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Redundancy and modularity in membrane-associated dissimilatory nitrate reduction in Bacillus.

Heylen K, Keltjens J - Front Microbiol (2012)

Bottom Line: The genomes of two phenotypically denitrifying type strains of the genus Bacillus were sequenced and the pathways for dissimilatory nitrate reduction were reconstructed.In addition to the already characterized menaquinol/cyt c-dependent nitric oxide reductase (Suharti et al., 2001, 2004) of which the encoding genes could be identified now, evidence for another novel nitric oxide reductase (NOR) was found.Also, our analyses confirm earlier findings on branched electron transfer with both menaquinol and cytochrome c as reductants.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Department of Biochemistry and Microbiology, University of Ghent Gent, Belgium.

ABSTRACT
The genomes of two phenotypically denitrifying type strains of the genus Bacillus were sequenced and the pathways for dissimilatory nitrate reduction were reconstructed. Results suggest that denitrification proceeds in the periplasmic space and in an analogous fashion as in Gram-negative organisms, yet with the participation of proteins that tend to be membrane-bound or membrane-associated. A considerable degree of functional redundancy was observed with marked differences between B. azotoformans LMG 9581(T) and B. bataviensis LMG 21833(T). In addition to the already characterized menaquinol/cyt c-dependent nitric oxide reductase (Suharti et al., 2001, 2004) of which the encoding genes could be identified now, evidence for another novel nitric oxide reductase (NOR) was found. Also, our analyses confirm earlier findings on branched electron transfer with both menaquinol and cytochrome c as reductants. Quite unexpectedly, both bacilli have the disposal of two parallel pathways for nitrite reduction enabling a life style as a denitrifier and as an ammonifying bacterium.

No MeSH data available.


Multiple sequence alignment of NrfA from B. azotoformans LMG 9581T, B. bataviensis LMG 21833T, and other bacteria. Heme-binding motifs are highlighted in red (white lettering), the lysine at the catalytic heme is highlighted in green, active site residues are printed in blue, distal heme-ligating histidines for hemes 2–6 are printed in red [according to Einsle et al. (2000)]. Bs, Bacillus selenitireducens MLS10 (Bsel_1305); Dv, Desulfovibrio vulgaris DP4 (PDB 2J7A); Dd, Desulfovibrio desulfuricans (PDB 10AH_A). PDB, protein database accession number.
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FA1: Multiple sequence alignment of NrfA from B. azotoformans LMG 9581T, B. bataviensis LMG 21833T, and other bacteria. Heme-binding motifs are highlighted in red (white lettering), the lysine at the catalytic heme is highlighted in green, active site residues are printed in blue, distal heme-ligating histidines for hemes 2–6 are printed in red [according to Einsle et al. (2000)]. Bs, Bacillus selenitireducens MLS10 (Bsel_1305); Dv, Desulfovibrio vulgaris DP4 (PDB 2J7A); Dd, Desulfovibrio desulfuricans (PDB 10AH_A). PDB, protein database accession number.

Mentions: Ammonium-forming nitrite-reducing NrfHA are encoded by BAZO_03250-03255 (Table 2). The nrfHA operon organization is similar to that described for several bacteria capable of DNRA such as Campylobacter jejuni, Desulfovibrio vulgaris, Geobacter sulfurreducens (Simon, 2002), and Desulfitobacterium hafniense DCB-2 (Kim et al., 2012). NrfA is a periplasmic pentaheme c protein in which four cytochromes c are involved in electron transfer while the fifth one has a catalytic function. Their presence in the amino acid sequence is characterized by CXXCH (electron transfer) and CXXCK (catalysis) motifs. BAZO_03255, indeed, shows these motifs and it displays also all other sequence features of NrfA proteins of which crystal structures are available (Bamford et al., 2002; Cunha et al., 2003; Rodrigues et al., 2006a) (Figure A1). Consistent with the localization of all known NrfA's, BAZO_03255 contains an N-terminal signal sequence for protein export. Known NrfA's are soluble proteins, but the LipoP program predicts BAZO_03255 to be a lipoprotein. As far as could be checked, the lipoprotein nature might also hold for NrfA's from other Firmicutes, namely Bacillus selenitireducens MLS10 (Bsel_1305) and Desulfitobacterium hafniense DCB-2 (Dhaf_4234). NrfH is a membrane-bound tetraheme cytochrome c belonging to the NapC/NirT family of menaquinol oxidases (Simon et al., 2000). The comparison with NrfH from the Deltaproteobacterium Desulfovibrio vulgaris, of which the structure has been resolved (Rodrigues et al., 2006b, 2008), establishes the conservation in BAZO_03250 of all relevant amino acids implemented with structuring the N-terminal TMH, quinol binding, and ligation of the four hemes c. Taken together, our observations indicate that both NrfA and NrfH are functional proteins in B. azotoformans. As a membrane-bound complex, NrfAH would facilitate in B. azotoformans menaquinol oxidation coupled with the reduction of nitrite making ammonium. One may note that both proteins are rich in heme c molecules. In this respect it is interesting that nrfAH is linked to a gene cluster (ccmEFHABC; BAZO_03265-BAZO_03300) encoding six out of eight proteins of the System I cytochrome c biogenesis machinery (Kranz et al., 2009).


Redundancy and modularity in membrane-associated dissimilatory nitrate reduction in Bacillus.

Heylen K, Keltjens J - Front Microbiol (2012)

Multiple sequence alignment of NrfA from B. azotoformans LMG 9581T, B. bataviensis LMG 21833T, and other bacteria. Heme-binding motifs are highlighted in red (white lettering), the lysine at the catalytic heme is highlighted in green, active site residues are printed in blue, distal heme-ligating histidines for hemes 2–6 are printed in red [according to Einsle et al. (2000)]. Bs, Bacillus selenitireducens MLS10 (Bsel_1305); Dv, Desulfovibrio vulgaris DP4 (PDB 2J7A); Dd, Desulfovibrio desulfuricans (PDB 10AH_A). PDB, protein database accession number.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475470&req=5

FA1: Multiple sequence alignment of NrfA from B. azotoformans LMG 9581T, B. bataviensis LMG 21833T, and other bacteria. Heme-binding motifs are highlighted in red (white lettering), the lysine at the catalytic heme is highlighted in green, active site residues are printed in blue, distal heme-ligating histidines for hemes 2–6 are printed in red [according to Einsle et al. (2000)]. Bs, Bacillus selenitireducens MLS10 (Bsel_1305); Dv, Desulfovibrio vulgaris DP4 (PDB 2J7A); Dd, Desulfovibrio desulfuricans (PDB 10AH_A). PDB, protein database accession number.
Mentions: Ammonium-forming nitrite-reducing NrfHA are encoded by BAZO_03250-03255 (Table 2). The nrfHA operon organization is similar to that described for several bacteria capable of DNRA such as Campylobacter jejuni, Desulfovibrio vulgaris, Geobacter sulfurreducens (Simon, 2002), and Desulfitobacterium hafniense DCB-2 (Kim et al., 2012). NrfA is a periplasmic pentaheme c protein in which four cytochromes c are involved in electron transfer while the fifth one has a catalytic function. Their presence in the amino acid sequence is characterized by CXXCH (electron transfer) and CXXCK (catalysis) motifs. BAZO_03255, indeed, shows these motifs and it displays also all other sequence features of NrfA proteins of which crystal structures are available (Bamford et al., 2002; Cunha et al., 2003; Rodrigues et al., 2006a) (Figure A1). Consistent with the localization of all known NrfA's, BAZO_03255 contains an N-terminal signal sequence for protein export. Known NrfA's are soluble proteins, but the LipoP program predicts BAZO_03255 to be a lipoprotein. As far as could be checked, the lipoprotein nature might also hold for NrfA's from other Firmicutes, namely Bacillus selenitireducens MLS10 (Bsel_1305) and Desulfitobacterium hafniense DCB-2 (Dhaf_4234). NrfH is a membrane-bound tetraheme cytochrome c belonging to the NapC/NirT family of menaquinol oxidases (Simon et al., 2000). The comparison with NrfH from the Deltaproteobacterium Desulfovibrio vulgaris, of which the structure has been resolved (Rodrigues et al., 2006b, 2008), establishes the conservation in BAZO_03250 of all relevant amino acids implemented with structuring the N-terminal TMH, quinol binding, and ligation of the four hemes c. Taken together, our observations indicate that both NrfA and NrfH are functional proteins in B. azotoformans. As a membrane-bound complex, NrfAH would facilitate in B. azotoformans menaquinol oxidation coupled with the reduction of nitrite making ammonium. One may note that both proteins are rich in heme c molecules. In this respect it is interesting that nrfAH is linked to a gene cluster (ccmEFHABC; BAZO_03265-BAZO_03300) encoding six out of eight proteins of the System I cytochrome c biogenesis machinery (Kranz et al., 2009).

Bottom Line: The genomes of two phenotypically denitrifying type strains of the genus Bacillus were sequenced and the pathways for dissimilatory nitrate reduction were reconstructed.In addition to the already characterized menaquinol/cyt c-dependent nitric oxide reductase (Suharti et al., 2001, 2004) of which the encoding genes could be identified now, evidence for another novel nitric oxide reductase (NOR) was found.Also, our analyses confirm earlier findings on branched electron transfer with both menaquinol and cytochrome c as reductants.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Department of Biochemistry and Microbiology, University of Ghent Gent, Belgium.

ABSTRACT
The genomes of two phenotypically denitrifying type strains of the genus Bacillus were sequenced and the pathways for dissimilatory nitrate reduction were reconstructed. Results suggest that denitrification proceeds in the periplasmic space and in an analogous fashion as in Gram-negative organisms, yet with the participation of proteins that tend to be membrane-bound or membrane-associated. A considerable degree of functional redundancy was observed with marked differences between B. azotoformans LMG 9581(T) and B. bataviensis LMG 21833(T). In addition to the already characterized menaquinol/cyt c-dependent nitric oxide reductase (Suharti et al., 2001, 2004) of which the encoding genes could be identified now, evidence for another novel nitric oxide reductase (NOR) was found. Also, our analyses confirm earlier findings on branched electron transfer with both menaquinol and cytochrome c as reductants. Quite unexpectedly, both bacilli have the disposal of two parallel pathways for nitrite reduction enabling a life style as a denitrifier and as an ammonifying bacterium.

No MeSH data available.