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Vol. 180, Issue 13, 3483-3485, July 1, 1998
NOTE
The fnr Gene of Bacillus
licheniformis and the Cysteine Ligands of the C-Terminal FeS
Cluster
Anette
Klinger1,
Jan
Schirawski1,
Philippe
Glaser2, and
Gottfried
Unden1*
1 Institut für Mikrobiologie und
Weinforschung, Johannes Gutenberg Universität, 55099 Mainz,
Germany,1 and
2 Laboratoire de Genomique
des Microorganismes Pathogenes, Institut Pasteur, Paris,
France2
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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.
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ARTICLE |
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. 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. 1) contained the 3' end of a
gene homologous to fnrBs. The missing 5' region
was obtained by inverse PCR (Fig. 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. 1)
(1, 6).
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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 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
fnrBs. 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.
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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. 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.
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Fig. 2.
Comparison of FnrBl as predicted from the
fnrBl gene to FnrBs. Identical (:)
and similar (.) amino acid residues are shown. Helices  E
and 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 fnrBs has the accession
no. Y16671 (EMBL database).
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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-X
2-Cys229-X
4-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
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-X
2-Cys23-X
5-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 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.
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
fnrBl 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
fnrBl 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 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.
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Table 1
Complementation of an fnr mutant of
B. subtilis 168 by fnrBl
introduced by transformation with plasmid
pMW93 (fnrBl+)
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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 plus
nitrate (20 mM each) under anaerobic conditions.
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ACKNOWLEDGMENTS |
This work was supported by Deutsche Forschungsgemeinschaft, the
Naturwissenschaftlich-Medizinisches Forschungszentrum
(Universität Mainz), and the Fonds der Chemischen Industrie.
We are grateful to D. Jahn (Freiburg) for supplying B. subtilis strains.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie und Weinforschung, Becherweg 15, D-55099 Mainz, Germany. Phone: 49-6131-393550. Fax:
49-6131-392695. E-mail: Unden{at}mail.uni-mainz.de.
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Copyright © 1998 by American Society for Microbiology
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