CraA, a major facilitator superfamily efflux pump associated with chloramphenicol resistance in Acinetobacter baumannii.

Acinetobacter baumannii has been increasingly associated with hospital-acquired infections, and the presence of multidrug resistance strains is of great concern to clinicians. A. baumannii is thought to possess a great deal of intrinsic resistance to several antimicrobial agents, including chloramphenicol, although the mechanisms involved in such resistance are not well understood. In this work, we have identified a major facilitator superfamily efflux pump present in most A. baumannii strains, displaying strong substrate specificity toward chloramphenicol.

The open reading frame present in the nrdEF cluster of Corynebacterium ammoniagenes is a transcriptional regulator belonging to the GntR family.

In this work we have identified the cagntR gene, present between the nrdE and nrdF genes of Corynebacterium ammoniagenes, as a transcriptional regulator belonging to the GntR family. This gene encodes a transcriptional factor actively transcribed in the opposite direction relative to the nrdHIEF operon. It is expressed in a cell-culture-dependent fashion and, although the members of this family have been reported to regulate transcription of genes found within their vicinity, we have shown that cagntR is not involved in the transcriptional regulation of either nrdE or nrdF. The role of this regulator, however, remains unknown.

Corynebacterium ammoniagenes class Ib ribonucleotide reductase: transcriptional regulation of an atypical genomic organization in the nrd cluster.

Ribonucleotide reductases (RNRs) are a family of complex enzymes that play an essential role in all organisms because they catalyse de novo synthesis of deoxyribonucleotides required for DNA replication and repair. Three different classes of RNR have been described according to their metal cofactors and organic radicals. Class Ib RNR is encoded in four different genes (nrdH, nrdI, nrdE and nrdF) organized in an operon. The authors previously cloned and sequenced the genes encoding the active class Ib RNR of Corynebacterium ammoniagenes and showed that these genes are clustered in an atypical nrdEF region, which differs from that of other known class Ib enzymes because of an intergenic sequence (1171 bp) present between nrdE and nrdF. This study investigated the transcriptional organization and regulation of this nrd region by RT-PCR. Three different and independent mRNA were found (nrdHIE, nrdF and an ORF present in the intergenic region), each one being transcribed from its own promoter and being essential for normal growth. The ratio of nrdF to nrdHIE mRNA was 9.1, as determined by competitive RT-PCR; the expression of both nrdHIE and nrdF was found to be dependent on the culture growth phase, and was induced in the presence of hydroxyurea, manganese and hydrogen peroxide. This is believed to be the first direct evidence for a manganese-dependent transcriptional regulation of nrd genes. These results suggest a common and coordinated regulation of the different nrd genes, despite their being transcribed from independent promoters.

Occurrence of multiple ribonucleotide reductase classes in gamma-proteobacteria species.

Ribonucleotide reductase (RNR) is central to de novo synthesis of deoxyribonucleotides and essential for all living cells. Three classes have been described; class I is oxygen dependent and represented by two subclasses, Ia (NrdAB) and Ib (NrdEF); class II (NrdJ) is indifferent to oxygen; and class III (NrdDG) is oxygen sensitive. More than one class can be found in an organism, reflecting the oxygen status of its environment. We have investigated, by using PCR and Southern blot, the occurrence of the different classes among species of the gamma-Proteobacteria. Class III are present in all species tested, but the presence of the other classes varies. Some species contain one unique additional enzyme, class Ia, Ib, or II, whereas others contain two additional enzymes, class Ia and Ib, or class Ia and II.

Application of an easy and reliable method for sulfolipid-I detection in the study of its distribution in Mycobacterium tuberculosis strains.

Sulfolipid-I (SL-I) is a specific Mycobacterium tuberculosis glycolipid that has been involved in the mechanisms of tuberculoid infection. Until now, a limited number of M. tuberculosis strains have been studied to ascertain their SL-I content, mainly due to the laborious techniques of purification used: DEAE-cellulose column chromatography (DEAE) or extensive solvent extractions. We designed a two-dimensional thin layer chromatographic (2D-TLC) system which allows the easy and reliable detection of SL-I in small amounts of M. tuberculosis-free glycolipid extracts without previous purification. A characteristic SL-I signal was clearly identified by a differential cresyl violet metachromatic stain. Seven clinical isolates, M. tuberculosis H(37)Ra, H(37)Rv and Canetti strains were tested by DEAE and the 2D-TLC system. Identical results were found using both methodologies. The 2D-TLC methodology devised could be applied to a large number of strains to ascertain easily the distribution of SL-I in the strains of M. tuberculosis species.

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein.

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.

The anaerobic (class III) ribonucleotide reductase from Lactococcus lactis. Catalytic properties and allosteric regulation of the pure enzyme system.

Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase. Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S](+) cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated. Also the FeS cluster was easily lost from NrdG. The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.

Ribonucleotide reduction in Pseudomonas species: simultaneous presence of active enzymes from different classes.

Three separate classes of ribonucleotide reductases exist in nature. They differ widely in protein structure. Class I enzymes are found in aerobic bacteria and eukaryotes; class II enzymes are found in aerobic and anaerobic bacteria; class III enzymes are found in strict and facultative anaerobic bacteria. Usually, but not always, one organism contains only one or two (in facultative anaerobes) classes. Surprisingly, the genomic sequence of Pseudomonas aeruginosa contains sequences for each of the three classes. Here, we show by DNA hybridization that other species of Pseudomonas also contain the genes for three classes. Extracts from P. aeruginosa and P. stutzeri grown aerobically or microaerobically contain active class I and II enzymes, whereas we could not demonstrate class III activity. Unexpectedly, class I activity increased greatly during microaerobic conditions. The enzymes were separated, and the large proteins of the class I enzymes were obtained in close to homogeneous form. The catalytic properties of all enzymes are similar to those of other bacterial reductases. However, the Pseudomonas class I reductases required the continuous presence of oxygen during catalysis, unlike the corresponding Escherichia coli enzyme but similar to the mouse enzyme. In similarity searches, the amino acid sequence of the class I enzyme of P. aeruginosa was more related to that of eukaryotes than to that of E. coli or other proteobacteria, with the large protein showing 42% identity to that of the mouse, suggesting the possibility of a horizontal transfer of the gene. The results raise many questions concerning the physiological function and evolution of the three classes in Pseudomonas species.

[Alternatives to population screening for breast cancer. Is the chance detection useful?]. / Alternatives al cribado poblacional de cáncer de mama. Es útil la detección oportunística?

OBJECTIVES: 1. To evaluate the characteristics of the demand, coverage and acceptability of random detection of breast cancer at a health centre (HC) between January 1995 and December 1996. 2. To describe the cases of breast cancer diagnosed since the setting up in 1988 of random demands for screening mammographies. DESIGN: Crossover study. SETTING: La Mina Primary Care Centre, Barcelona. PATIENTS: A sample of 340 women of a population of 2796 women between 45 and 75 with a clinical history at the HC were studied. MEASUREMENTS AND MAIN RESULTS: The profile of women with neoplasms was: aged 59 +/- 8.5; 279 women attended, 273 medicine, 132 medicine and gynaecology, and 6 gynaecology. Mammographies requested were: 143 (118 through gynaecology, 12 medicine and 13 outside the centre), which was 51% of women who attended and 42% of the sample. The reason for mammography was screening (129) and symptoms (14). Screening mammographies actually done (acceptance) were 116 out of 129 (90%). Sample coverage was 34% (116/340); and 42% of the women who attended (116/279). Most studied age-group was 50-64 (coverage 45%, n = 176). 11 cases of breast cancer were diagnosed: age 54.3 +/- 9.1; 1 in situ, 3 < 10 mm, 2 between 10 and 20 mm, 4 > 20 mm, 1 without data. Diagnosis to treatment delay: 33.7 +/- 0 days. At time of writing 8 women were free of illness. CONCLUSIONS: Through random detection 34% coverage was achieved with good acceptance. Most neoplasms are diagnosed in their early stages. The general practitioner should be more attentive to periodic requests for mammographies.

The manganese-containing ribonucleotide reductase of Corynebacterium ammoniagenes is a class Ib enzyme.

Ribonucleotide reductases (RNRs) are key enzymes in living cells that provide the precursors of DNA synthesis. The three characterized classes of RNRs differ by their metal cofactor and their stable organic radical. We have purified to near homogeneity the enzymatically active Mn-containing RNR of Corynebacterium ammoniagenes, previously claimed to represent a fourth RNR class. N-terminal and internal peptide sequence analyses clearly indicate that the C. ammoniagenes RNR is a class Ib enzyme. In parallel, we have cloned a 10-kilobase pair fragment from C. ammoniagenes genomic DNA, using primers specific for the known class Ib RNR. The cloned class Ib locus contains the nrdHIEF genes typical for class Ib RNR operon. The deduced amino acid sequences of the nrdE and nrdF genes matched the peptides from the active enzyme, demonstrating that C. ammoniagenes RNR is composed of R1E and R2F components typical of class Ib. We also show that the Mn-containing RNR has a specificity for the NrdH-redoxin and a response to allosteric effectors that are typical of class Ib RNRs. Electron paramagnetic resonance and atomic absorption analyses confirm the presence of Mn as a cofactor and show, for the first time, insignificant amounts of iron and cobalt found in the other classes of RNR. Our discovery that C. ammoniagenes RNR is a class Ib enzyme and possesses all the highly conserved amino acid side chains that are known to ligate two ferric ions in other class I RNRs evokes new, challenging questions about the control of the metal site specificity in RNR. The cloning of the entire NrdHIEF locus of C. ammoniagenes will facilitate further studies along these lines.

B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli.

The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

nrdD and nrdG genes are essential for strict anaerobic growth of Escherichia coli.

In Escherichia coli ribonucleotide reduction is catalyzed by two separate enzymes during aerobic and anaerobic growth. The aerobic enzyme is coded by the nrdAB genes, the anaerobic enzyme by nrdDG. We now show that knock-out mutants of either nrdD or nrdG cannot grow during strict anaerobiosis, achieved by inclusion of sodium sulfide in the medium. Interestingly, these mutants grow well under microaerophilic conditions by overproducing the aerobic enzyme. Under such conditions wild-type bacteria turn off nrdAB and switch on nrdDG.

Cloning of the Rhodobacter sphaeroides hisL gene: unifunctionality of the encoded protein and lack of linkage to other his genes.

The Rhodobacter sphaeroides 2.4.1 hisI gene, which encodes a phosphoribosyl-AMP-cyclohydrolase that catalyses the third step in the histidine biosynthetic pathway, has been isolated from a genomic library of this phototrophic bacterium by complementation of an Escherichia coli hisI mutant. Analysis of the nucleotide sequence of the R. sphaeroides hisI gene reveals that it encodes a deduced product of 119 aa with a predicted molecular mass of 13.4 kDa. In contrast to the situation in E. coli, the R. sphaeroides hisI gene encodes a unifunctional protein and it is not linked to the hisE gene. The absence of a single histidine operon like that of E. coli was confirmed by PFGE experiments and complementation analysis of a R. sphaeroides hisI mutant that was constructed by marker exchange. The location of hisI in the R. sphaeroides genome has been determined to be at map co-ordinate 2275 +/- 20 of chromosome l.

The ribonucleotide reductase system of Lactococcus lactis. Characterization of an NrdEF enzyme and a new electron transport protein.

Escherichia coli contains the genetic information for three separate ribonucleotide reductases. Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD- mutant of L. lactis grew well under standard anaerobic growth conditions. The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also differ in their allosteric regulation by dATP.

Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria.

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.

Two different operons for the same function: comparison of the Salmonella typhimurium nrdAB and nrdEF genes.

By using a P22 phage-mediated cloning system, the nrdAB genes of Salmonella typhimurium (St), encoding a ribonucleotide reductase (RR) of class I, have been isolated. The coding regions of the St nrdAB operon show a very high identity with those of the homologous operon of Escherichia coli (Ec). Nevertheless, there are significant differences in their promoter regions since, although the promoters of both operons present two DnaA boxes, these boxes are located downstream from the transcription start point in St, being upstream in Ec. Moreover, the deduced amino-acid sequences of the St nrdAB showed a very limited overall identity (28%) with the products of St nrdEF, which encode a second class-I RR. Expression of St nrdAB and nrdEF is inducible by hydroxyurea, an inhibitor of RR activity. Alignment of the promoter regions of the nrdAB and nrdEF operons of both St and Ec reveals the presence of a consensus sequence. St is the first organism from which two different RR belonging to the same biochemical class are known.

Cloning and sequencing of the gyrA gene from the plant pathogen Erwinia carotovora.

The gyrA gene of Erwinia carotovora subsp. carotovora has been cloned and sequenced. The deduced protein possessed 86% identity with the Escherichia coli GyrA protein. E. carotovora gyrA was also shown to complement an E. coli gyrA43ts mutation.

A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.

The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the R1 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 (R1E and R2F). They correspond to the R1 and R2 proteins. Each protein is a homodimer. Together they catalyze the reduction of CDP to dCDP, using dithiothreitol or reduced glutaredoxin, but not thioredoxin, as an electron donor. CDP reduction is strongly stimulated by low concentrations of dATP, presumably acting as an allosteric effector. Protein R2F contains an antiferromagnetically coupled dinuclear iron center and a tyrosyl free radical. The E. coli and S. typhimurium chromosome thus have maintained the information for a potentially active additional class I ribonucleotide reductase, whose role in vivo is as yet unknown. The allosteric regulation of this enzyme differs from that of the normally expressed reductase.

Cloning and sequencing of the genes from Salmonella typhimurium encoding a new bacterial ribonucleotide reductase.

A plasmid library of Salmonella typhimurium was used to complement a temperature-sensitive nrdA mutant of Escherichia coli. Complementation was obtained with two different classes of plasmids, one carrying the E. coli nrdAB-like genes and the second containing an operon encoding a new bacterial ribonucleotide reductase. Plasmids harboring these new reductase genes also enable obligately anaerobic nrdB::Mud1 E. coli mutants to grow in the presence of oxygen. This operon consists of two open reading frames, which have been designated nrdE (2,145 bp) and nrdF (969 bp). The deduced amino acid sequences of the nrdE and nrdF products include the catalytically important residues conserved in ribonucleotide reductase enzymes of class I and show 25 and 28% overall identity with the R1 and R2 protein, respectively, of the aerobic ribonucleoside diphosphate reductase of E. coli. The 3' end of the sequenced 4.9-kb fragment corresponds to the upstream region of the previously published proU operon of both S. typhimurium and E. coli, indicating that the nrdEF genes are at 57 min on the chromosomal maps of these two bacterial species. Analysis of the nrdEF and proU sequences demonstrates that transcription of the nrdEF genes is in the clockwise direction on the S. typhimurium and E. coli maps.

Insertion sequence IS200 fingerprinting of Salmonella typhi: an assessment of epidemiological applicability.

When Pst I-generated digests of genomic DNA from each of the type strains of 49 of the Vi phage types of Salmonella typhi were probed with a PCR-amplified IS200 gene probe, all strains were found to possess at least 11 IS200 elements carried on fragments in the range 24.2-1.2 kb. Fourteen fingerprints were identified but two patterns designated IS200Sty1 and IS200Sty2 predominated. In one strain, a plasmid-mediated IS200 element was identified. When IS200 fingerprinting was applied to epidemiologically-unrelated strains of S. typhi isolated in Ecuador, 3 patterns were identified in 10 strains belonging to 9 different phage types. It is concluded that Vi phage typing remains the method of choice for the primary differentiation of S. typhi but that IS200 fingerprinting may be of limited use in laboratories which do not have access to phage typing.