No evidence of inhibition of horizontal gene transfer by CRISPR-Cas on evolutionary timescales.

The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)-Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR-Cas maintenance and one of the causes of the patchy distribution of CRISPR-Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR-Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR-Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.

A MLVA genotyping scheme for global surveillance of the citrus pathogen Xanthomonas citri pv. citri suggests a worldwide geographical expansion of a single genetic lineage.

MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/).

How to identify CRISPRs in sequencing data.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA sequences composed of a succession of repeats (23-50 bp long) separated by unique sequences called spacers. CRISPRs together with a set of genes called cas for CRISPR associated, constitute a defence mechanism against invasion by foreign sequences. We describe protocols and bioinformatics tools that allow the identification of CRISPRs, their comparison and their component determination (the direct repeats and the spacers). A schematic representation of the spacer organization can be produced, allowing an easy comparison between strains.

BRASERO: A Resource for Benchmarking RNA Secondary Structure Comparison Algorithms.

The pairwise comparison of RNA secondary structures is a fundamental problem, with direct application in mining databases for annotating putative noncoding RNA candidates in newly sequenced genomes. An increasing number of software tools are available for comparing RNA secondary structures, based on different models (such as ordered trees or forests, arc annotated sequences, and multilevel trees) and computational principles (edit distance, alignment). We describe here the website BRASERO that offers tools for evaluating such software tools on real and synthetic datasets.

Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans.

The function of seven paralogues phylogenetically related to the Saccharomyces cerevisiae Fur4p together with a number of functionally related transporters present in Aspergillus nidulans has been investigated. After deletion of the cognate genes we checked the incorporation of radiolabelled substrates, utilization of nitrogen sources, resistance to toxic analogues and supplementation of auxotrophies. FurA and FurD encode allantoin and uracil transporters respectively. No function was found for FurB, FurC, FurE, FurF and FurG. As we failed to identify Fur-related transporters for uridine, pyridoxine or thiamine, we deleted other possible candidates for these functions. A FCY2-like gene carrying in its 5' UTR a putative thiamine pyrophosphate riboswitch, and which encodes a protein similar to the pyridoxine transporter of yeast (Tpn1p), does not encode either a major thiamine or a pyridoxine transporter. CntA, a member of the concentrative nucleoside transporter family, is a general nucleoside permease, while no function was found for PnpA, a member of the equilibrative transporter family. Phylogenetic analysis shows that within the ascomycetes, the same transport activity could be catalysed by totally unrelated proteins and that within the Fur subfamily convergent evolution towards uracil and allantoin transport activity has occurred at least three and two independent times respectively.

The tightly regulated promoter of the xanA gene of Aspergillus nidulans is included in a helitron.

In Aspergillus nidulans the xanA gene codes for a xanthine alpha-ketoglutarate-dependent dioxygenase, an enzyme only present in the fungal kingdom. The 5' region of this gene, including its putative promoter and the first 54 codons of the open reading frame, together with the first intron is duplicated in the genome. This duplication corresponds to a helitron, a eukaryotic element proposed to transpose replicatively by the rolling circle mechanism. We show that the regulation of xanA conforms to that of other genes of the purine degradation pathway, necessitating the specific UaY transcription factor and the AreA GATA factor. The promoter of the duplicated region is active ectopically and the difficulty in detecting an mRNA from the duplicated region is at least partially due to nonsense-mediated decay. Comparative genomic data are only consistent with the hypothesis that the 5' region of xanA pre-existed the helitron insertion, and that a 'secondary helitron' was generated from an insertion 5' to it and a pre-existing 3' consensus sequence within the open reading frame. It is possible to propose a role of helitrons in promoter shuffling and thus in recruiting new genes into specific regulatory circuits.

Differential physiological and developmental expression of the UapA and AzgA purine transporters in Aspergillus nidulans.

In this article we study the cellular expression of UapA and AzgA, the two major purine transporters of Aspergillus nidulans, by constructing strains expressing, from their native promoters, fully functional fluorescent (UapA-sGFP, AzgA-sGFP) or immunological (UapA-His) chimeric transporters. Epifluorescence microscopy and immunodetection showed that under different physiological conditions and during Aspergillus development: (i) UapA and AzgA expression in the plasma membrane becomes evident early during germination and remains at a significant basal level in mycelium, (ii) Neither of the two transporters is expressed in the stalk, the vesicle, the phialides and the conidiospores, but surprisingly, UapA is specifically and strongly expressed in the periphery of metulae, (iii) Both transporters are expressed in ascogenous hyphae and in hülle cells but not in cleistothecia or ascospores, (iv) Purine induction leads to approximately 4-fold increase in UapA and AzgA protein content in mycelium, compatible with an analogous increase at the transcriptional level, (v) Ammonium leads to removal of UapA, but not of AzgA, from the plasma membrane by sorting of the protein to the vacuole.

Convergent evolution of hydroxylation mechanisms in the fungal kingdom: molybdenum cofactor-independent hydroxylation of xanthine via alpha-ketoglutarate-dependent dioxygenases.

The xanthine oxidases and dehydrogenases are among the most conserved enzymes in all living kingdoms. They contain the molybdopterin cofactor Moco. We show here that in the fungi, in addition to xanthine dehydrogenase, a completely different enzyme is able to catalyse the oxidation of xanthine to uric acid. In Aspergillus nidulans this enzyme is coded by the xanA gene. We have cloned the xanA gene and determined its sequence. A deletion of the gene has the same phenotype as the previously known xanA1 miss-sense mutation. Homologues of xanA exist only in the fungal kingdom. We have inactivated the cognate gene of Schizosaccharomyces pombe and this results in strongly impaired xanthine utilization as a nitrogen source. We have shown that the Neurospora crassa homologue is functionally equivalent to xanA. The enzyme coded by xanA is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase which shares a number of properties with other enzymes of this group. This work shows that only in the fungal kingdom, an alternative mechanism of xanthine oxidation, not involving Moco, has evolved using the dioxygenase scaffold.

The AzgA purine transporter of Aspergillus nidulans. Characterization of a protein belonging to a new phylogenetic cluster.

The azgA gene of Aspergillus nidulans encodes a hypoxanthine-adenine-guanine transporter. It has been cloned by a novel transposon methodology. The null phenotype of azgA was defined by a number of mutations, including a large deletion. In mycelia, the azgA gene is, like other genes of purine catabolism, induced by uric acid and repressed by ammonium. Its transcription depends on the pathway-specific UaY zinc binuclear cluster protein and the broad domain AreA GATA factor. AzgA is not closely related to any other characterized membrane protein, but many close homologues of unknown function are present in fungi, plants, and prokaryotes but not metazoa. Two of three data bases and the phylogeny presented in this article places proteins of this family in a cluster clearly separated (but perhaps phylogenetically related) from the NAT family that includes other eukaryotic and prokaryotic nucleobase transporters. Thus AzgA is the first characterized member of this family or subfamily of membrane proteins.