Joshua Ramsay

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing–activated transfer of a mobile genetic element

Publisher: Proceedings of the National Academy of Sciences

Date: 18-03-2015

DOI: 10.1073/PNAS.1501574112

Abstract: Integrative and conjugative elements (ICEs) facilitate horizontal transfer of multiple genetic determinants. Here we show that a programmed ribosomal frameshift (PRF) contributes to the regulation of ICE transfer. The low-frequency PRF fuses the coding sequences of two genes, resulting in a single-protein Frameshifted excision activator (FseA) that activates ICE excision. An antiactivator, QseM, known to disrupt the quorum-sensing regulator TraR, also disrupted FseA. The evolved PRF site, together with the dual-target antiactivator, QseM, likely provides robust suppression of ICE transfer in the face of the inherent biological noise of quorum-sensing autoinduction. This work illustrates how a complex multipartite regulatory system has assembled through evolution to form a robust genetic toggle to control gene transcription and translation at both single-cell and cell-population levels.

An in vitro study into the antimicrobial and cytotoxic effect of Acticoat™ dressings supplemented with chlorhexidine

Publisher: Elsevier BV

Date: 06-2022

DOI: 10.1016/J.BURNS.2021.09.019

Rhizopine biosensors for plant‐dependent control of bacterial gene expression

Publisher: Wiley

Date: 04-12-2022

DOI: 10.1111/1462-2920.16288

Abstract: Engineering signalling between plants and microbes could be exploited to establish host‐specificity between plant‐growth‐promoting bacteria and target crops in the environment. We previously engineered rhizopine‐signalling circuitry facilitating exclusive signalling between rhizopine‐producing ( RhiP ) plants and model bacterial strains. Here, we conduct an in‐depth analysis of rhizopine‐inducible expression in bacteria. We characterize two rhizopine‐inducible promoters and explore the bacterial host‐range of rhizopine biosensor plasmids. By tuning the expression of rhizopine uptake genes, we also construct a new biosensor plasmid pSIR05 that has minimal impact on host cell growth in vitro and exhibits markedly improved stability of expression in situ on RhiP barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub‐population of Azorhizobium caulinodans cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing RhiP barley roots. However, these bacteria were mildly defective for colonization of RhiP barley roots compared to the wild‐type parent strain. This work provides advancement towards establishing more robust plant‐dependent control of bacterial gene expression and highlights the key challenges remaining to achieve this goal.

Draft Genome Sequence of Serratia sp. Strain ATCC 39006, a Model Bacterium for Analysis of the Biosynthesis and Regulation of Prodigiosin, a Carbapenem, and Gas Vesicles

Publisher: American Society for Microbiology

Date: 26-12-2013

DOI: 10.1128/GENOMEA.01039-13

Abstract: Serratia sp. strain ATCC 39006 is a Gram-negative bacterium and a member of the Enterobacteriaceae that produces various bioactive secondary metabolites, including the tripyrrole red pigment prodigiosin and the β-lactam antibiotic 1-carbapenen-2-em-3-carboxylic acid (a carbapenem). This strain is the only member of the Enterobacteriaceae known to naturally produce gas vesicles, as flotation organelles. Here we present the genome sequence of this strain, which has served as a model for analysis of the biosynthesis and regulation of antibiotic production.

Crystallographic and X-ray scattering study of RdfS, a recombination directionality factor from an integrative and conjugative element

Publisher: International Union of Crystallography (IUCr)

Date: 27-09-2022

DOI: 10.1107/S2059798322008579

Abstract: The recombination directionality factors from Mesorhizobium spp. (RdfS) are involved in regulating the excision and transfer of integrative and conjugative elements. Here, solution small-angle X-ray scattering, and crystallization and preliminary structure solution of RdfS from Mesorhizobium japonicum R7A are presented. RdfS crystallizes in space group P 2 1 2 1 2 1 , with evidence of eightfold rotational crystallographic/noncrystallographic symmetry. Initial structure determination by molecular replacement using ab initio models yielded a partial model (three molecules), which was completed after manual inspection revealed unmodelled electron density. The finalized crystal structure of RdfS reveals a head-to-tail polymer forming left-handed superhelices with large solvent channels. Additionally, RdfS has significant disorder in the C-terminal region of the protein, which is supported by the solution scattering data and the crystal structure. The steps taken to finalize structure determination, as well as the scattering and crystallographic characteristics of RdfS, are discussed.

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element

Publisher: Oxford University Press (OUP)

Date: 14-12-2021

DOI: 10.1093/NAR/GKAB1217

Abstract: Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer.

RNA-seq reveals the RNA binding proteins, Hfq and RsmA, play various roles in virulence, antibiotic production and genomic flux in Serratia sp. ATCC 39006

Publisher: Springer Science and Business Media LLC

Date: 22-11-2013

DOI: 10.1186/1471-2164-14-822

Abstract: Serratia sp. ATCC 39006 (S39006) is a Gram-negative enterobacterium that is virulent in plant and animal models. It produces a red-pigmented trypyrrole secondary metabolite, prodigiosin (Pig), and a carbapenem antibiotic (Car), as well as the exoenzymes, pectate lyase and cellulase. Secondary metabolite production in this strain is controlled by a complex regulatory network involving quorum sensing (QS). Hfq and RsmA (two RNA binding proteins and major post-transcriptional regulators of gene expression) play opposing roles in the regulation of several key phenotypes within S39006. Prodigiosin and carbapenem production was abolished, and virulence attenuated, in an S39006 ∆ hfq mutant, while the converse was observed in an S39006 rsmA transposon insertion mutant. In order to define the complete regulon of Hfq and RsmA, deep sequencing of cDNA libraries (RNA-seq) was used to analyse the whole transcriptome of S39006 ∆ hfq and rsmA ::Tn mutants. Moreover, we investigated global changes in the proteome using an LC-MS/MS approach. Analysis of differential gene expression showed that Hfq and RsmA directly or indirectly regulate (at the level of RNA) 4% and 19% of the genome, respectively, with some correlation between RNA and protein expression. Pathways affected include those involved in antibiotic regulation, virulence, flagella synthesis, and surfactant production. Although Hfq and RsmA are reported to activate flagellum production in E. coli and an adherent-invasive E. coli hfq mutant was shown to have no flagella by electron microscopy, we found that flagellar production was increased in the S39006 rsmA and hfq mutants. Additionally, deletion of rsmA resulted in greater genomic flux with increased activity of two mobile genetic elements. This was confirmed by qPCR and analysis of rsmA culture supernatant revealed the presence of prophage DNA and phage particles. Finally, expression of a hypothetical protein containing DUF364 increased prodigiosin production and was controlled by a putative 5′ cis -acting regulatory RNA element. Using a combination of transcriptomics and proteomics this study provides a systems-level understanding of Hfq and RsmA regulation and identifies similarities and differences in the regulons of two major regulators. Additionally our study indicates that RsmA regulates both core and variable genome regions and contributes to genome stability.

Population genomics of Australian indigenous Mesorhizobium reveals diverse nonsymbiotic genospecies capable of nitrogen-fixing symbioses following horizontal gene transfer

Publisher: Microbiology Society

Date: 05-01-2023

DOI: 10.1099/MGEN.0.000918

Abstract: Mesorhizobia are soil bacteria that establish nitrogen-fixing symbioses with various legumes. Novel symbiotic mesorhizobia frequently evolve following horizontal transfer of symbiosis-gene-carrying integrative and conjugative elements (ICESyms) to indigenous mesorhizobia in soils. Evolved symbionts exhibit a wide range in symbiotic effectiveness, with some fixing nitrogen poorly or not at all. Little is known about the genetic ersity and symbiotic potential of indigenous soil mesorhizobia prior to ICESym acquisition. Here we sequenced genomes of 144 Mesorhizobium spp. strains cultured directly from cultivated and uncultivated Australian soils. Of these, 126 lacked symbiosis genes. The only isolated symbiotic strains were either exotic strains used previously as legume inoculants, or indigenous mesorhizobia that had acquired exotic ICESyms. No native symbiotic strains were identified. Indigenous nonsymbiotic strains formed 22 genospecies with phylogenomic ersity overlapping the ersity of internationally isolated symbiotic Mesorhizobium spp. The genomes of indigenous mesorhizobia exhibited no evidence of prior involvement in nitrogen-fixing symbiosis, yet their core genomes were similar to symbiotic strains and they generally lacked genes for synthesis of biotin, nicotinate and thiamine. Genomes of nonsymbiotic mesorhizobia harboured similar mobile elements to those of symbiotic mesorhizobia, including ICESym-like elements carrying aforementioned vitamin-synthesis genes but lacking symbiosis genes. Diverse indigenous isolates receiving ICESyms through horizontal gene transfer formed effective symbioses with Lotus and Biserrula legumes, indicating most nonsymbiotic mesorhizobia have an innate capacity for nitrogen-fixing symbiosis following ICESym acquisition. Non-fixing ICESym-harbouring strains were isolated sporadically within species alongside effective symbionts, indicating chromosomal lineage does not predict symbiotic potential. Our observations suggest previously observed genomic ersity amongst symbiotic Mesorhizobium spp. represents a fraction of the extant ersity of nonsymbiotic strains. The overlapping phylogeny of symbiotic and nonsymbiotic clades suggests major clades of Mesorhizobium erged prior to introduction of symbiosis genes and therefore chromosomal genes involved in symbiosis have evolved largely independent of nitrogen-fixing symbiosis.

Identification and characterisation of fosfomycin resistance in Escherichia coli urinary tract infection isolates from Australia

Publisher: Elsevier BV

Date: 10-2020

DOI: 10.1016/J.IJANTIMICAG.2020.106121

Assembly and transfer of tripartite integrative and conjugative genetic elements

Publisher: Proceedings of the National Academy of Sciences

Date: 12-10-2016

DOI: 10.1073/PNAS.1613358113

Abstract: Integrative and conjugative elements (ICEs) are one of the most prevalent but least-characterized families of mobile genetic elements in bacteria. We identified a family of ICEs that exists as three separate parts integrated within the single chromosomes of symbiotic mesorhizobia. These “tripartite ICEs,” through a series of chromosomal recombinations mediated by integrase proteins, assemble into a single circular ICE. Following transfer to nonsymbiotic mesorhizobia, tripartite ICEs integrate and disassemble into three parts in the recipient genome and exconjugant mesorhizobia gain the ability to form a symbiosis with legumes. These discoveries expand our appreciation of the potential for gene transfer in bacteria and demonstrate how mobile genetic elements can dramatically manipulate the bacterial genome.

A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

Publisher: Frontiers Media SA

Date: 22-12-2015

DOI: 10.3389/FMICB.2015.01442

Evolution of a 72-Kilobase Cointegrant, Conjugative Multiresistance Plasmid in Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates from the Early 1990s

Publisher: American Society for Microbiology

Date: 11-2019

DOI: 10.1128/AAC.01560-19

Abstract: Horizontal transfer of plasmids encoding antimicrobial resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s, the first CA-MRSA strain isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline, and penicillin resistance genes on plasmid pWBG753 (∼30 kb).

Complete Genome Sequence of Stenotrophomonas maltophilia AB550, an Environmental Solar Radiation- and Multidrug-Resistant Strain Isolated in Western Australia

Publisher: American Society for Microbiology

Date: 09-08-2018

DOI: 10.1128/MRA.00914-18

Abstract: We report here the complete genome sequence of Stenotrophomonas maltophilia AB550, a multidrug- and solar radiation-resistant strain isolated from the effluents of an urban wastewater treatment plant in Western Australia. The genome consists of a single 4.9-Mb chromosome.

Comparative analysis of integrative and conjugative mobile genetic elements in the genus Mesorhizobium

Publisher: Microbiology Society

Date: 04-10-2021

DOI: 10.1099/MGEN.0.000657

Abstract: Members of the Mesorhizobium genus are soil bacteria that often form nitrogen-fixing symbioses with legumes. Most characterised Mesorhizobium spp. genomes are ~8 Mb in size and harbour extensive pangenomes including large integrative and conjugative elements (ICEs) carrying genes required for symbiosis (ICESyms). Here, we document and compare the conjugative mobilome of 41 complete Mesorhizobium genomes. We delineated 56 ICEs and 24 integrative and mobilizable elements (IMEs) collectively occupying 16 distinct integration sites, along with 24 plasmids. We also demonstrated horizontal transfer of the largest (853,775 bp) documented ICE, the tripartite ICE M spSym AA22 . The conjugation systems of all identified ICEs and several plasmids were related to those of the paradigm ICESym ICE Ml Sym R7A , with each carrying conserved genes for conjugative pilus formation ( trb ), excision ( rdfS ), DNA transfer ( rlxS ) and regulation ( fseA ). ICESyms have likely evolved from a common ancestor, despite occupying a variety of distinct integration sites and specifying symbiosis with erse legumes. We found extensive evidence for recombination between ICEs and particularly ICESyms, which all uniquely lack the conjugation entry-exclusion factor gene trbK . Frequent duplication, replacement and pseudogenization of genes for quorum-sensing-mediated activation and antiactivation of ICE transfer suggests ICE transfer regulation is constantly evolving. Pangenome-wide association analysis of the ICE identified genes potentially involved in symbiosis, rhizosphere colonisation and/or adaptation to distinct legume hosts. In summary, the Mesorhizobium genus has accumulated a large and dynamic pangenome that evolves through ongoing horizontal gene transfer of large conjugative elements related to ICE Ml Sym R7A .

Symbiosis islands of Loteae-nodulating Mesorhizobium comprise three radiating lineages with concordant nod gene complements and nodulation host-range groupings

Publisher: Microbiology Society

Date: 09-2020

DOI: 10.1099/MGEN.0.000426

Abstract: Mesorhizobium is a genus of soil bacteria, some isolates of which form an endosymbiotic relationship with erse legumes of the Loteae tribe. The symbiotic genes of these mesorhizobia are generally carried on integrative and conjugative elements termed symbiosis islands (ICESyms). Mesorhizobium strains that nodulate Lotus spp. have been ided into host-range groupings. Group I (GI) strains nodulate L. corniculatus and L. japonicus ecotype Gifu, while group II (GII) strains have a broader host range, which includes L. pedunculatus . To identify the basis of this extended host range, and better understand Mesorhizobium and ICESym genomics, the genomes of eight Mesorhizobium strains were completed using hybrid long- and short-read assembly. Bioinformatic comparison with previously sequenced mesorhizobia genomes indicated host range was not predicted by Mesorhizobium genospecies but rather by the evolutionary relationship between ICESym symbiotic regions. Three radiating lineages of Loteae ICESyms were identified on this basis, which correlate with Lotus spp. host-range grouping and have lineage-specific nod gene complements. Pangenomic analysis of the completed GI and GII ICESyms identified 155 core genes (on average 30.1 % of a given ICESym). In idual GI or GII ICESyms carried erse accessory genes with an average of 34.6 % of genes unique to a given ICESym. Identification and comparative analysis of NodD symbiotic regulatory motifs – nod boxes – identified 21 branches across the NodD regulons. Four of these branches were associated with seven genes unique to the five GII ICESyms. The nod boxes preceding the host-range gene nodZ in GI and GII ICESyms were disparate, suggesting regulation of nodZ may differ between GI and GII ICESyms. The broad host-range determinant(s) of GII ICESyms that confer nodulation of L. pedunculatus are likely present amongst the 53 GII-unique genes identified.

Complete Genome Sequences of Three of the Earliest Community-Associated Methicillin-Resistant Staphylococcus aureus Strains Isolated in Remote Western Australia

Publisher: American Society for Microbiology

Date: 16-09-2021

DOI: 10.1128/MRA.00797-21

Abstract: Initially reported in Western Australia in the 1980s, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a major cause of S. aureus infections globally. We report the complete genome sequences of three of the earliest CA-MRSA strains isolated from remote Australian Indigenous communities in the Kimberley region of Western Australia.

Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements

Publisher: Public Library of Science (PLoS)

Date: 22-03-2018

DOI: 10.1371/JOURNAL.PGEN.1007292

DUF2285 is a novel helix-turn-helix domain variant that orchestrates both activation and antiactivation of conjugative element transfer in proteobacteria

Publisher: Oxford University Press (OUP)

Date: 29-05-2023

DOI: 10.1093/NAR/GKAD457

Abstract: Horizontal gene transfer is tightly regulated in bacteria. Often only a fraction of cells become donors even when regulation of horizontal transfer is coordinated at the cell population level by quorum sensing. Here, we reveal the widespread ‘domain of unknown function’ DUF2285 represents an ‘extended-turn’ variant of the helix-turn-helix domain that participates in both transcriptional activation and antiactivation to initiate or inhibit horizontal gene transfer. Transfer of the integrative and conjugative element ICEMlSymR7A is controlled by the DUF2285-containing transcriptional activator FseA. One side of the DUF2285 domain of FseA has a positively charged surface which is required for DNA binding, while the opposite side makes critical interdomain contacts with the N-terminal FseA DUF6499 domain. The QseM protein is an antiactivator of FseA and is composed of a DUF2285 domain with a negative surface charge. While QseM lacks the DUF6499 domain, it can bind the FseA DUF6499 domain and prevent transcriptional activation by FseA. DUF2285-domain proteins are encoded on mobile elements throughout the proteobacteria, suggesting regulation of gene transfer by DUF2285 domains is a widespread phenomenon. These findings provide a striking ex le of how antagonistic domain paralogues have evolved to provide robust molecular control over the initiation of horizontal gene transfer.

Statin therapy causes gut dysbiosis in mice through a PXR-dependent mechanism

Publisher: Springer Science and Business Media LLC

Date: 09-08-2017

DOI: 10.1186/S40168-017-0312-4

Complete Genome Sequence of Community-Associated Methicillin-Resistant Staphylococcus aureus Sequence Type 1, SCC mec IV[2B], Isolated in the 1990s from Northern Western Australia

Publisher: American Society for Microbiology

Date: 16-09-2021

DOI: 10.1128/MRA.00796-21

Abstract: Sequence type 1 (ST1) methicillin-resistant Staphylococcus aureus (MRSA) type IV[2B] has become one of the most common community-associated MRSA clones in Australia. We report the complete genome sequence of one of the earliest isolated Australian S. aureus ST1-MRSA-IV strains, WBG8287, isolated from an Indigenous Australian patient living in the remote Kimberley Region of Western Australia.

Delineation of the integrase-attachment and origin-of-transfer regions of the symbiosis island ICEMlSymR7A

Publisher: Elsevier BV

Date: 07-2019

DOI: 10.1016/J.PLASMID.2019.102416

Abstract: Integrative and conjugative elements (ICEs) are chromosomally-integrated mobile genetic elements that excise from their host chromosome and transfer to other bacteria via conjugation. ICEMlSym

University Of Otago

Location: New Zealand

View Organisation

University Of Western Australia

Location: Australia

View Organisation

Curtin University

Location: Australia

View Organisation

University Of Cambridge

Location: United Kingdom of Great Britain and Northern Ireland

View Organisation

University Of Otago Health Sciences Career Development Programme Postdoctoral Fellowship

Start Date: 2011

End Date: 2013

Funder: University of Otago

Silencing Unwanted Expression In Molecular Circuits Using Naturally Evolved Solutions

Start Date: 2017

End Date: 2020

Funder: Marsden Fund

Redefining Antibiotic Resistance Plasmid Transfer In Staphylococcus Aureus

Start Date: 2018

End Date: 2020

Funder: National Health and Medical Research Council

Increasing The Effectiveness Of Nitrogen Fixation In Pulses Through Improved Rhizobial Strains In The GRDC Western Region

Start Date: 2018

End Date: 2023

Funder: Grains Research and Development Corporation

Herchel Smith Postdoctoral Fellowship

Start Date: 2008

End Date: 2011

Funder: University of Cambridge

From Powders To Proteins: Improving Diffraction Science In Western Australia

Start Date: 2017

End Date: 2017

Funder: Australian Research Council

Harnessing Horizontal Gene Transfer For Sustainable Nitrogen Fixation

Start Date: 2017

End Date: 2017

Funder: Australian Research Council

Returning WA Rapid Acquisition Fluorescent Microscopy To The Cutting Edge.

Start Date: 2019

End Date: 2020

Funder: Australian Research Council

ARC Future Fellowships - Grant ID: FT170100235

Start Date: 01-2018

End Date: 01-2024

Amount: $780,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE230100156

Start Date: 2023

End Date: 12-2023

Amount: $390,195.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE200100122

Start Date: 08-2020

End Date: 08-2021

Amount: $620,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100199

Start Date: 04-2017

End Date: 12-2018

Amount: $700,000.00

Funder: Australian Research Council