Antony T. Vincent

Chloroplast phylogenomic analysis of chlorophyte green algae identifies a novel lineage sister to the Sphaeropleales (Chlorophyceae)

BackgroundThe class Chlorophyceae (Chlorophyta) includes morphologically and ecologically diverse green algae. Most of the documented species belong to the clade formed by the Chlamydomonadales (also called Volvocales) and Sphaeropleales. Although studies based on the nuclear 18S rRNA gene or a few combined genes have shed light on the diversity and phylogenetic structure of the Chlamydomonadales, the positions of many of the monophyletic groups identified remain uncertain. Here, we used a chloroplast phylogenomic approach to delineate the relationships among these lineages.ResultsTo generate the analyzed amino acid and nucleotide data sets, we sequenced the chloroplast DNAs (cpDNAs) of 24 chlorophycean taxa; these included representatives from 16 of the 21 primary clades previously recognized in the Chlamydomonadales, two taxa from a coccoid lineage (Jenufa) that was suspected to be sister to the Golenkiniaceae, and two sphaeroplealeans. Using Bayesian and/or maximum likelihood inference methods, we analyzed an amino acid data set that was assembled from 69 cpDNA-encoded proteins of 73 core chlorophyte (including 33 chlorophyceans), as well as two nucleotide data sets that were generated from the 69 genes coding for these proteins and 29 RNA-coding genes. The protein and gene phylogenies were congruent and robustly resolved the branching order of most of the investigated lineages. Within the Chlamydomonadales, 22 taxa formed an assemblage of five major clades/lineages. The earliest-diverging clade displayed Hafniomonas laevis and the Crucicarteria, and was followed by the Radicarteria and then by the Chloromonadinia. The latter lineage was sister to two superclades, one consisting of the Oogamochlamydinia and Reinhardtinia and the other of the Caudivolvoxa and Xenovolvoxa. To our surprise, the Jenufa species and the two spine-bearing green algae belonging to the Golenkinia and Treubaria genera were recovered in a highly supported monophyletic group that also included three taxa representing distinct families of the Sphaeropleales (Bracteacoccaceae, Mychonastaceae, and Scenedesmaceae).ConclusionsOur phylogenomic study advances our knowledge regarding the circumscription and internal structure of the Chlamydomonadales, suggesting that a previously unrecognized lineage is sister to the Sphaeropleales. In addition, it offers new insights into the flagellar structures of the founding members of both the Chlamydomonadales and Sphaeropleales.

Detection of Variants of the pRAS3, pAB5S9, and pSN254 Plasmids in Aeromonas salmonicida subsp. salmonicida: Multidrug Resistance, Interspecies Exchanges, and Plasmid Reshaping

ABSTRACT The ubiquitous water-borne Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a worldwide disease in fish farms. Plasmids carrying antibiotic resistance genes have already been described for this bacterium. The aim of the present study was to identify and characterize additional multidrug resistance plasmids in A. salmonicida subsp. salmonicida. We sequenced the plasmids present in two multiple antibiotic-resistant isolates using high-throughput technologies. We also investigated 19 other isolates with various multidrug resistance profiles by genotyping PCR and assessed their resistance to tetracycline. We identified variants of the pAB5S9 and pSN254 plasmids that carry several antibiotic resistance genes and that have been previously reported in bacteria other than A. salmonicida subsp. salmonicida, which suggests a high level of interspecies exchange. Genotyping analyses and the antibiotic resistance profiles of the 19 other isolates support the idea that multiple versions of pAB5S9 and pSN254 exist in A. salmonicida subsp. salmonicida. We also identified variants of the pRAS3 plasmid. The present study revealed that A. salmonicida subsp. salmonicida harbors a wide variety of plasmids, which suggests that this ubiquitous bacterium may contribute to the spread of antibiotic resistance genes in the environment.

Next-generation sequencing (NGS) in the microbiological world: How to make the most of your money

The Sanger sequencing method produces relatively long DNA sequences of unmatched quality and has been considered for long time as the gold standard for sequencing DNA. Many improvements of the Sanger method that culminated with fluorescent dyes coupled with automated capillary electrophoresis enabled the sequencing of the first genomes. Nevertheless, using this technology to sequence whole genomes was costly, laborious and time consuming even for genomes that are relatively small in size. A major technological advance was the introduction of next-generation sequencing (NGS) pioneered by 454 Life Sciences in the early part of the 21th century. NGS allowed scientists to sequence thousands to millions of DNA molecules in a single machine run. Since then, new NGS technologies have emerged and existing NGS platforms have been improved, enabling the production of genome sequences at an unprecedented rate as well as broadening the spectrum of NGS applications. The current affordability of generating genomic information, especially with microbial samples, has resulted in a false sense of simplicity that belies the fact that many researchers still consider these technologies a black box. In this review, our objective is to identify and discuss four steps that we consider crucial to the success of any NGS-related project. These steps are: (1) the definition of the research objectives beyond sequencing and appropriate experimental planning, (2) library preparation, (3) sequencing and (4) data analysis. The goal of this review is to give an overview of the process, from sample to analysis, and discuss how to optimize your resources to achieve the most from your NGS-based research. Regardless of the evolution and improvement of the sequencing technologies, these four steps will remain relevant.

Diversity of antibiotic-resistance genes in Canadian isolates of Aeromonas salmonicida subsp. salmonicida : dominance of pSN254b and discovery of pAsa8

The bacterium Aeromonas salmonicida subsp. salmonicida is a common pathogen in fish farms worldwide. Since the antibiotic resistance of this bacterial species is on the increase, it is important to have a broader view on this issue. In the present study, we tested the presence of known plasmids conferring multi-drug resistance as well as antibiotic resistance genes by a PCR approach in 100 Canadian A. salmonicida subsp. salmonicida isolates. Our study highlighted the dominance of the conjugative pSN254b plasmid, which confers multi-drug resistance. We also identified a new multi-drug plasmid named pAsa8, which has been characterized by a combination of sequencing technologies (Illumina and Oxford nanopore). This new plasmid harbors a complex class 1 integron similar to the one of the Salmonella genomic island 1 (SGI1) found in Salmonella enterica and Proteus mirabilis. Consequently, in addition to providing an update on the A. salmonicida subsp. salmonicida isolates that are resistant to antibiotics, our data suggest that this bacterium is potentially an important reservoir of drug resistance genes and should consequently be monitored more extensively. In addition, we describe a screening method that has the potential to become a diagnostic tool that is complementary to other methods currently in use.

Variants of a genomic island in Aeromonas salmonicida subsp. salmonicida link isolates with their geographical origins

Aeromonas salmonicida subsp. salmonicida is a fish pathogen. Analysis of its genomic characteristics is required to determine the worldwide distribution of the various populations of this bacterium. Genomic alignments between the 01-B526 pathogenic strain and the A449 reference strain have revealed a 51-kb chromosomal insertion in 01-B526. This insertion (AsaGEI1a) has been identified as a new genomic island (GEI) bearing prophage genes. PCR assays were used to detect this GEI in a collection of 139 A. salmonicida subsp. salmonicida isolates. Three forms of this GEI (AsaGEI1a, AsaGEI1b, AsaGEI2a) are now known based on this analysis and the sequencing of the genomes of seven additional isolates. A new prophage (prophage 3) associated with AsaGEI2a was also discovered. Each GEI appeared to be strongly associated with a specific geographic region. AsaGEI1a and AsaGEI2a were exclusively found in North American isolates, except for one European isolate bearing AsaGEI2a. The majority of the isolates bearing AsaGEI1b or no GEI were from Europe. Prophage 3 has also a particular geographic distribution and was found only in North American isolates. We demonstrated that A. salmonicida subsp. salmonicida possesses unsuspected elements of genomic heterogeneity that could be used as indicators to determine the geographic origins of isolates of this bacterium.

Draft genome sequences of two Aeromonas salmonicida subsp. salmonicida isolates harboring plasmids conferring antibiotic resistance

The bacterium Aeromonas salmonicida is the etiological agent of furunculosis, a widespread fish disease causing important economic losses to the fish farming industry. Antibiotic treatments in fish farms may be challenging given the existence of multidrug-resistant isolates of this bacterium. Here, we report the draft genome sequences of the 2004-05MF26 and 2009-144K3 isolates, which harbor plasmids conferring antibiotic resistance. Both isolates also carry the large plasmid pAsa5, which is known to encode a type three secretion system (TTSS) and the pAsal1 plasmid which has the aopP gene producing a TTSS effector. These two isolates are good representatives of the plasmid diversity in A. salmonicida subsp. salmonicida.

Diversity and Homogeneity among Small Plasmids of Aeromonas salmonicida subsp. salmonicida Linked with Geographical Origin.

Furunculosis, which is caused by Aeromonas salmonicida subsp. salmonicida, is a major salmonid disease in fish farms worldwide. Several plasmids found in this bacterium confer phenotypes such drug resistance and virulence. Small plasmids (pAsa1, pAsa2, pAsa3, and pAsal1) related to ColE1- and ColE2-type replicons are usually present in its normal plasmidome. In the present study, with the objective to investigate if these plasmids display particularities related to the origin of the isolates bearing them, a total of 153 isolates, including 78 new and 75 previously described, were analyzed for the presence of small plasmids by PCR and DNA restriction fragment profiling. A geographical dichotomy between Canadian and European isolates for their propensity to do not have pAsa3 or pAsal1 was found. In addition, the genotyping analysis led to the identification of two European isolates harboring an unusual pAsal1. An investigation by next-generation sequencing (NGS) of these two isolates shed light on two pAsal1 variants (pAsal1C and pAsal1D). As with pAsal1B, another pAsal1 variant previously described, these two new variants bore a second insertion sequence (ISAS5) in addition to the usual ISAS11. The characterization of these variants suggested that they could predominate over the wild-type pAsal1 in stressful conditions such as growth at temperatures of 25°C and above. To obtain a comprehensive portrait of the mutational pressure on small plasmids, 26 isolates whose DNA had been sequenced by NGS were investigated. pAsa3 and pAsal1 were more prone to mutations than pAsa1 and pAsa2, especially in the mobA gene, which encodes a relaxase and a primase. Lastly, the average copy number of each plasmid per cell was assessed using raw sequencing data. A clear trend with respect to the relative proportion per cell of each plasmid was identified. Our large-scale study revealed a geographical dichotomy in small plasmid repertoire in addition to a clear trend for pAsa3 and pAsal1 to be more frequently altered. Moreover, we present the discovery of two new variants of pAsal1: pAsal1C and pAsal1D.

AsaGEI2b: a new variant of a genomic island identified in the Aeromonas salmonicida subsp. salmonicida JF3224 strain isolated from a wild fish in Switzerland.

Aeromonas salmonicida subsp. salmonicida is the causal agent of furunculosis in salmonids. We recently identified a group of genomic islands (AsaGEI) in this bacterium. AsaGEI2a, one of these genomic islands, has almost exclusively been identified in isolates from North America. To date, Aeromonas salmonicida subsp. salmonicida JF3224, a strain isolated from a wild brown trout (Salmo trutta) caught in Switzerland, was the only European isolate that appeared to bear AsaGEI2a. We analyzed the genome of JF3224 and showed that the genomic island in JF3224 is a new variant of AsaGEI, which we have called AsaGEI2b. While AsaGEI2b shares the same integrase gene and insertion site as AsaGEI2a, it is very different in terms of many other features. Additional genomic investigations combined with PCR genotyping revealed that JF3224 is sensitive to growth at 25°C, leading to insertion sequence-dependent rearrangement of the locus on the pAsa5 plasmid that encodes a type three secretion system, which is essential for the virulence of the bacterium. The analysis of the JF3224 genome confirmed that AsaGEIs are accurate indicators of the geographic origins of A. salmonicida subsp. salmonicida isolates and is another example of the susceptibility of the pAsa5 plasmid to DNA rearrangements.

Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids.

Freedom in bioinformatics

1 Institut de Biologie Integrative et des Systemes, Universite Laval, Quebec City, QC, Canada 2 Departement de Biochimie, de Microbiologie et de Bio-informatique, Faculte des Sciences et de Genie, Universite Laval, Quebec City, QC, Canada 3 Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Quebec, Quebec City, QC, Canada *Correspondence: antony.vincent.1@ulaval.ca