Search

Candida albicans is a dimorphic fungus that converts from a yeast form to a hyphae form during infection. This switch requires the formation of actin cable to coordinate polarized cell growth. It's known that nucleation of this cable requires a multiprotein complex localized at the tip called the polarisome, but the mechanisms underpinning this process were unclear. Here, we found that C. albicans Aip5, a homolog of polarisome component ScAip5 in Saccharomyces cerevisiae that nucleates actin polymerization and synergizes with the formin ScBni1, regulates actin assembly and hyphae growth synergistically with other polarisome proteins Bni1, Bud6, and Spa2. The C terminus of Aip5 binds directly to G-actin, Bni1, and the C-terminal of Bud6, which form the core of the nucleation complex to polymerize F-actin. Based on insights from structural biology and molecular dynamic simulations, we propose a possible complex conformation of the actin nucleation core, which provides cooperative positioning and supports the synergistic actin nucleation activity of a tri-protein complex Bni1-Bud6-Aip5. Together with known interactions of Bni1 with Bud6 and Aip5 in S. cerevisiae, our findings unravel molecular mechanisms of C. albicans by which the tri-protein complex coordinates the actin nucleation in actin cable assembly and hyphal growth, which is likely a conserved mechanism in different filamentous fungi and yeast.

Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry.

Global transcriptional regulator downstream RpfR (GtrR) is a key downstream regulator for quorum-sensing signaling molecule cis-2-dodecenoic acid (BDSF). As a bacterial enhancer-binding protein (bEBP), GtrR is composed of an N-terminal receiver domain, a central ATPases associated with diverse cellular activities (AAA+) ATPase σ54 -interaction domain, and a C-terminal helix-turn-helix DNA-binding domain. In this work, we solved its AAA+ ATPase domain in both apo and GTP-bound forms. The structure revealed how GtrR specifically recognizes GTP. In addition, we also revealed that GtrR has moderate GTPase activity in vitro in the absence of its activation signal. Finally, we found the residues K170, D236, R311, and R357 in GtrR that are crucial to its biological function, any single mutation leading to completely abolishing GtrR activity. Ministry of Education (MOE) This research was funded by the Ministry of Education (MOE) of Singapore, Tier II grant MOE2019-T2-2- 099 (to Y-GG) and Tier 1 grant RG108/20 (to Y-GG).

Profilins are abundant cytosolic proteins that are universally expressed in eukaryotes and that regulate actin filament elongation by binding to both monomeric actin (G-actin) and formin proteins. The atypical profilin Arabidopsis AtPRF3 has been reported to cooperate with canonical profilin isoforms in suppressing formin-mediated actin polymerization during plant innate immunity responses. AtPRF3 has a 37-amino acid-long N-terminal extension (NTE), and its suppressive effect on actin assembly is derived from enhanced interaction with the polyproline (Poly-P) of the formin AtFH1. However, the molecular mechanism remains unclear. Here, we solved the crystal structures of AtPRF3Δ22 and AtPRF3Δ37, as well as AtPRF2 apo form and in complex with AtFH1 Poly-P at 1.5-3.6 A resolutions. By combining these structures with molecular modeling, we found that AtPRF3Δ22 NTE has high plasticity, with a primary "closed" conformation that can adopt an open conformation that enables Poly-P binding. Furthermore, using molecular dynamics simulation and free-energy calculations of protein-protein binding, along with experimental validation, we show that the AtPRF3Δ22 binds to Poly-P in an adaptive manner, thereby enabling different binding modes that maintain the interaction through disordered sequences. Together, our structural and simulation results suggest that the dynamic conformational changes of the AtPRF3 NTE upon Poly-P binding modulate their interactions to fine-tune formin-mediated actin assembly.

A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown. We identify a previously functionally unknown polarisome member, actin-interacting-protein 5 (Aip5), which promotes actin assembly synergistically with formin Bni1. Aip5-C terminus is responsible for its activities by interacting with G-actin and Bni1. Through N-terminal intrinsically disordered region, Aip5 forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions. The molecular dynamics and reversibility of Aip5 condensates are regulated by scaffolding protein Spa2 via liquid-liquid phase separation both in vitro and in vivo. In the absence of Spa2, Aip5 condensates hamper cell growth and actin cable structures under stress treatment. The present study reveals the mechanisms of actin assembly for polarity establishment and the adaptation in stress conditions to protect actin assembly by protein phase separation., The polarisome is a dynamic protein complex that nucleates F-actin for polarized yeast growth, but its regulation is unclear. Here, the authors report that the polarisome protein Aip5 undergoes Spa2-mediated phase separation in physiological and stress conditions, potentially for regulating actin assembly.

Significance Bacterial infection has been extensively investigated; however, little is known about how bacterial pathogens timely shut down infecting machinery after successful infections. Here, a previously unknown sucrose–SghR/SghA–SAG–SA signaling axis was identified which controls the timing to shut off bacterial virulence expression and fine-tune host immune response. Sucrose, salicylic acid (SA), and its storage form SAG are small chemicals produced in plants whereas SghR is a bacterial sensor of sucrose and SghA is a bacterial enzyme that releases SA from SAG. Given that SA is an imperative signaling molecule in defense against a variety of microbial pathogens, these results depict a previously unknown 2-way chemical signaling cross-talk during microbe–host coevolution and shed mechanistic insights into host–bacteria interaction., It is highly intriguing how bacterial pathogens can quickly shut down energy-costly infection machinery once successful infection is established. This study depicts that mutation of repressor SghR increases the expression of hydrolase SghA in Agrobacterium tumefaciens, which releases plant defense signal salicylic acid (SA) from its storage form SA β-glucoside (SAG). Addition of SA substantially reduces gene expression of bacterial virulence. Bacterial vir genes and sghA are differentially transcribed at early and later infection stages, respectively. Plant metabolite sucrose is a signal ligand that inactivates SghR and consequently induces sghA expression. Disruption of sghA leads to increased vir expression in planta and enhances tumor formation whereas mutation of sghR decreases vir expression and tumor formation. These results depict a remarkable mechanism by which A. tumefaciens taps on the reserved pool of plant signal SA to reprogram its virulence upon establishment of infection.

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants. Ministry of Education (MOE) Accepted version This work was supported by a Tier II grant MOE2019-T2-2-099 from the Ministry of Education (MOE) of Singapore (Y.-G.G.), and ARC FT and DP grants (DP190101941; FT160100218), Villum Investigator (Project ID: 25915) and Novo Nordisk Laureate (NNF19OC0056076) grants (SP).

Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans. Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs. Published version

Carbonic anhydrases (CAs) are a well characterized family of metalloenzymes that are highly efficient in facilitating the interconversion between carbon dioxide and bicarbonate. Recently, CA activity has been associated with the LCIB (limiting CO2-inducible protein B) protein family, which has been an interesting target in aquatic photosynthetic microorganisms. To gain further insight into the catalytic mechanism of this new group of CAs, the X-ray structure of a highly active LCIB homolog (PtLCIB3) from the diatomPhaeodactylum tricornutumwas determined. The CA activities of PtLCIB3, its paralog PtLCIB4 and a variety of their mutants were also measured. It was discovered that PtLCIB3 has a classic β-CA fold and its overall structure is highly similar to that of its homolog PtLCIB4. Subtle structural alterations between PtLCIB3 and PtLCIB4 indicate that an alternative proton-shuttle cavity could perhaps be one reason for their remarkable difference in CA activity. A potential alternative proton-shuttle route in the LCIB protein family is suggested based on these results.

A chitinase gene (RmChiA) encoding 445 amino acid (aa) residues from a fungus Rhizomucor miehei was cloned and overexpressed in Escherichia coli. Two kinds of RmChiA crystal forms, with space groups P32 2 1 and P1, were obtained by sitting-drop vapor diffusion and the structures were determined by X-ray diffraction. The overall structure of RmChiA monomer, which is the first structure of bacterial-type chitinases from nonpathogenic fungi, adopts a canonical triosephosphate isomerase (TIM) barrel fold with two protruding chitinase insertion domains. RmChiA exhibited a unique NxDxE catalytical motif and a real active site tunnel structure, which are firstly found in GH family 18 chitinases. The motif had high structural homolog with the typical DxDxE motif in other GH family 18 chitinases. The tunnel is formed by two unusual long loops, containing 15 aa and 45 aa respectively, linked by a disulfide bond across the substrate-binding cleft. Mutation experiments found that opening the roof of tunnel structure increased the hydrolysis efficiency of RmChiA, but the thermostability of the mutants decreased. Moreover, the tunnel structure endowed RmChiA with the exo-chitinase character.

Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia, the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.

YajQ, a binding protein of the universal bacterial second messenger cyclic di-GMP (c-di-GMP), affects virulence in several bacterial pathogens, including Xanthomonas campestris. In this bacterium, YajQ interacts with the transcription factor LysR. Upon c-di-GMP binding, the whole c-di-GMP-YajQ-LysR complex is found to dissociate from DNA, resulting in virulence gene regulation. Here, we identify a YajQ-LysR-like system in the bacterial biocontrol agent Lysobacter enzymogenes OH11 that secretes an antifungal antibiotic, heat-stable antifungal factor (HSAF) against crop fungal pathogens. We show that the YajQ homologue, CdgL (c-di-GMP receptor interacting with LysR) affects expression of the HSAF biosynthesis operon by interacting with the transcription activator LysR. The CdgL-LysR interaction enhances the apparent affinity of LysR to the promoter region upstream of the HSAF biosynthesis operon, which increases operon expression. Unlike the homologues CdgL (YajQ)-LysR system in X. campestris, we show that c-di-GMP binding to CdgL seems to weaken CdgL-LysR interactions and promote the release of CdgL from the LysR-DNA complex, which leads to decreased expression. Together, this study takes the YajQ-LysR-like system from bacterial pathogens to a crop-protecting bacterium that is able to regulate antifungal HSAF biosynthesis via disassembly of the c-di-GMP receptor-transcription activator complex. Published version

Profilin functions with formin in actin assembly, a process that regulates multiple aspects of plant development and immune responses. High-level eukaryotes contain multiple isoforms of profilin, formin, and actin, whose partner-specific interactions in actin assembly are not completely understood in plant development and defense responses. To examine the functionally distinct interactions between profilin and formin, we studied all five Arabidopsis profilins and their interactions with formin by using both in vitro biochemical and in vivo cell biology approaches. Unexpectedly, we found a previously undescribed negative regulatory function of AtPRF3 in AtFH1-mediated actin polymerization. The N-terminal 37 residues of AtPRF3 were identified to play a predominant role in inhibiting formin-mediated actin nucleation via their high affinity for the formin polyproline region and their triggering of the oligomerization of AtPRF3. Both in vivo and in vitro mechanistic studies of AtPRF3 revealed a universal mechanism in which the weak interaction between profilin and formin positively regulates actin assembly by ensuring rapid recycling of profilin, whereas profilin oligomerization negatively regulates actin polymerization. Upon recognition of the pathogen-associated molecular pattern, the gene transcription and protein degradation of AtPRF3 are modulated for actin assembly during plant innate immunity. The prf3 Arabidopsis plants show higher sensitivity to the bacterial flagellum peptide in both the plant growth and ROS responses. These findings demonstrate a profilin-mediated actin assembly mechanism underlying the plant immune responses. MOE (Min. of Education, S’pore) Accepted version

Cell 139, 1084-1095 (2009). doi:10.1016/j.cell.2009.11.015, Published by Cell Press, [Cambridge, Mass.]

Succinic semialdehyde (SSA), an important metabolite of γ-aminobutyric acid (GABA), is a ligand of the repressor AttJ regulating the expression of the attJ-attKLM gene cluster in the plant pathogen Agrobacterium tumefaciens . While the response of A. tumefaciens to GABA and the function of attKLM have been extensively studied, genetic and physiological responses of A. tumefaciens to SSA remain unknown. In combination with microarray and genetic approaches, this study sets out to explore new roles of the SSA-AttJKLM regulatory mechanism during bacterial infection. The results showed that SSA plays a key role in regulation of several bacterial activities, including C 4 -dicarboxylate utilization, nitrate assimilation, and resistance to oxidative stress. Interestingly, while the SSA relies heavily on the functional AttKLM in mediating nitrate assimilation and oxidative stress resistance, the compound could regulate utilization of C 4 -dicarboxylates independent of AttJKLM. We further provide evidence that SSA controls C 4 -dicarboxylate utilization through induction of an SSA importer and that disruption of attKLM attenuates the tumorigenicity of A. tumefaciens . Taken together, these findings indicate that SSA could be a potent plant signal which, together with AttKLM, plays a vital role in promoting the bacterial prosurvival abilities during infection. IMPORTANCE Agrobacterium tumefaciens is a plant pathogen causing crown gall diseases and has been well known as a powerful tool for plant genetic engineering. During the long history of microbe-host interaction, A. tumefaciens has evolved the capabilities of recognition and response to plant-derived chemical metabolites. Succinic semialdehyde (SSA) is one such metabolite. Previous results have demonstrated that SSA functions to activate a quorum-quenching mechanism and thus to decrease the level of quorum-sensing signals, thereby avoiding the elicitation of a plant defense. Here, we studied the effect of SSA on gene expression at a genome-wide level and reported that SSA also promotes bacterial survival during infection. These findings provide a new insight on the biological significance of chemical signaling between agrobacteria and plant hosts.

The crystallization of the novel virulence factors SghA and SghR is reported., Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction.

No abstract

The quorum quenching system of Agrobacterium tumefaciens is specifically activated upon entering the stationary phase. Evidence has shown that this system includes two key components: the IclR-type transcriptional factor AttJ (also named as BlcR) and the AHL-lactonase AttM (also named as BlcC). At exponential phase, AttJ binds to the promoter region of attM and thus suppresses the expression of attM. At stationary phase, however, the small molecule SSA directly binds to AttJ and relieves its inhibition of AttJ and thereby triggers the expression of attM. While the regulation of AttM has been extensively investigated, little is known about the regulation of AttJ. In this study, we demonstrated the D101 amino acid of AttJ is essential for the AttJ function. In vitro, the variant protein of AttJD101H appeared to be readily aggregated. In vivo, the D101H mutation in AttJ entirely abolished the inhibitory activity of AttJ and overexpressed attM in A. tumefaciens A6. In addition, D101H mutation led to an overexpression of attJ, indicating an auto-regulatory mechanism for the attJ regulation. Put together, these findings demonstrate that D101 is an important amino acid for the transcription activity of AttJ and the transcription of attJ is regulated by a negative feedback loop. These results expand previous biochemical characterization of AttJ and provide new mechanistic insights into the regulation of quorum quenching in A. tumefaciens.

The atypical Gβ-like/RACK1 Gib2 protein promotes cAMP signalling that plays a central role in regulating the virulence of Cryptococcus neoformans. Gib2 contains a seven-bladed β transducin structure and is emerging as a scaffold protein interconnecting signalling pathways through interactions with various protein partners. Here, we present the crystal structure of Gib2 at a 2.2-Å resolution. The structure allows us to analyse the association between Gib2 and the ribosome, as well as to identify the Gib2 amino acid residues involved in ribosome binding. Our studies not only suggest that Gib2 has a role in protein translation but also present Gib2 as a physical link at the crossroads of various regulatory pathways important for the growth and virulence of C. neoformans.

RNA methyltransferase is responsible for transferring methyl and resulting in methylation on the bases or ribose ring of RNA, which existed widely but mostly remains an open question. A recombinant protein PH1948 predicting RNA methyltransferase from Pyrococcus horikoshii OT3 has been crystallized. The crystals of selenomethionyl PH1948 belong to space group C2, with unit-cell parameters a=207.0 A, b=43.1 A, c=118.2 A, b=92.1 degrees , and diffract X-rays to 2.2 A resolution. The V(M) value was determined to be 2.8 A3/Da, indicating the presence of four protein molecules in the asymmetric unit.

The biosynthetic pathway of the off-flavour terpenoid alcohol 2-methyliso­borneol (2-MIB) requires geranyl pyrophosphate methyltransferase (GPPMT) to methylate GPP before the cyclization reaction. GPPMT is the first example of an S-adenosyl-l-methionine-dependent methyltransferase that acts on general intermediates such as geranyl pyrophosphate and farnesyl pyrophosphate in isoprenoid biosynthetic pathways. In this study, recombinant GPPMT was overproduced, purified and crystallized in the absence and presence of cofactor, cofactor analogue and substrate. Well diffracting crystals of apo GPPMT containing one molecule in the asymmetric unit were obtained and the structure of this form was solved by the molecular-replacement method. Two crystal forms of the tertiary complex with GPP and sinefungin were also obtained. Structure analysis of these crystals is currently under way in order to understand the enzyme reaction mechanism.

SCO6571 protein from Streptomyces coelicolor A3(2) was overexpressed and purified using Rhodococcus erythropolis as an expressing host. Crystals of selenomethionine-substituted SCO6571 have been obtained by vapor diffusion method. SCO6571 crystals diffract to 2.3 A and were found to belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters a = 84.5, b = 171.6, c = 184.8 A. Six molecules in the asymmetric unit give a crystal volume per protein mass (V(M)) of 2.97 A (3) Da(-1) and solvent content of 58.6 %. The structure was solved by the single wavelength anomalous diffraction (SAD) method. SCO6571 is a TIM-barrel fold protein that assembles into a hexameric molecule with D(3) symmetry.

Bacterial and archaeal 2'-5' RNA ligases, members of the 2H phosphoesterase superfamily, catalyze the linkage of the 5' and 3' exons via a 2'-5'-phosphodiester bond during tRNA-precursor splicing. The crystal structure of the 2'-5' RNA ligase PH0099 from Pyrococcus horikoshii OT3 was solved at 1.94 A resolution (PDB code 1vgj). The molecule has a bilobal alpha+beta arrangement with two antiparallel beta-sheets constituting a V-shaped active-site cleft, as found in other members of the 2H phosphoesterase superfamily. The present structure was significantly different from that determined previously at 2.4 A resolution (PDB code 1vdx) in the active-site cleft; the entrance to the cleft is wider and the active site is easily accessible to the substrate (RNA precursor) in our structure. Structural comparison with the 2'-5' RNA ligase from Thermus thermophilus HB8 also revealed differences in the RNA precursor-binding region. The structural differences in the active-site residues (tetrapeptide motifs H-X-T/S-X) between the members of the 2H phosphoesterase superfamily are discussed.