research article contrasted reactivity to oxygen...

Research ArticleContrasted Reactivity to Oxygen Tensions in Frankia spStrain CcI3 throughout Nitrogen Fixation and Assimilation

Faten Ghodhbane-Gtari12 Karima Hezbri1 Amir Ktari1 Imed Sbissi1

Nicholas Beauchemin2 Maher Gtari12 and Louis S Tisa2

1 Laboratoire Microorganismes et Biomolecules Actives Universite Tunis El Manar (FST) and Universite Carthage (INSAT)Campus Universitaire 2092 Tunis Tunisia

2 Department of Molecular Cellular amp Biomedical Sciences University of New Hampshire 46 College Road DurhamNH 03824-2617 USA

Correspondence should be addressed to Louis S Tisa louistisaunhedu

Received 18 April 2014 Revised 28 April 2014 Accepted 15 May 2014 Published 28 May 2014

Academic Editor Ameur Cherif

Copyright copy 2014 Faten Ghodhbane-Gtari et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Reconciling the irreconcilable is a primary struggle in aerobic nitrogen-fixing bacteria Although nitrogenase is oxygen and reactiveoxygen species-labile oxygen tension is required to sustain respiration In the nitrogen-fixing Frankia various strategies have beendeveloped through evolution to control the respiration and nitrogen-fixation balance Here we assessed the effect of differentoxygen tensions on Frankia sp strain CcI3 growth vesicle production and gene expression under different oxygen tensionsBoth biomass and vesicle production were correlated with elevated oxygen levels under both nitrogen-replete and nitrogen-deficient conditions The mRNA levels for the nitrogenase structural genes (nifHDK) were high under hypoxic and hyperoxicconditions compared to oxic conditions The mRNA level for the hopanoid biosynthesis genes (sqhC and hpnC) was also elevatedunder hyperoxic conditions suggesting an increase in the vesicle envelope Under nitrogen-deficient conditions the hup2 mRNAlevels increased with hyperoxic environment while hup1 mRNA levels remained relatively constant Taken together these resultsindicate that Frankia protects nitrogenase by the use of multiple mechanisms including the vesicle-hopanoid barrier and increasedrespiratory protection

1 Introduction

The genus Frankia is comprised of nitrogen-fixing actinobac-teria that are able to establish a mutualistic symbiosis witha variety of dicotyledonous host plants that results in theestablishment of a root nodule structure [1ndash6] The bacterianourish their host plant with combined nitrogen and theplants provide in return carbon and energy This symbiosisallows actinorhizal host plants to colonize nutrient-poorsoils Besides its life style within the host plant these bacteriaare members of soil community although less informationis known about this life style [7] Under arid tropic andsubtropic conditions of North Africa actinorhizal plants areessentially represented by fast growing and highly toleranttrees from the family Casuarinaceae [8]

Under atmospheric oxygen conditions Frankia activelyfixes dinitrogen to ammoniumwithin the root nodules of the

host plants and aerobically in culture [9ndash15] The oxygen-labile nitrogenase enzyme is localized within specializedthick-walled structures termed vesicles that are formed inplanta and in vitro [2 16ndash18] Their shape is strain depen-dent and host-plant-influenced Vesicles act as specializedstructures for the nitrogen fixation process and are formedterminally on short side branches of hyphae that have aseptum near their base The mature vesicle is surroundedby an envelope that extends down the stalk of the vesiclepast the basal septum which separates the vesicle from thehypha The envelope surrounding the vesicle is composedof multilaminated lipid layers containing primarily bacterio-hopanetetrol and its derivatives [19ndash22] It is believed that thislipid envelope acts as an oxygen diffusion barrier to protectthe nitrogenase enzyme from oxygen inactivation [19]

Unlike other actinorhizal plants Frankia found withinthe root nodules of Casuarina and Allocasuarina plants are

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 568549 8 pageshttpdxdoiorg1011552014568549

2 BioMed Research International

devoid of symbiotic vesicle structures [23 24] A positivecorrelationwas observed between the differentiation of intra-cellular hyphae and the lignifications of the host-infected cellwalls [23] In several actinorhizal nodules a low oxygen ten-sion was shown to be consistent with the high concentrationsof hemoglobin [2] Frankia are known to produce truncatedhemoglobins [25ndash27] Besides hemoglobins Frankia possesshydrogenases that may act as oxygen-scavenging enzymes[28] Sequencing of several Frankia genomes [29ndash34] hasprovided insight on the physiology and opened up newgenomics tools for these microbes These databases havebeen used in transcriptomics [35ndash37] and proteomics studies[38ndash40] on these bacteria The aim of the present studywas to investigate the expression levels for several selectedgenes involved under different oxygen concentration for theCasuarina compatible Frankia sp strain CcI3 These geneswere involved in the following functions nitrogen fixationand assimilation hopanoid biosynthesis hydrogen uptakeand oxidative stress

2 Materials and Methods

21 Culture Conditions and Experimental Design Frankia spstrain CcI3 [41] was grown and maintained at 28∘C in basalMP growth medium with 50mM propionate and 50mMNH4Cl as carbon and nitrogen sources respectively as

described previously [42]In all experimental procedures Frankia cells were grown

for 7 days in 250mL cylindrical bottles with a workingMP medium volume of 50mL with and without NH

4Cl for

nitrogen-deficient and nitrogen-replete conditions respec-tively Three sets of oxygen tensions were considered oxic(atmospheric condition) hypoxic (reduced partial pressureof oxygen) and hyperoxic (elevated oxygen levels) Hypoxicconditions were generated by placing the cultures in Brewerrsquosjar that contained reduced partial pressures of oxygen by theuse of gas packets (BBL GasPak BBL CampyPak System)For this system water interacts with catalyst in the packetgenerating a reduced partial pressure of oxygen within thechamber Hyperoxic conditions were generated by continu-ously air-sparging the cultures via an aquarium pump

22 Growth Assessment and Vesicle Count For dry weightdeterminations cell cultures were collected on tarred mem-brane filters (type HA 045 um pore size Millipore Corp)The filters were placed in a Petri dish over desiccant anddried at 90∘C to constant weight [43] In parallel proteincontent was measured Briefly cell samples were solubilizedby heating for 15min at 90∘C in 10NNaOHand total proteinswere measured using BCA method [44]

Vesicle numberswere determined as previously described[45 46] Briefly cells were sonicated for 30 s with a Braunmodel 350 sonifier under power setting of 3 using microtipprobe This treatment disrupted the mycelia and releasedvesicles The numbers of vesicles were counted by usinga Petroff-Hausser counting chamber with a phase-contrastmicroscope at magnification of 400x

23 Determination of Ammonia Ammonium concentrationwas determined in cell-free media usingmodified protocol ofBerthelotrsquos reagent [47]

24 RNA Extraction RT-PCRs and Q-PCR For these exper-iments all solutions and materials were DEPC-treated toprevent RNA degradation RNA extractions were performedby the TritonX100method as previously described [48] RNAsamples were treated with DNase I (New England Biolabs)according to the manufacturerrsquos recommendations RNAsamples were quantified with a Nanodrop 2000c spectropho-tometer (Thermo Scientific) and stored at minus80∘C until useThe cDNA synthesis was performed using hexamer primers400 ng RNA and SuperScript III reverse transcriptase (Invit-rogen) according to the manufacturerrsquos recommendationsThe cDNA was quantified by a Nanodrop 2000c spectropho-tometer diluted to 10 ng120583L working stocks in DNAse-freeRNAse-free H

2O and stored at minus20∘C until use

Frankia gene expression analyses were performed byqRT-PCR using specific primers (Table 1) and SYBR GreenPCR Master Mix (Applied Biosystems) as described previ-ously [49] Briefly each 25 120583L reaction contained 50 ng tem-plate cDNA 300 nM of the forward and reverse primer mixand SYBRGreen PCRMasterMix Parameters for the AgilentMP3000 were as follows (1) 95∘C for 15min (2) 40 cycles of95∘C for 15 s and 60∘C for 30 s and (3) thermal disassociationcycle of 95∘C for 60 s 55∘C for 30 s and incremental increasesin temperature to 95∘C for 30 s Reactions were performedin triplicates and the comparative threshold-cycle methodwas used to quantify gene expression The results werestandardized with rpsA expression levels Relative expression(fold changes) was determined by the Pfafflmethod [50] withthe control as the calibrator Two biological replicates of thetriplicate samples were averaged

3 Results

31 Growth and Vesicle Production under Different OxygenPressures Figure 1 shows the effect of oxygen on the growthyield of Frankia sp strain CcI3 Under nitrogen-repleteconditions (NH

4) the biomass of cells grown under hyper-

oxic conditions was greater than both cultures grown underoxic and hypoxic conditions Under nitrogen-deficient (N

2)

conditions the biomass correlated with the oxygen level withthe hyperoxic conditions generating the greatest biomassFurthermore vesicle production under nitrogen-deficient(N2) conditions positively correlated with oxygen tension

Cells under hyperoxic (air-sparged) conditions produced 26-and 54-fold more vesicles (650 plusmn 041 times 106mg) than oxic(245 plusmn 029 times 106mg) and hypoxic (120 plusmn 036 times 106mg)conditions respectively Analysis of ammonia metabolism byFrankia CcI3 indicates that it was correlated with oxygentension With nitrogen-replete conditions hyperoxic condi-tions resulted in the highest ammonia consumption followedby oxic condition and lastly hypoxic condition (Figure 1(c))Under nitrogen-deficient conditions the level of ammo-nium ions increased under lower oxygen tension This leveldecreased with corresponding increases in oxygen tension

BioMed Research International 3

Table 1 Primers used in this study

Locus tag Gene Gene identity Sequence

francci3 4488 nifH Nitrogenase reductase iron-sulfur protein 51015840-CGACAACGACATGAAGACC-3101584051015840-CTTGCCGATGATGCTCTC-31015840

francci3 4487 nifD Nitrogenase molybdenum-iron protein alpha chain 51015840-AAGGACATCGTCAACATCAGCCAC-3101584051015840-AACTGCATCGCGGCGAAGTTATTC-31015840

francci3 4486 nifK Nitrogenase molybdenum-iron protein beta chain 51015840-TGACGACGACTCCGGAAACAAACA-3101584051015840-TGTGGTAGACCTCGTCCTTGAACA-31015840

francci3 4496 hup1 Nickel-dependant hydrogenase large subunit 51015840-AACAAATCTGCGACGTCACGGTCA-3101584051015840-ACTCTCGATCCATTCACCGCAGTA-31015840

francci3 1076 hup2 Uptake hydrogenase large subunit 51015840-TGGAAGGTCAACTGGCTGGAGAA-3101584051015840-ATGTCTAGGCAGTACCGGAGGAAGAA-31015840

francci3 1149 hboO Truncated hemoglobin 51015840-GGGACGCCTGGCTGAAGA-3101584051015840-CCAGAGCTGCCTGTCGAGATC-31015840

francci3 2581 hboN Truncated hemoglobin 51015840-CACCCCTCTTTGCCAACCG-3101584051015840-GGTGGTTTCCGTCGGGAC-31015840

francci3 0823 sqhC Squalene hopene cyclase 51015840-TGCAATGGCTGCTGGACAA-3101584051015840-TGCCGTAGACGTGGTTGAT-31015840

francci3 0819 hpnC Squalene synthase 51015840-AACTTCCCGGTCTCGCCGTT-3101584051015840-AACGCGTTGAAGTGGAAACGAACC-31015840

francci3 2949 katA Catalase 51015840-ACATGCCGGTGTTCTTCATTCAGG-3101584051015840-ACATCATCATGTGGCATCGACTCGG-31015840

francci3 2817 sodA Superoxide dismutase 51015840-GTGCCAATGACACCCTTGAGAAGA-3101584051015840-AGTGGAGAATATGCCCGGAAAGGT-31015840

francci3 3012 gltD Glutamate synthase small subunit 51015840-TGCATGCGACGAACAACTTCCC-3101584051015840-ATGATGCTGACCTCGATCTGCTTG-31015840

francci3 3013 gltB Glutamate synthase large subunit 51015840-CGTGCTGAAGGTGATGTCCAAGAT-3101584051015840-AAATAGGCGTCGATCAGTTCCTGG-31015840

francci3 3142 glnA Glutamine synthetase type I 51015840-ATGACCCGATCACCAAGGAACAGT-3101584051015840-GGGTTGTAGTCATAACGGACATCG-31015840

francci3 3143 glnA Glutamine synthetase type II 51015840-AACTTCTCCACCAGGCAGACGAT-3101584051015840-AGAACTTGTTCCACGGAGCTGTCT-31015840

francci3 4059 glnA Glutamine synthetase catalytic region 51015840-TACAACATCGACTACGCGCTTTCC-3101584051015840-ATACCGGAACACGATCTCGAACTG-31015840

francci3 1057 rpsA 30S ribosomal protein S1 51015840-CGAAGTCCGTTCCGAGTTC-3101584051015840-CGCCGAAGTTGACGATGG-31015840

Locus tag and gene designationwere determined from the IntegratedMicrobial Genomes System (IMG) at the Joint Genome Institute (httpsimgjgidoegov)[51]

32 Expression of Nitrogen Fixation and Assimilation Genesunder Different Oxygen Pressures The effect of oxygen on theexpression of several genes involved in nitrogen fixation andassimilation was measured by detecting changes in mRNAlevels via qRT-PCR (Figure 2) For nitrogen-deficient con-ditions the level of structural nitrogenase genes (nifHDK)mRNA increased gt10-fold under hyperoxic and hypoxicconditions compared to oxic condition (Figure 2(a)) Undernitrogen-replete conditions the expression levels for thesegenes were very low and there was no change with differentoxygen tensions

The Frankia genome contains two glutamate synthasegenes (gltB and gltD) encoding the large and small subunits ofthe enzymeThese two glutamate synthase geneswere studiedfor their expression levels under three oxygen tensions ThemRNA levels of the gltB gene were reduced except underhyperoxic and nitrogen-replete conditions (Figure 2(b)) ThegltD mRNA levels increased slightly (13ndash25-fold) under thedifferent nitrogen and oxygen conditions There were four

glutamine synthetase orthologs found within the Frankia spstrain CcI3 genome We were able to follow the expressionof three of these glnA genes (Figure 2(c)) The level offrancci3 3143 mRNA was controlled by nitrogen Under alloxygen conditions francci3 3143mRNA levels increased 10ndash15-fold under nitrogen-deficient (N

2) conditions Both high

and low oxygen tensions increased the level of francci3 3143mRNA The level of francci3 3142 mRNA was decreasedunder nitrogen-deficient (N

2) conditions and showed 7-fold

increase under hyperoxic under nitrogen-replete conditionsThe levels of francci3 4059mRNA remained constant exceptunder hyperoxic conditions in which levels increased 15-fold Under hyperoxic conditions the levels of francci3 4059mRNA were controlled by nitrogen status and increasedapproximately 2-3-fold from nitrogen-replete (NH

4) condi-

tions

33 Expression of Genes Known to Protect Nitrogenase fromOxygen and Reactive Oxygen Species The biosynthesis of


0

005

01

015

02

025

03D

ry w

eigh

t (m

gm

L)

LN2 LNH4 NN2 NNH4 HN2 HNH4

(a)

0

002

004

006

008

01

012

014

016

Prot

ein

(mg

mL)


(b)

0

10

20

30

40

50

60

70

Am

mon

ium

ions

(mg

L)


(c)

Figure 1 Biomass yields of Frankia sp strain CcI3 grown under nitrogen fixation (N2

) and nitrogen-replete (NH4

) at hypoxic (L) oxic (N)and hyperoxic (H) conditions as estimation by (a) dry weight and (b) total protein and determination of (c) ammonium ion concentrations

hopanoids has been correlated with vesicle development[19] The effect of oxygen tension on the expression of thesqualene synthase (hpnC) and squalenephytoene cyclase(sqhC) genes was examined (Figure 2(d)) Under nitrogen-replete conditions (NH

4) the level ofmRNA for sqhC showed

a 2-fold increase for hyperoxic conditions A smaller increasewas observed for hpnC mRNA levels In general sqhC andhpnC were expressed constitutively with comparable mRNAlevels for hypoxic and oxic levels Under nitrogen-deficient(N2) conditions the mRNA levels of both genes (sqhC and

hpnC) increased 2- and 15-fold respectivelyThe Frankia CcI3 genome contains two hydrogenase

operons [30 52 53] We tested the effects of oxygen ten-sion and nitrogen status of their gene expression levels(Figure 2(e)) Under nitrogen-replete (NH

4) conditions the

level of mRNA for hup2 increased proportionally with thelevel of oxygen present while the level of mRNA for hup1only increased under hyperoxic conditionsThe expression ofhup2 was influenced by the nitrogen status of the cells and bythe oxygen levels Under both conditions hup2 mRNA levelsincreased but hup1 expression remained constant

The effect of oxygen tension and nitrogen status wasinvestigated on the expression of two truncated hemoglobins(hboO and hboN) The level of mRNA of hboO and hboNincreased under hyperoxic condition for both nitrogen con-ditions (Figure 2(f)) Under nitrogen-replete (NH

4) condi-

tions mRNA levels for hboO increased proportionally to

the oxygen tension levels Under hypoxic nitrogen-deficientconditions mRNA levels for hboN increased about 15-fold

The effects of oxygen tension and nitrogen status on theexpression levels of two oxygen defense enzymes catalase(katA) and superoxide dismutase (sodA) were also tested(Figure 2(g)) Under hyperoxic conditions the mRNA levelsof katA increased 65- and 8-fold under nitrogen-deficient(N2) andnitrogen-replete (NH

4) conditions respectivelyThe

expression of the sodAgene appeared to be constitutive underall oxygen tensions and both nitrogen statuses

4 Discussion

Without a doubt the vesicle is the most characteristicmorphogenetic structure produced by Frankia [1] Vesiclesare functionally analogous to cyanobacterial heterocystsproviding unique specialized cells that allow nitrogen fixationunder aerobic condition [54 55] In this study the growthof Frankia strain CcI3 was evaluated under three oxygentensions The results indicate that growth increased withelevated oxygen tensions (Figure 1) confirming the aerobicnature of themicrobe Although the dry weightmeasurementincreased the total protein values were reduced under hyper-oxic nitrogen-deficient (N

2) conditions This result would

imply that the cells were producing other metabolic productsunder this condition and a similar level of protein comparedto hypoxic nitrogen-deficient (N

2) conditionThus this result


0

50

100

150

200

250

nifHnifD

nifK


(a)

0

05

1

15

2

25

3

gltDgltB


(b)

05

101520253035

francci3 3142francci3 3143

francci3 4059


(c)

0

1

2

3

4

5

6

sqhChpnC


(d)

0

5

10

15

20

25

30

hup2hup1


(e)

005

115

225

335

445

5

HboOHboN


(f)

0123456789

SodAKatA


(g)

Figure 2 Relative gene expression (fold change) in response to hyperoxic and hypoxic conditions Frankia cultures were grown undernitrogen-replete (NH

4

) or nitrogen-deficient (N2

) conditions These cultures were exposed to oxic (N) hyperoxic (H) and hypoxic (L)conditions as described in Section 2 Experimental gene expression was normalized to the rpsA housekeeping gene and compared to thecalibrator (NH

4

oxic conditions) The following genes were analyzed (a) nifHDK (b) gltB and gltD (c) glnA genes (d) hpnC and sqhC (e)hup1 and hup2 (f) hboN and hboO and (g) sodA and katA

suggests that part of the respiration was uncoupled providingsome oxygen protection Frankia contains two respiratorysystems and a cyanide-insensitive system was proposed tohelp protect nitrogenase from oxygen inactivation [46]Withother aerobic nitrogen-fixing bacteria increased respiratoryrates in response to elevated oxygen tensions help maintainlow levels of intracellular oxygen protecting nitrogenase frominactivation [56 57] Under nitrogen-deficient (N

2) condi-

tions vesicles were produced and correlated with oxygen

tensions The numbers of vesicles produced per mg dryweight increased with elevated oxygen levels These resultsconfirm those obtained previously [58 59]

In our study we investigated the effects of oxygen ongene expression for a variety of functional genes involved innitrogen fixation nitrogen assimilation and protection fromoxygen and other reactive oxygen species [60] The levelsof expression for the structural nitrogenase genes (nifHDK)indicate a concordant profile with clear induction under


nitrogen-deficient (N2) conditions Transcriptome studies

on Frankia sp strain CcI3 under nitrogen-deficient andnitrogen-replete conditions also show an increase in nifHDKgene expression [35 36] The levels of nifHDK mRNAshowed an increase under hypoxic and hyperoxic conditionsindicating that nitrogenase induction was influenced byoxygen levels

The hopanoid envelope has been postulated to beinvolved in the protection of nitrogenase from oxygeninactivation [19] We found that mRNA levels of squalenesynthase (hpnC) and squalene-hopene cyclase (sqhC) genesincreased in response to oxygen tension under nitrogen-deficient conditions but remained constant under nitrogen-replete conditions (Figure 2(d)) The results correlate withthe increase in vesicle envelope observed under high oxygenlevels [61] Nalin et al [62] found only a slightly higherhopanoid content under nitrogen-deficient conditions sug-gesting remobilization rather than nascent biosynthesis Fur-thermore the Frankia sp strain CcI3 transcriptome profilesunder nitrogen-deficient and nitrogen-replete conditions didnot show any significant differences in hopanoid biosyntheticgenes [35 36] However these studies were performed underone oxygen tension while our study has investigated threedifferent oxygen tensions

Analysis of the nitrogen assimilation genes (gltB gltDand glnA) is a bit more complex The Frankia CcI3 genomecontained several homologues of glnA The mRNA level offrancci3 3143 correlated the best with nitrogen regulationbeing increased under nitrogen-deficient conditions Tran-scriptome studies have shown that francci3 3143 expressionincreased significantly under nitrogen-fixing conditions [3536] while all of the other homologues remained consistentThis result would suggest that this gene encoded primarynitrogen scavenging enzyme The levels of expression werealso influenced by elevated oxygen tensions during increasednitrogenase activityThe expression levels of the gltB and gltDappear to be less influenced by oxygen tension These effectsseemed in agreement with the ammonia metabolism resultsthat showed an increase in consumption under hyperoxicconditions

Our results on hemoglobin gene expression correlate withprevious results [48] that showed no increase in hboN andhboO expression in response to nitrogen status increasedunder low oxygen tension However our results conflict inresponse to oxygen We found that both hboN and hboOmRNA levels increased under hyperoxic conditions The useof the more sensitive qRT-PCR in our study compared to RT-PCR is the best explanation for these differences

Frankia possesses two uptake hydrogenase systems [5253] One of them has been correlated with symbiotic growthand the other to free-living conditions [53] Our results showthat hup2 gene expression was influenced by nitrogen statussuggesting that it was associated with vesicle productionwhile hup1 gene expression was relatively constant Thelevels of hup2 mRNA increased proportionally with oxygentensions suggesting potential oxygen protection mechanismAnoxic conditions have no effect on hydrogenase geneexpression by Frankia CcI3 but increased by 30 for Frankia

alni ACN14a [60] We did not test anoxic conditions in ourstudy

Increased oxygen tension can lead to elevated oxidativestress conditions We investigated the influence of oxygentensions on reactive oxidative stress genes While sodAexpression levels were constitutive katA gene expressionincreased under hyperoxic conditions In general our resultsconfirm those of Steele and Stowers [63] which examinedenzymatic activity levels They reported an increase in cata-lase activity in cultures derepressed for nitrogen fixationcompared to ammonium-grown cultures

Conflict of Interests

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

Acknowledgments

Louis S Tisa was supported in part by Agriculture and FoodResearch Initiative Grant 2010-65108-20581 from the USDANational Institute of Food and Agriculture Hatch GrantNH530 and the College of Life Sciences and Agricultureat the University of New Hampshire Durham NH USAThis is scientific contribution number 2556 from the NHAgricultural Experimental Station Maher Gtari and FatenGhodhbane-Gtari were supported in part by a VisitingScientist and Postdoctoral Scientist Program administered bythe NH AES at the University of New Hampshire

References

[1] D R Benson and W B Silvester ldquoBiology of Frankia strainsactinomycete symbionts of actinorhizal plantsrdquoMicrobiologicalReviews vol 57 no 2 pp 293ndash319 1993

[2] K Huss-Danell ldquoActinorhizal symbioses and their N2

fixationrdquoNew Phytologist vol 136 no 3 pp 375ndash405 1997

[3] L GWall ldquoThe actinorhizal symbiosisrdquo Journal of Plant GrowthRegulation vol 19 no 2 pp 167ndash182 2000

[4] C Santi D Bogusz and C Franche ldquoBiological nitrogen fixa-tion in non-legume plantsrdquo Annals of Botany vol 111 no 5 pp743ndash767 2013

[5] J Schwencke andM Caru ldquoAdvances in actinorhizal symbiosishost plant-Frankia interactions biology and applications inarid land reclamation A reviewrdquo Arid Land Research andManagement vol 15 no 4 pp 285ndash327 2001

[6] A Sellstedt and K H Richau ldquoAspects of nitrogen-fixing actin-obacteria in particular free-living and symbiotic FrankiardquoFEMS Microbiology Letters vol 342 no 2 pp 179ndash186 2013

[7] E E Chaia L G Wall and K Huss-Danell ldquoLife in soil by theactinorhizal root nodule endophyte Frankia A reviewrdquo Symbi-osis vol 51 no 3 pp 201ndash226 2010

[8] M Gtari and J O Dawson ldquoAn overview of actinorhizal plantsin Africardquo Functional Plant Biology vol 38 no 8-9 pp 653ndash6612011

[9] J D Tjepkema W Ormerod and J G Torrey ldquoVesicle for-mation and acetylene reduction activity in Frankia sp CPI1cultured in defined nutrient mediardquo Nature vol 287 no 5783pp 633ndash635 1980


[10] J D TjepkemaW Ormerod and J G Torrey ldquoFactors affectingvesicle formation and acetylene reduction (nitrogenase activity)in Frankia sp CpI1rdquo Canadian Journal of Microbiology vol 27no 8 pp 815ndash823 1981

[11] D Gauthier H G Diem and Y Dommergues ldquoIn vitro nitro-gen fixation by 2 actinomycete strains isolated from Casuarinanodulesrdquo Applied and Environmental Microbiology vol 41 pp306ndash308 1981

[12] M A Murry Z Zhongze and J G Torrey ldquoEffect of O2

onvesicle formation acetylene reduction and O

2

-uptake kineticsin Frankia sp HFPCc13 isolated from Casuarina cunninghami-anardquo Canadian Journal of Microbiology vol 31 no 9 pp 804ndash809 1985

[13] Z Zhongze and J G Torrey ldquoBiological and cultural character-istics of the effective Frankia strain HFPCcl3 (Actinomycetales)from Casuarina cunninghamiana (Casuarinaceae)rdquo Annals ofBotany vol 56 no 3 pp 367ndash378 1985

[14] M S Fontaine S A Lancelle and J G Torrey ldquoInitiation andontogeny of vesicles in cultured Frankia sp strain HFPArI3rdquoJournal of Bacteriology vol 160 no 3 pp 921ndash927 1984

[15] A J P Burggraaf and W A Shipton ldquoStudies on the growth ofFrankia isolates in relation to infectivity and nitrogen fixation(acetylene reduction)rdquo Canadian Journal of Botany vol 61 no11 pp 2774ndash2782 1983

[16] T M Meesters ldquoLocalization of nitrogenase in vesiclesof Frankia sp Cc117 by immunogoldlabelling on ultrathincryosectionsrdquo Archives of Microbiology vol 146 no 4 pp 327ndash331 1987

[17] T M Meesters S T Van Genesen and A D L AkkermansldquoGrowth acetylene reduction activity and localization of nitro-genase in relation to vesicle formation in Frankia strains Cc117and Cp12rdquo Archives of Microbiology vol 143 no 2 pp 137ndash1421985

[18] T M Meesters W M Vanvliet and A D L AkkermansldquoNitrogenase is restricted to the vesicles in Frankia strainEAN1pecrdquo Physiologia Plantarum vol 70 no 2 pp 267ndash2711987

[19] A M Berry O T Harriott R A Moreau S F Osman D RBenson andA D Jones ldquoHopanoid lipids compose the Frankiavesicle envelope presumptive barrier of oxygen diffusion tonitrogenaserdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 90 no 13 pp 6091ndash6094 1993

[20] A M Berry R A Moreaua and A D Jones ldquoBacterio-hopanetetrol abundant lipid in Frankia cells and in nitrogen-fixing nodule tissuerdquo Plant Physiology vol 95 no 1 pp 111ndash1151991

[21] O T Harriott L Khairallah and D R Benson ldquoIsolation andstructure of the lipid envelopes from the nitrogen-fixing vesiclesof Frankia sp strain CpI1rdquo Journal of Bacteriology vol 173 no6 pp 2061ndash2067 1991

[22] H C Lamont W B Silvester and J G Torrey ldquoNile red fluo-rescence demonstrates lipid in the envelope of vesicles fromN

2

-fixing cultures of Frankiardquo Canadian Journal of Microbiologyvol 34 no 5 pp 656ndash660 1988

[23] R H Berg and L McDowell ldquoEndophyte differentiation inCasuarina actinorhizaerdquo Protoplasma vol 136 no 2-3 pp 104ndash117 1987

[24] R H Berg and L Mcdowell ldquoCytochemistry of the wall ofinfected-cells in Casuarina actinorhizaerdquo Canadian Journal ofBotany vol 66 no 10 pp 2038ndash2047 1988

[25] V Coats C R Schwintzer and J D Tjepkema ldquoTruncatedhemoglobins in FrankiaCcI3 effects of nitrogen source oxygen

concentration and nitric oxiderdquo Canadian Journal of Microbi-ology vol 55 no 7 pp 867ndash873 2009

[26] J D Tjepkema ldquoHemoglobins in the nitrogen-fixing rootnodules of actinorhizal plantsrdquoCanadian Journal of Botany vol61 no 11 pp 2924ndash2929 1983

[27] J D Tjepkema R E Cashon J Beckwith and C R SchwintzerldquoHemoglobin in Frankia a nitrogen-fixing actinomyceterdquoApplied and Environmental Microbiology vol 68 no 5 pp2629ndash2631 2002

[28] A Sellstedt P Reddell and P Rosbrook ldquoThe occurrenceof hemoglobin and hydrogenase in nodules of 12 Casuarina-Frankia symbiotic associationsrdquo Physiologia Plantarum vol 82no 3 pp 458ndash464 1991

[29] F Ghodbhane-Gtari N Beauchemin D Bruce et al ldquoDraftgenome sequence of Frankia sp strain CN3 an atypicalnon-infective (Nod-) ineffective (Fix-) isolate from Coriarianepalensisrdquo Genome Announcements vol 1 no 2 Article IDe0008513 2013

[30] P Normand P Lapierre L S Tisa et al ldquoGenome character-istics of facultatively symbiotic Frankia sp strains reflect hostrange and host plant biogeographyrdquo Genome Research vol 17no 1 pp 7ndash15 2007

[31] A Sen N Beauchemin D Bruce et al ldquoDraft genome sequenceof Frankia sp strain QA3 a nitrogen-fixing actinobacteriumisolated from the root nodule of Alnus nitidardquo GenomeAnnouncements vol 1 no 2 Article ID e0010313 2013

[32] S RMansour R Oshone S G Hurst KMorrisW KThomasand L S Tisa ldquoDraft genome sequence of Frankia sp strainCcI6 a salt-tolerant nitrogen-fixing actinobacterium isolatedfrom the root nodule of Casuarina cunninghamianardquo GenomeAnnouncements vol 2 no 1 Article ID e01205-13 2014

[33] I Nouioui N Beauchemin M N Cantor et al ldquoDraftgenome sequence of Frankia sp strain BMG512 a nitrogen-fixing actinobacterium isolated from Tunisian soilsrdquo GenomeAnnouncements vol 1 no 4 Article ID e00468-13 2013

[34] L G Wall N Beauchemin M N Cantor et al ldquoDraft genomesequence of Frankia sp strain BCU110501 a nitrogen-fixingactinobacterium isolated from nodules of Discaria trinevisrdquoGenome Announcements vol 1 no 4 Article ID e00503-132013

[35] D M Bickhart and D R Benson ldquoTranscriptomes of Frankiasp strain CcI3 in growth transitionsrdquoBMCMicrobiology vol 11article 192 2011

[36] H-I Lee A J Donati D Hahn L S Tisa and W-SChang ldquoAlteration of the exopolysaccharide production andthe transcriptional profile of free-living Frankia strain CcI3under nitrogen-fixing conditionsrdquo Applied Microbiology andBiotechnology vol 97 no 24 pp 10499ndash10509 2013

[37] N Alloisio C Queiroux P Fournier et al ldquoThe Frankiaalni symbiotic transcriptomerdquoMolecular Plant-Microbe Interac-tions vol 23 no 5 pp 593ndash607 2010

[38] N Alloisio S Felix J Marechal et al ldquoFrankia alni proteomeunder nitrogen-fixing and nitrogen-replete conditionsrdquo Physi-ologia Plantarum vol 130 no 3 pp 440ndash453 2007

[39] J E Mastronunzio and D R Benson ldquoWild nodules can bebroken proteomics of Frankia in field-collected root nodulesrdquoSymbiosis vol 50 no 1-2 pp 13ndash26 2010

[40] J E Mastronunzio Y Huang and D R Benson ldquoDiminishedexoproteome of Frankia spp in culture and symbiosisrdquo Appliedand Environmental Microbiology vol 75 no 21 pp 6721ndash67282009


[41] Z Zhang M F Lopez and J G Torrey ldquoA comparison ofcultural characteristics and infectivity of Frankia isolates fromroot nodules of Casuarina speciesrdquo Plant and Soil vol 78 no1-2 pp 79ndash90 1984

[42] N J Beauchemin T Furnholm J Lavenus et al ldquoCasuarinaroot exudates alter the physiology surface properties and plantinfectivity of Frankia sp strain CcI3rdquo Applied and Environmen-tal Microbiology vol 78 no 2 pp 575ndash580 2012

[43] L Tisa M Mcbride and J C Ensign ldquoStudies of growthand morphology of Frankiastrains EAN1pec EuI1c CpI1 andACN1AGrdquo Canadian Journal of Botany vol 61 no 11 pp 2768ndash2773 1983

[44] P K Smith R I Krohn G T Hermanson et al ldquoMeasurementof protein using bicinchoninic acidrdquo Analytical Biochemistryvol 150 no 1 pp 76ndash85 1985

[45] L S Tisa and J C Ensign ldquoComparative physiology of nitroge-nase activity and vesicle development for Frankia strains CpI1ACN1AG EAN1pec and EUN1frdquoArchives ofMicrobiology vol 147no 4 pp 383ndash388 1987

[46] L S Tisa and J C Ensign ldquoThe calcium requirement forfunctional vesicle development and nitrogen fixation by Frankiastrains EAN1pec and CpI1rdquoArchives of Microbiology vol 149 no1 pp 24ndash29 1987

[47] E D Rhine G K Sims R L Mulvaney and E J PrattldquoImproving the Berthelot reaction for determining ammoniumin soil extracts and waterrdquo Soil Science Society of AmericaJournal vol 62 no 2 pp 473ndash480 1998

[48] J Niemann and L S Tisa ldquoNitric oxide and oxygen regulatetruncated hemoglobin gene expression in Frankia strain CcI3rdquoJournal of Bacteriology vol 190 no 23 pp 7864ndash7867 2008

[49] F Perrine-Walker P Doumas M Lucas et al ldquoAuxin carrierslocalization drives auxin accumulation in plant cells infectedby Frankia in Casuarina glauca actinorhizal nodulesrdquo PlantPhysiology vol 154 no 3 pp 1372ndash1380 2010

[50] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquoNucleic Acids Research vol 29 no9 article e45 2001

[51] V M Markowitz F Korzeniewski K Palaniappan et al ldquoTheintegrated microbial genomes (IMG) systemrdquo Nucleic AcidsResearch vol 34 pp D344ndashD348 2006

[52] P Normand C Queiroux L S Tisa et al ldquoExploring thegenomes of Frankiardquo Physiologia Plantarum vol 130 no 3 pp331ndash343 2007

[53] M Leul P Normand and A Sellstedt ldquoThe organizationregulation and phylogeny of uptake hydrogenase genes inFrankiardquo Physiologia Plantarum vol 130 no 3 pp 464ndash4702007

[54] N A Noridge and D R Benson ldquoIsolation and nitrogen-fixing activity of Frankia sp strain CpI1 vesiclesrdquo Journal ofBacteriology vol 166 no 1 pp 301ndash305 1986

[55] L S Tisa and J C Ensign ldquoIsolation and nitrogenase activity ofvesicles from Frankia sp strain EAN1pecrdquo Journal of Bacteriol-ogy vol 169 no 11 pp 5054ndash5059 1987

[56] A Hochman and R H Burris ldquoEffect of oxygen on acetylenereduction by photosynthetic bacteriardquo Journal of Bacteriologyvol 147 no 2 pp 492ndash499 1981

[57] H Dalton and J R Postgate ldquoEffect of oxygen on growth ofAzotobacter chroococcum in batch and continuous culturesrdquoJournal ofGeneralMicrobiology vol 54 no 3 pp 463ndash473 1968

[58] M AMurry Z Zhongze and J G Torrey ldquoEffect of O2

on vesi-cle formation acetylene reduction and O

2

-uptake kinetics in

Frankia spHFPCcI3 isolated fromCasuarina cunninghamianardquoCanadian Journal of Microbiology vol 31 no 9 pp 804ndash8091985

[59] Z Zhongze M A Murry and J G Torrey ldquoCulture condi-tions influencing growth and nitrogen fixation in Frankia spHFPCcI3 isolated from Casuarinardquo Plant and Soil vol 91 no 1pp 3ndash15 1986

[60] K H Richau R L Kudahettige P Pujic N P Kudahettigeand A Sellstedt ldquoStructural and gene expression analyses ofuptake hydrogenases and other proteins involved in nitrogenaseprotection in Frankiardquo Journal of Biosciences vol 38 no 4 pp703ndash712 2013

[61] R Parsons W B Silvester S Harris W T Gruijters andS Bullivant ldquoFrankia vesicles provide inducible and absoluteoxygen protection for nitrogenaserdquo Plant Physiology vol 83 no4 pp 728ndash731 1987

[62] R Nalin S R Putra A-M Domenach M Rohmer F Gour-biere and A M Berry ldquoHigh hopanoidtotal lipids ratio inFrankiamycelia is not related to the nitrogen statusrdquoMicrobiol-ogy vol 146 no 11 pp 3013ndash3019 2000

[63] D B Steele and M D Stowers ldquoSuperoxide dismutase andcatalase in Frankiardquo Canadian Journal of Microbiology vol 32no 5 pp 409ndash413 1986

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


devoid of symbiotic vesicle structures [23 24] A positivecorrelationwas observed between the differentiation of intra-cellular hyphae and the lignifications of the host-infected cellwalls [23] In several actinorhizal nodules a low oxygen ten-sion was shown to be consistent with the high concentrationsof hemoglobin [2] Frankia are known to produce truncatedhemoglobins [25ndash27] Besides hemoglobins Frankia possesshydrogenases that may act as oxygen-scavenging enzymes[28] Sequencing of several Frankia genomes [29ndash34] hasprovided insight on the physiology and opened up newgenomics tools for these microbes These databases havebeen used in transcriptomics [35ndash37] and proteomics studies[38ndash40] on these bacteria The aim of the present studywas to investigate the expression levels for several selectedgenes involved under different oxygen concentration for theCasuarina compatible Frankia sp strain CcI3 These geneswere involved in the following functions nitrogen fixationand assimilation hopanoid biosynthesis hydrogen uptakeand oxidative stress

2 Materials and Methods

21 Culture Conditions and Experimental Design Frankia spstrain CcI3 [41] was grown and maintained at 28∘C in basalMP growth medium with 50mM propionate and 50mMNH4Cl as carbon and nitrogen sources respectively as

described previously [42]In all experimental procedures Frankia cells were grown

for 7 days in 250mL cylindrical bottles with a workingMP medium volume of 50mL with and without NH

4Cl for

nitrogen-deficient and nitrogen-replete conditions respec-tively Three sets of oxygen tensions were considered oxic(atmospheric condition) hypoxic (reduced partial pressureof oxygen) and hyperoxic (elevated oxygen levels) Hypoxicconditions were generated by placing the cultures in Brewerrsquosjar that contained reduced partial pressures of oxygen by theuse of gas packets (BBL GasPak BBL CampyPak System)For this system water interacts with catalyst in the packetgenerating a reduced partial pressure of oxygen within thechamber Hyperoxic conditions were generated by continu-ously air-sparging the cultures via an aquarium pump

22 Growth Assessment and Vesicle Count For dry weightdeterminations cell cultures were collected on tarred mem-brane filters (type HA 045 um pore size Millipore Corp)The filters were placed in a Petri dish over desiccant anddried at 90∘C to constant weight [43] In parallel proteincontent was measured Briefly cell samples were solubilizedby heating for 15min at 90∘C in 10NNaOHand total proteinswere measured using BCA method [44]

Vesicle numberswere determined as previously described[45 46] Briefly cells were sonicated for 30 s with a Braunmodel 350 sonifier under power setting of 3 using microtipprobe This treatment disrupted the mycelia and releasedvesicles The numbers of vesicles were counted by usinga Petroff-Hausser counting chamber with a phase-contrastmicroscope at magnification of 400x

23 Determination of Ammonia Ammonium concentrationwas determined in cell-free media usingmodified protocol ofBerthelotrsquos reagent [47]

24 RNA Extraction RT-PCRs and Q-PCR For these exper-iments all solutions and materials were DEPC-treated toprevent RNA degradation RNA extractions were performedby the TritonX100method as previously described [48] RNAsamples were treated with DNase I (New England Biolabs)according to the manufacturerrsquos recommendations RNAsamples were quantified with a Nanodrop 2000c spectropho-tometer (Thermo Scientific) and stored at minus80∘C until useThe cDNA synthesis was performed using hexamer primers400 ng RNA and SuperScript III reverse transcriptase (Invit-rogen) according to the manufacturerrsquos recommendationsThe cDNA was quantified by a Nanodrop 2000c spectropho-tometer diluted to 10 ng120583L working stocks in DNAse-freeRNAse-free H

2O and stored at minus20∘C until use

Frankia gene expression analyses were performed byqRT-PCR using specific primers (Table 1) and SYBR GreenPCR Master Mix (Applied Biosystems) as described previ-ously [49] Briefly each 25 120583L reaction contained 50 ng tem-plate cDNA 300 nM of the forward and reverse primer mixand SYBRGreen PCRMasterMix Parameters for the AgilentMP3000 were as follows (1) 95∘C for 15min (2) 40 cycles of95∘C for 15 s and 60∘C for 30 s and (3) thermal disassociationcycle of 95∘C for 60 s 55∘C for 30 s and incremental increasesin temperature to 95∘C for 30 s Reactions were performedin triplicates and the comparative threshold-cycle methodwas used to quantify gene expression The results werestandardized with rpsA expression levels Relative expression(fold changes) was determined by the Pfafflmethod [50] withthe control as the calibrator Two biological replicates of thetriplicate samples were averaged

3 Results

31 Growth and Vesicle Production under Different OxygenPressures Figure 1 shows the effect of oxygen on the growthyield of Frankia sp strain CcI3 Under nitrogen-repleteconditions (NH

4) the biomass of cells grown under hyper-

oxic conditions was greater than both cultures grown underoxic and hypoxic conditions Under nitrogen-deficient (N

2)

conditions the biomass correlated with the oxygen level withthe hyperoxic conditions generating the greatest biomassFurthermore vesicle production under nitrogen-deficient(N2) conditions positively correlated with oxygen tension

Cells under hyperoxic (air-sparged) conditions produced 26-and 54-fold more vesicles (650 plusmn 041 times 106mg) than oxic(245 plusmn 029 times 106mg) and hypoxic (120 plusmn 036 times 106mg)conditions respectively Analysis of ammonia metabolism byFrankia CcI3 indicates that it was correlated with oxygentension With nitrogen-replete conditions hyperoxic condi-tions resulted in the highest ammonia consumption followedby oxic condition and lastly hypoxic condition (Figure 1(c))Under nitrogen-deficient conditions the level of ammo-nium ions increased under lower oxygen tension This leveldecreased with corresponding increases in oxygen tension





























4) condi-

tions



0

005

01

015

02

025

03D

ry w

eigh

t (m

gm

L)


(a)

0

002

004

006

008

01

012

014

016

Prot

ein

(mg

mL)


(b)

0

10

20

30

40

50

60

70

Am

mon

ium

ions

(mg

L)


(c)









4) conditions the



4) condi-






4 Discussion






0

50

100

150

200

250

nifHnifD

nifK


(a)

0

05

1

15

2

25

3

gltDgltB


(b)

05

101520253035


francci3 4059


(c)

0

1

2

3

4

5

6

sqhChpnC


(d)

0

5

10

15

20

25

30

hup2hup1


(e)

005

115

225

335

445

5

HboOHboN


(f)

0123456789

SodAKatA


(g)


4



4



2) condi-















Acknowledgments


References
















2












2









































2

-uptake kinetics in














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























4) condi-

tions



0

005

01

015

02

025

03D

ry w

eigh

t (m

gm

L)


(a)

0

002

004

006

008

01

012

014

016

Prot

ein

(mg

mL)


(b)

0

10

20

30

40

50

60

70

Am

mon

ium

ions

(mg

L)


(c)









4) conditions the



4) condi-






4 Discussion






0

50

100

150

200

250

nifHnifD

nifK


(a)

0

05

1

15

2

25

3

gltDgltB


(b)

05

101520253035


francci3 4059


(c)

0

1

2

3

4

5

6

sqhChpnC


(d)

0

5

10

15

20

25

30

hup2hup1


(e)

005

115

225

335

445

5

HboOHboN


(f)

0123456789

SodAKatA


(g)


4



4



2) condi-















Acknowledgments


References
















2












2









































2

-uptake kinetics in














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

005

01

015

02

025

03D

ry w

eigh

t (m

gm

L)


(a)

0

002

004

006

008

01

012

014

016

Prot

ein

(mg

mL)


(b)

0

10

20

30

40

50

60

70

Am

mon

ium

ions

(mg

L)


(c)









4) conditions the



4) condi-






4 Discussion






0

50

100

150

200

250

nifHnifD

nifK


(a)

0

05

1

15

2

25

3

gltDgltB


(b)

05

101520253035


francci3 4059


(c)

0

1

2

3

4

5

6

sqhChpnC


(d)

0

5

10

15

20

25

30

hup2hup1


(e)

005

115

225

335

445

5

HboOHboN


(f)

0123456789

SodAKatA


(g)


4



4



2) condi-















Acknowledgments


References
















2












2









































2

-uptake kinetics in














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

50

100

150

200

250

nifHnifD

nifK


(a)

0

05

1

15

2

25

3

gltDgltB


(b)

05

101520253035


francci3 4059


(c)

0

1

2

3

4

5

6

sqhChpnC


(d)

0

5

10

15

20

25

30

hup2hup1


(e)

005

115

225

335

445

5

HboOHboN


(f)

0123456789

SodAKatA


(g)


4



4



2) condi-















Acknowledgments


References
















2












2









































2

-uptake kinetics in














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Acknowledgments


References
















2












2









































2

-uptake kinetics in














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2












2









































2

-uptake kinetics in














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















2

-uptake kinetics in














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article contrasted reactivity to oxygen...

Documents