Alteromonas fortis sp. nov., a non-flagellated bacterium specialized in the degradation of iota-carrageenan, and emended description of the genus Alteromonas

International audienceStrain 1T, isolated in the seventies from the thallus of the carrageenophytic red algae Eucheumaspinosum collected in Hawaii, USA, was retrospectively characterized using phenotypic,phylogenetic and genomic methods. Bacterial cells were Gram-stain-negative, strictly aerobic,non-flagellated, coccoid, ovoid or rod-shaped, and grew at 10-42 °C (optimum 20-25 °C), atpH 5.5-10 (optimum pH 6-9) and with 2-12 % NaCl (optimum 2-4 %). Strain 1T grew on theseaweed polysaccharides i-carrageenan, laminarin and alginic acid as sole carbon sources. Themajor fatty acids (>10 %) were C16:0, C18:1 ω7c and summed feature 3 (C16:1w7c and/or iso-C15:02OH) and significant amounts of C16:0 N alcohol (6.7 %) and 10 methyl C17:0 (8.6 %) were alsopresent. The only respiratory quinone was Q-8, and major polar lipids werephosphatidylethanolamine, phosphatidylglycerol and an unknown aminolipid. Phylogeneticanalyses based on 16S rRNA gene sequence comparisons showed that the bacterium is affiliatedto the genus Alteromonas (family Alteromonadaceae, class Gammaproteobacteria). Strain 1Texhibits 16S rRNA gene sequence similarity values of 98.8-99.2 % to the type strains ofAlteromonas mediterranea and Alteromonas australica respectively, and of 95.7-98.6 % tothose of the other species of the genus Alteromonas. The DNA G+C content of strain 1T is 43.9mol%. Based on the genome sequence of strain 1T, DNA-DNA hybridization predictions by theaverage nucleotide identity (ANI) and Genome-to-Genome Distance Calculations (GGDC)between strain 1T and other members of the genus Alteromonas showed values of 70-80 %, andbelow 26 %, respectively. The phenotypic, phylogenetic and genomic analyses show that strain 1T is distinct from species of the genus Alteromonas with validly published names and that itrepresents a novel species of the genus Alteromonas, for which the name Alteromonas fortis sp.nov. is proposed. The type strain is 1T (= ATCC 43554T = CIP XXXX)

Evolutionary Evidence of Algal Polysaccharide Degradation Acquisition by Pseudoalteromonas carrageenovora 9T to Adapt to Macroalgal Niches

About half of seaweed biomass is composed of polysaccharides. Most of these complex polymers have a marked polyanionic character. For instance, the red algal cell wall is mainly composed of sulfated galactans, agars and carrageenans, while brown algae contain alginate and fucose-containing sulfated polysaccharides (FCSP) as cell wall polysaccharides. Some marine heterotrophic bacteria have developed abilities to grow on such macroalgal polysaccharides. This is the case of Pseudoalteromonas carrageenovora 9T (ATCC 43555T), a marine gammaproteobacterium isolated in 1955 and which was an early model organism for studying carrageenan catabolism. We present here the genomic analysis of P. carrageenovora. Its genome is composed of two chromosomes and of a large plasmid encompassing 109 protein-coding genes. P. carrageenovora possesses a diverse repertoire of carbohydrate-active enzymes (CAZymes), notably specific for the degradation of macroalgal polysaccharides (laminarin, alginate, FCSP, carrageenans). We confirm these predicted capacities by screening the growth of P. carrageenovora with a large collection of carbohydrates. Most of these CAZyme genes constitute clusters located either in the large chromosome or in the small one. Unexpectedly, all the carrageenan catabolism-related genes are found in the plasmid, suggesting that P. carrageenovora acquired its hallmark capacity for carrageenan degradation by horizontal gene transfer (HGT). Whereas P. carrageenovora is able to use lambda-carrageenan as a sole carbon source, genomic and physiological analyses demonstrate that its catabolic pathway for kappa- and iota-carrageenan is incomplete. This is due to the absence of the recently discovered 3,6-anhydro-D-galactosidase genes (GH127 and GH129 families). A genomic comparison with 52 Pseudoalteromonas strains confirms that carrageenan catabolism has been recently acquired only in a few species. Even though the loci for cellulose biosynthesis and alginate utilization are located on the chromosomes, they were also horizontally acquired. However, these HGTs occurred earlier in the evolution of the Pseudoalteromonas genus, the cellulose- and alginate-related loci being essentially present in one large, late-diverging clade (LDC). Altogether, the capacities to degrade cell wall polysaccharides from macroalgae are not ancestral in the Pseudoalteromonas genus. Such catabolism in P. carrageenovora resulted from a succession of HGTs, likely allowing an adaptation to the life on the macroalgal surface

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota

International audienceSulfated glycans are ubiquitous nutrient sources for microbial communities that have co-evolved with eukaryotic hosts. Bacteria metabolise sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognise their glycan substrate remain poorly understood. Here, we utilise structural biology to determine how sulfatases from the human gut microbiota recognise sulfated glycans. We reveal 7 new carbohydrate sulfatase structures span four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent the first structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity towards related but distinct glycan targets. Collectively, these data reveal that Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: https://www.springernature.com/gp/open-research/policies/accepted-manuscript-term

A single sulfatase is required to access colonic mucin by a gut bacterium

International audienceHumans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel diseas

Rapid protein evolution, organellar reductions, and invasive intronic elements in the marine aerobic parasite dinoflagellate Amoebophrya spp

Background: Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (similar to 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. Results: We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. Conclusion: These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage

Rapid protein evolution, organellar reductions, and invasive intronic elements in the marine aerobic parasite dinoflagellate Amoebophrya spp

BACKGROUND : Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (~ 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. RESULTS : We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. CONCLUSION : These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage.ADDITIONAL FILE 1: FIGURE S1. Phylogeny of Alveolata. Proteomes from 89 alveolates genomes and transcriptome assemblies from the MMETSP project (https://zenodo.org/record/257026/files/) were used to create orthologous groups using orthofinder v2.2 with the diamond BLAST similarity search. Single ortholog alignments were pruned using PhyloTreePruner v.1.0 (minimum taxa to keep 44 and support value 0.9) and realigned using mafft v7 and filtered with Gblocks v.0.91b (−b5 = a -p = n). Filtered alignments were concatenated using seqCat.pl and a phylogenetic tree was produced under Maximum Likelihood framework using RAxML v8.2.9 with the PROTGAMMALGF model of sequence evolution and 101 bootstraps. Asterics represent support values of 95 and above. A detailed method can be found in Kayal et al. 2018 BMC Evol. Biol. (https://doi.org/10.1186/s12862-018-1142-0). The full tree can be found at http://mmo.sb-roscoff.fr/jbrowseAmoebophrya/. FIGURE S2. SSU rDNA sequence identity (in percentage, relative to A25 and A120 compared to other species). FIGURE S3. Distribution of k-mer in A25 and A120 genomes. FIGURE S4. Classification of repeated elements in 3 Amoebophrya genomes (AT5, A25, and A120) using REPET. The x-axis represents the cumulated number of bases of repeated elements in the genome. FIGURE S5. Conserved motif of the putative splice leader (SL) in A25 and A120. FIGURE S6. Alignments of gene encoding the putative spliced leader (SL) gene in A25 and A120. FIGURE S7. Gene orientation change rate in 3 Amoebophrya genomes. FIGURE S8. Number of orthologs genes shared by selected taxa. FIGURE S9. Boxplot of the dN/dS ratios of orthologous genes between A25 and A120, calculated using the model average method (MA). FIGURE S10. Synteny dot-plot obtained by comparison between Amoebophrya A25 and AT5 genomes. FIGURE S11. Synteny dot-plot obtained by comparison between Amoebophrya A120 and AT5 genomes. FIGURE S12. Intron length distribution. FIGURE S13. GC content distribution. FIGURE S14. Multiple alignments of U2 snRNAs. FIGURE S15. Multiple alignments of U4 snRNAs. FIGURE S16. Multiple alignments of U5 snRNAs. FIGURE S17. Multiple alignments of U6 snRNAs. FIGURE S18. Secondary structure of Amoebophrya snRNA. FIGURE S19. Example of introner elements (IEs) in Amoebophrya. FIGURE S20. Distribution the direct repeats with size ranging between 3 and 8 nucleotides in A25. FIGURE S21. Distribution of the direct repeats with size ranging between 3 and 8 nucleotides in A120. FIGURE S22. Composition of direct repeats in introners elements. The diversity in composition of the three (a, b, c) most abundant of direct repeats in introner elements in A25 (up) and A120 (down). FIGURE S23. Terminal inverted repeat locations around the splicing sites in A25 and A120. The position of inverted repeats according to the location of the splice sites in A25 and A120. Left, the inverted repeats of A120 are located at 1–5 the nucleotides upstream and downstream of the splice sites. Right, the inverted repeats of A25 are located at the 1–6 nucleotides in upstream and downstream of the splice sites. FIGURE S24. The flowchart for the in silico search of introner elements. FIGURE S25. Hierarchical clustering analysis (pairwise similarity and OrthoMCL) of all intron families and of the inverted repeats in A25 and A120. FIGURE S26. Percentage of genes with assigned functions in relation with introns composition. FIGURE S27. Difference in the proportion of IEs-containing-genes compared to their KEGG assignment in A25 and A120. FIGURE S28. Distribution of conserved introns. TABLE S1. RCC number, date and site of isolation of strains considered in this study. TABLE S2. Metrics of Nanopore runs for the two Amoebophrya strains. TABLE S3. Search for pathways involved in plastidial functions that are entirely independent of plastid-encoded gene content. TABLE S4. Number of the different types of introns identified in A25 and A120 genomes. TABLE S5. Search for RNA editing in A25 and A120 introns. TABLE S6. Putative Amoebophrya A25 and A120 snRNP homologs. TABLE S7. Classification into families of non-canonical introns in A25 and A120. TABLE S8. RNAseq read assembly statistics of Amoebophrya A25 and A120 corresponding samples from the different time of infection and to the freeliving stage (dinospore only). TABLE S9. Total number of contigs belonging to samples from different stages of infection and the proportion of them that were aligned against the genomes of both Amoebophrya A25 and A120. ND corresponds to “not determined” when no measurement was done. TABLE S10. Metabolic pathway screened in A25 and A120 proteomes.This research was funded by the ANR (Agence Nationale de la Recherche) Grant ANR-14-CE02-0007 HAPAR, the CEA and the Région Bretagne (RC doctoral grant ARED PARASITE 9450 and EK postdoctoral grant SAD HAPAR 9229), and the CNRS (X-life SEAgOInG).http://www.mdpi.com/journal/biomedicinesam2022BiochemistryGeneticsMicrobiology and Plant Patholog

Caractérisation moléculaire d'une sulfatase, d'une kappa-carraghénase et d'une iota-carraghénase chez deux bactéries marines : Alteromonas carrageenovora et Cytophaga Drobachiensis

Genomic libraries from three carrageenan-degrading marine bacteria Alteromonas carrageenovora, Alteromonas fortis and Cytophaga drobachiensis were realised to study carrageenase genes and their protein structures. A gene encoding a sulfatase from A. carrageenovora (ATCC 43555) which is not inhibited by inorganic sulfate, was cloned. The nucleotide sequence of the sulfatase gene (atsA) was determined. This study suggests that this sulfatase is not the glycosulfatase which desulfates the end-products obtained upon cleavage of kappa-carrageenan by the kappa-carrageenase but is a arylsulfatase. Sequence comparisons did not reveal any homology with know proteins.The nucleotide and deduced amino acid sequences are reported for the structural gene cgkA for kappa-carrageenase (Mr 44 412 Da) from the marine bacterium Alteromonas carrageenovora. The cgkA gene encodes a protein of 397 amino acids, and a signal pectide of 25 amino acids were found. These results suggest that the protein would cut twice to be exported. The enzyme is a new member of glycosyl-hydrolases family 16 which comprises glucanases from various sources and the ß-agarase from Streptomyces coelicolor. It is proposed that residue Glu163 in the kappa-carrageenase from A. carrageenovora and Glu155 in the ß-agarase from S. coelicolor are important for catalysis. These latter data show that proteins with similar tertiary structure hydrolyse differents substrates. The DNA fragment for the iota-carrageenase from C. drobachiensis encodes a protein with a molecular mass nearly similar to the exported protein. However, the N-terminus does not directly follows the peptide-signal. This suggest that, here also, the protein would be cut twice before being exported. There is no Shine-Dalgarno sequence, but the Omega and Epsilon sequences, observed upstream of the ORF, could initiate the traduction. No homology was found with other protein sequences.Afin d'étudier la structure des carraghénases bactériennes, des banques génomiques ont été construites à partir des bactéries marines carraghénolytiques Alteromonas carrageenovora, Alteromonas fortis et Cytophaga drobachiensis. Un gène codant pour une sulfatase (atsA) dans A. carrageenovora (ATCC 43555), non inhibée par le sulfate inorganique, a été cloné. Sa séquence nucléotidique a été déterminée. L'étude de cette sulfatase démontre qu'elle n'est pas la glycosulfatase qui désulfate les produits terminaux d'hydrolyse issus de l'action de la kappa-carraghénase, mais est une arylsulfatase supplémentaire dans cette bactérie. La protéine déduite de la séquence du gène ne montre aucune homologie avec d'autres protéines connues. Les séquences nucléotidique et protéique du gène cgkA codant la kappa-carraghénase de A. carrageenovora (Mr 44412 Da) sont présentées. Ce gène code pour une protéine de 397 acides aminés et un peptide signal de 25 acides aminés a été identifié. L'enzyme pourrait être coupé deux fois lors de son excrétion. La kappa-carraghénase montre des homologies avec diverses glucanases de la famille 16 des glycosidases ainsi qu'avec la ß-Glu155 dans la ß-agarase de Streptomyces coelicolor. Les résidus Glu163 dans la kappa-carraghénase et Glu155 dans la ß-agarase seraient catalytiques. Ces résultats indiquent que des protéines ayant des structures tertiaires similaires peuvent avoir des spécificités de substrat différentes. L'insert de la iota-carraghénase de C. drobachiensis code pour une protéine qui aurait une masse moléculaire approximativemet égale à celle de l'enzyme excrétée. Cependant, son extrémité NH2 ne fait pas directement suite au peptide signal. Ceci suggère que, là aussi, une seconde coupure pourrait être nécessaire à son excrétion. Il n'y a pas de séquence de Shine-Dalgorno en amont du gène de la iota-carraghénase mais les séquences appelées Oméga et Epsilon pourraient initier la traduction. Aucune homologie avec d'autres protéines connues n'a été trouvée

Biotechnological potential of the microflora associated with the brown alga Ascophyllum nodosum

Bacteria associated with algae are underexplored despite their huge biodiversity and the fact that they differ markedly from those living freely in seawater. These bacterial communities are known to represent great potential for the production of diverse bioactive compounds, such as specific glycoside hydrolases, as they interact in multiple complex ways with their host. Furthermore, enzymes from marine bacteria have original properties, like cold-adapted, halotolerant and highly stable, which are constantly searched out by bio-industries. The aim of our study was to identify bacteria, associated with the brown alga Ascophyllum nodosum, showing diverse polysaccharolytic activities. To isolate cultivable microorganisms, algal thalli of Ascophyllum nodosum were swabbed with sterile cotton tips and marine agar plates were inoculated. Three-hundred isolated bacteria were screened for agarase, kappa- and iota-carrageenase, and sulfatase activities on specific marine media. Thirty-two bacteria with polysaccharolytic activities were isolated and a part of their 16S rDNA (8F-1492R) were amplified and sequenced. Twenty-seven were classified as Flavobacteriia and five as Gammaproteobacteria. Putative new strains and species of Zobellia, Maribacter, Cellulophaga, Shewanella, Glaciecola, Pseudoalteromonas and Colwellia were identified by phylogenetic analysis. All those genera are well-known to colonize algal surface but only some of them are famous to degrade algal polysaccharides (Zobellia, Maribacter, Cellulophaga, and Pseudoalteromonas). However, all those novel bacterial strains/species showed multiple and diverse enzymatic activities (agarase, iota-and kappa-carrageenase, cellulase, beta-glucosidase, sulfatase and/or amylase activities). Genomics libraries with their DNA were constructed in Escherichia coli and Bacillus subtilis and are screened to identify the genes coding for the observed enzymatic activities. Those novel glycoside hydrolases from unknown marine bacteria should have original and innovative properties with great biotechnological potential