The UlaG protein familly defines novel structural and functional motifs grafted on an ancient RNase fold

Background: Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT). A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-b-lactamase (MBL) enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL) protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent)metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. Results: Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gramnegative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk of the MBL superfamily by duplication, neofunctionalization and fixation. Conclusions: Our results suggest that the forerunner of UlaG was present as an RNA metabolizing enzyme in the last common ancestor, and that the modern descendants of that ancestral gene have a wide phylogenetic distribution and functional roles. We propose that the UlaGL family evolved new metabolic roles among bacterial and possibly archeal phyla in the setting of a close association with metazoans, such as in the mammalian gastrointestinal tract or in animal and plant pathogens, as well as in environmental settings. Accordingly, the major evolutionary forces shaping the UlaGL family include vertical inheritance and lineage-specific duplication and acquisition of novel metabolic functions, followed by HGT and numerous lineage-specific gene loss events.

The awr gene family encodes a novel class of Ralstonia solanacearum type III effectors displaying virulence and avirulence activities

We present here the characterization of a new gene family, awr, found in all sequenced Ralstonia solanacearum strains and in other bacterial pathogens. We demonstrate that the five paralogues in strain GMI1000 encode type III-secreted effectors and that deletion of all awr genes severely impairs its capacity to multiply in natural host plants. Complementation studies show that the AWR (alanine-tryptophanarginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (¿awr1-5) exhibits enhanced pathogenicity on Arabidopsis plants. A gain-of-function approach expressing AWR in Pseudomonas syringae pv. tomato DC3000 proves that this is likely due to effector recognition, because AWR5 and AWR4 restrict growth of this bacterium in Arabidopsis. Transient overexpression of AWR in nonhost tobacco species caused macroscopic cell death to varying extents, which, in the case of AWR5, shows characteristics of a typical hypersensitive response. Our work demonstrates that AWR, which show no similarity to any protein with known function, can specify either virulence or avirulence in the interaction of R. solanacearum with its plant hosts.

Sistema de secreción de tipo III en Aeromonas mesòfilas

Aeromonas hydrophila és un bacil gram-negatiu, patogen oportunista d’animal i humans. La patogènesi d’A. Hydrophila és multifactorial. A fi d'identificar gens implicats en la virulència de la soca PPD134/91 d’A. hydrophila, vam realitzar experiments de substracció gènica, que van dur a la detecció de 22 fragments d’ADN que codificaven 19 potencials factors de virulencia, incloent un gen que codificava una proteïna de sistema de secreció de tipus III (T3SS). La importància creixent del T3SS en la patogènesi de diversos bacteris, ens va dur a identificar i analitzar l'agrupació gènica del T3SS de les soques AH-1 i AH-3 d’A. hydrophila. La inactivació dels gens de T3SS aopB i aopD d’A. hydrophila AH-1, i ascV d’A. hydrophila AH-3, comporta una disminució de la citotoxicitat, un increment de la fagocitosi, i una reducció de la virulència en diferents models animals. Aquests resultats demostren que el T3SS és necessari per a la patogenicitat. També vam clonar i seqüenciar una ADP-ribosiltransferasa (AexT) a la soca AH-3 d’A. hydrophila, i vam demostrar que aquesta toxina és translocada via el T3SS, sistema que al seu torn sembla ser induïble in vitro en condicions de depleció de calci. El mutant en el gen aexT de la soca AH-3 d’A. hydrophila va mostrar una lleugera reducció de la virulència, assajada amb diferents mètodes. Mitjançant l'ús de diferents sondes d’ADN, vam determinar la presència del T3SS en soques tant clíniques com ambientals de diferents espècies del gènere Aeromonas: A. hydrophila, A. veronii, i A. caviae, i la codistribució d'aquesta agrupació gènica i el gen aexT. Finalment, amb la finalitat d'estudiar la regulació transcripcional de l'agrupació gènica de T3SS i de l’efector AexT A. hydrophila AH-3, vam aïllar els promotors predits per l’operó aopN-aopD i el gen aexT, i els vam fusionar amb el gen reporter gfp (Green Fluorescence Protein). A més, vam demostrar que l'expressió d'ambdós promotors depèn de diferents components bacterians, com per exemple el sistema de dos components PhoP/PhoQ, el sistema de quorum sensing AhyI/AhyR, o el complex piruvat deshidrogenasa.