Journal of Molecular Biology
Volume 432, Issue 17, 7 August 2020, Pages 4840-4855
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A Two-Component System Acquired by Horizontal Gene Transfer Modulates Gene Transfer and Motility via Cyclic Dimeric GMP

https://doi.org/10.1016/j.jmb.2020.07.001Get rights and content

Highlights

  • A two-component system (TCS) was characterized in Rhodobacter capsulatus.

  • In this TCS, Rcc00620 is phosphorylated by Rcc00621.

  • Rcc00620 ~ P degrades c-di-GMP and stimulates gene transfer and motility.

  • The TCS was acquired by horizontal gene transfer (HGT) from a distant bacterium.

  • A TCS acquired by HGT is active and regulates gene transfer in the new location.

Abstract

Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important intracellular signaling molecule that affects diverse physiological processes in bacteria. The intracellular levels of c-di-GMP are controlled by proteins acting as diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that synthesize and degrade c-di-GMP, respectively. In the alphaproteobacterium Rhodobacter capsulatus, flagellar motility and gene exchange via production of the gene transfer agent RcGTA are regulated by c-di-GMP. One of the R. capsulatus proteins involved in this regulation is Rcc00620, which contains an N-terminal two-component system response regulator receiver (REC) domain and C-terminal DGC and PDE domains. We demonstrate that the enzymatic activity of Rcc00620 is regulated through the phosphorylation status of its REC domain, which is controlled by a cognate histidine kinase protein, Rcc00621. In this system, the phosphorylated form of Rcc00620 is active as a PDE enzyme and stimulates gene transfer and motility. In addition, we discovered that the rcc00620 and rcc00621 genes are present in only one lineage within the genus Rhodobacter and were acquired via horizontal gene transfer from a distantly related alphaproteobacterium in the order Sphingomonadales. Therefore, a horizontally acquired regulatory system regulates gene transfer in the recipient organism.

Introduction

Two-component systems (TCS) are widely used by bacteria to sense and respond to various internal and external stimuli [[1], 2., 3.]. A classic TCS comprises a sensor histidine kinase and a response regulator, proteins that are multidomain in nature, and cognate pairs are often encoded by neighboring genes [4]. The chemistry of the TCS involves the sequential phosphorylation of two different amino acids that, for the majority, are a histidine within the kinase and an aspartic acid within the response regulator, and they are therefore also referred to as histidyl-aspartyl phosphorelay systems. In response to a specific signal, the kinase becomes activated and autophosphorylates on the histidine residue using ATP as the phosphodonor. The phosphate is subsequently transferred to the aspartic acid in the N-terminal receiver (REC) domain of the cognate response regulator, causing a conformational change that activates a C-terminal output/effector domain for the appropriate cellular response [1]. Different response regulators contain different types of output domains, such as helix-turn-helix DNA-binding, enzymatic, or protein/ligand-binding domains, and the activities of these domains are regulated based on the phosphorylation state of the REC domain [[5], 6., 7.]. Many response regulators trigger adaptive responses through the direct alteration of gene expression [8], but > 5% are predicted to be involved in cyclic dimeric guanosine monophosphate (c-di-GMP) biosynthesis and/or degradation, indicating frequent TCS regulation of the cellular levels of this important signaling molecule [9].

C-di-GMP acts as a second messenger in bacteria, with changes in its levels affecting various processes in different organisms, such as motility, aggregation behaviors, and developmental transitions [[9], 10., 11.]. Diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes mediate the synthesis and degradation of c-di-GMP, respectively. DGCs contain a GGDEF motif in their active site, which catalyzes the cyclization of c-di-GMP from two GTP molecules [12]. PDEs contain EAL or HD-GYP motifs and break c-di-GMP into 5′-phosphoguanylyl-(3′–5′)-guanosine (5′-pGpG) or two GMP, respectively [13., 14., 15.]. Proteins containing both GGDEF and EAL domains are also common. In some of these proteins, one of the two domains is enzymatically inactive and serves as a regulatory site by binding to the GTP or c-di-GMP substrates [11]. Others are bifunctional enzymes, such as BphG1 in Rhodobacter sphaeroides [16], MSDGC-1 in Mycobacterium smegmatis [17], and ScrC in Vibrio parahaemolyticus [18], but it is still not completely clear if/how these two activities are reciprocally regulated and what determines the overall activity of such proteins. However, these proteins typically also contain additional sensory/signaling domains, such as Per-ARNT-Sim (PAS) or REC, that can affect the c-di-GMP-related domains in response to various stimuli [9].

Rhodobacter capsulatus is a purple non-sulfur photosynthetic bacterium that belongs to the class Alphaproteobacteria. It has been studied with respect to various cellular functions [19], especially phototrophy, and it is also known for being the first organism found to exchange DNA via a gene transfer agent (GTA) [20]. GTAs, which are now known to be produced by multiple species of bacteria and one archaeon, resemble bacteriophages but they package small fragments of the producing cell's genome (4–14 kb, depending on the GTA) that is transferred to other cells (reviewed by Lang et al. [21., 22., 23.] and Stanton [24]). Production of the R. capsulatus GTA (RcGTA) is affected by two regulators with clear evolutionary connections to phages [25,26] as well as multiple cellular regulatory systems (reviewed by Lang et al. [21]), including quorum sensing via the GtaI-GtaR proteins [27,28] and the CckA-ChpT-CtrA histidyl-aspartyl phosphorelay [29,30]. A transcriptomic study focused on CtrA showed that more than 20 genes encoding predicted signal transduction and transcription-regulating proteins were affected by the loss of this response regulator [31]. These included nine proteins predicted to be involved in c-di-GMP signaling, and four of these were subsequently shown to affect RcGTA production as well as flagellar motility by altering c-di-GMP levels [32]. One of these four proteins, Rcc00620, possesses a response regulator REC domain and both GGDEF and EAL domains and was shown to act as a positive regulator of RcGTA production by acting as a PDE enzyme [32]. However, this protein acted as a DGC in Escherichia coli [32]. This differential activity in the two bacteria led us to speculate that the activity of one or both domains might be regulated by the phosphorylation status of the N-terminal REC domain and through the action of a cognate histidine kinase protein.

In this study, we used genetic manipulations, site-directed mutagenesis, and in vitro phosphorylation assays to determine if the c-di-GMP-modulating protein Rcc00620 acts as a response regulator in a TCS with a putative cognate histidine kinase, Rcc00621. We evaluated the role of the Rcc00620 REC domain and Rcc00621 in regulating the enzymatic activity of Rcc00620 in R. capsulatus by quantifying RcGTA production, cell motility, and c-di-GMP levels in relevant strains, and via E. coli c-di-GMP indicator assays. We also investigated the evolutionary history of the two genes and their protein motif conservation across alphaproteobacterial species. We show that these genes were horizontally acquired by an ancestral Rhodobacter from within the alphaproteobacterial order Sphingomonadales and encode a functional TCS where the c-di-GMP-related enzymatic activity of Rcc00620 is regulated through phosphorylation of its REC domain by the histidine kinase Rcc00621.

Section snippets

Rcc00620 and Rcc00621 functional domains

The previously studied Rcc00620 protein is encoded in a two-gene operon with a gene (rcc00621) predicted to encode a histidine kinase protein (Figure 1(a)). We performed a detailed analysis of the conserved domains in the two proteins (Figure 1(a)). Rcc00620 contains an N-terminal REC domain, a central GGDEF (DGC) domain, and a C-terminal EAL (PDE) domain. For Rcc00621, all typical histidine kinase domains were identified: the HAMP domain, the H-box that contains the histidine

Rcc00620 and Rcc00621 form a TCS involved in the regulation of c-di-GMP levels, RcGTA production, and flagellar motility

In R. capsulatus, Rcc00620 and Rcc00621 contain all of the conserved domains and residues required to act as TCS response regulator and histidine kinase, respectively (Figure 1). These domains in both proteins have been well conserved throughout evolution, suggesting this is an important regulatory system in multiple species and genera within the class Alphaproteobacteria. The disruption of rcc00621 and both rcc00620–621 resulted in decreased gene transfer activity (Figure 2), with the changes

Concluding Remarks

In this study, we delineated the role of a TCS involved in the regulation of c-di-GMP levels in R. capsulatus, and the consequent effects on gene transfer and flagellar motility. The Rcc00620 protein possesses a REC-DGC-PDE multidomain architecture, which is one of the most common domain architectures among c-di-GMP signaling proteins. Here, by assaying RcGTA production, motility and c-di-GMP levels in R. capsulatus and using c-di-GMP indicator assays in E. coli, we show that the enzymatic

Sequence and phylogenetic analyses

The SMART [53,54] and Expasy-Prosite [55] databases were used to identify functional domains in the protein sequences, and TMpred [56] and Phobius [57] were used to predict transmembrane domains.

rcc00620 and rcc00621 homologs were identified using the BLASTn [58] online tool with adjusted scoring parameters (Match/Mismatch Scores: 1/− 1; Gap Costs: Existence 2, Extension 1), and searches were performed within the “non-redundant” and “whole-genome shotgun contigs” (wgs) databases using the

CRediT authorship contribution statement

Purvikalyan Pallegar:Conceptualization, Data curation, Formal analysis, Writing - original draft.Marta Canuti:Data curation, Formal analysis, Writing - original draft.Evan Langille:Data curation, Formal analysis, Writing - review & editing.Lourdes Peña-Castillo:Writing - review & editing, Funding acquisition.Andrew S. Lang:Conceptualization, Writing - review & editing, Funding acquisition, Project administration.

Acknowledgments

This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada to A.S.L. (grant numbers RGPIN-2012-341561 and RGPIN-2017-04636) and L.P.-C. (grant number RGPIN-2011-402087). P.P. was partially supported by funding from the Memorial University of Newfoundland School of Graduate Studies.

Declarations of Competing Interest

None.

References (79)

  • M. Levet-Paulo et al.

    The atypical two-component sensor kinase Lpl0330 from Legionella pneumophila controls the bifunctional diguanylate cyclase-phosphodiesterase Lpl0329 to modulate bis-(3′–5′)-cyclic dimeric GMP synthesis

    J. Biol. Chem.

    (2011)
  • H. Wang et al.

    The CtrA phosphorelay integrates differentiation and communication in the marine alphaproteobacterium Dinoroseobacter shibae

    BMC Genomics

    (2014)
  • S.F. Altschul et al.

    Basic local alignment search tool

    J. Mol. Biol.

    (1990)
  • D.A. Ryjenkov et al.

    The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in enterobacteria

    J. Biol. Chem.

    (2006)
  • P. Prentki et al.

    In vitro insertional mutagenesis with a selectable DNA fragment

    Gene

    (1984)
  • A.M. Stock et al.

    Two-component signal transduction

    Annu. Rev. Biochem.

    (2000)
  • R. Gao et al.

    Biological insights from structures of two-component proteins

    Annu. Rev. Microbiol.

    (2009)
  • Y. Shiro et al.

    Structural basis of the signal transduction in the two-component system

    Adv. Exp. Med. Biol.

    (2008)
  • J. Perry et al.

    Receptor domains of two-component signal transduction systems

    Mol. BioSyst.

    (2011)
  • T. Krell et al.

    Bacterial sensor kinases: diversity in the recognition of environmental signals

    Annu. Rev. Microbiol.

    (2010)
  • U. Römling et al.

    Cyclic di-GMP: the first 25 years of a universal bacterial second messenger

    Microbiol. Mol. Biol. Rev.

    (2013)
  • U. Jenal et al.

    Cyclic di-GMP: second messenger extraordinaire

    Nat. Rev. Microbiol.

    (2017)
  • R. Hengge

    Principles of c-di-GMP signalling in bacteria

    Nat. Rev. Microbiol.

    (2009)
  • R. Paul et al.

    Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain

    Genes Dev.

    (2004)
  • R. Simm et al.

    GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessibility to motility

    Mol. Microbiol.

    (2004)
  • A.D. Tischler et al.

    Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation

    Mol. Microbiol.

    (2004)
  • U. Römling et al.

    Discovery of the second messenger cyclic di-GMP

    Methods Mol. Biol.

    (2017)
  • M. Kumar et al.

    Cyclic di-GMP: a second messenger required for long-term survival, but not for biofilm formation, in Mycobacterium smegmatis

    Microbiology

    (2008)
  • R.B.R. Ferreira et al.

    Vibrio parahaemolyticus ScrC modulates cyclic dimeric GMP regulation of gene expression relevant to growth on surfaces

    J. Bacteriol.

    (2008)
  • H. Strnad et al.

    Complete genome sequence of the photosynthetic purple nonsulfur bacterium Rhodobacter capsulatus SB 1003

    J. Bacteriol.

    (2010)
  • B. Marrs

    Genetic recombination in Rhodopseudomonas capsulata

    Proc. Natl. Acad. Sci.

    (1974)
  • A.S. Lang et al.

    The distribution, evolution, and roles of gene transfer agents in prokaryotic genetic exchange

    Ann. Rev. Virol.

    (2017)
  • A.S. Lang et al.

    Gene transfer agents: phage-like elements of genetic exchange

    Nat. Rev. Microbiol.

    (2012)
  • A.P. Hynes et al.

    Functional and evolutionary characterization of a gene transfer agent’s multilocus “genome”

    Mol. Biol. Evol.

    (2016)
  • P.C.M. Fogg

    Identification and characterization of a direct activator of a gene transfer agent

    Nat. Commun.

    (2019)
  • A.L. Schaefer et al.

    Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production

    J. Bacteriol.

    (2002)
  • M.M. Leung et al.

    The GtaR protein negatively regulates transcription of the gtaRI operon and modulates gene transfer agent (RcGTA) expression in Rhodobacter capsulatus

    Mol. Microbiol.

    (2012)
  • A.S. Lang et al.

    Genetic analysis of a bacterial genetic exchange element: the gene transfer agent of Rhodobacter capsulatus

    Proc. Natl. Acad. Sci. U. S. A.

    (2000)
  • R.G. Mercer et al.

    Regulatory systems controlling motility and gene transfer agent production and release in Rhodobacter capsulatus

    FEMS Microbiol. Lett.

    (2012)
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