SPR measurements with CcpA, HPrSerP or CrhP each from B. subtilis or xylAcre, were analysed using a BIAcoreX instrument operated at 25 °C (BIAcoreX, Uppsala, Sweden). For the analysis of protein–protein interactions CcpA was immobilized by amine coupling on the carboxylated dextran matrix of a CM5 sensorchip (Biacore AB) in flowcell Fowcell 1 contained TetR from E. coli and was used as a reference. For immobilization on the activated chip matrix (injection of 35 µL of a mixture containing 50 mmN-hydroxysuccinimide and 200 mmN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride in desalted, sterile water) the proteins were injected at 500 nm concentrations in 10 mm sodiumacetate, pH 5. After coupling of the proteins the residual activated carboxyl groups were deactivated by injection of 1 m ethanolamine hydrochloride/NaOH, pH 8.5. Both proteins, CcpA and TetR-B/D, were adjusted to equal immobilization levels of 1700–2100 RU on different sensorchips. During immobilization and interaction analyses HBS/EP buffer (0.01 m Hepes pH 7.4, 0.15 m NaCl, 3 mm EDTA, 0.005% polysorbate) purchased from Biacore was used as a running buffer at a flowrate of 5 µL·min−1. For the interaction analyses, the injected analyte volume was adjusted to the amount needed for a constant response difference indicating the equilibrium of interaction of CcpA with HPrSerP or CrhP. The concentration of the complex is measured directly as the steady state response [R(eq)] in SPR. As the analyte is constantly replenished during sample injection, the concentration of free analyte is equal to the bulk analyte concentration. The equilibrium constants were determined by Langmuir fits of plots from the steady state response vs. the analyte concentrations. Evaluation was done using the Langmuir equation for 1 : 1 ligand binding of the program sigmaplot™8.0 (SPSS Inc., Chicago, IL, USA). Each equilibrium constant and deviations were determined from three different titrations. For interaction analyses of CcpA with xylAcre we immobilized amino-modified 26-meric DNA (see preparation of cre DNA) containing the xylAcre or a nonspecific DNA sequence on the surface of Biacore CM5 chips. We used a new method for coupling of amino-modified DNA to Biacore CM5 chips. This method uses cetyltrimethylammoniumbromide (CTAB) micelles as carriers to immobilize DNA on the carboxymethylated dextran matrix (H. Sjöbom, Biacore AB, Uppsala, Sweden, personal communication). We coupled hybridized nonspecific DNA in flowcell 1 and xylAcre containing DNA in flowcell 2 by injection of mixtures containing 5 µm of amino-modified DNA, 0.6 mm CTAB in 10 mm Hepes at a pH of 7.4 over a CM5 chip that was activated as described above. During coupling we used HBS-N (10 mm Hepes, 150 mm NaCl) as a running buffer at a flow rate of 5 µL·min−1. After deactivation of residual activated carboxyl groups as described above ≈ 280 RU DNA remained stably attached to the chip, but only ≈ 30–60 RU were functional as calculated from the maximum response of CcpA-HPrSerP binding to xylAcre. For all CcpA–cre interaction analyses HBS-EP buffer purchased from Biacore AB was used as a running buffer. The mass transport limitation was tested by alteration of flow rates. A flow rate of 40 µL·min−1 was suitable for all experiments to minimize mass transport. To regenerate the chip surface the dissociation of the CcpA–HPrSer46P complex was stopped by injection of 80 µL HBS-EP buffer at 40 µL·min−1 after each injection. Fits showed that concentrations > 30 nm CcpA or CcpA–HPrSerP complex, which saturate the cre coupled to the chip, result in biphasic sensorgrams. We analysed only sensorgrams from 1 nm to 30 nm CcpA in the presence of HPrSerP or HPrSerP and FBP. The titrations for the kinetic measurements have been carried out twice for each protein complex, CcpA–HPrSerP or CcpA–HPrSerP–FBP. FBP (Fluka) F-6-P, Glc6-P or Glc1-P (Sigma) were diluted immediately before each experiment in HBS-EP buffer to 100 mm stock solutions and if necessary adjusted to pH 7.4. In order to prevent bulk effects the HBS-EP running buffer was adjusted to the concentration of these compounds and then supplied with HPrSerP if required.