Role of a Putative Cyclic Di-GMP Forming Locus MSMEG_2196 in Mycobacterium Smegmatis

<p>Cyclic di-guanosine-monophosphate (c-di-GMP) has been recognized as a second messenger in bacteria controlling multiple cellular processes such as biofilm formation, motility, and virulence. Proteins containing GGDEF and EAL domains are engaged in the synthesis and degradation, respectively, of cyclic di-GMP. Some bacteria contain multiple proteins with GGDEF and EAL domains. The genome of Mycobacterium tuberculosis encodes only one protein (Rv1354c) which contains a GGDEF domain. This protein also contains a tandem EAL. The function of this protein in mycobacteria has not yet been determined. In this study, the orthologue of Rv1354c was investigated in Mycobacterium smegmatis (MSMEG_2196). The expression of MSMEG_2196 in M. smegmatis was altered by constructing sense and antisense expressing strains. The effect of the altered expression of MSMEG_2196 on M. smegmatis was tested under carbon, oxygen, phosphorous, and nitrogen limited growth conditions. There was no significant effect on growth in either the antisense or sense expressing strains grown under nutrient-rich, or carbon-, or oxygen-, or phosphorous limitation conditions. However, a growth effect was observed in the antisense expressing strain when grown under nitrogen-limited conditions. In particular, at mid stationary-phase (1,800 min) the MSMEG_2196 antisense strain had an OD600 value of 0.60, compared to that of the control M. smegmatis/pMind strain (OD600 value of 1.09). These results were further confirmed by the low colony forming units measures observed in MSMEG_2196 antisense strain. Proteomic analysis was carried out on the MSMEG_2196 antisesne expressing strain grown in the nitrogen-limited condition. Proteins that were differentially expressed were identified by mass spectrometry. A number of the proteins that were down-regulated in the antisense expressing strain are important in the survival of the bacteria under nitrogen-limited conditions. This study indicates a role for MSMEG_2196 in growth or survival of mycobacteria under nitrogen-limitations.</p>

Role of a Putative Cyclic Di-GMP Forming Locus MSMEG_2196 in Mycobacterium Smegmatis

<p>Cyclic di-guanosine-monophosphate (c-di-GMP) has been recognized as a second messenger in bacteria controlling multiple cellular processes such as biofilm formation, motility, and virulence. Proteins containing GGDEF and EAL domains are engaged in the synthesis and degradation, respectively, of cyclic di-GMP. Some bacteria contain multiple proteins with GGDEF and EAL domains. The genome of Mycobacterium tuberculosis encodes only one protein (Rv1354c) which contains a GGDEF domain. This protein also contains a tandem EAL. The function of this protein in mycobacteria has not yet been determined. In this study, the orthologue of Rv1354c was investigated in Mycobacterium smegmatis (MSMEG_2196). The expression of MSMEG_2196 in M. smegmatis was altered by constructing sense and antisense expressing strains. The effect of the altered expression of MSMEG_2196 on M. smegmatis was tested under carbon, oxygen, phosphorous, and nitrogen limited growth conditions. There was no significant effect on growth in either the antisense or sense expressing strains grown under nutrient-rich, or carbon-, or oxygen-, or phosphorous limitation conditions. However, a growth effect was observed in the antisense expressing strain when grown under nitrogen-limited conditions. In particular, at mid stationary-phase (1,800 min) the MSMEG_2196 antisense strain had an OD600 value of 0.60, compared to that of the control M. smegmatis/pMind strain (OD600 value of 1.09). These results were further confirmed by the low colony forming units measures observed in MSMEG_2196 antisense strain. Proteomic analysis was carried out on the MSMEG_2196 antisesne expressing strain grown in the nitrogen-limited condition. Proteins that were differentially expressed were identified by mass spectrometry. A number of the proteins that were down-regulated in the antisense expressing strain are important in the survival of the bacteria under nitrogen-limited conditions. This study indicates a role for MSMEG_2196 in growth or survival of mycobacteria under nitrogen-limitations.</p>

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

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.

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.

Activation mechanism of a small prototypic Rec-GGDEF diguanylate cyclase

Diguanylate cyclases 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, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. 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 diguanylate cyclases. Full-length crystal structures reveal 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 diguanylate cyclases with N-terminal coiled-coil linkers.

Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.

Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies.

Activation mechanism of a small prototypic Rec-GGDEF diguanylate cyclase

Diguanylate 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.

Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa

&lt;p&gt;The molecule c-di-GMP is a bacterial second messenger that controls various processes such as motility or biofilm formation in bacteria [1]. To synthesize and degrade c-di-GMP, enzymes called diguanylate cyclases (DGC) containing a GGDEF-domain and phosphodiesterases (PDE) containing an EAL-domain or HD-GYP-domain are important [1, 2].&lt;em&gt; Pseudomonas aeruginosa&lt;/em&gt;, a model organism for biofilm formation and dispersion, encodes for 18 GGDEF, 5 EAL, 16 GGDEF / EAL, and 3 HD-GYP-domain-containing proteins [3].&lt;br /&gt;One of the GGDEF / EAL-containing proteins is NbdA. This protein also harbors an N-terminal membrane anchored MHYT-domain, that is predicted to be a sensor for NO, CO or O&lt;sub&gt;2&lt;/sub&gt; [4]. In this work, recombinant and affinity purified NbdA was tested for its PDE activity. Three different methods were used to measure the PDE activity of NbdA: a bis-pNPP-assay in which the conversion of the pseudosubstrate bis-pNPP into p-nitrophenol was detected spectroscopically, an HPLC-analysis of an enzymatic assay with the native substrate c-di-GMP, and a MANT-c-di-GMP-assay in which a fluorescently labeled form of the presumed substrate c-di-GMP was utilized.&lt;br /&gt;To establish these methods, the two known phosphodiesterases, PdeH from &lt;em&gt;Escherichia coli&lt;/em&gt; [5] and RocR from &lt;em&gt;P. aeruginosa&lt;/em&gt; [6], were also produced and tested. Subsequently, three variants of NbdA were investigated: the full-length version and two truncated versions of the protein. Activity was further assessed using functional complementation of an &lt;em&gt;E. coli&lt;/em&gt; phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity &lt;em&gt;in vivo&lt;/em&gt;.&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt; &lt;p&gt;[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.&lt;/p&gt; &lt;p&gt;[2] R&amp;#246;mling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629&amp;#8211;639.&lt;/p&gt; &lt;p&gt;[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547&amp;#8211;12555.&lt;/p&gt; &lt;p&gt;[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17&amp;#8211;23.&lt;/p&gt; &lt;p&gt;[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434&amp;#8211;2446.&lt;/p&gt; &lt;p&gt;[6] Chen et al. (2012) Chen, M.W., Kotaka, M., Vonrhein, C., Bricogne, G., Rao, F., Chuah, M.L.C., Svergun, D., Schneider, G., Liang, Z.-X., Lescar, J.&amp;#160; (2012). Signaling. J. Bacteriol. 194: 4837&amp;#8211;4846&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt;

Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3′,3′-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.