review article bioenergetics and the role of soluble...

Review ArticleBioenergetics and the Role of SolubleCytochromes c for Alkaline Adaptation in Gram-NegativeAlkaliphilic Pseudomonas

T Matsuno12 and I Yumoto12

1Laboratory of Environmental Microbiology Graduate School of Agriculture Hokkaido University Kita-ku Sapporo 060-8589 Japan2Bioproduction Research Institute National Institute of Advanced Industrial Science and Technology (AIST)2-17-2-1 Tsukisamu-Higashi Toyohira-ku Sapporo 062-8517 Japan

Correspondence should be addressed to I Yumoto iyumotoaistgojp

Received 25 April 2014 Revised 27 November 2014 Accepted 29 November 2014

Academic Editor Stanley Brul

Copyright copy 2015 T Matsuno and I Yumoto This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Very few studies have been conducted on alkaline adaptation of Gram-negative alkaliphiles The reversed difference of H+concentration across themembrane will make energy production considerably difficult for Gram-negative as well as Gram-positivebacteria Cells of the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21119879 grown at pH 10 under low-aerationintensity have a soluble cytochrome c content that is 36-fold higher than that of the cells grown at pH 7 under high-aerationintensity Cytochrome c-552 content was higher (64 in all soluble cytochromes c) than those of cytochrome c-554 and cytochromec-551 In the cytochrome c-552-dificient mutant grown at pH 10 under low-aeration intensity showed a marked decrease in 120583max[hminus1] (40) andmaximum cell turbidity (25) relative to those of the wild type Considering the high electron-retaining abilities ofthe three soluble cytochromes c the deteriorations in the growth of the cytochrome c-552-deficient mutant could be caused by thesoluble cytochromes c acting as electron storages in the periplasmic space of the bacteriumThese electron-retaining cytochromescmay play a role as electron and H+ condenser which facilitate terminal oxidation at high pH under air-limited conditions whichis difficult to respire owing to less oxygen and less H+

1 Introduction

Microorganisms are widely distributed in various environ-ments in nature such as in cold regions in Antarctica in areasof high temperature and pressure such as in black smokerchimneys in the deep sea and in environments with highosmotic pressure such as the Dead Sea These microorgan-isms living in extreme environments are called extremophiles[1] Extremophiles have provided us with enzymes that canwork under corresponding growth conditions [2] Amongsuch extremophiles there are microorganisms living at highpH such as above pH 10 [3ndash9] Alkaliphilic Bacillus specieshave been isolated for the investigation of their physiologicalfunction of adaptation to high pHs and for the utilizationof their enzymes [3] While most extremophiles are livingin specific environments for example psychrophiles are

living in permanently cold environments such as the polarregions alkaliphiles that is alkaliphilic Bacillus species arewildly distributed not only in specific environments relatedto alkaline conditions such as alkaline soda lakes indigofermentation fluid and the intestinal tracts of termites butalso in environments not related to alkaline conditions suchas ordinary soil deep sea and seawater [5 7] This prob-ably indicates that there are very small niches of alkalinitydistributed in ordinary environments It is also consideredthat the evolutionary adaptation is easier from neutralophilesto alkaliphiles than from mesophiles to thermophiles It isexpected that whole enzyme systems should change frommesophilic enzymes to thermophilic enzymes for the evolu-tionary change from mesophiles to thermophiles

Alkaliphilic Bacillus species have been isolated and stud-ied for their environmental adaptation as typical alkaliphiles

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 847945 14 pageshttpdxdoiorg1011552015847945

2 BioMed Research International

[5 7] The physiology of alkaline adaptation has been exten-sively studied on alkaliphilic species [4 6 8 9] Firstly oneof the most important strategies for alkaline adaptation isprotection from alkalinity outside of their cells It has beenreported that cells of Bacillus halodurans C-125 produceacidic substances such as teichuronopeptide and teichuronicacid on their surface [10ndash12] On the other hand cells ofBacillus pseudofirmus OF4 produce the cell surface proteinSlpA and polyglutamic acid on their surface [13] These areacidic substances that attract H+ and repel OHminus in the extra-cellular environment Protons (H+) are usually used in solutetransport systems and torque for flagella rotation AlkaliphilicBacillus species adopt strategies for saving H+ usage forsolute transport systems and torque for flagella rotationTheyemploy sodium ion solute uptake and the flagella rotationsystemThe above-mentioned alkaline avoidance barrier andH+ usage system are not well investigated in Gram-negativealkaliphiles

In addition to the avoidance of a harmful extracellularenvironment and lack of the necessary ions for solute trans-port and flagella rotation bioenergetics is one of the mostfundamental issues for survival in alkaline environments[4 6 9 14 15] It is considered that H+-deficient conditionshave an extremely negative impact on energy production inmicroorganisms because F

1F0-ATP synthase is driven by H+

as the torque According to Peter Michellrsquos chemiosmotictheory the H+ motive force (Δ119901) for driving F

1F0-ATP

synthase consists of a transmembrane pH gradient (ΔpH)(intracellular pH minus extracellular pH) and a membraneelectrical potential (Δ120595) (minus inside + outside) [16] Fromthe calculation of medium pH as the outer membrane pHATP production is impossible theoretically It is predictedthat alkaliphiles possesses mechanisms that compensate forthe negative factor that is H+-deficient condition for energyproduction

Although numbers of reported Gram-negative alka-liphiles are less than those of Gram-positive alkaliphilesboth types of alkaliphiles are distributed in soda lakes [7]Therefore it is very important to consider the bioenergeticproblems in Gram-negative alkaliphiles to elucidate thepossible environmental adaptation mechanisms that mighthave led to these alkaliphiles to possess a different type ofcell surface membrane structure It can be recognized thatthe lack of transmembrane pH gradient makes energy pro-duction difficult for not only the Gram-positive but also theGram-negative bacteria Although there are many reports onthe environmental adaptation of the Gram-positive bacteriathose of the Gram negative bacteria are very few In additionseveral reviews on the bioenergetics of alkaliphilic Bacillusspecies have been published [4 6 9 14 15] These includerespiratory components and bioenergetics parameters andthe relationship among them However there has been noreview paper on bioenergetics in Gram-negative alkaliphilicbacteria Gram-negative bacteria possess an outer membraneand a periplasmic space These structures do not exist inGram-positive bacteria The periplasmic space is betweenthe cytoplasmic membrane and the outer membrane It hasbeen reported that the periplasmic space comprises 40 of

the total volume of the cells and it includes solutes and pro-teins that are involved in a wide variety of functions rangingbetween nutrient binding transport electron transport andalteration of substances toxic to the cell [17] Therefore itis expected that the periplasmic space plays an importantrole not only in the protection against an extracellularalkaline environments but also in retaining key proteins forbioenergetics adaptation to alkaline environments In thisreview we discuss the characteristics and physiological func-tions of soluble cytochromes c in the periplasmic space foradaptation to alkaline environments Furthermore we alsodiscuss the differences between Gram-negative and Gram-positive alkaliphiles from the bioenergetics viewpoints

2 Alkaliphilic Pseudomonas

Gram-negative alkaliphilic bacteria are less well known thanGram-positive ones It is probably because most colonieson standard alkaline-containing media such as Horikoshirsquosmedium are identified as Bacillus species when we try toisolate alkaliphilic bacteria from terrestrial environmentssuch as soils and manure [5 7] However if we try toisolate alkaliphiles from aquatic environments or particularsubstances such as polychlorinated biphenyl (PCB)- [18 19]or hydrocarbon- [20] containing media the possibility ofisolating alkaliphiles other than Bacillus species such asPseudomonas species becomes higher Pseudomonas speciesare widely distributed in nature such as soils freshwaterseawater and terrestrial and marine animals and plants [21]Some Pseudomonas species are able to survive in organicsolvents such as toluene [22 23] It is considered that Pseu-domonas species are a metabolically and genetically diversebacterial group Their functions are related to the circulationof carbons and nitrogen on Earth by decomposing vari-ous organic substances and reducing nitrogen compoundsOn the other hand some of the Pseudomonas species arepathogenic to humans plants and fish Considering theirenvironmental distribution and metabolic and genetic diver-sity it is no wonder that numerous alkaliphilic Pseudomonasspecies exist in various taxonomic groups It has been knownthat Pseudomonas pseudoalcaligenes can grow on media withhigh pH [18 19] We have isolated Pseudomonas alcaliphilaAL15-21T from seawater obtained from the coast of RumoiHokkaido Japan by using a general medium for the isolationof alkaliphiles which contains peptone yeast extract and ametal mixture [24] A scanning electron microscopy imageof the cells of platinumpalladium-coated P alcaliphilaAL15-21

T grown at pH 10 shows a rough surface whereas the cellsgrown at pH 7 show a smooth surface (Figure 1) We havealso isolated Pseudomonas toyotomiensis from a surface soilimmersed in hydrocarbon-containing hot spring water usinga medium containing hydrocarbon as the sole carbon source[20] P pseudoalcaligenes P alcaliphila and P toyotomiensisare on the same node in the phylogenetic tree based on the16S rRNA gene sequence (Figure 2) Pseudomonasmendocinaand Pseudomonas oleovorans are also located on the samenodewith these alkaliphilicPseudomonas species and as far aswe have examined they alsowere able to grow at high pH (pH

BioMed Research International 3

(a) (b)

Figure 1 Scanning electron microscopy image of cells of platinumpalladium-coated P alcaliphilaAL15-21T grown (a) at pH 10 showing therough surface of the cells and (b) at pH 7 showing the smooth surface of the cells Bar 180 120583m

001

704539

786

525

781973

999

969690 663

796857

604907

521

879630

Cellvibrio ostraviensis LMG 19434T (AJ493583)

Pseudomonas putida DSM 291T (Z76667)

Pseudomonas fragi IFO 3458T (AB021413)

Pseudomonas cichorii LMG 2162T (Z76658)

Pseudomonas viridiflava LMG 2352T (Z76671)

Pseudomonas balearica DSM 6083T (U26418)

Pseudomonas stutzeri ATCC 17587T (U25431)

Pseudomonas chloritidismutans DSM 13592T (AY017341)

Pseudomonas xanthomarina KMM 1447T (AB176954)

Pseudomonas agarici LMG 2112T (Z76652)

Pseudomonas aeruginosa LMG 1242T (Z76651)

Pseudomonas citronellolis NBRC 103043T (AB681923)

Pseudomonas nitroreducens IAM 1439T (D84021)

Pseudomonas multiresinivorans NBRC 103160T (AB681968)

Pseudomonas alcaligenes LMG 1224T (Z76653)

Pseudomonas resinovorans ATCC 14235T (AB021373)

Pseudomonas otitidis MCC 10330T (AY953147)

Pseudomonas tuomuerensis 78-123T (DQ868767)

Pseudomonas toyotomiensis HT-3T (AB453701)Pseudomonas alcaliphila AL15-21T (AB030583)

Pseudomonas oleovorans subsp lubricantis ATCC BAA-1494T (DQ842018)Pseudomonas pseudoalcaligenes LMG 1225

T (Z76666)

Pseudomonas oleovorans DSM 1045T (Z76665)

Pseudomonas indoloxydans IPL-1T (DQ916277)

Pseudomonas mendocina LMG 1223T (Z76664)

Figure 2 Neighbour-joining phylogenetic tree derived from 16S rRNA gene sequence data showing the position of P alcaliphila AL15-21Tand other related Pseudomonas species Numbers at branch points are bootstrap percentages based on 1000 replicates Bar 001 changes pernucleotide position The red bracket indicates that Pseudomonas species can grow at pH 10

10) Therefore it is considered that this phylogenetic groupbelongs to alkaliphiles Nine strains of alkaliphiles belongingto the genus Pseudomonas were isolated from a Hungariansoda lake [25] They are closely related to P alcaliphila and Ppseudoalcaligenes with 16S rRNA gene sequence similaritieshigher than 98 Recently a novel alkaliphilic Pseudomonas

species ldquoPseudomonas yangmingensisrdquo has been isolatedfrom a sample of soil mixed with water from a hot sulfurspring in Taiwan [26]This strain does not belong to the nodethat includes P alcaliphila This suggests that the possibilitythat undiscovered alkaliphilic Pseudomonas species still existin nature


3 Bioenergetic Background

31 Chemiosmotic Theory and Growth There is less informa-tion on alkaliphilic Pseudomonas species than on alkaliphilicBacillus species First wewould like to describe the elucidatedbioenergetics mechanisms in alkaliphilic Bacillus species asbackground references on Pseudomonas speciesThe effect ofgrowth pH on the growth characteristics and bioenergeticsparameters has been studied in the steady state under pH-controlled culture at various pHs Generation times of 54and 38min were obtained at medium pHs controlled at75 and 106 respectively in the facultatively alkaliphilicbacterium Bacillus pseudofirmus OF4 [27] The obligatealkaliphilic bacterium Bacillus clarkii strain K24-IU exhibitsa higher growth rate (120583max = 033 hminus1) than the neutralophilicbacterium Bacillus subtilis IAM 1026 (120583max = 026 hminus1) inbatch culture [28]These growth characteristics of alkaliphilicBacillus species contradict the deficiency of bioenergeticsparameter owing to the reversed transmembrane pHgradientin alkaliphilic Bacillus in alkaline conditions

It is considered that F1F0-ATP synthase produces ATP

energized by Δ119901 across the cell membrane [16] Accordingto the formula in Peter Mitchellrsquos chemiosmotic theory Δ119901production derived based on the Gibbs free energy can becalculated as

Δ119901 = Δ120595 minus 119885ΔpH 119885 =23119877119879

119865= 59 (1)

where Δ120595 is the electrical potential across the membrane(minus inside + outside) and ΔpH is the transmembrane pHgradient (intracellular pHminus extracellular pH)The value119885 = 59 can be calculated as 23 (coefficient for exchange fromnatural legalism to common legalism) times gas constant (119877 =8315 Jmol) times absolute temperature (298K = 25∘C)Faradayconstant (119865 = 96485 kj[vsdotmol]) Although the Δ120595 valuesof alkaliphilic Bacillus species are relatively higher thanthose of neutralophilic bacteria they cannot compensate forthe reversed ΔpH for ATP production theoretically if themedium pH is regarded as the extracellular pH [4 6 15]Therefore if we consider the medium pH as the extracellularpH it is impossible to produce ATP by the theoreticallycalculated Δ119901 Therefore it is expected that the pH of theouter surface of the membrane is lower than the mediumpH The evidence proved that the same alkaliphilic Bacillusspecies exhibit a higher growth rate and extent even duringgrowth at pH 10 as described above [29]

32 Proton Behavior at Outer Surface of Membrane underNeutralophilic Condition It seems that the H+ transfer alongthe outer surface of the membrane under high pH is difficultowing to the predicted diffusion of H+ attracted by theforce from the H+-deficient bulk environment In the neu-tralophilic environment the rapid movement of translocatedH+ by the respiratory chain or bacteriorhodopsin to thechannel gate of F

1F0-ATP synthase on the outer surface of

the membrane has been reported In most experiments therapid transfer of H+ on the outer surface of the cellular mem-brane has been demonstrated using a fluoresceine indicatorparticularly attached to the cysteine residue in the native

protein [30 31] The H+ transfer on the outer surface ofthe membrane was enhanced by the existence of a proton-collecting antenna consisting of carboxylate and histidineresidues [32 33] On the other hand in a report the H+attraction at the outer surface of the membrane is accountedfor by the H+-attracting force owing to the negative charge ofa membrane phospholipid [34] As reported previously evenin the neutralophilic condition H+ transfer along the outersurface of themembrane is clearly a fundamental mechanismfor the effective production of energy Therefore it must notbe true that the pH at the outer surface of the membrane isthe same as that of the medium It has been reported thatalkaliphilic Bacillus species generate a larger transmembraneelectrical gradient (Δ120595 = minus180 sim minus210mV) [4 6 15] thanthe neutralophilic Bacillus subtilis (Δ120595 = minus122 sim minus130mV)[35 36] Although it has been considered that Δ119901 calculatedfrom themedium pH (Δ119901 = caminus40) is not sufficient for ATPproduction the large Δ120595may play a role in compensating forthe adverse H+ concentration in the environment

33 Behavior of TranslocatedH+ in Alkaliphilic Bacillus PeterMitchell (1968) advocated the H+ well concept that accountsfor the bioenergetics equivalence of the chemical (ΔpH) andelectrical (Δ120595) components of the H+ motive force (Δ119901) TheH+ well was defined as an H+-conducting crevice passingdown into the membrane dielectric and able to accumulateH+ in response to the generation of either Δ120595 or ΔpH TheH+ well concept implies that an H+ in such a well wouldsense both the surface pH changes and the changes in Δ120595The outer surface of the membrane senses the changes in Δ120595andor in surface pH and would drive H+ either inside oroutside the well Mulkidjanian advocated that the H+ wellis constructed on the basis of the desolvation penalty fortransferring H+ into the membrane core [37] Retardationof the H+ translocated by the respiratory chain on the outersurface of themembrane was demonstrated by B clarkiiK24-1U grown at pH 10 In addition the retardation of H+ at theouter surface of membrane was disrupted by valinomycinThis indicates that there is possibility that anH+ trap occurredbecause H+ on the outer surface of the membrane senses thepresence of Δ120595 [29]

4 Electron Transfer Coupled to H+

Transfer in Cytochrome c

It has been considered that cytochrome c is solely an electrontransfer protein H+-coupled electron transfer in cytochromec has been demonstrated by Murgida and Hildebrandt [38]The electron transfer dynamics of horse heart cytochromec of self-assembled monolayers (SAM) adsorbed on an Agelectrode formed by different 120596-carboxyl alkanethiols ofdifferent chain lengths (C

2ndashC16) was monitored by changing

the electrode potential The electron transfer rate betweenthe cytochrome c and the Ag electrode was slower whenD2O (33 sminus1) rather than H

2O (132 sminus1) and was used as

the aqueous solution when the 120596-carboxyl alkanethiol chainlength was short (C

2 distance between the cytochrome c and

the electrode 63 A) On the other hand there is no difference


in the electron transfer rate (0073ndash0074 sminus1) regardless ofwhether D

2O or H

2O was used as the aqueous solution

when the 120596-carboxyl alkanethiol chain length was long (C16

24 A) It is considered that theH+-exchange-coupled electrontransfer may be a rate-limiting step only if the velocityof electron transfer between the cytochrome c and the Agelectrode is high This phenomenon has not been observedfor cytochrome c in solution Therefore it is consideredthat the H+ transfer rate between the cytochrome c and theaqueous solution is affected by the change in the electricfield when the electron was taken in and out during theelectron-transfer-coupledH+ transferThis suggested that theH+ transfer rate between the cytochrome c and the aqueoussolution is affected by the change in the transmembranepotential (Δ120595 andor ΔpH) during the electron-transfer-coupled H+ transfer in biological membranes

5 Bioenergetics and Function ofthe Cytochrome c in Alkaliphilic Bacillus

Alkaliphilic Bacillus spp have several strategies for cuttingcorners of H+ usage in several functions such as solutetransport and torque of flagella rotation by using Na+ [6]However they use H+ as the torque of F

1F0-ATP synthase

rotation Therefore they may have several strategies in ordernot to waste the precious H+ across the membrane TheH+ transfer or recycle event will happen in the vicinity ofmembrane as mentioned above Cytochrome c may occur atan important function for the efficient use ofH+ at the vicinityof outer surface membrane On the other hand alkaliphilicBacillus spp possess larger Δ120595 This suggests that Δ120595 hasimportant functions including the compensation of deficientΔpH The presence of cytochrome c has been consideredimportant in alkaliphilic Bacillus species for adaptationto alkaline environments Mutants of obligate alkaliphileswhich cannot grow under alkaline condition exhibit a muchlower cytochrome c content than the wild type strain [39]Facultative alkaliphiles such as Bacillus pseudofirmus OF4and Bacillus cohnii YN-2000 exhibit a higher cytochrome ccontent when they are grown at alkaline pH than when theyare grown at neutral pH [40 41] However the induction ofcytochrome c is not always observed in all alkaliphilicBacillusspecies grown at high pH [42] Membrane-anchor-less low-molecular-mass cytochromes c have been isolated fromalkaliphilicBacillus species (including Sporosarcina pasteurii)and characterized [41 43ndash45] All these cytochromes c weredetermined to have low redox potential (+47sim+95mV) com-pared with those from neutralophiles (+178mV in Bacillussubtilis) [46] by redox titration The low redox potential ofcytochromes c produces a large redox potential differencebetween its electron accepter cytochrome a in the terminaloxidaseThis large redox potentialmay play an important rolein electron transfer between cytochrome c and cytochromec oxidase at high Δ120595 across the cellular membrane Thesecytochromes c from alkaliphiles exhibit acidic nature (pI =40ndash42) compared with those from neutralophilic Bacillusspecies (pI = 52ndash97) [4] It has been reported that the outersegments of several membrane proteins exhibit extensively

reduced amounts of basic amino acids and increased amountsof acidic amino acids Membrane-bound cytochromes c inGram-positive bacteria also exist at the outer surface ofmembrane The acidic nature of these proteins will result ina negative charge to attract H+ and expel OHminus at the outersurface of the cellular membrane

Membrane-bound cytochromes c are considered to bindto the cellularmembrane via a diacylglyceryl-cysteinemoietyAlthough several diacylglyceryl-cysteinemoieties attached tocytochromes c have been isolated from several Gram-positivebacteria there has been no report on the diacylglyceryl-cysteine moiety attached to cytochromes c from alkali-philic Bacillus Ogami et al purified and characterized a cyto-chrome c with a diacylglyceryl-cysteine moiety attachednamely cytochrome c-550 fromanobligate alkaliphilicBacil-lus clarkiiK24-1U [28] Cytochrome c-550 binds to fatty acidsof C15 C16 and C

17chain lengths via glycerol-Cys18 The

cytochrome c is an acidic protein (predicted pI = 41 based onamino acid composition) It contains only two basic aminoacids including histidine of the heme c axial ligand Thiscytochrome c contains a conspicuous amino acid sequence ofGly22ndashAsn34 in comparison with the cytochromes c of otherneutralophilic or facultative alkaliphilicBacillus species Sim-ilar amino acid sequences have been observed only for obli-gate alkaliphiles The amino acid sequence of Gly22ndashAsn34location corresponds to the intermediate location betweenN-terminal and first 120572-helical structure from the N-terminalIt can be predicted that the sequence locates the surface ofthe cytochrome c molecule The amino acid sequences arerich in asparagine (Asn) residue An Asn residue locatedin H+ transfer pass way has been reported in heme-copperoxidase [47] The conspicuous amino acid sequence mayhave an important function in retaining or transferring H+from the outer surface of the membrane under alkaline pHcondition by its contribution for the formation of H-boundnetwork on the outer surface of themembraneThemidpointredox potential (E

1198987) of cytochrome c-550 as determined by

redox titration was +83mV On the other hand the midpointredox potential (E∘1015840) value at pH 7 determined by cyclicvoltammetric measurement was +7mV [28] It is consideredthat the redox potential of cytochrome c-550 becomes evenlower under a changing electric field when electrons aretaken up and out This may be related to the midpoint redoxpotential change of cytochrome c depending on theΔ120595 acrossthe cellular membrane In addition although it is not certainwhether the ambient pH affects the midpoint redox potentialof cytochrome c-550 there is a possibility that the ambient pHor ΔpH affects the midpoint redox potential of cytochromec-550 This produces an even larger redox potential differ-ence between cytochrome c-550 and the electron acceptercytochrome a The translocation of H+ in presence of a largerΔ120595 can be predicted The cytochrome c faction that lowersthe redox potential in the presence of a large Δ120595 may playan important role in translocating the positively charged H+from the intracellular to extracellular side of the membrane

It is considered that the regulation of H+-coupled elec-tron transfer of cytochrome c plays an important role inthe adaptation of alkaliphilic Bacillus species to alkaline


552

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

417

523

412

418

Cytochrome c-552

(a)

554

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

525

417

418

413

Cytochrome c-554

(b)

551

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

522

415 410

415

Cytochrome c-551

(c)

Figure 3 Absorption spectra of resting (red line) reduced (blue line) and oxidized (green line) cytochrome c-552 (a) cytochrome c-554 (b)and cytochrome c-551 (c) from P alcaliphila AL15-21T showing 120572 120573 and Soret bands The protein was reduced with sodium dithionite andoxidized with potassium ferricyanide

environments From the facts described above the redoxstate (presence or absence of electron) amino acid sequenceacidic nature of cytochrome cΔ120595ΔpH (or ambient pH) andredox potential change may affect the H+ behavior in H+-coupled electron transfer in cytochrome c

Although alkaliphilic Bacillus species produce a largeamount of cytochrome c during their growth at high pHthe respiratory rates are not high This fact suggests thatcytochrome c stores electrons rather than accelerates electrontransport in the respiratory chain In addition the charac-teristics of cytochrome c together with electron storage inthe cytochrome c and Δ120595 are related to H+ accumulation Ithas been reported that facultative alkaliphilic Bacillus cohniiYN-2000 produces higher amounts of a nonproteinaceoussubstance at pH 10 than at pH 7 [47] This nonproteinaceoussubstance plays a role as a substituent of cytochrome c oxidasein terminal oxidation in the respiratory chain [48] It isconsidered that it does not have a function in the electron-transfer-coupled H+ transfer in the respiratory chain Thesefacts described above suggested that the respiratory chain ofalkaliphilic Bacillus species not only has a function in theelectron-coupled H+ transfer across the membrane but alsoin the electron and H+ storage for ATP production Hencethere is a possibility that the cytochrome c accumulated inlarge amounts on the outer surface of membrane functions asan electron and H+ condenser

6 Production of SolubleCytochromes c and Their GeneralCharacteristics in P alcaliphila

As described above Gram-negative bacteria have a periplas-mic space whereas Gram-positive bacteria do not have

a periplasmic space Therefore Gram-negative bacteria havesoluble cytochrome c whereas Gram-positive bacteria onlyhave membrane-bound cytochrome c The cytochrome ccontents in the soluble fraction at pH 7ndash10 under low-and high-aeration intensities in Gram-negative facultativealkaliphilic P alcaliphila AL 15-21T have been estimated[49] The cytochrome c content in the soluble fraction ishigher in the cells grown at pH 10 than at pH 7 under low-aeration intensity In addition the cytochrome c content ishigher under low-aeration intensity than under high-aerationintensity The highest of cytochrome c content is observedin the cells grown at pH 10 under low-aeration intensityThe cytochrome c content in the cells grown at pH 10 underlow-aeration intensity (047 plusmn 005 nmolsdotmg proteinminus1) was36-fold higher than that in the cells grown at pH 7 underhigh-aeration intensity (013 plusmn 001 nmolsdotmg proteinminus1) Inthe soluble fraction of P alcaliphila AL-15-21T three typesof soluble cytochrome c namely cytochrome c-552 [49]cytochrome c-554 and cytochrome c-551 have been found

61 Properties of Cytochrome c-552 Cytochrome c-552 com-prises 64 of the total soluble cytochrome c (Figure 3) Theremaining cytochromes c c-551 and c-552 comprise theremaining 36Therefore cytochrome c-552 is themajor sol-uble cytochrome c in P alcaliphila The absorption spectrumof the resting state of cytochrome c-552 is very similar to thatof the fully reduced state (Figure 4)The 120572 120573 and Soret peaksof the purified cytochrome c-552 in the resting state are at 552523 and 417 nm respectively On the other hand the peaks ofits fully reduced state are at 552 (120572) 523 (120573) and 418 (Soret)nm In its fully oxidized form the Soret peak is at 412 nmIts molecular mass determined by sodium dodecyl sulfate-(SDS-) poly acrylamide gel electrophoresis (PAGE) is 75 kDa


0

005

01

015

02

0

02

04

06

08

0 100 200 300 400

Abso

rban

ce (4

15

nm)

Fraction number

NaC

l (M

)

Peak 1

Peak 2

Figure 4 Elution profile of anion-exchange chromatography (QAE-Toyopearl) loaded on the cell extract of P alcaliphila AL15-21Tgrown at pH 10 under low aeration intensity The cell extract wasprepared by cell disruption using a French press The first (peak1) and second (peak 2) peaks indicate cytochrome c-552 and amixture of cytochromes c other than cytochrome c-552 (cytochromec-554 and c-551) respectively The percentages relative to totalcytochromes c of peaks 1 and 2 are 64 and 36 respectively

This mass is smaller than those of typical cytochromes c forexample soluble cytochromes c purified from Pseudomonasaeruginosa are reported to be 9ndash15 kDa Although the pI ofcytochrome c-552 predicted on the basis of its amino acidcomposition is 57 the pI determined by isolectric focusingis 43 Although its pI is slightly higher than those of thecytochromes c of alkaliphilic Bacillus species (pI = 34ndash40)the acidic nature of cytochrome c-552 is similar to those fromalkaliphilic Bacillus The midpoint redox potentials (E

1198987) of

cytochrome c-552 as determined by redox titration and cyclicvoltammetry are +228 and +224mV respectively Althoughthese values are much higher than those of cytochromec from alkaliphilic Bacillus species (+47mVndash+95mV) theyare lower than those of cytochrome c-550 and c-552 fromneutralophilic Pseudomonas aeruginosa that is +280mV and+286mV respectively

The gene coding for cytochrome c-552 reveals that itconsists of 96 amino acids with 19 residues as the signalpeptide located at the N-terminal sequence The amino acidsequence deduced from the gene sequence revealed a sin-gle heme-binding sequence Cys-X-X-Cys-His (monohemecytochrome c) The amino acid sequence of cytochrome c-552 exhibited a relatively high similarity with the amino acidsequence encoded in the genes ofPseudomonas speciesTheseare assigned as putative cytochrome c from the determinedfull genome sequence of Pseudomonas species Thereforethe proteins produced from these genes have never beenpurified and characterized The amino acid sequence ofcytochrome c-552 exhibited similaritywith those of putativelyassigned cytochromes c as follows Pseudomonas mendocinaputative cytochrome c (gene accession number in Gen-bankEMBLDDBJABP82930 916 identity) Pseudomonasaeruginosa putative cytochrome c (ABR84170 698) andPseudomonas fluorescens putative cytochrome c (ABA71798

615) The high sequence similarity between cytochrome c-552 and cytochrome c of Pmendocinamay reflect the phylo-genetic relationship between P alcaliphila and PmendocinaAs described above P mendocina can also grow at pH 10There is a possibility that the presence of a cytochrome csimilar to cytochrome c-552 plays an important role in theadaptation to alkaline environments On the basis of phyloge-netic analysis based on the amino acid sequence cytochromec-552 is classified as small cytochrome c

5belonging to group

4 in class I cytochrome c (Figure 5)

62 Properties of Cytochrome c-551 Theabsorption spectrumof the resting state of cytochrome c-551 is identical to that ofthe fully reduced state (Figure 4) The 120572 120573 and Soret peaksof the purified cytochrome c-551 in the resting state are at 551522 and 415 nm respectively The peaks of its fully reducedstate are at 551 (120572) 522 (120573) and 415 (Soret) nm In its fullyoxidized form the Soret peak is at 410 nmThemolecularmassdetermined by SDS-PAGE is 19 kDa Its pI predicted on thebasis of the amio acid composition is 54

The gene sequence encoding cytochrome c-551 is locateddownstream of the gene sequence encoding cytochromec-552 The gene coding for cytochrome c-551 reveals thatit consists of 201 amino acids with 20 residues as thesignal peptide located at N-terminal sequence The aminoacid sequence deduced from the gene sequence revealedtwo heme-binding sequences Cys-X-X-Cys-His (dihemecytochrome c) The amino acid sequence of cytochromec-551 exhibited similarity with that of Pseudomonas men-docina cytochrome c

4(gene accession number in Gen-

bankEMBLDDBJABP82931 980 identity) On the basisof phylogenetic analysis based on the amino acid sequencecytochrome c-551 is classified as cytochrome c

4belonging to

group 3 in class I cytochrome c (Figure 5)

63 Properties of Cytochrome c-554 The absorption spec-trumof the resting state of cytochrome c-554 is very similar tothat of the fully reduced state (Figure 4) The 120572 120573 and Soretpeaks of the purified cytochrome c-554 in the resting stateare at 554 525 and 418 nm respectivelyThe peaks of its fullyreduced state are at 554 (120572) 525 (120573) and 417 (Soret) nm In itsfully oxidized form the Soret peak is at 413 nm Its molecularmass determined by SDS-PAGE is 11 kDa Its pI predicted onthe basis of the amino acid composition is 55

The gene coding for cytochrome c-554 reveals that itconsists of 137 amino acids with 21 residues as the signalpeptide located at N-terminal sequence The deduced aminoacid sequence from the gene sequence revealed a sin-gle heme-binding sequence Cys-X-X-Cys-His (monohemecytochrome c)The amino acid sequence of cytochrome c-554exhibited similarity with those of P mendocina cytochromec5(gene accession number in GenbankEMBLDDBJP00121

931 identity) and P aeruginosa cytochrome c5(gene acces-

sion number in GenbankEMBLDDBJ Q9HTQ4 706identity) On the basis of phylogenetic analysis based onthe amino acid sequence cytochrome c-554 is classified ascytochrome c

5belonging to group 4 in class I cytochrome c

(Figure 5)


Group 4 cytochrome c5

Group 2 cytochrome c6Group 5 cytochrome c8

Group 6 Bacilluslow-molecular-weight cytochrome c



Group 1 subunit IIin cytochrome c oxidase

Putative cytochrome c

Small cytochrome c5in Pseudomonas spp

Pseudomonas alcaliphila



91

9836

89

81

83

100

100

100

100

91

77

9467

9958

54

32

49

7653

58

66

02

Pseudomonas aeruginosa PAO1 cyt c (AAG08876)Pseudomonas aeruginosa UCBPP-PA14 cyt c (ABJ14877)

Pseudomonas aeruginosa PA7 cyt c (ABR84170)Pseudomonas putida KT2440 cyt c (ANN68938)

Pseudomonas fluorescens Pf0-1 cyt c (ABA71798)Pseudomonas fluorescens Q8r1-96 cyt c (AAO13003)

Pseudomonas putida F1 cyt c (ABQ76317)Pseudomonas putida GB-1 cyt c (ABY96056)

Pseudomonas mendocina ymp cyt c (ABP96056)

Shewanella putrefaciens Scy A (O52685)Shewanella violacea cyt cA (Q9RHJ6)

Azotobacter vinelandii cyt c5 (AAC45922)Pseudomonas aeruginosa cyt c5 (Q9HTQ4)

Pseudomonas mendocina cyt c5 (P00121)Chlamydomonas reinhardtii cyt c 6 (P08197)

Pseudomonas aeruginosa PAO1 c-551 (P00099)Bacillus subtilis cyt c-550 (P24469)Bacillus subtilis cyt c-551 (O34594)

Pseudomonas mendocina ymp cyt c4 (ABP82931)

Pseudomonas fulva 12-X cyt c4 (AEF20250)Pseudomonas entomophila L48 cyt c4 (CAK13073)

Phaeospirillum fulvum cyt c2 (P00086)Bacillus cohnii YN-2000 llc (Q59231)

cyt -552 (EF178295)

cyt -551 (EF178295)

cyt -554

c

c

c

Figure 5 Phylogenetic tree for cytochromes c of P alcaliphilaAL15-21T based on the amino acid sequenceThese are indicated by red arrowsThe phylogenetic tree was constructed by the neighbour-joiningmethodThe numbers at nodes are bootstrap values based on 1000 replicatesBar 001 changes per nucleotide positionThe red bracket indicates the small cytochrome c

5in Pseudomonas speciesThe yellowmarking and

blue bold arrow indicate putative small cytochrome c5observed in the determined whole genome sequence

7 Redox Properties of Cytochrome c-552

Cytochrome c-552 a small cytochrome c5belonging to group

4 in class I cytochrome c has been firstly purified and char-acterized However further characterization and clarificationof the physiological function of cytochrome c-552 is difficultbecause purification of a large amount of the protein isdifficult given that the amount of expressed protein fromP alcaliphila is very small in comparison with the amountof concomitantly existing background proteins Thereforeif purification is started from a large amount of cells thefirst purification step is very difficult to manage owing tothe large amount of protein To obtain a large amount ofpurified cytochrome c-552 a recombinant cytochrome c-552expression system was constructed by the transformation ofthe plasmid (pKK223-3[c552]) carrying the cytochrome c-552gene and the plasmid (pEC86) carrying the gene cluster ofccmA-H requiring the assembly of a c-type cytochrome inEscherichia coli under anaerobic growth condition The yieldof cytochrome c-552 from recombinant E coli is 28mgsdotLminus1of culture medium which is 764-fold larger than that fromnative cells Analytical data such as absorption spectra E

1198987

molecular mass determined by SDS-PAGE and MALDI-TOFMS and N-terminal amino acid sequence exhibited that

the recombinant protein is exactly the same as the nativecytochrome c-552

Changes in the redox potential (E∘1015840) depending on thepH were estimated using the recombinant cytochrome c-552The E∘1015840 values at pHs 6 7 and 8 are 223 224 and 226mVrespectively (average 224mV)On the other hand theE∘1015840 val-ues at pHs 9 and 10 are 217 and 218mV respectively (average218mV) Although these determined values are similar to oneanother the E∘1015840 values are slightly decreased at pHs 9 and 10in comparison to those of pHs 6ndash8 This small change maycontribute electron retaining ability of cytochrome c-552 atpHs 9-10 The E

1198987of cytochrome c-552 is lower than that of

cytochrome c-550 of neutralophilic Pseudomonas aeruginosaAlthough the high affinity of electrons in cytochrome c-552cannot be accounted for only by the E∘1015840 values the lower E∘1015840value of cytochrome c-552 than that of the neutralophile mayhave a meaning in adjusting the electron transfer betweencytochrome c-552 and its electron acceptor cytochrome coxidase in the adapting to physiological conditions

The reduction rate of cytochrome c-552 was estimatedusing native cytochrome c-552 by estimating the reductionrate under anaerobic condition (pHs 85 [100mM Tris-HClbuffer] and 10 [100mM glycine-NaOH buffer] and the pres-ence or absence of 10120583MTMPD) [49] Cytochrome c-552was


almost fully reduced after 40 h of incubation at pH 85 whileit was fully reduced after 4 h at pH 10 in the presence ofTMPD The reduction rate exhibited a first-order reactionwith reaction constants of 007 and 056 hminus1 at pHs 85 and 10respectivelyThe reduction of cytochrome c-552 occurs at pH85 only in the presence of TMPD while it is observed at pH10 even in the absence of TMPD Cytochrome c-552 is almostfully reduced at pH 10 in the absence of TMPDwith a reactionconstant of 004 hminus1 These results indicated that the electronmediator TMPD accelerates the reduction of cytochrome c-552 and glycine reduces cytochrome c at pH 10

The oxidation rate of cytochrome c-552 at pHs 6ndash10is estimated using 100mM buffers as follows phosphate-NaOH buffer for pHs 6 and 7 Tris-HCl buffer for pH 8 andsodium carbonate buffer for pHs 9 and 10 [50] Cytochromec-552 oxidized very slowly at pHs 8ndash10 with the slowest atpH 8 while it oxidized rapidly at pHs 6-7 On the otherhand the oxidation rates of horse heart cytochrome c arehigh at pHs 6ndash10 The results described above indicate thatcytochrome c-552 possesses the distinctive ability to retainits reduced state at pHs 8ndash10 As described above theelectron-transfer-coupled H+ transfer in cytochrome c hasbeen reported The electron-transfer-coupled H+ transfer ispossible if cytochrome c-552 retention of H+ on the basis ofthe negative charge of electron in its reduced state A highconcentration of cytochrome c-552 in the periplasmic spacewill contribute to the retention of H+ in the extracellularmembrane space

8 Role of Cytochrome c-552 inRespiratory System

For the search of an electron acceptor of cytochrome c-552 cytochrome c oxidase was purified from P alcaliphilaThe cytochrome c oxidase obtained from the solubilizedmembrane of P alcaliphila is a cb-type cytochrome c oxidasewhich shows an absorption Soret peak at 412 nm in theoxidized state In the reduced state it shows a Soret peakat 426 nm with a distinct shoulder at 418 nm In the visibleregion peaks at 558 553 529 and 523 nm are observed in thereduced form The peaks at 418 (shoulder) 523 and 553 nmare characteristic of ferrous c-type hemes On the otherhand the peaks at 426 529 and 558 nm are characteristicof ferrous b-type hemes The cb-type cytochrome c oxidaseshows a higher absorption intensity for b-type hemes thanfor c-type hemes whereas Pseudomonas stutzeri cytochromecbb3oxidase shows a higher absorption intensity for c-type

hemes [51] Actually Pseudomonas stutzeri cytochrome cbb3

oxidase contains two b-type and three c-type hemes The cb-type cytochrome c oxidase exhibits bands showingmolecularmasses of 55 42 and 32 kDa in SDS-PAGE Pseudomonasstutzeri cytochrome cbb

3oxidase exhibits bands showing

molecular masses of 41 35 and 26 kDa [52] while themolecular masses predicted from the gene sequence are53 197 34984 and 23463Da These molecular masses arevery similar to those of the cytochrome cbb

3oxidase from

P mendocina The molecular masses of subunits of thecytochrome cbb

3oxidase from P mendocina predicted from

the gene sequence are 53 34 22 and 6 kDa (CP000680-2580-2583) The cytochrome c oxidase activity of the cb-typecytochrome c oxidase was measured spectrophotometricallyusing recombinant P alcaliphila cytochrome c-552 and horseheart cytochrome c as the electron donors The cytochromec oxidase activities were 96 and 82 120583molsdotminminus1sdotmgminus1 whenP alcaliphila cytochrome c-552 and horse heart cytochromec were used as the electron donors respectively

9 Contribution of Cytochrome c-552 forAlkaline Adaptation

To understand the physiological function of cytochrome c-552 in P alcaliphila AL15-21T [53] an antibiotic-marker-less cytochrome c-552-deficient mutant has been constructedfollowing the method of Choi and Schweizer [54] A mutantfragment containing the 51015840 and 31015840 flanking regions of thetarget gene that is chromosomal cytochrome c-552 geneplus an antibiotic resistance maker gene GmR originatedfrom the pPS856 plasmid is contracted by a PCR overlapextension The resulting mutant fragment was cloned intothe Gateway vector pDONR 221 by BP clonase reactionand then recombined into the Gateway-compatible genereplacement suicide vector pEX18ApGW by LR clonasereaction (Figure 6)The plasmid-borne deletion allele is thentransformed into P alcaliphila AL15-21T by electroporationand recombined into the bacterial chromosome The mutantfragment is integrated into the target gene by a single- ordouble-crossover event Merodiploids via the integration ofthe suicide plasmid by a single-crossover event are resolvedby sucrose selection in the presence of the gentamycinfor deletion of the wild type gene Finally the antibioticresistance marker gene (GmR) is subsequently deleted fromthe chromosome by Flp recombination reaction

The growth characteristics of wild type P alcaliphilaAL15-21T and the cytochrome c-552 deletion mutant wereexamined at pHs 10 and 7 under low- and high-aerationintensities (Figure 7) The maximum specific growth rate(120583max [h

minus1]) and maximum cell turbidity (OD660max) of the

two strains were estimated Although the growth under thelow-aeration intensity is inferior to that under the high-aeration intensity initial growth rate is higher under the low-aeration intensity This is probably related to the high oxygentension that inhibits the growth initiation Significant dif-ferences in growth parameters concomitant with differencesin the growth curve are observed at pH 10 under the low-aeration intensity between wild type P alcaliphila AL15-21T(120583max [hminus1] = 0911 OD

660max = 027) and the cytochromec-552 deletion mutant (120583max [hminus1] = 0709 OD

660max =021) The 120583max (hminus1) and OD

660max of the deletion mutantdecreased by 22 and 25 respectively relative to those ofthe wild type It is considered that this large difference isattributed to the large difference in the expression level ofcytochrome c A slight decreasewas observed in themutant incomparison with the wild type under high-aeration intensityin the 120583max (h

minus1) during growth at pH 10 The 120583max (hminus1) is

0475 and OD660max is 206 in the wild type whereas the 120583max


FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB

P alcaliphila AL15-21T chromosome

Cyt c-552 gene

c-552 gene

Gm

Gm

Gm

1st homologous recombination

Plasmid to generate deletion mutant

Δ

Δ

Gm resistant gene was deleted by FlpFRThomogeneous DNA recombination

2nd homologous recombination

Figure 6 Scheme of producing unmarked cytochrome c-552-deficient mutant of P alcaliphilaAL15-21T by homologous recombinationThemutant fragment generated by overlap extension PCR is cloned into the pDONR221 and then recombined into the pEX18ApGW plasmid bygateway-recombinational cloningThe resulting suicide vector is transferred to P alcaliphilaAL15-21TThe plasmid-borne deletionmutationis exchanged with the corresponding part in the chromosomeThemerodiploid state is resolved by sacB-mediated sucrose counterselection inthe presence of gentamycin Finally the gentamycin resistance marker (GmR) is deleted by FlpFRF homologues DNA recombination sacBBacillus subtilis levansucrase-encoding gene Cd carbenicillin resistance marker

(hminus1) is 0416 (12 decrease) and OD660max is 206 in the

mutant No significant difference in the 120583max (hminus1) between

the two strainswas observed during growth at pH7 regardlessof the aeration intensityThe 120583max (h

minus1) is 1063 andOD660max

is 053 in the wild type whereas the 120583max (hminus1) is 1063 and

OD660max is 050 in the mutant during growth under low-

aeration intensityThe120583max (hminus1) is 0693 andOD

660max is 218in thewild type whereas the120583max (h


is 217 in the mutant grown under high aeration intensityUnder these conditions cytochrome c-552 can be substitutedby other soluble cytochromes c such as cytochrome c-551and cytochrome c-554 for the function of electron transferto the terminal oxidase cytochrome cb As described abovethe largest amount of soluble cytochrome c production wasobserved at pH 10 under low-aeration intensity among thepH range of 7ndash10 under low- and high-aeration intensitiesThe difference in 120583max

(hminus1) and OD660max can be attributed to the large differ-

ence in the expression level of cytochrome c In other wordsthe contribution of cytochrome c-552 becomes high if theamount of total soluble cytochrome c is enhanced at pH 10under low-aeration intensity in the wild type

The oxygen consumption rate of the P alcaliphila AL15-21

T cell suspensionwas determined No significant differencein the growth between the wild type and the cytochromec-552 deletion mutant was observed at pH 7 The oxygenconsumption rates of the mutant showed a 14 increase anda 31 decrease relative to those of the wild type On the otherhand a significant promotion of the oxygen consumptionrate between the two strains grown at pH 10 was observedregardless of aeration intensity The oxygen consumptionrate is 884 plusmn 27 natom Osdotminminus1sdotmg proteinminus1 in the mutantwhereas it is 790 plusmn 16 natom Osdotminminus1sdotmg proteinminus1 in thewild type under low-aeration intensity It indicates a 12


0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)

Figure 7 Time course of growth of P alcaliphilaAL15-21T (wild type blue symbols) and cytochrome c-552 deletionmutant derived fromwildtype (mutant red symbols) grown under (a) high- and (b) low-aeration intensities at pH 7 (squares) and pH 10 (circles) The reproducibilityof the results was ascertained by performing three independent experiments

increase in the mutant relative to that in the wild typeUnder the high-aeration intensity at pH 10 the rate is 1106 plusmn69 natom Osdotminminus1sdotmg proteinminus1 in the mutant whereas it is948 plusmn 53 natom Osdotminminus1sdotmg proteinminus1 (a 17 increase) inthe wild type These results suggest that cytochrome c-552acts as an electron reserve rather than a promoter of electrontransfer in the respiratory chain This is in accordance withthe characteristics of cytochrome c-552 that exhibits highelectron- retaining ability at high pH

The generation of H+ motive force by the respiratorychain may be difficult under alkaline pH (pH 10) andorunder low-aeration intensity because it denotes a transportsubstrate on H+-less andor a terminal electron acceptor onO2-less condition Cytochrome c-552 plays a role in retaining

electrons concomitant with H+ by the electric charge ofelectrons and characteristics of the protein that is pKa of thesurface amino acid sequence of the protein Thus the accu-mulation of cytochrome c-552 in the periplasmic space resultsin the storage of electrons and H+ in the periplasmic space inthe bacterium We hypothesize that cytochrome c-552 in theperiplasmic space plays the role of ldquocondenserrdquo of H+ andorelectrons for the respiratory system This system may workgiven the pool of H+ andor electrons using the vast periplas-mic space and may partially link ordinary electron-flow-dependent H+ transfer across the membrane It is consideredthat the system using cytochrome c-552 in periplasmic spacehas an advantage for the functioning of the respiratory systemunder high-pH andor low-aeration intensity

10 Conclusions and Perspective

As mentioned above developments of a barrier such as anacidic substance teichuronopeptide teichuronic acid and

acidic amino acid chains around the cell surface to avoidalkaline pH are observed in alkaliphilic Bacillus Althoughthe composition of the cell barrier in P alcaliphila is notwell understood the components and specific structure canbe observed by scanning electron microscopy (Figure 1) Thecells of strain grown at pH 10 exhibit a rough surface of thecells whereas cells grown at pH 7 exhibit smooth surfaceTherefore it is considered that protection of the interfacefrom the outer environment is fundamentally very importantin many alkaliphilic bacteria whether Gram-negative orGram-positive bacteria

Numerous of alkaliphilic Bacillus species have beenknown as authorized species Although alkaliphilic Bacillusspecies exhibit diverse ecological distributions and diversephylogenetic positions they exhibit a distinctive phylogeneticposition among Bacillus species Although not so many alka-liphilic Pseudomonas species are authorized the consistencyof the distinctive position of alkaliphilic Pseudomonas speciesis also observed These genera include numerous species andexhibit a wide range of distribution and exquisite adaptabilityto various environments in natureThese results indicate thatthere is a relationship between the evolutionary process andthe gain in adaptability to alkaline environments

From the bioenergetics viewpoint transmembrane Δ120595which contributes to the H+ attraction at the outer surfaceof the cellular membrane and sustains effective energy pro-duction plays one of the most important roles in alkalineadaptation in alkaliphilic Bacillus species In addition it isconsidered that certain alkaliphilic Bacillus species produceda large amount of membrane-bound cytochrome c on theouter surface of membrane to support the electron trans-fer from the outer surface of the membrane to the innermembrane direction under a large transmembrane Δ120595 [29]Furthermore cytochrome c may play a role in regulating


Cytochrome c-552Cytochrome c-554Cytochrome c-551

Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O

Intramembrane side high pH (low concentration of H+)under air-limited condition

Figure 8 Hypothetical model of cytochrome c-552 function in respiratory system of P alcaliphila AL15-21T The function of cytochromec-552 as an electron and H+ reserve enhances the function of terminal oxidation (cytochrome cb) at pH 10 under low-aeration intensity (H+-less and O

2-less condition) Owing to the large negative charge of electron H+ is attracted by the reduced cytochrome c-552 Thus owing to

the large electron and H+ capacity of enhanced amount of cytochrome c-552 the cytochrome cb terminal oxidase can transfer electrons andF1F0-ATP synthase can translocate H+ at pH 10 under low-aeration intensity The hypothetical model shows that cytochrome c-552 in large

capacity in the periplasmic space plays the role of an ldquoelectron and H+ condenserrdquo in terminal oxidation and F1F0-ATP synthase activation

H+ transfer on the outer surface of the membrane via asurface protein function sensing of Δ120595 and H+ attractionby retaining electrons in H+ and as an electron condenserconstructed on the membrane

On the other hand although the contribution of Δ120595in alkaliphilic Pseudomonas species is unknown these bac-teria utilize cytochrome c which exhibits excellent elec-tron retaining ability and a vast periplasmic space for notonly accumulating H+ to protect against an extracellu-lar alkaline environment but also as an H+ and electroncondenser (Figure 8) Thus the differences in bioenergeticalkali-adaptation strategy betweenGram-positive andGram-negative bacteria using a function of H+ and electron con-denser related to the function of cytochrome c are attributedto the membrane surface structure Owing to the phylogenicdiversity of alkaliphilic Bacillus species diverse variationsin alkaline adaptation mechanisms probably exist in thisGram-negative bacterial group In the near future numerousalkaliphilic Pseudomonas species will be discovered It isexpected that variations in alkaline adaptation mechanismsexist among alkaliphilic Pseudomonas species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] K Horikoshi ldquoPrologue definition categories distributionorigin and evolution pioneering studies and emerging studiesand emerging fields of extremophilesrdquo in Extremophiles Hand-book K Horikoshi G Antranikian A T Bull F T Robb andK O Stetter Eds 15 p 3 Springer Tokyo Japan 2011

[2] G Antranikian and K Egorova ldquoExtremophiles a uniqueresource of biocatalysts for industrial biotechnologyrdquo in Phys-iology and Biochemistry of Extremophiles C Gerday and NGlansdorff Eds pp 361ndash406 ASM Press Washington DCUSA 2007

[3] K Horikoshi Alkaliphiles Kodansha Tokyo Japan SpringerBerlin Germany 2006

[4] T Goto T Matsuno M Hishinuma-Narisawa et al ldquoCyto-chrome c and bioenergetic hypothetical model for alkaliphilicBacillus spprdquo Journal of Bioscience and Bioengineering vol 100no 4 pp 365ndash379 2005

[5] I Yumoto ldquoEnvironmental and taxonomic biodiversities ofGram-positive alkaliphilesrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 295ndash310ASM Press Washington DC USA 2007

[6] T A Krulwich D B Hicks T Swartz and M Ito ldquoBioener-getic adaptations that support alkaliphilyrdquo in Physiology andBiochemistry of Extremophiles C Gerday and N GlansdorffEds pp 311ndash329 ASM Press Washington DC USA 2007

[7] I Yumoto K Hirota and K Yoshimune ldquoEnvironmen-tal distribution and taxonomic diversity of alkaliphilesrdquo in


Extremophiles Handbook K Horikoshi G Antranikian A TBull F T Robb and K O Stetter Eds pp 55ndash79 SpringerTokyo Japan 2011

[8] K Horikoshi ldquoGeneral physiology of alkaliphilesrdquo in Extre-mophiles Handbook K Horikoshi G Antranikian A T BullF T Robb and K O Stetter Eds pp 99ndash118 Springer TokyoJapan 2011

[9] T A Krulwich J Liu M Morino M Fujisawa M Ito andD B Hicks ldquoAdaptive mechanisms of extreme alkaliphilesrdquo inExtremophiles Handbook K Horikoshi G Antranikian A TBull F T Robb and K O Stetter Eds pp 119ndash139 SpringerTokyo Japan 2011

[10] R Aono M Ito and K Horikoshi ldquoOccurrence of teichu-ronopeptide in cell walls of group 2 alkaliphilic Bacillus spprdquoJournal of General Microbiology vol 139 no 11 pp 2739ndash27441993

[11] R AonoM Ito K N Joblin and K Horikoshi ldquoA high cell wallnegative charge is necessary for the growth of the alkaliphileBacillus lentus C-125 at elevated pHrdquoMicrobiology vol 141 no11 pp 2955ndash2964 1995

[12] R Aono M Ito and T Machida ldquoContribution of the cellwall component teichuronopeptide to pH homeostasis andalkaliphily in the alkaliphile Bacillus lentus C-125rdquo Journal ofBacteriology vol 181 no 21 pp 6600ndash6606 1999

[13] R Gilmour P Messner A A Guffanti et al ldquoTwo-dimensionalgel electrophoresis analyses of pH-dependent protein expres-sion in facultatively alkaliphilic Bacillus pseudofirmus OF4lead to characterization of an S-layer protein with a role inalkaliphilyrdquo Journal of Bacteriology vol 182 no 21 pp 5969ndash5981 2000

[14] I Yumoto ldquoBioenergetics of alkaliphilic Bacillus spprdquo Journal ofBioscience and Bioengineering vol 93 no 4 pp 342ndash353 2002

[15] I Yumoto ldquoElectron transport system in alkaliphilic Bacillusspprdquo Recent Research Developments in Bacteriology vol 1 pp131ndash149 2003

[16] P Mitchell ldquoCoupling of phosphorylation to electron andhydrogen transfer by a chemi-osmotic type of mechanismrdquoNature vol 191 no 4784 pp 144ndash148 1961

[17] G Seltman and O Holst The Bacterial Cell Wall SpringerBerlin Germany 2002

[18] K Taira J Hirose S Hayashida and K Furukawa ldquoAnalysisof bph operon from the polychlorinated biphenyl-degradingstrain of Pseudomonas pseudoalcaligenes KF707rdquoThe Journal ofBiological Chemistry vol 267 no 7 pp 4844ndash4853 1992

[19] S Fedi M Carnevali F Fava A Andracchio S Zappoli and DZannoni ldquoPolychlorinated biphenyl degradation activities andhybridization analyses of fifteen aerobic strains isolated from aPCB-contaminated siterdquo Research in Microbiology vol 152 no6 pp 583ndash592 2001

[20] K Hirota K Yamahira K Nakajima Y Nodasaka HOkuyama and I Yumoto ldquoPseudomonas toyotomiensis sp nova psychrotolerant facultative alkaliphile that utilizes hydro-carbonsrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 61 no 8 pp 1842ndash1848 2011

[21] N J Palleroni ldquoGenus I Pseudomonas Migula 1894 237119860119871rdquoin Bergyrsquos Manual of Systematic BacteriologymdashVolume 2 TheProteobacteria Part B The Gammaproteobacteria D J BrennerN R Krieg J T Staley and G M Garrity Eds pp 323ndash379Springer New York NY USA 2nd edition 2005

[22] Y Sardessai and S Bhosle ldquoTolerance of bacteria to organicsolventsrdquo Research in Microbiology vol 153 no 5 pp 263ndash2682002

[23] Y Ni L Song X Qian and Z Sun ldquoProteomic analysisof Pseudomonas putida reveals an organic solvent tolerance-related gene mmsBrdquo PLoS ONE vol 8 no 2 Article ID e558582013

[24] I Yumoto K Yamazaki M Hishinuma et al ldquoPseudomonasalcaliphila sp nov a novel facultatively psychrophilic alka-liphile isolated from seawaterrdquo International Journal of System-atic and Evolutionary Microbiology vol 51 no 2 pp 349ndash3552001

[25] A K Borsodi AMicsinai A Rusznyak et al ldquoDiversity of alka-liphilic and alkalitolerant bacteria cultivated fromdecomposingreed rhizomes in aHungarian soda lakerdquoMicrobial Ecology vol50 no 1 pp 9ndash18 2005

[26] B-TWong andD-J Lee ldquoPseudomonas yangmingensis sp novan alkaliphilic denitrifying species isolated from a hot springrdquoJournal of Bioscience and Bioengineering vol 117 no 1 pp 71ndash742014

[27] A A Guffanti andD B Hicks ldquoMolar growth yields and bioen-ergetic parameters of extremely alkaliphilic Bacillus species inbatch cultures and growth in a chemostat at pH 105rdquo Journalof General Microbiology vol 137 no 10 pp 2375ndash2379 1991

[28] S Ogami S Hijikata T Tsukahara et al ldquoA novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U expression molecular features and properties of redoxpotentialrdquo Extremophiles vol 13 no 3 pp 491ndash504 2009

[29] K Yoshimune H Morimoto Y Hirano J Sakamoto HMatsuyama and I Yumoto ldquoThe obligate alkaliphiles Bacillusclarkii K24-1U retains extruded protons at the beginning ofrespirationrdquo Journal of Bioenergetics and Biomembrane vol 42no 2 pp 111ndash116 2010

[30] P Scherrer U Alexiev TMarti H G Khorana andM P HeynldquoCovalently bound pH-indicator dye at selected extracellularor cytoolasmic sites in bacteriorhodopsin 1 Proton migrationalong the surface of bacteriorhodopsin micelles and delayedtransfer from surface to bulkrdquo Biochemistry vol 33 no 46 pp13684ndash13692 1994

[31] U Alexiev ldquoCovalently bound pH-indicator dyes at selectedextracellular or cytoplasmic sites in bacteriorhodopsin 2 Rota-tional orientation of helices D and e and kinetic correlationbetween M formation and proton release in bacteriorhodopsinmicellesrdquo Biochemistry vol 33 no 46 pp 13693ndash13699 1994

[32] M Gutman and E Nachliel ldquoThe dynamics of proton exchangebetween bulk and surface groupsrdquo Biochimica et BiophysicaActamdashBioenergetics vol 1231 no 2 pp 123ndash138 1995

[33] Y Marantz O Einarsdottir E Nachliel and M GutmanldquoProton-collecting properties of bovine heart cytochrome coxidase kinetic and electrostatic analysisrdquoBiochemistry vol 40no 50 pp 15086ndash15097 2001

[34] T H Haines and N A Dencher ldquoCardiolipin a proton trap foroxidative phosphorylationrdquo FEBS Letters vol 528 no 1ndash3 pp35ndash39 2002

[35] J I Shioi S Matsuura and Y Imae ldquoQuantitative measure-ments of proton motive force and motility in Bacillus subtilisrdquoJournal of Bacteriology vol 144 no 3 pp 891ndash897 1980

[36] T Hirabayashi T Goto H Morimoto K Yoshimune HMatsuyama and I Yumoto ldquoRelationship between rates ofrespiratory proton extrusion and ATP synthesis in obligatelyalkaliphilic Bacillus clarkiiDSM 8720119879rdquo Journal of Bioenergeticsand Biomembranes vol 44 no 2 pp 265ndash272 2012

[37] A Y Mulkidjanian ldquoProton in the well and through the desol-vation barrierrdquo Biochimica et Biophysica Acta Bioenergetics vol1757 no 5-6 pp 415ndash427 2006


[38] D H Murgida and P Hildebrandt ldquoProton-coupled electrontransfer of cytochrome crdquo Journal of the American ChemicalSociety vol 123 no 17 pp 4062ndash4068 2001

[39] R J Lewis S Belkina and T A Krulwich ldquoAlkalophiles havemuch higher cytochrome contents than conventional bacteriaand than their own non-alkalophilic mutant derivativesrdquo Bio-chemical and Biophysical Research Communications vol 95 no2 pp 857ndash863 1980

[40] A A Guffanti O Finkelthal D B Hicks et al ldquoIsolationand characterization of new facultatively alkalophilic strains ofBacillus speciesrdquo Journal of Bacteriology vol 167 pp 766ndash7731986

[41] I Yumoto Y Fukumori and T Yamanaka ldquoPurification andcharacterization of two membrane-bound 119888-type cytochromesfrom a facultative alkalophilic Bacillusrdquo Journal of Biochemistryvol 110 no 2 pp 267ndash273 1991

[42] I Yumoto K Nakajima and K Ikeda ldquoComparative study oncytochrome content of alkaliphilic Bacillus strainsrdquo Journal ofFermentation and Bioengineering vol 83 no 5 pp 466ndash4691997

[43] M W Davidson K A Gray D B Knaff and T A KrulwichldquoPurification and characterization of two soluble cytochromesfrom the alkalophile Bacillus firmus RABrdquo Biochimica et Bio-physica ActamdashBioenergetics vol 933 no 3 pp 470ndash477 1988

[44] I H M Vandenberghe Y Guisez S Ciurli S Benini andJ J Van Beeumen ldquoCytochrome c-553 from the alkalophilicbacterium Bacillus pasteurii has the primary structure charac-teristics of a lipoproteinrdquo Biochemical and Biophysical ResearchCommunications vol 264 no 2 pp 380ndash387 1999

[45] S Benini A Gonzalez W R Rypniewski K S Wilson J J VanBeeumen and S Ciurli ldquoCrystal structure of oxidized Bacilluspasteurii cytochrome c

553at 097-A resolutionrdquo Biochemistry

vol 39 no 43 pp 13115ndash13126 2000[46] C Von Wachenfeldt and L Hederstedt ldquoPhysico-chemical

characterisation of membrane-bound and water-soluble formsof Bacillus subtilis cytochrome c-550rdquo European Journal ofBiochemistry vol 212 no 2 pp 499ndash509 1993

[47] P Brzezinski and G Larsson ldquoRedox-driven proton pumpingby heme-copper oxidasesrdquo Biochimica et Biophysica ActamdashBioenergetics vol 1605 no 1ndash3 pp 1ndash13 2003

[48] A Higashibata T Fujiwara and Y Fukumori ldquoStudies onthe respiratory system in alkaliphilic Bacillus a proposed newrespiratory mechanismrdquo Extremophiles vol 2 no 2 pp 83ndash921998

[49] TMatsunoNMorishita K Yamazaki et al ldquoCytochrome c-552from gram-negative alkaliphilic Pseudomonas alcaliphila AL15-21119879 alters the redox properties at high pHrdquo Journal of Bioscienceand Bioengineering vol 103 no 3 pp 247ndash254 2007

[50] T Matsuno Y Mie K Yoshimune and I Yumoto ldquoPhysio-logical role and redox properties of a small cytochrome 119888

5

cytochrome 119888-552 from alkaliphile Pseudomonas alcaliphilaAL15-21119879rdquo Journal of Bioscience and Bioengineering vol 108 no6 pp 465ndash470 2009

[51] R S Pitcher M R Cheesman and N JWatmough ldquoMolecularand spectroscopic analysis of the cytochrome cbb

3oxidase from

Pseudomonas stutzerirdquoThe Journal of Biological Chemistry vol277 no 35 pp 31474ndash31483 2002

[52] A Urbani S Gemeinhardt A Warne and M SarasteldquoProperties of the detergent solubilised cytochrome c oxidase(cytochrome cbb

3) purified from Pseudomonas stutzerirdquo FEBS

Letters vol 508 no 1 pp 29ndash35 2001

[53] T Matsuno K Yoshimune and I Yumoto ldquoPhysiologicalfunction of soluble cytochrome c-552 from alkaliphilic Pseu-domonas alcaliphila AL15-21119879rdquo Journal of Bioenergetics andBiomembranes vol 43 no 5 pp 473ndash481 2011

[54] K-H Choi andH P Schweizer ldquoAn improvedmethod for rapidgeneration of unmarked Pseudomonas aeruginosa deletionmutantsrdquo BMCMicrobiology vol 5 article 30 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


[5 7] The physiology of alkaline adaptation has been exten-sively studied on alkaliphilic species [4 6 8 9] Firstly oneof the most important strategies for alkaline adaptation isprotection from alkalinity outside of their cells It has beenreported that cells of Bacillus halodurans C-125 produceacidic substances such as teichuronopeptide and teichuronicacid on their surface [10ndash12] On the other hand cells ofBacillus pseudofirmus OF4 produce the cell surface proteinSlpA and polyglutamic acid on their surface [13] These areacidic substances that attract H+ and repel OHminus in the extra-cellular environment Protons (H+) are usually used in solutetransport systems and torque for flagella rotation AlkaliphilicBacillus species adopt strategies for saving H+ usage forsolute transport systems and torque for flagella rotationTheyemploy sodium ion solute uptake and the flagella rotationsystemThe above-mentioned alkaline avoidance barrier andH+ usage system are not well investigated in Gram-negativealkaliphiles

In addition to the avoidance of a harmful extracellularenvironment and lack of the necessary ions for solute trans-port and flagella rotation bioenergetics is one of the mostfundamental issues for survival in alkaline environments[4 6 9 14 15] It is considered that H+-deficient conditionshave an extremely negative impact on energy production inmicroorganisms because F

1F0-ATP synthase is driven by H+

as the torque According to Peter Michellrsquos chemiosmotictheory the H+ motive force (Δ119901) for driving F

1F0-ATP

synthase consists of a transmembrane pH gradient (ΔpH)(intracellular pH minus extracellular pH) and a membraneelectrical potential (Δ120595) (minus inside + outside) [16] Fromthe calculation of medium pH as the outer membrane pHATP production is impossible theoretically It is predictedthat alkaliphiles possesses mechanisms that compensate forthe negative factor that is H+-deficient condition for energyproduction

Although numbers of reported Gram-negative alka-liphiles are less than those of Gram-positive alkaliphilesboth types of alkaliphiles are distributed in soda lakes [7]Therefore it is very important to consider the bioenergeticproblems in Gram-negative alkaliphiles to elucidate thepossible environmental adaptation mechanisms that mighthave led to these alkaliphiles to possess a different type ofcell surface membrane structure It can be recognized thatthe lack of transmembrane pH gradient makes energy pro-duction difficult for not only the Gram-positive but also theGram-negative bacteria Although there are many reports onthe environmental adaptation of the Gram-positive bacteriathose of the Gram negative bacteria are very few In additionseveral reviews on the bioenergetics of alkaliphilic Bacillusspecies have been published [4 6 9 14 15] These includerespiratory components and bioenergetics parameters andthe relationship among them However there has been noreview paper on bioenergetics in Gram-negative alkaliphilicbacteria Gram-negative bacteria possess an outer membraneand a periplasmic space These structures do not exist inGram-positive bacteria The periplasmic space is betweenthe cytoplasmic membrane and the outer membrane It hasbeen reported that the periplasmic space comprises 40 of

the total volume of the cells and it includes solutes and pro-teins that are involved in a wide variety of functions rangingbetween nutrient binding transport electron transport andalteration of substances toxic to the cell [17] Therefore itis expected that the periplasmic space plays an importantrole not only in the protection against an extracellularalkaline environments but also in retaining key proteins forbioenergetics adaptation to alkaline environments In thisreview we discuss the characteristics and physiological func-tions of soluble cytochromes c in the periplasmic space foradaptation to alkaline environments Furthermore we alsodiscuss the differences between Gram-negative and Gram-positive alkaliphiles from the bioenergetics viewpoints

2 Alkaliphilic Pseudomonas

Gram-negative alkaliphilic bacteria are less well known thanGram-positive ones It is probably because most colonieson standard alkaline-containing media such as Horikoshirsquosmedium are identified as Bacillus species when we try toisolate alkaliphilic bacteria from terrestrial environmentssuch as soils and manure [5 7] However if we try toisolate alkaliphiles from aquatic environments or particularsubstances such as polychlorinated biphenyl (PCB)- [18 19]or hydrocarbon- [20] containing media the possibility ofisolating alkaliphiles other than Bacillus species such asPseudomonas species becomes higher Pseudomonas speciesare widely distributed in nature such as soils freshwaterseawater and terrestrial and marine animals and plants [21]Some Pseudomonas species are able to survive in organicsolvents such as toluene [22 23] It is considered that Pseu-domonas species are a metabolically and genetically diversebacterial group Their functions are related to the circulationof carbons and nitrogen on Earth by decomposing vari-ous organic substances and reducing nitrogen compoundsOn the other hand some of the Pseudomonas species arepathogenic to humans plants and fish Considering theirenvironmental distribution and metabolic and genetic diver-sity it is no wonder that numerous alkaliphilic Pseudomonasspecies exist in various taxonomic groups It has been knownthat Pseudomonas pseudoalcaligenes can grow on media withhigh pH [18 19] We have isolated Pseudomonas alcaliphilaAL15-21T from seawater obtained from the coast of RumoiHokkaido Japan by using a general medium for the isolationof alkaliphiles which contains peptone yeast extract and ametal mixture [24] A scanning electron microscopy imageof the cells of platinumpalladium-coated P alcaliphilaAL15-21

T grown at pH 10 shows a rough surface whereas the cellsgrown at pH 7 show a smooth surface (Figure 1) We havealso isolated Pseudomonas toyotomiensis from a surface soilimmersed in hydrocarbon-containing hot spring water usinga medium containing hydrocarbon as the sole carbon source[20] P pseudoalcaligenes P alcaliphila and P toyotomiensisare on the same node in the phylogenetic tree based on the16S rRNA gene sequence (Figure 2) Pseudomonasmendocinaand Pseudomonas oleovorans are also located on the samenodewith these alkaliphilicPseudomonas species and as far aswe have examined they alsowere able to grow at high pH (pH


(a) (b)


001

704539

786

525

781973

999

969690 663

796857

604907

521

879630





















T (Z76666)












Δ119901 = Δ120595 minus 119885ΔpH 119885 =23119877119879

119865= 59 (1)


















2O or H






1F0-ATP synthase










552

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

417

523

412

418

Cytochrome c-552

(a)

554

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

525

417

418

413

Cytochrome c-554

(b)

551

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

522

415 410

415

Cytochrome c-551

(c)









0

005

01

015

02

0

02

04

06

08

0 100 200 300 400

Abso

rban

ce (4

15

nm)

Fraction number

NaC

l (M

)

Peak 1

Peak 2



1198987) of




5belonging to group






4belonging to







(Figure 5)













91

9836

89

81

83

100

100

100

100

91

77

9467

9958

54

32

49

7653

58

66

02













cyt -552 (EF178295)

cyt -551 (EF178295)

cyt -554

c

c

c







1198987
















3oxidase from















FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)


001

704539

786

525

781973

999

969690 663

796857

604907

521

879630





















T (Z76666)












Δ119901 = Δ120595 minus 119885ΔpH 119885 =23119877119879

119865= 59 (1)


















2O or H






1F0-ATP synthase










552

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

417

523

412

418

Cytochrome c-552

(a)

554

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

525

417

418

413

Cytochrome c-554

(b)

551

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

522

415 410

415

Cytochrome c-551

(c)









0

005

01

015

02

0

02

04

06

08

0 100 200 300 400

Abso

rban

ce (4

15

nm)

Fraction number

NaC

l (M

)

Peak 1

Peak 2



1198987) of




5belonging to group






4belonging to







(Figure 5)













91

9836

89

81

83

100

100

100

100

91

77

9467

9958

54

32

49

7653

58

66

02













cyt -552 (EF178295)

cyt -551 (EF178295)

cyt -554

c

c

c







1198987
















3oxidase from















FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Δ119901 = Δ120595 minus 119885ΔpH 119885 =23119877119879

119865= 59 (1)


















2O or H






1F0-ATP synthase










552

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

417

523

412

418

Cytochrome c-552

(a)

554

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

525

417

418

413

Cytochrome c-554

(b)

551

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

522

415 410

415

Cytochrome c-551

(c)









0

005

01

015

02

0

02

04

06

08

0 100 200 300 400

Abso

rban

ce (4

15

nm)

Fraction number

NaC

l (M

)

Peak 1

Peak 2



1198987) of




5belonging to group






4belonging to







(Figure 5)













91

9836

89

81

83

100

100

100

100

91

77

9467

9958

54

32

49

7653

58

66

02













cyt -552 (EF178295)

cyt -551 (EF178295)

cyt -554

c

c

c







1198987
















3oxidase from















FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2O or H






1F0-ATP synthase










552

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

417

523

412

418

Cytochrome c-552

(a)

554

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

525

417

418

413

Cytochrome c-554

(b)

551

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

522

415 410

415

Cytochrome c-551

(c)









0

005

01

015

02

0

02

04

06

08

0 100 200 300 400

Abso

rban

ce (4

15

nm)

Fraction number

NaC

l (M

)

Peak 1

Peak 2



1198987) of




5belonging to group






4belonging to







(Figure 5)













91

9836

89

81

83

100

100

100

100

91

77

9467

9958

54

32

49

7653

58

66

02













cyt -552 (EF178295)

cyt -551 (EF178295)

cyt -554

c

c

c







1198987
















3oxidase from















FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


552

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

417

523

412

418

Cytochrome c-552

(a)

554

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

525

417

418

413

Cytochrome c-554

(b)

551

Abs

orba

nce

10

08

06

04

02

0

12

Wavelength (nm)350 400 450 500 550 600

522

415 410

415

Cytochrome c-551

(c)









0

005

01

015

02

0

02

04

06

08

0 100 200 300 400

Abso

rban

ce (4

15

nm)

Fraction number

NaC

l (M

)

Peak 1

Peak 2



1198987) of




5belonging to group






4belonging to







(Figure 5)













91

9836

89

81

83

100

100

100

100

91

77

9467

9958

54

32

49

7653

58

66

02













cyt -552 (EF178295)

cyt -551 (EF178295)

cyt -554

c

c

c







1198987
















3oxidase from















FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

005

01

015

02

0

02

04

06

08

0 100 200 300 400

Abso

rban

ce (4

15

nm)

Fraction number

NaC

l (M

)

Peak 1

Peak 2



1198987) of




5belonging to group






4belonging to







(Figure 5)













91

9836

89

81

83

100

100

100

100

91

77

9467

9958

54

32

49

7653

58

66

02













cyt -552 (EF178295)

cyt -551 (EF178295)

cyt -554

c

c

c







1198987
















3oxidase from















FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













91

9836

89

81

83

100

100

100

100

91

77

9467

9958

54

32

49

7653

58

66

02













cyt -552 (EF178295)

cyt -551 (EF178295)

cyt -554

c

c

c







1198987
















3oxidase from















FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










3oxidase from















FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


FRT

FRT

FRT

FRT

FRT

FRT

FRT

Cb

Cb

sacB

sacB


Cyt c-552 gene

c-552 gene

Gm

Gm

Gm



Δ

Δ



















0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0001

001

01

1

10

0 5 10 15 20 25

OD660

Time (h)

(a)

0001

001

01

1

0 5 10 15 20 25

OD660

Time (h)

(b)












Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Electron flow

C

C

CC

C

C

C

C

C

C

C

H+

H+

H+

H+H+

H+

H+H+

H+

H+H+H+

Cytochrome cb

O2 + 4H+ 2H2O










References
























































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















































5



3oxidase from














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article bioenergetics and the role of soluble...

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