A GGDEF domain serves as a spatial on-switch for a phosphodiesterase by direct interaction with a polar landmark protein

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bi-modal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here we show how PdeB polar recruitment is mediated by direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the C-terminal FimV domain of the polar landmark protein HubP. We demonstrate that this interaction is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. We further show that PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations that is generated by differences in abundance and temporal polar appearance of PdeB as well as by bi-modal distribution after cell fission orchestrates the population behavior with respect to cell-surface interaction and environmental spreading.

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The GGDEF Domain of the Phosphodiesterase PdeB in Shewanella putrefaciens Mediates Recruitment by the Polar Landmark Protein HubP

Journal of Bacteriology ◽

10.1128/jb.00534-18 ◽

2019 ◽

Vol 201 (7) ◽

Cited By ~ 5

Author(s):

Florian M. Rossmann ◽

Tim Rick ◽

Devid Mrusek ◽

Lasse Sprankel ◽

Anja K. Dörrich ◽

...

Keyword(s):

Cell Division ◽

Bacterial Species ◽

Direct Interaction ◽

Shewanella Putrefaciens ◽

Marker Protein ◽

Content Type ◽

Cell Pole ◽

Activity Measurements ◽

Ggdef Domain

ABSTRACT Bacteria commonly exhibit a high degree of cellular organization and polarity which affect many vital processes such as replication, cell division, and motility. In Shewanella and other bacteria, HubP is a polar marker protein which is involved in proper chromosome segregation, placement of the chemotaxis system, and various aspects of pilus- and flagellum-mediated motility. Here, we show that HubP also recruits a transmembrane multidomain protein, PdeB, to the flagellated cell pole. PdeB is an active phosphodiesterase and degrades the second messenger c-di-GMP. In Shewanella putrefaciens, PdeB affects both the polar and the lateral flagellar systems at the level of function and/or transcription in response to environmental medium conditions. Mutant analysis on fluorescently labeled PdeB indicated that a diguanylate cyclase (GGDEF) domain in PdeB is strictly required for HubP-dependent localization. Bacterial two-hybrid and in vitro interaction studies on purified proteins strongly indicate that this GGDEF domain of PdeB directly interacts with the C-terminal FimV domain of HubP. Polar localization of PdeB occurs late during the cell cycle after cell division and separation and is not dependent on medium conditions. In vitro activity measurements did not reveal a difference in PdeB phosphodiesterase activities in the presence or absence of the HubP FimV domain. We hypothesize that recruitment of PdeB to the flagellated pole by HubP may create an asymmetry of c-di-GMP levels between mother and daughter cells and may assist in organization of c-di-GMP-dependent regulation within the cell. IMPORTANCE c-di-GMP-dependent signaling affects a range of processes in many bacterial species. Most bacteria harbor a plethora of proteins with domains which are potentially involved in synthesis and breakdown of c-di-GMP. A potential mechanism to elicit an appropriate c-di-GMP-dependent response is to organize the corresponding proteins in a spatiotemporal fashion. Here, we show that a major contributor to c-di-GMP levels and flagellum-mediated swimming in Shewanella, PdeB, is recruited to the flagellated cell pole by the polar marker protein HubP. Polar recruitment involves a direct interaction between HubP and a GGDEF domain in PdeB, demonstrating a novel mechanism of polar targeting by the widely conserved HubP/FimV polar marker.

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The small GTPase MglA together with the TPR domain protein SgmX stimulates type IV pili formation inM. xanthus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004722117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23859-23868 ◽

Cited By ~ 1

Author(s):

Anna Potapova ◽

Luís Antonío Menezes Carreira ◽

Lotte Søgaard-Andersen

Keyword(s):

Direct Interaction ◽

Bound State ◽

Small Gtpase ◽

Type Iv Pili ◽

Polar Localization ◽

Cell Pole ◽

Type Iv ◽

Direction Of Movement ◽

Underlying Mechanisms ◽

Tpr Domain

Bacteria can move across surfaces using type IV pili (T4P), which undergo cycles of extension, adhesion, and retraction. The T4P localization pattern varies between species; however, the underlying mechanisms are largely unknown. In the rod-shapedMyxococcus xanthuscells, T4P localize at the leading cell pole. As cells reverse their direction of movement, T4P are disassembled at the old leading pole and then form at the new leading pole. Thus, cells can form T4P at both poles but engage only one pole at a time in T4P formation. Here, we address how this T4P unipolarity is realized. We demonstrate that the small Ras-like GTPase MglA stimulates T4P formation in its GTP-bound state by direct interaction with the tetratricopeptide repeat (TPR) domain-containing protein SgmX. SgmX, in turn, is important for polar localization of the T4P extension ATPase PilB. The cognate MglA GTPase activating protein (GAP) MglB, which localizes mainly to the lagging cell pole, indirectly blocks T4P formation at this pole by stimulating the conversion of MglA-GTP to MglA-GDP. Based on these findings, we propose a model whereby T4P unipolarity is accomplished by stimulation of T4P formation at the leading pole by MglA-GTP and SgmX and indirect inhibition of T4P formation at the lagging pole by MglB due to its MglA GAP activity. During reversals, MglA, SgmX, and MglB switch polarity, thus laying the foundation for T4P formation at the new leading pole and inhibition of T4P formation at the new lagging pole.

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Formation of a complex between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris

Microbiology Research ◽

10.4081/mr.2018.7601 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Shi-qi An ◽

Ji-liang Tang

Keyword(s):

Virulence Factors ◽

Xanthomonas Campestris ◽

Adaptor Protein ◽

Physical Interaction ◽

Diguanylate Cyclase ◽

Domain Interaction ◽

Ggdef Domain ◽

Interdomain Interactions ◽

Xanthomonas Campestris Pv Campestris ◽

Two Hybrid

RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), RpfG together with the sensor kinase RpfC regulates the synthesis of a range of virulence factors as a response to the cell-cell Diffusible Signaling Factor (DSF). RpfG regulates many different virulence factors by divergent pathways. Physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. This is a dynamic interaction that depends upon DSF signaling and involves the conserved GYP motif in the HD-GYP domain. Here we use synthetic peptide overlay technology and yeast two-hybrid analysis in conjunction with alanine substitution mutagenesis to define a motif within the GGDEF domain proteins required for interaction. We show that regulation of motility by the GGDEF domain proteins depends upon this motif. Furthermore, we show by Y2H that both GGDEF domain proteins bind a specific PilZ domain adaptor protein, and this interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signaling influences motility through the interaction of proteins that affect pilus action. The motif required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions may be of broad relevance.

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Changes in the Min Oscillation Pattern before and after Cell Birth

Journal of Bacteriology ◽

10.1128/jb.00364-10 ◽

2010 ◽

Vol 192 (16) ◽

pp. 4134-4142 ◽

Cited By ~ 24

Author(s):

Jennifer R. Juarez ◽

William Margolin

Keyword(s):

Cell Division ◽

Fluorescent Protein ◽

Time Lapse ◽

The Other ◽

Cell Pole ◽

Daughter Cells ◽

Division Site ◽

Before And After ◽

Dividing Cells ◽

Green Fluorescent

ABSTRACT The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.

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Multiple conformations facilitate PilT function in the type IV pilus

Nature Communications ◽

10.1038/s41467-019-13070-z ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Matthew McCallum ◽

Samir Benlekbir ◽

Sheryl Nguyen ◽

Stephanie Tammam ◽

John L. Rubinstein ◽

...

Keyword(s):

Single Particle ◽

Protein Complexes ◽

Comprehensive Model ◽

Type Iv Pilus ◽

Electron Cryomicroscopy ◽

X Ray ◽

Functional Studies ◽

Type Iv ◽

Coevolution Analysis ◽

Multiple Conformations

AbstractType IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C2 symmetry; however, most of these ATPases crystallize with either C3 or C6 symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C2,C3, and C6 conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.

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Conserved genomic neighborhood is a strong but no perfect indicator for a direct interaction of microbial gene products

BMC Bioinformatics ◽

10.1186/s12859-019-3200-z ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Robert Esch ◽

Rainer Merkl

Keyword(s):

Gene Product ◽

Protein Complexes ◽

Sequence Similarity ◽

False Positive Rate ◽

Direct Interaction ◽

Interaction Partner ◽

Gene Products ◽

Genomic Context ◽

Positive Rate ◽

Genomic Neighborhood

Abstract Background The order of genes in bacterial genomes is not random; for example, the products of genes belonging to an operon work together in the same pathway. The cotranslational assembly of protein complexes is deemed to conserve genomic neighborhoods even stronger than a common function. This is why a conserved genomic neighborhood can be utilized to predict, whether gene products form protein complexes. Results We were interested to assess the performance of a neighborhood-based classifier that analyzes a large number of genomes. Thus, we determined for the genes encoding the subunits of 494 experimentally verified hetero-dimers their local genomic context. In order to generate phylogenetically comprehensive genomic neighborhoods, we utilized the tools offered by the Enzyme Function Initiative. For each subunit, a sequence similarity network was generated and the corresponding genome neighborhood network was analyzed to deduce the most frequent gene product. This was predicted as interaction partner, if its abundance exceeded a threshold, which was the frequency giving rise to the maximal Matthews correlation coefficient. For the threshold of 16%, the true positive rate was 45%, the false positive rate 0.06%, and the precision 55%. For approximately 20% of the subunits, the interaction partner was not found in a neighborhood of ± 10 genes. Conclusions Our phylogenetically comprehensive analysis confirmed that complex formation is a strong evolutionary factor that conserves genome neighborhoods. On the other hand, for 55% of the cases analyzed here, classification failed. Either, the interaction partner was not present in a ± 10 gene window or was not the most frequent gene product.

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Formation of a Bazooka–Stardust complex is essential for plasma membrane polarity in epithelia

The Journal of Cell Biology ◽

10.1083/jcb.201006029 ◽

2010 ◽

Vol 190 (5) ◽

pp. 751-760 ◽

Cited By ~ 65

Author(s):

Michael P. Krahn ◽

Johanna Bückers ◽

Lars Kastrup ◽

Andreas Wodarz

Keyword(s):

Plasma Membrane ◽

Protein Complexes ◽

Pdz Domain ◽

Phosphorylation Site ◽

Direct Interaction ◽

Epithelial Polarity ◽

Kinase C ◽

Discs Large ◽

Evolutionarily Conserved ◽

Functional Hierarchy

Apical–basal polarity in Drosophila melanogaster epithelia depends on several evolutionarily conserved proteins that have been assigned to two distinct protein complexes: the Bazooka (Baz)–PAR-6 (partitioning defective 6)–atypical protein kinase C (aPKC) complex and the Crumbs (Crb)–Stardust (Sdt) complex. These proteins operate in a functional hierarchy, in which Baz is required for the proper subcellular localization of all other proteins. We investigated how these proteins interact and how this interaction is regulated. We show that Baz recruits Sdt to the plasma membrane by direct interaction between the Postsynaptic density 95/Discs large/Zonula occludens 1 (PDZ) domain of Sdt and a region of Baz that contains a phosphorylation site for aPKC. Phosphorylation of Baz causes the dissociation of the Baz–Sdt complex. Overexpression of a nonphosphorylatable version of Baz blocks the dissociation of Sdt from Baz, causing phenotypes very similar to those of crb and sdt mutations. Our findings provide a molecular mechanism for the phosphorylation-dependent interaction between the Baz–PAR-3 and Crb complexes during the establishment of epithelial polarity.

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Agrobacterium tumefaciens VirB6 Protein Participates in Formation of VirB7 and VirB9 Complexes Required for Type IV Secretion

Journal of Bacteriology ◽

10.1128/jb.185.9.2867-2878.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2867-2878 ◽

Cited By ~ 74

Author(s):

Simon J. Jakubowski ◽

Vidhya Krishnamoorthy ◽

Peter J. Christie

Keyword(s):

Agrobacterium Tumefaciens ◽

Protein Complexes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Type Iv Secretion ◽

Wild Type ◽

Mutant Proteins ◽

Type Iv ◽

Inner Membrane Protein ◽

Substrate Transfer

ABSTRACT This study characterized the contribution of Agrobacterium tumefaciens VirB6, a polytopic inner membrane protein, to the formation of outer membrane VirB7 lipoprotein and VirB9 protein multimers required for type IV secretion. VirB7 assembles as a disulfide cross-linked homodimer that associates with the T pilus and a VirB7-VirB9 heterodimer that stabilizes other VirB proteins during biogenesis of the secretion machine. Two presumptive VirB protein complexes, composed of VirB6, VirB7, and VirB9 and of VirB7, VirB9, and VirB10, were isolated by immunoprecipitation or glutathione S-transferase pulldown assays from detergent-solubilized membrane extracts of wild-type A348 and a strain producing only VirB6 through VirB10 among the VirB proteins. To examine the biological importance of VirB6 complex formation for type IV secretion, we monitored the effects of nonstoichiometric VirB6 production and the synthesis of VirB6 derivatives with 4-residue insertions (VirB6.i4) on VirB7 and VirB9 multimerization, T-pilus assembly, and substrate transfer. A virB6 gene deletion mutant accumulated VirB7 dimers at diminished steady-state levels, whereas complementation with a plasmid bearing wild-type virB6 partially restored accumulation of the dimers. VirB6 overproduction was correlated with formation of higher-order VirB9 complexes or aggregates and also blocked substrate transfer without a detectable disruption of T-pilus production; these phenotypes were displayed by cells grown at 28°C, a temperature that favors VirB protein turnover, but not by cells grown at 20°C. Strains producing several VirB6.i4 mutant proteins assembled novel VirB7 and VirB9 complexes detectable by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two strains producing the D60.i4 and L191.i4 mutant proteins translocated IncQ plasmid and VirE2 effector protein substrates in the absence of a detectable T pilus. Our findings support a model that VirB6 mediates formation of VirB7 and VirB9 complexes required for biogenesis of the T pilus and the secretion channel.

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A non-radioactive mass spectrometry based differential radial capillary action of ligand assay (DRaCALA) to assess ligand binding to proteins

10.33774/chemrxiv-2021-nj2ws ◽

2021 ◽

Author(s):

Annika Cimdins-Ahne ◽

Alexey Chernobrovkin ◽

Roman Zubarev ◽

Ute Römling

Keyword(s):

Mass Spectrometry ◽

Ligand Binding ◽

Chronic Infection ◽

Second Messengers ◽

Physicochemical Characteristics ◽

Diguanylate Cyclase ◽

Capillary Action ◽

Ggdef Domain ◽

Nucleotide Binding Proteins ◽

Bacterial Domain

Binding of ligands to macromolecules changes their physicochemical characteristics. Cyclic di-GMP and other cyclic di-nucleotides are second messengers involved in motility/sessility and acute/chronic infection life style transition. Although the GGDEF domain encoding preferentially a diguanylate cyclase represents one of the most abundant bacterial domain superfamilies, the number of cyclic di-GMP receptors falls short. To facilitate screening for cyclic di-nucleotide binding proteins, we describe a non-radioactive, MALDI-TOF based modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that YciRFec101, but not the YciRTOB1 variant of the diguanylate cyclase/phosphodiesterase YciR binds cyclic di-GMP.

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Activation mechanism of a small prototypic Rec-GGDEF diguanylate cyclase

10.1101/2020.08.28.271692 ◽

2020 ◽

Author(s):

Raphael D. Teixeira ◽

Fabian Holzschuh ◽

Tilman Schirmer

Keyword(s):

Sensory Input ◽

Coiled Coil ◽

Activation Mechanism ◽

Diguanylate Cyclase ◽

Histidine Kinases ◽

Diguanylate Cyclases ◽

Component Systems ◽

Switch Mechanism ◽

Two Component Systems ◽

Ggdef Domain

AbstractDiguanylate cyclases (DGCs) synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of DGCs. Full-length crystal structures revealed that BeF3- pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many DGCs with N-terminal coiled-coil linkers.

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