Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae

<p>Abstract</p> <p>Background</p> <p>The outermost layer of the bacterial surface is of crucial importance because it is in constant interaction with the host. Glycopeptidolipids (GPLs) are major surface glycolipids present on various mycobacterial species. In the fast-grower model organism <it>Mycobacterium smegmatis</it>, GPL biosynthesis involves approximately 30 genes all mapping to a single region of 65 kb.</p> <p>Results</p> <p>We have recently sequenced the complete genomes of two fast-growers causing human infections, <it>Mycobacterium abscessus </it>(CIP 104536T) and <it>M. chelonae </it>(CIP 104535T). We show here that these two species contain genes corresponding to all those of the <it>M. smegmatis </it>"GPL locus", with extensive conservation of the predicted protein sequences consistent with the production of GPL molecules indistinguishable by biochemical analysis. However, the GPL locus appears to be split into several parts in <it>M. chelonae </it>and <it>M. abscessus</it>. One large cluster (19 genes) comprises all genes involved in the synthesis of the tripeptide-aminoalcohol moiety, the glycosylation of the lipopeptide and methylation/acetylation modifications. We provide evidence that a duplicated acetyltransferase (<it>atf1 </it>and <it>atf2</it>) in <it>M. abscessus </it>and <it>M. chelonae </it>has evolved through specialization, being able to transfer one acetyl at once in a sequential manner. There is a second smaller and distant (<it>M. chelonae</it>, 900 kb; <it>M. abscessus</it>, 3 Mb) cluster of six genes involved in the synthesis of the fatty acyl moiety and its attachment to the tripeptide-aminoalcohol moiety. The other genes are scattered throughout the genome, including two genes encoding putative regulatory proteins.</p> <p>Conclusion</p> <p>Although these three species produce identical GPL molecules, the organization of GPL genes differ between them, thus constituting species-specific signatures. An hypothesis is that the compact organization of the GPL locus in <it>M. smegmatis </it>represents the ancestral form and that evolution has scattered various pieces throughout the genome in <it>M. abscessus </it>and <it>M. chelonae</it>.</p

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic

Non Mycobacterial Virulence Genes in the Genome of the Emerging Pathogen Mycobacterium abscessus

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a “mycobacterial” gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the “non mycobacterial” factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients

DomainSieve: a protein domain-based screen that led to the identification of dam-associated genes with potential link to DNA maintenance.

MOTIVATION: The Dam methyltransferase (DamMT) activity, broadly distributed in association with restriction endonucleases, as part of the restriction-modification defense systems, has evolved to become intimately associated with essential biological functions in a few organisms. In Escherichia coli, DamMT is involved in multiple aspects of DNA maintenance, replication initiation, daughter chromosome segregation, DNA mismatch repair, gene expression control, etc. The participation of DamMT in such a diverse set of functions required that other genes adapted, or emerged through evolution, in response to the DamMT-induced modification of the genomic environment. One example is SeqA, a protein that senses the methylation status of the origin of replication of the chromosome to control the timing of replication initiation. Interestingly, seqA is only present in a few DamMT-specifying proteobacteria. This observation led us to hypothesize that other genes, specifying related functions, might also be found in these organisms. To test this hypothesis, we implemented a large-scale comparative genomic screen meant to identify genes specifying DNA methylation sensing domains, probably involved in DNA maintenance functions. RESULTS: We carried out a phylogenetic analysis of DamMT, identifying two contrasting behaviors of the protein. Based on this phylogeny, we defined precisely a set of genomes, in which the protein activity is likely to be involved in DNA maintenance functions, the 'resident' dam genomes. We defined a second set of genomes, in which DamMT is not resident. We developped a new tool, 'DomainSieve', in order to screen these two sets for protein domains that are strictly associated with 'resident' dam genomes. This approach was rewarding and generated a list of genes, among which some, at least, specify activities with clear linkage to DamMT-dependent DNA methylation and DNA maintenance. AVAILABILITY: DomainSieve is implemented as a web resource and is accessible at http://stat.genopole.cnrs.fr/ds/

Identification of genomic features using microsyntenies of domains: Domain teams

The detection, across several genomes, of local conservation of gene content and proximity considerably helps the prediction of features of interest, such as gene fusions or physical and functional interactions. Here, we want to process realistic models of chromosomes, in which genes (or genomic segments of several genes) can be duplicated within a chromosome, or be absent from some other chromosome(s). Our approach adopts the technique of temporarily forgetting genes and working directly with protein “domains” such as those found in Pfam. This allows the detection of strings of domains that are conserved in their content, but not necessarily in their order, which we refer to as domain teams. The prominent feature of the method is that it relaxes the rigidity of the orthology criterion and avoids many of the pitfalls of gene-families identification methods, often hampered by multidomain proteins or low levels of sequence similarity. This approach, that allows both inter- and intrachromosomal comparisons, proves to be more sensitive than the classical methods based on pairwise sequence comparisons, particularly in the simultaneous treatment of many species. The automated and fast detection of domain teams, together with its increased sensitivity at identifying segments of identical (protein-coding) gene contents as well as gene fusions, should prove a useful complement to other existing methods

Domestication of Lambda Phage Genes into a Putative Third Type of Replicative Helicase Matchmaker

International audienceAt the onset of the initiation of chromosome replication, bacterial replicative helicases are recruited and loaded on the DnaA-oriC nucleoprotein platform, assisted by proteins like DnaC/DnaI or DciA. Two orders of bacteria appear, however, to lack either of these factors, raising the question of the essentiality of these factors in bacteria. Through a phylogenomic approach, we identified a pair of genes that could have substituted for dciA. The two domesticated genes are specific of the dnaC/dnaI-and dciA-lacking organisms and apparently domesticated from lambdoid phage genes. They derive from kO and kP and were renamed dopC and dopE, respectively. DopE is expected to bring the replicative helicase to the bacterial origin of replication, while DopC might assist DopE in this function. The confirmation of the implication of DopCE in the handling of the replicative helicase at the onset of replication in these organisms would generalize to all bacteria and therefore to all living organisms the need for specific factors dedicated to this function

Genetic diversity and population structure of Tenacibaculum maritimum, a serious bacterial pathogen of marine fish: from genome comparisons to high throughput MALDI-TOF typing

International audienceAbstractTenacibaculum maritimum is responsible for tenacibaculosis, a devastating marine fish disease. This filamentous bacterium displays a very broad host range and a worldwide geographical distribution. We analyzed and compared the genomes of 25 T. maritimum strains, including 22 newly draft-sequenced genomes from isolates selected based on available MLST data, geographical origin and host fish. The genome size (~3.356 Mb in average) of all strains is very similar. The core genome is composed of 2116 protein-coding genes accounting for ~75% of the genes in each genome. These conserved regions harbor a moderate level of nucleotide diversity (~0.0071 bp−1) whose analysis reveals an important contribution of recombination (r/m ≥ 7) in the evolutionary process of this cohesive species that appears subdivided into several subgroups. Association trends between these subgroups and specific geographical origin or ecological niche remains to be clarified. We also evaluated the potential of MALDI-TOF-MS to assess the variability between T. maritimum isolates. Using genome sequence data, several detected mass peaks were assigned to ribosomal proteins. Additionally, variations corresponding to single or multiple amino acid changes in several ribosomal proteins explaining the detected mass shifts were identified. By combining nine polymorphic biomarker ions, we identified combinations referred to as MALDI-Types (MTs). By investigating 131 bacterial isolates retrieved from a variety of isolation sources, we identified twenty MALDI-Types as well as four MALDI-Groups (MGs). We propose this MALDI-TOF-MS Multi Peak Shift Typing scheme as a cheap, fast and an accurate method for screening T. maritimum isolates for large-scale epidemiological surveys