Ignasi Roca

NrdI Essentiality for Class Ib Ribonucleotide Reduction in Streptococcus pyogenes

The Streptococcus pyogenes genome harbors two clusters of class Ib ribonucleotide reductase genes, nrdHEF and nrdF*I*E*, and a second stand-alone nrdI gene, designated nrdI2. We show that both clusters are expressed simultaneously as two independent operons. The NrdEF enzyme is functionally active in vitro, while the NrdE*F* enzyme is not. The NrdF* protein lacks three of the six highly conserved iron-liganding side chains and cannot form a dinuclear iron site or a tyrosyl radical. In vivo, on the other hand, both operons are functional in heterologous complementation in Escherichia coli. The nrdF*I*E* operon requires the presence of the nrdI* gene, and the nrdHEF operon gained activity upon cotranscription of the heterologous nrdI gene from Streptococcus pneumoniae, while neither nrdI* nor nrdI2 from S. pyogenes rendered it active. Our results highlight the essential role of the flavodoxin NrdI protein in vivo, and we suggest that it is needed to reduce met-NrdF, thereby enabling the spontaneous reformation of the tyrosyl radical. The NrdI* flavodoxin may play a more direct role in ribonucleotide reduction by the NrdF*I*E* system. We discuss the possibility that the nrdF*I*E* operon has been horizontally transferred to S. pyogenes from Mycoplasma spp.

Clinical impact and molecular basis of antimicrobial resistance in non-baumannii Acinetobacter

Species of Acinetobacter other than Acinetobacter baumannii are involved in nosocomial infections. Acinetobacter lwoffii, Acinetobacter genomospecies 3 and Acinetobacter genomospecies 13TU are found in community- and nosocomial-acquired infections as well as in neonatal intensive care units. The non-baumannii Acinetobacter are normally highly susceptible to ciprofloxacin, ampicillin/sulbactam, gentamicin and tigecycline. Carbepenems show good activity although resistant isolates have been reported. Resistance to β-lactams other than carbapenems is associated with overexpression of chromosomal cephalosporinases and extended-spectrum β-lactamase acquisition, whereas resistance to carbapenems involves acquisition of carbapenemases. Quinolone resistance is related to gyrA and/or parC mutations but overexpresion of efflux proteins also plays an important role. With the development of novel and more accurate typing methodologies, an increase in infections caused by non-baumannii Acinetobacter might be observed in the future.

Ribonucleotide reductases of Salmonella typhimurium: transcriptional regulation and differential role in pathogenesis.

Ribonucleotide reductases (RNRs) are essential enzymes that carry out the de novo synthesis of deoxyribonucleotides by reducing ribonucleotides. There are three different classes of RNRs (I, II and III), all having different oxygen dependency and biochemical characteristics. Salmonella enterica serovar Typhimurium (S. Typhimurium) harbors class Ia, class Ib and class III RNRs in its genome. We have studied the transcriptional regulation of these three RNR classes in S. Typhimurium as well as their differential function during infection of macrophage and epithelial cells. Deletion of both NrdR and Fur, two main transcriptional regulators, indicates that Fur specifically represses the class Ib enzyme and that NrdR acts as a global repressor of all three classes. A Fur recognition sequence within the nrdHIEF promoter has also been described and confirmed by electrophoretic mobility shift assays (EMSA). In order to elucidate the role of each RNR class during infection, S. Typhimurium single and double RNR mutants (as well as Fur and NrdR mutants) were used in infection assays with macrophage and epithelial cell lines. Our results indicate class Ia to be mainly responsible for deoxyribonucleotide production during invasion and proliferation inside macrophages and epithelial cells. Neither class Ib nor class III seem to be essential for growth under these conditions. However, class Ib is able to maintain certain growth in an nrdAB mutant during the first hours of macrophage infection. Our results suggest that, during the early stages of macrophage infection, class Ib may contribute to deoxyribonucleotide synthesis by means of both an NrdR and a Fur-dependent derepression of nrdHIEF due to hydrogen peroxide production and DNA damage associated with the oxidative burst, thus helping to overcome the host defenses.

Mycobacterium smegmatis displays the Mycobacterium tuberculosis virulence-related neutral red character when expressing the Rv0577 gene.

Neutral red staining is a cytochemical reaction that has been found to be related to Mycobacterium tuberculosis virulence and, therefore, the component involved in it is thought to be a virulence factor. To study the molecular basis of this reaction we constructed an M. tuberculosis cosmid library in Mycobacterium smegmatis and selected recombinant neutral red positive clones. Heterologous complementation identified Rv0577 as the gene responsible for this trait and we have also shown that it is expressed as a single polycistronic unit together with Rv0576 which could also be involved in the neutral red staining.

Evaluation of a Loop-Mediated Isothermal Amplification-Based Methodology To Detect Carbapenemase Carriage in Acinetobacter Clinical Isolates

ABSTRACT Carbapenem-resistant Acinetobacter baumannii is a major source of nosocomial infections worldwide and is mainly associated with the acquisition of OXA-type carbapenemases and, to a lesser extent, metallo-β-lactamases (MBLs). In this study, 82 nonepidemiologically related Acinetobacter strains carrying different types of OXA or MBL enzymes were tested using the Eazyplex system, a loop-mediated isothermal amplification (LAMP)-based method to rapidly detect carbapenemase carriage. The presence/absence of carbapenem-hydrolyzing enzymes was correctly determined for all isolates in <30 min.

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.