Accessory genome contributes to the virulence and resistance of the ocular isolate of Pseudomonas aeruginosa: A complete genome analysis

Bacteria can acquire an accessory genome through the horizontal transfer of genetic elements from non-parental lineages. This leads to rapid genetic evolution allowing traits such as antibiotic resistance and virulence to spread through bacterial communities. The study of complete genomes of bacterial strains helps to understand the genomic traits associated with virulence and antibiotic resistance. We aimed to investigate the complete accessory genome of an ocular isolate of P. aeruginosa. We obtained the complete genome of the ocular isolate strain PA34 of P. aeruginosa utilising genome sequence reads from Illumina and Oxford Nanopore Technology followed by PCR to close any identified gaps. In-depth genomic analysis was performed using various bioinformatics tools. The phenotypic properties of susceptibility to heavy metals and cytotoxicity were determined to confirm expression of certain traits. The complete genome of PA34 includes a chromosome of 6.8 Mbp and two plasmids of 95.4 Kbp (pMKPA34-1) and 26.8 Kbp (pMKPA34-2). PA34 had a large accessory genome of 1,213 genes and had 543 unique genes not present in other strains. These exclusive genes encoded features related to metal and antibiotic resistance, phage integrase and transposons. At least 24 GIs were predicated in the complete chromosome, of which two were integrated into novel sites. Eleven GIs carried virulence factors or replaced pathogenic genes. A bacteriophage carried the aminoglycoside resistance gene (aac(3)-IId). The two plasmids carried other six antibiotic resistance genes. The large accessory genome of this ocular isolate plays a large role in shaping its virulence and antibiotic resistance

Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Murray, S. A., Ruvindy, R., Kohli, G. S., Anderson, D. M., & Brosnahan, M. L. Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1. Scientific Reports, 9(1), (2019): 14532, doi:10.1038/s41598-019-51074-3.Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L−1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.We thank the Australian Research Council for Funding (FT120100704). We thank Chowdhury Sarowar for the toxicity measurements, at the Sydney Institute of Marine Science. Support to MB and DA was provided by MIT Sea Grant (NA14OAR4170077) and the Woods Hole Center for Oceans and Human Health (National Science Foundation award OCE-1840381 and National Institute of Environmental Health Sciences award 1-P01-ES028938–01). We are grateful for assistance from David Kulis, Claire Mullen, and Isaac Rosenthal for assistance in the collection and processing of Salt Pond samples

Comparative genomics of clinical strains of Pseudomonas aeruginosa strains isolated from different geographic sites

© 2018, The Author(s). The large and complex genome of Pseudomonas aeruginosa, which consists of significant portions (up to 20%) of transferable genetic elements contributes to the rapid development of antibiotic resistance. The whole genome sequences of 22 strains isolated from eye and cystic fibrosis patients in Australia and India between 1992 and 2007 were used to compare genomic divergence and phylogenetic relationships as well as genes for antibiotic resistance and virulence factors. Analysis of the pangenome indicated a large variation in the size of accessory genome amongst 22 stains and the size of the accessory genome correlated with number of genomic islands, insertion sequences and prophages. The strains were diverse in terms of sequence type and dissimilar to that of global epidemic P. aeruginosa clones. Of the eye isolates, 62% clustered together within a single lineage. Indian eye isolates possessed genes associated with resistance to aminoglycoside, beta-lactams, sulphonamide, quaternary ammonium compounds, tetracycline, trimethoprims and chloramphenicols. These genes were, however, absent in Australian isolates regardless of source. Overall, our results provide valuable information for understanding the genomic diversity of P. aeruginosa isolated from two different infection types and countries

De novo assembly and characterisation of transcriptomes from Amphidinium species.

Amphidinium Claparede et Lachmann is one of the most abundant and diverse dinoflagellate genera found in marine benthic, sand dwelling and pelagic habitats. Species of Amphidinium have been widely studied for their potential to produce natural products, in particular, long chain and linear polyketide and macrolide compounds. More than 40 compounds produced by different species of Amphidinium have been isolated. These compounds with varying bioactivities are being investigated for their efficacy as antimicrobial and/or therapeutic agents. Some compounds produced by Amphidinium species have bioactivities against fish gill cells, and may lead to fish kills. Structural elucidation of many toxic compounds produced by dinoflagellates suggest that they may be based on a polyether ladder backbone, and that polyketide synthase (PKS) enzymes may have a role in their biosynthesis. The aim of our study was to screen Amphidinium species for the production of polyketide compounds and test their toxicity. We conducted a transcriptomic sequencing study to determine the presence of PKS genes (both mono-functionally expressed catalytic domains and genes encoding multimodular PKS enzyme complexes) in Amphidinium. The ketosynthase domain encoding transcripts were found to form distinct clades in the phylogenetic analysis, in comparison to similar genes from other organisms. We also show the presence of genes encoding six key enzymes essential for fatty acid production in Amphidinium. The results presented here are a step forward towards recognising the genes encoding critical steps in toxin biosynthesis and designing tools to monitor for and mitigate human illnesses due to harmful algal blooms

Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa

Glutathione (GSH) is the most abundant antioxidant in all living organisms. Previously, we have shown that a deletion mutant in the glutathione synthetase gene (ΔgshB) decreases the expression of type III secretion system (T3SS) genes of Pseudomonas aeruginosa. However, the mechanism remains elusive. In this study, a comprehensive transcriptomic analysis of the GSH-deficient mutant ΔgshAΔgshB was used to elucidate the role of GSH in the pathogenesis of P. aeruginosa. The data show that the expression of genes in T3SS, type VI secretion system (T6SS) and some regulatory genes were impaired. ΔgshAΔgshB was attenuated in a mouse model of acute pneumonia, swimming and swarming motilities, and biofilm formation. Under T3SS inducing conditions, GSH enhanced the expression of T3SS in both wild-type PAO1 and ΔgshAΔgshB, but not in Δvfr. Genetic complementation of Δvfr restored the ability of GSH to induce the expression of T3SS genes. Site-directed mutagenesis based substitution of cysteine residues with alanine in Vfr protein abolished the induction of T3SS genes by GSH, confirming that GSH regulates T3SS genes through Vfr. Exposure to H2O2 decreased free thiol content on Vfr, indicating that the protein was sensitive to redox modification. Importantly, GSH restored the oxidized Vfr to reduced state. Collectively, these results suggest that GSH serves as an intracellular redox signal sensed by Vfr to upregulate T3SS expression in P. aeruginosa. Our work provides new insights into the role of GSH in P. aeruginosa pathogenesis

BRIDGING FUNCTIONAL AND PHYLOGENETIC DIVERSITY OF MARINE HETEROTROPHIC PROTISTS VIA SINGLE-CELL TRANSCRIPTOMICS

The comprehensive description of unicellular heterotrophic protists is essential for understanding the functioning of marine ecosystems and defining evolutionary relationships within marine microbial communities. For that reason, new insights into the functional genes of key protists, such as ciliates and dinoflagellates, are needed to complement the increasing taxonomic complexity and bridge the gap between various eco-functional processes in the ocean. In this study, single-cell transcriptomic sequencing proved to be an efficient method to create a snapshot of expressed genes of unicellular heterotrophs. We sequenced 65 single-cell transcriptomes from 20 fresh field samples collected from Sub-Arctic and North Sea waters. These 13 ciliate and 52 dinoflagellate transcriptomes will generally contribute to a greater understanding of functional and evolutionary processes of these marine protists. Further, we generated multi-gene phylogenies of several dozen genes to unravel the relationships of these heterotrophic taxa to other dinoflagellates and ciliates, respectively. These approaches also helped to elucidate the evolution of functional genes and traits for these understudied essential groups. Additionally, the datasets were incorporated into our metatranscriptomic reference database to fill the gap (of approx. 50%) of genomic information of heterotrophic organisms and their functional processes. Overall, identifying the phylogenetic relationships and functional diversity of heterotrophic and mixotrophic protists will clarify paramount marine microbial food web processes and provide clues to the system's sensitivity to climate change

Evolutionary distinctiveness of fatty acid and polyketide synthesis in eukaryotes

© 2016 International Society for Microbial Ecology All rights reserved. Fatty acids, which are essential cell membrane constituents and fuel storage molecules, are thought to share a common evolutionary origin with polyketide toxins in eukaryotes. While fatty acids are primary metabolic products, polyketide toxins are secondary metabolites that are involved in ecologically relevant processes, such as chemical defence, and produce the adverse effects of harmful algal blooms. Selection pressures on such compounds may be different, resulting in differing evolutionary histories. Surprisingly, some studies of dinoflagellates have suggested that the same enzymes may catalyse these processes. Here we show the presence and evolutionary distinctiveness of genes encoding six key enzymes essential for fatty acid production in 13 eukaryotic lineages for which no previous sequence data were available (alveolates: dinoflagellates, Vitrella, Chromera; stramenopiles: bolidophytes, chrysophytes, pelagophytes, raphidophytes, dictyochophytes, pinguiophytes, xanthophytes; Rhizaria: chlorarachniophytes, haplosporida; euglenids) and 8 other lineages (apicomplexans, bacillariophytes, synurophytes, cryptophytes, haptophytes, chlorophyceans, prasinophytes, trebouxiophytes). The phylogeny of fatty acid synthase genes reflects the evolutionary history of the organism, indicating selection to maintain conserved functionality. In contrast, polyketide synthase gene families are highly expanded in dinoflagellates and haptophytes, suggesting relaxed constraints in their evolutionary history, while completely absent from some protist lineages. This demonstrates a vast potential for the production of bioactive polyketide compounds in some lineages of microbial eukaryotes, indicating that the evolution of these compounds may have played an important role in their ecological success

Diversity and genetics of Australasian dinoflagellates, including Gambierdiscus spp. the causative agent of Ciguatera Fish Poisoning

Marine microbial protists from benthic habitats include species of the genera Gambierdiscus, which can produce polyketide toxins with ecosystem-wide impacts and impacts on human health. Knowledge of the presence, diversity, effects and distribution of benthic dinoflagellates and their toxins is vital both for basic ecological research and for managing potential impacts on human health. However, at present such information is limited. This study designed novel pyrosequencing-based tools that utilised 18S rRNA and mitochondrial cytochrome b barcoding marker genes. Using these tools, the largest set of dinoflagellate cytochrome b gene sequences to date was developed, and dinoflagellate species richness within marine benthic samples from five tropical marine benthic sites were determined. These tools were applied to samples from a temperate marine benthic site from southern NSW and the species Gambierdiscus carpenteri was found to be highly abundant throughout the region. This was unexpected, given that it is primarily considered a tropical species. The significance of finding this species is discussed in the context of long term monitoring data and its potential for toxin production.Species of Gambierdiscus produce two major toxin groups, ciguatoxins (CTXs) and maitotoxins (MTXs), that can accumulate via marine food chains in seafood and ultimately cause ciguatera fish poisoning (CFP) in humans. While the role of CTXs is well established in CFP, little is known about the role of MTXs. This work showed experimentally that MTXs can accumulate in liver and muscle tissues of carnivorous fish (Pagrus auratus). The levels of MTX detected in fish tissues showed for the first time that MTX may accumulate in fish at levels harmful for human consumption. In order to investigate the genetic basis of toxin biosynthesis in species of Gambierdiscus, two comprehensive transcriptomic libraries for the species Gambierdiscus australes and Gambierdiscus belizeanus, which produce MTXs were sequenced and assembled. Data analysis discovered three hundred and seven genes that formed eight clades within a phylogeny of dinoflagellate polyketide synthesis genes, and may be involved in MTX biosynthesis. This is the first step towards identifying prospective genes that may be involved in MTX biosynthesis, and provides a basis for the future development of molecular tools to assess the risk of CFP

Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates

A group of marine dinoflagellates (Alveolata, Eukaryota), consisting of similar to 10 species of the genus Alexandrium, Gymnodinium catenatum and Pyrodinium bahamense, produce the toxin saxitoxin and its analogues (STX), which can accumulate in shellfish, leading to ecosystem and human health impacts. The genes, sxt, putatively involved in STX biosynthesis, have recently been identified, however, the evolution of these genes within dinoflagellates is not clear. There are two reasons for this: uncertainty over the phylogeny of dinoflagellates; and that the sxt genes of many species of Alexandrium and other dinoflagellate genera are not known. Here, we determined the phylogeny of STX-producing and other dinoflagellates based on a concatenated eight-gene alignment. We determined the presence, diversity and phylogeny of sxtA, domains A1 and A4 and sxtG in 52 strains of Alexandrium, and a further 43 species of dinoflagellates and thirteen other alveolates. We confirmed the presence and high sequence conservation of sxtA, domain A4, in 40 strains (35 Alexandrium, 1 Pyrodinium, 4 Gymnodinium) of 8 species of STX-producing dinoflagellates, and absence from non-producing species. We found three paralogs of sxtA, domain A1, and a widespread distribution of sxtA1 in non-STX producing dinoflagellates, indicating duplication events in the evolution of this gene. One paralog, clade 2, of sxtA1 may be particularly related to STX biosynthesis. Similarly, sxtG appears to be generally restricted to STX-producing species, while three amidinotransferase gene paralogs were found in dinoflagellates. We investigated the role of positive (diversifying) selection following duplication in sxtA1 and sxtG, and found negative selection in clades of sxtG and sxtA1, clade 2, suggesting they were functionally constrained. Significant episodic diversifying selection was found in some strains in clade 3 of sxtA1, a clade that may not be involved in STX biosynthesis, indicating pressure for diversification of function. (C) 2015 Elsevier Inc. All rights reserved