Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)

The Escherichia coli arginine repressor (ArgR) is an l -arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.     

Regulatory gene mutations affecting arginine biosynthesis in Escherichia coli

Summary Mutants of Escherichia coli with altered regulation of arginine biosynthesis were isolated. The alterations in all of the mutants with increased levels of the biosynthetic enzymes were found to map in the argR locus. The mutants were grouped into three classes based on their effect on the regulatory behavior. Complementation studies with stable merodiploid strains demonstrated that the derepressed synthesis in the mutants was recessive to wild-type regulation.     

DNA-binding site of lac repressor probed by dimethylsulfate methylation of lac operator.

In order to compare the structures of the DNA-binding sites on variants of the lac repressor, we have studied the influence of these variants on the dimethylsulfate methylation of the lac operator. Since a bound protein changes the availability of specific purines in the operator to this chemical attack, comparisons of the methylation patterns will show similarities or differences in the protein DNA contacts. We compared lac repressor, induced lac repressor (repressor bound to the gratuitous inducer isopropyl-β-d-thiogalactoside), mutant repressors having increased operator affinities (X86, I12 and the X86-I12 double mutant) and repressor peptides (long headpiece, residues 1 to 59 and short headpiece. residues 1 to 51). All of these repressors and repressor peptides exhibit the same general pattern of protection and enhancement in the operator; however, the short headpiece pattern differs most from that of the repressor while the induced repressor and the long headpiece show intermediate patterns that are strikingly similar to each other. The mutant repressors do not show an isopropyl-β-d-thiogalactoside effect but otherwise are almost indistinguishable from wild-type repressor. These results demonstrate that all molecules bind to the operator using basically the same protein-DNA contacts; they imply that (1) most and possibly all repressor contacts to operator lie within amino acids 1 to 51, (2) inducer weakens many contacts rather than totally disrupting one or even a few and (3) the tight-binding mutants do not make additional contacts to the DNA.These results are consistent with a model in which the amino-terminal portions of two repressor monomers make the DNA contacts. We show that one can understand the affinity of binding as related to the accuracy of the register of the two amino-terminal portions along the DNA. Furthermore, the action of inducer and the behaviour of the tight binding mutants can be accomodated within a two-state model in which the strongly or weakly binding states correspond to structures in which the amino-terminal regions are rigidly or loosely held with respect to each other.     

Control of arg gene expression in Salmonella typhimurium by the arginine repressor from Escherichia coli K-12

Summary The regulation of synthesis of arg enzymes in Salmonella typhimurium by the arginine repressor of Escherichia coli K-12 has been reevaluated using a strain of S. typhimurium in which the argR gene was rendered nonfunctional by inserting the translocatable tetracyclineresistance element Tn10 into the argR gene. In contrast to previous studies, the introduction of the argR ⁺ allelle of E. coli on an F-prime factor to the argR::Tn10 S. typhimurium strain reduced the synthesis of arg enzymes to essentially wild-type levels. The elevated levels of arg enzymes observed in other hybrid merodiploids may have been the consequence of the formation of hybrid repressor molecules. The readily scoreable phenotype of tetracycline resistance facilitated establishing linkage of cod and argR (0.6% cotransduction) by P22 phage-mediated transduction.     

DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state

The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site-specific recombination at cer and related plasmid recombination sites. The hexameric wild-type protein shows L -arginine-dependent DNA binding. In this work, ArgR mutants that are defective in trimer–trimer interactions and bind DNA as trimers in an L -arginine-independent manner are isolated and characterized. Whereas the wild-type ArgR hexamer exhibits high-affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half-sites), the trimeric mutants bind to and footprint three adjacent half-sites of this 'idealized' substrate. Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site-specific recombination. In the absence of L -arginine, residual wild-type ArgR-binding occurs as trimers. The binding of an N-terminal 77-amino-acid DNA-binding domain to idealized ARG boxes is also characterized.     

Computational design of a substrate specificity mutant of a protein

The wild-type trp repressor of E. coli bound 5-methoxytryptophan, a Trp analogue, less tightly than Trp. A mutant repressor (Val⁵⁸→Ala) that should bind 5-methoxytryptophan preferentially to Trp was computationally designed by free-energy calculations accompanied by free-energy decomposition. The designed mutant was demonstrated by experiments to bind 5-methoxytryptophan more tightly than Trp, consistent with the computational prediction. This success indicates the usefulness of free energy decomposition in protein design. Proteins 26:459–464 © 1996 Wiley-Liss, Inc.     

Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins

The recA protein of E coli participates in several diverse biological processes and promotes a variety of complex in vitro reactions. A careful comparison of the phenotypic behavior of E coli recA mutations to the biochemical properties of the corresponding mutant proteins reveals a close parallel both between recombination phenotype and DNA strand exchange and renaturation activities, and between inducible phenomena and repressor cleavage activity. The biochemical alterations manifest by the mutant recA proteins are reflected in the strength of their interaction with ssDNA. The defective mutant recA proteins fail to properly assume the high-affinity DNA-binding state that is characteristic of the wild-type protein and, consequently, form less stable complexes with DNA. The mutant proteins displaying an 'enhanced' activity bind ssDNA with approximately the same affinity as the wild-type protein but, due to altered protein-protein interactions, they associate more rapidly with ssDNA. These changes proportionately affect the ability of recA protein to compete with SSB protein, to interact with dsDNA, and, perhaps, to bind repressor proteins. In turn, the DNA strand exchange, DNA renaturation, and repressor cleavage activities mirror these modifications.     

Single‐chain 434 repressors with altered DNA‐binding specificities

Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA — 6 base-pairs — NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An invivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator. Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10–50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target.This revised version was published online in October 2005 with corrections to the Cover Date.     

精氨酸代谢调控蛋白ArgR调控嗜热链球菌胞外多糖合成

[目的] 研究精氨酸代谢调控蛋白ArgR对嗜热链球菌胞外多糖（EPS）合成的调控作用。[方法] 利用大肠杆菌异源表达嗜热链球菌ArgR蛋白,通过尿素变性-复性和Ni²⁺亲和层析纯化。采用凝胶电泳迁移（EMSA）和生物膜层干涉（BLI）分析ArgR和eps基因簇中P_epsA启动子的相互作用和动力学信息。构建过表达和弱化argR基因菌株,利用苯酚-硫酸法测定其合成EPS差异。[结果] 大肠杆菌异源表达的ArgR为包涵体,使用尿素变性-复性纯化可获得2.95 mg/mL可溶性蛋白;EMSA和BLI结果显示ArgR和启动子P_epsA有特异性结合,且结合因解离水平低而稳定;过表达argR基因可显著降低嗜热链球菌EPS合成,而弱化argR基因则提高EPS合成。[结论] 本研究表明ArgR能特异性结合嗜热链球菌eps基因簇启动子,并负调控EPS生物合成。     

Acetylhistidine as substrate for acetylornithinase: a new system for the selection of arginine regulation mutants in Escherichia coli

Summary The utilisation of acetylhistidine by histidine auxotrophs of E. coli K-12 was found to require a functioning acetylornithinase. The growth, on acetylhistidine-containing media, of his mutants possessing this enzyme was inhibited by arginine or its precursors acetylornithine, ornithine and citrulline. Mutants able to overcome this inhibition belonged to two classes: those (selected on acetylhistidine+acetylornithine or arginine) in which the arginine biosynthetic enzymes were repressible, as in the parent strains; and those (selected on acetylhistidine+acetylornithine, ornithine, citrulline or arginine) in which these enzymes were formed at high, non-repressible levels. The altered properties of the first class were shown genetically not to result from mutation in the argR or argECBH regions; the data are consistent with the second class carrying mutations at the argR locus.It is supposed that arginine, ornithine or citrulline, by repressing the formation of acetylornithinase, diminish the rate at which acetylhistidine can be utilised (although an acetylhistidine uptake system under arginine control would equally explain the results); non-repressible mutants would escape this effect. The kinetic properties, in crude extracts, of acetylornithinase from the parent strains and from members of each mutant class, with acetylornithine and acetylhistidine as substrates, were investigated. It was tentatively concluded that, in accord with the genetic results, the first class do not possess an acetylornithinase altered to make it function better with acetylhistidine as substrate. It is suggested that arginine may affect acetylhistidine utilisation by affecting its uptake in a manner not shared with ornithine or citrulline, as well as by repressing proteins of the arginine system, and that this arginine-specific effect is inoperative in the first class of mutants. The nature of the changes leading to ability to grow on acetylhistidine+acetylornithine remains unknown. Possible applications of these findings to the selection of hitherto undiscovered but potentially informative mutant types are discussed.     

Isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric DNA sequences containing the TTAA and TTAC subsites.

A novel single-chain (sc) protein framework containing covalently dimerized DNA-binding domains (DBD) of the phage 434 repressor was used to construct combinatorial mutant libraries in order to isolate mutant DBDs with altered specificities. The library members contain one wild-type DBD and one mutant domain with randomized amino acids in the DNA-contacting region. Based on previous studies, the mutant sc derivatives are expected to recognize a general ACAA-6 bp-NNNN sequence, where ACAA is contacted by the wild-type and NNNN by the mutant domain. In principle, any sequence can stand for NNNN and serve as a selection target. Here an in vivo library screening method was used to isolate mutant sc repressors that interact with an asymmetric operator containing the TTAA target. Several mutants showed high affinity in vitro binding to operators containing the target and strong (up to 80-fold) preference for the TTAA target over the wild-type TTGT. Specificity studies revealed that certain mutants bound with substantially higher affinities (K(d) approximately 10(-11)M) to operators containing the TTAC sequence, a close homolog of the TTAA target. Thus, we have fortuitously isolated mutant sc repressors that show up to a several hundred-fold preference for TTAC over TTGT.     

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

Summary The nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argF _po), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argF _po. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argF _po —strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru^–/Oru^– phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.Abbreviations Arg^– arginine auxotrophy - Aru arginine utilization - Oru ornithine utilization     

Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties.

Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition. In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR. A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the alpha2 and alpha3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression. Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo. In E. coli, the wild-type B. stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E. coli host cells bearing B. stearothermophilusargR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied. All mutants affected in the DNA-binding domain lost this super-repressor behaviour. Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression. Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant. Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling.     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.