For this purpose the cbbR gene was cloned and expressed in E. coli. Purified CbbR was used to prepare antisera (anti-CbbR antibodies) whose activity was checked by Western blotting
against purified CbbR (data not shown). Biotin-labeled promoter DNA for the EMSA assays was prepared by PCR using primers specified in Table 2 and whose locations within the four operons are shown in Figure. 2. Results show that CbbR was able to retard the promoter regions of the cbb1, cbb2 and cbb3 operons but not the cbb4 operon (Figure 3). When a 50-fold molar excess of unlabelled fragment was included in the binding assay Staurosporine retardation of the labelled fragments was abolished. Furthermore, the addition of anti-CbbR antibodies to the reaction produced a supershift in migration, indicating that the shift was caused specifically by the binding of CbbR. Figure 3 Binding of CbbR to the promoter regions www.selleckchem.com/products/AZD1152-HQPA.html of the operons cbb1-4 using the EMSA assay in the presence (+) or absence (-) of competing 50× excess of unlabelled probe DNA (P[50x]) or antibodies to CbbR (anti-CbbR). Abbreviations: P*, probe DNA; S, shift; SS, supershift. Binding of CbbR to the predicted promoter regions of operons cbb1-3 suggests that it is involved in their regulation. The reason for the failure Compound C of CbbR to retard the DNA fragment containing the predicted promoter
of the cbb4 operon is not known. Perhaps this fragment requires the presence of additional factors for CbbR binding that are not present in the in vitro cocktail used for the EMSA analysis. Alternatively, the predicted CbbR binding site is not functional. Gene organization of the cbb operons The cbb3 operon includes
not only genes involved in carbon assimilation but also harbors genes with similarity to trpE and trpG that are predicted to encode the components I and II of anthranilate synthase, the first enzyme of the tryptophan biosynthesis pathway. Anthranilate synthase catalyzes the conversion of chorismate to anthranilate with the concomitant release of pyruvate [38, 39]. In some cases, this conversion can be accomplished by TrpE alone . In order to determine if the association between trpEG and the cbb genes is restricted to A. ferrooxidans, an examination of gene organization was carried out next in all sequenced genomes of facultative and obligate autotrophic proteobacteria. Twenty-six proteobacterial organisms (11 α-, 7 β- and 8 γ-) were analyzed, including 10 obligate autotrophs. Linkage between trpE/G and cbbE and/or cbbZ was found in all sequenced obligate autotrophs, all of which belong to the β- or γ-proteobacteria divisions (Figure 4, Table 4), whereas only 4 out of 14 facultative heterotrophs were detected with this clustering. These four exceptions are found only in the β- or γ-proteobacteria and none in the α-proteobacterial division (Figure 4, Table 4).