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Cross-utilization of β-galactosides and cellobiose in Geobacillus stearothermophilus

2020; Elsevier BV; Volume: 295; Issue: 31 Linguagem: Inglês

10.1074/jbc.ra120.014029

1083-351X

Smadar Shulami, A. Zehavi, Valery Belakhov, R. Salama, Shifra Lansky, Timor Baasov, G. Shoham, Yuval Shoham,

Microbial Metabolic Engineering and Bioproduction

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-β-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-β-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system. Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-β-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-β-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system. Geobacillus stearothermophilus T-1 is a thermophilic, Gram-positive, soil bacterium, which is capable of utilizing plant cell wall–derived polysaccharides, including xylan, arabinan, and galactan (1Shulami S. Gat O. Sonenshein A.L. Shoham Y. The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6.J. Bacteriol. 1999; 181 (10368143): 3695-370410.1128/JB.181.12.3695-3704.1999Crossref PubMed Google Scholar, 2Shulami S. Raz-Pasteur A. Tabachnikov O. Gilead-Gropper S. Shner I. Shoham Y. The l-arabinan utilization system of Geobacillus stearothermophilus.J. Bacteriol. 2011; 193 (21460081): 2838-285010.1128/JB.00222-11Crossref PubMed Scopus (44) Google Scholar, 3Tabachnikov O. Shoham Y. Functional characterization of the galactan utilization system of Geobacillus stearothermophilus.FEBS J. 2013; 280 (23216604): 950-96410.1111/febs.12089PubMed Google Scholar). Utilization of these polysaccharides includes extracellular and intracellular hemicellulolytic enzymes, ABC sugar-transport systems, carbohydrate-sensing systems, sugar metabolism enzymes, and regulatory proteins (4Bravman T. Mechaly A. Shulami S. Belakhov V. Baasov T. Shoham G. Shoham Y. Glutamic acid 160 is the acid-base catalyst of β-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase.FEBS Lett. 2001; 495 (11322958): 115-11910.1016/S0014-5793(01)02371-7Crossref PubMed Scopus (41) Google Scholar, 5Zaide G. Shallom D. Shulami S. Zolotnitsky G. Golan G. Baasov T. Shoham G. Shoham Y. Biochemical characterization and identification of catalytic residues in α-glucuronidase from Bacillus stearothermophilus T-6.Eur. J. Biochem. 2001; 268 (11358519): 3006-301610.1046/j.1432-1327.2001.02193.xCrossref PubMed Scopus (43) Google Scholar, 6Shallom D. Belakhov V. Solomon D. Shoham G. Baasov T. Shoham Y. Detailed kinetic analysis and identification of the nucleophile in α-l-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase.J. Biol. Chem. 2002; 277 (12221104): 43667-4367310.1074/jbc.M208285200Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 7Hövel K. Shallom D. Niefind K. Belakhov V. Shoham G. Baasov T. Shoham Y. Schomburg D. Crystal structure and snapshots along the reaction pathway of a family 51 α-l-arabinofuranosidase.EMBO J. 2003; 22 (14517232): 4922-493210.1093/emboj/cdg494Crossref PubMed Scopus (116) Google Scholar, 8Golan G. Shallom D. Teplitsky A. Zaide G. Shulami S. Baasov T. Stojanoff V. Thompson A. Shoham Y. Shoham G. Crystal structures of Geobacillus stearothermophilus α-glucuronidase complexed with its substrate and products: mechanistic implications.J. Biol. Chem. 2004; 279 (14573597): 3014-302410.1074/jbc.M310098200Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). A major challenge for soil bacteria in the natural environment is to sense the scarce carbon sources and compete for these sources with nearby microorganisms. Indeed, we have demonstrated that G. stearothermophilus employs a unique strategy for the efficient utilization of polysaccharides in its immediate environment. First, it utilizes two- or three-component systems to sense minute amounts of mono- or disaccharides in the surroundings, which signal the presence of the corresponding polysaccharides (2Shulami S. Raz-Pasteur A. Tabachnikov O. Gilead-Gropper S. Shner I. Shoham Y. The l-arabinan utilization system of Geobacillus stearothermophilus.J. Bacteriol. 2011; 193 (21460081): 2838-285010.1128/JB.00222-11Crossref PubMed Scopus (44) Google Scholar, 9Shulami S. Zaide G. Zolotnitsky G. Langut Y. Feld G. Sonenshein A.L. Shoham Y. A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus.Appl. Environ. Microbiol. 2007; 73 (17142383): 874-88410.1128/AEM.02367-06Crossref PubMed Scopus (56) Google Scholar). The two- or three-component sensing systems then activate dedicated ABC sugar transporters that transfer the sugars into the cell and induce the corresponding systems for expressing extracellular endo-type glycoside hydrolases that partially degrade the high-molecular-weight polysaccharides into short (usually decorated) oligosaccharides. Additional ABC transporters for oligosaccharides transfer the large oligosaccharides into the cell, and those are further hydrolyzed into sugar monomers by a battery of specific intracellular glycoside hydrolases (10Teplitsky A. Mechaly A. Stojanoff V. Sainz G. Golan G. Feinberg H. Gilboa R. Reiland V. Zolotnitsky G. Shallom D. Thompson A. Shoham Y. Shoham G. Structure determination of the extracellular xylanase from Geobacillus stearothermophilus by selenomethionyl MAD phasing.Acta Crystallogr. D Biol. Crystallogr. 2004; 60 (15103129): 836-84810.1107/S0907444904004123Crossref PubMed Scopus (46) Google Scholar, 11Czjzek M. Ben David A. Bravman T. Shoham G. Henrissat B. Shoham Y. Enzyme-substrate complex structures of a GH39 beta-xylosidase from Geobacillus stearothermophilus.J. Mol. Biol. 2005; 353 (16212978): 838-84610.1016/j.jmb.2005.09.003Crossref PubMed Scopus (54) Google Scholar, 12Solomon V. Teplitsky A. Shulami S. Zolotnitsky G. Shoham Y. Shoham G. Structure-specificity relationships of an intracellular xylanase from Geobacillus stearothermophilus.Acta Crystallogr. D Biol. Crystallogr. 2007; 63 (17642511): 845-85910.1107/S0907444907024845Crossref PubMed Scopus (55) Google Scholar, 13Alalouf O. Balazs Y. Volkinshtein M. Grimpel Y. Shoham G. Shoham Y. A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.J. Biol. Chem. 2011; 286 (21994937): 41993-4200110.1074/jbc.M111.301051Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Salama R. Alalouf O. Tabachnikov O. Zolotnitsky G. Shoham G. Shoham Y. The abp gene in Geobacillus stearothermophilus T-6 encodes a GH27 β-l-arabinopyranosidase.FEBS Lett. 2012; 586 (22687242): 2436-244210.1016/j.febslet.2012.05.062Crossref PubMed Scopus (15) Google Scholar, 15Lansky S. Salama R. Solomon H.V. Feinberg H. Belrhali H. Shoham Y. Shoham G. Structure-specificity relationships in Abp, a GH27 β-l-arabinopyranosidase from Geobacillus stearothermophilus T6.Acta Crystallogr. D Biol. Crystallogr. 2014; 70 (25372689): 2994-301210.1107/S139900471401863XCrossref PubMed Scopus (9) Google Scholar, 16Solomon H.V. Tabachnikov O. Lansky S. Salama R. Feinberg H. Shoham Y. Shoham G. Structure-function relationships in Gan42B, an intracellular GH42 β-galactosidase from Geobacillus stearothermophilus.Acta Crystallogr. D Biol. Crystallogr. 2015; 71 (26627651): 2433-244810.1107/S1399004715018672Crossref PubMed Scopus (16) Google Scholar). This utilization strategy allows the bacterium to (a) react rapidly to presence of potential polysaccharides such as xylan, arabinan, and galactan in the immediate environment; (b) transfer efficiently the degradation products into the bacterium cell; and (c) almost exclusively utilize the degraded decorated oligosaccharides because these are rarely imported by other organisms. Essentially the same strategy was recently demonstrated for the utilization of yeast mannan by the human gut Bacteroidetes and was coined a "selfish mechanism" (17Cuskin F. Lowe E.C. Temple M.J. Zhu Y. Cameron E. Pudlo N.A. Porter N.T. Urs K. Thompson A.J. Cartmell A. Rogowski A. Hamilton B.S. Chen R. Tolbert T.J. Piens K. et al.Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.Nature. 2015; 517 (25567280): 165-16910.1038/nature13995Crossref PubMed Scopus (246) Google Scholar). In addition to the complete utilization machinery for xylan, arabinan, and galactan, G. stearothermophilus also possesses scavenging mechanisms for the utilization of mono- or disaccharides that are often found in the surroundings, resulting from the extensive degradation of the corresponding polysaccharides by other soil microorganisms. In this case, the sugars are imported by the bacterium into the cell via a different type of transporters, the phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The PTS systems usually use phosphoenolpyruvate as the phosphoryl donor for sugar phosphorylation, together with three essential catalytic entities, termed enzyme I, enzyme II, and HPr (heat-stable, histidine-phosphorylatable protein) (18Saier Jr., M.H. The bacterial phosphotransferase system: new frontiers 50 years after its discovery.J. Mol. Microbiol. Biotechnol. 2015; 25 (26159069): 73-7810.1159/000381215Crossref PubMed Scopus (31) Google Scholar). During their import via the PTS systems, sugars are simultaneously phosphorylated at the C6 hydroxyl group of the terminal sugar unit at the nonreducing end and are further cleaved (inside the cell) by dedicated 6-phospho-β-glycosidases/galactosidases (19Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42 (24270786): D490-D49510.1093/nar/gkt1178Crossref PubMed Scopus (3271) Google Scholar). In the present study, we identified in G. stearothermophilus strain T-1 such PTS systems for cellobiose and galactose and biochemically characterized the corresponding enzymes involved. These are the 6-phospho-β-gal Gan1D, belonging to glycoside hydrolase (GH) family GH1, and the 6-phospho-β-glycosidase Cel1A, belonging also to family GH1. In addition, we demonstrate that the bacterium can utilize the β-galactosides lactose and galactosyl-glycerol, using the cellobiose-PTS system together with the bifunctional 6-phospho-β-gal, Gan1D. As part of a systematic search for hemicellulolytic utilization systems in G. stearothermophilus, we identified in strain T-1 a 4.3-kb gene cluster, which appears to be involved in the utilization of cellobiose (Fig. 1A). The cluster was identified via bioinformatics analysis of the genome sequence of strains T-1 and includes an operon of four genes and a transcriptional regulator gene. Based on sequence homology, the celBCD genes encode for a complete PTS system. The CelD protein is homologous to proteins from the lactose/cellobiose EIIC family that bind cellobiose or lactose. CelC and CelB are the EIIA and EIIB domains, respectively, which transfer the phosphoryl group from HPr to the transported sugar. The fourth gene in this operon, celA, encodes for a 6-phospho-β-glucosidase, Cel1A, which shows sequence homology to a number of GH1 6-phospho-glucosidases, including those from Streptococcus pyogenes (51% sequence identity), Lactobacillus plantarum (32%), and Streptococcus mutans (32%). Adjacent to the operon lies the celR gene encoding for a transcriptional regulator belonging to the GntR family. The genes for the EI and HPr proteins are located elsewhere on the chromosome. G. stearothermophilus T-1 is unable to grow on cellulose but grows very well on cellobiose. To test whether the celBCD, celA and celR genes are involved in cellobiose utilization, we measured the corresponding mRNA levels in cultures grown on cellobiose, xylose, or glucose. Total mRNA was extracted from mid-exponential phase cultures, and the cDNA was amplified with primers specific to celA, celB, celC, celD, and celR as well as for the isocitrate dehydrogenase gene, ict, that was used for normalization. Relative expression was measured by real-time RT-PCR, as presented in Fig. 1B. The expression levels of the corresponding genes were 7–15-fold higher in the cellobiose-grown culture, as compared with cultures grown on xylose or glucose. The relatively high levels of celA mRNA compared with the celBCD genes may reflect differences in mRNA stability. These results suggest that the celBCDA operon is induced by cellobiose and thus highly likely to be involved in cellobiose utilization. The biochemical activity of Cel1A was determined using different chromogenic and natural substrates (Table 1). Cel1A exhibited a significant catalytic activity on substrates with glucose 6-phosphate at the glycon moiety and had no detectable activity toward unphosphorylated substrates. These results suggest that Cel1A is a 6-phospho-β-glucosidase, with high specificity toward glucose 6-phosphate as the glycon moiety. The effect of temperature on Cel1A activity was determined at pH 7.0, using the chromogenic substrate oNP-β-d-glucopyranoside 6-phosphate (oNPβGlc6P). The optimal temperature in a 20-min reaction was 65 °C (Fig. 2A). The thermal stability was determined after incubating Cel1A at temperatures between 30 and 85 °C for 10 min The enzyme was stable at temperatures below 65 °C and lost over 90% of its activity at 75 °C (Fig. 2B). The activity of Cel1A was determined at different pH values in the range of 3.5–9.5 (Fig. 2C). The pH profile is a typical bell-shaped curve, which may reflect the ionization of the two catalytic carboxylates. The enzyme was most active at the pH range of 6.5–8 and lost about 90% of its activity at pH 3.5 and 9.5. Based on sequence alignment of Cel1A with other retaining GH1 enzymes, the putative acid/base and nucleophile catalytic residues are Glu-174 and Glu-373, respectively. These residues were substituted with alanine, and the effect of the mutations on the activity of the enzyme was determined using oNPβGlc6P as the substrate. The Michaelis–Menten catalytic constants of the Cel1A-E174A catalytic mutant toward oNPβGlc6P were 0.01 mm, 2.1 s−1, and 2.1 × 105 s−1m−1 for Km, kcat, and kcat/Km, respectively. The kcat value was about 50-fold lower, compared with the WT, and the Km was 10-fold lower, compared with the WT, suggesting the accumulation of a glycosyl-enzyme intermediate. Such results are expected for an acid-base mutant because the relatively high reactivity of the o-nitrophenol leaving group (pKa 7.22) elevates the rate of the first glycosylation step even without a proton assistant. The second deglycosylation step remains considerably slow due to the loss of the base required for the activation of the catalytic water molecule (20Yu W.L. Jiang Y.L. Pikis A. Cheng W. Bai X.H. Ren Y.M. Thompson J. Zhou C.Z. Chen Y. Structural insights into the substrate specificity of a 6-phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.J. Biol. Chem. 2013; 288 (23580646): 14949-1495810.1074/jbc.M113.454751Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The alanine replacement of the Glu-373 nucleophile resulted in a nondetectable activity toward oNPβGlc6P, reflecting the inability of this mutant to bind the substrate and stabilize the first transition state. In similar retaining glycoside hydrolases, the nucleophile is usually involved in a strong interaction with the C2 hydroxyl group of the glycone sugar (21Shallom D. Leon M. Bravman T. Ben-David A. Zaide G. Belakhov V. Shoham G. Schomburg D. Baasov T. Shoham Y. Biochemical characterization and identification of the catalytic residues of a family 43 β-d-xylosidase from Geobacillus stearothermophilus T-6.Biochemistry. 2005; 44 (15628881): 387-39710.1021/bi048059wCrossref PubMed Scopus (79) Google Scholar).Table 1Michaelis–Menten catalytic constants for the hydrolysis of synthetic and natural substrates by Cel1A. The reactions were performed at 40 °C, in 100 mm citric acid-Na2HPO4 buffer, pH 7Substratekcat (s−1)Km (mm)kcat/Km (s−1·M−1)oNPβGlc6P1060.224.8 × 105oNPβGal6PNDaND, nondetectable.NDNDCellobiose 6-phosphate605.11.2 × 104Lactose 6-phosphateNDNDNDoNPβGlcNDNDNDa ND, nondetectable. Open table in a new tab We have demonstrated previously that G. stearothermophilus T-1 consumes galactan efficiently, utilizing the galactan utilization gene cluster, ganREFGBA (3Tabachnikov O. Shoham Y. Functional characterization of the galactan utilization system of Geobacillus stearothermophilus.FEBS J. 2013; 280 (23216604): 950-96410.1111/febs.12089PubMed Google Scholar). Following bioinformatics analysis of the bacterium genome sequence, we identified a new cluster that appears to be involved in galactose utilization. This new 12.5-kb cluster is composed of genes encoding for a putative three-component regulatory sensing system (GalPST2), an ABC sugar transport system (GalE2F2G2), a regulatory protein (GanR2), and two 6-phospho-glycosidases (Gan4C and Gan1D) (Fig. 3). GalP exhibits significant sequence similarity to periplasmic sugar-binding proteins with a 27-amino acid signal peptide at its N terminus. GalS exhibits characteristic features of bacterial histidine kinase proteins, including two transmembrane (TM) helices (TM1, residues 7–25; TM2, residues 177–197) flanking an extracellular domain (residues 26–176) and a conserved C-terminal cytoplasmic region containing the ATP-binding kinase domain. Downstream from the galS gene lies the galT2 gene, which encodes for a protein with high sequence similarity to response regulators. As in the case for many response regulators, GalT2 has a predicted two-domain architecture, with an N terminus signal receiver domain (REC), linked to a C terminus effector domain (22West A.H. Stock A.M. Histidine kinases and response regulator proteins in two-component signaling systems.Trends Biochem. Sci. 2001; 26 (11406410): 369-37610.1016/S0968-0004(01)01852-7Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). The N-terminal signal receiver domain (positioned at residues 5–118) shares a significant homology with the Rec superfamily of the REC domains, whereas the C-terminal domain (residues 157–225) contains a putative helix-turn-helix motif, resembling the AraC-type DNA-binding domain (23Gallegos M.T. Schleif R. Bairoch A. Hofmann K. Ramos J.L. Arac/XylS family of transcriptional regulators.Microbiol. Mol. Biol. Rev. 1997; 61 (9409145): 393-41010.1128/.61.4.393-410.1997Crossref PubMed Scopus (618) Google Scholar). Taken together, these observations suggest that the galPST2 and galE2F2G2 clusters encode for a three-component sensing system and an ABC galactose transport system, respectively. Based on genome sequence analysis, strain T-1 encodes for four sugar-specific PTS systems, two for the disaccharides, cellobiose and trehalose, and two for the monosugars, mannitol and galactose (Fig. 4A). To determine whether the galPST2 and galE2F2G2 gene clusters are involved in galactose utilization and to identify the PTS system for galactose, the mRNA levels of the galPST2, galE2F2G2, and the four sugar-PTS systems were measured in cultures grown in the presence of galactose and related sugars (Fig. 4). The expression of galE2 was 5-fold higher on galactose-grown cultures, compared with cultures grown on either glucose, xylose, or galactan (Fig. 4B). These results support our original suggestion that the galE2F2G2 operon constitutes an ABC galactose transporter. The expression of the galP gene, which is part of a three-component sensing system, appears to be relatively low and constant, regardless of the carbon source used (Fig. 4B). Although these results cannot correlate the system to galactose, it is expected that the sensing systems will be expressed constitutively. The mRNA levels of the four PTS systems were also measured in cultures grown on either galactose or glucose (Fig. 4C). The expression level of the ptsA gene in galactose-grown culture was about 9-fold higher than for a culture grown on glucose, suggesting that ptsA is part of the galactose-PTS transporter, dedicated to galactose import. Taken together, these results suggest that G. stearothermophilus T-1 has two transport systems for galactose. The putative three-component system for galactose, GalPST2, and the galactose ABC transport system, GalE2F2G2, both have dedicated sugar-binding proteins (GalP and GalE2, respectively) that are tethered to the local membrane. The ability of these proteins to bind galactose was confirmed by isothermal titration calorimetry (ITC). The calorimetric titration curves are shown in Fig. 5, and the thermodynamic binding parameters are summarized in Table 2. These results demonstrate that GalE2 and GalP are able to bind galactose quite tightly, with dissociation constants in the micromolar range, KD = 1.4 µm and KD = 6.1 µm, respectively. Similar titrations of GalP with cellobiose and lactose did not result in a significant enthalpy change. Surprisingly, however, both proteins bind glucose in similar affinities to galactose. Nevertheless, although glucose seems to bind these proteins quite tightly, it is rather unlikely that it can activate the corresponding sensing systems, because the expression of the operons of these systems is practically unaffected by glucose (Fig. 4B).Table 2Thermodynamic parameters for the binding of galactose or glucose to GalE2 and GalPProteinLigandnKD (µm)ΔHB (kcal mol−1)TΔSB (kcal mol−1)ΔGB (kcal mol−1)GalE2Galactose1.121.4−5.8 ± 0.30.3−6.1Glucose1.043.1−7.0 ± 0.40.8−7.8GalPGalactose0.656.1−10 ± 0.6−2.6−12.6Glucose0.714.2−9.0 ± 0.2−1.3−10.2 Open table in a new tab The fact that the potential three-component sensing system for galactose (galPST2) is located adjacent to a putative ABC transport system (galE2F2G2) suggests that the sensing system is functionally linked to this transport system and that GalT2 is therefore a response regulator that regulates the expression of the transporter. This type of adjacent arrangement of gene clusters was found in G. stearothermophilus also for the arabinose and xylotriose utilization systems (2Shulami S. Raz-Pasteur A. Tabachnikov O. Gilead-Gropper S. Shner I. Shoham Y. The l-arabinan utilization system of Geobacillus stearothermophilus.J. Bacteriol. 2011; 193 (21460081): 2838-285010.1128/JB.00222-11Crossref PubMed Scopus (44) Google Scholar, 9Shulami S. Zaide G. Zolotnitsky G. Langut Y. Feld G. Sonenshein A.L. Shoham Y. A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus.Appl. Environ. Microbiol. 2007; 73 (17142383): 874-88410.1128/AEM.02367-06Crossref PubMed Scopus (56) Google Scholar). Sequence analysis of the promoter region of galE2 revealed a putative −35 sequence (TTGATA), which is a relatively close match to the σA consensus sequence, TTGACA. This −35 sequence is separated by 18 bp from the potential −10 region (CAACAT), which differs from the Bacillus subtilis consensus, TATAAT, by three nucleotides (24Jarmer H. Larsen T.S. Krogh A. Saxild H.H. Brunak S. Knudsen S. Sigma A recognition sites in the Bacillus subtilis genome.Microbiology. 2001; 147 (11535782): 2417-242410.1099/00221287-147-9-2417Crossref PubMed Scopus (63) Google Scholar). The upstream region of the −35 of the galE2 promoter contains two direct repeats, CAAAAAAGT, separated by 11 bp, which may function as the recognition sequences for the response regulator GanlT2 (Fig. 6A). This putative binding site, upstream of the –35 region, can allow direct interaction of the activator with the C-terminal domain of the α subunit of RNA polymerase (25Rhodius V.A. Busby S.J. Positive activation of gene expression.Curr. Opin. Microbiol. 1998; 1 (10066477): 152-15910.1016/S1369-5274(98)80005-2Crossref PubMed Scopus (113) Google Scholar, 26Barnard A. Wolfe A. Busby S. Regulation at complex bacterial promoters: how bacteria use different promoter organizations to produce different regulatory outcomes.Curr. Opin. Microbiol. 2004; 7 (15063844): 102-10810.1016/j.mib.2004.02.011Crossref PubMed Scopus (116) Google Scholar). To test whether GalT2 can bind the galE2 promoter region, we utilized gel mobility shift assays. These studies indicated that the GalT2 protein (using N-His6-GalT2) can bind significantly to a 113-bp DNA fragment containing the two direct repeats discussed above, and a nearly complete shift was obtained in the presence of 0.3 µm GalT2 (Fig. 6B). This relatively high concentration of GalT2 (reflecting a relatively weak protein-DNA binding) may originate from the fact that the protein was not fully phosphorylated, as often observed for other response regulators (27Buschiazzo A. Trajtenberg F. Two-component sensing and regulation: how do histidine kinases talk with response regulators at the molecular level?.Annu. Rev. Microbiol. 2019; 73 (31226026): 507-52810.1146/annurev-micro-091018-054627Crossref PubMed Scopus (28) Google Scholar). To test whether the phosphorylation of GalT2 increases significantly its binding to DNA, we phosphorylated GalT2 in vitro, using Mg2+ and acetyl phosphate as the phosphate donor. As shown by size-exclusion chromatography, such phosphorylation changed the oligomeric state of GalT2 in solution, transforming it from a monomer (not phosphorylated) to a dimer (phosphorylated) (Fig. 6C), as usually observed for related response regulators. Indeed, the fully phosphorylated GalT2 protein (in its dimeric form) gave a complete shift at 0.07 µm (Fig. 6D), demonstrating a significant increase in its DNA-binding capabilities. These results further support the identification of GalT2 as a response regulator, as such regulators usually change their conformation upon phosphorylation, allowing them to enhance their binding to their target DNA segments (28Kenney L.J. Response-regulator phosphorylation and activation: a two-way street? Response.Trends Microbiol. 2000; 8 (10754570): 155-15610.1016/S0966-842X(00)01708-XAbstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 29Bogel G. Schrempf H. Ortiz de Orué Lucana D. DNA-binding characteristics of the regulator SenR in response to phosphorylation by the sensor histidine autokinase SenS from Streptomyces reticuli.FEBS J. 2007; 274 (17617222): 3900-391310.1111/j.1742-4658.2007.05923.xCrossref PubMed Scopus (14) Google Scholar). The 12.5-kb galactose utilization cluster also contains the gan1D gene, which is expressed in cultures grown on galactose or galactan (Fig. 4B). We have previously described the 3D crystal structure of Gan1D, as well as its catalytic mutants complexed with substrates and products (30Lansky S. Zehavi A. Belrhali H. Shoham Y. Shoham G. Structural basis for enzyme bifunctionality—the case of Gan1D from Geobacillus stearothermophilus.