the groel gene as an additional marker for finer differentiation of ‘candidatus phytoplasma...

Annals of Applied Biology ISSN 0003-4746

R E S E A R C H A R T I C L E

The groEL gene as an additional marker for finer differentiationof ‘Candidatus Phytoplasma asteris’-related strainsJ. Mitrovic1, S. Kakizawa2, B. Duduk1, K. Oshima2, S. Namba2 & A. Bertaccini3

1 Institute of Pesticides and Environmental Protection, Belgrade, Serbia

2 Department of Agricultural and Environmental Biology, The University of Tokyo, Tokyo, Japan

3 DiSTA, Plant Pathology, Alma Mater Studiorum, University of Bologna, Bologna, Italy

Keywords16S rDNA; aster yellows phytoplasmas;

genetic marker; groEL gene.

CorrespondenceDr. B. Duduk, Institute of Pesticides and

Environmental Protection, Belgrade

11080, Serbia.

Email: [email protected]

Received: 6 December 2010; revised version

accepted: 2 March 2011.

doi:10.1111/j.1744-7348.2011.00472.x

Abstract

Phytoplasma classification established using 16S ribosomal groups and‘Candidatus Phytoplasma’ taxon are mainly based on the 16S rDNA propertiesand do not always provide molecular distinction of the closely related strainssuch as those in the aster yellows group (16SrI or ‘Candidatus Phytoplasmaasteris’-related strains). Moreover, because of the highly conserved natureof the 16S rRNA gene, and of the not uncommon presence of 16S rDNAinteroperon sequence heterogeneity, more variable single copy genes, suchas ribosomal protein (rp), secY and tuf, were shown to be suitable fordifferentiation of closely related phytoplasma strains. Specific amplificationof fragments containing phytoplasma groEL allowed studying its variabilityin 27 ‘Candidatus Phytoplasma asteris’-related strains belonging to different16SrI subgroups, of which 11 strains were not studied before and 8 morewere not studied on other genes than 16S rDNA. The restriction fragmentlength polymorphism (RFLP) analyses of the amplified fragments confirmeddifferentiation among 16SrI-A, I-B, I-C, I-F and I-P subgroups, and showedfurther differentiation in strains assigned to 16SrI-A, 16SrI-B and 16SrI-Csubgroups. However, analyses of groEL gene failed to discriminate strainsin subgroups 16SrI-L and 16SrI-M (described on the basis of 16S rDNAinteroperon sequence heterogeneity) from strains in subgroup 16SrI-B. Onthe contrary, the 16SrI unclassified strain ca2006/5 from carrot (showinginteroperon sequence heterogeneity) was differentiable on both rp and groELgenes from the strains in subgroup 16SrI-B. These results indicate thatinteroperon sequence heterogeneity of strains AY2192, PRIVA (16SrI-L), AVUT(16SrI-M) and ca2006/5 resulted in multigenic changes – one evolutionary stepfurther – only in the latter case. Phylogenetic analyses carried out on groELare in agreement with 16Sr, rp and secY based phylogenies, and confirmed thedifferentiation obtained by RFLP analyses on groEL amplicons.

Introduction

The phytoplasma classification was established usingrestriction fragment length polymorphism (RFLP) anal-yses with a number of restriction enzymes on 1200 bpamplicons in their 16S rDNA (Lee et al., 1993, 1998b).Obtained phytoplasma 16Sr groups have been shownto be consistent with the groups (clades) defined byphylogenetic analysis of near-full-length 16S rRNA genesequences, confirming the phylogenetical validity ofthis grouping that was first assessed by Southern blot

hybridisation assays with random cloned DNA probes

(Lee et al., 1992). However, this classification, that led

to the introduction of ‘Candidatus’ status for phyto-

plasmas (IRPCM, 2004), does not always provide the

molecular distinction necessary for phytoplasma strain

characterisation for epidemiological studies towards dis-

ease control.

Numerous different diseases in various plant species

and insect vectors were described and attributed to

phytoplasmas belonging to different 16SrI subgroups

Ann Appl Biol 159 (2011) 41–48 © 2011 The Authors 41Annals of Applied Biology © 2011 Association of Applied Biologists

The groEL gene as an additional marker J. Mitrovic et al.

described as ‘Ca. P. asteris’-related phytoplasmas (Leeet al., 2006). Because of the highly conserved nature of the16S rRNA gene and of the not uncommon presence of 16SrDNA interoperon sequence heterogeneity (Schneider &Seemuller, 1994; Liefting et al., 1996; Lee et al., 1998a;Jomantiene et al., 2002; Davis et al., 2003; Duduk et al.,2009), some additional tools for phylogenetic analysesand finer strain differentiation of this heterogeneousgroup are needed. More variable single copy genes, suchas ribosomal protein (rpl22 and rpS3), secY and tuf, werealready reported to be suitable for finer differentiationof aster yellows and other phytoplasmas (Marcone et al.,2000; Lee et al., 2004, 2006, 2010).

The groEL is a conserved gene already used forprokaryotic organism strain characterisation (Desai et al.,2009; Vermette et al., 2010). It is proposed that GroEL mayact as an adhesin–invasin in Mycoplasma and speculatedthat its function in Mollicutes could be that of a virulencefactor (Clark & Tillier, 2010). GroEL was therefore studiedin this work to evaluate its effectiveness towards detectingvariability in 27 ‘Ca. P. asteris’-related strains assigned todifferent 16SrI subgroups and originated from differenthosts and geographical areas.

Materials and methods

Sample selection

Twenty-seven ‘Ca. P. asteris’-related strains belongingto different 16SrI subgroups were employed (Table 1);available aster yellows phytoplasma 16S ribosomal DNAsequences were retrieved from the NCBI’s sequencedatabase, while for those unavailable in the database (11strains), sequencing of P1/P7 amplicons was performed(see below).

All the employed strains were maintained in periwinkleor available as extracted nucleic acids from periwinkle(Bertaccini, 2010), except samples PopD, ca2006/1,ca2006/5, ca2006/9, and MBSColombia that wereobtained from naturally infected plant hosts.

Primer design

Several publically available sequences of the 3.6 kbfragments of ‘Candidatus Phytoplasma asteris’-relatedstrains (Accession numbers: AB167357, AB124808, AB-124806, AB124807, AB124809, AB124810, AB124811,AB242231, AB242232, AB242233, AB242234, AB-242235, AB242236 and AB242237), containing groES,groEL, amp and nadE genes, two full genome sequencesof ‘Ca. P. asteris’-related strains (Accession numbers:CP000061 and AP006628) and two full genomesequences of ‘Ca. P. mali’ and ‘Ca. P. australiense’ (Acces-sion numbers: CU469464 and AM422018) allowed design

of primers that specifically amplify DNA fragments (1397bp long) inside the phytoplasma groEL gene (1611 bp,total) of ‘Ca. P. asteris’-related strains.

Both primers were designed inside the groEL. Forwardprimer AYgroelF (5′-GGCAAAGAAGCAAGAAAAG-3′)was based on the sequence starting 21 bp from the5′ end, while the reverse primer AYgroelR (5′-TTTAAGGGTTGTAAAAGTTG-3′) was based on the sequenceending 193 bp from the 3′ end of the groEL gene. Theprimer AYgroelF was designed on the basis of a groEL geneportion universal for phytoplasmas, while the AYgroelRwas designed on the basis of the portion specific only foraster yellows phytoplasmas.

Amplification of 16S ribosomal DNA and of partialgroEL gene

Direct PCR assays with the universal phytoplasma primerpair P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995)and nested PCR on obtained amplicons with primer pairR16F2/R2 (Lee et al., 1995) were carried out. Direct PCRassays, for partial amplification of groEL gene, werecarried out using AYgroelF/R primer pair on the 27aster yellows phytoplasma strains as well as on stolburfrom pepper (STOL) in periwinkle (ribosomal group16SrXII-A), ‘Ca. P. japonicum’ in Hydrangea sp. (16SrXII),tomato big bud from Australia (TBB) in periwinkle(16SrII-D), flavescence doree in grapevine (FD) fromSerbia (16SrV-C) and European stone fruit yellows inpeach (ESFY) from Serbia (16SrX-B).

Each 25 μL PCR reaction mix contained 20 ng templateDNA, 1× PCR Master Mix (Fermentas, Vilnius, Lithuania)and 0.4 μM of each primer. Samples lacking DNA wereemployed as negative controls. Thirty-five PCR cycleswere performed, for amplifications with P1/P7 andR16F27R2 primers, under described conditions (Schaffet al., 1992). Thirty-five PCR cycles were performedfor AYgroelF/R primers under the following conditions:1 min (2 min for the first cycle) for denaturation step at94◦C, 2 min for annealing at 55◦C and 3 min (10 minfor the last cycle) for primer extension at 72 C. A6 μL of PCR products was separated in 1% agarose gel,stained with ethidium bromide and visualised with UVtransilluminator.

Restriction fragment length polymorphism analyses of16S ribosomal DNA and of partial groEL gene

The 16SrI subgrouping, for the 11 strains not identifiedin the literature, were determined with RFLP analysesusing TaqI, HhaI and TruI (Fermentas, Vilnius, Lithuania)restriction enzymes on R16F2/R2 amplicons. The groELI(groEL gene RFLP group I) RFLP subgrouping wasperformed with AluI (Fermentas, Vilnius, Lithuania) and

42 Ann Appl Biol 159 (2011) 41–48 © 2011 The AuthorsAnnals of Applied Biology © 2011 Association of Applied Biologists

J. Mitrovic et al. The groEL gene as an additional marker

Table 1 Aster yellows-related reference phytoplasma strains employed and results of RFLP analyses on groEL amplicons

RFLP Subgroup Classification

groEL

Phytoplasma

strain Acronym

Associated

Disease Country 16S rDNAa groELa

16SrIb Subgroup

Literature groELTruI groELAluI

groELI

Subgroup

CHRYM Chrysanthemum

yellows

Germany AY265214 AB599692 AMarcone et al.

(2000)

3 3 I

ca2006/1 Carrot yellows Serbia EU215424 AB599708 ADuduk et al.

(2009)

3 3

PVM Plantago

virescence

Germany AY265216 AB599706 ABertaccini

et al. (2000)

3 3

NJ-AY New Jersey aster

yellows

NJ, USA HM590622 AB599703 ALee et al.

(2005)

3 3

GD Grey dogwood

stunt

NY, USA DQ112021 AB599694 ALee et al.

(2006)

5 6 II

AVUT Aster yellows Germany AY265209 AB599686 B(M)Marcone et al.

(2000)

1 1 III

AY2192 Aster yellows Germany AY180957 AB599687 B(L)Marcone et al.

(2000)

1 1

PRIVA Primrose

virescence

Germany AY265210 AB599705 B(L)Marcone et al.

(2000)

1 1

AY-W American aster

yellows

FL, USA HM590617 AB599691 BBertaccini

et al. (2000)

1 1

DIV Diplotaxis

virescence

Spain HM590618 AB599693 BBertaccini

et al. (2000)

1 1

PrG Primula green

yellows

UK HM590623 AB599696 BThis work

1 1

PYR Peach yellows Italy HM590624 AB599697 BThis work

1 1

RV Oilseed rape

virescence

France HM590625 AB599698 BBertaccini

et al. (2000)

1 1

GLAWC Gladiolus

witches

broom

the Netherlands HM590619 AB599700 BBertaccini

et al. (2000)

1 1

NA Periwinkle

virescence

Italy HM590621 AB599702 BThis work

1 1

SAY Aster yellows CA, USA AY222063 AB599707 BBertaccini

et al. (2000)

1 1

ca2006/9 Carrot yellows Serbia EU215426 AB599709 BDuduk et al.

(2009)

1 1

AY-27 Aster yellows Canada HM467127 AB599688 BLee et al.

(1993)

1 1

AY-J Aster yellows France HM590616 AB599689 BThis work

2 2 IV



Table 1 Continued

RFLP Subgroup Classification

groEL

Phytoplasma

strain Acronym

Associated

Disease Country 16S rDNAa groELa

16SrIb Subgroup

Literature groELTruI groELAluI

groELI

Subgroup

ca2006/5 Carrot yellows Serbia EU215425GQ175789 AB599711 B(?)Duduk et al. (2009)

2 2

MBS Colombia Maize bushy

stunt

Colombia HQ530152 AB599712 BDuduk et al. (2008)

2 8 V

CA Carrot yellows Italy HM448473 AB599690 CBertaccini et al. (2000)

4 4 VI

LEO Leontodon

yellows

Italy HM590620 AB599701 CBertaccini et al. (2000)

4 4

KVF Clover phyllody France HQ530150 AB599695 CMarcone et al. (2000)

4 5 VII

PPT Potato purple

top

France HQ530151 AB599704 CMarcone et al. (2000)

4 5

A-AY Apricot chlorotic

leafroll

Spain AY265211 AB599699 FLee et al. (2004)

3 5 VIII

PopD Populus decline Serbia HM590626 AB599710 PThis work

6 7 IX

aValues in bold indicate accession numbers of sequences obtained in this paper.bLetters in parenthesis indicate 16SrI subgroups determined based on one operon.

TruI restriction enzymes. All restriction products wereseparated in 8% polyacrylamide gel and stained andvisualised as described above.

Sequencing and sequence analyses of 16S ribosomalDNA and of partial groEL gene

The direct P1/P7-amplified products were purified usingQiagen PCR purification kit (Qiagen GmbH, Hilden,Germany), sequenced in both directions with the twoforward primers P1 and R16F2 (Lee et al., 1995)and the reverse primer P7 and deposited in theNCBI (Table 1). Obtained sequences together with thesequences retrieved from the NCBI were aligned andtrimmed to contain near full 16S rDNA.

DNA fragments containing groEL gene of analysed phy-toplasma strains were amplified by long and accurate PCR(LA-PCR) using LA Taq DNA polymerase (TaKaRa Bio,Shiga, Japan) with ES-1/Nad-2 primers, and sequenceddirectly with 11 primers ES-1, ES-2, EL-1, EL-3, EL-F1w,Amp-N1, Amp-3, Amp-C1, Nad-1, Nad-2 and Nad-3 aspreviously described (Kakizawa et al., 2006). Nucleotidesequences were determined using the dideoxynucleotidechain termination method with an automatic DNAsequencer (ABI PRISM 3100 Genetic Analyzer, AppliedBiosystems Japan, Tokyo, Japan) according to themanufacturer’s instructions. The groEL gene sequenceswere obtained from all AY phytoplasmas analysed andwere deposited in the NCBI (Table 1). Sequences of groEL

homologue genes of these fragments were aligned usingCLUSTALX program (Thompson et al., 1997) and adjustedmanually. A phylogenetic tree was constructed basedon the 27 aster yellows strains sequenced, ‘Ca. P. mali’and ‘Ca. P. australiense’ using MEGA version 4 (Tamuraet al., 2007). Acholeplasma laidlawii was designated as theoutgroup. Nucleotide and amino acid sequence identityvalues were calculated for aster yellows-related phyto-plasmas using the same program.

Results and discussion

Amplification and RFLP analyses of 16S ribosomalDNA

The 16S rDNA of all tested AY phytoplasmas, and of theother phytoplasma strains belonging to diverse ribosomalgroups were successfully amplified using P1/P7 primerpair, confirming presence of target DNA. The AY phyto-plasmas, for which 16SrI subgroups were not describedin literature, were amplified by using primers R16F2/R2in nested PCR assays and the amplicons were subjectedto RFLP analyses using TaqI, HhaI and TruI restrictionenzymes; restriction profiles (data not shown) allowedaffiliation of these strains to the 16SrI subgroups (Table 1).

Amplification and RFLP analyses of partial groEL gene

Using AYgroelF/R primer pair in direct PCR, expectedlength amplicons (about 1.4 kb) of partial groEL gene



Figure 1 Polyacrylamide gel 8% showing the TruI and AluI restriction fragment length polymorphism patterns on groEL amplified with AYgroelF/R primer

pair of selected phytoplasma strains belonging to 16SrI and groELI subgroups studied; see Table 1 for strain abbreviations; �X174, marker phiX174

HaeIII digested; fragment sizes in base pairs from top to bottom: 310, 281, 271, 234, 194, 118 and 72 (left) and 1353, 1078, 872, 603, 310, 281, 271, 234,

194, 118 and 72 (right).

were obtained with all employed AY phytoplasmas. No

amplification was obtained with STOL, ‘Ca. P. japonicum’,

FD, TBB and ESFY phytoplasmas. This indicates to some

extent specificity of these primers to 16SrI phytoplasma

group, probably because of sequence specificity of the

reverse primer (AYgroelR). The obtained amplicons were

subjected to RFLP analyses with TruI and AluI restriction

enzymes which yielded six and eight different restriction

profiles, respectively (Fig. 1; Table 1). These restriction

profiles allowed comprehensive differentiation of AY phy-

toplasmas on the basis of groEL gene to nine groELI RFLP

subgroups (I–IX) compared to the five 16SrI subgroups.

In particular, phytoplasmas in 16SrI-A and I-C

subgroups each showed two different groEL RFLP profiles,

while phytoplasmas in 16SrI-B showed three. Also,

the strains from 16SrI-F and I-P ribosomal subgroups

were differentiated from all other strains by groEL RFLP

analyses which is in agreement with 16S rDNA group

I subgrouping. Namely, the strain PopD, belonging to

16SrI-P subgroup, had unique groEL RFLP profiles. The

strain A-AY, belonging to 16SrI-F subgroup, shared groEL

RFLP profiles with other subgroups, but its combination

was unique, therefore, on the basis of its collective RFLP

profile, it was assigned to a different groELI subgroup.

However, strains belonging to 16SrI-L (AY2192, PRIVA),

and I-M (AVUT) subgroups were not differentiated from

16SrI-B strains. On the other hand, the phytoplasmas

assigned to 16SrI-L and I-M subgroups were already

described as closely related to 16SrI-B subgroup, and were

differentiated from it only for the presence of 16S rDNA

interoperon sequence heterogeneity (Marcone et al.,2000). As well, phytoplasmas included in these subgroupscould not be differentiated from 16SrI-B phytoplasmasbased on the RFLP analyses of tuf (Marcone et al., 2000),rpl22 and rpS3 (Lee et al., 2007), and secY (Lee et al., 2006)genes. On the contrary, strain ca2006/5, which also differsfrom 16SrI-B phytoplasmas for the sequence of one rDNAoperon, clearly represents, together with strain AY-J, aseparate groEL RFLP subgroup and was differentiableon both rpl22 and rpS3 (Duduk et al., 2009), and groELgenes from the strains in subgroup 16SrI-B. Theseresults indicate that interoperon sequence heterogeneityof strains AY2192 and PRIVA (16SrI-L), AVUT (16SrI-M) and ca2006/5 resulted in multigenic changes – oneevolutionary step further – only in the latter case, whereresults from rp and groEL differentiation are congruent.

The AluI restriction enzyme could differentiate allgroELI subgroups except subgroup groELI-VIII, for thedifferentiation of which appliance of both restrictionenzymes is needed.

These results show that RFLP analysis of theAYgroelF/R amplified fragments is a suitable and reliableadditional tool for differentiation of closely related,but not always clearly distinguishable, aster yellowsphytoplasma strains.

Comparative sequence analyses of 16S rDNAand groEL genes

Considering the limitations of 16S rDNA analysis inthe phylogenetic study of closely related aster yellows



phytoplasmas (Lee et al., 2006; Martini et al., 2007) and

to further evaluate the groEL gene usefulness, compara-

tive sequence analyses of AY-strains were conducted on

16S rDNA sequences, obtained both in this work and

from NCBI GenBank, and on groEL sequences obtained

in this work.

The average nucleic acid similarity of 16S rDNA of

examined AY-strains was 99.5% ranging from 98.7% to

99.5% among the 16SrI subgroups and from 99.1% to

100% inside the subgroups. The lowest similarities of

16S rDNA (98.7–99.3%) were observed between PopD

phytoplasma (16SrI-P) and the strains from the other

16SrI subgroups.

On the other hand, the average nucleic acid similar-

ity of groEL of examined AY-strains was 98.1% ranging

from 93.8% to 98.1% and from 98% to 100% among

and inside the 16SrI subgroups, respectively. Again the

lowest similarities of groEL (93.8–94.8%) were detected

between PopD phytoplasma (16SrI-P) and the strains

from the other 16SrI subgroups. The average similarity of

groEL predicted amino acid sequences was 98.8% ranging

from 96.0% to 97.9% and from 97.7% to 100% among

and inside the 16SrI subgroups, respectively, while for

PopD phytoplasma it was extremely lower ranging from

94.7% to 96.2%.

Regarding the intragroup similarities, strain GD showed

to be the most distinct member of a 16SrI subgroup,

showing significantly lower similarity to other members

of 16SrI-A subgroup, in both 16S rDNA and groEL

sequences. The 16S rDNA and groEL nucleic acid sequence

similarity between GD and other members of the same

subgroup ranged from 99.1% to 99.5% and from 98% to

98.1%, respectively, while for the other 16SrI subgroups,

intragroup similarity ranged from 99.4% to 100% and

from 99.7% to 100%, respectively.

These results show that the groEL has lower intergroup

sequence similarities than 16S rDNA and therefore has

more resolution and definition power in separating

closely related aster yellows strains as confirmed by the

phylogenetic analysis (Fig. 2).

Phylogenetic analyses based on the groEL nucleotide

sequences (data not shown) delineated the same

lineages as those obtained with amino acid sequences.

Phylogenetic trees based on groEL gene sequences had

basically similar topology to that of 16S rDNA, and were

highly congruent to those of rpl22 and rpS3, secY and

Figure 2 Phylogenetic tree constructed by parsimony analyses of predicted groEL gene amino acid sequences (left) and 16S rDNA sequences (right)

of 27 aster yellows strains, ‘Ca. P. mali’ and ‘Ca. P. australiense’ employing Acholeplasma laidlawii as the outgroup. Aster yellows phytoplasma strains

are described in Table 1. Numbers on the branches are bootstrap values obtained for 1000 replicates (only values above 50% are shown). Restriction

fragment length polymorphism classifications by 16S rDNA and groEL gene are shown on the right side of the trees.



tuf genes (Marcone et al., 2000; Lee et al., 2004, 2006).However, in the phylogenetic tree based on the groELamino acid sequences 16SrI-A and I-B subgroups weredivided into two clusters each.

Particularly, strain GD (16SrI-A) clustered separatelyfrom the rest of 16SrI-A strains, while strains MBSColom-bia and AY-J (16SrI-B), together with ca2006/5 (16SrI,subgroup not assigned), formed a lineage separate fromthe other 16SrI-B strains. 16SrI-L and I-M subgroups clus-tered together with the majority of 16SrI-B strains, as itwas shown also by the 16S rDNA, rpl22 and rpS3, secY andtuf genes based phylogeny (Marcone et al., 2000; Lee et al.,2004, 2006). The 16SrI-C strains and A-AY (16SrI-F)formed two distinct clusters in agreement with data avail-able for the other genes. PopD (16SrI-P) formed separatelineage in agreement with 16S rDNA-based phylogeny.

Both RFLP and sequence analyses of the groEL showedthat GD strain is distinct from other members of 16SrI-Asubgroup and this is in agreement with reported analysesof the same strain on secY and rpl22 and rps3 genes (Leeet al., 2004, 2006; Martini et al., 2007).

The seven lineages delineated by phylogenetic analysesbased on groEL are consistent with groEL RFLP subgroup-ing, except that the division of 16SrI-C on subgroupsgroELI-VI and groELI-VII is not supported by phylogenydata. In fact, on the groEL sequences there is only onebase pair difference between these groELI subgroups atposition 1206 (AluI restriction site) producing synony-mous codons, and therefore not implying amino aciddifferences.

Phylogenetic analyses carried out on full groEL gene(1610 bp) confirmed the finer differentiation by RFLPanalyses on specifically amplified and sequenced 1397 bpfragments.

Conclusions

The developed method is reliable for the differentiationof aster yellows strains not only from phytoplasmacollection in periwinkle, but also from field collectedstrains as for the samples examined from carrot andpopulus from Serbia and corn from Colombia. Thestudy was carried out on 27, the largest number of‘Ca. P. asteris’-related strains so far, of which 11 strainswere not studied before and 8 more were not studiedon other genes than 16S rDNA. The RFLP analyses ongroEL amplicons confirmed the differentiation in 16SrDNA among 16SrI-A, I-B, I-C, I-F and I-P subgroups,and showed further differentiation in strains assigned to16SrI-A, 16SrI-B and 16SrI-C subgroups, substantially inagreement with other genes reported for phytoplasmadifferentiation (rpl22 and rpS3 and secY genes). Analysesof groEL gene failed to discriminate strains in subgroups

16SrI-L and 16SrI-M (described on the basis of 16SrDNA interoperon sequence heterogeneity) from strainsin subgroup 16SrI-B, which is in full agreement withresults reported on tuf, rp and secY genes (Marcone et al.,2000; Lee et al., 2006, 2007, 2010).

Acknowledgements

This research was supported by: project TR31043 fromthe Ministry of Science and Technological Development,Republic of Serbia; Grants-in-Aid for Scientific Research,Funding Program for Next Generation World-LeadingResearchers, Japan, and Program for Promotion ofBasic Research Activities for Innovative Bioscience(PROBRAIN) of Japan.

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