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

Abstract

In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X₂-Cys229-X₄-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the B. licheniformis gene to an fnr mutant of B. subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth.

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 (Fnr_Ec) 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 (Fnr_Bs) 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 Fnr_Bs 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 Fnr_Ec (1). Fnr_Bs 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 Fnr_Ec. Since Fnr_Bs is the only Fnr protein of this type known so far, the fnr gene of Bacillus licheniformis (fnr_Bl) was isolated.

fnr_Bs-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 fnr_Bs gene. The probe (Fig. 1) comprised major parts of the fnr_Bs 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 fnr_Bs probe. The cloned fragment of the positive clones (pMW72) (Fig. 1) contained the 3′ end of a gene homologous to fnr_Bs. The missing 5′ region was obtained by inverse PCR (Fig. 1). The DNA region obtained comprised the fnr_Bs-homologous gene, located within two incomplete open reading frames similar to the narK and ywiC genes of B. subtilis (Fig. 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 fnr_Bs probe and the sequences of the fragments contained in pMW72 and pMW93 are shown. The probe was generated by PCR from genomic DNA of B. subtilis with the oligonucleotides oBsfnr3 (5′-GCA GAA GAG CTT TAT CTG ATT CAA TC-3′) and oBsfnr4 (5′-GCA ATT TTC ACA CTC AAT CTC ACA TC-3′). Plasmid pMW72 is a derivative of pBluescriptII KS⁻ carrying a 911-bp ClaI fragment of the genomic DNA of B. licheniformis, which hybridized with probe fnr_Bs. The fragment of pMW93 was obtained by PCR of genomic DNA of B. licheniformis with the oligonucleotides oBlikom1 (5′-CGT GAT CTA GAT CGT CCA AAA CGA AGG-3′), which introduces an XbaI restriction site, and oBlikom2 (5′-GCT CAG TCG ACA CTG TGC TTC ATG TCC TTG TTT G-3′), which introduces a SalI restriction site. The generated PCR fragment (945 bp) was cloned into the XbaI and SalI sites of pDG148 (14), resulting in pMW93. The fragment generated by inverse PCR (iPCR) was produced from genomic PstI fragments after ligation and PCR amplification with the oligonucleotides oBlifnr1 (5′-TGC GTG CTC ATC CAT TTC ATA AAC TC-3′) and oBlifnr4 (5′-CGA TTA TCT TAA TCG ACA GTT TCC TCC-3′). The nucleotide sequences were determined by the dideoxy chain termination method with fluorescently labeled nucleotides.

Properties of Fnr_Bl.

The supposed fnr_Bl gene encodes a protein of 237 amino acid residues with high sequence identity (81%) and similarity (97%) to Fnr_Bs (Fig. 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 fnr_Bl and Fnr_Bl, respectively.

FIG. 2.

Comparison of Fnr_Bl as predicted from the fnr_Bl gene to Fnr_Bs. Identical (:) and similar (.) amino acid residues are shown. Helices α_E and α_F of the helix-turn-helix motif are represented by shading. Comparison was performed with the Align program of the GeneStream SSearch network server with default parameters. Conserved (↓) and nonconserved (⇑) cysteine residues are marked. The nucleotide sequence of fnr_Bs has the accession no. Y16671 (EMBL database).

In Fnr_Bl only five cysteine residues are found, all in conserved positions relative to those of Fnr_Bs. The C-terminal cluster consists of only three residues (Cys226, Cys229, and Cys234) (Fig. 2). Cys223 of Fnr_Bs is replaced by an arginine residue in Fnr_Bl. 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 α_E of the protein and points away from the other cysteine residues, according to the supposed three-dimensional structure (1). Thus, in Fnr_Bl 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 Fnr_Ec (Cys20-X₂-Cys23-X₅-Cys29). This C-terminal cysteine cluster might be characteristic for Bacillus-type Fnr.

Function of Fnr_Bl as an O₂-sensitive transcriptional regulator.

Fnr_Bs is supposed to function as a transcriptional activator of nitrate metabolism (nar genes) under anaerobic conditions in a manner similar to that of Fnr_Ec (1, 6). In B. licheniformis, too, nitrate reductase is produced only under anoxic conditions (Table 1). It was tested whether fnr_Bl is able to restore nitrate reductase activity in an fnr_Bs mutant. The fnr_Bs mutant was no longer able to grow with nitrate under anaerobic conditions in a mineral medium (Fig. 3) due to the lack of nitrate reductase activity (Table 1), in agreement with earlier findings (1). Transformation of the mutant with a plasmid carrying the fnr_Bl gene (pMW93) (Fig. 1) fully restored nitrate reductase activity in the transformed strain (Table 1). Growth of the transformed strain on nitrate was complemented by the fnr_Bl gene too, but not completely (Fig. 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 fnr_Bl. The regulation by Fnr_Bl was oxygen sensitive, since nitrate reductase activity in the transformed strain was negligible after aerobic growth. The experiments therefore suggest that Fnr_Bl functions as an oxygen-sensitive transcriptional regulator in a manner similar to that of Fnr_Bs.

TABLE 1.

Complementation of an fnr mutant of B. subtilis 168 by fnr_Bl introduced by transformation with plasmid pMW93 (fnr_Bl⁺)

Strain	Nitrate reductase (U/g of protein)a		Increase (fold)
	Aerobic growth	Anaerobic growth
B. subtilis 168	1.6	131	82
B. subtilis 168 fnr::Spcr	0.2	2.6	13
B. subtilis 168 fnr::Spcr (pMW93)	≤0.2	180	900
B. licheniformis	1.4	134	96

^aMeasured in cell extracts with reduced benzyl viologen (averages of three independent assays) (9). Growth occurred in mineral medium with Casamino Acids (9) and 20 mM glucose (aerobic growth) or 20 mM glucose plus 20 mM nitrate (anaerobic growth). 

FIG. 3.

Growth of B. licheniformis (•), B. subtilis 168 (▴), B. subtilis 168 fnr::Spc^r (◊), and B. subtilis 168 fnr::Spc^r transformed (2) with plasmid pMW93 (⧫) in mineral medium supplemented with Casamino Acids (9) and glucose plus nitrate (20 mM each) under anaerobic conditions.