BvgS is a hybrid sensor-kinase harboring several cytoplasmic domains that mediate a complex phospho-transfer cascade . It also contains three potential perception domains, two periplasmic Venus flytrap (VFT) find more domains in tandem and a cytoplasmic Per/ArnT/Sim (PAS) domain followed by the kinase domain . We have established that the second VFT domain, VFT2, binds nicotinate and related negative modulator molecules . BvgS is the prototype for VFT-containing sensor-kinases mostly found in Proteobacteria whose molecular mechanisms are poorly
understood. In this work, we characterized the PAS domain of BvgS (PASBvg). PAS domains are structurally conserved, 100- to 120-residue-long signaling modules with sensory and regulatory functions, present in kinases, chemoreceptors and other types of proteins in all branches of the phylogenetic tree [7, 8]. They are composed of a central, five-stranded anti-parallel β sheet flanked by α helices. Many PAS domains appear to form www.selleckchem.com/HDAC.html dimers in vitro and in vivo. A subset of PAS domains harbors heme, flavine nucleotide or other cofactors for perception of physical parameters such as light or O2. Some cytoplasmic PAS domains appear to modulate signal transmission rather than HSP990 supplier to directly perceive a signal [8, 10, 11]. Finally, some PAS domains, including the periplasmic ‘PDC’ (PhoP/DcuS/CitA) domains found in many bacterial TCS sensor-kinases
bind small chemical ligands, which triggers signal transduction [12–15]. Although the presence of a PAS domain in BvgS has been recognized for over 20 years [16, 17], its role is still unknown. Here, we show that this domain is required for transmission of signals from the periplasm. Methods Strains and plasmids The sequence coding for the PAS core domain was amplified by PCR using the PAScore
UP and PAScore LO oligonucleotides as primers (see Additional file 1: Table S1). The amplicon was inserted in pCRII-TOPO (Invitrogen) and sequenced. It was then introduced as a BamHI-HindIII fragment into the corresponding sites of pQE-30 (Qiagen). The resulting plasmid encodes the PASBvg core with an N-terminal His tag. Next, two longer constructs were prepared using the primers PAS His UP and PAS His LO and PAS GB1 UP and PAS GB1 LO. The first amplicon was introduced into pQE30 as a BglII-HindIII fragment, and the other was introduced Galeterone into pGEV2  as a BamHI-XhoI fragment. The first plasmid codes for PASBvg flanked by its N- and C-terminal helices and with an N-terminal 6-His tag. The second codes for a fusion between the GB1 domain and the same BvgS fragment. Finally, sequences coding for PASBvg recombinant proteins of various lengths were amplified by PCR using a combination of the following primers: PAS N1UP, PAS N2UP or PAS N3UP and PAS C1LO, PAS C2LO or PAS C3LO (Additional file 1: Table S1). The amplicons were restricted as BsaI fragments, introduced into the corresponding sites of the pASK-IBA35+ vector (IBA) and sequenced.