ORCID Profile
0000-0003-4675-6009
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Publisher: Wiley
Date: 04-12-2022
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.
Publisher: Frontiers Media SA
Date: 23-06-2021
DOI: 10.3389/FMICB.2021.690439
Abstract: 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 erse 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 ulge 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.
Publisher: Springer Science and Business Media LLC
Date: 23-11-2020
Publisher: Cold Spring Harbor Laboratory
Date: 13-04-2022
DOI: 10.1101/2022.04.13.488174
Abstract: Due to the costly energy demands of N 2 fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the N 2 -fixing catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity NH 3 assimilation by glutamine synthetase (GS), preventing excess release of excess NH 3 for plants. Diazotrophic bacteria can be engineered to excrete NH 3 by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH 3 to non-target plants. Here, we attempted two strategies to control GS regulation and NH 3 excretion in our model cereal symbiont Azorhizobium caulinodans Ac LP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P II homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyltransferases (uATs) in a Δ glnE mutant of Ac LP which permitted strong GS shutdown and excretion of NH 3 derived from N 2 fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter P nifH , permitting GS shutdown and NH 3 excretion specifically under microaerobic conditions, the same cue that initiates N 2 fixation, then deleted nifA and transferred a rhizopine-inducible nifA L94Q/D95Q -rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N 2 fixation with NH 3 excretion. In future, this highly sophisticated and multi-layered control circuitry could be used to activate N 2 fixation and NH 3 excretion specifically by Ac LP colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop, and preventing interaction with non-target plants. Inoculation of cereal crops with associative “diazotrophic” bacteria that convert atmospheric N 2 to NH 3 could be used to sustainably improve delivery of nitrogen in agriculture. However, due to the costly energy demands of N 2 fixation, natural diazotrophic bacteria have evolved to conserve energy by preventing excess production of NH 3 and release to the plants. Diazotrophs can be engineered for excess NH 3 production and release, however genetic control is required to minimise growth penalties and prevent unintended provision of NH 3 to non-target weed species. Here, we engineer control of N 2 fixation and NH 3 excretion in response to the signalling molecule rhizopine which is produced by transgenic barley. This control could be used to establish plant host-specific activation of N 2 fixation and NH 3 release following root colonisation in the field, minimising bacterial energy requirements in the bulk soil and preventing provision of NH 3 to non-target plants.
Publisher: Proceedings of the National Academy of Sciences
Date: 12-10-2016
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.
Publisher: American Society for Microbiology
Date: 31-08-2017
Abstract: 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).
Publisher: American Society for Microbiology
Date: 12-02-2021
DOI: 10.1128/AEM.02558-20
Abstract: Symbiotic N 2 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 maximize fixed N 2 inputs into farming systems. Symbiosis genes for Mesorhizobium spp. are often carried chromosomally within mobile gene clusters called ICEs.
Publisher: Public Library of Science (PLoS)
Date: 21-06-2022
DOI: 10.1371/JOURNAL.PGEN.1010276
Abstract: Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH 3 ) assimilation by glutamine synthetase (GS), preventing excess release of excess NH 3 for plants. Diazotrophic bacteria can be engineered to excrete NH 3 by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH 3 to non-target plants. Here, we tested two strategies to control GS regulation and NH 3 excretion in our model cereal symbiont Azorhizobium caulinodans Ac LP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P II homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a Δ glnE mutant of Ac LP which permitted strong GS shutdown and excretion of NH 3 derived from N 2 fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter P nifH , permitting GS shutdown and NH 3 excretion specifically under microaerobic conditions, the same cue that initiates N 2 fixation, then deleted nifA and transferred a rhizopine nifA L94Q/D95Q -rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N 2 fixation and NH 3 excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N 2 fixation and NH 3 excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.
Publisher: American Society for Microbiology
Date: 30-06-2016
Abstract: We report the complete genome sequence of Mesorhizobium ciceri strain CC1192, an efficient nitrogen-fixing microsymbiont of Cicer arietinum (chickpea). The genome consists of 6.94 Mb distributed between a single chromosome (6.29 Mb) and a plasmid (0.65 Mb).
Publisher: American Society for Microbiology
Date: 30-06-2016
Abstract: We report the complete genome sequence of Mesorhizobium ciceri bv. biserrulae strain WSM1284, a nitrogen-fixing microsymbiont of the pasture legume Biserrula pelecinus . The genome consists of 6.88 Mb distributed between a single chromosome (6.33 Mb) and a single plasmid (0.55 Mb).
Publisher: Cold Spring Harbor Laboratory
Date: 04-2021
DOI: 10.1101/2021.03.31.437999
Abstract: Accurate quantification 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. Because a low oxygen environment that may not be conducive to plant growth is essential for optimal stability and function of the N-fixing catalyst nitrogenase, quantification of N fixation is routinely carried out on “free-living” bacteria grown in the absence of a host plant. Such experiments may not ulge the true extent of N fixation occurring 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 planta acetylene reduction assay, utilising the model cereal barley as a host, to quantify associative N fixation by 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. Exploiting “nitrogen-fixing” bacteria that reduce atmospheric dinitrogen into ammonia as inoculants of cereal crops has great potential to alleviate current inputs of environmentally deleterious fertiliser nitrogen and drive more sustainable crop production. Accurately quantifying plant-associative bacterial nitrogen fixation is central to the development of such inoculant bacteria, but most assays fail to adequately reproduce the conditions of plant root systems. In this work, we have validated and optimised a simple in planta assay to accurately quantify N fixation in bacteria occupying the root and surrounding soil of the model cereal barley. This assay represents a benchmark for quantification of plant-associative bacterial N fixation.
Publisher: Wiley
Date: 15-02-2021
Abstract: 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 ersity 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.
Publisher: Public Library of Science (PLoS)
Date: 22-03-2018
Publisher: Proceedings of the National Academy of Sciences
Date: 11-04-2022
Abstract: Inoculation of cereals with diazotrophic (N 2 -fixing) bacteria offers a sustainable alternative to the application of nitrogen fertilizers in agriculture. While natural diazotrophs have evolved multilayered regulatory mechanisms that couple N 2 fixation with assimilation of the product NH 3 and prevent release to plants, genetic modifications can permit excess production and excretion of NH 3 . However, a lack of stringent host-specificity for root colonization by the bacteria would allow growth promotion of target and nontarget plants species alike. Here, we exploit synthetic transkingdom signaling to establish plant host-specific control of the N 2 -fixation catalyst nitrogenase in Azorhizobium caulinodans occupying barley roots. This work demonstrates how partner-specific interactions can be established to avoid potential growth promotion of nontarget plants.
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.PLASMID.2017.06.001
Abstract: 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, β 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 (ICEMlSym
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2020
End Date: 2023
Funder: Biotechnology and Biological Sciences Research Council
View Funded ActivityStart Date: 2015
End Date: 2018
Funder: Grains Research and Development Corporation
View Funded ActivityStart Date: 2019
End Date: 2021
Funder: Royal Commission for the Exhibition of 1851
View Funded Activity