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xaxa是什么意思? - 知乎

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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Jessica_Batterton_Cr

2021年5月21日

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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?

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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

 

人才队伍

概况

研究员

兼职研究员

产业研究员

副研究员、高级工程师

 

您现在的位置：首页 > 中文 > 人才库 > 研究员

 

研究员

 

姓名  

刘君

性别  

男

专家类别  

N/A

职称  

研究员

学历  

博士研究生

电话  

N/A

传真  

N/A

电子邮件  

liu_jun@tib.cas.cn

地址  

天津空港经济区西七道32号

邮编  

300308

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：