The argRB of Escherichia coli is rare in isolates obtained from natural sources

A single nucleotide polymorphism between Escherichia coli strains K12 and B is known to alter the mechanism by which the arginine repressor regulates arginine biosynthesis, from a regulated system in E. coli K12 to a deregulated system in E. coli B. Laboratory experiments have demonstrated that the different regulatory strategies are selectively favored under different environmental conditions. In this study we analyzed 537 E. coli strains and show that the argR allele in E. coli B, which causes deregulation, is rare in isolates obtained from natural sources. Moreover, sequence analysis of 85 strains shows no evidence of selection at the arginine repressor locus. This illustrates that analysis of sequence data is insufficient to detect selection of uncommon alleles in rare environments.     

The arginine repressor of Escherichia coli.

This review tells the story of the arginine repressor of Escherichia coli from the time of its discovery in the 1950s until the present. It describes how the research progressed through physiological, genetic, and biochemical phases and how the nature of the repressor and its interaction with its target sites were unraveled. The studies of the repression of arginine biosynthesis revealed unique features at every level of the investigations. In the early phase of the work they showed that the genes controlled by the arginine repressor were scattered over the linkage map and were not united, as in other cases, in a single operon. This led to the concept of the regulon as a physiological unit of regulation. It was also shown that different alleles of the arginine repressor could result in either inhibition of enzyme formation, as in E. coli K-12, or in stimulation of enzyme formation, as in E. coli B. Later it was shown that the arginine repressor is a hexamer, whereas other repressors of biosynthetic pathways are dimers. As a consequence the arginine repressor binds to two palindromic sites rather than to one. It was found that the arginine repressor not only acts in the repression of enzyme synthesis but also is required for the resolution of plasmid multimers to monomers, a completely unrelated function. Finally, the arginine repressor does not possess characteristic structural features seen in other prokaryotic repressors, such as a helix-turn-helix motif or an antiparallel beta-sheet motif. The unique features have sustained continuous interest in the arginine repressor and have made it a challenging subject of investigation.     

Biosynthesis of bacterial glycogen. Kinetic studies of a glucose-1-phosphate adenylyltransferase (EC 2.7.7.27) from a glycogen-deficient mutant of Escherichia coli B.

An Escherichia coli B mutant, SG14, accumulates glycogen at 28% the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to alpha- and beta-amylosis, chain length determination, and I2-complex absorption spectra. The SG14 mutant contains normal glycogen synthase and branching enzyme activity but has an ADP-glucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and requires a 12-fold higher concentration of fructose-P2 or a 26 fold higher concentration of pyridoxal-P than the parent type enzyme for 50% of maximal allosteric activation. TPNH, an effective activator of the E. coli B enzyme, does not activate the SG14 ADP-glucose pyrophosphorylase. Other studies show that for the SG14 enzyme the concentrations of ATP and Mg2+ in the synthesis direction and the concentrations of ADP-glucose and PPi in the pyrophosphorolysis direction required to give 50% of maximal activity are 3- to 6-fold higher than those observed for the parent E. coli B ADP-glucose pyrophosphorylase. The Km for alpha-glucose-1-P at saturating to half-saturating concentrations of the activator, fructose-P2, are about the same for both enzymes. However, in the presence of no activator, the concentration of glucose-1-P required for half-maximal activity is about 1.8-fold higher for the SG14 enzyme. Thus SG14 ADP-glucose pyrophosphorylase has lower affinity for its substrates than does the parent enzyme. Previously the SG14 enzyme had been shown to be less sensitive to inhibition by 5'-AMP than the E. coli B enzyme. This ensensitivity to inhibition renders the SG14 enzyme less responsive to energy charge than the E. coli B ADP-glucose pyrophosphorylase. On the basis of the above results and taking into account the reported concentrations of fructose-P2, of pyridoxal-P, and of the adenine nucleotide pool and its energy charge in E. coli strains, it is concluded that furctose-P2 is the important physiological allosteric activator of E. coli ADP-glucose pyrophosphorylase. Furthermore, the 1.7-fold increased rate of accumulation of glycogen observed when E. coli B or SG14 shifts from exponential phase to stationary phase of growth in nitrogen-limiting media can be accounted for by the 2.4-fold increase of the levels of the glycogen biosynthetic enzymes, glycogen synthase, and ADP-glucose pyrophosphorylase. Thus both allosteric regulation of the ADP-glucose pyrophosphorylase as well as the genetic regulation of the biosynthesis of the glycogen biosynthetic enzymes are involved in the regulation of glycogen accumulation in E. coli B.     

Ornithine transcarbamylase from Salmonella typhimurium: purification, subunit composition, kinetic analysis, and immunological cross-reactivity.

Ornithine transcarbamylase (OTCase) was purified to hemogeneity from a derepressed strain of Salmonella typhimurium. The optimal pH for enzyme activity is 8.0. The molecular weight of the enzyme was calculated to be 116,000, based on measurements of the sedimentation coefficient by sucrose gradient ultracentrifugation and the Stokes radius by gel filtration. Polyacrylamide gel electrophoresis of cross-linked OTCase in the presence of sodium dodecyl sulfate showed that the enzyme is composed of three identical subunits. The molecular weight of the monomer was determined to be 39,000. Steady-state kinetics indicate that the reaction mechanism is sequential. The limiting Michealis constants for carbamylphosphate and ornithine were determined to be 0.06 and 0.2 mM, respectively. The dissociation constant for carbamylphosphate was 0.02 mM. Product and dead-end inhibition patterns are consistent with an ordered Bi Bi mechanism, in which carbamylphosphate is the first substrate added and phosphate is the last product released. OTCase activity was inhibited by arginine, but relatively high concentrations were required for significant inhibition. The inhibition by arginine might be physiologically significant in the regulation of carbamlphosphate utilization; a single carbamylphosphate synthetase is responsible for the synthesis of carbamylphosphate for both arginine and pyrimidines in S. typhimurium and the inhibition by argine might serve to divert carbamlphosphate to the synthesis of pyrimidines when arginine is present at high concentrations. The crossreaction of OTCases from different microorganisms with purified antibodies raised against the homogeneous OTCase from S. typhimurium was investigated. The results of immunotitration and immunodiffusion experiments revealed a high degree of identity between the enzymes form S. typhimurium and Esherichia coli B and W. In these three cases, a single gen (argl) encodes OTCase. Wild-type E. coli K-12 and strain 3000 X 111, which carry two OTCase genes (argI, argF), also revealed similar cross-reactivity, supporting the hypothesis that argF is the product of a relatively recent duplication. The activity of OTCase from Bacillus subtilis was partially inhibited by antibodies against the enzyme from S. typhimurium, indicating unusual conservation of primary structure among widely different taxonomic groups. OTCase from Saccharomyces cerevisiae, whose molecular weight and primary structure are similar to those of the enzyme from S. typhimurium, was without detectable cross-reactivity.     

Structure-function relationship of arginyl-tRNA synthetase from Escherichia coli: isolation and characterization of the argS mutation MA5002.

The Escherichia coli K12 argS MA5002 mutant appears to have a functionally altered arginyl-tRNA synthetase (ArgRS). The gene coding for this enzyme was isolated from E. coli genomic DNA using the PCR procedure and inserted into a pUC18 multicopy vector. Sequencing revealed that it differs from the wildtype ArgRS structural gene only by one mutation: a replacement of a C by an A residue which results in substitution of an arginine by a serine at position 134, located two residues downstream from the HVGH consensus sequence. As compared to the genomic enzyme level, this recombinant vector, containing the mutated gene, produces in E. coli JM103, about 100 times as much modified ArgRS. This enzyme was obtained nearly pure after only two chromatographic steps; it exhibits a 4-6 times as low activity and a 5 times as high Km value for ATP as the wildtype enzyme in the aminoacylation and ATP-PPi reactions; Km values for arginine and tRNAArg remained unaltered. The position of this mutation and its effect on enzymatic properties suggest the implication of arginine 134 in ATP binding as well as in the activation catalytic process.     

The nucleotide recognized by the Escherichia coli A restriction and modification enzyme

The nucleotide recognition sequence for the restriction-modification enzyme of Escherichia coli A (EcoA) has been determined to be GAG-7N-GTCA. This sequence is fairly similar, but distinctly different from the two other type I restriction enzyme recognition sites known for E. coli B and E. coli K12, respectively. N6-adenosine methylation has been observed at nucleotide positions 2 and 12 within that sequence after modification by EcoA. As a reference point for mapping the single EcoA site in lambda, the position of lambda point mutation Oam29 has been determined also.     

Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains.

Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.     

Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides.

In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.     

Roles of arginine and canavanine in the synthesis and repression of ornithine transcarbamylase by Escherichia coli

Conditions were found under which the processes of repression and derepression of ornithine transcarbamylase were separated from the process of enzyme synthesis. After 10 min of arginine deprivation followed by the addition of 2 to 200 mug of l-arginine per ml, a number of strains of Escherichia coli exhibited a significant burst of ornithine transcarbamylase synthesis which lasted 3 to 4 min before the onset of repression. The rapid increase of enzyme activity was shown to require protein synthesis, and was not due to a slow uptake of arginine or induction of an arginine-inducible ornithine transcarbamylase. The capacity of E. coli to synthesize the burst of ornithine transcarbamylase reached a maximum after 10 min of arginine deprivation and then remained constant. The observed increase in enzyme synthesis may reflect the level of unstable messenger ribonucleic acid (RNA) for ornithine transcarbamylase present in the cell at the time protein synthesis was reinitiated. After the addition of arginine in the absence of protein synthesis, the burst of ornithine transcarbamylase decayed with a half-life of about 3 min. The data implied that arginine prevents synthesis of new messenger RNA that can translate this enzyme. Repression of ornithine transcarbamylase by l-canavanine (100 to 200 mug/ml) was observed, and no active enzyme was formed in the presence of this analogue. The action of canavanine as a repressor was distinguished from the inhibitory effect of this compound on protein synthesis.     

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.     

Insulin action on Escherichia coli. Regulation of the adenylate cyclase and phosphotransferase enzymes.

Insulin on Escherichia coli was studied using wild type E. coli B/r and K12 strains and a number of phosphoenolpyruvate phosphotransferase mutants. In vivo, the effects of insulin on the differential rate of tryptophanase synthesis, the rate of alpha-methylglucoside uptake and the rate of growth on glucose were determined in E. coli B/r. In vitro, the effect of insulin on the adenylate cyclase and the phosphotransferase activities was determined using toluenized cell preparations of E. coli B/r, E. coli K12 and phosphotransferase mutant strains. The specificity of insulin action on E. coli was determined using glucagon, vasopressin and somatropin as well as insulin antisera. Results show the specific action of insulin on E. coli, inhibiting tryptophanase induction and adenylate cyclase activity, while stimulating growth on glucose and uptake and phosphorylation of alpha-methylglucoside.     

Dual regulation by arginine of the expression of the Escherichia coli argECBH operon.

The correlation between the level of messenger ribonucleic acid (mRNA) specific for the argECBH gene cluster (argECBH mRNA) measured by ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization and the rates of synthesis of N-acetylornithine deacetylase (argE enzyme) and of argininosuccinate lyase (argH enzyme) of Escherichia coli strain K-12 were determined for steady-state growth with and without added L-arginine and during the transition periods between these two states. During the transient period after arginine removal (transient derepression), the synthesis of enzymes argE and argH was initially three to five times greater than the steady-state derepressed rate finally reached 50 min later. The level of argECHB mRNA correlated well both quantitatively and temporally with the rates of enzyme synthesis during this transition. The level of in vivo charged arginyl-transfer RNA (tRNAarg), monitored simultaneously, was initially only 5 to 10% and gradually increased to a final level of 80% after 45 min. During the transient period after arginine addition (transient repression), the rates of synthesis of enzymes argE and argH decreased to almost zero and gradually reached steady-state repressed rates after about 180 min. The argECBH mRNA level remained constant at the steady-state repressed level throughout transient repression, revealing a discontinuity between the level of this mRNA and rates of enzyme synthesis. A similar discrepancy was noted during the transition after ornithine addition. In vivo charged tRNAarg remained constant at 80% during this transition. After removal of arginine, the zero-level transient enzyme synthesis developed after only 7.5 min of arginine deprivation and was maximum after 30 min. The results suggest an accumulation of a molecule regulated by arginine that plays a role in transient repression. Our data indicate that arginyl-tRNA synthetase is not this molecule since its synthesis was unaffected by arginine. The ratios of steady-state argE and argH enzyme synthesis without arginine to that with arginine were 12 and 20, respectively, whereas the similar ratio for argECBH mRNA was 2 to 3. The repressed level of argECBH mRNA was not affected by attempts to repress or derepress the ppc+ gene (carried on the DNA used for hybridization), and the repressed level of argECBH mRNA was lowered about 50% in cells carrying an internal argBH deletion. These data taken together indicate the presence of an excess of untranslated argECBH mRNA during both transient and steady-state repression by arginine. Thus, a second regulatory mechanism, not yet defined, appears to play an important role in arginine regulation of enzyme synthesis.     

Expression of the Arginine Regulon of Escherichia coli W: Evidence for a Second Regulatory Gene

Effect of the M (modifier) gene of Escherichia coli W on the expression of wild-type structural genes of four arginine biosynthetic enzymes was studied by examining enzyme activity in cell-free extracts of cultures grown in minimal medium and medium containing arginine. The mutant M gene was originally identified as causing arginine-induced synthesis of acetylornithine delta-transaminase in a strain deficient for the enzyme. The strains used in this study received the mutant M gene by recombination. Noncoordinate repression has been demonstrated for two more enzymes of the arginine regulon of E. coli W and the M(-) gene increases the degree of noncoordinate repression for the regulon. Mutation of the M gene results in altered regulation of acetylornithine delta-transaminase, ornithine transcarbamylase, and acetylornithinase. In addition, a decreased growth rate is observed. It is proposed that the M gene is a regulatory gene. A model is presented to explain the data which involves changes in operator-repressor affinity for the structural genes and possibly for the gene controlling arginyl transfer ribonucleic acid synthetase.     

Isolation from Klebsiella and characterization of two rcs genes that activate colanic acid capsular biosynthesis in Escherichia coli

Two genes, designated rcsA (regulation of capsule synthesis) and rcsB, that had been cloned from the chromosome of Klebsiella aerogenes (K. pneumoniae) capsular serotype K21 were capable of activating expression of colanic acid capsular polysaccharide in Escherichia coli K12. The Klebsiella rcsA gene encoded a polypeptide of 23 kDa that was required for the induction of a mucoid phenotype at less than or equal to 30 degrees C but not at greater than or equal to 37 C. The Klebsiella rcsB locus encoded no apparent polypeptides and was not capable by itself of causing the overproduction of colanic acid. However, when present in the same cell with rcsA, either in cis or in trans, rcsB caused expression of mucoidy in E. coli at all growth temperatures. These findings are best explained if the Klebsiella rcsA gene product acts as a positive regulator of colanic acid biosynthesis in E. coli and that activity of this protein is in turn subject to regulation by Lon protease. The Klebsiella rcsB locus may exert its effect by preferentially binding a negative regulator of capsular biosynthesis, possibly Lon itself. DNA sequences homologous to the Klebsiella K21b rcsA and rcsB genes were found in the genomes of all other capsular serotypes of klebsiellae examined, including K2, K12, K36 and K43. However, there was no homology between such genes and the chromosome of E. coli. The ability of these rcs genes to induce a mucoid phenotype explains the apparent conjugative transfer from klebsiellae to E. coli of the ability to produce K21 or other Klebsiella capsular polysaccharides that are structurally and antigenically related to colanic acid.     

Sequences required for regulation of arginine biosynthesis promoters are conserved between Bacillus subtilis and Escherichia coli

The region required for regulation of a previously characterized arginine-regulatable promoter upstream from the argC gene in the argCAEBD-cpa-argF cluster of Bacillus subtilis was defined by integration of argC-lacZ translational fusions into the chromosome at a site distant from the arginine loci. Some sequence similarity was detected between the argC regulatory region and the well-characterized Escherichia coli arginine operators (ARG boxes). This similarity was shown to be functional in vivo in that the B. subtilis repressor regulated the E. coli arginine genes, but the E. coli repressor, even when encoded by a multicopy plasmid, could not repress the B. subtilis argC promoter. In vitro binding studies using purified repressors on DNA fragments encoding operators from both E. coli and B. subtilis demonstrated interactions by both proteins.