Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray.

The genus Brucella contains bacteria producing a zoonosis of large sanitary and economical impact. The complete nucleotide sequence of eight Brucella isolates is currently available. This information can be used for high throughput approaches to the biology of this genus such as the construction of comprehensive collections of ORF clones or ORFeomes. The ORFeome of Brucella melitensis was a first contribution to this goal. Using the Brucella ORFeome as starting material we have amplified each ORF and printed them in duplicate onto coated glass slides along with the appropriate positive and negative controls. Quality control of the microarray was performed by image analysis after ethidium bromide staining. This Brucella DNA microarray was used to determine the global transcriptional profile of Brucella abortus grown under laboratory conditions. Two sets of genes representing strongly and poorly expressed genes have been defined. The occurrence of several genes of the same operon in the same data set has been taken as additional proof of the significance of the results. The two sets have been validated by RT-PCR of retrotranscribed RNA. Among the more abundant transcripts we found ribosomal proteins, Krebs cycle and oxidative phosphorylation enzymes. virB, flagellar components and other genes related with virulence and intracellular growth were in the poorly transcribed set. This report demonstrated the usefulness of the ORFeome for the construction of a PCR product microarray for the analysis of global gene expression in Brucella and also applicable to other microorganisms. The results provided here represent a comprehensive description of the global transcriptional profile of B. abortus grown under laboratory conditions and, at the same time, validate the use of this Brucella microarray for the study of the biology and pathogenesis of Brucella through the analysis of gene expression under any experimental conditions.

Detection of plasmid-mediated quinolone resistance genes in clinical isolates of Enterobacter spp. in Spain.

We have studied by PCR and DNA sequencing the presence of the qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA, intI1, and ISCR1 genes in 200 clinical isolates of Enterobacter cloacae (n = 153) and E. aerogenes (n = 47) consecutively collected between January 2004 and October 2005 in two hospitals located in Santander (northern Spain) and Seville (southern Spain). Mutations in the quinolone resistance-determining region of gyrA and parC also were investigated in organisms containing plasmid-mediated quinolone resistance genes. The isolates had different resistant phenotypes, including AmpC hyperproduction, extended-spectrum beta-lactamase production, resistance or decreased susceptibility to quinolones, and/or resistance to aminoglycosides. Among the 116 E. cloacae isolates from Santander, qnrS1, qnrB5, qnrB2, and aac(6')-Ib-cr were detected in 22 (19%), 1 (0.9%), 1 (0.9%), and 3 (2.6%) isolates, respectively. Twenty-one, 17, and 2 qnrS1-positive isolates also contained bla(LAP-1), intI1, and ISCR1, respectively. A qnrB7-like gene was detected in one E. aerogenes isolate from Santander. No plasmid-mediated quinolone resistance gene was detected in the isolates from Seville. The qnrS1-containing isolates corresponded to four pulsed-field gel electrophoresis patterns and showed various levels of resistance to quinolones. Six isolates were susceptible to nalidixic acid and presented reduced susceptibility to ciprofloxacin. The qnrS1 gene was contained in a conjugative plasmid of ca. 110 kb, and when the plasmid was transferred to recipient strains that did not have a specific mechanism of quinolone resistance, the ciprofloxacin MICs ranged from 0.047 to 0.125 microg/ml.

First detection of the OXA-40 carbapenemase in P. aeruginosa isolates, located on a plasmid also found in A. baumannii.

The aim of this work was to carry out the molecular investigation of the OXA-40 carbapenemase detected in two isolates of Pseudomonas aeruginosa resistant to imipenem. The sequence showed 100% of homology with the gene previously described in Acinetobacter baumannii. Hybridization experiments located the gene on a plasmid also found in the OXA-40 control strain of A. baumannii.

Identification of a streptogramin A acetyltransferase gene in the chromosome of Yersinia enterocolitica.

Streptogramins are polypeptide antibiotics inhibiting protein synthesis by the prokaryotic ribosome. Gram-positive organisms are susceptible to streptogramins, while most gram-negative bacteria are intrinsically resistant. We have found a genomic fragment from a Yersinia enterocolitica isolate with an open reading frame coding for a polypeptide similar to the virginiamycin acetyltransferases found in various plasmids from gram-positive bacteria. The susceptible Escherichia coli strain DB10 was transformed to resistance to the type A streptogramins and to mixed (A + B) streptogramins upon introduction of a plasmid containing that gene. In addition, we showed streptogramin acetylating activity in vitro dependent on the presence of the Y. enterocolitica sat gene. Southern blot hybridization experiments showed that the sat gene was present in all the Y. enterocolitica isolates examined. These data together show that the gene in the Y. enterocolitica chromosome encoded an active streptogramin acetyltransferase. The deduced sequence of the Y. enterocolitica Sat protein was close to those of sat gene products found in gram-positive bacteria and cyanobacteria, suggesting a common evolutionary origin.

The IntI1 integron integrase preferentially binds single-stranded DNA of the attC site.

IntI1 integrase is a member of the prokaryotic DNA integrase superfamily. It is responsible for mobility of antibiotic resistance cassettes found in integrons. IntI1 protein, as well as IntI1-COOH, a truncated form containing its carboxy-terminal domain, has been purified. Electrophoretic mobility shift assays were carried out to study the ability of IntI1 to bind the integrase primary target sites attI and aadA1 attC. When using double-stranded DNA as a substrate, we observed IntI1 binding to attI but not to attC. IntI1-COOH did not bind either attI or attC, indicating that the N-terminal domain of IntI1 was required for binding to double-stranded attI. On the other hand, when we used single-stranded (ss) DNA substrates, IntI1 bound strongly and specifically to ss attC DNA. Binding was strand specific, since only the bottom DNA strand was bound. Protein IntI1-COOH bound ss attC as well as did the complete integrase, indicating that the ability of the protein to bind ss aadA1 attC was contained in the region between amino acids 109 and 337 of IntI1. Binding to ss attI DNA by the integrase, but not by IntI1-COOH, was also observed and was specific for the attI bottom strand, indicating similar capabilities of IntI1 for binding attI DNA in either double-stranded or ss conformation. Footprinting analysis showed that IntI1 protected at least 40 bases of aadA1 attC against DNase I attack. The protected sequence contained two of the four previously proposed IntI1 DNA binding sites, including the crossover site. Preferential ssDNA binding can be a significant activity of IntI1 integrase, which suggests the utilization of extruded cruciforms in the reaction mechanisms leading to cassette excision and integration.

The defect in the metabolism of erythritol of the Brucella abortus B19 vaccine strain is unrelated with its attenuated virulence in mice.

The role of the defect in erythritol catabolism in the attenuated virulence of Brucella abortus B19 vaccine strain in mice was investigated by means of five different strains: (i) the erythritol sensitive B19 vaccine strain; (ii) a natural erythritol tolerant (NET) mutant obtained spontaneously from B19; (iii) an erythritol resistant derivative from B19 (FJS19) obtained by gene replacement of the deleted ery region; (iv) the erythritol resistant B. abortus 2308 reference virulent strain; and (v) an erythritol sensitive mutant (227 strain) obtained from strain 2308 by transposon insertion in the chromosomal ery region. Besides virulence for mice, erythritol oxidation as well as other phenotypic markers were tested in all the strains. The 2308 and FJS19 strains grew in the presence of erythritol and oxidized the sugar, whereas the B19 and 227 strains did not. The NET strain grew in presence of erythritol but was unable to oxidize it. The B19 vaccine strain and its two erythritol resistant derivatives, NET and FJS19, showed similar residual virulence and splenic time courses in mice. Moreover, the virulent strain 2308 and its erythritol sensitive derivative (227 strain) exhibited similar levels of splenic infection. Altogether, these results demonstrate that the genetic region implicated in erythritol catabolism is not related to the low virulence exhibited by B19 in mice.

A hot spot in plasmid F for site-specific recombination mediated by Tn21 integron integrase.

Integron In2 integrase (IntI1)-mediated site-specific recombination between two primary sites occurs at a high frequency, while that between a primary and a secondary site occurs at frequencies around 10,000 times lower. Secondary sites consist of a pentanucleotide with only two fully conserved residues (GWTMW). The analysis of IntI1-mediated recombinants in the plasmid pOX38 revealed the existence in this plasmid of a site used at a frequency intermediate between those of primary and secondary sites. Analysis of this site showed two potentially relevant structural features: first, a set of two consensus pentanucleotides, separated by 5 bp and in opposite orientations, forming what will be called a double site; and second, a longer sequence with some extent of sequence symmetry with the double site at its 3' end. A recombinant plasmid, pSU18P, containing a double site was constructed. Examination of R388-pSU18P recombinants showed that double sites were used preferentially over single pentanucleotides by IntI1. Comparisons of the nucleotide sequences of known 59-bp elements showed that in most cases there was a double site at each element end. Mutagenesis of the F hot spot was carried out to make it look more like the consensus 59-bp element. The improved sites showed recombination frequencies and specificities almost comparable to those observed at IntI1 primary sites.

Improvement of the Brucella abortus B19 vaccine by its preparation in a glycerol based medium.

The Brucella abortus B19 vaccine strain differs from other Brucella strains in its sensitivity to erythritol. However, erythritol tolerant (Eri(t)) mutants arise from sensitive cultures of B19 at high rate, and may cause persistence and/or abortion when the vaccine is inoculated on adult cattle. Twelve different batches of B19 have been examined for the presence of Eri(t) mutants. All contained Eri(t) variants at a proportion ranging from 10(-4) to 10(-6). In order to eliminate these mutants from the vaccine cultures, we have developed a minimal medium with glycerol as the sole carbon source, named MMG30. Growth of the parental strain B19 (erythritol sensitive) in this medium was fairly good compared with the growth of its Eri(t) derivatives. Culture of the 12 different batches of B19 in liquid MMG30 produced up to a thousandfold decrease in the proportion of Eri(t) mutants present in the vaccine cultures. Use of this medium to grow B19 could represent an easy and considerable improvement of the vaccine, by the reduction of the presence of potentially dangerous Eri(t) mutants.

Gene integration in the Escherichia coli chromosome mediated by Tn21 integrase (Int21).

A replication-thermosensitive, pSC101-derived plasmid containing the int gene and RHS-2 from the integron in Tn21 and a kanamycin resistance marker has been constructed and used to obtain Tn21 integrase (Int21)-mediated plasmid integration in the Escherichia coli chromosome. Colonies carrying an integrated plasmid were obtained after growth at 42 degrees C. Southern hybridization and PCR experiments indicated that they contained the plasmid specifically integrated through the RHS into different positions in the E. coli chromosome. Nucleotide sequence determination of the plasmid-chromosome junctions showed that integration sites in the chromosome were pentanucleotides with the sequence described for Int21 secondary sites.

An inverted repeat in the Tn21 integron determines the production of stem-loop DNA.

A 580-bp-long stem-loop DNA (sl-DNA) molecule was produced by a recombinant plasmid containing complete int and aadA genes from the transposon Tn21 cloned in the vector pUC18. A 50-bp-long inverted repeat found near the int gene stop codon could be involved in the generation of the sl-DNA molecule. cloning of the dhfrII gene between the plasmid replication origin and this IR produced an extended sl-DNA including the dhfrII gene. Tn21 recombination hot spot 2 was also able to direct the production of sl-DNA.

The Brucella abortus vaccine strain B19 carries a deletion in the erythritol catabolic genes.

Brucella abortus B19, an avirulent strain obtained by spontaneous mutation, is used worldwide as a vaccine for the control of bovine brucellosis. B19 differs from other B. abortus strains in its sensitivity to erythritol. We took advantage of a previously obtained erythritol sensitive Tn5 insertion mutant of B. abortus 2308 to clone the chromosomal region containing erythritol catabolic genes from this representative pathogenic strain and from the vaccine strain B19. Physical mapping with restriction endonucleases and nucleotide sequence determination revealed the existence of a 702 bp long deletion, occurring between two short direct repeats, in the chromosome of B19. This deletion rendered the B19 strain sensitive to erythritol. Two oligonucleotides whose sequences flank this deletion provided an easy method to differentiate B19 from all other B. abortus isolates.

Secondary-sites for integration mediated by the Tn21 integrase.

The integrase encoded by the integron of the transposon Tn21 can mediate the site-specific fusion of two plasmids if there is a recombination hot spot (59 bp element) in one of them and the sequence GWTMW in the other. The use of this latter, loosely defined site explains how antibiotic-resistance genes could first become associated with integrons.

Internucleosomal DNA fragmentation and programmed cell death (apoptosis) in the interdigital tissue of the embryonic chick leg bud.

In this work we have attempted to characterize the programmed cell death process in the chick embryonic interdigital tissue. Interdigital cell death is a prominent phenomenon during limb development and has the role of sculpturing the digits. Morphological changes in the regressing interdigital tissue studied by light, transmission and scanning electron microscopy were correlated with the occurrence of internucleosomal DNA fragmentation, evaluated using agarose gels. Programming of the cell death process was also analyzed by testing the chondrogenic potential of the interdigital mesenchyme, in high density cultures. Our results reveal a progressive loss of the chondrogenic potential of the interdigital mesenchyme, detectable 36 hours before the onset of the degenerative process. Internucleosomal DNA fragmentation was only detected concomitant with the appearance of cells dying with the morphology of apoptosis, but unspecific DNA fragmentation was also present at the same time. This unspecific DNA fragmentation was explained by a precocious activation of the phagocytic removal of the dying cells, confirmed in the tissue sections. From our observations it is suggested that programming of cell death involves changes before endonuclease activation. Further, cell surface changes involved in the phagocytic uptake of the dying cells appear to be as precocious as endonuclease activation.

Nucleotide sequence of the ampC-ampR region from the chromosome of Yersinia enterocolitica.

The nucleotide sequence of a 3.1-kb region from the chromosome of the Yersinia enterocolitica O:5b strain IP97 containing the gene for an inducible chromosomal cephalosporinase has been determined. The cephalosporinase gene was homologous to other enterobacterial chromosomal cephalosporinase genes, and it was accompanied by a gene homologous to the regulatory ampR gene. The arrangement of genes in the Y. enterocolitica ampCR unit was identical to that in the Enterobacter cloacae and Citrobacter freundii ampCR units.

Nucleotide sequence of a new class A beta-lactamase gene from the chromosome of Yersinia enterocolitica: implications for the evolution of class A beta-lactamases.

The nucleotide sequence has been determined of a 1400 bp fragment from the chromosome of Yersinia enterocolitica containing the gene for beta-lactamase I. An ORF of 882 bp was identified, which could code for a polypeptide of 294 amino acids, closely related to other beta-lactamases of molecular class A. Amino acids 1-30 could constitute a signal peptide. The mature protein would be 264 amino acids long with a calculated pI of 6.2. Alignment of the amino acid sequence of the class A beta-lactamases suggested the existence of two subgroups in the same class, and this is discussed in the context of the evolution of the enzymes.

Cloning of chromosomal beta-lactamase genes from Yersinia enterocolitica.

Two beta-lactamase genes present in the chromosome of Yersinia enterocolitica have been cloned individually into the plasmid pACY184 and expressed in Escherichia coli. The gene for broad-spectrum beta-lactamase I ('A') was cloned from a strain belonging to the O:3 serotype, and the gene for (cephalosporinase) beta-lactamase II ('B') was cloned from a strain of the O:5b serotype. The properties of the beta-lactamases expressed in E. coli are similar to those previously described in Y. enterocolitica.

Nucleotide sequence and intracellular location of the product of the fosfomycin resistance gene from transposon Tn2921.

An 863-base-pair DNA fragment containing the fosfomycin resistance gene from transposon Tn2921 was sequenced. Analysis of the sequence revealed the presence of a single open reading frame that encoded the FOS polypeptide of 16 kilodaltons from Tn2921. Minicell fractionation studies showed that the FOS protein was located in the bacterial cytoplasm.

Structure and mobilization of an ampicillin and gentamicin resistance determinant.

A DNA segment originally found in an epidemic plasmid of Escherichia coli encoding an aminoglycoside-(3)-N-acetyltransferase gene (aacC5) and a TEM-type beta-lactamase gene was characterized. The two genes were adjacent and constituted a single transcriptional unit. In addition, these genes were simultaneously mobilized through the action of an insertion sequence related to IS26, IS140, and IS15-delta. This DNA segment is a composite transposon which has been called Tn2922.