scholarly journals PdhS, an Old-Pole-Localized Histidine Kinase, Recruits the Fumarase FumC in Brucella abortus

2010 ◽  
Vol 192 (12) ◽  
pp. 3235-3239 ◽  
Author(s):  
Johann Mignolet ◽  
Charles Van der Henst ◽  
Cécile Nicolas ◽  
Michaël Deghelt ◽  
Delphine Dotreppe ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Histidine Kinase ◽  
Sinorhizobium Meliloti ◽  
Caulobacter Crescentus ◽  
Bacterial Pathogen ◽  
The Other ◽  
Yeast Two Hybrid ◽  
Two Hybrid

ABSTRACT The bacterial pathogen Brucella abortus was recently demonstrated to recruit the essential cytoplasmic histidine kinase PdhS to its old pole. Here, we report identification of the fumarase FumC as a specific partner for the N-terminal “sensing” domain of PdhS, using an ORFeome-based yeast two-hybrid screen. We observed that FumC and PdhS colocalize at the old pole of B. abortus, while the other fumarase FumA is not polarly localized. FumC is not required for PdhS localization, and polar FumC localization is not FumA dependent. FumC homologs are not polarly localized in Sinorhizobium meliloti and Caulobacter crescentus, suggesting that polar recruitment of FumC by PdhS is evolutionarily recent.

scholarly journals Interaction Network and Localization of Brucella abortus Membrane Proteins Involved in the Synthesis, Transport, and Succinylation of Cyclic β-1,2-Glucans

2015 ◽  
Vol 197 (9) ◽  
pp. 1640-1648 ◽  
Author(s):  
Leticia S. Guidolin ◽  
Susana M. Morrone Seijo ◽  
Francisco F. Guaimas ◽  
Diego J. Comerci ◽  
Andrés E. Ciocchini
Keyword(s):  
Membrane Proteins ◽  
Hybrid System ◽  
Brucella Abortus ◽  
Interaction Network ◽  
Fluorescence Analysis ◽  
The Other ◽  
Content Type ◽  
Membrane Complex ◽  
Heterotypic Interactions ◽  
Two Hybrid

ABSTRACTCyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that play an important role in the virulence and interaction ofBrucellawith the host. Once synthesized in the cytoplasm by the CβG synthase (Cgs), CβG are transported to the periplasm by the CβG transporter (Cgt) and succinylated by the CβG modifier enzyme (Cgm). Here, we used a bacterial two-hybrid system and coimmunoprecipitation techniques to study the interaction network between these three integral inner membrane proteins. Our results indicate that Cgs, Cgt, and Cgm can form both homotypic and heterotypic interactions. Analyses carried out with Cgs mutants revealed that the N-terminal region of the protein (Cgs region 1 to 418) is required to sustain the interactions with Cgt and Cgm as well as with itself. We demonstrated by single-cell fluorescence analysis that inBrucella, Cgs and Cgt are focally distributed in the membrane, particularly at the cell poles, whereas Cgm is mostly distributed throughout the membrane with a slight accumulation at the poles colocalizing with the other partners. In summary, our results demonstrate that Cgs, Cgt, and Cgm form a membrane-associated biosynthetic complex. We propose that the formation of a membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating their synthesis with the transport and modification.IMPORTANCEIn this study, we analyzed the interaction and localization of the proteins involved in the synthesis, transport, and modification ofBrucella abortuscyclic β-1,2-glucans (CβG), which play an important role in the virulence and interaction ofBrucellawith the host. We demonstrate that these proteins interact, forming a complex located mainly at the cell poles; this is the first experimental evidence of the existence of a multienzymatic complex involved in the metabolism of osmoregulated periplasmic glucans in bacteria and argues for another example of pole differentiation inBrucella. We propose that the formation of this membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating synthesis with the transport and modification.

scholarly journals Identification and Comprehensive Structural and Functional Analyses of the EXO70 Gene Family in Cotton

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1594
Author(s):  
Ya-Qian Zhu ◽  
Lu Qiu ◽  
Lu-Lu Liu ◽  
Lei Luo ◽  
Xin-Pei Han ◽  
...  
Keyword(s):  
Gene Family ◽  
Upland Cotton ◽  
The Other ◽  
Biological Processes ◽  
Polar Growth ◽  
Yeast Two Hybrid ◽  
Plant Cell Division ◽  
Vital Component ◽  
Functional Analyses ◽  
Two Hybrid

The EXO70 gene is a vital component of the exocytosis complex and participates in biological processes ranging from plant cell division to polar growth. There are many EXO70 genes in plants and their functions are extensive, but little is known about the EXO70 gene family in cotton. Here, we analyzed four cotton sequence databases, identified 165 EXO70 genes, and divided them into eight subgroups (EXO70A–EXO70H) based on their phylogenetic relationships. EXO70A had the most exons (≥11), whereas the other seven each had only one or two exons. Hence, EXO70A may have many important functions. The 84 EXO70 genes in Asian and upland cotton were expressed in the roots, stems, leaves, flowers, fibers, and/or ovules. Full-length GhEXO70A1-A cDNA was homologously cloned from upland cotton (Gossypium hirsutum, G. hirsutum). Subcellular analysis revealed that GhEXO70A1-A protein was localized to the plasma membrane. A yeast two-hybrid assay revealed that GhEXO70A1-A interacted with GhEXO84A, GhEXO84B, and GhEXO84C. GhEXO70A1-A silencing significantly altered over 4000 genes and changed several signaling pathways related to metabolism. Thus, the EXO70 gene plays critical roles in the physiological functions of cotton.

scholarly journals The Agrobacterium T-DNA Transport Pore Proteins VirB8, VirB9, and VirB10 Interact with One Another

2000 ◽  
Vol 182 (3) ◽  
pp. 758-763 ◽  
Author(s):  
Anath Das ◽  
Yong-Hong Xie
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transfer Function ◽  
Outer Membrane ◽  
Dna Transfer ◽  
The Other ◽  
Yeast Two Hybrid ◽  
Dna Transport ◽  
Interaction Domains ◽  
Two Hybrid

ABSTRACT The VirB proteins of Agrobacterium tumefaciens form a transport pore to transfer DNA from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10. Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another. In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself. These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore. Fusions that contain small, defined segments of the proteins were used to define the interaction domains of VirB8 and VirB9. All interaction domains of both proteins mapped to the N-terminal half of the proteins. Two separate domains at the N- and C-terminal ends of VirB9 are involved in its homotypic interaction, suggesting that VirB9 forms a higher oligomer. We observed that the alteration of serine at position 87 of VirB8 to leucine abolished its DNA transfer function. Studies on the interaction of the mutant protein with the other VirB proteins showed that the VirB8S87L mutant is defective in interaction with VirB9. The mutant, however, interacted efficiently with VirB8 and VirB10, suggesting that the VirB8-VirB9 interaction is essential for DNA transfer.

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
M. V. Borca ◽  
E. A. Vuono ◽  
E. Ramirez-Medina ◽  
P. Azzinaro ◽  
K. A. Berggren ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interaction ◽  
Hybrid Approach ◽  
Classical Swine Fever ◽  
Host Protein ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Viral Virulence ◽  
Glycoprotein E2 ◽  
Two Hybrid

ABSTRACT The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

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