An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus

Abstract

Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eightyfour proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors. IMPORTANCEIn this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

Target highlights in CASP13: Experimental target structures through the eyes of their authors

“…The Bd2924:C10‐CoA complex provided structural insights into the interactions Bd2924 makes with its substrate. The C10‐CoA ligand bound into a long narrow tunnel that runs deep into the protein beneath the bound FAD molecule, similarly to that described for other ACAD structures . Interestingly, only one molecule of C10‐CoA per dimer could be confidently placed into the electron density maps, which corresponded to the conformation of Trp428 beneath the re ‐face of the FAD molecule.…”

“…The tetrameric assembly of divergent ACADs appears to block the proposed docking site of electron transferring flavoprotein (ETF) . Interestingly, we were unable to detect any dehydrogenation activity; likewise no significant dehydrogenase activity has been reported for other divergent ACADs . Moreover, the structure of Bd2924 reveals that the chemistry of the conserved active site residue Glu429 may be altered by participating in a hydrogen bond with Asn171.…”

“…Here we investigated a putative ACAD, Bd2924, from the obligate predatory bacterium, B. bacteriovorus . Our initial interest arose in Bd2924 when it was first identified as a potential novel cyclic di‐GMP (cdG) interacting protein . CdG is an almost universally important bacterial secondary messenger molecule influencing growth and a range of behaviors such as motility, virulence, biofilm formation, cell cycle progression and (in Bdellovibrio ) control of predation …”

“…The tertiary structure of Bd2924 forms a biologically relevant homo‐tetramer, composed of a dimer of dimers, which was later confirmed to be the predominant species in solution by size exclusion chromatography experiments. Bd2924 shares the common ACAD fold, and is most closely related to the divergent ACADs, AidB, and ACDH‐11 . Bd2924 can be divided into four distinct domains: an N‐terminal α‐helical domain that is interrupted by a single β‐sheet projection (residues 3‐168), a central β‐sheet domain formed by two orthogonal four‐stranded antiparallel β‐sheets (residues 169‐285), a central α‐helical domain (residues 286‐449) and a short C‐terminal α‐helical domain (residues 450‐505) (Figure A).…”

Bdellovibrioet organismes apparentés

“…Le pôle d'invasion relierait donc la détection de la proie au système de signalisation du di-GMP cyclique. Ce système semble former un réseau étendu chez B. bacteriovorus qui pourrait affecter de nombreuses fonctions [30]. De plus, merRNA, un ARN très fortement exprimé dans la PA et rapidement dégradé pendant la TP et au début de la PC, coderait un riboswitch 12 du diGMP cyclique [18].…”