6533b870fe1ef96bd12d07c9

RESEARCH PRODUCT

The fnr Gene of Bacillus licheniformis and the Cysteine Ligands of the C-Terminal FeS Cluster

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Many of the O2-responsive gene regulators of bacteria are members of the fumarate nitrate reductase-cyclic AMP receptor protein family of transcriptional regulators (12, 13, 15, 17) with predicted structures similar to those of the cyclic AMP receptor protein (11). The Fnr (stands for fumarate nitrate reductase regulator) protein from Escherichia coli (FnrEc) controls the expression of a variety of genes, mainly of anaerobic respiration and metabolism (5, 13). It contains a N-terminal cluster of three essential cysteine residues which are supposed to bind together with Cys122 a [4Fe 4S]2+ cluster which is required for O2 sensing (4, 7, 8, 10, 16). A wide variety of gram-negative bacteria contain an Fnr of this type (for an overview, see references 13, 15, and 17). The few examples known of Fnr-like proteins from gram-positive bacteria like Flp from Lactobacillus casei and Fnr from Bacillus subtilis (FnrBs) show characteristic differences with respect to the cysteine residues (1, 3). Flp contains only two Cys residues, which have been suggested to be oxidized from the dithiol to the disulfide state by O2 (3). In FnrBs the sensory N-terminal Cys cluster of E. coli appears to be replaced by a C-terminal extension with a cluster of four Cys residues similar to that from FnrEc (1). FnrBs contains six cysteine residues, and it is assumed that three Cys residues from the C terminus together with one Cys residue from the central part of the protein bind a polynuclear FeS center, as occurs with FnrEc. Since FnrBs is the only Fnr protein of this type known so far, the fnr gene of Bacillus licheniformis (fnrBl) was isolated. fnrBs-like genes in B. licheniformis and Bacillus megaterium. Genomic DNAs of Bacillus and Paenibacillus strains were analyzed by Southern blotting with a probe derived from the fnrBs gene. The probe (Fig. ​(Fig.1)1) comprised major parts of the fnrBs gene, including sequences corresponding to the C-terminal Cys cluster. The probe hybridized to DNA fragments of B. licheniformis and B. megaterium genomic DNAs. With genomic DNAs from Paenibacillus (formerly Bacillus) macerans and Paenibacillus polymyxa, no hybridization signal was detected. From a partial gene bank containing 1-kb ClaI fragments of B. licheniformis DNA, positive colonies were identified by colony hybridization with the fnrBs probe. The cloned fragment of the positive clones (pMW72) (Fig. ​(Fig.1)1) contained the 3′ end of a gene homologous to fnrBs. The missing 5′ region was obtained by inverse PCR (Fig. ​(Fig.1).1). The DNA region obtained comprised the fnrBs-homologous gene, located within two incomplete open reading frames similar to the narK and ywiC genes of B. subtilis (Fig. ​(Fig.1)1) (1, 6). FIG. 1 fnr locus of B. licheniformis. The numbers (base pairs) give the sizes of the corresponding genes and intergenic regions. The location of the fnrBs probe and the sequences of the fragments contained in pMW72 and pMW93 are shown. The probe was generated ... Properties of FnrBl. The supposed fnrBl gene encodes a protein of 237 amino acid residues with high sequence identity (81%) and similarity (97%) to FnrBs (Fig. ​(Fig.2)2) and includes the allosteric domain and a DNA-binding helix-turn-helix domain. The DNA-binding region is followed by a short C-terminal sequence with a cysteine cluster. Due to their similarities to the gene and protein from B. subtilis, the gene and protein from B. licheniformis are designated fnrBl and FnrBl, respectively. FIG. 2 Comparison of FnrBl as predicted from the fnrBl gene to FnrBs. Identical (:) and similar (.) amino acid residues are shown. Helices αE and αF of the helix-turn-helix motif are represented by shading. Comparison was performed with the Align ... In FnrBl only five cysteine residues are found, all in conserved positions relative to those of FnrBs. The C-terminal cluster consists of only three residues (Cys226, Cys229, and Cys234) (Fig. ​(Fig.2).2). Cys223 of FnrBs is replaced by an arginine residue in FnrBl. Lack of Cys223 was confirmed by repeated independent PCR amplification from genomic DNA and sequencing of the fragments. For the binding of polynuclear FeS clusters, four cysteine residues are required. It is suggested that Cys71 and the three Cys residues from the C-terminal cluster (Cys226-X2-Cys229-X4-Cys234) serve this function. Cys184 is in a unfavorable position for liganding of the presumptive FeS cluster. The residue is located in the presumptive DNA-binding helix αE of the protein and points away from the other cysteine residues, according to the supposed three-dimensional structure (1). Thus, in FnrBl only four Cys residues remain as potential ligands for the cluster. The spacing of the C-terminal Cys residues is similar to that in the N terminus of FnrEc (Cys20-X2-Cys23-X5-Cys29). This C-terminal cysteine cluster might be characteristic for Bacillus-type Fnr. Function of FnrBl as an O2-sensitive transcriptional regulator. FnrBs is supposed to function as a transcriptional activator of nitrate metabolism (nar genes) under anaerobic conditions in a manner similar to that of FnrEc (1, 6). In B. licheniformis, too, nitrate reductase is produced only under anoxic conditions (Table ​(Table1).1). It was tested whether fnrBl is able to restore nitrate reductase activity in an fnrBs mutant. The fnrBs mutant was no longer able to grow with nitrate under anaerobic conditions in a mineral medium (Fig. ​(Fig.3)3) due to the lack of nitrate reductase activity (Table ​(Table1),1), in agreement with earlier findings (1). Transformation of the mutant with a plasmid carrying the fnrBl gene (pMW93) (Fig. ​(Fig.1)1) fully restored nitrate reductase activity in the transformed strain (Table ​(Table1).1). Growth of the transformed strain on nitrate was complemented by the fnrBl gene too, but not completely (Fig. ​(Fig.1).1). This result suggests that functions other than that of nitrate reductase which are required for growth by nitrate respiration are not restored completely by fnrBl. The regulation by FnrBl was oxygen sensitive, since nitrate reductase activity in the transformed strain was negligible after aerobic growth. The experiments therefore suggest that FnrBl functions as an oxygen-sensitive transcriptional regulator in a manner similar to that of FnrBs. TABLE 1 Complementation of an fnr mutant of B. subtilis 168 by fnrBl introduced by transformation with plasmid pMW93 (fnrBl+) FIG. 3 Growth of B. licheniformis (•), B. subtilis 168 (▴), B. subtilis 168 fnr::Spcr (◊), and B. subtilis 168 fnr::Spcr transformed (2) with plasmid pMW93 (⧫) in mineral medium supplemented with Casamino Acids (9) and glucose ...