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.

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.

sulA-independent division inhibition in his-constitutive strains of Salmonella typhimurium.

Constitutive expression of the S. typhimurium histidine operon causes multiple phenotypic changes including strong filamentation. However, the SOS regulatory network is not involved in the inhibition of cell division. The possibility of SOS-independent activation of sulA gene transcription has been ruled out using sulA-lacZ fusions. These results suggest the existence of a pathway of division inhibition unrelated to sulA and not regulated by the SOS system.

Cyclic AMP stimulates transcription of the structural gene of the outer-membrane protein OmpA of Escherichia coli.

To analyze the effect of cyclic AMP on the expression of the ompA gene of Escherichia coli, encoding the outer-membrane protein OmpA, a fusion between this gene and the lacZ gene was constructed in vitro by using a promoter-probe plasmid. The results obtained indicated that the presence of glucose in the culture medium decreased the transcription of the ompA gene. Likewise, cya and crp mutants exhibited lower levels of ompA gene expression than the wild-type strain. Furthermore, the addition of cyclic AMP increased the expression of the ompA gene in both cya and wild-type strains but not in a crp mutant. All these data show that the cyclic AMP receptor protein-cyclic AMP complex positively modulates ompA transcription in E. coli K-12.

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.

Nucleotide sequence of the methoxyneurosporene dehydrogenase gene from Rhodobacter sphaeroides: comparison with other bacterial carotenoid dehydrogenases.

The nucleotide sequence of the 1794-bp fragment containing the crtD gene from Rhodobacter sphaeroides 2.4.1 encoding for methoxyneurosporene dehydrogenase has been determined. A 63% sequence identity was found when compared with the nucleotide sequence of the crtD gene from Rhodobacter capsulatus. A putative regulatory palindromic motif present in the crtD gene from R. capsulatus also exists in this gene from R. sphaeroides. The translated open reading frame of the crtD gene of R. sphaeroides has identified a polypeptide of 495 amino acids which shares a 56% sequence identity with the same CrtD protein of R. capsulatus. The N- and C-termini of these CrtD proteins present a high degree of similarity with the N- and C-termini of other carotenoid dehydrogenases including those encoded by crtI genes. This is in good agreement with the previously hypothesized homology between CrtI and CrtD proteins.

Insertion sequence IS200 can differentiate drug-resistant and drug-sensitive Salmonella typhi of Vi-phage types E1 and M1.

The type strains of Vi-phage types E1, M1 and A of Salmonella typhi, together with drug-resistant and drug-sensitive strains of phage types E1 and M1 isolated in 1992 from patients associated with India or Pakistan, and a drug-resistant strain of phage type A isolated in South Africa in 1991, were characterised with respect to the presence of plasmids conferring resistance to antimicrobial drugs and their chromosomal insertion sequence IS200 profiles. The three type strains, the drug-sensitive strains of Vi-phage types E1 and M1, and a strain of phage type M1 resistant to ampicillin and trimethoprim but not to chloramphenicol, did not contain plasmids. In contrast, for strains of phage types E1 and M1 resistant to chloramphenicol, ampicillin and trimethoprim, and for the drug-resistant strain of phage type A, the complete spectrum of resistance was encoded by high molecular mass plasmids belonging to the H1 incompatibility group. Characterisation of IS200 profiles demonstrated that at least 13 IS200 copies were distributed on the chromosome of all strains tested. Although the IS200 profiles of the type strains of Vi-phage types A, E1 and M1 were identical, it was possible to distinguish between drug-sensitive and drug-resistant strains of Vi-phage types E1 and M1 isolated from patients infected in India and Pakistan by this method. It was concluded that although IS200 typing is not as discriminatory as phage typing for the primary subdivision of S. typhi, it may be useful for certain epidemiological investigations and, in particular, for investigating the origins of strains with multiple drug resistance.

5-Azacytidine: survival and induction of the SOS response in Escherichia coli K-12.

Survival and induction of the SOS system by 5-azacytidine, an analog of cytidine, were studied in Escherichia coli K-12. This compound did not produce any effect on the viability of dcm and dam dcm mutants. Furthermore, recA430 and lexA1 strains (both mutations interfere with LexA repressor cleavage but not recombination proficiency) were more resistant than the wild-type strain of E. coli K-12. In contrast, recBC and recA13 mutants were more sensitive to 5-azacytidine than the wild type. Transient exposure of E. coli to 5-azacytidine for 60 min induced both recA-dependent inhibition of cell division and induction of lambda prophage in Dcm+ strains but not in Dcm- mutants. Expression of both functions was dependent on recBC exonuclease. On the other hand, 5-azacytidine was unable to trigger the induction of umuCD and mucB genes and no amplification of RecA protein synthesis in either Dcm+ or Dcm- strains was observed. These last results are in agreement with previously reported data suggesting that there is a discrimination in the expression of the several SOS functions and that some SOS genes may be induced without amplification of RecA protein synthesis.

Evidence for a specific regulation of recA gene transcription in Escherichia coli.

The kinetics of the recA, sfiA and umuDC genes transcription were studied during a double SOS-inducing treatment in Escherichia coli cells using several strains carrying lacZ gene fusions. A transient inhibition in recA, but not in sfiA or umuDC promoted beta-galactosidase synthesis was detected after successive UV-irradiations. Results obtained with a recA--lacZ fusion introduced in several DNA-repair mutants demonstrated that neither a lower LexA inactivation nor a decrease in the production of the inducing signal are the events through which the successive UV-irradiation promoted the arrest of recA transcription. On the contrary, a specific UV-dose-dependent delay appears to be the reason for the inhibition of the recA gene transcription in cells irradiated twice.

Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells.

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.

[Monitoring of intracranial pressure with intraparenchymal fiberoptic transducer. Technical aspects and clinical reliability]. / Monitorage de la pression intracrânienne par capteur intraparenchymateux à fibres optiques: aspects techniques et fiabilité clinique.

A fiberoptic intracranial pressure transducer (Camino) was assessed prospectively in 100 patients. In all, 122 sensors were inserted intraparenchymally at the bedside, without the help of a neurosurgeon. Before the procedure, patients were given 2 to 4 mg of phenoperidine. The scalp was opened over a few millimeters in the frontal paramedian area. A burr holc was made with a 2 mm bit. The dura mater was opened and a hollow screw inserted in the diploë. When the zero of the transducer had been obtained, a 5 cm length was inserted within the screw. The transducer was then about 5 mm deep within cerebral parenchyma. The procedure took an average of about 15 min. An intracerebral haematoma around the transducer occurred five times. One had to be drained surgically. There were no infectious complications. The daily baseline drift was about 0.3 mmHg. The system seemed to be reliable: there was close agreement between the intracranial pressure (ICP), neurological status and CT scan findings. In trauma cases, there was also good correlation between mean ICP and the basal cistern obliteration score, finally, ICP became equivalent to mean arterial blood pressure in all brain dead patients. It is concluded that this system may be used in all cases where ICP requires to be monitored, even when the lateral ventricles are no longer visible, or when craniotomy has been performed. This will most probably result in a more extended use of ICP monitoring in neurosurgical intensive care.

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.

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.

Modulating action of cyclic AMP on the expression of two SOS genes in Escherichia coli K-12.

Ultraviolet irradiation and cyclic AMP treatment produce a synergistic effect on the induction of the cle1 gene (coding for bacteriocin ColE1) in wild-type strains of Escherichia coli. On the other hand, cyclic AMP does not affect the uv-mediated induction of the recA, sfiA, and umuDC genes. Growth in the presence of glucose or glycerol does not affect the factor of amplification of the expression of the cle1 gene in uv-irradiated cells of the wild-type strain. Although, in cultures not treated with uv, the basal level of cle1 induction is about twice as high in cell grown grown with glycerol as in those using glucose as carbon source. In recA mutants neither simultaneous nor separate treatments with either cyclic AMP or uv irradiation induced transcription of the cle1 gene. Moreover, cyclic AMP induced a slight increase in cle1 gene expression in uv-irradiated cya strains, but not in the crp mutants. Nevertheless, the pattern of the uv-mediated induction of other SOS genes, such as umuDC, was the same in the cya and crp mutants, as in their parental wild-type strains. Furthermore, the uv-mediated induction of lambda prophage was decreased after either addition of cyclic AMP or growth in cultural conditions where the level of this nucleotide was low.

Molecular fingerprinting of Salmonella typhimurium by IS200-typing as a tool for epidemiological and evolutionary studies.

The aim of this work was to develop and evaluate a molecular typing strategy for Salmonella based on hybridization of chromosomal DNA with two different probes derived from insertion sequence IS200. Probe IS200-TT was specifically constructed for this study as a trimer of a 112 pb TaqI-TaqI fragment of IS200. Among several restriction enzymes evaluated, two were selected: EcoRI, which cuts the insertion sequence in two pieces, each one complementary to one of the probes used, and PstI, a restriction enzyme with no recognition site into IS200. With several combinations of these restrictions enzymes and probes, 43 Salmonella typhimurium strains were analyzed for copy number and location of IS200, as well as reproducibility and stability of the patterns. IS200 types have been shown to be stable, both in strains isolated from different patients implicated in the same salmonellosis outbreak and in strains isolated from the same patient at different times or from different specimens. The discriminatory power of the method has been 0.91 to 0.94. As a comparison, S. typhimurium strains were also ribotyped. Discriminatory power of the ribotypes oscillated between 0.44 and 0.55, depending on the enzyme used, and achieved a 0.78 value when the information obtained with two restriction enzymes was combined. Moreover, IS200 typing was able to differentiate among a group of S. typhimurium strains which were identical by ribotype and enzymatic electrophoretic mobility. These results enable us to conclude that, for the stability, reproducibility and discriminatory power of the patterns generated, IS200 probes can be a very useful tool in the molecular typing of S. typhimurium.

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.

Regulation of ubiG gene expression in Escherichia coli.

To study the regulation of the expression in Escherichia coli of the ubiG gene, which codes for the last enzyme in the pathway of ubiquinone biosynthesis, a fusion between the ubiG and lacZ genes was constructed in vitro. The results showed that (i) the expression of the ubiG gene was higher under aerobic conditions than under anaerobic growth conditions, (ii) the presence of glucose in the culture medium decreased the transcription of the ubiG gene, and (iii) cya and crp mutants exhibited lower levels of ubiG gene expression than the wild-type strain. The addition of cyclic AMP increased the expression of the ubiG gene in both cya and wild-type strains but not in a crp mutant. This fact suggests that the cyclic AMP receptor protein-cyclic AMP complex positively modulates ubiG gene transcription. It was also determined that the transcription of the ubiG gene was in the counterclockwise direction on the E. coli map.

Distribution of insertion sequence IS200 in Salmonella and Shigella.

Two DNA probes for the detection of insertion sequence IS200 by either Southern blotting or colony hybridization were constructed. One of the probes is a 300 bp EcoRI-HindIII fragment of IS200 cloned onto pBluescript KS(+); the other is a tail-to-tail dimer of the same fragment cloned onto pUC19. A survey of the presence of IS200 among enteric bacteria revealed that more than 90% of the pathogenic or food-poisoning isolates of Salmonella spp. examined contained one or more copies of insertion sequence IS200, with the exception of the subgenus I serovar S. agona in which IS200 is not found. Although insertion sequence IS200 was first considered a Salmonella-specific element, it also exists in many isolates of Shigella sonnei and Shigella flexneri, but not in Shigella dysenteriae.

Measurement of in vivo expression of nrdA and nrdB genes of Escherichia coli by using lacZ gene fusions.

By using a promoter probe plasmid we investigated expression of the linked nrdA and nrdB genes coding for the two different subunits of the ribonucleoside diphosphate reductase enzyme of Escherichia coli. For this reason, nrdA-lacZ, nrdAB-lacZ and nrdB-lacZ fusions were constructed. Results obtained indicate that the nrdB gene has a promoter from which it may be transcribed independently of the nrdA gene. Furthermore, the nrdB gene may also be transcribed from the nrdA promoter. The expression of the nrdB gene is about 14-fold higher from the nrdA promoter than from its own promoter. The induction of both nrdA and nrdB genes by DNA-damaging agents in the wild-type strain as well as in several SOS mutants was also studied; nrdA gene expression was increased by these treatments in RecA+, RecA-, and LexAInd- strains, although in both RecA- and LexAInd- mutants the nrdA gene expression was considerably lower than that in RecA+ cells. nrdB gene expression was stimulated by DNA damage only when its transcription was from the nrdA promoter, but there was no effect when nrdB was transcribed from its own promoter. In addition, the basal level of nrdA-lacZ and nrdAB-lacZ fusions was reduced in strains containing either RecA- and LexAInd- mutations or a multicopy plasmid carrying the lexA+ gene, whereas the presence of a LexA51Def mutation increased the constitutive expression of both fusions. On the contrary, the basal level of the nrdB-lacZ fusion remained constant in all these strains. Together these results indicate that induction of the SOS response enhances expression of the nrd genes from the nrdA promoter.