Rapid dissemination of host metabolism-manipulating genes via integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.

Genomic characterization of a unique Panton-Valentine leucocidin-positive community-associated methicillin-resistant Staphylococcus aureus lineage increasingly impacting on Australian indigenous communities.

In 2010 a single isolate of a trimethoprim-resistant multilocus sequence type 5, Panton-Valentine leucocidin-positive, community-associated methicillin-resistant Staphylococcus aureus (PVL-positive ST5 CA-MRSA), colloquially named WA121, was identified in northern Western Australia (WA). WA121 now accounts for ~14 % of all WA MRSA infections. To gain an understanding of the genetic composition and phylogenomic structure of WA121 isolates we sequenced the genomes of 155 WA121 isolates collected 2010-2021 and present a detailed genomic description. WA121 was revealed to be a single clonally expanding lineage clearly distinct from sequenced ST5 strains reported outside Australia. WA121 strains were typified by the presence of the distinct PVL phage φSa2wa-st5, the recently described methicillin resistance element SCCmecIVo carrying the trimethoprim resistance (dfrG) transposon Tn4791, the novel ß-lactamase transposon Tn7702 and the epidermal cell differentiation inhibitor (EDIN-A) plasmid p2010-15611-2. We present evidence that SCCmecIVo together with Tn4791 has horizontally transferred to Staphylococcus argenteus and evidence of intragenomic movement of both Tn4791 and Tn7702. We experimentally demonstrate that p2010-15611-2 is capable of horizontal transfer by conjugative mobilization from one of several WA121 isolates also harbouring a pWBG749-like conjugative plasmid. In summary, WA121 is a distinct and clonally expanding Australian PVL-positive CA-MRSA lineage that is increasingly responsible for infections in indigenous communities in northern and western Australia. WA121 harbours a unique complement of mobile genetic elements and is capable of transferring antimicrobial resistance and virulence determinants to other staphylococci.

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

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 diversity 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 diversity overlapping the diversity 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 diversity amongst symbiotic Mesorhizobium spp. represents a fraction of the extant diversity of nonsymbiotic strains. The overlapping phylogeny of symbiotic and nonsymbiotic clades suggests major clades of Mesorhizobium diverged prior to introduction of symbiosis genes and therefore chromosomal genes involved in symbiosis have evolved largely independent of nitrogen-fixing symbiosis.

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

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.

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

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 ICEMspSymAA22. The conjugation systems of all identified ICEs and several plasmids were related to those of the paradigm ICESym ICEMlSymR7A, 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 diverse 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 ICEMlSymR7A.

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

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.

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

Sequence type 1 (ST1) methicillin-resistant Staphylococcus aureus (MRSA) SCCmec 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.

Evolution of diverse effective N2-fixing microsymbionts of Cicer arietinum following horizontal transfer of the Mesorhizobium ciceri CC1192 symbiosis integrative and conjugative element.

Rhizobia are soil bacteria capable of forming N2-fixing symbioses with legumes, with highly effective strains often selected in agriculture as inoculants to maximize symbiotic N2 fixation. When rhizobia in the genus Mesorhizobium have been introduced with exotic legumes into farming systems, horizontal transfer of symbiosis Integrative and Conjugative Elements (ICEs) from the inoculant strain to soil bacteria has resulted in the evolution of ineffective N2-fixing rhizobia that are competitive for nodulation with the target legume. In Australia, Cicer arietinum (chickpea) has been inoculated since the 1970's with Mesorhizobium ciceri sv. ciceri CC1192, a highly effective strain from Israel. Although the full genome sequence of this organism is available, little is known about the mobility of its symbiosis genes and the diversity of cultivated C. arietinum-nodulating organisms. Here, we show the CC1192 genome harbors a 419-kb symbiosis ICE (ICEMcSym1192) and a 648-kb repABC-type plasmid pMC1192 carrying putative fix genes. We sequenced the genomes of 11 C. arietinum nodule isolates from a field site exclusively inoculated with CC1192 and showed they were diverse unrelated Mesorhizobium carrying ICEMcSym1192, indicating they had acquired the ICE by environmental transfer. No exconjugants harboured pMc1192 and the plasmid was not essential for N2 fixation in CC1192. Laboratory conjugation experiments confirmed ICEMcSym1192 is mobile, integrating site-specifically within the 3' end of one of the four ser-tRNA genes in the R7ANS recipient genome. Strikingly, all ICEMcSym1192 exconjugants were as efficient at fixing N2 with C. arietinum as CC1192, demonstrating ICE transfer does not necessarily yield ineffective microsymbionts as previously observed.Importance Symbiotic N2 fixation is a key component of sustainable agriculture and in many parts of the world legumes are inoculated with highly efficient strains of rhizobia to maximise fixed N2 inputs into farming systems. Symbiosis genes for Mesorhizobium spp. are often encoded chromosomally within mobile gene clusters called Integrative and Conjugative Elements or ICEs. In Australia, where all agricultural legumes and their rhizobia are exotic, horizontal transfer of ICEs from inoculant Mesorhizobium strains to native rhizobia has led to the evolution of inefficient strains that outcompete the original inoculant, with the potential to render it ineffective. However, the commercial inoculant strain for Cicer arietinum (chickpea), M. ciceri CC1192, has a mobile symbiosis ICE (ICEMcSym1192) which can support high rates of N2 fixation following either environmental or laboratory transfer into diverse Mesorhizobium backgrounds, demonstrating ICE transfer does not necessarily yield ineffective microsymbionts as previously observed.

Population Genomics of Bacterial Plant Pathogens.

Population genomics is transforming our understanding of pathogen biology and evolution, and contributing to the prevention and management of disease in diverse crops. We provide an overview of key methods in bacterial population genomics and describe recent work focusing on three topics of critical importance to plant pathology: (i) resolving pathogen origins and transmission pathways during outbreak events, (ii) identifying the genetic basis of host specificity and virulence, and (iii) understanding how pathogens evolve in response to changing agricultural practices.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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

Mesorhizobium is a genus of soil bacteria, some isolates of which form an endosymbiotic relationship with diverse 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 divided 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). Individual GI or GII ICESyms carried diverse 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.

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

Of 1033 Escherichia coli urinary tract infection isolates collected from females >12 years of age in Australia in 2019, only 2 isolates were resistant to fosfomycin with a minimum inhibitory concentration (MIC) of >256 mg/L. Despite having different multilocus sequence types, the two isolates harboured an identical plasmid-encoded fosA4 gene. The fosA4 gene has previously been identified in a single clinical E. coli isolate cultured in Japan in 2014. Each fosfomycin-resistant isolate harboured two conjugative plasmids that possessed an array of genes conferring resistance to aminoglycosides, ß-lactams, macrolides, quinolones, sulfonamides and/or trimethoprim.

The ecological genetics of Pseudomonas syringae from kiwifruit leaves.

Interactions between commensal microbes and invading pathogens are understudied, despite their likely effects on pathogen population structure and infection processes. We describe the population structure and genetic diversity of a broad range of co-occurring Pseudomonas syringae isolated from infected and uninfected kiwifruit during an outbreak of bleeding canker disease caused by P. syringae pv. actinidiae (Psa) in New Zealand. Overall population structure was clonal and affected by ecological factors including infection status and cultivar. Most isolates are members of a new clade in phylogroup 3 (PG3a), also present on kiwifruit leaves in China and Japan. Stability of the polymorphism between pathogenic Psa and commensal P. syringae PG3a isolated from the same leaf was tested using reciprocal invasion from rare assays in vitro and in planta. P. syringae G33C (PG3a) inhibited Psa NZ54, while the presence of Psa NZ54 enhanced the growth of P. syringae G33C. This effect could not be attributed to virulence activity encoded by the Type 3 secretion system of Psa. Together our data contribute toward the development of an ecological perspective on the genetic structure of pathogen populations.

Origin and Evolution of the Kiwifruit Canker Pandemic.

Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa). In order to strengthen understanding of population structure, phylogeography, and evolutionary dynamics, we isolated Pseudomonas from cultivated and wild kiwifruit across six provinces in China. Based on the analysis of 80 sequenced Psa genomes, we show that China is the origin of the pandemic lineage but that strain diversity in China is confined to just a single clade. In contrast, Korea and Japan harbor strains from multiple clades. Distinct independent transmission events marked introduction of the pandemic lineage into New Zealand, Chile, Europe, Korea, and Japan. Despite high similarity within the core genome and minimal impact of within-clade recombination, we observed extensive variation even within the single clade from which the global pandemic arose.

Evolution of copper resistance in the kiwifruit pathogen Pseudomonas syringae pv. actinidiae through acquisition of integrative conjugative elements and plasmids.

Horizontal gene transfer can precipitate rapid evolutionary change. In 2010 the global pandemic of kiwifruit canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) reached New Zealand. At the time of introduction, the single clone responsible for the outbreak was sensitive to copper, however, analysis of a sample of isolates taken in 2015 and 2016 showed that a quarter were copper resistant. Genome sequences of seven strains showed that copper resistance - comprising czc/cusABC and copABCD systems - along with resistance to arsenic and cadmium, was acquired via uptake of integrative conjugative elements (ICEs), but also plasmids. Comparative analysis showed ICEs to have a mosaic structure, with one being a tripartite arrangement of two different ICEs and a plasmid that were isolated in 1921 (USA), 1968 (NZ) and 1988 (Japan), from P. syringae pathogens of millet, wheat and kiwifruit respectively. Two of the Psa ICEs were nearly identical to two ICEs isolated from kiwifruit leaf colonists prior to the introduction of Psa into NZ. Additionally, we show ICE transfer in vitro and in planta, analyze fitness consequences of ICE carriage, capture the de novo formation of novel recombinant ICEs, and explore ICE host-range.