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A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts [1]

['Julian A. Bustamante', 'Department Of Biology', 'San Francisco State University', 'San Francisco', 'California', 'United States Of America', 'Josue S. Ceron', 'Ivan Thomas Gao', 'Hector A. Ramirez', 'Milo V. Aviles']

Date: 2023-11

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.

Symbiotic bacteria that live in the roots of legume plants produce biologically accessible nitrogen compounds, offering a more sustainable and environmentally sound alternative to industrial fertilizers generated from fossil fuels. Understanding the multitude of factors that contribute to successful interaction between such bacteria and their plant hosts can help refine strategies for improving agricultural output. In addition, because disease-causing microbes share many genes with these beneficial bacteria, unraveling the cellular mechanisms that facilitate host invasion can reveal ways to prevent and treat infectious diseases. In this report we show that two genes in the model bacterium Sinorhizobium meliloti contribute to effective symbiosis by helping the cells adapt to living in host plants. This finding furthers knowledge about genetic factors that regulate interactions between microbes and their hosts.

Funding: Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award Number SC3-GM096943 to J.C.C. and National Science Foundation (NSF), Division of Integrative Organismal Systems, under Rules of Life Award Number 2015870 to S.R.L. NIH Award Number T34-GM008574 (MARC) provided support for J.A.B., J.S.C., R.C., R.L.-M., and J.N.; R25-GM048972 (Bridge to the Doctorate) supported H.A.R.; R25-GM059298 (MBRS-RISE) supported J.S.-M.; R25-GM050078 (Bridges to the Baccalaureate) supported J.S.C. and R.C.; T34-GM145400 (U-RISE) supported R.C.; and UL1-GM118985, TL4-GM118986, RL5-GM118984 (SF BUILD) supported B.K.R.M. NSF REU DBI Award Number 1156452 provided summer funding for R.L.-M. and R.B., and NSF DBI Award Number 1548297 provided summer funding for H.A.R. R.R.M. was supported by the Beckman Scholars Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

One of the genes previously identified in a transposon-based screen as necessary for S. meliloti resistance against NCR247 in culture is SMc03872 (jspA), predicted to encode a periplasmic protease conserved in alpha-proteobacteria and shown to confer a competitive advantage during symbiosis with alfalfa [ 63 ]. jspA was also identified in a genetic selection for suppressors that ameliorated the osmosensitivity of a podJ null mutant [ 44 ]. That work demonstrated that PodJ is a conserved polarity factor that contributes to cell envelope integrity and EPS-I production in S. meliloti, and that deletion of jspA or SMc00067 (lppA), both encoding putative lipoproteins, reduced EPS-I levels. Here we show that jspA and lppA jointly influence EPS-I production by lowering the steady-state levels of periplasmic ExoR and thus activating the ExoS-ChvI signal transduction pathway ( Fig 1 ). This regulation contributes to competitive fitness during host colonization, suggesting that jspA and lppA facilitate transition to a gene expression pattern more suitable for the host environment.

One key subset of the regulon induced upon ExoS-ChvI activation is the exo genes, responsible for synthesis of succinoglycan, or EPS-I, originally characterized in S. meliloti strain Rm1021 as the only symbiotically active EPS [ 48 – 51 ]. An increase in EPS-I production, usually concomitant with a decrease in flagellar motility [ 30 , 44 ], represents a physiological transition from saprophytic to endosymbiotic, as EPS-I contributes to successful interaction between compatible symbiotic partners. Mutants that lack EPS-I or synthesize variants with altered structures (for example, absence of succinylation) exhibit defects in the initiation or elongation of infection threads, while changes in EPS-I levels can influence symbiotic efficiency [ 52 – 56 ]. Thus, both the quality and quantity of EPS-I matter during infection. EPS-I may serve as a recognition signal, particularly for suppressing host defenses [ 57 ]. While no plant receptor for S. meliloti EPS-I has been identified so far [ 58 ], EPS-I does enhance tolerance of various environmental assaults [ 59 , 60 ], including those encountered during host colonization, such as acidity, oxidative stress, and antimicrobial peptides [ 61 – 65 ].

Irrespective of the specific triggers, the S. meliloti ExoR-ExoS-ChvI system influences a multitude of physiological activities, including exopolysaccharide (EPS) production, motility, biofilm formation, cell envelope maintenance, and nutrient utilization, befitting its pivotal regulation of symbiotic development [ 17 , 18 , 21 , 26 ]. Initial transcriptome profiles of S. meliloti exoS::Tn5 and exoR::Tn5 mutants revealed altered expression of hundreds of genes [ 21 , 26 ], but subsequent interrogation that included identification of genomic regions bound by ChvI winnowed the direct targets of the response regulator down to 64, many known to participate in physiological activities described above [ 30 , 36 ]. Perhaps illustrating the complex interaction of regulatory pathways and the difficulty of signal deconvolution, a significant fraction of ChvI targets also changed expression with other published perturbations [ 30 ], including acid stress [ 37 – 39 ], antimicrobial peptide treatment [ 40 ], phosphate starvation [ 41 ], cyclic nucleotide accumulation [ 42 ], overexpression of SyrA [ 43 ], and mutations in podJ, cbrA, ntrY, and emrR [ 44 – 47 ].

Schematic diagram shows relationship of pathway components, their subcellular locations, and impact on expression of representative genes. Pointed and blunt arrowheads represent positive and negative regulation, respectively. Solid arrows indicate previously demonstrated, direct interactions. Results from this study suggest that, in response to cell envelope stress such as exposure to acidic pH, JspA and LppA negatively regulate ExoR via proteolysis. As a typical pair of histidine kinase and response regulator, ExoS and ChvI are presumed to function as homodimers [ 13 , 30 ]; for simplicity, the diagram does not show that.

Multiple factors found to be critical for S. meliloti to form mutualistic symbiosis have been shown to contribute to host infection in related pathogens, such as Brucella spp., suggesting mechanistic parallels between mutualism and pathogenesis [ 7 ]. One such shared mechanism is the ExoS-ChvI two-component phosphorelay pathway, conserved across many alpha-proteobacteria, particularly in Rhizobiales (synonym Hyphomicrobiales) ( Fig 1 ) [ 11 , 12 ]. ExoS is a membrane-bound histidine kinase with a periplasmic sensor domain, while ChvI is its cognate response regulator [ 13 ]. Mutations in ExoS and ChvI, as well as their orthologs in related endosymbionts, impair host colonization [ 14 – 22 ]. A third component of the S. meliloti signaling system, ExoR, acts as a periplasmic repressor of ExoS via physical association [ 19 , 21 ]. ExoR is regulated by proteolysis [ 23 – 25 ], and binding to ExoS protects it from degradation [ 19 ]. Mutations in ExoR also disrupt symbiosis [ 19 , 21 , 26 – 28 ].

Rhizobia-legume symbioses account for a substantial proportion of terrestrial nitrogen fixation, reducing molecular dinitrogen to a more bioavailable form such as ammonia [ 1 , 2 ]. The mutualistic relationship requires complex communication and coordination between two compatible partners [ 3 , 4 ], as well as bacterial adaptation to the “stresses” of the host plant environment [ 5 , 6 ]. The alpha-proteobacterium Sinorhizobium meliloti and its hosts, including Medicago sativa (alfalfa) and M. truncatula (barrel medic), emerged as models for nitrogen-fixing root nodule symbiosis [ 7 ]. Here, compounds released by the host plant induce bacterial production of signaling molecules called Nod factors, required for eliciting formation of root nodules [ 8 ]. Nodule colonization begins with bacterial cells invading the root hair via plant cell wall-derived tunnels called infection threads, followed by release into plant cells, in which the rhizobia differentiate into “bacteroids” capable of fixing nitrogen in exchange for carbon from the host [ 2 , 9 , 10 ].

Results

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010776

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