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1 1 CitA (citrate) and DcuS (C4-dicarboxylate) sensor kinases in thermophilic 2 Geobacillus kaustophilus and G. thermodenitrificans 3 4 Running title: CitA sensor kinases of Geobacillus 5 6 7 Sabrina Graf, Constanze Broll, Juliane Wissig, Alexander Strecker, Maria Parowatkin, 8 Gottfried Unden* 9 10 Institute for Microbiology and Wine Research, Johannes Gutenberg University Mainz, 55099 11 Mainz, Germany 12 13 14 15 16 *For correspondence: 17 Dr. G. Unden, University of Mainz, Institute for Microbiology and Wine Research 18 Becherweg 15, 55099 Mainz, Germany 19 Phone: +49-6131-3923550 20 [email protected] 21 2 1 The thermophilic Geobacillus thermodenitrificans and Geobacillus kaustophilus are able to 2 use citrate or C4-dicarboxylates like fumarate or succinate as the substrates for growth. The 3 genomes of the sequenced Geobacillus strains (9 strains) each encoded a two-component 4 system of the CitA family. The sensor kinase of G. thermodenitrificans (termed CitAGt) was 5 able to replace CitA of E. coli (CitAEc) in a heterologous complementation assay restoring 6 expression of the CitAEc dependent citC-lacZ reporter gene and anaerobic growth on citrate. 7 Complementation was specific for citrate. The sensor kinase of G. kaustophilus (termed 8 DcuSGk) was able to replace DcuSEc of E. coli. It responded in the heterologous expression 9 system to C4-dicarboxylates and to citrate, suggesting that DcuSGk is like DcuSEc a C4- 10 dicarboxylate sensor with a side-activity for citrate. DcuSGk required unlike the homologous 11 DctS from B. subtilis no binding protein for function in the complementation assay. Thus the 12 thermophilic G. thermodenitrificans and G. kaustophilus contain citrate and C4-dicarboxylate 13 sensor kinases of the CitA and DcuS-type, respectively, and retain function and substrate 14 specificity under mesophilic growth conditions in E. coli. 15 16 17 3 1 Introduction 2 Bacteria of various taxa are able to grow at the expense of C4-dicarboxylates or citrate 3 (Scheu et al., 2010; Unden & Kleefeld, 2004; Kröger 1980; Kröger et al., 1992; Bott, 1997). 4 The metabolic pathways for aerobic and anaerobic growth on C4-dicarboxylates and citrate 5 are well characterized whereas induction of the corresponding pathways has been studied 6 only in a limited number of bacteria. Induction of C4-dicarboxylate metabolism is 7 accomplished by three types of two-component sensor systems, DcuS-DcuR, DctSRc-DctR and 8 DctBsm-DctR. Citrate catabolism is induced by the citrate responsive two-component system 9 CitA-CitB. DcuS-DcuR of enteric bacteria with sensor kinase DcuS represents the prototype of 10 a C4-dicarboxylate responsive two components system (Janausch et al., 2002b; Scheu et al., 11 2010; Zientz et al., 1998; Golby et al. 1999). The DcuS sensor kinases constitute together 12 with the citrate sensor CitA the CitA family of histidine kinases. The C4-dicarboxylate sensor 13 kinases DctSRc from Rhodobacter capsulatus and DctBSm from Sinorhizobium meliloti, on the 14 other hand, are members of the FixL and NtrB families of sensor kinases, respectively 15 (Hamblin et al., 1993; Reid & Poole, 1998; Valentini & Lapouge, 2013; Janausch et al., 2002b; 16 Scheu et al., 2010). 17 Sensors of the CitA family of γ-proteobacteria are membrane integral and have a common 18 domain structure (Bott, 1997; Scheu et al., 2010). Sensing of the substrates is achieved by an 19 extracytoplasmic PASP type sensor domain that is flanked by transmembrane helices TM1 20 and TM2 (Kaspar et al., 1999; Pappalardo et al., 2003; Kneuper et al., 2005). On the 21 cytoplasmic side, TM2 is followed by a second PAS domain (PASC) that transmits the signal to 22 the C-terminal kinase domain. DcuS of E. coli (DcuSEc) responds to C4-dicarboxylates like 23 fumarate, succinate or L-malate, and with lower sensitivity to citrate (Zientz et al., 1998; 24 Golby et al., 1999; Kneuper et al., 2005). DcuS requires the tranporters DctA under aerobic 4 1 or DcuB under anaerobic conditions as co-regulators (Davies et al., 1999; Steinmetz et al., 2 2014; Witan et al., 2012; Kleefeld et al., 2009). B. subtilis contains the sensor kinase DctSBs 3 resembling DcuSEc in domain composition and function (Asai et al., 2000; Graf et al., 2014). 4 DctSBs requires like DcuSEc the transporters DctABs, and an extra-cytoplasmic substrate 5 binding protein for function and response to the C4-dicarboxylates (Graf et al., 2014). 6 The citrate sensor CitA is found in bacteria that are able to use citrate as the C- and energy 7 source. CitA is highly specific for citrate and defined by the prototypic CitA sensor kinases of 8 Klebsiella and E. coli (Bott et al., 1995; Bott, 1997; Kaspar et al., 1999). CitA of the 9 proteobacteria, Corynebacterium and the homologous CitS of B. subtilis share the domain 10 composition with the DcuS sensor kinases (Bott et al., 1995; Brocker et al., 2009; Yamamoto 11 et al., 2000). The CitA protein of E. coli functions unlike DcuSEc as a stand-alone sensor 12 without the need for accessory proteins (Scheu et al., 2012). 13 Screening the genome sequences of thermophilic bacteria revealed that various Geobacillus 14 and Deinococcus strains encode sensor kinases of the CitA family with features indicating 15 that C4-dicarboxylate (DcuS-type) and citrate (CitA-type) sensor kinases are present in 16 different strains. Thus sensor kinases from G. thermodenitrificans and G. kaustophilus 17 representing CitA- and DcuS-type sensor kinases, respectively, were selected for 18 characterization and comparison to the corresponding sensor kinases from proteobacteria. 19 G. thermodenitrificans and G. kaustophilus are thermophilic with temperature optima at 60 20 and 55°C, respectively (Narzina et al., 2001). The bacteria that are known to grow on citrate, 21 were tested for growth on C4-dicarboxylates. For identification as CitA or DcuS-type systems, 22 heterologous complementation was used in dcuS or citA deficient strains of E. coli. The E. coli 23 strains were tested for gain of growth on citrate and C4-dicarboxylates, and for the 24 expression of DcuS and CitA dependent reporter genes in response to C4-dicarboxylates or 5 1 citrate. 2 3 Methods 4 Bacteria and molecular genetics methods. Geobacillus strains, derivates of E. coli and 5 plasmids used are listed in Table 1. The molecular methods were performed according to 6 standard procedures (Sambrook & Russel, 2001). All plasmids were isolated via GeneJETTM 7 Plasmid Miniprep Kit and PCR products were purified using the GeneJETTM PCR Purification 8 Kit (Fermentas). Oligonucleotides were synthesized by Eurofins MWG. For transformation of 9 E. coli electroporation (Dower et al., 1988) or heat shock were applied. Antibiotics were used 10 at the following concentrations: 100 µg/mL ampicillin, 20 µg/mL chloramphenicol, 50 µg/mL 11 kanamycin and 15 µg/mL tetracycline. When two or more antibiotics were used the 12 concentrations were halved. 13 Bioinformatics. Blastp and DELTA blast were used for screening all non-redundant GenBank 14 sequences (including cDNA sequence translations, PDB, SwissProt, PIR and PRF data bases, 15 but excluding environmental samples from whole genome shotgun sequencing projects, 16 with 69 159 658 sequences (version 2015/07/14). 17 Overproduction and isolation of His6-DcuSGk and His6-CitAGt, and autophosphorylation. 18 DcuSGk and His6-CitAGt were overproduced from pMW817 or pMW960, respectively, in E. coli 19 C43(DE3) as N-terminal His6 fusion proteins after cloning of dcuSGk and citAGt in pET28a 20 (Table 1). The proteins were isolated from the membrane fraction of the bacteria by 21 extraction with 2 % Empigen in purified by Ni-NTA chromatography in 0.04% LDAO 22 containing buffer by the procedure described by Janausch et al. (2002a) for DcuSEc. 13.5 mg 23 per liter of DcuSGk and 4 mg per liter of CitAGt were purified. For the autophosphorylation 6 1 assay, the proteins were reconstituted in liposomes (protein:lipid ration 1:20, w/w) 2 produced from E. coli phospholipids (E. coli Polar Lipid Extract, Avanti Polar Lipids) as 3 described by Janausch et al. (2002a). Prior to reconstitution, the liposomes were frozen in 4 liquid N2 and thawed at room temperature three times. Then the liposomes were 5 destabilized by Triton X-100 (detergent:lipid ratio of 2.5 (Rigaud et al., 1988, 1995), mixed 6 with the protein solution and the detergent was removed by Bio-Beads SM-2 (Bio-Rad) 7 (Holloway, 1973; Janausch et al., 2002a). The proteoliposomes were sedimented by 8 ultracentrifugation, dissolved in buffer and stored in liquid N 2 (Janausch et al., 2002a). 9 Autophosphorylation of the proteins was tested with different concentrations of [γ33P]-ATP 10 (see Fig. 5) and varying times of incubation. The liposomes were then dissolved in SDS- 11 containing buffer, subjected to SDS-PAGE. Radioactivity associated with the bands of DcuS or 12 CitA was determined by exposure of the gels to a Phosphor imager plate (BAS-MP2040, 13 Fujifilm) and evaluated by Fluorescent Imagereader FLA7000 (Fujifilm). For quantitative 14 evaluation slices of the SDS PAGE with the labelled proteins were digested and counted for 15 radioactivity by scintillation counting. From the radioactivity, the specific radioactivity and 16 the amount of DcuS or CitA used in the experiment, the labeling was calculated as described 17 by Janausch et al., (2002a). 18 ß-galactosidase assays. Expression of the dcuB-lacZ and citC-lacZ reporter gene fusions was 19 measured as the ß-galactosidase activity of exponential growing E. coli (ΔOD578 nm 0.5 to 0.8). 20 Cells were cultivated in 96-deep-well plates, anaerobically at 37 °C under an atmosphere of 21 N2 in enriched mineral (eM9) medium supplemented with acid-hydrolyzed Casamino acids 22 (0.1 %), L-tryptophan (0.005 %), and glycerol (50 mmol/L) plus dimethyl sulfoxide (DMSO) 23 (20 mmol/L) as the substrates. The effectors (citrate, glucose, fumarate, L-malate and 24 succinate (20 mmol/L)) were included as indicated. β-Galactosidase activity (Miller, 1992) 7 1 was quantified in 96-well-microtiter plates (Monzel et al., 2013). Optical density (570 nm) 2 and β-galactosidase activity (415 nm) were measured in a volume of 250 µl per well. For cell 3 permeabilization, 200 µl cell culture were mixed with 800 µl buffer (0.1 mol/L potassium 4 phosphate, 10 mmol/L potassium chloride, 1 mmol/L magnesium chloride, 0.005% (w/v) 5 cetyltrimethylammonium bromide, 0.0025 % (w/v) sodium deoxycholate and 0.027 % (v/v) 6 2-mercaptoethanol). For the assay, 150 µl of the permeabilized cells were incubated with 30 7 µl of ortho-nitrophenyl-ß-galactoside solution (0.4 % w/v) at 30 °C. After 20 min the reaction 8 was stopped with 70 µl sodium carbonate (1 mol/L). The β-galactosidase activity was 9 determined in triplicate in three induction experiments. 10 Growth experiments. Growth experiments with G. kaustophilus and G. thermodenitrificans 11 were performed in supplemented White minimal medium (White, 1972). For G. kaustophilus 12 the White-medium (WM-Gk) was enriched with acid-hydrolyzed Casamino acids (0.1 %), L- 13 tryptophan (0,01 %) and 0,1 % yeast extract. G. thermodenitrificans was cultivated in WM- 14 medium enriched with acid-hydrolyzed Casamino acids (0.1 %), L-tryptophan (0.01 %), 0.1 % 15 meat extract and 0.5 % sodium chloride (WM-Gt). For growth the media were supplemented 16 with 20 mM of the substrates as indicated. The bacteria were grown at 60 °C (G. 17 thermodenitrificans) or 55 °C (G. kaustophilus). For anaerobic growth the media were 18 supplemented with 50 mM glucose with or without the indicated electron acceptor (50 mM 19 or as indicated). Growth experiments with E. coli were performed anaerobically at 37°C in 20 enriched M9 medium (Kim et al. 2007) containing gluconate (3 mM), glycerol (50 mM) and 21 dimethyl sulfoxide as growth substrates plus effector (20 mM) as indicated. 22 23 24 Results 8 1 Genes encoding two-component systems of the CitA family in Geobacillus 2 The genomes of the Geobacillus strains that are available in databases were blasted for 3 genes encoding members of the CitA family of sensor kinases with the sequences of DcuS 4 and CitA of E. coli as the query. All strains (9) encoded homologs of the CitA family 5 (similarities >51 %; identity >29 %) with the respective domain composition. The sensor 6 kinases were members either of the DcuS (G. kaustophilus, G. subterraneus, and G. 7 thermoglucosidasius), CitA (G. thermodenitrificans, G. vulcani, G. stearothermophilus, G. 8 caldoxylosilyticus, G. thermoleovorans) sensor kinases according to sequence similarity. The 9 corresponding genes of G. thermodenitrificans and G. kaustophilus encoding citA and dcuS 10 like homologs, respectively, were selected for analysis. The G. thermodenitrificans genes 11 GTNG_1840 and GTNG_1839 (Fig. 1A) are arranged in a predicted operon and code for 12 proteins with similarity to CitA and CitB from E. coli. The genes are located downstream of 13 gene cluster tctCBA coding for proteins similar to a tripartite tricarboxylate transport system. 14 In G. kaustophilus a gene cluster of dcuS dcuR like genes was located upstream of genes for a 15 dicarboxylate binding protein similar to the binding protein DctB of B. subtilis and a C4- 16 dicarboxylate transporter DctA (Fig. 1A). The effector binding regions of the PASP domains of 17 DcuSEc and CitAKp are known (Gerharz et al., 2003; Reinelt et al., 2003; Kneuper et al., 2005; 18 Cheung & Hendrickson, 2008). The corresponding domains in the proteins of G. kaustophilus 19 and G. thermodenitrificans show 67% and 65% similarity, respectively to the domains of 20 DcuSEc and CitAEc. The domains reveal signature sequences specific for C4-dicarboxylate 21 (DcuSEc or DctSBs) or citrate (CitA) binding (Fig. 1B). The signature sequence is composed of 22 residues that are common for for CitA- and DcuS-type sensors, and additional residues (M122, 23 S144, K152 and S167, numbering of K. pneumonia CitA) that are specific for CitA-like proteins. 24 The conserved residues are mostly ligands for citrate or C4-dicarboxylate binding in the 9 1 citrate or L-malate co-crystals of the CitA and DcuS PASP domains (Reinelt et al., 2003; 2 Cheung & Hendrickson, 2008). The sensor kinase from G. thermodenitrificans contained nine 3 of the CitA specific residues, whereas that of G. kaustophilus carried the C4-dicarboxylate 4 motif and lacked the CitA specific residues. The similarities suggest that G. 5 thermodenitrificans encodes a citrate sensor of the CitA-type (CitAGt) and G. kaustophilus a 6 C4-dicarboxylate sensor of the DcuS-type (DcuSGk). 7 8 Growth on C4-dicarboxylates and citrate 9 Citrate, various sugars and polyols have been shown among others to support growth of G. 10 thermodenitrificans (strain DSM 466) and G. kaustophilus (Nazina et al., 2004; Manachini et 11 al., 2000). In the experiment of Fig. 2 the bacteria were tested for growth on C4- 12 dicarboxylates that had not been used as substrates before. Under aerobic conditions 13 succinate or fumarate supported growth of G. thermodenitrificans but the final cell densities 14 were significantly lower than on glucose. Anaerobic conditions allowed only poor growth on 15 glucose which was neither stimulated by fumarate nor nitrate. G. kaustophilus grew under 16 aerobic conditions with C4-dicarboxylates including L-malate, and the cell densities reached 17 54 % or more of growth on glucose (Fig. 2B). Under anaerobic conditions, glucose enabled 18 significant growth, provided that nitrate was included as an electron acceptor. Overall, G. 19 kaustophilus and G. thermodenitrificans are able to use C4-dicarboxylates for aerobic growth 20 in addition to the citrate that was demonstrated earlier as a substrate. The lack of growth on 21 L-malate by G. thermodenitrificans might be caused by the absence of specific L-malate 22 transporters of the MaeN and YflS type. The transporters are essential in some bacilli for L- 23 malate utilization since L-malate is not transported by the general C4-dicarboxylate 24 transporter DctA in the bacteria (Tanaka et al. 2003). 10 1 2 Citrate specific complementation of E. coli citA mutants by the citAGt gene of Geobacillus 3 The supposed citAGt and dcuSGk genes (or CitAGt and DcuSGk proteins) of G. 4 thermodenitrificans and G. kaustophilus, respectively, were tested for their ability to 5 complement citA or dcuS deficient strains of E. coli. The supposed CitAGt was tested in the E. 6 coli citA mutant for its capacity to restore expression of the CitA-CitB dependent citC-lacZ 7 reporter gene fusion (Fig. 3A) and anaerobic growth on citrate (Fig. 3B). The coding regions 8 of citA and citB of E. coli overlap by 32 bp, and therefore inactivation of citA causes lack of 9 citB gene translation. For complementation by citAGt, the bacteria were therefore supplied 10 by citAGt and by full length citBEc on plasmid, and both genes were under the control of 11 inducible promoters. 12 In the citA citB negative strain the expression of the citC-lacZ reporter drops to background 13 levels (Fig. 3A). When citAGt was supplied on plasmid (together with citBEc), citrate caused 14 induction of citC-lacZ expression that exceeded the induction by complementation with 15 citAEc. Complementation by both citA variants required the presence of citBEc (not shown) 16 and for both variants the induction was specific for citrate whereas fumarate or L-malate 17 produced only a low response. Thus the citAGt gene encodes a sensor kinase which is able to 18 substitute CitAEc, and CitAGt is citrate specific in the heterologous system. The E. coli citA 19 mutant has lost anaerobic growth on citrate whereas anaerobic growth by fumarate 20 respiration is retained as expected (Fig. 3B). CitAGt restored the growth on citrate with an 21 efficiency similar to CitA of E. coli (Fig. 3B) indicating that citrate transport and citrate lyase 22 are produced in the presence of CitAGt. The heterologous complementation of CitAEc by 23 CitAGt suggests that CitAGt functionally interacts with CitBEc. Overall, the data shows that 24 CitAGt is a citrate specific sensor that is able to replace CitAEC in the heterologous system and 11 1 interacts with CitBEc. This finding is in agreement with the citrate dependent growth of the 2 bacteria, but the missing response of CitAGt to fumarate suggests that the growth of G. 3 thermodenitrificans on C4-dicarboxylates is constitutive or independent from CitAGt. 4 The citAGt gene was also used to test complementation of a dcuS deficient strain of E. coli 5 (Fig. 3C). Plasmid encoded citAGt allowed induction of the DcuS-DcuR dependent dcuB-lacZ 6 reporter gene at high levels which even exceeded that by plasmid encoded dcuS of E. coli. 7 However, restoration of dcuB-lacZ expression was maximal with citrate when CitAGt was 8 present, whereas restoration was maximal with fumarate in the presence of DcuSEc. 9 Therefore CitAGt is apparently able to interact with and phosphorylate DcuR. Remarkably, in 10 the CitAGt-DcuREc containing bacteria the specificity for the stimulus (citrate) is that of the 11 senor CitAGt. DcuR retains the specificity for its target (dcuB promoter) demonstrating that 12 the heterologous complementation involves a cross-talk between a CitA sensor kinase and a 13 DcuR response regulator. There is, however, no cross-talk between the E.coli CitAEc and 14 DcuREc (Fig. 3C) since CitAEc provided on plasmid is not able to complement DcuS deficiency. 15 16 Complementation of dcuS of E. coli by dcuS of G. kaustophilus 17 In the same way complementation of an E.coli dcuS mutant was tested by a plasmid 18 encoding the supposed DcuSGk protein (Fig. 4A). The test strain (E. coli IMW260) is deficient 19 of dcuS but proficient for chromosomally encoded dcuR, and contains a (DcuS-DcuR 20 dependent) dcuB-lacZ reporter gene fusion. The strain lacks dcuB-lacZ expression (Fig. 4A). 21 Complementation with plasmid encoded dcuSEc restored expression of dcuB-lacZ by C4- 22 dicarboxylates like fumarate and L-malate, and in agreement with earlier reports to lower 23 extents by citrate (Zientz et al., 1998; Krämer et al., 2007). Plasmid encoded dcuSGk was able 24 to restore expression of dcuB-lacZ, but the effector specificity was significantly different. 12 1 Thus L-malate and citrate stimulated the expression most efficiently, followed by fumarate. 2 Therefore DcuSGk is similar to DcuSEc (and different from the CitA proteins) by the broad 3 specificity, but is has a high sensitivity to citrate as well, exceeding that for fumarate. 4 DcuSEc shows a high fumarate independent background activity in the expression of dcuB- 5 lacZ when the transporters DctA or DcuB are missing. The transporters function as co- 6 regulators of DcuS and infer responsiveness for C4-dicarboxylates to DcuS (Davies et al., 7 1999; Kleefeld et al., 2009; Witan et al., 2012; Steinmetz et al., 2014). Thus in 8 complementation by DcuSGk, a high background activity of induction of dcuB-lacZ was 9 observed in the absence of inducer (C4-dicarboxylates) (Fig. 4A), resembling the situation 10 when in DcuS+ strains transporters DcuB or DctA are deleted. This observation might be an 11 indication that DcuSGk requires like DcuSEc a co-regulator for adopting the ground-state and 12 for full C4-dicarboxylate responsiveness, and that DctAEc cannot serve this function entirely. 13 Deletion of DcuS causes diminished aerobic growth of E. coli on fumarate or L-malate by 14 decreased expression of dctA encoding the aerobic C4-dicarboxylate transporter DctA 15 (Davies et al., 1999). Thus the decreased growth of the dcuS mutant on fumarate or L-malate 16 was restored by plasmid encoded dcuSEc (Fig. 4B) and to nearly the same extent by dcuSGk. 17 Therefore DcuSGk is able to restore the aerobic growth deficiency of a dcuS mutant by 18 activating dctA expression in E. coli. 19 The data altogether indicate that DcuSGk is a typical DcuS-type sensor kinase. Some sensor 20 kinases of this type, exemplified by DctSBs of B. subtilis require the function of an 21 extracytoplasmic binding protein in addition (Asai et al., 2000; Graf et al., 2014). Thus in 22 agreement with earlier data (Graf et al., 2014) DctSBs alone is not able to complement for 23 DcuSEc deficiency and to restore dcuB-lacZ expression in the presence of fumarate or L- 24 malate (Fig. 4A). This finding is in contrast to the efficient complementation by DcuS Gk 13 1 indicating that the latter functions independent of a binding protein. 2 3 DcuSGk phosphorylation 4 For an initial characterization of one of the thermophilic sensor kinases, 5 autophosphorylation of DcuSGk was studied after purification and reconstitution of the 6 protein in liposomes. DcuSGk was overproduced heterologously in E. coli as a His6-DcuSGk 7 fusion protein. The protein was solubilized from the membrane fraction with detergent 8 LDAO and purified to near homogeneity by Ni-NTA-chromatography (Fig. 5A). The purified 9 DcuSGk showed only very weak autophosphorylation in the presence of [γ33P]ATP. After 10 incorporation into membranes produced from E. coli phospholipids, the protein was 11 autophosphorylated efficiently (Fig. 5B). The degree of autophosphorylation exceeded that 12 of DcuSEc when treated under comparable conditions at 37°C. For a more quantitative study, 13 autophosphorylation of DcuSGk and of DcuSEc was performed in the presence of increasing 14 concentrations of [γ33P]ATP (Fig. 5C). The degree of phosphorylation was determined using 15 the specific radioactivity of the ATP mixture, and after separating the protein by SDS PAGE 16 from non-bound [33P]. The radioactivity and phosphorylation in the bands corresponding to 17 DcuSEc or DcuSGk was calculated from radioactivity incorporated and the specific radioactivity 18 as described earlier for DcuSEc (Janausch et al., 2002a). Phosphorylation reached saturation 19 at high concentrations of [γ33P]ATP. With 10 mM [γ33P]ATP about 18% of the DcuSGk was 20 phosphorylated whereas only 2.2% of the DcuSEc were phosphorylated after reaching 21 maximal phosphorylation. The concentrations for half-maximal phosphorylation were 22 approx. 43 µM and 420 µM ATP for DcuSGk and DcuSEc, indicating that DcuSGk when 23 produced in E. coli and tested at 37°C is active and exceeds DcuSEc in activity and affinity. 24 14 1 Relation of CitAGt and DcuSGk to sensor kinases of the CitA-, DctSRc- and DctBSm-families. 2 The three major classes of C4-dicarboxylate sensor kinases are represented by the CitA/DcuS, 3 DctSRc and the DctBSm sensor kinases. The CitA family is characterized by sequence similarity 4 to CitAKp and the domain composition with a PASP, two TM helices, PASC and the kinase 5 domain (Bott et al., 1995; Zientz et al., 1998). DcuSGk and CitAGt are by domain composition 6 and sequence similarity members of the CitA family. The DctSRc type sensor kinases are 7 defined by the Rhodobacter capsulatus DctSRc that belongs to the FixL family of sensor 8 kinases. DctSRc type sensor kinases are found in R. capsulatus (α-proteobacteria) and 9 Aromatoleum aromaticum (β-proteobacteria) (Hamblin et al., 1993; Trautwein et al. 2012; 10 Scheu et al. 2010). DctSRc has a predicted domain composition similar to CitA or DcuS with 11 two transmembrane helices, a periplasmic and a cytoplasmic PAS domain, and the C- 12 terminal kinase (UniProt (Magrane & Consortium, 2011)). The periplasmic PAS domains of 13 DcuSEc and DctSRc, however, are only distantly related (Golby et al., 1999; Krämer et al., 14 2007). 15 The γ-proteobacteria Vibrio cholerae and Pseudomonas aeruginosa, and the α- 16 proteobacterium Sinorhizobium meliloti contain C4-dicarboxylate sensor kinases of the 17 DctBSm type (Reid & Poole, 1998; Valentini et al., 2011). DctBSm belong to the NtrB family of 18 sensor kinases (Janausch et al., 2002b; Scheu et al., 2010; Valentini et al., 2011) and contains 19 tandem extracytoplasmic PASP domains with low similarity to DcuSEc and CitAKp. The 20 cytoplasmic part is composed of a coiled coil CC domain and the kinase. 21 For a more detailed analysis the sequences of the extracytoplasmic PASP domains of the C4- 22 dicarboxylate or tricarboxylate sensor kinases were clustered by CLANS (Frickey & Lupas, 23 2004) using the sequences of the PASP domains of DcuSEc, DctSRc, DctSBs and DctBSm in the 24 PSI-Blast. Sequences with an E-value cutoff of 10 (default) and 10 iterations were used 15 1 resulting in the CitA/DcuS, DctSRc, DctB clusters of sensor kinases and two clusters of 2 guanylate cyclases (Fig. 6). CitA/DcuS represents the largest group and contains the 3 prototypic DcuS and CitA sensor kinases from enteric bacteria. DcuSGk of G. kaustophilus and 4 CitAGt of G. thermodenitrificans and the sensor kinases termed DctS (including DctSBs) and 5 CitS of the Gram-positive Bacilli (Yamamoto et al., 2000), Lactobacilli and Clostridia are 6 members of the CitA/DcuS cluster. The MalK malate sensor kinases from Bacilli (Tanaka et al. 7 2003), Streptomyces and Clostridia, and MaeK of Lactobacillus casei (Landete et al., 2010) 8 are part of the CitA/DcuS cluster as well (Fig. 6). Additionally, four strains of the 9 Deinococcus/Thermus group and two Meiothermus strains contained CitA homologs. 10 Remarkably, all of the sensor kinases of the CitA family that were characterized so far cluster 11 in one subgroups within this family (Fig. 6). The bacteria of the second subgroup (left-hand 12 side within the CitA/DcuS cluster) without characterized CitA or DcuS proteins are mostly 13 from the Actinomycetales group of grampositive bacteria. The proteins of the DctSRc cluster 14 represent the smallest group and comprise the DctS proteins of the proteobacteria. 15 The sequence similarity of the PASP domains of DcuSEc and other members within the CitA 16 family is typically higher than 50% (e.g. CitAEc:DcuSEc 62%, CitSBc:DcuSEc 59% similarity), 17 whereas that of DcuSEc with DctSRc is as low as 35%. In agreement with their separate 18 clustering, the PASP domain DctBSm (distal PASP of the PASP tandem structure) shares only 48 19 and 35% similarity with the domains of DcuSEc and DctSRc, respectively 20 21 22 Discussion 23 In Geobacilli two-component systems of the CitA/DcuS family are wide-spread and are in 24 domain composition and sequence similar to the DcuS and CitA proteins from 25 proteobacteria. The cluster analysis shows that the DcuS/CitA group which was originally 16 1 defined by the proteins of proteobacteria, includes many two-component systems of Gram- 2 positive bacteria including those from lactobacilli, bacilli, clostridia, corynebacteria, 3 geobacilli, deinococcus and the actinomycetales. Indeed, a large number of the known C4- 4 dicarboxylate/citrate sensor kinases in this group are from grampositive bacteria, whereas 5 the C4-dicarboxylate sensor kinases from the proteobacteria are found in the DctSRc and 6 DctBSm clusters as well. 7 Heterologous complementation of a dcuS deficient mutant of E. coli by dcuS of G. 8 kaustophilus confirmed the functional similarity of DcuSGk with DcuSEc. In the heterologous 9 complementation DcuSGk showed functional interaction with DcuREc. Interestingly, the 10 mesophilic growth conditions in E. coli and replacement of the Geobacillus lipid composition 11 by the E. coli lipids obviously had no severe effects on the function of the Geobacillus DcuSGk 12 and CitAGt in E. coli. 13 The substrate specificities of DcuSGk was in agreement with that expected by the presence of 14 the C4-dicarboxylate (‘DcuS’) signature in the binding sites as defined in Fig. 1. Thus DcuSGk is 15 a typical DcuS sensor with broad specificity to C4-dicarboxylates and to citrate. G. 16 kaustophilus is able to grow aerobically on C4-dicarboxylates and on citrate. The lack of an 17 additional CitA-CitB system, suggests that DcuSGk-DcuRGk is responsible for induction of both 18 metabolic systems, or that citrate metabolism is constitutively induced. Genes encoding a 19 fumarase (FumC) and DctA next to the genes for the two-component system support the 20 role of DcuSGk-DcuRGk in the control of C4-dicarboxylate metabolism. 21 DcuS-type sensor kinases require transporters like DctA or DcuB as co-regulators, and DcuSEc 22 and DctSBs are in the permanent ON state when the transporters are missing (Davies et al. 23 1999; Kleefeld et al. 2009; Witan et al. 2012; Steinmetz et al. 2014). The high background 24 activity of dcuB-lacZ expression after complementation by DcuSGk in the absence of 17 1 effectors, indicates that DcuSGk requires also a transporter for adjusting the OFF or C4- 2 dicarboxylate responsive state. The E. coli transporters presumably cannot fully replace the 3 G. kaustophilus transporters in this respect. Sensor kinase DctSBs of B. subtilis requires an 4 additional extracytoplasmic binding protein for function (Asai et al., 2000; Graf et al., 2014). 5 The high activity of DcuSGk in the absence of a binding protein indicates that DcuSGk functions 6 independent of an extracytoplasmic binding protein. Overall, it appears that the DcuS-like 7 protein in G. kaustophilus shares many properties with the corresponding sensor kinases of 8 E. coli and that it is independent of an extracytoplasmic binding protein known from bacilli. 9 CitAGt, on the other hand, has the typical properties of a CitA type sensor kinase and the 10 signature of a citrate binding site. Remarkably, cross-talk between non-cognate systems 11 (CitAGt and DcuREc) was observed whereas in the homologous system (CitAEc with DcuREc) 12 cross-talk was lacking in agreement with earlier suggestions (Scheu et al., 2012). Therefore in 13 the heterologous system obviously some specificity in the sensor kinase/response regulator 14 interaction is lost. 15 The data suggests that the CitAGt-CitBGt two-component system is suitable for inducing 16 metabolism by citrate and growth on citrate which is supported by the gene cluster tctABC 17 that is located adjacent to the citAB genes and encodes a citrate transporter. Genome 18 analysis shows that only the CitA type sensor is present in G. thermodenitrificans but no 19 DcuS type sensor kinase. Therefore the relatively weak growth on C4-dicarboxylates depends 20 on constitutive expression of the corresponding metabolism, or the function of an additional 21 unknown system. 22 23 24 Acknowledgements. Financial support by a grant of Deutsche Forschungsgemeinschaft to 18 1 GU (UN49/17-1) is gratefully acknowledged. We are grateful to Drs. J. Schultz, A. Lupas and J. 2 Baßler (Tübingen) for introduction to and help with CLANS. 3 4 5 References 6 7 Asai, K., Baik, S. 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(A) Genes and (putative) 5 encoded proteins: citT, citrate/succinate antiporter; citCDEFXG, citrate-lyase; citA, citrate 6 sensor kinase CitA; citB, citrate response regulator CitB; ydbD, manganese catalase; dctA, C4- 7 dicarboxylate transporter DctA; dctB, C4-dicarboxylate-binding protein DctB; dctR, C4- 8 dicarboxylate response regulator DctR, dctS, C4-dicarboxylate sensor kinase DctS; fumC, 9 fumarate hydratase FumC; GTNG_1838 probable transporter (40 % identitiy to transporter 10 MleP of Oenococcus oeni); tctABC, tripartite tricarboxylate transport system. (B) Signature 11 sequences in the ligand binding sites in the periplasmic PASP domains of DcuS and CitA 12 proteins. Residues involved in L-malate/fumarate or citrate binding (Cheung & Hendrickson, 13 2008; Sevvana et al., 2008; Gerharz et al., 2003; Kneuper et al., 2005) are highlighted: 14 Orange, L-malate/fumarate binding sites; blue, citrate binding sites; grey, residues 15 equivalent for fumarate and the citrate binding in DcuSEc and CitAKp/Ec. Sequence alignments 16 were performed with DcuSEc (P0AEC8), DcuSGk (Q5L2I6), DcuSGtg (A0A0F4E3S3), DctSBs 17 (P96601), CitAEc (P77510), CitAKp (P52687),CitAGt (A4IPE6), and CitAGst (A0A087LI38) using 18 Clustal Omega (Sievers et al., 2011). Ec, Escherichia coli; Gk, G. kaustophilus; Gtg, G. 19 thermoglucosidasius; Bs, B. subtilis; Kp, K. pneumoniae; Gt, G. thermodenitrificans; Gst, G. 20 stearothermophilus. 21 22 Figure 2. Growth of G. thermodenitrificans (A) and G. kaustophilus (B) on glucose and 23 carboxylates. The bacteria were grown at 60 °C (G. thermodenitrificans) or 55 °C (G. 24 kaustophilus) in enriched White minimal medium. The medium was enriched with 0.1% AHC, 23 1 0.01% L-tryptophane, 0.5% NaCl and 0.1% meat extract (G. thermodenitrificans), or with 2 0.1% yeast extract (G. kaustophilus). Growth substrates were added at 20 mM (citrate 5 3 mM) for aerobic growth as indicated, or 50 mM (citrate 5 mM) for anaerobic growth. 4 Growth was measured as the OD578nm after incubation for 7 hours (G. thermodenitrificans) or 5 16 hours (G. kaustophilus). Cit* indicates positive growth reaction on citrate agar. 6 7 Figure 3. Complementation of citAEc (A and B) and dcuSEc (C) by citAGt. For 8 complementation of citAEc (A+B), strain E. coli IMW549 (citA and citC-lacZ) was transformed 9 with a plasmid encoding CitAGt (pMW1652) and CitBEc (pMW1653) or CitA/BEc (pMW1599). 10 The citC-lacZ reporter gene expression was measured as indicated in the methods section. 11 For complementation of dcuSEc (C), strain E. coli IMW260 (dcuS and ΦdcuB-lacZ) was 12 transformed with a plasmid encoding DcuSEc (pMW151), CitAGt (pMW1652), or CitAEc 13 (pMW1651), and dcuB-lacZ reporter gene expression was measured. Reporter gene activities 14 (mean ± standard deviation) are the average of three biological replicates, and of at least 15 four independent measurements. In the complementation studies the strains were grown 16 anaerobically in eM9 medium containing gluconate (3 mM), glycerol (50 mM) and dimethyl 17 sulfoxide as growth substrates plus the effector (20 mM) citrate (black bars), fumarate (grey 18 bars), or without effector (white bar). 19 20 Figure 4. Complementation of dcuSEc by dcuSGk as tested by gene expression (A) and 21 growth (B). For testing complementation of dcuS dependent gene expression (A), E. coli 22 IMW260 (dcuS and ΦdcuB-lacZ) was grown anaerobically in enriched M9 medium with 23 glycerol/gluconate/DMSO as the basic substrates plus one of the inducers (20 mM each) 24 citrate (black bar), fumarate (light grey bar), malate (grey bars), or without (white bar). For 24 1 complementation, strain E. coli IMW260 was transformed with plasmids encoding DcuSEc 2 (pMW151), or DcuSGk (pMW817), or DctSBs (pMW1558). Reporter gene activities (mean ± 3 standard deviation) are the average of three biological replicates, and of at least four 4 independent measurements. For growth complementation (B), the same strain IMW260 5 strain was transformed plasmids encoding either DcuSEc (pMW151), or DcuSGk (pMW817). 6 Bacterial growth is given as the final OD578nm after growth on fumarate or L-malate for the 7 DcuS-negative strain and after complementation with DcuSEc or DcuSGk. 8 9 Figure 5. Purification (A), reconstitution in liposomes and autophosphorylation of His6- 10 DcuSGk and CitAGt (B, C). (A) DcuSGk and CitAGt solubilized and purified from E. coli 11 BL21DE3pMW817 and BL21DE3pMW960, respectively, were separated by SDS-PAGE (12% 12 acrylamide) and stained with Coomassie Blue (A). The SDS-PAGE shows protein marker, His6- 13 DcuSGk (15 µg) His6-CitAGt (10 µg) as eluted from the Ni-NTA-agarose column. (B) 14 Autophosphorylation of purified (Sol) and reconstituted (Rec) DcuSGk and DcuSEc. His6-DcuSGk 15 and His6-DcuSEc were reconstituted in E. coli liposomes. Samples of purified (Sol) and 16 reconstituted (Rec) protein (5 µg each) were incubated for 30 min with 0.1 µM [γ33P]ATP in 17 the presence of fumarate (20 mM). The samples were subject to SDS-PAGE (protein stain, 18 lower part of (B)) and autoradiography (upper part of (B)). (C) Phosphorylation of DcuSGk and 19 DcuSEc as a function of ATP concentration. DcuSGk or DcuSEc in proteoliposomes was 20 incubated with [γ33P]ATP (0.1 µM to 10 mM) for 30 min. The proteoliposomes were 21 dissolved in SDS sample buffer and subject to SDS-PAGE. After slicing the gel, the 22 radioactivity in the protein bands was determined by scintillation counting, and the level of 23 DcuS phosphorylation was calculated from radioactivity and protein content (Janausch et al., 24 2002a). 25 1 2 Figure 6. Cluster analysis of C4-dicarboxylate sensor kinases based on the sequences of 3 DcuSEc, DcuSRc and DctBSm. The sequences of the binding (PASP) domains of the proteins 4 were used for the PSI-Blast analysis. Sequences of the PSI-Blast searches with an E-value 5 cutoff of 10 were combined and used for cluster analysis by CLANS (Frickey & Lupas, 2004). 6 The CitA cluster is presented in an extended version, and specific members are shown: 1 7 (red), MaeK of Lactobacillus casei (Landete et al., 2010); 2, (green) CitAGt of G. 8 thermodenitrificans; 3 (magenta), CitS of Clostridium ultunense; 4 (brown), CitAKp of K. 9 pneumonia; 5 (light green) DcuSEc; 6 (blue), MalK of C. intestinale; 7 (yellow) DctSBs; 8 10 (orange), DcuSGk of G. kaustophilus; 9 (light blue), DcuS of Deinococcus geothermalis, 10 11 (purple) Corynebacterium variabile. 12 26 1 2 Table 1: Strains and plasmids used in this study Strain or plasmid Strains Geobacillus kaustophilus DSM7263 Geobacillus thermodenitrificans DSM466 Escherichia coli K-12 BL21(DE3) C43(DE3) IMW260 IMW549 Plasmids pET28a pBAD33 pME6010 pMW151 pMW817 pMW960 pMW1558 pMW1599 pMW1601 pMW1651 pMW1652 pMW1653 3 4 5 Genotype or characteristic(s) Ref or source Wildtype Nazina 2001 Nazina 2001 Wildtype et al., et al., F-, ompT, hsdSBgal1, dcmλDE3, IPTGinducible chromosomal T7-RNA pol Mutant of BL21(DE3), for expression of membrane proteins MC4100, λ(Φ(dcuB'-'lacZ)hyb, bla+, Δ lacZ, dcuS::camR IMW279 (citA::kanR), λ(Φ(citC'-'lacZ) hyb, ampR Studier & Moffat, 1986 Miroux & Walker, 1996 Zientz et al., 1998 Overexpression plasmid, His6-tag, kanR Expression plasmid with pBR322 ori, arabinose induction, camR Low-copy plasmid, 8.3 kb, tetR Novagen Guzman et al., 1995 Heeb et al., 2000 pET28a with dcuSEc-his6 kanR pET28a with dcuSGk-his6 kanR pET28a with citAGt-his6 kanR pHT304 with dctSBs with own promotor and ribosome binding site, ampR (E. coli) eryR (B. subtilis) pME6010, with citA citB-his6 behind citAEc promoter, tetR pME6010 with intergenic region upstream of citAEc pMW1601, with his6-citAEc behind citAEc promoter, tetR pMW1601, with his6-citAGt behind citAEc promoter, tetR pBAD33, with citBEc-his6 and N-terminal ribosome-binding-site, camR This study This study This study This study Scheu et al., 2012 This study This study This study This study This study