Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Laventie, Benoît-Joseph; Nesper, Jutta; Ahrné, Erik; Schmidt, Alexander; Jenal, Urs

doi:10.3791/51404

Cited by 13 publications

(11 citation statements)

References 24 publications

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“…Capture experiments were carried out as described earlier (18,19). All experiments were performed in 200-l 12-tube PCR strips (Thermo Scientific).…”

Section: Bacterialmentioning

confidence: 99%

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

et al. 2016

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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.

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“…Capture experiments were carried out as described earlier (18,19). All experiments were performed in 200-l 12-tube PCR strips (Thermo Scientific).…”

Section: Bacterialmentioning

confidence: 99%

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

et al. 2016

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“…Indeed, P. aeruginosa protein PA2989 (which has a typical RxxxR motif but the [D/N] residue of the second motif is replaced by Ser, Fig. S3), has been experimentally shown to bind c-di-GMP (47, 48). In contrast, in PA2989 homologs from some other Pseudomonas species, these motifs are disrupted (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural conservation and diversity of PilZ-related domains

Galperin

Chou

2019

Preprint

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The widespread bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a 1 variety of processes, including protein secretion, motility, cell development, and biofilm 2 formation. C-di-GMP-dependent responses are often mediated by its binding to the 3 cytoplasmic receptors that contain the PilZ domain. We present here comparative 4 structural and sequence analysis of various PilZ-related domains and describe three 5 principal types of them: (i) the canonical PilZ domain, whose structure includes a six-6 stranded beta-barrel and a C-terminal alpha-helix; (ii) an atypical PilZ domain that 7 contains two extra alpha-helices and forms stable tetramers, and (iii) divergent PilZ-8 related domains, which include the eponymous PilZ protein and PilZN (YcgR_N) and 9 PilZNR (YcgR_2) domains. We refine the second c-di-GMP binding motif of PilZ as 10 [D/N]hSxxG and show that the hydrophobic residue h of this motif interacts with a cluster 11 of conserved hydrophobic residues, helping maintain the PilZ domain fold. We describe 12 several novel PilZN-type domains that are fused to the canonical PilZ domains in specific 13 taxa, such as spirochetes, actinobacteria, cellulosolytic clostridia, deltaproteobacteria, 14 and aquificae. We propose that the evolution of three major groups of PilZ domains 15 included (i) fusion of pilZ with other genes, which produced Alg44, cellulose synthase, and 16 other multidomain proteins; (ii) insertion of a ~200-bp fragment, which resulted in the 17 formation of tetramer-forming PilZ proteins, and (iii) tandem duplication of pilZ genes, 18 which led to the formation of PilZ dimers and YcgR-like proteins. 19 IMPORTANCE 20Cyclic di-GMP is a ubiquitous bacterial second messenger that regulates motility, biofilm 21 formation and virulence of many bacterial pathogens. The PilZ domain is a widespread c-22 di-GMP receptor that binds c-di-GMP through its RxxxR and [D/N]xSxxG motifs; some PilZ 23 domains lack these motifs and are unable to bind c-di-GMP. We used structural and 24 sequence analysis to assess the diversity of PilZ-related domains and define their common 25 features. We show that the hydrophobic residue h in the second motif is highly conserved; 26 it may serve as a readout for c-di-GMP binding. We describe three principal classes of 27 PilZ-related domains: canonical, tetramer-forming, and divergent PilZ domains, and 28 propose the evolutionary pathways that led to the emergence of these PilZ types. 29 30

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“…A new aspect of control for the transition between planktonic and biofilm behaviors was presented by Benoît-Joseph Laventie (Biozentrum, Basel, Switzerland), who described a new c-di-GMP effector in P. aeruginosa, identified using capture-compound mass spectrometry (10). This effector, FimA, mediates pilus-mediated attachment and biofilm formation.…”

Section: Bacterial Adhesion Factors and The Planktonic To Biofilm Lifmentioning

confidence: 99%

Biofilms 2015: Multidisciplinary Approaches Shed Light into Microbial Life on Surfaces

et al. 2016

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The 7th ASM Conference on Biofilms was held in Chicago, Illinois, from 24 to 29 October 2015. The conference provided an international forum for biofilm researchers across academic and industry platforms, and from different scientific disciplines, to present and discuss new findings and ideas. The meeting covered a wide range of topics, spanning environmental sciences, applied biology, evolution, ecology, physiology, and molecular biology of the biofilm lifestyle. This report summarizes the presentations with regard to emerging biofilm-related themes.

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Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Cited by 13 publications

References 24 publications

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

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

Structural conservation and diversity of PilZ-related domains

Biofilms 2015: Multidisciplinary Approaches Shed Light into Microbial Life on Surfaces

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