From the
The genes of Bacillus stearothermophilus K1041 encoding
cytochrome b
Cytochrome b is the central catalytic subunit of
ubiquinol:cytochrome c oxidoreductase (or bc
In contrast to the
respiratory chain of Gram-negative bacteria, little is known about that
of Gram-positive bacteria. 1) Thermophilic Bacillus PS3
contains both four-subunit cytochrome c oxidase and
four-subunit bc
We succeeded to clone the
genes for cytochrome b
Most of this DNA was cloned in M13 phage, and
the clone was named bc15. Using this bc15 as the probe, about 10,000
recombinants of the B. stearothermophilus K1041 library in
EMBL-3 were screened. Only one plaque was truly positive to hybridize
with the probe. The DNA prepared from the phage lysate infected with
this plaque and cut by restriction enzymes showed that 4 kilobase pairs
( SalI cut) and 1.6 kilobase pairs ( SalI and
EcoRI double cut) fragments hybridized the probe. This
1.6-kilobase pair fragment was subcloned into pUC119 and sequenced.
Fig. 1
shows the map of the genes coding for cytochrome
b
The conclusion that
Bacillus cytochrome bc
The PS3 gene lacks 54 base pairs for the
N-terminal region (see Fig. 2), and thus total amino acid numbers are
223 in the K1041 gene and 205 in the PS3 gene. The codon usage number
is shown as K1041/PS3.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Mie Shugyo for skillful technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
( Bacillus cytochrome b is referred to as cytochrome
b
for its resemblance to plastid
b
) and subunit IV of the quinol:cytochrome c oxidoreductase ( bc
complex) were cloned and
sequenced. For preparation of the probe for cloning, polymerase chain
reaction was carried out using oligonucleotide mixtures targeting for
N-terminal regions of cytochrome bc and subunit IV of the
thermophilic Bacillus PS3. The deduced amino acid sequences
contained 224 residues of 25,425 daltons for cytochrome
b
and 173 residues of 19,371 daltons for
subunit IV, and both open reading frames were separated by 67 base
pairs. Cytochrome b and subunit IV contained 4 and 3
hydrophobic transmembrane segments, respectively, indicating that the
fourth segment of subunit IV (eighth segment of cytochrome b)
is lacking. Four histidine residues supposed to ligand two protohemes
were conserved, but the two His in the fourth segment were separated by
14 amino acid residues like cytochrome b
, not like
mitochondrial cytochrome b. The residues that might have
conferred the two quinol-binding sites were mostly conserved, but
especially the third His residue in the fourth segment of mitochondrial
cytochrome b was replaced by Arg in Bacillus cytochrome b
as in cytochrome
b
. These characteristics and quantitative
comparison of the protein sequences indicate that this Bacillus sequence is unique and meanwhile rather close to the
cyanobacteria-plastids type than the purple bacteria-mitochondria type.
complex) in the respiratory chain of mitochondria and aerobic
bacteria
(1, 2, 3) . A similar cytochrome named
cytochrome b
is known to be present in the
homologous b
f complex of the
photosynthetic electron transfer chain of cyanobacteria and
chloroplasts
(3, 4, 5) . The cytochromes
b/ b
are transmembrane proteins containing
two protohemes responsible for vectorial electron transfer. The
molecular mass of cyanobacterial and chloroplast cytochrome
b
determined by SDS-PAGE
(
)
(20-22 kDa) was much smaller than the mitochondrial
and purple bacterial ones (34-40 kDa)
(3, 4) , but
this is due to the fact that the C-terminal half of the cytochrome
b is translated separately in chloroplast and cyanobacteria
(6) , which is named subunit IV
(3) . The gene for
cytochrome b
( petB) is terminated at the
downstream of the fourth transmembrane segment (between helix D and E,
in cytochrome b), and the gene for subunit IV ( petD)
composed of three helices (equivalent for E, F, and G of cytochrome
b) sits downstream of the gene for cytochrome
b
, indicating that cytochrome b
plus subunit IV is equivalent to mitochondrial cytochrome b(2, 3, 4) .
complex forming a
menaquinol-oxidizing respiratory chain
(8, 9, 10) . 2) Bacillus subtilis(11, 12, 13) and Bacillus cereus(14) grown under regular conditions use quinol oxidases
containing cytochrome aa
-600 and Cu
instead of cytochrome c oxidase, while any cytochrome
bc
- b
f complex has
not been reported
(13) . The thermophilic Bacillus
bc
complex is composed of 29-kDa cytochrome
c
23-kDa Rieske iron-sulfur protein, 21-kDa
cytochrome b
, and 14-kDa subunit IV
(9) . The absorption spectrum of PS3 ferrocytochrome
c
has a shoulder at 548 nm in addition to the peak
at 553 nm like cytochrome f, and two protohemes in 21-kDa
cytochrome b
shows very similar
absorption maxima, not like mitochondrial cytochromes b-562
and b-566
(9) . These characteristics suggest that
quinol:cytochrome c oxidoreductase of Gram-positive bacteria
is rather similar to cytochrome b
f complex. The history of a living creature and protein molecule may
be written in DNA, and the gene for quinol reductase of a thermophilic
Bacillus may contribute elucidation of the role of quinol
reductase in mesophilic Bacillus having quinol oxidase
(cytochrome aa
-600).
and subunit IV of
Bacillusstearothermophilus K1041 using PCR. This is
the first report of the gene structure and the deduced protein sequence
of the genes for the cytochrome b of Gram-positive bacteria.
Comparison of the protein sequence indicates closer similarity to
cyanobacterial and plastid cytochrome b
rather than to those of purple bacteria and mitochondria, as well
as several unique characteristics.
Materials
T4-DNA ligase, Klenow
fragment, DNA polymerase of Thermusaquaticus ( Taq polymerase), restriction enzymes, plasmid vectors
pUC18 and pUC19, and M13 bacteriophages mp18 and mp19 were obtained
from Takara Shuzo Co. (Kyoto, Japan).
[-
P]dCTP and Hybond N
(nylon membranes for DNA blotting) were purchased from Amersham
Corp. Butyl-toyopearl (butylfractogel) was a product of Tosoh Co.
(Tokyo). Cytochrome bc
complex from the
thermophilic Bacillus PS3 was prepared as previously described
(9) . Separation of each subunit of the complex was carried out
by SDS-PAGE
(15) .
Molecular Cloning
The methods used for
molecular cloning were based on those of Maniatis et al.(16) . Purified genomic DNA of B. stearothermophilus K1041 was partially digested with Sau3AI, fractionated by
gradient centrifugation, and ligated to the BamHI site of the
vector EMBL-3. This library was screened using the probe
containing the gene for cytochrome b of Bacillus PS3
under the hybridization condition at 55 °C in 5
SSC. This
PS3 gene was prepared by cloning the PCR product synthesized with the
sense [5`-TGGCG(A/C/G/T)GA(C/T)AT(A/C/T)GC(A/G/C/T)GA] and
antisense oligonucleotides
[5`-CA(A/G)TT(C/T)TC(A/G)AA(C/T)TTCAT] as the primers
(Fig. 1) and PS3 DNA as the template. These primers are targeted
against N-terminal peptides of PS3 cytoochrome
b
and subunit IV, respectively, although
these oligonucleotides are not completely redundant (see
for the codon usage). The PCR was performed according to
the manufacturer's protocol in a thermal cycler (Perkin-Elmer
Corp.) with PS3 DNA (1 ng). A combination of 94, 45, and 72 °C (2
min each) was repeated 25 times. The PCR product was centrifuged in a
cup of a Centricon (Amicon, Beverly, MA) to remove primers and
mononucleotides, dephosphorylated by calf intestine alkaline
phosphatase (2 µg), and ligated to the M13 mp19 vector cleaved with
SmaI. One clone in M13 ( bc15) contained the sense
primer site but no antisense primer site, and the deduced amino acid
sequence showed that the cloned gene really encoded the cytochrome
b
protein composed of 230 amino acid
residues, including four conserved histidines as ligands for the hemes.
Figure 1:
Sequence
strategy and a map of B. stearothermophilus chromosome in the
region encoding cytochrome b and subunit IV. The
area subcloned, from SalI (the 5`-end of the EMBL3) to
EcoRI and its vicinity, is shown.
Sequencing of DNA and Peptide
Nucleotide
sequencing was carried out by the chain termination method
(17) using [-
P]dCTP. Peptide
sequences were determined by the Edman degradation method using an
Applied Biosystems model 473A gas-phase sequencer with specimen
transferred to polyvinylidene fluoride membrane after SDS-PAGE. The
sequence data were analyzed with a software program (GENETYX 2.0.0).
Cloning of B. stearothermophilus Genes Encoding
Cytochrome b
The N-terminal protein sequences of cytochrome b and subunit IV of cytochrome bc1 complex from the thermophilic
Bacillus PS3 were analyzed to be MLNKLYDWVDERLDITPLWRDIADHE-
and MKFENTGL-, respectively. We then prepared oligonucleotides as
primers, expecting that the structure gene for subunit IV may sit after
the gene for cytochrome b + Subunit
IV
as in the
operon of cytochrome b
f complexes in
chloroplast and cyanobacteria
(7) . PCR with the sense and
antisense primers in the presence of total PS3 DNA produced a DNA of
about 700 base pairs.
and subunit IV of the
bc
complex and the sequence strategy.
DNA and Amino Acid Sequences
Fig. 2
shows the DNA and deduced amino acid sequences of this
SalI- EcoRI fragment from B. stearothermophilus K1041. The open reading frame for cytochrome b consists
of 672 bases, and the deduced protein is composed of 224 amino acid
residues having 25,425 Da. Likewise, the open reading frame for subunit
IV starts at position 934 and terminates with TGA at position 1447,
coding 173 amino acid residues of 19,371 Da. These values are higher
than the apparent molecular masses of 21 kDa for PS3 cytochrome c and 14 kDa for the subunit IV estimated from SDS-PAGE
(8) .
However, this kind of discrepancy is often observed for highly
hydrophobic membrane proteins as is also observed in subunits of
cytochrome c oxidase. The amino acid sequence of the
N-terminal region of PS3 cytochrome b coincides with the DNA
sequence reading from the initiation codon at position 191, indicating
that no processing occurs at the N-terminal region. The N-terminal MKF
was shown with peptide sequencing of PS3 subunit IV. The sequences at
C-terminal region has not been confirmed. The two structure genes are
separated by a relatively long nucleotide sequence composed of 68
bases, although neither terminator nor promotor structure is found, and
thus the gene for cytochrome b and subunit IV seems to form an
operon. A promotor-like structure ( dotted in Fig. 2) is
present in the 5`-upstream region of the gene for cytochrome
b, but we have not found a terminator
structure in 3`-downstream region of the figure after the stop codon
for subunit IV.
Figure 2:
DNA and deduced amino acid sequences of
B. stearothermophilus b and subunit IV. The
putative Shine-Dalgarno sequences are boxed. The nucleotides
that may constitute the putative promotor region are
dotted.
Structure of Cytochrome
b
Fig. 3
shows the hydropathy
plots of cytochrome band subunit IV.
The hydrophobicity of the segment marked ``IV'' was not so
strong in B. stearothermophilus cytochrome
b
, and this segment might sit on the
surface of the lipid bilayer or membrane protein as claimed by Rao and
Argos
(18) and Croft et al.(19) . On the
contrary, the numbers of hydrophobic segments of subunit IV are three
as in subunits IV of cyanobacteria and plastids
(2, 3, 7) . It is thus likely that total numbers
of membrane-spanning hydrophobic segments of cytochrome
b
+ subunit IV are seven as in the
cyanobacteria and plastids and not eight as in cytochromes b of purple bacteria and mitochondria.
Figure 3:
Hydropathy profile of cytochrome b and subunit IV of B. stearothermophilus. The procedure of
Kyte and Doolittle (41) was used for calculation with a window of 17
residues.
Fig. 4
shows the
alignment of B. stearothermophilus sequences of cytochrome
band subunit IV with the corresponding
sequences of four typical groups, mitochondrial b, purple
bacterial b, cyanobacterial b
+ IV,
and chloroplast b
+ IV. Four heme-binding
histidine residues are conserved: two His residues, respectively, in
segments B and D in a pairwise fashion. Two His residues in segment D
are separated by 14 residues as in cytochrome b
instead of 13 in cytochrome b. Cytochrome b ( b
+ IV) is the catalytic subunit of the
bc
complex playing central roles for vectorial
oxidation of quinol. The Q-cycle mechanism, originally proposed by P.
Mitchell
(20) , is now crucially supported by several
experiments
(21, 22) . The mechanism needs for
cytochrome b ( b
+ IV) to possess two
hemes and two quinone-binding sites
(2, 3, 4, 5) . Four histidine residues
are conserved: two each in hydrophobic segments B and D as marked
(&cjs1372;). The quinol-oxidizing (Qo) site sits close to outside of
the cytoplasmic membrane and is known to be susceptible to myxothiazol
and 2-alkyl-4-hydroxyquinoline N-oxide, while the
quinone-reducing (Qi) site close to cytoplasm is known to be antimycin
A-sensitive and to possess a stabilized semiquinone in the
mitochondrial and purple bacterial complexes
(2, 23) .
The Bacillus enzyme was insensitive to antimycin A as
cytochrome b
f complexes
(9) ,
indicating that the Qi sites of these complexes are somewhat different
from that of the mitochondrial bc
complex. In
fact, His-202 in the yeast cytochrome b just after the fourth
His residue in segment D was replaced by Arg in the thermophilic
bacilli as in Nostoc and maiza plastid sequences. An important
role of His-217 as a residue conferring Qi site was reported with the
Rhodobacter capsulatus enzyme
(24) . On the contrary,
the Bacillus enzyme is susceptible to
2-alkyl-4-hydroxyquinoline N-oxide and myxothiazol, which is
consistent with the present finding that the several residues in the
loop cd as well as -PEWY- and -LR- in the loop ef of subunit IV are
also conserved in the thermophilic Bacillus as in cytochrome
b
and subunit IV.
Figure 4:
Multiple alignment of cytochrome
b/ b + subunit IV. Identical or
conserved (L = I = V, K = R, D = E, Q
= N) residues among cytochrome b or b
+ IV are boxed. Identical and conserved residues
among all cytochrome b/ b
+ IV are
indicated by asterisks on the top and are also
boxed. 1, yeast Saccharomyces cerevisiae cytochrome b (41); 2, Rhodopseudomonas
viridis cytochrome b (42); 3, B.
stearothermophilus cytochrome b (present work);
4, Nostoc PCC7906 cytochrome b
(7); 5, maiza chloroplast cytochrome b
(43). The alignment starts at the 1st ( 1), 13th
( 2), and 4th ( 3-5) amino acid
residues.
It is also generally
confirmed that the amino acid sequences of Bacillus are mostly
in common with those of the b group, and only a
few are in common with cytochrome b group, as shown in
Fig. 4
. gives percentage identities for the
cytochrome b sequences. B. stearothermophilus cytochrome b
is more similar to
Nostoc b
and maiza plastid b
than the corresponding Rhodopseudomonas viridis and
yeast mitochondrial sequences (A). The same tendency was also observed
in subunit IV (B), but the similarity of Bacillus subunit IV
to cyanobacterial and plastid subunit IV was not so high as the former.
Thus, the comparison of the percentage identity shows closeness of the
Bacillus cytochrome b to those of cyanobacterial and
plastidal counterparts rather than to the mitochondria-purple bacteria
group, although it is different from the other four groups.
Characteristics of Bacillus Cytochrome
b
Purified
cytochrome bc + IV
complex from the thermophilic
Bacillus PS3 showed that its cytochrome
b
was rather small (21 kDa by SDS-PAGE)
and contained the fourth subunit
(9) . Bacillus cytochrome b
contained two
protohemes showing similar values of E
and
absorption maxima of the
band
(9) like plastid cytochrome
b
. The present result of cloning and sequencing of
the genes for the thermophilic Bacillus enzyme is in accord
with the above protein characterization. 1) The open reading frame for
Bacillus cytochrome b
encodes
224 residues, and the initiation codon for subunit IV (173 residues) is
separated by 67 base pairs. 2) The third hydrophobic segment in subunit
IV (segment H) is lacking in Bacillus subunit IV. 3) Two His
residues in Bacillus segment D are separated by 14 amino acid
residues as in cyanobacterial and plastid enzymes, not 13 residues as
in mitochondrial and purple bacterial counterparts. 4) Arg residue is
present in the Bacillus enzyme as in cyanobacteria cytochrome
b
but not His-202 as in yeast cytochrome
b, which is supposed to confer inhibitor sensitivity on the Qi
site
(24) . This difference may explain the inhibitor
sensitivity. 5) Lys-228 conserved among antimycin-susceptible
cytochrome b is replaced by Asn as in cytochrome
b
. The K228I mutant of yeast was shown to be
antimycin A resistant
(7, 25) .
( b
f) complex is not so similar to
purple bacterial-mitochondrial enzyme and rather close to
cyanobacterial enzyme is in accord with the case of cytochrome c oxidase; the cta operons of Bacillus PS3
(26, 27) , B. subtilis(28) ,
Bacillus firmus(29) , and Synechococcus vulcanus(30, 31) encode subunit
II
(2) -I
(14) -III
(5) , whereas the operons of
purple bacteria such as of Paracoccus denitrificans(32) and Rhodopseudomonas sphaeroides(33) encode subunit II
(2) -I
(12) -III
(7) .
The numbers in parentheses are the numbers of hydrophobic segments.
This difference may be explained by assuming that the places of the
stop codon of subunit I are different in the two groups
(34) .
It is also noteworthy that analyses of 16 S rRNA showed a close
relationship between Gram-positive bacteria and cyanobacteria
(35) .
Use of B. stearothermophilus K1041
In
this work, we used B. stearothermophilus K1041, which was
recently isolated from soil and shown to be transformable by
electroporation
(36, 37) . The bacterium grows up to 65
°C. The doubling time in the complex medium is about 15 min. The
thermophilic Bacillus PS3 separated from a hot spring in Japan
and able to grow up to 75 °C is also similar to B.
stearothermophilus. The present data (Fig. 2) indicate that
their differences in DNA and amino acid sequences of cytochrome
bare minor; only 62 bases and 8 amino
acids are replaced out of 620 bases (corresponding to nucleotides
245-864 in Fig. 2) and 206 amino acids. The amino acid
replacements are caused by three single-base changes and five
double-base changes, while 49 base changes do not affect the amino acid
sequence.
Codon Usage of the Thermophilic Bacilli
gives the codon usage in cytochromes b of B.
stearothermophilus and the thermophilic Bacillus PS3. The
latter is a partial sequence due to PCR. Its sequence has been
previously reported
(38) , and the protein sequence has been
aligned
(4) . The codon usage of both bacteria is very similar.
There is a tendency for G or C to be preferentially chosen at the third
position of four-codon-using amino acids such as Gly, Ala, Val, Thr,
and Pro, as we have observed in the PS3 genes for cytochrome c oxidase
(27) and H-ATP synthase
(39) . This tendency may be a little milder in K1041 than in
PS3; K1041 does not use GGC for Gly so frequently as PS3. Tolner et
al.
(40) , however, reported a preference for A/T over G/C
in the third letter of genes encoding
Na
/H
-coupled L-glutamate
symport proteins of B. stearothermophilus ATCC 7954 and
Bacillus caldotenax.
Conclusion and Perspectives
This is the
first report of the cloning and sequencing of the genes for cytochrome
b ( bplus subunit IV) of
cytochrome c reductase of Gram-positive bacteria. The
structure and sequence homologies of the genes indicate they are
similar to those of cyanobacteria rather than to those of purple
bacteria and mitochondria. The gene seems useful to clarify the
presence and roles of cytochrome b ( b
plus subunit IV) in mesophilic Bacilli such as B.
subtilis, in which the presence of cytochrome
b
and cytochrome c reductase
was postulated
(13) , but nothing has been known about it
probably because of its rare presence due to the fact that quinol
oxidase (cytochrome aa
-600) is mainly working in
the B. subtilis respiratory chain
(11, 12) .
Table: Comparative matrices for cytochrome
b/b + IV proteins
Table: Comparison of codon usage of the cytochrome
b gene of B. stearothermophilus K1041
with that of PS3
/EMBL Data Bank with accession number(s) D45410.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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