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

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

Discovery and characterisation of tripartite Integrative & Conjugative Elements

Bacterial integrative & conjugative elements (ICEs) are chromosomally-integrated DNA islands that excise to form circular molecules capable of horizontal self-transmission via conjugation (cell-to-cell contact). Symbiosis ICEs, such as ICEMlSymR7A of Mesorhizobium loti, are a group of ICEs that carry genes enabling rhizobial bacteria to engage in N2-fixing symbioses with leguminous plants. Transfer of symbiosis ICEs can convert non-symbiotic rhizobia into legume symbionts in a single evolutionary step. In this thesis, a novel form of “tripartite” ICE (ICE3) is reported that exists as three entirely separated regions of DNA residing in the chromosomes of genetically diverse N2-fixing Mesorhizobium spp. These ICE3 regions did not excise independently, rather through multiple recombinations with the host chromosome they formed a single contiguous region of DNA prior to excision and conjugative transfer. Upon integration into a recipient chromosome, the ICE3 recombined the recipient chromosome to disassemble into the tripartite form. These recombination reactions were catalysed by three Integrase proteins IntG, IntM, and IntS, acting on three associated integrase attachment sites. The “excisive” recombination reactions (i.e. assembly and excision) were stimulated by three recombination directionality factors RdfG, RdfM, and RdfS. Expression of ICE3 transfer and conjugation genes were found to be 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. A model for ICE3 recombination and transfer is presented in this thesis. The conservation of gene content between symbiosis ICE and ICE3 indicated that these elements share a common evolutionary history. However, the persistence of ICE3 structure in diverse mesorhizobia is perplexing due to its seemingly unnecessary complexity. Bioinformatic comparisons of ICE and ICE3 indicated that the tripartite configuration itself may provide selective benefits to the element, including enhanced host range, host stability and resistance to destabilization by tandem insertion of competing integrative elements. In congruency with ICEMlSymR7A, ICE3 acquisition can convey upon recipients the ability to form N2-fixing symbiosis with the host-legume of the ICE3 donor. Interestingly, the effectiveness of N2-fixation may be impaired. The consequences of the emergence of sub-optimal N2-fixing strains following ICE3 transfer in agriculture is discussed. If ICE3 transfer poses a barrier for future inoculation success, the elucidation of the mechanism of ICE3 assembly, excision, and transfer will allow for the development of strategies for management

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

Transcriptional regulation of <i>rdfG</i> and <i>rdfM</i> by RdfS.

<p>β-galactosidase assays [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007292#pgen.1007292.ref067" target="_blank">67</a>] were performed for (A) WSM1271 and (B) R7ANS carrying either control vector pPR3 or pPR3-<i>rdfS</i> (constitutively expressing <i>rdfS</i>) together with one of three RDF promoter-<i>lacZ</i> fusion constructs cloned into the pSDz vector. Assays were performed with six biological replicates and mean β-galactosidase activity values (Relative Fluorescent Units/s/OD₆₀₀) were compared by Bonferroni adjusted student’s t-tests. SD is denoted by error bars.</p

Quorum sensing activation of ICE<i>Mc</i>Sym¹²⁷¹ promoters.

<p>Overlayed relative read coverage (or sequencing depth) plots represent standardised values for the mean number of reads mapped to the positive strand of the regions shown in this figure from the three unfiltered QS+ (grey) and QS- (black) transcriptome libraries of WSM1271. QS+ strains were induced for QS by overexpressing both <i>traI1</i> and <i>traR1</i> from the plasmids pPR3-<i>traI1</i> and pSDz<i>-traR1</i>, respectively, whereas the QS- control strains carried the parent vectors pPR3 and pSDz. The mean read depth for the (A) <i>traI2-msi172-msi171</i>, and (B) <i>rdfS-traF-msi107</i> and <i>rlxS</i> regions of ICE<i>Mc</i>Sym¹²⁷¹ in QS- transcriptome libraries were almost non-existent relative to that of the QS+ strain (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007292#pgen.1007292.s008" target="_blank">S1 Dataset</a> for a full list of TPM values from the filtered reads). A magnified view of reads mapping to the promoter region and the DNA sequence is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007292#pgen.1007292.s004" target="_blank">S4 Fig</a>. These plots were produced using Integrated Genome Browser [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007292#pgen.1007292.ref073" target="_blank">73</a>].</p

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

qPCR measurement of excisive ICE<i>Mc</i>Sym¹²⁷¹ recombination.

<p>Measurements represent the mean percentage of WSM1271 chromosomes in stationary-phase cultures harbouring each excisive Int-mediated recombination product (<i>attB</i>_<i>S</i>, <i>attP</i>_<i>S</i>, <i>attB</i>_<i>G</i>, <i>attP</i>_<i>G</i>, <i>attP</i>_<i>M</i>, <i>and attP</i>_<i>M</i>) determined by qPCR [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007292#pgen.1007292.ref019" target="_blank">19</a>]. Where appropriate, plasmids carried by WSM1271 (here abbreviated as 1271) are listed in brackets after the strain name (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007292#pgen.1007292.t003" target="_blank">Table 3</a> for a description of plasmids). Values for each of the assay types <i>attB</i>_<i>S</i>, <i>attP</i>_<i>S</i>, <i>attB</i>_<i>G</i>, <i>attP</i>_<i>G</i>, <i>attP</i>_<i>M</i>, and <i>attP</i>_<i>M</i> site were individually compared between strains within the same panel (panel A, B, or C) using ANOVA and Fisher’s LSD test controlling for type I error using the Bonferroni adjustment. Groups of values from the same assay type and in the same panel that are not significantly different from each other have the same letter (a, b, c, d, e, f or g) indicated above. Expression from the IPTG inducible promoter of pSDz constructs were not induced with IPTG as they exhibit leaky expression without induction in TY medium used for assays. (A) Involvement of <i>rdfG</i> and <i>rdfM</i> in excisive recombination. (B) Quorum-sensing induction of excisive recombination. (C) Involvement of <i>rdfS</i> in excisive recombination.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Quorum-sensing induced/repressed ICE<i>Mc</i>Sym¹²⁷¹-encoded genes.

<p>Quorum-sensing induced/repressed ICE<i>Mc</i>Sym¹²⁷¹-encoded genes.</p