Asunción Seoane

Characterization of the Urease Operon of Brucella abortus and Assessment of Its Role in Virulence of the Bacterium

ABSTRACT Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

Identification of a Streptogramin A Acetyltransferase Gene in the Chromosome of Yersinia enterocolitica

ABSTRACT 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 aYersinia 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 thesat 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 theY. enterocolitica Sat protein was close to those ofsat gene products found in gram-positive bacteria and cyanobacteria, suggesting a common evolutionary origin.

Evaluation of the effects of erythritol on gene expression in Brucella abortus

Bacteria of the genus Brucella have the unusual capability to catabolize erythritol and this property has been associated with their virulence mainly because of the presence of erythritol in bovine foetal tissues and because the attenuated S19 vaccine strain is the only Brucella strain unable to oxydize erythritol. In this work we have analyzed the transcriptional changes produced in Brucella by erythritol by means of two high throughput approaches: RNA hybridization against a microarray containing most of Brucella ORFs constructed from the Brucella ORFeome and next generation sequencing of Brucella mRNA in an Illumina GAIIx platform. The results obtained showed the overexpression of a group of genes, many of them in a single cluster around the ery operon, able to co-ordinately mediate the transport and degradation of erythritol into three carbon atoms intermediates that will be then converted into fructose-6P (F6P) by gluconeogenesis. Other induced genes participating in the nonoxidative branch of the pentose phosphate shunt and the TCA may collaborate with the ery genes to conform an efficient degradation of sugars by this route. On the other hand, several routes of amino acid and nucleotide biosynthesis are up-regulated whilst amino acid transport and catabolism genes are down-regulated. These results corroborate previous descriptions indicating that in the presence of erythritol, this sugar was used preferentially over other compounds and provides a neat explanation of the the reported stimulation of growth induced by erythritol.

Tandem Amplification of a 28-Kilobase Region from the Yersinia enterocolitica Chromosome Containing the blaA Gene

ABSTRACT Most Yersinia enterocolitica strains are resistant to β-lactam antibiotics due to the production of one or two chromosomally encoded β-lactamases. Strain Y56 is a Y. enterocolitica O:3 serotype natural isolate that is resistant to moderate amounts of penicillins and that produces a single class A β-lactamase. To select mutants with increased levels of resistance to β-lactam antibiotics, strain Y56 was grown on plates containing increasing amounts of ampicillin, and variants resistant to up to 500 μg of ampicillin per ml were obtained. Chromosomal DNA from hyperresistant isolates was analyzed by Southern hybridization with a blaA-specific probe to detect gene rearrangements. The use of pulsed-field gel electrophoresis revealed that the increase in the resistance level correlated with the amplification in tandem of a DNA fragment of about 28 kb containing the blaA gene. The phenotype of these isolates was not stable, and they recovered the basal low resistance level when the ampicillin used for selection was withdrawn from the growth medium. This loss of resistance was followed by the recovery of the original chromosomal structure. To understand this amplification process, the 28-kb amplification unit was cloned, and the ends were sequenced. The analysis of these sequences did not reveal the presence of either repeats or transposable elements to explain this process. However, we found short sequences similar to some DNA gyrase target sequences that have been described. In addition, we observed that the frequency of appearance of ampicillin-hyperresistant isolates by amplification of the blaA locus was lowered in the presence of the gyrase inhibitor novobiocin. These findings suggest that the DNA gyrase could be involved in this amplification event.