 83 min produced elevated levels of L-threonine deaminase. One of the strains, MSR91, isolated as resistant to valine plus glycyl-valine, was chosen for more detailed study. The locus in strain MSR91 conferring resistance was located in four factor crosses between ilvE and rbs, and is in or near the ilvO gene postulated to be a site controlling the expression of the ilvEDA genes. Synthesis of the ilvEDA gene products in strain MSR91 is constitutive and derepressed approximately 200-fold relative to the parent strain, indicating that the genetic regulatory effects of the ilvA538 allele have been suppressed. Strain MSR91 should be suitable for use in purification of the ilvA538 gene product, since enzyme synthesis is fully derepressed and the suppressor mutation is clearly not located within the ilvA gene.     

Role for Free Isoleucine or Glycyl-Leucine in the Repression of Threonine Deaminase in Escherichia coli

In Escherichia coli, the three branched-chain amino acid activating enzymes appear to be essential for multivalent repression of the isoleucine- and valine-forming enzymes. The results of experiments with a mutant, strain CU18, having an altered threonine deaminase, indicate that free isoleucine and some form of threonine deaminase (the product of the ilvA gene) are also involved in multivalent repression. This strain exhibits abnormally high derepressibility but normal repressibility of its ilv gene products, and its threonine deaminase is inhibited only by high concentrations of isoleucine. In strain CU18, the isoleucine analogue, thiaisoleucine, is incapable of replacing isoleucine in the multivalent repression of the ilv genes, whereas the analogue can fully replace the natural amino acid in repression in other strains examined. The dipeptide, glycyl-leucine, which, like isoleucine, is a heterotropic negative effector of threonine deaminase but is not a substrate for isoleucyl-transfer ribonucleic acid synthetase, can completely prevent the accumulation of threonine deaminase-forming potential during isoleucine starvation in strains with normal threonine deaminases. It can not, however, prevent such accumulation in strain CU18 or in other strains in which threonine deaminase is insensitive to any concentration of isoleucine.     

Isolation and characterization of ilvA, ilvBN, and ilvD mutants of Caulobacter crescentus.

Caulobacter crescentus strains requiring isoleucine and valine (ilv) for growth were shown by transduction and pulsed-field gel electrophoresis to contain mutations at one of two unlinked loci, ilvB and ilvD. Other C. crescentus strains containing mutations at a third locus, ilvA, required either isoleucine or methionine for growth. Biochemical assays for threonine deaminase, acetohydroxyacid synthase, and dihydroxyacid dehydratase demonstrated that the ilvA locus encodes threonine deaminase, the ilvB locus encodes acetohydroxyacid synthase, and the ilvD locus encodes dihydroxyacid dehydratase. C. crescentus strains resistant to the herbicide sulfometuron methyl, which is known to inhibit the action of certain acetohydroxyacid synthases in a variety of bacteria and plants, were shown to contain mutations at the ilvB locus, further suggesting that an acetohydroxyacid synthase gene resides at this locus. Two recombinant plasmids isolated in our laboratory, pPLG389 and pJCT200, were capable of complementing strains containing the ilvB and ilvD mutations, respectively. The DNA in these plasmids hybridized to the corresponding genes of Escherichia coli and Serratia marcescens, confirming the presence of ilvB-like and ilvD-like DNA sequences at the ilvB and ilvD loci, respectively. However, no hybridization was observed between any of the other enteric ilv genes and C. crescentus DNA. These results suggest that C. crescentus contains an isoleucine-valine biosynthetic pathway which is similar to the corresponding pathway in enteric bacteria but that only the ilvB and ilvD genes contain sequences which are highly conserved at the DNA level.     

F'-plasmid transfer from Escherichia coli to Pseudomonas fluorescens.

Various F' plasmids of Escherichia coli K-12 could be transferred into mutants of the soil strain 6.2, classified herein as a Pseudomonas fluorescens biotype IV. This strain was previously found to receive Flac plasmid (N. Datta and R.W. Hedges, J. Gen Microbiol. 70:453-460, 1972). ilv, leu, met, arg, and his auxotrophs were complemented by plasmids carrying isofunctional genes; trp mutants were not complemented or were very poorly complemented. The frequency of transfer was 10(-5). Subsequent transfer into other P. fluorescens recipients was of the same order of magnitude. Some transconjugants were unable to act as donors, and these did not lose the received information if subcultured on nonselective media. Use of F' plasmids helped to discriminate metabolic blocks in P. fluorescens. In particular, metA, metB, and argH mutants were so distinguished. In addition, F131 plasmid carrying the his operon and a supD mutation could partially relieve the auxotrophy of thr, ilv, and metA13 mutants, suggesting functional expression of E. coli tRNA in P. fluorescens. In P. fluorescens metA Rifr mutants carrying the F110 plasmid, which carried the E. coli metA gene and the E. coli rifs allele, sensitivity to rifampin was found to be dominant at least temporarily over resistance. This suggests interaction of E. coli and P. fluorescens subunits of RNA polymerase. his mutations were also complemented by composite P plasmids containing the his-nif region of Klebsiella pneumoniae (plasmids FN68 and RP41). nif expression could be detected by acetylene reduction in some his+ transconjugants. The frequency of transfer of these P plasmids was 5 X 10(-4).     

Molecular Studies on Entry Exclusion in Escherichia coli Minicells

Minicells produced by abnormal cell division in a strain of Escherichia coli (K-12) have been employed here to investigate the phenomenon of "entry exclusion." When purified minicells from strains containing F' or R factors, or both, are mated with radioactive thymidine-labeled Hfr or R(+) donors, the recipient minicells can be conveniently separated from normal-sized donors following mating, and the products of conjugation can be analyzed in the absence of donors and of further growth of the recipients. Transmissible plasmids or episomes are transferred less efficiently to purified minicells derived from strains carrying similar or related elements than to strains without them. Measurement of deoxyribonucleic acid (DNA) degradation and determination of weight-average molecular weights following transfer indicate that degradation of transferred DNA or transfer of smaller pieces cannot account for the comparative reduction in transfer to entry-excluding recipients. Therefore, we conclude that entry exclusion operates to prevent the physical entry of DNA into recipients expressing the exclusion phenotype. The R-produced repressor (product of the drd(+) gene), which represses fertility (i.e., ability to act as donor), reduces exclusion mediated by R or F factor, or both, in matings between strains carrying homologous elements. Furthermore, the data suggest that the presence of the F pilus or F-like R pilus on recipient cells ensures maximum expression of the exclusion phenotype but is not essential for its expression. In contrast to previous suggestions, we found no evidence for a reduction of entry exclusion attributable to the DNA temperature-sensitive chromosomal mutation dnaB(TS).     

Effect of a leu-linked mutation on the valine sensitivity of acetohydroxy acid synthase activity in Escherichia coli.

A spontaneous leu-linked mutation (ilvH2015) in Escherichia coli K-12 made the strain resistant to 1 mM valine and l mM glycylvaline (Val-r) and caused the isoleucine and valine biosynthetic enzyme, acetohydroxy acid synthase, to be less sensitive to feedback inhibition by valine than the wild-type enzyme. Transfer of the ilvDAC deletion into a strain carrying ilvH2015 abolished the effect of the marker on the acetohydroxy acid synthase and rendered it as sensitive to valine as the enzyme in the isogenic control strain without the Val-r marker under both repressing and limiting conditions. In contrast, auxotrophy caused by transfer of an ilvC lesion into the Val-r strain did not interfere with the effect of ilvH2015 on valine sensitivity of acetohydroxy acid synthase. In addition, the presence of the Val-r marker produced minor but significant pleiotropic effects on several other isoleucine and valine biosynthetic enzymes but did not cause derepression of the ilv gene cluster. These studies suggest some type of interaction between a product produced by a gene close to leu and the isoleucine and valine biosynthetic enzymes.     

Procedure for Identifying Nonsense Mutations

A method has been devised for the rapid identification of nonsense mutations (UAG, UAA, UGA codons) in Salmonella. The mutations to be tested are reverted, and the revertants are replica-printed onto lactose plates spread with lawns of tester strains. These tester strains contain F' lac episomes with nonsense mutations in the episomal Z gene. The revertants are infected with the episome from the tester strain lawn. Because S. typhimurium is unable to ferment lactose, only those revertants which have nonsense suppressors are able to grow on lactose. If colonies appear on the lactose plate, it may be concluded that the original strain carries a nonsense mutation, since nonsense suppressors suppress the mutant phenotype.