Metabolism of arginine-specific messenger ribonucleic acid in Escherichia coli K-12.

Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of the level of messenger RNA (mRNA) transcribed from seven of the nine genes of the arginine regulon of Escherichia coli K-12. The quantity of RNA complexing with each of the separated DNA strands of the argA, argF, argE, and argCBH operons carried on specialized transducing phages was measured. The derepressed:repressed ratio of mRNA formed in vivo was found to vary between about 3 and 4 when measured by hybridization to DNA isolated from specialized transducing phages carrying the argA, argE, argCBH, argF, and argI operons.     

DNA cleavage of lambda and phi 80 transducing phages carrying the argA, argECBH, argF and argI operons of Escherichia coli K-12 with the restriction endonucleases EcoRI, SmaI and HindIII.

DNA isolated from each of the seven arginine transducing phages lambdaargA2cI857susS7, phi80ppc argECBH, phi80argF, phi80argF ilambdacI857, lambdaargF2, lambdaargF23 and lambdaargI valScI857susS7 has been specifically cleaved by the restriction endonucleases EcoRI, SmaI and HindIII. The DNA fragments resulting from single, and in some cases, double endonuclease digests were separated by electrophoresis in agarose and also in polyacrylamide gel. The electrophoretic patterns thus obtained were compared with those produced by digestion of DNA isolated from the corresponding lambda and phi80 parental phages. The majority of cleavage sites produced by the action of these restriction enzymes on arginine transducing DNA have been physically mapped.     

Isolation and characterization of lambda transducing bacteriophages for argF, argI and adjacent genes.

Two genes for ornithinetranscarbamylase exist in strain Escherichia coli K-12, argI, at 85 min, and argF, at 7 min. In an attempt to compare the deoxyribonucleic acid material of these two genes, the lambda transducing phages carrying a portion of the argI region, lambda dvalS argI, lambda pvalS, and lambda dvalS pyrB, and of the argF region, lambda dargF, have been isolated. Their structure, including that of phi 80dargF previously isolated, was studied by the method of heteroduplex mapping. In this paper, the results of this mapping are reported.     

In vitro synthesis of ornithine transcarbamylase.

An in vitro system for the synthesis of ornithine transcarbamylase (OTCase) was established using iS-30 extract from E. coli MDS6-2(lambda) and DNA of a lambda transducing phage carrying argI and argF genes. This in vitro synthesis was completely dependent on the additon of DNA, and was sensitive to chloramphenicol and rifampicin. Radioisotopic analysis confirmed that the synthesized enzyme catalyzes the carbamylation of ornithine to citrulline. In the in vitro system the repression and derepression of OTCase synthesis could be observed by mixing iS-30 extracts prepared from argR+ and argR- cells. A remarkable maturation effect could be observed for the FFF enzyme, but not for the III enzyme. This system is considered to reflect the in vivo situation, and should therefore be useful for investigations on the regulation of OTCase synthesis in vivo.     

Escherichia coli ornithine carbamolytransferase isoenzymes: evolutionary significance and the isolation of lambdaargF and lambdaargI transducing bacteriophages.

Among the Enterobacteriaceae, Escherichia coli K-12 is the only strain known to have two structural genes (argF and argI) for ornithine carbamoyltransferase. The two gene products interact to form a family of four functional isoenzymes, respectively designated FFF, FFI, FII, and III. The FFF and III isoenzymes exhibit nearly identical kinetic parameters in the conditions applied. FFF is more thermolabile than III; this allows the straightforward characterization of new transducing phages carrying either argF or argI. The bearing of the available information regarding ornithine carbamoyltransferase isoenzymes on the evolution of the ancestral E. coli chromosome is reconsidered.     

Cloning of the argF gene encoding the ornithine carbamoyltransferase from Corynebacterium glutamicum.

The argF gene encoding ornithine carbamoyl-transferase (OTCase; EC2.1.3.3) has been cloned from Corynebacterium glutamicum by transforming the Escherichia coli arginine auxotroph with the genomic DNA library. The cloned DNA also complements the E. coli argG mutant, suggesting a clustered organization of the genes in the genome. We have determined the DNA sequence of the minimal fragment complementing the E. coli argF mutant. The coding region of the cloned gene is 957 nucleotides long with a deduced molecular mass of about 35 kDa polypeptide. The enzyme activity and size of the expressed protein in the E. coli auxotroph carrying the argF gene revealed that the cloned gene indeed codes for OTCase. Analysis of the amino acid sequence of the predicted protein revealed a strong similarity to the corresponding protein of other bacteria.     

Homologous control sites and DNA transcription starts in the related argF and argI genes of Escherichia coli K12

The argF and argI genes code for similar proteins able to assemble into hybrid isoenzymes and are therefore thought to share a common origin. We show here that the nucleotide sequence of the promoter and operator regions of these two genes are highly homologous. DNA regions preceding the control sites also present significant homologies. The results support the notion of divergent evolution of the two genes from a common ancestor. Like argE and argCBH , argF and argI are controlled by a repressor molecule recognizing a family of similar operator sites. Attenuation appears to play no role in this regulation.     

Characterization of P1argF derivatives from Escherichia coli K12 transduction. I. IS1 elements flank the argF gene segment

Summary Specialized transducing derivatives of the temperate bacteriophage P1 (P1std) are selected by transduction into recipients with deletions in the corresponding genes (Stodolsky 1973). When Escherichia coli K12 strains are used as donors in such transduction experiments, P1argF derivatives can be selected. The argF gene is unique to these strains (Glansdorff et al. 1967). Under these experimental conditions P1argF are formed with frequencies 10,000 times greater than other P1std. The majority of the P1argF derivatives that have been analyzed are indistinguishable by cleavage analyses. One such derivative, P1argF5 has been characterized in detail. Heteroduplex analysis against P1, P7, and P1CmO identified an 11 kb insertion of DNA precisely at the naturally occurring IS1 locus of P1. Cleavage analysis with EcoRI, BamHI and PstI confirmed this finding. To further define the argF insertion, a P1Cm13argF derivative was constructed having the IS1 sequences of Cm13 and argF in opposite orientation. Intrastrand annealing of P1Cm13argF5 DNA established that the argF segment is flanked by directly repeated IS1 sequences. The IS1-argF-IS1 segment is desigmated Tn2901. The assignment of the map position of the argF gene within the 11 kb insert of P1argF5 is discussed. The evolutionary significance of this finding and a model for P1argF formation is also presented.     

Isolation, cloning and characterization of argF gene DNA from Escherichia coli K-12.

A 1650 base pair (BP) fragment carrying the entire argF structural gene with its associated control regions was isolated from an EcoRI/BamHI digest of phi80argFilambda cI857 DNA. This segment was cloned using the EcoRI and BamHI cleavage sites in the plasmid pBR322. A preliminary restriction map of the argF region was prepared. RNA polymerase binding studies indicated that the argF promoter is located approx. 30 base pairs from the EcoRI terminus of the cloned DNA segment.     

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.     

Isolation and characterization of the specialized transducing bacteriophages phi80dargF and lambdah80cI857 dargF: specific cleavage of arginine transducing deoxyribonucleic acid by the endonucleases EcoRI and SmaR.

The directed transposition of argF to the tonB locus of the Escherichia coli chromosome and the subsequent isolation of the specialized transducing phage phi80dargF is described. The structure of this phage has been has been determined. A hybrid lambdah80cI857dargF phage has been constructed. Deoxyribonucleic acid isolated from these and their parent bacteriophages has been specifically cleaved by the endonucleases EcoRI and SmaR; the unique deoxyribonucleic acid fragments thus obtained have been resolved and analyzed by electrophoresis in agarose gel.     

Specialized Transducing Phages Derived from Salmonella Phage P22

Salmonella phage P22 has been used in the construction of three sorts of specialized transducing phage: P22 proAB, P22 proABlac and P22 argF. The bacterial genes carried are derived from E. coli K12. Since E. coli and Salmonella chromosomes recombine very poorly, E. coli genes cannot be transduced into Salmonella recipients by P22's generalized transduction mechanism. Therefore, stable inheritance of E. coli material provides a means of detecting specialized transduction. Formation of these phages was possible because the P22 prophage recognizes an attachment site in the E. coli F' prolac episome. Salmonella strains carrying the F' prolac episome can be lysogenized by P22 so as to leave the prophage inserted into the E. coli material of the F' factor. Improper prophage excision can then lead to formation of P22 specialized phages carrying E. coli genetic material.     

Biochemical and genetic studies with arginine and proline auxotrophs of Neisseria gonorrhoeae

The prevalence of specific arginine biosynthesis gene defects was studied for 319 arginine-requiring clinical isolates of Neisseria gonorrhoeae by using the ability of the strains to utilize intermediates of arginine biosynthesis. Only 11% of the uracil-requiring strains defective in the carbamylation of ornithine to yield citrulline had a defective carbamoylphosphate synthetase gene (carAB). Strains defective in carAB were of auxotype CUH. The other strains (89%) having a dual requirement for citrulline and uracil, which were mostly of auxotype PCU, were defective in the ornithine transcarbamoylase gene (argF). Over 90% of the strains were defective either in argJ (174 strains) or in argF (126 strains). Three argininosuccinate-requiring strains (i.e., defective in argG) of auxotype PAU were identified. Some of the arginine auxotrophs of N. gonorrhoeae defective in carAB, argJ, argF, or argG were complemented by genetic transformation with DNA from recombinant bacteriophages carrying characterized gonococcal arginine biosynthesis genes. Gene defects in proA (five strains) and in proB (six strains) were identified by gonococcal transformation assays with recombinant bacteriophages or plasmids carrying proline biosynthesis genes from N. gonorrhoeae. None of the 11 proline-requiring strains tested was defective in proC.     

Transduction of plasmid determinants in Staphylococcus aureus and Escherichia coli.

Buoyant density analysis of transducing lysates derived from Staphylococcus aureus and Escherichia coli indicated that phage particles bearing plasmid determinants contain a quantity of DNA equivalent to that found in the lytic particles. Transducing particles that bear plasmid determinants smaller than viral DNA must therefore contain a quantity of DNA in excess of a single plasmid genome. In the E. coli P1vir system, a dependence upon host-mediated recombination for the transduction of small plasmids, but not for large R factors or chromosomal genes, was observed. However, no evidence for the involvement of such functions in the transduction of S. aureus plasmids was obtained. Although the origin of the additional DNA in plasmid transducing particles has not been identified, circumstantial evidence has been presented in the staphylococcal system indicating that transducing particles carrying a small tetracycline plasmid are not formed by the wrapping of multiple copies of this plasmid DNA.     

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: high frequency of aberrant prophage excision.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanisms. Based on the properties of the DNA packaging mechanism of phage P22 a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10(-2)/PFU)- and proA,B (10(-3)/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.     

Binding of the arginine repressor of Escherichia coli K12 to its operator sites.

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.