Sensor Domain of Histidine Kinase VxrA of Vibrio cholerae- A Hairpin-swapped Dimer and its Conformational Change.

VxrA and VxrB are cognate histidine kinase (HK) - response regulator (RR) pairs of a two-component signaling system (TCS) found in Vibrio cholerae, a bacterial pathogen that causes cholera. The VxrAB TCS positively regulates virulence, the Type VI Secretion System, biofilm formation, and cell wall homeostasis in V. cholerae, providing protection from environmental stresses and contributing to the transmission and virulence of the pathogen. The VxrA HK has a unique periplasmic sensor domain (SD) and, remarkably, lacks a cytoplasmic linker domain between the second transmembrane helix and the dimerization and histidine phosphotransfer (DHp) domain, indicating that this system may utilize a potentially unique signal sensing and transmission TCS mechanism. In this study, we have determined several crystal structures of VxrA-SD and its mutants. These structures reveal a novel structural fold forming an unusual ß hairpin-swapped dimer. A conformational change caused by relative rotation of the two monomers in a VxrA-SD dimer could potentially change the association of transmembrane helices and, subsequently, the pairing of cytoplasmic DHp domains. Based on the structural observation, we propose a putative scissor-like closing regulation mechanism for the VxrA HK.IMPORTANCE V. cholerae has a dynamic life cycle, which requires rapid adaptation to changing external conditions. Two-component signal transduction (TCS) systems allow V. cholerae to sense and respond to these environmental changes. The VxrAB TCS positively regulates a number of important V. cholerae phenotypes, including virulence, the Type Six Secretion System, biofilm formation, and cell wall homeostasis. Here, we provide the crystal structure of the VxrA sensor histidine kinase sensing domain and propose a mechanism for signal transduction. The cognate signal for VxrAB remains unknown, however, in this work we couple our structural analysis with functional assessments of key residues to further our understanding of this important TCS.

A Cytological F1 RNAi Screen for Defects in Drosophila melanogaster Female Meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, our lab has screened the VALIUM22 collection produced by the Harvard TRiP Project, which contains RNAi constructs targeting genes known to be expressed in the germline in a vector optimized for germline expression. By driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4), we can test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we can identify defects associated with genes whose knockdown results in sterility or causes other errors besides nondisjunction. We screened this collection to identify genes that disrupt either of two phenotypes when knocked down: the ability of meiotic chromosomes to congress to a single mass at the end of prometaphase, and the sequestration of Mps1-GFP to ooplasmic filaments in response to hypoxia. After screening >1450 lines of the collection, we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

A cytological F1 RNAi screen for defects in Drosophila melanogaster female meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, we have screened the VALIUM22 collection of RNAi constructs that target germline-expressing genes in a vector optimized for germline expression by driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4). This allowed us to test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we could identify defects in sterile females. After screening >1,450 lines of the collection for two different defects (chromosome congression and the hypoxic sequestration of Mps1-GFP to ooplasmic filaments), we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.