Na
+
-coupled ATP synthase. This enzyme exhibits low ATP synthesis activity and is
thought to rather play a role in the establishment of an SMF for sodium-coupled
solute uptake and motility [69]. A reason for the preferential use of proton-coupled
ATP synthases in most alkaliphiles might relate to the overwhelming pressure to
maintain pH homeostasis, as the proton uptake during ATP synthesis contributes
to the acidification of the cytoplasmic environment [67]. This suggestion is also
re-enforced by the fact that most alkaliphile ATP synthases exhibit very low or
absent hydrolytic activity. For instance, the ATP synthase from the
thermoalkaliphilic Bacillus sp. TA2.A1 contains two salt bridges in the beta subunit
that prevent the protein from rotating in the ATP hydrolytic direction, thus
preventing the outwards flux of protons to the bulk medium [70].
6.1 NADH and Succinate Dehydrogenases
Studies of B. pseudofirmus have demonstrated that it contains two distinct types of
NADH dehydrogenases, NDH-2A and NDH-2B [41]. NDH-2A shows significant
orthology with the type 2 NDH from Bacillus subtilis, YjlD (37% identity), while
NDH-2B shows significant homology (56%) to a putative NADH dehydrogenase
from Halobacillus dabanensis that has been shown to couple Na
+
/H
+ antiport
activity to the respiration chain [64]. Both dehydrogenases are localized in the
cytoplasm and show very different spectra of activity, with NDH-2A being predominantly active against NADH, while NDH-2B also shows activity against NADPH
and d-NADH with ferricyanide being the primary acceptor. It is hypothesized that
NDH-2A plays the primary role in providing the entry point for electrons to the
respiratory chains [64].
Bacillus YN-1 was found to contain a type 2 NDH homodimer which contains
one FAD and shows homology to a thioredoxin reductase from Escherichia coli
[71]. In turn, the NDH-2 from the thermoalkaliphilic bacterium Caldalkalibacillus
thermarum is an FAD and NADH utilizing homodimer composed of subunits with
both membrane-anchoring and catalytic domains, where binding sites for NADH
and quinone do not overlap [72].
6.2 Cytochromes
In addition to the antiporters, B. halodurans also contains two bo3-type cytochrome
c oxidase genes that are absent from the neutrophilic B. subtilis [13]. C-type
cytochromes from alkaliphiles can be distinguished by being low midpoint potential
electron carriers (+50 to +100 mV) compared to their neutrophilic counterparts
(+180 to +250 mV) and by having low isoelectric points [41]. B pseudofirmus was
shown to express four distinct heme-containing membrane peptides, all of which
have counterparts in B. subtilis [41]. One of these is subunit II of the terminal oxidase
Genomics of Alkaliphiles
149
+
-coupled ATP synthase. This enzyme exhibits low ATP synthesis activity and is
thought to rather play a role in the establishment of an SMF for sodium-coupled
solute uptake and motility [69]. A reason for the preferential use of proton-coupled
ATP synthases in most alkaliphiles might relate to the overwhelming pressure to
maintain pH homeostasis, as the proton uptake during ATP synthesis contributes
to the acidification of the cytoplasmic environment [67]. This suggestion is also
re-enforced by the fact that most alkaliphile ATP synthases exhibit very low or
absent hydrolytic activity. For instance, the ATP synthase from the
thermoalkaliphilic Bacillus sp. TA2.A1 contains two salt bridges in the beta subunit
that prevent the protein from rotating in the ATP hydrolytic direction, thus
preventing the outwards flux of protons to the bulk medium [70].
6.1 NADH and Succinate Dehydrogenases
Studies of B. pseudofirmus have demonstrated that it contains two distinct types of
NADH dehydrogenases, NDH-2A and NDH-2B [41]. NDH-2A shows significant
orthology with the type 2 NDH from Bacillus subtilis, YjlD (37% identity), while
NDH-2B shows significant homology (56%) to a putative NADH dehydrogenase
from Halobacillus dabanensis that has been shown to couple Na
+
/H
+ antiport
activity to the respiration chain [64]. Both dehydrogenases are localized in the
cytoplasm and show very different spectra of activity, with NDH-2A being predominantly active against NADH, while NDH-2B also shows activity against NADPH
and d-NADH with ferricyanide being the primary acceptor. It is hypothesized that
NDH-2A plays the primary role in providing the entry point for electrons to the
respiratory chains [64].
Bacillus YN-1 was found to contain a type 2 NDH homodimer which contains
one FAD and shows homology to a thioredoxin reductase from Escherichia coli
[71]. In turn, the NDH-2 from the thermoalkaliphilic bacterium Caldalkalibacillus
thermarum is an FAD and NADH utilizing homodimer composed of subunits with
both membrane-anchoring and catalytic domains, where binding sites for NADH
and quinone do not overlap [72].
6.2 Cytochromes
In addition to the antiporters, B. halodurans also contains two bo3-type cytochrome
c oxidase genes that are absent from the neutrophilic B. subtilis [13]. C-type
cytochromes from alkaliphiles can be distinguished by being low midpoint potential
electron carriers (+50 to +100 mV) compared to their neutrophilic counterparts
(+180 to +250 mV) and by having low isoelectric points [41]. B pseudofirmus was
shown to express four distinct heme-containing membrane peptides, all of which
have counterparts in B. subtilis [41]. One of these is subunit II of the terminal oxidase
Genomics of Alkaliphiles
149