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xaxa是什么意思? - 知乎首页知乎知学堂发现等你来答切换模式登录/注册游戏xaxa是什么意思?今天在对峙2上把我的akr暗金法老借了一个外国友人玩，等结束他给我发了句xaxa，这是啥意思？显示全部 关注者3被浏览4,908关注问题写回答邀请回答好问题 1添加评论分享2 个回答默认排序知乎用户在西里尔字母中，X发H的音。所以希腊人或者俄罗斯人或其它使用西里尔字母的人写Xaxa,就是哈哈笑的意思。发布于 2020-07-27 02:03赞同 8添加评论分享收藏喜欢收起轻笑涉猎万物兴趣者 关注如果是外国人可能一种文字表情吧客家话里面谢可以近似读成xa(拼起来)xaxa＝谢谢所以那人是外国人!?xaxa也有阿司匹林的意思俄语里xaxa是哈哈的意思编辑于 2019-07-16 08:19赞同添加评论分享收藏喜欢收起
"Xaxaxaxa "是什么意思？ -关于希腊语 | HiNative
"Xaxaxaxa "是什么意思？ -关于希腊语 | HiNative
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2021年5月25日
Jessica_Batterton_Cr
2021年5月21日
英语 (美国)
希腊语
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Xaxaxaxa 是什么意思？
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siasialand
2021年5月21日
希腊语
In Greek "χαχαχαχα" is hahaha we use it when we laugh for sth in written speech.
In Greek "χαχαχαχα" is hahaha we use it when we laugh for sth in written speech.
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deltav
2021年5月23日
希腊语
Xaxaxaxa = hahahaha. The only difference is that in Greek we tend to swap the letter h to x, so instead of (h)a(h)a(h)a greeks replace the letter h with x and write (x)a(x)a(x)a instead :)
Xaxaxaxa = hahahaha. The only difference is that in Greek we tend to swap the letter h to x, so instead of (h)a(h)a(h)a greeks replace the letter h with x and write (x)a(x)a(x)a instead :)
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What does spamming "ax" mean in Russian?
I was watching a Russian twitch stream and after a crazy play happened I saw a bunch of comments like "axaxaxaxaxaxaxaxax" What exactly does this mean?
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Characterization of the Functionally Critical AXAXAXA and PXXEXXP Motifs of the ATP Synthase c-Subunit from an Alkaliphilic Bacillus - PMC
Characterization of the Functionally Critical
AXAXAXA and PXXEXXP Motifs of the
ATP Synthase c-Subunit from an Alkaliphilic
Bacillus - PMC
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J Biol Chem
v.284(13); 2009 Mar 27
PMC2659230
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J Biol Chem
v.284(13); 2009 Mar 27
PMC2659230
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J Biol Chem. 2009 Mar 27; 284(13): 8714–8725. doi: 10.1074/jbc.M808738200PMCID: PMC2659230PMID: 19176524Characterization of the Functionally Critical
AXAXAXA and PXXEXXP Motifs of the
ATP Synthase c-Subunit from an Alkaliphilic
Bacillus*S⃞Jun Liu, Makoto Fujisawa,1 David B. Hicks, and Terry A. Krulwich2Author information Article notes Copyright and License information PMC DisclaimerDepartment of Pharmacology and Systems Therapeutics, Mount Sinai School
of Medicine, New York, New York 100291Current address: 7th Lab, Faculty of Life Sciences, Toyo University, 1-1-1
Izumino, Itakura-machi, Ora-gun, Gunma 374-D193, Japan.2
To whom correspondence should be addressed: Dept. of Pharmacology &
Systems Therapeutics, Mount Sinai School of Medicine, One Gustave L. Levy
Place, New York, NY 10029. Tel.: 212-241-7280; Fax: 212-996-7214; E-mail:
ude.mssm@hciwlurk.yrret.
Received 2008 Nov 18; Revised 2008 Dec 30Copyright © 2009, The American Society for Biochemistry and
Molecular Biology, Inc.Associated DataSupplementary Materials
[Supplemental Data]
M808738200_index.html (1.2K)GUID: EC98A756-CF40-4D01-BBD1-A5E42B0EA58E
M808738200_Supplementary_Table_1.doc (44K)GUID: C51C9C6D-5146-41D8-84FA-B0A9DB520BB0
AbstractThe membrane-embedded rotor in the F0 sector of
proton-translocating ATP synthases is formed from hairpin-like
c-subunits that are protonated and deprotonated during energization
of ATP synthesis. This study focuses on two c-subunit motifs that are
unique to synthases of extremely alkaliphilic Bacillus species. One
motif is the AXAXAXA sequence found in the
N-terminal helix-1 instead of the GXGXGXG of
non-alkaliphiles. Quadruple A→G chromosomal mutants of alkaliphilic
Bacillus pseudofirmus OF4 retain 50% of the wild-type hydrolytic
activity (ATPase) but <18% of the ATP synthase capacity at high pH.
Consistent with a structural impact of the four alanine replacements, the
mutant ATPase activity showed enhanced inhibition by dicyclohexylcarbodiimide,
which blocks the helix-2 carboxylate. Single, double, or triple A→G
mutants exhibited more modest defects, as monitored by malate growth. The key
carboxylate is in the second motif, which is
P51XXE54XXP in extreme alkaliphiles
instead of the (A/G)XX(E/D)XXP found elsewhere. Mutation of
Pro51 to alanine had been shown to severely reduce malate growth
and ATP synthesis at high pH. Here, two Pro51 to glycine mutants of
different severities retained ATP synthase capacity but exhibited growth
deficits and proton leakiness. A Glu54 to Asp54 change
increased proton leakiness and reduced malate growth 79-90%. The
Pro51 and the Glu54 mutants were both more
dicyclohexylcarbodiimide-sensitive than wild type. The results highlight the
requirement for c-subunit adaptations to achieve alkaliphile ATP
synthesis with minimal cytoplasmic proton loss and suggest partial suppression
of some mutations by changes outside the atp operon.Proton-translocating ATP synthases transport protons down their
electrochemical gradient through the membrane-embedded F0
sector of the synthase. This facilitates use of the proton-motive force
produced by the respiratory chain to drive ATP synthesis by a rotary mechanism
during OXPHOS3 in both
prokaryotes and eukaryotes
(1-4).
The critical proton-binding carboxylate residues on the F0
rotor are in the C-terminal helix-2 of the hairpin-like c-subunit.
This subunit forms a ring that is composed of 10-15 c-subunit
monomers depending on the organism
(5-7).
Helix-1 packs on the inside of the c-ring and helix-2 on the outside.
Small residues in helix-1 are suggested to contribute to the tight packing
seen in the c-ring structure
(5), including a highly
conserved GXGXGXG sequence
(6,
8). On the outside of the ring,
a single a-subunit binds to the ring in bacteria, making sequential
contact with pairs of c-ring subunits as rotation of the ring
proceeds (9,
10). Protons are taken up from
the outside using a pathway involving a-subunit residues. These
residues are proposed to form an aqueous channel between the external surface
of the bacterial membrane and the entry point of protons on to the
c-ring within the membrane
(11,
12). The path of protons that
return from the c-ring and complete their downhill movement into the
cytoplasm is less-well defined and may be through a distinct aqueous channel
comprised of residues from the a-subunit
(13) or via a pathway that
involves the c-subunit
(14). Interaction of the
essential a-subunit arginine with the carboxylate of the
c-subunits is a key event in the protonation/deprotonation reactions
at the a/c interface during rotation of the c-ring
(15,
16). The functional properties
of the c-subunit carboxylate on helix-2 are also affected by residues
in helix-1 that are opposite the carboxylate. For example, mutations in
Ala24 and Ile28 in the Escherichia coli
c-subunit helix-1 make the ATP synthase resistant to
dicyclohexylcarbodiimide (DCCD), a reagent that specifically reacts with the
carboxylate (Asp61 in E. coli) resulting in inactivation
of the enzyme (17,
18).OXPHOS supported by the proton-coupled ATP synthase of alkaliphilic
Bacillus species at pH 10.5 presents a set of challenges with respect
to capture of protons, successful proton translocation to the cytoplasm, and
proton retention in the cytoplasm. B. pseudofirmus OF4 maintains a
cytoplasmic pH of 8.2 during growth in rigorously controlled continuous
culture at pH 10.5 in medium containing malate, a non-fermentable substrate
(19,
20). This successful pH
homeostasis is critical to growth at highly alkaline pH, so these organisms
require mechanisms to minimize proton leakiness or membrane potential
decreases that result in proton loss
(21). It has been suggested
that, without any specific adaptations of the OXPHOS machinery itself, the
challenges of proton capture in support of OXPHOS and pH homeostasis can be
overcome by global features that foster proton movement along the outer
surface of the membrane to the ATP synthase and to the antiporters that
support pH homeostasis (22).
However, we have shown that the pKa of the
c-subunit carboxylate (Glu54) of B. pseudofirmus
OF4 is higher than those reported for several other bacterial
c-subunit carboxylates, a possible adaptation for proton acquisition
and retention during rotation of the c-ring
(23).Another indication that specific adaptations of the proton-coupled ATP
synthase are involved in OXPHOS by extreme alkaliphiles is the presence of a
group of alkaliphile-specific residues and motifs in functionally important
regions of the a- and c-subunits of the synthases. The
sequences of these residues and motifs differ significantly from the consensus
sequences in non-alkaliphilic Bacillus species and the well studied
E. coli ATP synthase
(24,
25). In an initial mutagenesis
study, we changed six of the most striking sequence features of the
a- and c-subunits in the chromosome of genetically tractable
B. pseudofirmus OF4 to the sequences found in non-alkaliphilic
Bacillus megaterium
(26). B. megaterium
was chosen because its a- and c-subunit sequences have the
closest overall sequence similarity to those of the B. pseudofirmus
OF4 subunits among non-extremophilic Bacillus species. The results
supported the conclusion that the ability of alkaliphiles to capture and
productively translocate protons through the ATP synthase depends upon the
alkaliphile-specific motifs or residues.One of the features flagged by the initial mutagenesis study as crucial for
ATP synthesis at high pH was the Pro51 residue found in a
C-terminal helix-2 in extreme alkaliphiles but not in modest alkaliphiles such
as Bacillus sp. TA2.A1. Arechaga and Jones designated as an
“alkaliphile motif” the
P51XXEXXP57 sequence that begins with
Pro51, includes the proton-binding Glu54 residue, and
then ends with a second, conserved Pro57
(27) (see the alignment in
Fig. 1). In the only
mutagenesis study of an ATP synthase in its natural alkaliphile host, we
changed Pro51 of B. pseudofirmus OF4 to alanine, the
residue found in non-alkaliphilic Bacillus species
(Fig. 1). The cP51A
mutation resulted in a severe deficit in malate growth and ATP synthase
capacity at pH 10.5 and a much smaller deficit at pH 7.5
(26). The goals of the current
study were to expand the characterization of the motifs of the alkaliphile
c-subunit, the heart of the rotary ATP synthase, to a broader panel
of mutants. In the earlier study, a striking candidate for another
c-subunit motif of extreme alkaliphiles,
AXAXAXA, was not studied in detail, because, when
it was changed to the B. megaterium
GXGXGXG sequence, almost no ATP synthase was found
in the alkaliphile membrane
(26). Alkaliphile
c-subunits typically have substitution of two or more alanines for
the glycines of the conserved helix-1 GXGXGXG motif
found in non-alkaliphiles. The version of the motif with the full four
alanines is found only in the most extreme alkaliphiles, such as B.
pseudofirmus OF4 (Fig. 1).
In this study, we first constructed a double mutant of B.
pseudofirmus OF4 in which both the AXAXAXA
motif and the Pro51 of helix-2 were changed to the B.
megaterium sequence, because it was possible that more favorable helix
interactions in the double mutant would support a higher membrane level of
synthase. If not, mutagenesis of single and multiple alanine residues of the
AXAXAXA motif, rather than its entire replacement,
would be conducted. More limited replacement of the motif might support higher
synthase levels in the membrane so that we could evaluate whether the motif
has an important role in alkaliphile OXPHOS. We also investigated the effects
of additional mutations in the
P51XXEXXP57 motif, including: (i)
mutation of the unusual Pro51 to glycine, which is found in the
moderate alkaliphile and thermophilic Bacillus sp. TA2.A1 as well as
E. coli (Fig. 1); (ii)
mutation of Glu54 to aspartate; and (iii) mutation of conserved
Pro57 to glycine and alanine. The results establish a major role
for the AXAXAXA motif in alkaliphile OXPHOS and
greatly extend our appreciation of the adverse consequences of mutational
changes in the PXXEXXP motif.Open in a separate windowFIGURE 1.Alignment of c-subunit of the
F1F0-ATP synthase from
Bacillus species and E. coli. The shaded
organisms are the extreme alkaliphiles B. pseudofirmus OF4, B.
halodurans C-125, and B. alcalophilus, the more moderately
alkaliphilic B. clausii, and the moderate alkaliphile and thermophile
Bacillus sp. TA2.A1. Residue numbers are given for the B.
pseudofirmus OF4 (top) and the E. coli c-subunits
(bottom). The AXAXAXA motif and
PXXEXXP motif are shaded. An open box
shows the residues that align with the Gly17 residue, which
includes the Ala24 residue of E. coli. The NCBI gene
accession numbers for the data shown are: B. pseudofirmus OF4
(accession number {"type":"entrez-protein","attrs":{"text":"AAC08039","term_id":"142546"}}AAC08039); B. halodurans C-125 (accession number
NP_244626); B. alcalophilus (accession number {"type":"entrez-protein","attrs":{"text":"AAA22255","term_id":"142567"}}AAA22255); B.
clausii KSM-K16 (accession number YP_177348); Bacillus sp.
TA2.A1 (accession number {"type":"entrez-protein","attrs":{"text":"AAQ10085","term_id":"33329382"}}AAQ10085); B. megaterium (accession number
{"type":"entrez-protein","attrs":{"text":"AAA82521","term_id":"142556"}}AAA82521); Geobacillus kaustophilus HTA426 (accession number
YP_149216); B. subtilis subsp. subtilis str.168 (accession number
{"type":"entrez-protein","attrs":{"text":"NP_391567","term_id":"16080739"}}NP_391567); and E. coli K12 substr. DH10B (accession number
YP_001732558).EXPERIMENTAL PROCEDURESBacterial Strains, Mutant Construction, and Growth
Conditions—The wild-type and all mutant strains used in this study
are listed in Table 1. The
wild-type strain is a derivative of alkaliphilic B. pseudofirmus OF4
811M that has an EcoR1 site introduced into atpB in the same location
as found in all the mutants used in the study
(26). Mutations in the
atpE gene that encodes the c-subunit were constructed in a
cloned atpB-F fragment containing the EcoR1 site. The mutated
fragments were then introduced into a mutant strain of B.
pseudofirmus OF4 811M, designated ΔF0, that is
deleted in the F0-encoding genes (ΔatpB-F).
The two-step method used to regenerate the complete, mutant chromosomal
atp operons in the ΔF0 strain is described
in Ref. 26. Briefly,
mutagenesis of atpB-F fragments was conducted using the
GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), in the low
copy plasmid pMW118 (Nippon Gene, Toyama, Japan). The primers used for all the
mutations made in this study are listed in supplemental Table S1. The mutant
atpB-F constructs from pMW118 were digested with BamHI and KpnI and
then ligated with pG+host4 (Appligene, Pleasanton, CA) previously
digested with BamHI and KpnI. The ligation mixtures were used to transform
E. coli XL-1 Blue MRF (Promega, Madison, WI), and selection was on
Luria-Bertani broth containing 250 μg/ml erythromycin. Constructs in
pG+host4 with the correct mutant sequences were introduced into the
ΔF0 strain (ΔatpB-F). Homologous
recombination introduced the mutations into the chromosomal atp
locus. This recombination and concomitant loss of the pG+host4
plasmid were achieved by temperature selection methods described previously
(28). The
F0 segment of each mutant was entirely sequenced and
verified to have only the desired mutation(s). These and other DNA sequence
analyses were conducted at Genewiz, Inc. (South Plainfield, NJ). For each
mutation, a panel of candidate recombinant colonies was tested for growth on
malate at pH 7.5 and pH 10.5. For most of the mutants, all of the colonies of
a panel showed very similar growth, and a representative colony was chosen for
study. For a few mutants, the panel yielded two types of colonies
(e.g. one type that exhibited more growth on malate at pH 7.5 than
the other). For those mutants, a representative colony was chosen for each
type, and the entire atp operon was sequenced (see
“Results” and “Discussion” for further details).
Growth of wild-type and mutant strains on semi-defined glucose or malate
medium were determined as described previously
(26). Briefly, cells were
pre-grown on glucose at pH 7.5 and inoculated into comparable media with the
carbon source and pH indicated in the text or figures. For glucose growth, the
small amount of growth on yeast extract as the sole carbon source was
subtracted from total growth assessed by A600 after 14 h
at 30 °C. For malate growth, the small amount of growth exhibited by the
ΔF0 strain on malate was subtracted from the growth
of the other strains. All growth experiments were conducted in duplicate in at
least two independent experiments.TABLE 1B. pseudofirmus OF4 strains used in this studyStrain
Properties
Source
Wild type
Derivative of B. pseudofirmus OF4 811 m into which an
EcoRI site was introduced in atpB by silent mutation of nucleotides
484 and 487
Ref. 27
ΔFo
Deletion of atpB-F
Ref. 27Mutants in the c-subunit helix-1
motifacHx1
Substitution of wild-type atpE residues 15-23 (VAGAIAVAI) with
corresponding B. megaterium residues 15-23 (LGAGIGNGL)
Ref. 27cP51A
P51XXEXXP57→
A51XXEXXP57 Ref. 27cHx1-cP51A
Double cHx1 and cP51A mutant
This study
cA16G
A16XAXAXA22→
G16XAXAXA22 This study
cA18G
A16XAXAXA22→
A16XGXAXA22 This study
cA20G
A16XAXAXA22→
A16XAXGXA22 This study
cA22G
A16XAXAXA22→
A16XAXAXG22 This study
cA1620G
A16XAXAXA22→
G16XAXGXA22 This study
cA162022G
A16XAXAXA22→G16XAXGXG22 This study
cA4G
A16XAXAXA22→G16XGXGXG22 This study
cG17A
cG17A mutant
This study
Mutants in the c-subunit helix-2
motifacP51G
P51XXEXXP57→
G51XXEXXP57 This study
cE54D
P51XXEXXP57→
P51XXDXXP57 This study
cP57G
P51XXEXXP57→
P51XXEXXG57 This study
cP57A
P51XXEXXP57→
P51XXEXXA57
This study
Open in a separate windowaThe residues in boldface and underlined are mutated residues.Isolation of Everted Vesicles, Assays of Their
Octylglucoside-stimulated ATPase, and Assessments of the Membrane Content of
ATP Synthase Protein—Wild-type and mutant derivatives were grown at
30 °C in a semi-defined medium containing 0.1% yeast extract with mineral
salts and buffered with 0.1 m
Na2CO3/NaHCO3 at pH 10.5 or buffered with 0.1
m MOPS at pH 7.5 with 50 mm glucose or malate as the
major carbon and energy source
(26). Everted membrane
vesicles were prepared from overnight cultures of the wild-type strain and
mutant derivatives. The cells were washed with 50 ml of 50 mm
Tricine-NaOH, pH 8.0/5 mm MgCl2, pelleted, and
resuspended in 25 ml of French Press buffer containing 50 mm
Tricine-NaOH, pH 8.0/5 mm MgCl2, a protease inhibitor
tablet (Roche Applied Science), 1 mm phenylmethylsulfonyl fluoride,
and a trace amount of DNase I (Roche Applied Science). The cells were broken
in a French Press cell at 18,000 p.s.i. The broken cell suspensions were
centrifuged at 15,000 × g for 10 min to precipitate unbroken
cells and debris. The resulting supernatants were subjected to
ultracentrifugation at 250,000 × g (Beckman Ti-60 rotor for 1.5
h at 4 °C) to pellet the everted membrane vesicles. The everted vesicles
were suspended in 1 ml of 50 mm Tricine-NaOH, pH 8.0/5
mm MgCl2. These vesicles were used for determinations of
protein content, octylglucoside (OG)-stimulated ATPase (representing the total
uncoupled ATPase activity), and the levels of ATP synthase protein in the
membrane. Protein content for this and other experiments was measured by the
Lowry method using bovine serum albumin as the standard
(29). OG-stimulated ATPase
assays were conducted as described previously
(26). For assessment of the
ATP synthase content of the everted vesicle membranes, the protein complement
of the vesicles was fractionated on 12% SDS-PAGE gels
(30), and transferred
electrophoretically to nitrocellulose. Western analyses were carried out by
the chemiluminescence method according to the manufacturer's instructions
(Pierce). The β-subunit of the
F1F0 was detected using a monoclonal
antibody against the E. coli β-subunit of
F1F0 ATP synthase (Molecular Probes,
Eugene, OR). For purposes of quantitation, image analysis was performed using
ImageJ 1.40 software
(rsbweb.nih.gov/ij/).Assays of ATP Synthesis in ADP plus Pi-loaded Right-side-out
Membrane Vesicles—Wild-type and mutant derivatives were grown to an
A600 of 0.6 in the semi-defined medium containing 0.1
m Na2CO3/NaHCO3 at pH 10.5 with
glucose as the energy source or with Na2CO3-buffered
medium at pH 8.5 with malate as the energy source, as indicated in connection
with specific experiments. Right-side out (RSO) ADP plus Pi-loaded
membrane vesicles were prepared by methods described previously
(31). After the wash step, the
vesicles were suspended in 0.25 m sucrose, 20 mm
potassium phosphate, pH 8.3, 5 mm sodium phosphate, pH 8.3, and 5
mm MgCl2. ATP synthesis reactions were carried out at
room temperature as follows. The ADP plus Pi-loaded RSO membrane
vesicles were diluted 1:20, to 500 μg of protein/ml, into either pH 7.5
buffer containing 25 mm sodium phosphate, 0.25 m
sucrose, 5 mm MgCl2, and 200 mm
K2SO4 or into pH 10.5 buffer containing 25 mm
Na2CO3, 0.25 m sucrose, 5 mm
MgCl2, and 200 mm K2SO4, and ATP
synthesis was initiated by energizing the aerated vesicles with 10
mm ascorbate and 0.1 mm ascorbate-phenazine
methosulfate. Samples (200 μl) of the reaction mixtures were removed at 10
s and transferred to fresh tubes containing 50 μl of ice-cold 30%
perchloric acid. After neutralization, the ATP content was determined by the
luciferin-luciferase method
(32). For each experimental
set, samples were also taken of unenergized vesicles for assessment of
background ATP values. The amount of ATP synthesized was calculated from a
standard curve.Determination of the Cytoplasmic pH after a Shift in the External pH
from 8.5 to 10.5—pH shift experiments were conducted as described
previously (26,
28). Cells were grown
overnight (50 ml) at pH 8.5 in either malate- or glucose-containing medium, as
indicated in connection with specific experiments. An equal volume of fresh
medium was added to the overnight culture, and the cells were grown for three
more hours. Cells were harvested by centrifugation, washed with and then
resuspended in 3 ml of pH 8.5 buffer containing 100 mm
Na2CO3/NaHCO3, 1 mm
MgSO4, 1 mm KH2PO4. These
manipulations under de-energized conditions provide a period of equilibration
of the cytoplasmic pH with the buffer at pH 8.5. The A600
of the suspensions was adjusted to 20, and then each cell sample was diluted
1:25 into pH 10.5 buffer containing 100 mm
Na2CO3/NaHCO3 and 10 mm malate.
The radioactive probe, [14C]methylamine (1.7 μm with
60 mCi/mmol), was added to the dilution buffer. For controls for nonspecific
binding of the probe, a parallel set of samples also contained 10
μm gramicidin. After 10-min incubation, the diluted suspensions
were filtered through GF/F filters (Whatman). The filters were then dried, and
the radioactivity was assessed by liquid scintillation spectrometry. Values
for the cytoplasmic pH were calculated from duplicate measurements of the
outside pH and the transmembrane pH gradient (ΔpH). The ΔpH was
determined as described earlier from the measured distribution of radiolabeled
methylamine (33). At least
three independent assays were conducted.DCCD Inhibition of Unstimulated ATPase Activity—A malachite
green phosphate detection method was used to measure the low ATPase activity
of the ATP synthase in the absence of OG. The sensitivity of this method was
sufficient to give substantial absorbance readings throughout a time course of
0-30 min under the conditions described below. The time course was found to be
linear and yielded the same specific activity as was reported in an earlier
study (26). In that study, the
less sensitive LeBel et al. method
(34) was used for measuring
phosphate release, requiring longer ATPase reaction times and less dilution of
the DCCD-treated sample into the reaction mixture. The combination of these
two aspects of the LeBel-based DCCD inhibition assay meant that the DCCD was
probably diluted insufficiently to prevent further DCCD inhibition during the
enzyme assay. Thus, in the current study, the wild-type enzyme was inhibited
20-24% as compared with ∼50% determined previously. The assay procedure
was based on that previously described
(35-37).
The malachite green reagent contained malachite green (0.081%, w/v), polyvinyl
alcohol (2.32%, w/v), ammonium molybdate (5.72%, w/v, in 6 m HCl),
and water, mixed in the ratio 2:1:1:2. The reagent was used after it turned to
a golden-yellow color, from its initial dark brown color, as described. For
assays of DCCD sensitivity, everted membrane vesicles were preincubated with
10 μl of methanol (no DCCD) or 10 μl of 10 mm DCCD (dissolved
in methanol) to 100 μm final concentration in 1 ml at a
concentration of 2.5 mg of protein/ml for 30 min at room temperature in 20
mm MOPS-NaOH, pH 7.0, 5 mm MgCl2. Aliquots of
200 μl of the preincubation mixture were then diluted 10-fold into
pre-warmed assay buffer containing 50 mm Tricine-NaOH, pH 8.0, 5
mm MgCl2, 5 mm ATP. Reactions were carried
out for 10 min at 37 °C; preliminary experiments showed that the reaction
was linear for 30 min. The reactions were terminated by transferring 200 μl
of the reaction mixture to a fresh tube containing 800 μl of malachite
green reagent. After 1 min of color development, 100 μl of 34% sodium
citrate was added to stop further color development, and the tubes were
allowed to stand at room temperature for 15 min. The absorbance at 620 nm was
measured and compared with a Pi standard curve.RESULTSLevels of ATP Synthase in a Mutant with the B. megaterium GXGXGXG Motif
Replacing the Native Alkaliphile AXAXAXA Motif Are Not Significantly Enhanced
by Also Introducing a cP51A Mutation—In the initial mutagenesis
study of alkaliphile-specific c-subunit features, the
AXAXAXA sequence in the N-terminal helix-1, which
is V15AGAIAVAI23 in B.
pseudofirmus OF4 when including two flanking residues, was replaced with
the sequence found in the corresponding location of the B. megaterium
c-subunit,
L15GAGIGNGL23 (see
Fig. 1). Very little ATP
synthase was found in the membranes of that mutant. This made it impossible to
assess whether there were functional consequences of replacing the alternating
alanines of the alkaliphile helix-1 motif with glycines, as found in
non-alkaliphile c-subunits
(26). Here, we first tested
the possibility that the B. megaterium sequence would be more
compatible with normal levels of ATP synthase in the membrane if the
Pro51 of the PXXEXXP sequence in the
opposing helix-2 of the alkaliphile c-subunit was changed to alanine,
the residue found in B. megaterium. As shown in
Table 2, the double mutant grew
well on glucose at pH 10.5, but could not grow on malate at either pH 7.5 or
pH 10.5. The double mutant had a membrane β-subunit content (representing
ATP synthase protein level) that was <10% of the wild-type level. Thus the
low levels of the of the ATP synthase in the alkaliphile mutant with a
replacement of the helix-1 AXAXAXA region with the
complete B. megaterium sequence was not due to an effect of the
PXXEXXP motif on the opposing helix. Subsequent mutant
analyses (shown below) demonstrated that B. pseudofirmus OF4 mutants
with glycine(s) replacing one, two, three, or four alanines of the alkaliphile
motif all have significant membrane levels of ATP synthase. This suggested
that other residues, i.e. residues surrounding the alternating
alanines or glycines of helix-1, contributed to the deficit in ATP synthase in
the membrane, because some of them are different in B. pseudofirmus
OF4 than in B. megaterium. Those contributors could be the following:
Leu15, which is Val15 in the alkaliphile;
Ala17, which is Gly17 in the alkaliphile;
Asn21, which is Val21 in the alkaliphile; and/or
Leu23, which is Ile23 in the alkaliphile. To test the
idea that these altered residues contributed to the loss of ATP synthase in
the membrane with a total motif replacement, we constructed one additional
mutant in which only the Gly17 of the alkaliphile motif was
replaced by alanine, as is found in the same position in B.
megaterium. That single change resulted in a 35% reduction in the level
of ATP synthase in the membrane, relative to the wild-type level. This
significant reduction was consistent with the notion that the
“Xs” of the motif have an impact on membrane levels of
the enzyme (Table 2).TABLE 2A cHx1-cP51A double mutant is highly deficient in
growth on malate and β-subunit content, and a single cG17A
mutant alone exhibits significant defectsGrowtha,b
β-Subunit content
(Western)a,c
StrainGlucose
Malate
pH 7.5pH 10.5pH 7.5pH 10.5 %
Wild-type
100
100
100
100
100
cHx1
105 ± 18
107 ± 10
0
0
4 ± 0.1
cP51A
62 ± 9
89 ± 13
75 ± 6
23 ± 10
73 ± 4
cHx1-cP51A
110 ± 3
101 ± 3
0
0
6 ± 0.4
cG17A
103 ± 7
76 ± 13
55 ± 18
60 ± 11
65 ± 12
Open in a separate windowa% of wild-type.bAverage of two independent experiments ± S.D.cAverage of two vesicle preparations ± S.D.A Panel of AXAXAXA Mutations with Only A→G Changes
Reveals a Crucial Role of the Motif in OXPHOS at High pH—A new
mutant panel was constructed in the alkaliphile
AXAX-AXA sequence that changed the alternating
alanines without concomitant changes in residues surrounding the alanines. The
panel included mutants with the following A→G changes (see
Table 1): four single mutants,
cA16G, cA18G, cA20G, and cA22G; a double
mutant in the first and third alanines, cA1620G, which is the pattern
found in the alkaliphilic B. clausii c subunit; a triple mutant in
the first and the last two alanines, cA162022G; and the quadruple
mutant, cA16182022G, which we designated cA4G. In
preliminary studies of the quadruple mutant, we found two growth phenotypes
even though sequence analyses showed that the F0 sequence
of both types was identical and was the expected sequence. We chose a
representative of each phenotype for inclusion in further studies, designated
cA4G-1 and cA4G-2. The sequence of the entire atp
operon in these strains showed that there were no mutations other than the
four changes of alanine to glycine that we had introduced. All eight
alkaliphile mutants in the new panel had over 40% of the ATP synthase levels
found in wild type (Fig.
2A), in contrast to the alkaliphile mutants into which
the B. megaterium sequence had been introduced
(Table 2). The level of
OG-stimulated ATPase activity, relative to that of wild-type, paralleled the
ATP synthase content throughout the new mutant panel
(Fig. 2A). Because
OG-stimulated ATPase activity represents the total (uncoupled) hydrolytic
activity, the specific hydrolytic activity was not significantly altered by
any of the A→G mutations. All the mutants also grew well on glucose
(≥63% of wild type) both at pH 7.5 and pH 10.5
(Fig. 2B,
bottom). This was not the case for malate growth of all the mutants
(Fig. 2B,
top). All four mutant strains with only a single A→G
substitution had a reproducible but small growth deficit relative to wild type
on malate, with growth ≥76% and ≥66% of the wild-type at pH 7.5 and
10.5, respectively. The deficits in the double, triple, and quadruple mutants
were much greater, especially at pH 10.5. The double and triple A→G
replacement mutants exhibited malate growth that was 48-54% of wild type at pH
7.5 and only 23-27% at pH 10.5. The two quadruple mutant types, represented by
strains cA4G-1 and cA4G-2, exhibited poor growth on malate.
Both strains showed almost no growth on malate at pH 10.5 (7-9% of wild-type
malate growth with no statistical difference between the two strains). At pH
7.5, malate growth of cA4G-2 was 35% of wild type, whereas malate
growth of cA4G-1 was even worse, at only 9% of wild type.Open in a separate windowFIGURE 2.Functional characterization of helix-1 alanine mutants. A,
β-subunit content and OG-stimulated ATPase activity of mutant strains.
Strains were grown on glucose at pH 10.5. The values for the mutants are given
as % of wild type, with the wild type set at 100%. Values are the average of
determinations from at least two independent vesicle preparations, and the
error bars show the ± S.D. B, growth of wild-type and
mutant strains as a function of pH and carbon source. The values are the
average of duplicate determinations from at least two independent growth
experiments, and the error bars show the ±S.D. C, ATP
synthesis by ADP plus Pi-loaded RSO membrane vesicles of wild type
and the two types of quadruple A→G mutants. Vesicles were prepared from
cells grown on glucose at pH 10.5. Assays were initiated by the addition of
ascorbate-phenazine methosulfate as described under “Experimental
Procedures.” The values are the average of duplicate assays from at
least three independent vesicle preparations, and the error bars show
the ±S.D. D, DCCD inhibition of unstimulated ATPase activity
of wild-type and the two types of quadruple A→G mutants. Assays were
conducted as described under “Experimental Procedures.” Values are
the average of duplicate determinations from at least two independent vesicle
preparations, and the error bars show the ±S.D. The
numbers in parentheses above the gray columns indicate the %
DCCD inhibition.Further studies were conducted on the two quadruple alanine mutants
cA4G-1 and cA4G-2 to assess the correlation of their
phenotypes with their ATP synthesis activities (which we will call ATP
synthase activities throughout the text) and with sensitivity of their ATPase
activity to inhibition by DCCD. For in vitro assays of ATP synthesis,
ADP plus Pi-loaded RSO vesicles with an intravesicular pH of 8.3
were prepared from wild-type and mutant cells. ATP synthesis was determined
after energization of the vesicles with ascorbate-phenazine methosulfate at
external pH values of 7.5 and 10.5. The intravesicular pH of 8.3 was
comparable to the cytoplasmic pH of cells growing on malate at pH 10.5
(20). The shift of the
vesicles loaded with buffer at pH 8.3 to either pH 7.5 or 10.5 imposed a pH
gradient of 0.8 units, alkali in, at pH 7.5, whereas a 2.2-unit pH gradient,
acid in, was imposed at pH 10.5. Consequently, the bulk proton electrochemical
gradient was much higher at pH 7.5 in these experiments than at pH 10.5. The
wild-type ATP synthase activities at pH 10.5 were 38% of those at pH 7.5 for
vesicles prepared from glucose-grown cells
(Fig. 2C) and 45% of
those at pH 7.5 for vesicles prepared from malate-grown cells (see
Fig. 3B). Consistent
with the malate growth patterns at high pH, the ATP synthase activity of both
A4G-1 and A4G-2 at pH 10.5 was very low (12-20% of wild-type) and not
significantly different from each other, although that of A4G-1 always
exhibited a little higher activity (Fig.
2C). Surprisingly, the ATP synthase activities of the two
mutant strains at pH 7.5 were also not significantly different from each
other, at 62-81% of wild type, despite the better malate growth of the
cA4G-2 mutant at that pH (Fig. 2,
B and C).Open in a separate windowFIGURE 3.Functional characterization of the cP51G mutants.
A, growth of wild-type and the two types of cP51G mutants as
a function of pH and carbon source. Assays were conducted as described
earlier. The values are the average of duplicate determinations from at least
two independent growth experiments, and the error bars show the
±S.D. B, ATP synthesis by ADP plus Pi-loaded RSO
membrane vesicles of wild-type and the two types of cP51G mutants.
Vesicles were prepared from cells grown on malate at pH 8.5. Assays were
conducted as described under “Experimental Procedures.” The values
are the average of duplicate assays from at least two independent vesicle
preparations, and the error bars show the ±S.D. C,
DCCD inhibition of unstimulated ATPase of the wild-type and the two types of
cP51G mutants. Strains were grown on glucose at pH 7.5. Assays were
conducted as described under “Experimental Procedures.” Values are
the average of duplicate determinations from at least two independent vesicle
preparations, and the error bars show the ±S.D. The
numbers in parentheses above the gray columns indicate the %
DCCD inhibition. D, determination of the cytoplasmic pH after a shift
in the external pH from 8.5 to 10.5 of the wild type and the two types of
cP51G mutants. Cells were grown on malate at pH 8.5. Assays were
conducted as described as “Experimental Procedures.” Values are
the average of duplicate determinations from at least three independent
experiments, and the error bars show the ±S.D.We then probed whether there were differences in the sensitivity of
unstimulated ATPase activity to inhibition by DCCD between the two quadruple
mutant strains and the wild type. We had shown earlier that the unstimulated
hydrolytic activity of the purified alkaliphile ATP synthase reconstituted in
proteoliposomes resulted in proton pumping that was abolished by DCCD
pretreatment (and, additionally, that hydrolysis was stimulated by uncouplers)
(38). Thus this unstimulated
ATPase activity represents a coupled activity. DCCD inhibition of ATPase
activity is the most widely used method to evaluate the reactivity of the
crucial carboxylate with DCCD (see, e.g. Ref.
39). The extent of DCCD
inhibition is expected to correlate with access of the carbodiimide reagent to
the c-subunit carboxylate on helix-2, which in turn can be affected
by changes in c-subunit structure. Alternatively, mutations in helix
1 could cause structural changes that affect the pKa of
the carboxylate, resulting in altered reactivity with DCCD. The coupled ATPase
activity of the wild-type B. pseudofirmus OF4 enzyme was inhibited
20-24% by DCCD (24% in the case of the cA4G mutants,
Fig. 2D). The ATPase
of both quadruple mutants with the AXAXAXA changed
to GXGXGXG exhibited increased DCCD inhibition
relative to that of the wild-type enzyme (61-64% inhibition for both mutant
types, well over twice the sensitivity of wild type)
(Fig. 2D). The results
of the ATP synthase activity and DCCD inhibition of ATPase in the two
quadruple mutants were consistent with an important role of the
AXAX-AXA sequence for ATP synthase activity at pH
10.5 and with an impact of the quadruple mutation on accessibility of the
c-ring carboxylate to DCCD and/or its reactivity with DCCD.Mutants such as the two cA4G mutants that have major defects in
alkaline growth need to be assessed for their relative proton leakiness,
because a large pH gradient (more acid in the cytoplasm relative to the
outside medium) is required for robust alkaliphile growth at high pH
(21,
40). Our assay for this
property was to determine the effect of an alkaline shift in the outside pH on
the pH of the cytoplasm. Wild-type and quadruple mutant cells were grown on
glucose at pH 8.5 (because malate growth of cA4G-1 was poor), washed,
and equilibrated in pH 8.5 buffer and then shifted by dilution into malate and
sodium-containing buffer at pH 10.5. The cytoplasmic pH values 10 min after
the shift were 8.86 ± 0.04 for wild-type, 8.92 ± 0.01 for
cA4G-1, and 8.68 ± 0.03 for cA4G-2. This indicated
somewhat better pH homeostasis in cA4G-2 than in wild-type and
slightly worse pH homeostasis in cA4G-1 than in wild-type, but
neither mutant exhibited a large defect in pH homeostasis that would suggest
leakiness. We note that the glucose-grown wild-type cells in these experiments
showed a lower capacity for rigorous cytoplasmic pH homeostasis than is
observed with malate-grown wild-type cells; malate-grown wild-type cells
typically exhibit a cytoplasmic pH of 8.2-8.3 after such a shift
(40,
41) (see
“Discussion”).Analyses of PXXEXXP Mutants in Alkaliphile-specific Pro51,
the Carboxylate Glu54, and the Conserved Pro57 Show That
Changes Result in Diverse Defects in the Alkaliphile Setting—The
Pro51 that produces the
P51XXEXXP57 motif
(27) of extreme alkaliphiles
is important for OXPHOS at high pH. This conclusion is based on the poor
growth on malate and greatly reduced ATP synthase activity, at pH 10.5, of the
P51A mutant characterized in our initial mutagenesis study; alanine is the
residue found in the c-subunit 51-position in non-alkaliphilic
Bacillus species (Fig.
1) (25,
26). The first new mutant of
the PXXEXXP motif in this study again focused on
Pro51 of B. pseudofirmus OF4 and changed Pro51
to glycine. Glycine is present at the position equivalent to the
Pro51 of extreme alkaliphiles both in E. coli and in the
more moderately alkaliphilic and thermophilic Bacillus TA2.A1
(42)
(Fig. 1). Two types of
phenotypes were observed among the group of cP51G mutants that were
isolated. We chose strains that were representative of the two types and
designated them cP51G-1 and cP51G-2. Sequence analyses
showed that neither mutant had any mutations in the atp operon apart
from the change introduced in Pro51. Both mutant strains had
≥73% of the ATP synthase level and ≥78% of the OG-stimulated ATPase
activity of wild-type membranes (data not shown). cP51G-1 did not
exhibit significant growth deficits relative to wild-type on either glucose or
malate at either pH 7.5 or 10.5. By contrast, cP51G-2 also showed no
significant growth defect on either carbon source at pH 7.5 but exhibited a
large deficit in growth at pH 10.5 on both carbon sources, i.e.
exhibited a non-alkaliphilic phenotype. Growth of cP51G-2 was only
12-20% of wild-type at high pH (Fig.
3A). The ATP synthase activities of the two mutant types
did not correlate with their different growth phenotypes at pH 10.5, inasmuch
as cP51G-1 and cP51G-2 both had ≥80% of the wild-type ATP
synthase activity at pH 7.5 and had 69-76% of the wild-type activity at pH
10.5 (Fig. 3B).
Membrane vesicles of both of the mutant strains exhibited a >3.5-fold
increase in the unstimulated ATPase activity relative to wild type. Both
mutants also showed a >2-fold increase in the % DCCD inhibition of that
coupled ATPase activity, relative to wild type
(Fig. 3C). A highly
significant difference was observed between the two mutants and wild type in
pH shift experiments that were conducted on malate-grown cells. Wild type
exhibited a cytoplasmic pH of 8.3 10 min after a shift in external pH from 8.5
to 10.5, whereas the cytoplasmic pH values in the cP51G-1 and
cP51G-2 mutants were, respectively, 8.7 and 9.1
(Fig. 3D). These
assays also revealed the first difference between the two cP51G
mutant strains that could relate to their different growth phenotypes, because
the pH homeostasis defect of cP51G-2 was significantly greater than
that of cP51G-1 (Fig.
3D).The second mutation made in the PXXEXXP motif of B.
pseudofirmus OF4 was a change in the essential carboxylate,
Glu54, to aspartate. Replacement by Miller et al.
(43) of the corresponding
carboxylate residue, Asp61, with a glutamate in E. coli
was tolerated; the mutant retained ∼50% of the wild-type capacity for
growth on succinate. The cE54D mutant of B. pseudofirmus OF4
grew as well as wild type on glucose at either pH 7.5 or 10.5
(Fig. 4A, bottom) and
had wild-type levels of membrane ATP synthase β-subunit as well as
OG-stimulated ATPase activity (data not shown). However, the mutant had a
severe defect in malate medium, with growth yields of only 21 and 10% of
wild-type at pH 7.5 and 10.5, respectively
(Fig. 4A,
top). The ATP synthase activity assayed in vitro was not
significantly different from wild type at pH 7.5 despite the profound growth
defect on malate at this pH (Fig.
4B). At pH 10.5, the ATP synthase activity of the
cE54D mutant was 30% of the wild-type ATP synthase activity, thus
correlating better with the very poor growth of the mutant at pH 10.5
(Fig. 4B). Assays of
coupled ATPase activity showed that the activity in the cE54D mutant
in the absence of added DCCD was more than twice that found in wild type,
consistent with uncoupling. The inhibition by DCCD was ∼2.5 times that of
wild type (Fig. 4C).
Assessments of cytoplasmic pH homeostasis using the pH shift protocol had to
be conducted on glucose-grown cells because of the growth phenotype of the
mutant. The cytoplasmic pH 10 min after a pH 8.5→10.5 shift was 9.13
± 0.03 for the cE54D mutant compared with 8.86 ± 0.04
for the wild-type, suggestive of proton leakiness in the mutant that was
significantly greater than observed with the cA4G mutants under the
same conditions (Fig.
4D).Open in a separate windowFIGURE 4.Functional characterization of cE54D mutant. A,
growth of wild type and cE54D mutant as a function of pH and carbon
source. The values are the average of duplicate determinations from at least
two independent growth experiments, and the error bars show the
±S.D. B, ATP synthesis by ADP plus Pi-loaded RSO
membrane vesicles of wild type and the cE54D mutant. Vesicles were
prepared from cells grown with glucose at pH 10.5. Assays were conducted as
described under “Experimental Procedures.” The values are the
average of duplicate assays from at least three independent vesicle
preparations, and the error bars show the ±S.D. C,
DCCD inhibition of unstimulated ATPase activity of wild-type and the
cE54D mutant. Everted vesicles were prepared from strains grown on
glucose at pH 10.5. Values are the average of duplicate determinations from at
least two independent vesicle preparations, and the error bars show
the ±S.D. The numbers in parentheses above the gray
columns indicate the % DCCD inhibition. D, determination of the
cytoplasmic pH after a shift in the external pH from 8.5 to 10.5 of wild-type
and the cE54D mutant. Cells were grown with glucose at pH 8.5. Values
are the average of duplicate determinations from at least three independent
experiments, with the wild-type data set serving as the control for these
assays of cytoplasmic pH homeostasis as well as those of the cA4G
mutants. The error bars show the ±S.D.The second proline in the PXXEXXP motif is a conserved
proline, in both alkaliphiles and neutrophiles, three residues from
Glu54 on the C-terminal side. We constructed cP57A and
cP57G mutations to test the importance of this proline in the B.
pseudofirmus OF4 context. The cP57A mutant strain exhibited 76
and 73% of wild-type levels of enzyme and OG-stimulated ATPase activity,
respectively (Fig.
5A). The growth of cP57A on either glucose or
malate at either pH 7.5 or 10.5 was ≥64% of wild-type with only growth on
malate at pH 10.5 showing any deficit (Fig.
5B). Two types of cP57G mutants were identified
among the mutant isolates. Representative examples of the two types,
designated cP57G-1 and cP57G-2, were shown by sequencing to
have no mutations in the atp operon except for the cP57G
change. Both cP57G-1 and cP57G-2 had greatly reduced
membrane levels of the β-subunit of the ATP synthase and OG-stimulated
ATPase activity (Fig.
5A); the Western assays did not show a statistically
significant difference between the two mutants, but the ATPase activity of
cP57G-2 was a little lower than that of cP57G-1.
cP57G-1 grew as well as wild-type on glucose at pH 7.5 or 10.5, while
showing reduced malate growth, especially at pH 10.5
(Fig. 5B). The
phenotype of cP57G-2 was more extreme, in that it exhibited a growth
deficit on glucose as well as total absence of growth on malate (when
corrections were made for growth by the ΔF0 strain)
(Fig. 5B). ATP
synthase activity was assessed in the two cP57G mutants. Both mutants
showed similar results for ATP synthesis. cP57G-1 and
cP57G-2, respectively, exhibited 82 and 63% of wild-type activity at
pH 7.5, and 27 and 20% of wild-type at pH 10.5
(Fig. 5C). The results
indicated that the lower membrane levels of ATP synthase in the cP57G
relative to wild-type led to deficits in ATP synthase and malate growth that
were much larger at pH 10.5 than at pH 7.5. They were also larger in
cP57G-2 than in cP57G-1.Open in a separate windowFIGURE 5.Functional characterization of the cP57G and cP57A
mutants. A, β-subunit content and OG-stimulated ATPase
activity of mutant strains. Strains were grown with glucose at pH 10.5. The
values for the mutants are given as % of wild-type, with the wild-type set at
100%. Values are the average of determinations from at least two independent
vesicle preparations, and the error bars show the ±S.D.
B, growth of wild type, the two types of cP57G mutants, and
the cP57A mutant as a function of pH and carbon source. The values are the
average of duplicate determinations from at least two independent growth
experiments, and the error bars show the ±S.D. C. ATP
synthesis by ADP plus Pi-loaded RSO membrane vesicles of wild type
and two types of cP57G mutants. Vesicles were prepared from cells
grown on glucose at pH 10.5. Assays were conducted as described under
“Experimental Procedures.” The values are the average of duplicate
assays from at least three independent vesicle preparations, and the error
bars show the ±S.D.DISCUSSIONThe results presented here strongly support the importance of
alkaliphile-specific motifs for function of the ATP synthase
c-subunit in OXPHOS at high pH. At pH 10.5, the bulk proton-motive
force is much lower than at pH 7.5, because of the success with which
secondary antiporters maintain a cytoplasmic pH that is over two units lower
than the outside pH (21,
40,
44). We have long proposed
that sequestered proton translocation between proton-pumping respiratory chain
elements and the proton-consuming ATP synthase plays a critical role, perhaps
enhanced by high membrane levels of cardiolipin and an unusually high content
of amino acids with anionic side chains in external loops of many alkaliphile
membrane proteins (40).
Sequestration could be mediated by fast movement of protons along the membrane
surface as has been demonstrated in other systems
(45,
46) and by a “surface
proton-motive” force of greater magnitude than the bulk proton-motive
force (22,
47). It is further possible
that OXPHOS in some settings involves direct dynamic interactions between
respiratory chain complexes and the ATP synthase, as was recently shown for
alkaliphile cytochrome oxidase and ATP synthase in a reconstituted system
(48). This study extends
initial evidence that alkaliphile-specific motifs that are predicted to be
just outside the membrane surface
(26) or within the membrane
are adaptations of the ATP synthase machinery itself that are required for
OXPHOS in the alkaliphile context. These findings negate the suggestion that
properties of surface retention or rapid movement of protons can fully address
the bioenergetic challenge of OXPHOS by alkaliphiles at high pH without
adaptations in the synthase
(22,
47). The results here
demonstrate that the ATP synthase c-subunit is specially adapted to
achieve inward proton translocation that energizes ATP synthesis while
minimizing loss of protons from the rotor or cytoplasm to the alkaline bulk
phase.A major new finding is the demonstration that the alkaliphile-specific
AXAXAXA sequence of N-terminal helix-1 of the
c-subunit plays an indispensable role in ATP synthase at high pH and
hence is a functional motif. Replacement of only two of the four alanines with
glycines produced a significant deficit in non-fermentative growth on malate,
i.e. growth was 25% the wild-type level
(Fig. 2B). Replacement
of all the alanines with glycines resulted in ≥90% deficit in malate growth
and in ≥82% deficit in in vitro ATP synthase activity at high pH
relative to wild-type. Moreover, the findings suggest that the specific
residues that surround the alanines of the AXAXAXA
motif have functional impact since replacement of the four alanines as well as
the surrounding residues to those found in the B. megaterium
GXGXGXG motif led to reduced membrane levels of the
ATP synthase, but comparable reductions were not found in the cA4G
strains that had no changes in those additional residues
(Table 2).It is interesting to note that in studies by others of helix-1 of the
E. coli c-subunit, mutation of Ala24 (equivalent to
Gly17 in the alkaliphile) to serine or of Ile28
(equivalent to Val21 in the alkaliphile) to valine or threonine
increased resistance of the enzyme to DCCD
(17,
18). This contrasts with the
increased inhibition found here when mutations were introduced on a different
face of the helix, in a different frame of the neutrophile
GXGXGXG motif or alkaliphile
AXAXAXA. The GXGXGXG
motif that is present in non-alkaliphiles has been proposed by Vonck et
al. (8) to function in
accommodating the tight packing of the inner ring of N-terminal helices in the
c-ring of Ilyobacter tartaricus. In the alkaliphile setting,
changes of AXAXAXAto
GXGXGXG seemed to have “opened up” the
structure as indicated by the greater inhibition by DCCD
(Fig. 2D), if greater
accessibility of the carboxylate to DCCD accounts for this change. However, it
is possible that the structure of the quadruple mutants is still
“tight” and that the greater DCCD inhibition is caused by an
effect of the mutation on the pKa of the carboxylate that
increases its reactivity with DCCD. A change in the pKa
could perturb the protonation/deprotonation properties of the enzyme and thus
account for the poor synthesis. The cA4G strains show little or no
defect in pH shift experiments, consistent with retention of a relatively
tight structure at least as far as proton leakage is concerned. We hypothesize
that the less severe phenotype of cA4G-2 relative to cA4G-1
results from suppression by a change outside the atp operon. For
example, a modest increase in activity of a sodium-proton antiporter in
cA4G-2 could account for its slightly better pH homeostasis after a
shift to pH 10.5 and could also account for its better malate growth at pH
7.5, because a higher sodium motive force is needed to drive alkaliphile
transport systems that are coupled to sodium (e.g. malate transport)
at pH 7.5 than at pH 10.5 (21,
49).To the extent that the AXAXAXA motif has effects
on the packing of the c-subunits, it could be involved in supporting
formation of a c-ring that has a higher stoichiometry of
c-subunit monomers than that found in non-alkaliphilic
Bacillus species. A high c-subunit stoichiometry could be a
partial solution to the conundrum of robust alkaliphile OXPHOS at high pH
despite the lower bulk proton-motive force
(7,
50,
51). So far, however, elevated
c-subunit stoichiometries have been reported but were not associated
with AXAXAXA motifs. The tridecameric
c-ring of the moderate alkaliphile and thermophile Bacillus
sp. TA2.A1, which has a higher stoichiometry than that observed in several
non-alkaliphilic bacterial c-rings, has an unusual
GXSXGXS sequence in this position
(50). The role of that
N-terminal helix sequence in the c-subunit stoichiometry or in
synthase function at alkaline pH and/or elevated temperature has not yet been
reported for Bacillus TA2.A1. In two cyanobacteria,
Synechocystis PCC6803-14 and Spirulina
(Arthrospira) platensis, that have c-rotors with
even higher stoichiometries, c14 and
c15 rings, respectively, the c-subunit helix-1
motif has a GXGXGXG sequence
(7,
51). Therefore an elevated
c-subunit stoichiometry does not require the
AXAX-AXA sequence found in extreme alkaliphiles, at
least up to 15 monomers/ring. Thus, although direct evidence for or against an
effect of the quadruple mutation on c-subunit stoichiometry should be
sought, it seems more likely that the alkaliphile
AXAXAXA motif functions in some other way,
e.g. by influencing the pKa of the carboxylate on
the opposing helix.The second major finding of the current study is that mutational changes in
both the Pro51 and the Glu54 of the alkaliphile
PXXEXXP motif to residues that support functional ATP
synthases in non-alkaliphiles are not compatible with function of the
alkaliphile ATP synthase at high pH. The two types of cP51G mutants
showed a unique pattern among alkaliphile ATP synthase mutants studied to
date. The ATP synthase capacity of the two strains, in assays of ATP synthesis
in ADP plus Pi-loaded vesicles, was not severely compromised in
either cP51G-1 or cP51G-2
(Fig. 3B). One of the
mutant types, cP51G-1, also lacked major deficits in growth on both
malate and glucose relative to wild-type, although malate growth, especially
at pH 10.5, was much more variable than typically observed. By contrast, the
growth phenotype of the other mutant type, c-P51G-2, was severe at pH
10.5 on both glucose and malate, where the growth yields were 20 and 12%,
respectively, of wild-type yields (Fig.
3A). The ATPase activity in the absence of OG was greatly
increased in both cP51G mutant strains relative to wild type
(Fig. 3C), suggesting
that the ATPase was to some extent uncoupled in both mutants. This was
consistent with the proton leakiness evidenced by the large alkalinization (to
8.7 and 9.1) of the cytoplasm of both cP51G mutant strains after a
shift in the external pH from 8.5 to 10.5; the wild-type cytoplasmic pH was
8.3 after the shift (Fig.
3D). A slightly larger unstimulated ATPase activity and
cytoplasmic alkalinization were observed with cP51G-2 than with
cP51G-1, but the differences seem too small to account for the much
more severe phenotype of cP51G-2, especially at pH 10.5
(Fig. 3A). The effects
of the mutation that were observed in the cP51G-2 strain may be
suppressed by a change outside of the atp operon that ameliorates
those effects in cP51G-1. As with the cA4G mutants, it will
be of interest to identify the mechanism of such suppression, for which a
change in the antiporter activity profile would again be a candidate.Mutation of c-subunit Glu54 to aspartate yielded a
single mutant type whose growth on glucose was comparable to wild type at both
pH 7.5 and 10.5, but showed a large defect in malate growth at both pH values,
with a greater deficit at pH 10.5 than at pH 7.5
(Fig. 4A). The
cE54D mutant of the alkaliphile showed a larger deficit in
non-fermentative growth (10-21% of wild type) than the 50% reported for a
cD61E mutant in the carboxylate of the E. coli c-subunit
(43). The ATP synthase
activity of the alkaliphile cE54D mutant was also greatly reduced at
pH 10.5 relative to wild type (Fig.
4B). The cE54D mutant had a large increase in
unstimulated ATPase activity (that was accompanied by increased DCCD
inhibition) (Fig. 4C)
and also showed significant alkalinization of the cytoplasm in a pH shift
experiment (Fig. 4D)
relative to wild type. Such proton leakiness would be highly detrimental to
non-fermentative growth, especially at pH 10.5. It is possible that the change
in the pKa of the carboxylate also contributes to the
deficit in ATP synthesis and non-fermentative growth, in view of the high
pKa measured for the wild-type carboxylate
(23). In sum, the findings
with the cP51G and cE54D mutant strains underscore the role
of alkaliphile-specific features of the PXXEXXP motif in
protecting the native host from cytoplasmic proton loss. As with the helix-1
AXAXAXA mutations, more structural-functional
information on the alkaliphile c-ring will be required before we
fully understand all the current observations on the PXXEXXP
motif.The other new mutant in this study was in the conserved Pro57 of
the PXXEXXP motif. Mutation of this residue to alanine did
not produce a severe growth phenotype, whereas mutation to glycine produced
two mutant types with respect to the severity of their growth phenotypes. Both
types, represented by cP57G-1 and cP57G-2, exhibited major
defects in the ATP synthase content and hence also had deficits in the
OG-stimulated ATPase activity and in malate growth. Although the levels of
these parameters were only slightly lower in cP57G-2 than in
cP57G-1 (Fig. 5, A and
C), the former strain did not grow on malate at all and
also showed a modest growth deficit on glucose, whereas cP57G-1
showed no growth deficit on glucose and a smaller one on malate
(Fig. 5B). Because no
differences between the two mutants were found within the atp operon,
suppression of the more severe phenotype of cP51G-1 may involve a
change elsewhere. Clearly, though, the conserved Pro57 can be
changed to either alanine or glycine without complete loss of ATP synthase
activity.In this study, we have used both glucose-grown and malate-grown cells for
pH shift experiments in which we assessed effects of mutations on cytoplasmic
pH homeostasis as an indicator of proton leakiness in ATP synthase
F0 mutants. Glucose-grown cells had to be used for mutants
that could not grow well on malate even at pH 8.5, i.e. the
cA4G mutants and the cE54D mutant. We note that 10 min after
a shift of glucose-grown cells of wild-type from the pH 8.5 equilibration
buffer to the pH 10.5 buffer, the cytoplasmic pH was pH 8.86, whereas that of
malate-grown cells was pH 8.27. Glucose-grown cells have lower levels of
respiratory chain components and ATP synthase than malate-grown
cells.4 This probably
accounts for the reduced capacity for pH homeostasis following a sudden upward
pH shift. It is also likely that proton capture during OXPHOS plays a role in
pH homeostasis under alkaline conditions
(21). Up-regulation of ATP
synthase genes under alkali stress has been observed in E. coli
(52), Bacillus
subtilis (53), and
Desulfovibrio vulgaris
(54).Supplementary Material
[Supplemental Data]
Click here to view.
NotesThe nucleotide sequence(s) reported in this paper has been submitted to
the GenBank™/EBI Data Bank with accession number(s).*This work was supported, in whole or in part, by National
Institutes of Health Grant
GM28454 (to T. A. K.). The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The on-line version of this article (available at
http://www.jbc.org)
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研究员----中国科学院天津工业生物技术研究所
研究员----中国科学院天津工业生物技术研究所
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研究员
姓名
刘君
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liu_jun@tib.cas.cn
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天津空港经济区西七道32号
邮编
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简历
1994.9-1998.6, 南开大学, 微生物学专业, 学士学位
1999.9-2002.6, 南开大学, 生物化学和分子生物学专业，硕士学位
2002.9-2006.1, 中国科学院微生物研究所, 微生物学专业，博士学位
2006.3-2010.6，美国西奈山医学院，博士后
2010.7-2013.12, 美国西奈山医学院, 研究助理教授
2014.2-目前，中国科学院天津工业生物技术研究所，研究员
研究方向：
研究组定位于重要工业微生物的应用基础研究,围绕微生物生理和代谢工程开展以下研究:
1、系统解析重要工业微生物对环境胁迫的生理机制，在此基础上构建具有抗逆性能的工业菌种。
2、阐明重要工业微生物合成特定代谢产物的分子基础和调控机制，并利用代谢工程和系统生物学等技术实现工业菌种的遗传改造。
代表论著：
[1] Wang X, Yang H, Zhou W, Liu J, Xu N.2019. Deletion of cg1360 affects ATP synthase function and enhances the production of L-valine in Corynebacterium glutamicum. J Microbiol Biotechnol. Jul 30. doi: 10.4014/jmb.1904.04019.
[2] Wei L, Wang Q, Xu N, Cheng J, Zhou W, Han GQ, Jiang HF*, Liu J*, Ma YH.2019. High-level o-acetylhomoserine production in Escherichia coli through protein and metabolic engineering. ACS Synthetic Biology, 8(5):1153-1167.
[3] Xu N, Wei L, Liu J*. 2019. Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis. World J Microbiol Biotechnol, 35: 33. (Invited review)
[4] Wu Z, Wang J, Liu J, Wang Y, Bi C, Zhang X.2019. Engineering an electroactive Escherichia coli for the microbial electrosynthesis of succinate from glucose and CO2. Microb Cell Fact. 18(1):15. doi: 10.1186/s12934-019-1067-3.
[5] Xu N#, Lv HF#, Wei L, Ju JS, Liu J*, Ma YH. 2019. Impaired oxidative stress and sulfur assimilation contribute to acid tolerance of Corynebacterium glutamicum. Appl Microbiol Biotech, doi: 10.1007/s00253-018-09585-y.
[6] Wei L#, Wang H#, Xu N, Zhou W, Ju JS*, Liu J*, Ma YH. 2019. Metabolic engineering of Corynebacterium glutamicum for L-cysteine production. Appl Microbiol Biotech, doi: 10.1007/s00253-018-9547-7.
[7] Xu N, Zheng YY, Wang XC, Krulwich TA, Ma YH, Liu J*. 2018. The lysine 299 residue endows the multisubunit Mrp1 antiporter with dominant roles in Na+-resistance and pH homeostasis in Corynebacterium glutamicum. Appl Environ Microbiol, 84: e00110-18.
[8] Vaish M, Price-Whelan A, Reyes-Robles T, Liu J, Jereen A, Christie S, Alonzo F 3rd, Benson MA, Torres VJ, Krulwich TA.2018. Roles of Staphylococcus aureus Mnh1 and Mnh2 Antiporters in Salt Tolerance, Alkali Tolerance, and Pathogenesis. J Bacteriol.200(5). pii: e00611-17. doi: 10.1128/JB.00611-17.
[9] Wei L#, Xu N#, Cheng HJ, Wang YR, Han GQ, Ma YH, Liu J*. 2018. Promoter library-based module-combination (PLMC) technology for optimization of threonine biosynthesis in Corynebacterium glutamicum. Appl Microbiol Biotech, 102: 4117-30.
[10] Xu N, Wei L, Liu J*. 2017. Biotechnological advances and perspectives of gamma-aminobutyric acid production. World J Microbiol Biotechnol, 33: 64. (Invited review)
[11] Liu QD, Ma XQ, Cheng HJ, Xu N, Liu J*, Ma YH. 2017. Co-expression of L-glutamate oxidase and catalase in Escherichia coli to produce alpha-ketoglutaric acid by whole-cell biocatalyst. Biotechnol Lett, 39 (6):913-9.
[12] Xu N, Wang L, Cheng Hj, Liu Qd, Liu J*, Ma YH. 2016. In vitro functional characterization of the Na+/H+ antiporters in Corynebacterium glutamicum. FEMS Microbiol Lett, 363: fnv237.
[13] Liu QD, Cheng H, Ma X, Xu N，Liu J*, Ma YH 2016. Expression, characterization and mutagenesis of a novel glutamate decarboxylase from Bacillus megaterium. Biotechnol Lett, 38(7): 1107-13.
[14] Preiss L, Langer JD, Hicks DB, Liu J, Yildiz O, Krulwich TA, Meier T. 2014. The c-ring ion binding site of the ATP synthase from Bacillus pseudofirmus？OF4 is adapted to alkaliphilic lifestyle. Mol Microbiol. 92(5):973-84.
[15] Liu J, Ryabichko S, Bogdanov M, Fackelmayer OJ, Dowhan W, Krulwich TA. 2014. Cardiolipin is dispensable for oxidative phosphorylation and non-fermentative growth of alkaliphilic Bacillus pseudofirmus OF4. J Biol Chem. 289(5):2960-71.
[16] Preiss L, Klyszejko AL, Hicks DB, Liu J, Fackelmayer OJ, Yildiz ？, Krulwich TA, Meier T. 2013. The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4. Proc. Natl. Acad. Sci. USA. 110(19):7874-9.
[17] Liu J, Hicks DB, Krulwich TA. 2013. Roles of AtpI and two YidC-type proteins from alkaliphilc Bacillus pseudofirmus OF4 in ATP synthase assembly and non-fermentative growth. J. Bacteriol. 195(2): 220-30.
[18] Janto B, Ahmed A, Ito M, Liu J et al. 2011. The genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4. Environmental Microbiology. 13(12): 3289-3309.
[19] Liu J, Fackelmayer OJ, Hicks DB, Preiss L, Meier T, Sobie EA, Krulwich TA. 2011. Mutations in a helix-1 motif of the ATP synthase c-subunit of Bacillus pseudofirmus OF4 cause functional deficits and changes in the c-ring stability and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry. 50(24):5497-5506.
[20] Fujisawa M, Fackelmayer OJ, Liu J, Krulwich TA, Hicks DB. 2010. The ATP synthase a-subunit of extreme alkaliphiles is a distinct variant: mutations in the critical alkaliphile- specific residue Lys-180 and other residues that support alkaliphile oxidative phosphorylation. J. Biol. Chem. 15; 285(42):32105-15.
[21] Hicks DB, Liu J, Fujisawa M, Krulwich TA. 2010. F1Fo-ATP synthases of alkaliphilic bacteria: lessons from their adaptations. Biochim. Biophys. Acta. 1797(8):1362-1377.
[22] Guo Y, Xue Y, Liu J, Wang Q, Ma Y. 2009. Characterization and function analysis of a Halo-alkaline-adaptable Trk K+ uptake system in Alkalimonas amylolytica strain N10. Sci China C Life Sci. 52(10):949-57.
[23] Liu J, Fujisawa M, Hicks DB, Krulwich TA. 2009. Characterization of the functionally critical AXAXAXA and PXXEXXP motifs of the ATP synthase c-subunit from an alkaliphilic Bacillus. J. Biol. Chem. 284(13):8714-25.
[24] Liu J, Krulwich TA, Hicks DB. 2008. Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4. Biochim. Biophys. Acta. 1777(5):453-61.
[25] Wei Y, Liu J, Ma Y, Krulwich TA. 2007. Three putative cation/proton antiporters from the soda lake alkaliphile Alkalimonas amylolytica N10 complement an alkali-sensitive Escherichia coli mutant. Microbiology. 153:2168-2179.
[26] Yuan S, Ren P, Liu J, Xue Y, Ma Y, Zhou P. 2007. Lentibacillus halodurans sp. nov., a moderately halophilic bacterium isolated from a salt lake in Xin-Jiang, China. Int. J. Syst. Evol. Microbiol. 57(3):485-488.
[27] Wang N, Zhang Y, Wang Q, Liu J, Wang H, Xue Y, Ma Y. 2006. Gene cloning and characterization of a novel α-amylase from alkaliphilic Alkalimonas amylolytica. Biotechnol. J. 1(11):1258-65.
[28] Liu J, Xue Y, Wang Q, Wei Y, Swartz TH, Hicks DB, Ito M, Ma Y, Krulwich TA. 2005. The activity profile of the NhaD-type Na+(Li+)/H+ antiporter from the soda lake haloalkaliphile Alkalimonas amylolytica is adaptive for the extreme environment. J. Bacteriol. 187(22):7589-95.
Book Chapter
[29] Krulwich TA, Liu J, Morino M, Fujisawa M, Ito M, Hicks DB, 2010. Adaptive mechanisms of extreme alkaliphiles. In: Extremophiles Handbook. Horikoshi K, Antranikian G, Bull A, Robb F, Stetter K (eds), Springer, Heidelberg,PP. 120-139.
承担科研项目情况：
研究组承担国家合成生物学重点专项2项,中科院重点部署项目1项, 国家自然科学基金面上项目1项,青年基金3项,天津市自然科学基金2项。
获奖及荣誉：