Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins

Summary In Rhizobium leguminosarum bv. viciae, quorumsensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots

The cin and rai Quorum-Sensing Regulatory Systems in Rhizobium leguminosarum Are Coordinated by ExpR and CinS, a Small Regulatory Protein Coexpressed with CinI▿

To understand how the Rhizobium leguminosarum raiI-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce raiI. Since raiR (and raiI) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs

Development of an E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass

Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate ([CCIm][OAc]) for pretreatment of switchgrass biomass. [CCIm][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [CCIm]-tolerant E. coli strain, where [CCIm] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [CCIm]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [CCIm]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [CCIm]-tolerant E. coli strain so as to secrete this [CCIm]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process

Development of a native Escherichia coli induction system for ionic liquid tolerance.

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR', PydfO', and PydfA', were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA' and PmarR' based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA' and PmarR' in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR'-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR' can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains

Development of a Native <i>Escherichia coli</i> Induction System for Ionic Liquid Tolerance

<div><p>The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In <i>E. coli</i> this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by <i>eilA</i> from <i>Enterobacter lignolyticus</i>. In the present work, we used microarray analysis to identify native <i>E. coli</i> IL-inducible promoters and develop control systems for regulating <i>eilA</i> gene expression. Three candidate promoters, P<i>marR’,</i> P<i>ydfO’,</i> and P<i>ydfA’</i>, were selected and compared to the IPTG-inducible P<i>lacUV5</i> system for controlling expression of <i>eilA</i>. The P<i>ydfA’</i> and P<i>marR’</i> based systems are as effective as P<i>lacUV5</i> in their ability to rescue <i>E. coli</i> from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of P<i>ydfA’</i> and P<i>marR’</i> in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pP<i>marR’-eilA</i> outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as P<i>marR’</i> can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.</p></div

Relative abundance of <i>eilA</i> expression strains after competitive growth in pools with increasing [C₂mim]Cl concentrations.

<p>Cultures were pooled and grown over 48-specific primers. 10 µM IPTG was added to induce the pP<i>lacUV5-eilA</i> construct. qPCR results were normalized using <i>cat</i> (the gene conferring chloramphenicol resistance on the expression plasmid) as endogenous control. Error bars represent standard errors. Dark blue: pP<i>lacUV5-rfp,</i> red: pP-<i>eilA,</i> green: pP<i>ydfO’-eilA,</i> purple: pP<i>ydfA’-eilA,</i> light blue: pP<i>marR’-eilA,</i> orange: pP<i>lacUV5-eilA.</i></p