Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon.

Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically. Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes. Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC. The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway. The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.     

Multiplicity of leucine transport systems in Escherichia coli K-12

The major component of leucine uptake in Escherichia coli K-12 is a common system for l-leucine, l-isoleucine, and l-valine (LIV-I) with a Michaelis constant (K(m)) value of 0.2 muM (LIV-I system). The LIV-binding protein appears to be associated with this system. It now appears that the LIV-I transport system and LIV-binding protein also serve for the entry of l-alanine, l-threonine, and possibly l-serine. A minor component of l-leucine entry occurs by a leucine-specific system (L-system) for which a specific leucine-binding protein has been isolated. A mutant has been obtained that shows increased levels of the LIV-I transport activity and increased levels of both of the binding proteins. Another mutant has been isolated that shows only a major increase in the levels of the leucine-specific transport system and the leucine-specific binding protein. A third binding protein that binds all three branched-chain amino acids but binds isoleucine preferentially has been identified. The relationship of the binding proteins to each other and to transport activity is discussed. A second general transport system (LIV-II system) with a K(m) value of 2 muM and a relatively low V(max) can be observed in E. coli. The LIV-II system is not sensitive to osmotic shock treatment nor to growth of cells in the presence of leucine. This high K(m) system, which is specific for the branched-chain amino acids, can be observed in membrane vesicle preparations.     

Genome-wide expression profiling in Escherichia coli K-12.

We have established high resolution methods for global monitoring of gene expression in Escherichia coli. Hybridization of radiolabeled cDNA to spot blots on nylon membranes was compared to hybridization of fluorescently-labeled cDNA to glass microarrays for efficiency and reproducibility. A complete set of PCR primers was created for all 4290 annotated open reading frames (ORFs) from the complete genome sequence of E.coli K-12 (MG1655). Glass- and nylon-based arrays of PCR products were prepared and used to assess global changes in gene expression. Full-length coding sequences for array printing were generated by two-step PCR amplification. In this study we measured changes in RNA levels after exposure to heat shock and following treatment with isopropyl-beta-D-thiogalactopyranoside (IPTG). Both radioactive and fluorescence-based methods showed comparable results. Treatment with IPTG resulted in high level induction of the lacZYA and melAB operons. Following heat shock treatment 119 genes were shown to have significantly altered expression levels, including 35 previously uncharacterized ORFs and most genes of the heat shock stimulon. Analysis of spot intensities from hybridization to replicate arrays identified sets of genes with signals consistently above background suggesting that at least 25% of genes were expressed at detectable levels during growth in rich media.     

Molecular basis for modulated regulation of gene expression in the arginine regulon of Escherichia coli K-12.

We compare the nucleotide sequences of the regulatory regions of five genes or groups of genes of the arginine regulon of Escherichia coli K-12: argF, argI, argR, the bipolar argECBH operon and the carAB operon. All these regions harbour one or two copies of a conserved 18 bp sequence which appears to constitute the basic arginine operator sequence (ARG box). We discuss the influence of ARG box copy number, degree of dyad symmetry, base composition, and position relative to the cognate promoter site on the derepression-repression ratios of the genes of the regulon. A novel hypothesis, based on structural considerations, is also put forward to account for the absence ot attenuation control.     

Isolation of an Escherichia coli K-12 dnaE mutation as a mutator.

Using a papillation method, a large number of Escherichia coli K-12 mutator mutations have been isolated. Only one of these (out of 1,250) mutator mutations has proved to be conditionally lethal at high temperatures. In vivo complementation tests indicated that this mutation, dnaE9, lies in dnaE, the structural gene for DNA polymerase III. The dnaE9 polymerase was not thermolabile in vitro; however, it showed a slow decline in specific activity in vivo at the nonpermissive temperature. Cultures of this mutant exhibited a comparably slow shutoff of DNA synthesis on shift to a nonpermissive temperature. dnaE9 showed temperature-sensitive mutator activity, which is not dependent on recA.     

Map location of the ssd mutation in Escherichia coli K-12.

A pleiotropic mutation at the ssd locus was mapped at 86 min near rha. A mutation at the ssd locus resulted in elevated L-serine deaminase activity, inability to grow with succinate as the carbon source, and inability to grow anaerobic conditions.     

Chromosomal mutation for citrate utilization by Escherichia coli K-12.

A mutant strain of Escherichia coli K-12 that utilizes citrate as a sole source of carbon and energy was isolated. Citrate utilization arose as the consequence of two mutations in genes citA and citB, which are linked to the gal operon. The mutant strain expresses a semiconstitutive citrate transport system, and it utilizes both citrate and isocitrate as carbon and energy sources. It is capable of utilizing cis- and trans-aconitate, but only if it is preinduced by growth on citrate.     

Purine-mediated growth inhibition caused by a pyrE mutation in Escherichia coli K-12.

A purine-sensitive phenotype results from a previously described mutation in the structural gene (pyrE) for orotate phosphoribosyltransferase (OPT) in Escherichia coli K-12. OPT from both the mutant and the wild-type was partially inhibited by adenine and adenosine, although other purine derivatives were not effective for this inhibition. The Km values of the mutant OPT were 580 and 760 microM for orotate and 5'-phosphoribosyl-1'-pyrophosphate (PRib-PP), respectively, whereas the corresponding values for the wild-type OPT were 40 and 60 microM. The intracellular level of PRib-PP was decreased to less than 15% of the normal level when purine derivatives were added to exponentially growing cultures of both the parent and mutant strains. However, this decrease of the PRib-PP level was not found in strains derived from the mutant, in which the purine-sensitive phenotype was suppressed by a secondary mutation. The purine-sensitive phenotype was caused by retardation of the pyrimidine de novo pathway, when the intracellular level of PRib-PP was diminished by exogenously supplied purine derivatives.     

Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12

Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher K(i) for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.     

The acetohydroxy acid synthase III isoenzyme of Escherichia coli K-12: regulation of synthesis by leucine.

Acetohydroxy acid synthase III (AHAS III) is one of the three isoenzymes which catalyze the condensation reaction for the biosynthesis of the branched chain amino acids in $\text{Escherichia coli}$ K-12. The synthesis of this enzyme is repressed by leucine. As a consequence of this regulatory feature, strain PS1035, in which AHAS III is the only AHAS isoenzyme expressed, does not grow in minimal medium containing leucine. The other two branched chain amino acids, isoleucine and valine, do not have regulatory effects on AHAS III synthesis.     

Resolution of glpA and glpT loci into separate operons in Escherichia coli K-12 strains.

The independent insertion of bacteriophage Mu into the gene coding for anaerobic sn-glycerol 3-phosphate dehydrogenase (glpA) or into the genes coding for sn-glycerol 3-phosphate transport (glpT) suggested that these two closely linked loci are in separate operons.     

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.