The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the Rl and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S.typhimurium nrdE and nrdF genes (RlE and R2F Ribonucleotide reductases catalyze the synthesis of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis. At least three separate classes of enzymes are known, each with a distinct protein structure but all requiring a protein radical for catalysis (1). The long-studied aerobic Escherichia coli enzyme is the prototype for class I enzymes, also present in all higher organisms and some other microorganisms. E. coli genes nrdA and nrdB encode the a and 8 polypeptide chains, respectively, that form the Rl (a2) and R2 (132) proteins that constitute the enzyme (2). Each protomer of the Rl dimer (2 x 85.7 kDa) contains one substrate-binding site with redox-active thiols involved in the reduction of the substrate ribonucleoside diphosphate, and two separate types of allosteric sites: one, the activity site, controls the overall activity of the enzyme, with ATP as a positive effector and dATP as a negative effector; the other, the substrate-specificity site, controls the specificity of the enzyme, with ATP and dATP favoring pyrimidine reduction, dTTP favoring GDP reduction, and dGTP favoring ADP reduction (3).The R2 dimer (2 x 43.4 kDa) contains two dinuclear iron centers with associated stable tyrosyl free radicals, located at Tyr-122 of the polypeptide chain (4). The drug hydroxyurea scavenges this radical and thereby inactivates the enzyme. Class II and III enzymes lack the tyrosyl radical. Class II enzymes, with the Lactobacillus leichmanni enzyme as a prototype, employ adenosylcobalamin as a radical generator, whereas class III enzymes use S-adenosylmethionine together with iron for this purpose.Salmonella typhimurium contains an active class I enzyme with amino acid sequences 96.5% and 98.4% identical to the E. coli Rl and R2 proteins, respectively (A.J., unpublished results). Recent genetic evidence involving complementation of nrd mutants of E. coli suggested the presence in S. typhimurium of the genes, nrdE and nrdF, coding for a second class I enzyme (5). These genes are also present on the chromosome of E. coli but are, under standard growth conditions, expressed only when present on a plasmid. The amino acid sequences deduced for the corresponding proteins showed a limited identity with other class I enzymes but contained many of their catalytically important residues.We have purified and characterized the two proteins encoded by the cloned genes nrdE and nrdF from S. typhimurium. Each protein is a homodimer. Together they catalyze the reductio...
The induction of several SOS genes of Escherichia coli by fluoroquinolones has been studied. Three different SOS gene fusions (recA::lacZ, umuC::lacZ and sulA::lacZ) have been introduced into the E.coli MC1061 strain to study the induction of these SOS genes in the same genetic background. Data on the basal level of expression of these fusions, as well as their induction by mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine are presented. Using these strains, we have found that, like nalidixic acid, ofloxacin, enoxacin and ciprofloxacin are strong inducers of the SOS genes tested, umuC gene expression being the highest. Furthermore, fluoroquinolones produced a significant increase in the reversion of the base substitution hisG428 mutation in the TA102 Salmonella tester strain, while no effect was found in strains TA98, TA100, TA1537 and TA1535. These data indicate that the error-prone repair pathway can participate in mutagenesis induced by fluoroquinolones and also that the damage produced by these chemicals may be similar to that produced by nalidixic acid.
Salmonella typhimurium and Escherichia coli cells have two different class I ribonucleotide reductases encoded by the nrdEF and nrdAB operons. Despite the presence of one additional ribonucleotide reductase, the nrdAB-encoded enzyme is essential to the aerobic growth of the cell because nrdAB-defective mutants of both species are not viable in the presence of oxygen. Several factors controlling nrdAB gene transcription have been analysed intensively. Nothing is known about the expression of the nrdEF genes. To study this subject, and after cloning of E. coli nrdEF genes and sequencing of their 5' ends, the promoter of this operon has been identified by primer extension in both bacterial species. The +1 position was 691 bp and 692 bp upstream of the translational start points of the nrdE genes of S. typhimurium and E. coli, respectively. Downstream of the +1 position, and before the nrdE gene, two open reading frames (ORFs) of 81 and 136 amino acid residues are present in both bacteria. The synthesis of a polypeptide with a molecular mass of 9 kDa, corresponding to the first of these two ORFs, was observed by using the T7 RNA polymerase expression system. Comparison of the amino acid predicted sequence of this ORF reveals a significant similarity with glutaredoxin proteins. Competitive, reverse-transcription polymerase chain reaction experiments indicate that transcription from the nrdEF promoter normally takes place in wild-type cells. nrdEF transcription is increased by hydroxyurea, which inhibits class I ribonucleotide reductase activity, in both RecA+ and RecA- cells. nrdA(ts) mutants show a higher level of nrdEF transcription than wild-type cells at either the permissive or the restrictive temperature. nrdEF expression was unaffected by changes in DNA supercoiling whether caused by the introduction of either topA::Tn10 and hns::Tn10 mutations or by the inhibition of DNA gyrase with the antibiotic novobiocin. In contrast to the nrdAB genes, the nrdEF operon is not essential to the cells because nrdEF-defective mutants are viable under both aerobic and anaerobic conditions.
Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron-sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.
The quorum-sensing (QS) system present in the emerging nosocomial pathogen Stenotrophomonas maltophilia is based on the signaling molecule diffusible signal factor (DSF). Production and detection of DSF are governed by the rpf cluster, which encodes the synthase RpfF and the sensor RpfC, among other components. Despite a well-studied system, little is known about its implication in virulence regulation in S. maltophilia. Here, we have analyzed the rpfF gene from 82 S. maltophilia clinical isolates. Although rpfF was found to be present in all of the strains, it showed substantial variation, with two populations (rpfF-1 and rpfF-2) clearly distinguishable by the N-terminal region of the protein. Analysis of rpfC in seven complete genome sequences revealed a corresponding variability in the N-terminal transmembrane domain of its product, suggesting that each RpfF variant has an associated RpfC variant. We show that only RpfC-RpfF-1 variant strains display detectable DSF production. Heterologous rpfF complementation of ⌬rpfF mutants of a representative strain of each variant suggests that RpfF-2 is, however, functional and that the observed DSF-deficient phenotype of RpfC-RpfF-2 variant strains is due to permanent repression of RpfF-2 by RpfC-2. This is corroborated by the ⌬rpfC mutant of the RpfC-RpfF-2 representative strain. In line with this observations, deletion of rpfF from the RpfC-RpfF-1 strain leads to an increase in biofilm formation, a decrease in swarming motility, and relative attenuation in the Caenorhabditis elegans and zebrafish infection models, whereas deletion of the same gene from the representative RpfC-RpfF-2 strain has no significant effect on these virulence-related phenotypes. Q uorum sensing (QS) is a bacterial cell-cell communication process that allows bacteria to synchronize particular behaviors on a population-wide scale. Within current knowledge, QS in Stenotrophomonas maltophilia depends on the diffusible signal factor QS (DSF-QS) system, which is based mainly on the fatty acid DSF (cis-11-methyl-2-dodecenoic acid) (1, 2). DSF synthesis is fully dependent on RpfF, an enoyl coenzyme A hydratase encoded by the rpf (regulation of pathogenicity factors) cluster, a set of genes that includes all of the components necessary for the synthesis and detection of DSF molecules. In addition to RpfF, rpf encodes the aconitase RpfA, the fatty acid ligase RpfB, the two-component sensor-effector hybrid system RpfC, and the cytoplasmic regulator element RpfG (1, 2). The DSF-QS system was first described in the phytopathogen Xanthomonas campestris pv. campestris, where it plays an important role in virulence regulation (3). Since then, this system has been described in several members of the order Xanthomonadales, including the genera Xanthomonas, Xylella, and Stenotrophomonas, as well as in members of the order Burkholderiales (1, 3-5). The specific functions regulated by the DSF-QS system are dependent on the species, but it has been suggested that it controls several virulence-related phenotypes (6...
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