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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.


Physical map of qCuANOR type I (A) and qCuANOR type II (B) gene cluster in B. azotoformans LMG 9581T, B. bataviensis LMG 21833T, Caldalkalibacillus thermarum TA2.A1, Geobacillus thermodenitrificans NG80-2, and G. kaustophilus HTA426. Arrows show the direction of transcription. Open reading frames are drawn to scale. Homologous genes are shown in identical colors.
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Figure 2: Physical map of qCuANOR type I (A) and qCuANOR type II (B) gene cluster in B. azotoformans LMG 9581T, B. bataviensis LMG 21833T, Caldalkalibacillus thermarum TA2.A1, Geobacillus thermodenitrificans NG80-2, and G. kaustophilus HTA426. Arrows show the direction of transcription. Open reading frames are drawn to scale. Homologous genes are shown in identical colors.

Mentions: Besides the two qNORs, no less than four gene clusters are found in the genome of B. azotoformans encoding members of the HCO superfamily that share significant sequence homology with cytochrome c-dependent ba3 oxidase (CbaAB) from Thermus thermophilus (Figures A3, A4). This is a terminal oxidase that is structurally and functionally quite well investigated [see amongst others: Soulimane et al. (2000); Fee et al. (2008); Smirnova et al. (2008); Liu et al. (2012); von Ballmoos et al. (2012)]. ba3-type oxidase receives its electrons for O2 reduction from cyt c. The electrons are transferred via CuA in subunit II (CbaB), and cyt b in subunit I (CbaA) to the a3-CuA catalytic center in this subunit. In agreement herewith, all amino acids that had been structurally assigned to CuA binding and to electron transfer were conserved in the B. azotoformans subunits II (Figure A4). Importantly, the N-terminal sequence of BAZO_06394 (MHKSEKIWLTLSFGMIMGFM) is identical to the one of subunit II of qCuANOR published by Suharti et al. (2001). These authors (Suharti et al., 2004) also presented the N-terminal sequence of the large subunit (MTKKNTQEVVKEGREGIGTFIGVGIVGAV), but this was not found in the corresponding subunit I (BAZO_06399). Rather, the almost identical sequence—MATTKNTQEVVKEGREGIGTFIGVGIVGAV—was retrieved in the adjacent gene (contig42_104893_105033) (Figure 2A). This gene encodes a small (46 amino acids) membrane-bound peptide. Hence, it is very well conceivable that this peptide formed part of the enzyme preparation purified by Suharti et al. (2001, 2004). The comparison of the amino acid sequence of subunit I (BAZO_06399) with that of ba3 from T. thermophilus revealed that the tyrosine (Y223, T. thermophilus numbering in Figure A3) covalently binding a CuB-associated histidine (H219) was substituted by an asparagine in the B. azotoformans protein, in agreement with predictions made by Hemp and Gennis (2008) for NOR functionality (Figure A3). These findings identify BAZO_06394-BAZO_06399 as the dual-function quinol and cyt c-dependent qCuANOR described by Suharti et al. (2001, 2004). We may note that the genes coding for these three subunits are linked to two other ones, one (BAZO_06404) coding for a SenC/SCO1-type membrane-bound protein that has been implemented with the insertion of copper (CuA) into subunit I, and BAZO_06409 that is predicted to comprise six TMHs (Figure 2A). In protein databases, homologs of the latter are found being annotated as cyt c oxidase-associated membrane proteins. Furthermore, the presence of BAZO_06394-06399 qCuANOR is not restricted to B. azotoformans and B. bataviensis (see below), but close homologs sharing the same sequential features and the same cluster organization are found in various other Gram-positive bacteria, including Caldalkalibacillus thermarum TA2, but also in Nitrosomonas eutropha and other nitrifiers (Figure 2A and data not shown). In N. eutropha the particular NOR was designated sNOR (Stein et al., 2007). Transcriptome analysis suggested sNOR as a suitable candidate for aerobic NO reduction, next to NorBC (Cho et al., 2006).


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

Heylen K, Keltjens J - Front Microbiol (2012)

Physical map of qCuANOR type I (A) and qCuANOR type II (B) gene cluster in B. azotoformans LMG 9581T, B. bataviensis LMG 21833T, Caldalkalibacillus thermarum TA2.A1, Geobacillus thermodenitrificans NG80-2, and G. kaustophilus HTA426. Arrows show the direction of transcription. Open reading frames are drawn to scale. Homologous genes are shown in identical colors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Physical map of qCuANOR type I (A) and qCuANOR type II (B) gene cluster in B. azotoformans LMG 9581T, B. bataviensis LMG 21833T, Caldalkalibacillus thermarum TA2.A1, Geobacillus thermodenitrificans NG80-2, and G. kaustophilus HTA426. Arrows show the direction of transcription. Open reading frames are drawn to scale. Homologous genes are shown in identical colors.
Mentions: Besides the two qNORs, no less than four gene clusters are found in the genome of B. azotoformans encoding members of the HCO superfamily that share significant sequence homology with cytochrome c-dependent ba3 oxidase (CbaAB) from Thermus thermophilus (Figures A3, A4). This is a terminal oxidase that is structurally and functionally quite well investigated [see amongst others: Soulimane et al. (2000); Fee et al. (2008); Smirnova et al. (2008); Liu et al. (2012); von Ballmoos et al. (2012)]. ba3-type oxidase receives its electrons for O2 reduction from cyt c. The electrons are transferred via CuA in subunit II (CbaB), and cyt b in subunit I (CbaA) to the a3-CuA catalytic center in this subunit. In agreement herewith, all amino acids that had been structurally assigned to CuA binding and to electron transfer were conserved in the B. azotoformans subunits II (Figure A4). Importantly, the N-terminal sequence of BAZO_06394 (MHKSEKIWLTLSFGMIMGFM) is identical to the one of subunit II of qCuANOR published by Suharti et al. (2001). These authors (Suharti et al., 2004) also presented the N-terminal sequence of the large subunit (MTKKNTQEVVKEGREGIGTFIGVGIVGAV), but this was not found in the corresponding subunit I (BAZO_06399). Rather, the almost identical sequence—MATTKNTQEVVKEGREGIGTFIGVGIVGAV—was retrieved in the adjacent gene (contig42_104893_105033) (Figure 2A). This gene encodes a small (46 amino acids) membrane-bound peptide. Hence, it is very well conceivable that this peptide formed part of the enzyme preparation purified by Suharti et al. (2001, 2004). The comparison of the amino acid sequence of subunit I (BAZO_06399) with that of ba3 from T. thermophilus revealed that the tyrosine (Y223, T. thermophilus numbering in Figure A3) covalently binding a CuB-associated histidine (H219) was substituted by an asparagine in the B. azotoformans protein, in agreement with predictions made by Hemp and Gennis (2008) for NOR functionality (Figure A3). These findings identify BAZO_06394-BAZO_06399 as the dual-function quinol and cyt c-dependent qCuANOR described by Suharti et al. (2001, 2004). We may note that the genes coding for these three subunits are linked to two other ones, one (BAZO_06404) coding for a SenC/SCO1-type membrane-bound protein that has been implemented with the insertion of copper (CuA) into subunit I, and BAZO_06409 that is predicted to comprise six TMHs (Figure 2A). In protein databases, homologs of the latter are found being annotated as cyt c oxidase-associated membrane proteins. Furthermore, the presence of BAZO_06394-06399 qCuANOR is not restricted to B. azotoformans and B. bataviensis (see below), but close homologs sharing the same sequential features and the same cluster organization are found in various other Gram-positive bacteria, including Caldalkalibacillus thermarum TA2, but also in Nitrosomonas eutropha and other nitrifiers (Figure 2A and data not shown). In N. eutropha the particular NOR was designated sNOR (Stein et al., 2007). Transcriptome analysis suggested sNOR as a suitable candidate for aerobic NO reduction, next to NorBC (Cho et al., 2006).

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.