hns, rpoS and lrp mutations affect stationary-phase survival at high osmolarity.

The survival of enteric bacteria during stationary phase requires the expressions of different genes than those required for growth during log phase. Genes coding for functions protecting cells from environmental stress are expressed during the onset of stationary phase. Many promoters of these genes require sigma s, the product of the rpoS gene, for transcription. During stationary phase, we found that strains lacking the neutral, histone-like, DNA-binding protein H-NS (hns- rpoS- double mutants) are more viable at high osmolarity at 37 degrees C than hns+ rpoS- cells. We did not observe differential viability at high osmolarity at 30 degrees C or at normal osmolarity at 37 degrees C. We showed that this effect is due to the absence of H-NS. The leucine-responsive regulatory protein acts without H-NS to protect a strain containing an rpoS mutation from death in stationary phase at high osmolarity. We present evidence that hns- rpoS double mutants can synthesize the log phase osmotic shock protective system.

The H-NS protein modulates the activation of the ilvIH operon of Escherichia coli K12 by Lrp, the leucine regulatory protein.

The H-NS protein, the product of the hns gene, plays a central role in the cellular response of bacteria to environmental stresses such as modification of osmolarity and temperature. The leucine regulatory protein (Lrp) controls a wide array of operons both as an activator (e.g. ilvIH) and as a repressor. We demonstrate that H-NS can decrease the activity of Lrp in stationary phase and under conditions of high osmolarity. Strains containing hns mutations have higher levels of Lrp-activated ilvIH transcription, while strains carrying the hns+ allele on a pBR322 plasmid have lower activity of Lrp-directed ilvIH gene expression.

The role of H-NS in one carbon metabolism.

The H-NS protein of Escherichia coli regulates the expression of genes involved in many general processes such as osmoregulation and virulence. More recently, H-NS was shown to exert an effect on ilvIH gene expression in conjunction with the leucine responsive regulatory protein (Lrp). We show that H-NS is involved in the transcriptional regulation of the kbl/tdh operon, which is also Lrp regulated. Insertional inactivation of the hns gene results in two-fold derepression of the kbl/tdh operon. This level of expression is sufficient to suppress the auxotrophic requirements imposed by a glyA mutation. We show that expression of the kbl/tdh operon is temperature controlled and that this control is not mediated through H-NS action as has been shown for some other temperature controlled genes.

Activation of a cryptic pathway for threonine metabolism via specific IS3-mediated alteration of promoter structure in Escherichia coli.

The tdh operon of Escherichia coli consists of two genes whose products catalyze sequential steps in the formation of glycine and acetyl coenzyme A from threonine. The operation of the tdh pathway can potentially confer at least two capabilities on the cell: the first is to provide a biosynthetic source of glycine, serine, or both that is an alternative to the conventional (triose phosphate) pathway; the second is to enable cells to utilize threonine as the sole carbon source. The latter capability is referred to as the Tuc+ phenotype. In wild-type E. coli, the tdh operon is expressed at levels that are too low to bestow the Tuc+ phenotype, even in leucine-supplemented media, where the operon is induced eightfold. In eight Tuc+ mutants, the expression of the tdh operon was elevated 100-fold relative to the uninduced wild-type operon. The physical state of the DNA at the tdh locus in these Tuc+ strains was analyzed by Southern blotting and by DNA sequencing. In eight independent isolates the mobile genetic element IS3 was found to lie within the tdh promoter region in identical orientations. In six cases that were examined by DNA sequencing, IS3 occupied identical sites between the -10 and -35 elements of the tdh promoter. The transcription start points for the wild-type tdh promoter and one IS3-activated tdh promoter were identical. In effect, the repeatedly observed transposition event juxtaposed an IS3-borne -35 region and the tdh-specific -10 region, generating a hybrid promoter whose utilization led to elevated, constitutive expression of the tdh operon. This is the first case of promoter activation by IS3 where the site of transcription initiation is unaltered.

Synthesis of the isoleucyl- and valyl-tRNA synthetases and the isoleucine-valine biosynthetic enzymes in a threonine deaminase regulatory mutant of Escherichia coli K-12.

A mutation in the structural gene for threonine deaminase, ilvA538 , results in lower than normal levels of the isoleucyl, valyl- and leucyl-tRNA synthetases. Moreover, this regulatory mutation decreases the level of expression of the ilv biosynthetic operons and renders their expression non-responsive to limitations of the branched-chain amino acids. In this paper, we present in vitro evidence for the inhibition of isoleucyl- and valyl-tRNA synthetase activity by threonine deaminase and 2-ketobutyrate, the product of the threonine deaminase reaction, through the formation of a high molecular weight complex of the three molecules. Based on these results, we propose a model to explain the regulation of the isoleucyl- and valyt -tRNA synthetases in which transient inhibition of the synthetase enzyme activities by threonine deaminase and 2-ketobutyrate increases the expression of ileS and valS , the structural genes for isoleucyl- and valyt -tRNA synthetase, respectively. Further, the results suggest that the hyperattenuated expression of the ilv biosynthetic operons is due to an increased rate of complex formation of valyl and isoleucyl-tRNA synthetases and the altered form of threonine deaminase of the ilvA538 mutant strain.

Reversion of the effects of a threonine deaminase regulatory mutant by a mutation in ilvH in Escherichia coli K-12.

In a strain carrying an ilvA538 mutation, the ilvGEDA operon expression is decreased (hyperattenuated) and the activity and/or expression of isoleucyl- and valyl- tRNA synthetases is decreased. We have isolated two revertants of ilvA538 owing to mutations in the ilvH gene, whose product is acetohydroxy acid synthase III. The regulatory properties of these revertants are consistent with a dual role for threonine deaminase as an effector of the ilvGEDA operon and the isoleucyl- and valyl- tRNA synthetase structural genes.

A role for threonine deaminase in the regulation of alpha-acetolactate biosynthesis in Escherichia coli K12.

The flow of carbon to alpha-acetolactate is Escherichia coli K12 is shown to involve the endogenous pool of alpha-ketobutyrate (alpha-KB). In vivo, the acetohydroxy acid synthase (AHAS) isoenzymes have an affinity for alpha-KB sufficiently high that alpha-acetolactate production is severely limited when alpha K-B is supplied exogenously. The ability of threonine deaminase to make alpha-KB is correlated with the synthesis of the AHAS isoenzymes. Mutations in ilvA that alter the catalytic and allosteric properties of threonine deaminase affect alpha-KB production and the expression of the AHAS isoenzymes in a direct way. The ilv A538 mutation results in a feedback-hypersensitive threonine deaminase ans slow alpha-KB and AHAS production. A spontaneous revertant of an ilvA538 strain expressing a feedback-resistant threonine deaminase produces alpha-KB and AHAS more quickly. A physiological role for the activator (valine) site on threonine deaminase is proposed and valine is shown to increase alpha-KB production in vivo. Valine can thus regulate its own biosynthetic pathway without jeopardizing the production of isoleucine. The physiological implications of the role of alpha-KB in the biosynthesis of acetolactate are discussed.

A new map location for the ilvB locus of Escherichia coli.

We isolated, in E. coli K12, new alleles of the ilvB locus, the structural gene for acetolactate synthase isoenzyme I, and showed them to map at or near the ilvB619 site. The map position of the ilvB locus was redetermined because plasmids containing the ilvC-cya portion of the chromosome did not complement mutations at the ilvB locus. Furthermore, diploids for the ilvEDAC genes formed with these plasmids in an ilvHI background facilitated the isolation of the new ilvB alleles. The ilvB locus was remapped and found to be located at 81.5 minutes, between the uhp and dnaA loci. This location was determined by two- and three-point transductional crosses, deletion mapping and complementation with newly isolated plasmids. One of the new alleles of the ilvB gene is a mu-1 insertion. When present in the donor strain, this allele interferes with the linkage of genes flanking the mu-1 insertion, as well as the linkage of genes to either side of the mu-1 insertion.

Dual autogenous regulatory role of threonine deaminase in Escherichia coli K-12.

We describe the regulatory properties of two strains carrying either the ilvA624 or the ilvA625 mutations, located in the structural gene for threonine deaminase. Crude extracts of both these strains possess a threonine deaminase activity migrating on polyacrylamide gels, differently from the wild type enzyme. Growth studies demonstrate that these mutations do not cause a limitation of isoleucine biosynthesis, suggesting normal catalytic activity of deaminase. A regulatory consequence of the ilvA624 allele is a derepression of the isoleucine-valine biosynthetic enzymes, which is recessive to an ilvA+ allele. The ilvA625 mutation causes a derepression which is dominant in an ilvA625/ILVA+ diploid. We interpret these data assuming that threonine deaminase, previously shown to be an autogenous regulator of the ilv genes, lacks a repressor function in the ilvA624 mutant, while in the ilvA625 mutant it is a better activator than wild type threonine deaminase. The data are discussed in terms of a model requiring that threonine deaminase, or a precursor of it, is in equilibrium between two forms, one being an activator of gene expression and the other being a repressor.

Growth inhibition of Escherichia coli K-12 by L-valine: a consequence of a regulatory pattern.

We studied the production of the ilvG gene product, the valine resistant acetolactate synthase isoenzyme II, in an ilvO+ G+ ilvB ilvHI derivative of Escherichia coli K-12. This strain contains mutations in the structural genes for the valine sensitive acetolactate synthase isoenzymes I and III. We find that the ilvG gene is not expressed in this strain when gworn with either isoleucine and valine or with isoleucine, leucine and valine, or when limited for either isoleucine or valine. Since we previously found that the ilvG gene is expressed in an ilvO603 containing strain (Favre et al., 1976), we presume that the mechanism by which E. coli K-12 regulates the ilv gene cluster is responsible for the lack of ilvG expression in the ilvO+ strain. The valine sensitivity of E. Coli K-12 is a consequence of this regulatory pattern.

trans-Recessive mutation in the first structural gene of the histidine operon that results in constitutive expression of the operon.

The first enzyme for histidine biosynthesis, encoded in the hisG gene, is involved in regulation of expression of the histidine operon in Salmonella typhimurium. The studies reported here concern the question of how expression of the histidine operon is affected by a mutation in the hisG gene that alters the allosteric site of the first enzyme for histidine biosynthesis, rendering the enzyme completely resistant to inhibition by histidine. The intracellular concentrations of the enzymes encoded in the histidine operon in a strain carrying such a mutation on an episome and missing the chromosomal hisG gene are three- to fourfold higher than in a strain carrying a wild-type hisG gene on the episome. The histidine operon on such a strain fails to derepress in response to histidine limitation and fails to repress in response to excess histidine. Furthermore, utilizing other merodiploid strains, we demonstrate that the wild-type hisG gene is trans dominant to the mutant allele with respect to this regulatory phenomenon. Examination of the regulation of the histidine operon in strains carrying the feedback-resistant mutation in an episome and hisT and hisW mutations in the chromosome showed that the hisG regulatory mutation is epistatic to the hisT and hisW mutations. These data provide additional evidence that the first enzyme for histidine biosynthesis is involved in autogenous regulation of expression of the histidine operon.

Specific binding of the first enzyme for histidine biosynthesis to the DNA of histidine operon.

Studies were done to examine direct binding of the first enzyme of the histidine biosynthetic pathway (phosphoribosyltransferase) to 32P-labeled phi80dhis DNA and competition of this binding by unlabeled homologous DNA and by various preparations of unlabeled heterologous DNA, including that from a defective phi80 bacteriophage carrying the histidine operon with a deletion of part of its operator region. Our findings show that phosphoribosyltransferase binds specifically to site in or near the regulatory region of the histidine operon. The stability of the complex formed by interaction of the enzyme with the DNA was markedly decreased by the substrates of the enzyme and was slightly increased by the allosteric inhibitor, histidine. These findings are consistent with previous data that indicate that phosphoribosyltransferase plays a role in regulating expression of the histidine operon.

Complementation between different mutations in the ilvA gene of Escherichia coli K-12.

An ilvA mutation carried by a ø80i(lambda)dilv transducing phage complemented some ilvA mutations and did not complement others. Complementation was accompanied by appearance of threonine deaminase activity in vivo. These results divided the ilvA mutations into two sets which formerly appeared to define two cistrons.

The pi-histidine factor of Salmonella typhimurium: a demonstration that pi-histidine factor integrates into the chromosome.

The Salmonella typhimurium pi-histidine episome was identified by Ames et al. (2) in an unstable partial revertant of a deletion mutation-containing strain, hisG203. HisG203 lacks the histidine operator, promoter, and part of the first structural gene. In this paper, we study some properties of pi factor and demonstrate a low frequency of pi integration into the chromosome at or near the histidine region.