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.

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.

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).

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.