Stable, fluorescent markers for tracking synthetic communities and assembly dynamics.

BACKGROUND: After two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom. RESULTS: To address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots. CONCLUSIONS: DFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly. Video Abstract.

Rhizopine biosensors for plant-dependent control of bacterial gene expression.

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.

Engineered plant control of associative nitrogen fixation.

Engineering N2-fixing symbioses between cereals and diazotrophic bacteria represents a promising strategy to sustainably deliver biologically fixed nitrogen (N) in agriculture. We previously developed novel transkingdom signaling between plants and bacteria, through plant production of the bacterial signal rhizopine, allowing control of bacterial gene expression in association with the plant. Here, we have developed both a homozygous rhizopine producing (RhiP) barley line and a hybrid rhizopine uptake system that conveys upon our model bacterium Azorhizobium caulinodans ORS571 (Ac) 103-fold improved sensitivity for rhizopine perception. Using this improved genetic circuitry, we established tight rhizopine-dependent transcriptional control of the nitrogenase master regulator nifA and the N metabolism σ-factor rpoN, which drove nitrogenase expression and activity in vitro and in situ by bacteria colonizing RhiP barley roots. Although in situ nitrogenase activity was suboptimally effective relative to the wild-type strain, activation was specific to RhiP barley and was not observed on the roots of wild-type plants. This work represents a key milestone toward the development of a synthetic plant-controlled symbiosis in which the bacteria fix N2 only when in contact with the desired host plant and are prevented from interaction with nontarget plant species.

A Simple in situ Assay to Assess Plant-Associative Bacterial Nitrogenase Activity.

Assessment of plant-associative bacterial nitrogen (N) fixation is crucial for selection and development of elite diazotrophic inoculants that could be used to supply cereal crops with nitrogen in a sustainable manner. Although diazotrophic bacteria possess diverse oxygen tolerance mechanisms, most require a sub 21% oxygen environment to achieve optimal stability and function of the N-fixing catalyst nitrogenase. Consequently, assessment of N fixation is routinely carried out on "free-living" bacteria grown in the absence of a host plant and such experiments may not accurately divulge activity in the rhizosphere where the availability and forms of nutrients such as carbon and N, which are key regulators of N fixation, may vary widely. Here, we present a modified in situ acetylene reduction assay (ARA), utilizing the model cereal barley as a host to comparatively assess nitrogenase activity in diazotrophic bacteria. The assay is rapid, highly reproducible, applicable to a broad range of diazotrophs, and can be performed with simple equipment commonly found in most laboratories that investigate plant-microbe interactions. Thus, the assay could serve as a first point of order for high-throughput identification of elite plant-associative diazotrophs.

Deciphering bacterial mechanisms of root colonization.

Bacterial colonization of the rhizosphere is critical for the establishment of plant-bacteria interactions that represent a key determinant of plant health and productivity. Plants influence bacterial colonization primarily through modulating the composition of their root exudates and mounting an innate immune response. The outcome is a horizontal filtering of bacteria from the surrounding soil, resulting in a gradient of reduced bacterial diversity coupled with a higher degree of bacterial specialization towards the root. Bacteria-bacteria interactions (BBIs) are also prevalent in the rhizosphere, influencing bacterial persistence and root colonization through metabolic exchanges, secretion of antimicrobial compounds and other processes. Traditionally, bacterial colonization has been examined under sterile laboratory conditions that mitigate the influence of BBIs. Using simplified synthetic bacterial communities combined with microfluidic imaging platforms and transposon mutagenesis screening approaches, we are now able to begin unravelling the molecular mechanisms at play during the early stages of root colonization. This review explores the current state of knowledge regarding bacterial root colonization and identifies key tools for future exploration.

Engineering rhizobacteria for sustainable agriculture.

Exploitation of plant growth promoting (PGP) rhizobacteria (PGPR) as crop inoculants could propel sustainable intensification of agriculture to feed our rapidly growing population. However, field performance of PGPR is typically inconsistent due to suboptimal rhizosphere colonisation and persistence in foreign soils, promiscuous host-specificity, and in some cases, the existence of undesirable genetic regulation that has evolved to repress PGP traits. While the genetics underlying these problems remain largely unresolved, molecular mechanisms of PGP have been elucidated in rigorous detail. Engineering and subsequent transfer of PGP traits into selected efficacious rhizobacterial isolates or entire bacterial rhizosphere communities now offers a powerful strategy to generate improved PGPR that are tailored for agricultural use. Through harnessing of synthetic plant-to-bacteria signalling, attempts are currently underway to establish exclusive coupling of plant-bacteria interactions in the field, which will be crucial to optimise efficacy and establish biocontainment of engineered PGPR. This review explores the many ecological and biotechnical facets of this research.

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

Tripartite integrative and conjugative elements (ICE3) are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases-or recombination directionality factors-RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a "master controller" of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer.

Complete Genome Sequence of Mesorhizobium ciceri bv. biserrulae WSM1497, an Efficient Nitrogen-Fixing Microsymbiont of the Forage Legume Biserrula pelecinus.

We report here the complete genome sequence of Mesorhizobium ciceri bv. biserrulae strain WSM1497, the efficient nitrogen-fixing microsymbiont and commercial inoculant in Australia of the forage legume Biserrula pelecinus The genome consists of 7.2 Mb distributed across a single chromosome (6.67 Mb) and a single plasmid (0.53 Mb).

Evolutionary persistence of tripartite integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are generally regarded as regions of contiguous DNA integrated within a bacterial genome that are capable of excision and horizontal transfer via conjugation. We recently characterized a unique group of ICEs present in Mesorhizobium spp., which exist as three entirely separate but inextricably linked chromosomal regions termed α, ß and γ. These regions occupy three different recombinase attachment (att) sites; however, they do not excise independently. Rather, they recombine the host chromosome to form a single contiguous region prior to excision and conjugative transfer. Like the single-part ICE carried by M. loti R7A (ICEMlSymR7A), these "tripartite" ICEs (ICE3s) are widespread throughout the Mesorhizobium genus and enable strains to form nitrogen-fixing symbioses with a variety of legumes. ICE3s have likely evolved following recombination between three separate ancestral integrative elements, however, the persistence of ICE3 structure in diverse mesorhizobia is perplexing due to its seemingly unnecessary complexity. In this study, examination of ICE3s revealed that most symbiosis genes are carried on the large α fragment. Some ICE3-ß and γ regions also carry genes that potentially contribute to the symbiosis, or to persistence in the soil environment, but these regions have been frequently subjected to recombination events including deletions, insertions and recombination with genes located on other integrative elements. Examination of a new ICE3 in M. ciceri Ca181 revealed it has jettisoned the genetic cargo from its ß region and recruited a serine recombinase gene within its γ region, resulting in replacement of one of the three ICE3 integration sites. Overall the recombination loci appear to be the only conserved features of the ß and γ regions, suggesting that the tripartite structure itself provides a selective benefit to the element. We propose the ICE3 structure provides enhanced host range, host stability and resistance to destabilization by tandem insertion of competing integrative elements. Furthermore, we suspect the ICE3 tripartite structure increases the likelihood of gene capture from integrative elements sharing the same attachment sites.

Assembly and transfer of tripartite integrative and conjugative genetic elements.

Integrative and conjugative elements (ICEs) are ubiquitous mobile genetic elements present as "genomic islands" within bacterial chromosomes. Symbiosis islands are ICEs that convert nonsymbiotic mesorhizobia into symbionts of legumes. Here we report the discovery of symbiosis ICEs that exist as three separate chromosomal regions when integrated in their hosts, but through recombination assemble as a single circular ICE for conjugative transfer. Whole-genome comparisons revealed exconjugants derived from nonsymbiotic mesorhizobia received three separate chromosomal regions from the donor Mesorhizobium ciceri WSM1271. The three regions were each bordered by two nonhomologous integrase attachment (att) sites, which together comprised three homologous pairs of attL and attR sites. Sequential recombination between each attL and attR pair produced corresponding attP and attB sites and joined the three fragments to produce a single circular ICE, ICEMcSym1271 A plasmid carrying the three attP sites was used to recreate the process of tripartite ICE integration and to confirm the role of integrase genes intS, intM, and intG in this process. Nine additional tripartite ICEs were identified in diverse mesorhizobia and transfer was demonstrated for three of them. The transfer of tripartite ICEs to nonsymbiotic mesorhizobia explains the evolution of competitive but suboptimal N2-fixing strains found in Western Australian soils. The unheralded existence of tripartite ICEs raises the possibility that multipartite elements reside in other organisms, but have been overlooked because of their unusual biology. These discoveries reveal mechanisms by which integrases dramatically manipulate bacterial genomes to allow cotransfer of disparate chromosomal regions.