1H, 13C and 15N resonance assignments of the C-terminal DNA-binding domain of RstA protein from Klebsiella pneumoniae

Bacterial cells often use two-component signal transduction systems to regulate genes in response to environmental stimuli. The RstA/RstB system is a two-component regulatory system consisting of the membrane sensor, RstB, and its cognate response regulator RstA. The RstA of Klebsiella pneumoniae consists of a N-terminal receiver domain (NRD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of the response regulator induces a conformational change in the regulatory domain of RstA, which results in activation of the effector domain to regulate the downstream genes, including the ferrous iron transport system (Feo), at low-pH condition. Here we report the ¹H, ¹³C and ¹⁵N resonance assignments and secondary structure identification of the DBD of RstA from K. pneumoniae as a first step for unraveling the structural and functional relationship of the RstA/RstB two component system.     

Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element

The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1–119) and a C-terminal DNA-binding domain (DBD, residues 130–236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein–DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.     

Quantitative proteomic analysis of Salmonella enterica serovar Typhimurium under PhoP/PhoQ activation conditions

The PhoP/PhoQ two-component system plays a central regulatory role in the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium), and it can be activated by low Mg(2+) concentrations and sublethal concentrations of cationic antimicrobial peptides (CAMP). Therefore, these two PhoP/PhoQ activation signals are considered as in vivo environmental cues sensed by S. Typhimurium for adaptation and survival. In this work, we conducted a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic study to survey the proteomic changes of S. Typhimurium in response to low Mg(2+) concentrations or CAMP. We discovered that CAMP activated a portion of the PhoP/PhoQ regulatory network, whereas low Mg(2+) concentrations upregulated nearly all known members of this network, a number of previously unknown proteins, and some proteins regulated by IHF and RpoS. Systematic analysis following metabolic pathways revealed that low Mg(2+) concentrations selectively influenced proteins of certain metabolic functions while CAMP did not. Our study indicates that the low Mg(2+)-concentration condition may lead S. Typhimurium into a growth-control lifestyle, which provides new perspectives about Salmonella's adaptation to the host environment.