CitA (citrate) and DcuS (C4-dicarboxylate) sensor kinases in thermophilic Geobacillus kaustophilus and Geobacillus thermodenitrificans

Abstract

The thermophilic Geobacillus thermodenitrificans and Geobacillus kaustophilus are able to use citrate or C 4 -dicarboxylates like fumarate or succinate as the substrates for growth. The genomes of the sequenced Geobacillus strains (nine strains) each encoded a two-component system of the CitA family. The sensor kinase of G. thermodenitrificans (termed CitA Gt ) was able to replace CitA of Escherichia coli (CitA Ec ) in a heterologous complementation assay restoring expression of the CitA Ec -dependent citC-lacZ reporter gene and anaerobic growth on citrate. Complementation was specific for citrate. The sensor kinase of G. kaustophilus (termed DcuS Gk ) was able to replace DcuS Ec of E. coli. It responded in the heterologous expression system to C 4 -dicarboxylates and to citrate, suggesting that DcuS Gk is, like DcuS Ec , a C 4 -dicarboxylate sensor with a side-activity for citrate. DcuS Gk , unlike the homologous DctS from Bacillus subtilis, required no binding protein for function in the complementation assay. Thus, the thermophilic G. thermodenitrificans and G. kaustophilus contain citrate and C 4 -dicarboxylate sensor kinases of the CitA and DcuS type, respectively, and retain function and substrate specificity under mesophilic growth conditions in E. coli.

Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase

“…29 A BLAST search 30 identified CitA homologues in several Geobacillus species out of which the protein of G. thermoleovorans ( Figure S1) was chosen for further characterization. In the course of this study a highly similar histidine kinase of Geobacillus thermodenitrif icans was reported to function as citrate sensor, 31 thereby confirming that the chosen PAS domain was most likely specific for citrate. The DNA sequence encoding residues 31−161 of GtCitA was cloned into plasmid pIVEX2.4IN 32 to express the PAS domain N-terminally fused to His-tagged immunity protein 7 of Escherichia coli (Im7; see Figure S2).…”

“…29 A BLAST search 30 identified CitA homologues in several Geobacillus species out of which the protein of G. thermoleovorans (Figure S1) was chosen for further characterization. In the course of this study a highly similar histidine kinase of Geobacillus thermodenitrif icans was reported to function as citrate sensor, 31 thereby confirming that the chosen PAS domain was most likely specific for citrate.…”

CyaC, a redox‐regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme‐B membrane anchor domain

“…Nitrate was included to support anaerobic growth, glycerol and gluconate as carbon sources that support growth without strong glucose repression. For anaerobic conditions, plates were incubated under N 2 in anaerobic jars, β‐galactosidase activity was determined in 96‐well microtiter plates (Graf et al , ). β‐Galactosidase is given in Miller U, or MU (ΔA 420 min −1 ml −1 OD 570 −1 ) (Miller, ; Graf et al , ).…”

Mechanism of sensor kinase CitA transmembrane signaling

“…It is a member of the PAS domain containing HKs which comprise 30 % of all HKs ( 14 ). CitA belongs to a sensor kinase subfamily ( 15 ) of HKs, which can bind to citrate or C 4 -dicarboxylates. This class of sensor HK has two PAS domains, a periplasmic (or extracytoplasmic) ligand binding domain (PASp) and a cytoplasmic transmitter domain (PASc).…”