evidence of recent inter kingdom horizontal gene transfer between bacteria and candida is

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Open AcceResearch articleEvidence of recent interkingdom horizontal gene transfer between bacteria and Candida parapsilosisDavid A Fitzpatrick*, Mary E Logue and Geraldine Butler
Address: School of Biomolecular and Biomedical Science, Conway Institute, University College, Dublin, Belfield, Dublin 4, Ireland

Email: David A Fitzpatrick* - [email protected]; Mary E Logue - [email protected]; Geraldine Butler - [email protected]

* Corresponding author

AbstractBackground: To date very few incidences of interdomain gene transfer into fungi have beenidentified. Here, we used the emerging genome sequences of Candida albicans WO-1, Candidatropicalis, Candida parapsilosis, Clavispora lusitaniae, Pichia guilliermondii, and Lodderomyces elongisporusto identify recent interdomain HGT events. We refer to these as CTG species because theytranslate the CTG codon as serine rather than leucine, and share a recent common ancestor.

Results: Phylogenetic and syntenic information infer that two C. parapsilosis genes originate frombacterial sources. One encodes a putative proline racemase (PR). Phylogenetic analysis also infersthat there were independent transfers of bacterial PR enzymes into members of thePezizomycotina, and protists. The second HGT gene in C. parapsilosis belongs to the phenazine F(PhzF) superfamily. Most CTG species also contain a fungal PhzF homolog. Our phylogeny suggeststhat the CTG homolog originated from an ancient HGT event, from a member of theproteobacteria. An analysis of synteny suggests that C. parapsilosis has lost the endogenous fungalform of PhzF, and subsequently reacquired it from a proteobacterial source. There is evidence thatSchizosaccharomyces pombe and Basidiomycotina also obtained a PhzF homolog through HGT.

Conclusion: Our search revealed two instances of well-supported HGT from bacteria into theCTG clade, both specific to C. parapsilosis. Therefore, while recent interkingdom gene transfer hastaken place in the CTG lineage, its occurrence is rare. However, our analysis will not detect ancientgene transfers, and we may have underestimated the global extent of HGT into CTG species.

BackgroundLateral or horizontal gene transfer (HGT) is defined as theexchange of genes between different strains or species [1].HGT introduces new genes into a recipient genome thatare either homologous to existing genes, or belong toentirely new sequence families. Large-scale genomicsequencing of prokaryotes has revealed that gene transferis an important evolutionary mechanism for these organ-isms [2,3]. HGT has been linked to the acquisition of drugresistance by benign bacteria [4], and also to the gain of

genes that confer the ability to catabolize certain aminoacids that are important virulence factors [5]. Howeverthere is much debate as to whether lateral gene transfer isan ubiquitous influence throughout prokaryotic genomeevolution [6]. Until recently, the process of gene transferhas been assumed to be of limited significance to eukary-otes [7]. The availability of diverse eukaryotic genomesequence data is dramatically changing our views on theimportant role gene transfer can play in eukaryotic evolu-tion.

Published: 24 June 2008

BMC Evolutionary Biology 2008, 8:181 doi:10.1186/1471-2148-8-181

Received: 17 January 2008Accepted: 24 June 2008

This article is available from: http://www.biomedcentral.com/1471-2148/8/181

© 2008 Fitzpatrick et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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The rapid increase in fungal sequence data has promotedthis kingdom to the forefront of comparative genomics[8]. Whereas there is some documented evidence for HGTbetween fungal species [9-17] or from bacteria to fungi[18-28] [see additional file 1], overall very few incidenceshave been identified. There are two possible explanations:either gene transfer is indeed extremely rare amongstfungi, or it has not yet been thoroughly studied. Toaddress this question we investigated the frequency of suc-cessful recent interdomain HGT events between prokary-otes and yeast species belonging to the CTG clade. Wechose this course of action as we expect recent interdo-main HGT events to be more readily identified and sup-ported than more ancient transfers.

For the purposes of this study, we define CTG species asthe immediate relatives of C. albicans, including C. tropica-lis, C. parapsilosis, Clavispora lusitaniae, Pichia guilliermon-dii, and Lodderomyces elongisporus. These species have beencompletely sequenced, share a relatively recent commonancestor [29], and the codon CUG is translated as serinerather than leucine [30].

We used syntenic, phylogenetic and sequence based anal-yses to identify two cases of interdomain HGT betweenprokaryotes and C. parapsilosis, most likely involving theproteobacteria phylum. Our results suggest that extantCTG species do not readily take up exogenous DNA.

Results and discussionIdentification of horizontal gene transfer candidates through Blast database searchWe compared all available CTG gene sets against UniProtusing BlastP [31]. CTG genes with top database hits tobacterial species were identified as putative horizontallytransferred genes and the resultant Blast files wereinspected manually. A D. hansenii gene (protein ydhR pre-cursor) with a top database hit to a bacterial sequence wasnot considered for further analsyes as it has previouslybeen described [22]. After this process two genes from C.parapsilosis were considered for further analysis; oneencodes a putative proline racemase, and the secondencodes a member of the phenazine F superfamily.Related family members were identified by a secondround of database searching against GenBank to ensureall available genomic data was utilized.

Proline racemase phylogeny and characterizationThe C. parapsilosis gene (designated CPAG_02038) is mostsimilar to a proline racemase homolog from Burkholderiacenocepacia AU 1054 protein (66% pairwise identity; Fig-ure 1A). Amino acid racemases catalyze the interconver-sion of L- and D-amino acids by abstraction of the α-amino proton of the enzyme bound substrate [32].CPAG_02038 lies within a large contig and is also present

in a previously published genome survey of C. parapsilosis[33], suggesting its presence does not the result from con-tamination. We could not locate any related genes in anyother CTG genome (using BlastP or TBlastN). Familymembers are widely distributed throughout the prokaryo-tes however, and are also located within the Pezizomy-cotina.

We extracted 321 putative proline racemases from 207organisms, including members of the α, β, γ, and δ-pro-teobacteria, Actinobacteria, Fungi, Protozoa and Metazoa.Numerous species were found to have several familymembers [see additional file 2]; all were included forcomplete comparative purposes. A maximum likelihood(ML) phylogeny was reconstructed from an alignment ofall the PR proteins (Figure 2).

There are a large number of polytomies displayed in Fig-ure 2. These probably result from duplication of PR genesfollowed by diversifying selection, leading to a highdegree of sequence heterogeneity. For example, Agrobacte-rium tumefaciens str. C58 contains three PR homologs [seeadditional file 2], with an average amino acid pairwisepercentage identity of ~31%. Burkholderia cenocepacia AU1054 contains 2 proline racemase homologs [see addi-tional file 2], which are only 28% identical. To helpresolve the evolutionary history amongst PR homologs wereconstructed an additional ML phylogeny based on areduced dataset (Figure 3). We also reconstructed a Baye-sian phylogeny using the heterogeneous CAT site model.The CAT model can account for site-specific features ofsequence evolution and has been found to be more robustthan other methods against phylogenetic artifacts such aslong branch attraction [34]. The resultant Bayesian phyl-ogeny is highly congruent with the ML phylogeny (notshown).

The putative C. parapsilosis PR homolog lies in a stronglysupported (100% Bootstrap support (BP)) clade with Bur-kholderia species (Figures 2 &3 clade-A). Burkholderia are β-proteobacteria. However, no other β-proteobacteria, orindeed any other bacterial genus were found within clade-A (Figures 2 &3).

Although no PR homologs were identified in other CTGspecies, or indeed in any other of the Saccharomycotina,there are homologs in family members of the Pezizomy-cotina. A Pezizomycotina specific subclade is evident inour phylogeny containing Phaeosphaeria nodorum, Aspergil-lus niger and Gibberella zeae (Figures 2 &3 clade-B 100%BP). This subclade is found in a strongly supported cladewith members of the Actinobacteria (Figure 2 100% BP),containing Brevibacterium linens and an unclassifiedmarine actinobacterium and excluding Rubrobacter xylano-philus (Figure 2 87% BP). This suggests that these Pezizo-



mycotina species obtained their PR gene from theActinobacteridae subclass rather than the Rubrobacteri-dae subclass. This transfer event is another independentHGT event of a PR gene into fungi, and we hypothesize itoccurred early in the Pezizomycotina lineage, as it isshared by three distantly related species. Its patchyphyletic distribution suggests it has been subsequentlylost in other Pezizomycotina species.

There are also PR homologs in the Metazoans. These arefound in a eukaryote clade that also contains a number ofPezizomycotina representatives (Figures 2 &3 clade-C93% BP). Several scenarios can explain this phylogeneticpositioning. Firstly, the PR gene may have been present inthe last universal common ancestor of all eukaryotes buthas been differentially lost in all lineages except thoseleading to modern day Metazoa and Pezizomycotina.Alternatively, an ancient gene transfer from bacteria to thelast common ancestor (LCA) of Metazoa and Fungi couldhave occurred, with subsequent gene loss amongst differ-ent Metazoan and Fungal lineages. A third hypothesis is

that two independent gene transfers have occurred intothe Metazoan and Pezizomycotina lineages from unsam-pled bacterial donors. Finally, a transfer from unsampledbacteria into one of the eukaryote clades (either Metazoaor Pezizomycotina) may have occurred with subsequenttransfer from one eukaryotic group to the other.

A. niger, A. oryzae and G. zeae all contain multiple PRhomologs [see additional file 2]. One A. niger, one G. zeaeand the three A. oryzae PR homologs are nested in astrongly supported Pezizomycotina specific subclade (Fig-ures 2 &3 clade-D 100% BP). This subclade if foundwithin a larger predominately proteobacterial clade (Fig-ure 2 74% BP). This infers that there was an independentgene transfer event of a bacterial PR homolog into anancestral Pezizomycotina species.

The phylogenetic position of the C. parapsilosis PRhomolog (Figures 2 &3) resemble that described for theadenosine deaminase (ADA) gene in the Dekkera bruxel-lensis genome [21]. In that analysis, the authors suggest

A) An alignment of PR proteins from C. parapsilosis (CpPR CPAG_02038) and Burkholderia cenocepacia (BcPR) was generated with MUSCLEFigure 1A) An alignment of PR proteins from C. parapsilosis (CpPR CPAG_02038) and Burkholderia cenocepacia (BcPR) was generated with MUSCLE. These proteins are 66% identical B) An alignment of PhzF proteins from Candida parapsilosis (CpPhzF CPAG_03462) and Photorhabdus luminescens (PlPhzF), these are 61% identical.

(A) (B)

LSTEQMQTIANWTNLSETTFVFPATSDKALSSIQMQGIANWTNLSETTFILPAENPLA

YYVRIFTPQSELPFAGHPTIGTCHALLESYRVRIFTPGSELPFAGHPTIGTAHALLEA

LISAKDGVVVQECGAGLVKLTIVPG----LIQAREGRIVQECGAGLITLNVTERDEGQ

STSFELPDPVITPLSDTQINSLETDLGCALITFELPEPTITPLSSEQIDRLESILDCP

DKNLRPALVDVGARWIIARVADAKTVLSA DRALTPALIDVGARWIVAHTTGAEAVLAT

PAFPQLAKHNNELKATGVSIYGNWSD---PDYARLLEHDTQMNITGVCLYGAYHEGAE

HIEVRSFAPACGVDEDPVCGSGNGAVAAF DIEVRSFAPSCGVNEDPVCGSGNGSVAAF

RSHTN---EDKILKSSQGSVVNREGALKL RHHKVAMIDDKIVHSSQGKKLGRQGSVWL

ISNKKVLVGGDAVTCIEGKIRITHSDGKIFVGGSAVTCINGTITI-

MSSA-FKQVDVFTSKPFKGNPVAVIMDANMSLVPFKQVDVFTHRPFKGNPVAVVMDAQ

0 0

CpPhzFPlPhzF

89 90

CpPhzFPlPhzF

59 60

CpPhzFPlPhzF

29 30

CpPhzFPlPhzF

145 150

CpPhzFPlPhzF

115 120

CpPhzFPlPhzF

202 210

CpPhzFPlPhzF

175 180

CpPhzFPlPhzF

232 240

CpPhzFPlPhzF

259 270

CpPhzFPlPhzF

MNQDRLISTIETHTGGEPFRIVTSGLPRLKMKISRSLSTVEVHTGGEAFRIVTSGLPRLP

GDTIVAKRTWIKTHHDEIRKFLMYEPRGHAGDTIVQRRAWLKAHADEIRRALMFEPRGHA

DMYGGYLVDSVSDDADFGVIFLHNEGYSDHDMYGGYLTEPVSPNADFGVIFVHNEGYSDH

CGHGIIALASTAVKLGWVERTQPKTRVGIDCGHGVIALSTAAVELGWVQRTVPETRVGID

APCGFIEAFVKWDGEKVGNVRFVNVPSFMYAPCGFIEAFVQWDGEHAGPVRFVNVPSFIW

IKDATVDTPSFGEVIGDIAFGGAFYFYMNSRRDVSVDTPSFGTVTGDIAYGGAFYFYVDG

ASLDIPIGLSQVETLRRLGNEVKIAANKKYAPFDLPVRESAVEKLIRFGAEVKAAANATY

KVVHPEIAEINHVYGTIIDNISPDGGASQSPVVHPEIPEINHIYGTIIANAPRHAGSTQA

NVCIFADRQVDRSPTGSGTAGRAAQLFARGNCCVFADREVDRSPTGSGTGGRVAQLYQRG

ELQVGQVFTNESIVGSVFSAKVVKQVKYHGLLAAGDTLVNESIVGTVFKGRVLRETTVGD

FDAVIPEVEGNANVIGYANWIVDPDDEIGKFPAVIPEVEGSAHICGFANWIVDERDPLTY

GFLVREGFLVR-

0 0

CpPRBcPR

120120

CpPRBcPR

90 90

CpPRBcPR

60 60

CpPRBcPR

30 30

CpPRBcPR

210210

CpPRBcPR

180180

CpPRBcPR

150150

CpPRBcPR

270270

CpPRBcPR

240240

CpPRBcPR

300300

CpPRBcPR

330330

CpPRBcPR



that D. bruxellensis and Burkholderia species received theADA gene from a species not yet represented in the publicsequence databases. Our PR phylogeny suggests a similarevent may have occurred within clade-A, which contains

only C. parapsilosis and Burkholderia species (Figures 2 &3).Burkholderia species are known to have a genomic reper-toire that allows the transfer and receipt of exogenousDNA [35] and a number of studies have reported success-

Proline racemase maximum likelihood phylogenyFigure 2Proline racemase maximum likelihood phylogeny. The optimum model of protein substitution was found to be WAG+G. The number of gamma rate categories was 4 (alpha = 1.163). Bootstrap resampling (100 iterations) was undertaken and are displayed. For display purposes branches with less than 50% support were collapsed. Letters (A-E) in parentheses are used to distinguish clades and are discussed in the text. Branches are colored according to their taxonomy. Fungal branches and species names are colored green.

Burkholderia sp 383

Erwinia carotovora SCRI1043

Pseudomonas chlororaphis

Pseudomonas aeruginosa C3719

Ferroplasma acidarmanus fer1

Bacillus cereus W

Bacillus cereus E

33L

Bac

illus

ant

hrac

is s

tr A

2012

Bac

illus

ant

hrac

is s

tr A

mes

Bac

illus

cer

eus

AH

820

Bac

illus

thur

ingi

ensi

s st

r A

l Hak

amB

acill

us th

urin

gien

sis

str

9727

Bacillus cereus A

H187

Bacillus cereus G

9241

Bacillus cereus A

TC

C 10987

Bacillus cereus A

H1134

Bacillus cereus B

4264B

acillus cereus AT

CC

14579B

acillus cereus G9842

Bacillus w

eihenstephanensis KB

AB

4

Bacillus sp B

14905

Lysinibacillus sphaericus C3-41

Trypanosoma cruzi strain CL Brener

Trypanosoma cruzi strain C

L Brener

Clostridium

botulinum A str. ATC

C 3502

Clostridium

botulinum Bf

Clostridium

difficile 630

Clostridium

sp OhILA

s

Alkaliphilus m

etalliredigenes QY

MF

Clostridium

sticklandii

Dorea longicatena D

SM 13814

Clostridium

scindens ATCC

35704

Rhodobacter sphaeroides ATCC 17029

Rhodobacter sphaeroides 241

Rubrobacter xylanophilus DSM 9941

Brevibacterium linens BL2

Saccharopolyspora erythraea NRRL 2338

Streptomyces coelicolor A3(2)

Streptomyces averm

itilis MA4680

Clavibacter michiganensis strain NCPPB 382

Clavibacter michiganensis

Renibacterium salm

oninarum ATCC 33209


Haloarcula marismortui ATCC 43049

Methylobacterium nodulans ORS 2060

Methylobacterium sp 446

Bur

khol

deria

am

bifa

ria M

C40

6B

urkh

olde

ria m

ultiv

oran

s A

TC

C 1

7616

Mar

icau

lis m

aris

MC

S10

Rho

dosp

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m r

ubru

m A

TC

C 1

1170

Bur

khol

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xen

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LB40

0

Bor

dete

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DS

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2804

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ti M

AFF

3030

99

Bruc

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mel

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6M

Bruc

ella

abo

rtus

biov

ar 1

str

9941

Bruc

ella

sui

s 13

30

Bru

cella

sui

s A

TCC

2344

5

Och

roba

ctru

m a

nthr

opi A

TCC

4918

8

Aura

ntim

onas

sp

SI85

9A1

Hoe

flea

phot

otro

phic

a D

FL43

Oce

anib

ulbu

s in

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x HEL4

5

Oce

anico

la g

ranu

losu

s HTC

C2516

Fulvi

mar

ina

pela

gi H

TCC25

06

Sinor

hizo

bium

med

icae

WSM

419

Sinorh

izobiu

m m

elilot

i 102

1

Parac

occu

s de

nitri

fican

s PD12

22

Mic

rosc

illa

mar

ina

AT

CC

231

34

Alg

orip

hagu

s sp

PR

1

Rob

igin

itale

a bi

form

ata

HT

CC

2501

Fla

voba

cter

iale

s ba

cter

ium

HT

CC

2170

Kor

dia

algi

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OT

1

Psy

chro

flexu

s to

rqui

s A

TCC

700

755

Flav

obac

teria

les

bact

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m A

LC-1

Leeu

wen

hoek

iella

bla

nden

sis

ME

D21

7

Dok

doni

a do

ngha

ensi

s M

ED

134

Met

hylo

bact

eriu

m s

p 44

6

Par

acoc

cus

deni

trifi

cans

PD

1222

Agr

obac

teriu

m tu

mef

acie

ns s

tr C

58R

hizo

bium

legu

min

osar

um W

SM

2304

Rhi

zobi

um e

tli C

FN

42

Rhi

zobi

um le

gum

inos

arum

WS

M13

25

Rhi

zobi

um le

gum

inos

arum

bv

vici

ae 3

841

Mar

inom

onas

sp M

ED121

Hahell

a ch

ejuen

sis K

CTC 239

6

Vibrio

para

haem

olytic

us R

IMD 2

2106

33

Vibrio para

haemolyticu

s AQ3810

Vibrio sp

Ex2

5

Vibrio alginolyt

icus 1

2G01

Shewanella lo

ihica PV4

Shewanella amazonensis

SB2B

Shewanella pealeana ATCC700345

Shewanella halifaxensis HAW-EB4

Shewanella woodyi ATCC 51908

Shewanella sediminis HAWEB3

Shewanella benthica KT99Colw

ellia psy

chreryt

hraea 34H

Pseudoalte

romonas tunica

ta D2

Alteromonadales b

acteriu

m TW7

Bacillus cereus A

TC

C 10987

Bacillus cereus A

H187

Bacillus cereus A

H820

Bacillus anthracis str A

mes

Bacillus thuringiensis str 9727

Bacillus cereus N

VH

0597-99

Bacillus thuringiensis str A

l Hakam

Bacillus cereus E

33L

Bacillus cereus G

9241

Bacillus cereus A

TCC

14579

Bacillus cereus B

4264

Bacillus cereus A

H1134

Bacillus cereus G

9842

Bacillus w

eihenstephanensis KB

AB

4

Paracoccus denitrificans PD1222


Rhodococcus sp RHA1

Rhodococcus sp RHA1

Xanthomonas axonopodis pv citri str 3

06

Aspergillus oryzae

Aspergillus oryzae

Roseobacter sp MED193

Roseobacter sp SK20926

Rhizobium leguminosarum WSM2304

Solibacter usitatus Ellin6076

Roseiflexus castenholzii DSM13941

Mesorhizobium loti MAFF303099Rhodobacterales bacterium HTCC2150

Brucella suis 1330Brucella canis ATCC 23365

Brucella melitensis 16M

Brucella ovis ATCC25840

Ochrobactrum anthropi ATCC49188

Paracoccus denitrificans PD1222Rhizobium leguminosarum WSM2304

alpha proteobacterium BAL199

Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa C3719

Pseudomonas aeruginosa 2192

Pseudomonas aeruginosa UCBPPPA14

Pseudomonas aeruginosa PA7Silicibacter pomeroyi DSS3

Jannaschia sp CCS1

Psychromonas ingrahamii 37

Rhodobacterales bacterium HTCC2255

Roseovarius sp. TM1035

Roseovarius sp 217Roseobacter sp Azw3B

Sinorhizobium medicae WSM419

Sinorhizobium meliloti 1021

Agrobacterium tumefaciens str C58

Oceanibulbus indolifex HEL45

Oceanicola granulosus HTCC2516

Roseovarius nubinhibens ISM

Stappia aggregata IAM 12614

Silicibacter sp TM1040

Phaeobacter gallaeciensis 2.10

Phaeobacter gallaeciensis BS107

Pseudomonas putida W619

Agrobacterium tumefaciens str C58

Rhizobium etli CFN 42Rhizobium leguminosarum WSM2304


Rhizobium leguminosarum bv viciae 3841

Mesorhizobium sp BNC1


Burkholderia ambifaria MC406Burkholderia multivorans ATCC1761 6

Alkalilimnicola ehrlichei MLHE1

Reinekea sp MED297

Chromohalobacter salexigens DSM3043

Marinobacter algicola DG893

Marinobacter aquaeolei VT8

Marinobacter sp ELB17

Plesiocystis pacifica SIR-1

Verminephrobacter eiseniae EF012

Pseudomonas fluorescens Pf5

Serratia proteamaculans 568

Herpetosiphon aurantiacus ATCC23779

Photorhabdus luminescens subsp laum

ondii TTO1

Burkholderia pseudomallei DM

98

Psychrobacter cryohalolentis K5

Blastopirellula marina D

SM 3645

Gem

mata obscuriglobus U

QM

2246

Rhodopirellula baltica SH

1

Pseudom

onas aeruginosa C3719

Pseudom

onas phage D3

Pseudom

onas aeruginosa UC

BP

PP

A14

Pseudom

onas aeruginosa PA

O1

Pseudom

onas aeruginosa PA

7

Acinetobacter baumannii ATC

C 17978

Acinetobacter baumannii

Chrom

ohalobacter salexigens DS

M 3043

Chrom

obacterium violaceum

AT

CC

12472

Pseudom

onas fluorescens PfO

1

Pseudom

onas putida F1

Pseudom

onas putida KT

2440

Pseudom

onas putida GB

1

Pseudom

onas entomophila L48

Burkholderia phym

atum S

TM815

Burkholderia xenovorans LB

400

Burkholderia phytofirm

ans PsJN

Myxococcus xanthus D

K 1622

Xanthom

onas axonopodis str 306

Xanthom

onas campestris str8510

Xanthom

onas oryzae MA

FF

311018

Xanthom

onas oryzae pv oryzae KA

CC

10331X

anthomonas oryzae B

LS256

Xanthom

onas campestris str A

TC

C33913

Bur

khol

deria

thai

land

ensi

s B

t4

Bur

khol

deria

thai

land

ensi

s T

XD

OH

Bur

khol

deria

thai

land

ensi

s E

264

Bur

khol

deria

mal

lei A

TC

C 2

3344

Bur

khol

deria

mal

lei G

B8

hors

e 4

Bur

khol

deria

pse

udom

alle

i 171

0b

Bur

khol

deria

pse

udom

alle

i K96

243

Bur

khol

deria

pse

udom

alle

i 14

Bur

khol

deria

pse

udom

alle

i DM

98B

urkh

olde

ria th

aila

nden

sis

MS

MB

43

Bur

khol

deria

okl

ahom

ensi

s E

O14

7

Bur

khol

deria

cen

ocep

acia

MC

03

Bur

khol

deria

cen

ocep

acia

AU

105

4B

urkh

olde

ria s

p 38

3

Bur

khol

deria

am

bifa

ria M

C40

6

Bur

khol

deria

cep

acia

AM

MD

Bur

khol

deria

vie

tnam

iens

is G

4

Bur

khol

deria

mul

tivor

ans

AT

CC

176

16

Bur

khol

deria

ubo

nens

is B

u

Bur

khol

deria

dol

osa

AU

O15

8

Bur

khol

deria

dol

osa

AU

O15

8

Phaeobacter gallaeciensis B

S107

Phaeobacter gallaeciensis 2.10

Roseobacter sp M

ED

193

Roseobacter sp S

K20926

Stappia aggregata IA

M 12614

Marinom

onas sp MW

YL1

Roseovarius sp 217

Roseovarius sp. T

M1035

Silicibacter pom

eroyi DS

S3

Sili

ciba

cter

sp

TM

1040

Bacillu

s ce

reus

W

Bacillu

s ce

reus

AH82

0

Bacillu

s ce

reus

Bacillu

s cer

eus E

33L

Bacillu

s an

thra

cis s

tr Am

es

Bacillu

s th

urin

gien

sis s

tr Al H

akam

Baci

llus

cere

us A

H18

7

Baci

llus

cere

us A

TCC

145

79

Baci

llus

cere

us A

TCC

109

87

Baci

llus

cere

us G

9842

Baci

llus

thur

ingi

ensi

s AT

CC

356

46

Bac

illus

cer

eus

G92

41

Bac

illus

cer

eus

H30

81.9

7

Bac

illus

cer

eus

AH

1134

Bac

illus

thur

ingi

ensi

s st

r 972

7

Bac

illus

cer

eus

NV

H05

97-9

9

Bac

illus

wei

hens

teph

anen

sis

KB

AB

4

Bac

illus

thur

ingi

ensi

s A

TCC

3564

6

Plesiocystis pacifica SIR-1

Microscilla marina ATCC 23134

Flavobacteriales bacterium HTCC2170

Colwellia psychrerythraea 34H

Shewanella amazonensis SB2B

Shewanella loihica PV4

Shewanella sediminis HAWEB3

Shewanella woodyi ATCC 51908

Shewanella pealeana ATCC 700345

Shewanella halifaxensis HAW-EB4Roseiflexus sp RS1

Chloroflexus aurantiacus J10

Hoeflea phototrophica DFL4243

Nematostella vectensisNematostella vectensisNematostella vectensis

Strongylocentrotus purpuratusStrongylocentrotus purpuratusStrongylocentrotus purpuratus

Tetraodon nigroviridisDanio frankei

Silurana

Monodelphis domestica

Canis familiaris

Bos grunniens x Bos taurus

Equus caballus

Mus musculus x Rattus norvegicus

Mus musculus

Mus musculusMus musculus

Homo sapiens

Macaca mulatta

Pan troglodytes

Gallus gallus

Ornithorhynchus anatinus

Aspergillus nidulans FGSC A4Aspergillus nigerAspergillus terreus NIH2624Aspergillus fumigatus Af293Aspergillus fumigatus A1163

Neosartorya fischeri NRRL 181

Methylobacterium nodulans ORS 2060

Gibberella zeae PH1Aspergillus oryzae

Aspergillus niger

(C)

(D)

(E)

Burkholderia phymatum STM815

Burkholderia xenovorans LB400

Burkholderia

phytofirm

ans PsJ

N

Burkholderia cenocepacia AU 1054

Burkholderia cenocepacia PC184

Burkholderia cenocepacia M

C03

Burkholderia ambifaria MC406

Burkholderia

cepacia

AMMD

Burkholderia sp 383

Burkholderia

multiv

orans ATCC 1761

Burkholderia

ubonensis

Rubro

bacte

r xyla

noph

ilus D

SM 9

941

mar

ine a

ctino

bacte

rium

PHSC20

C1

Brevib

acte

rium

linen

s BL2

100

97

64

99

75

77

99

92

71

99

Phaeosphaeria

nodorum SN15

Asperg

illus n

iger

Gibber

ella z

eae P

H1

Candida parapsilosis

(A)

(B)

Fungiα-proteobacteria

γ-proteobacteriaActinobacteria

β-proteobacteria

miscellaneous bacteria

TrypanosomaMetazoaFirmicutes




Reduced Proline racemase maximum likelihood phylogeny with active site alignmentFigure 3Reduced Proline racemase maximum likelihood phylogeny with active site alignment. Bootstrap resampling (100 iterations) was undertaken and percentages are displayed. Fungal branches are shown in green. An alignment around the active site is also displayed. Clade letters in parentheses correspond to those in Figure 2. The phylogeny is rooted around the Meta-zoan/Pezizomycotina specific clade (clade-C), all members of this clade have a threonine at the active site. C. parapsilosis and its phylogenetic neighbors have a threonine instead of a cysteine at the active site (clade-A). A. oryzae, A. niger and G. zeae all con-tain cysteine at the active site (clade-D). A. flavus, A. oryzae, A. niger, A. nidulans and G. zeae also have cysteine at the active site (clade-C).

Brucella suisBrucella melitensisParacoccus denitrificansAgrobacterium tumefaciens

Silicibacter

Aspergillus oryzae

Aspergillus oryzaeAspergillus oryzae

Aspergillus niger

Gibberella zeae

Psychromonas ingrahamii

Brucella suisBrucella melitensis

Paracoccus denitrificans

Agrobacterium tumefaciens

Burkholderia cenocepaciaBurkholderia cenocepaciaBurkholderia multivorans

Burkholderia ambifariaBurkholderia cepacia

Burkholderia phymatum

Burkholderia xenovoransBurkholderia phytofirmans

Burkholderia cenocepacia


Burkholderia ambifaria

BurkholderiaBurkholderia cepacia

Burkholderia cenocepaciaBurkholderia cenocepacia

Burkholderia xenovoransBurkholderia phytofirmansBurkholderia phymatum

Pan troglodytesHomo sapiens

Mus musculusRattus norvegicus

Aspergillus niger

Neosartorya fischeriAspergillus fumigatus

Aspergillus nidulans100

100

100

95

55

55

100

100

100

55

95

100

7590

70

100

65

100

100

95

100

95

100

100

100

100

100

100 90

95

100

100

95

100

100

60

95

55

65

100

90

100

Silicibacter

Aspergillus terreus

Aspergillus flavus

100

100 Aspergillus niger Gibberella zeae

Phaeosphaeria nodorum

100

Aspergillus flavus

Aspergillus flavus

marine actinobacteriumBrevibacterium linens

DRSPTGSGVTARIALDRSPTGSGVTARIALDRSPTGSCVTARLALDRSPTGSCVAARVALDRSPTGSCVTARMALDRSPTGSCVTARMAL

DRSPTGSCVTARMALDRSPTGSCVIARMAL

DRSPTGSGTAGRAAQDRSPTGSGTGGRVAQDRSPTGSGTGGRVAQDRSPTGSGTGGRVAQDRSPTGSGTGGRVAQDRSPTGSGTGGRVAQDRSPTGSGTGGRVAQ

DRSPTGSGTGGRVAQDRSPTGSGTGGRVAQDRSPTGSGTAGRVAQ DRSPCGSGTCARIATDRSPCGTGTSARVAADRSPCGSGTASRIAVDRSPCGSGTCARIATDRSPCGSGTSARLAI

DRSPTGTALSARMAVDRSPCGTGTSARMAQDRSPCGTGTSARMAQDRSPCGTGTSARMAQDRSPCGTGSSGFVACDRSPCGTGTSAKLACDRSPCGTGTSAKLAC

DRSPCGTGTSAKLACDRSPCGTGTSAKLACDRSPCGTGTSAKLACDRSPCGTGTSAKVACDRSPCGTGTSAKVACDRSPCGTGTSAKVAC

DRSPTGTGCSARMAV

DRSPTGTGCSARMAV

DRSPTGTGCSARMAVDRCPTGTSVSARMAI

DRSPTGTAVSARMALDRSPCGTGSSSRLAI

DRCPCGTGSCARMAV

DRSPTGSGVTARIALDRSPTGSGVTARIAL

DRCPCGTGSSARMAVDRCPCGTGSSARMAV

DRSPCGTGSSARMAVDRSPCGTGSSARMAVDRSPCGTGSSARMAV

DRSPTGTGCSARMAV

(A)

(C)

(D)

(B)


ful gene transfers into Burkholderia species [36,37]. It ispossible therefore that there have been other successfulgene transfers into this bacterial lineage.

The vast majority of amino acids found in living cells cor-respond to the L-stereoisomer [38]. However, D-aminoacids are long known to be found in the cell walls of Grampositive and negative bacteria, where they are essentialcomponents of peptidoglycan [39]. Apart from low levelsof D-amino acids derived from spontaneous racemizationas a result of aging [40], it was assumed that only L-aminoacid enantiomers were present in eukaryotes [41]. How-ever, recent studies have reported the presence of numer-ous D-amino acids in an array of organisms, includingmammals [42]. The first eukaryotic (proline) amino acidracemase has recently been described from the humanpathogen Trypanosoma cruzi [43]. A high degree ofsequence similarity was observed between the T. cruzi andbacterial homologs [43]. Our phylogeny infers that T.cruzi obtained its PR homolog through interdomain HGTfrom a member of the Firmicutes (subclass Clostridia), asit is grouped beside members of this group with a highdegree of support (Figure 2 clade-E 96% BP). We per-formed database searches [44], against other Protozoangenomes including Trypanosoma brucei, Trypanosoma con-golense and Trypanosoma annulata. We failed to locate ahomolog in all species except for T. vivax.

Previous analysis has shown that T. cruzi and T. vivax arenot each others closest phylogenetic neighbors, relative tothe other species sampled [45]. This suggests an ancestralTrypanosoma gained the PR gene and multiple losses indifferent Trypanosoma lineages has subsequentlyoccurred.

Gene order around PR homologsThe C. parapsilosis PR homolog lies close to an ortholog(CPAG_02041) of orf19.1135 from C. albicans (Figure 4).The gene order to the left of this ORF is conserved in allCTG species, the order to the right is conserved in mostCTG species apart from C. parapsilosis and L. elongisporus.C. parapsilosis and L. elongisporus are closely related [29],and an examination of synteny suggests that the PR gene(together with a second ORF, cpar5437) were insertedbetween CPAG_2041 and CPAG_2037 (Figure 4).cpar5437 encodes a neutral amino acid (AA) transporter.The presence of an AA transporter beside the PR homologis interesting. If the putative proline racemase has a role inamino acid metabolism, then the presence of the trans-porter may be the result of an adaptive translocation toenhance the activity of the PR gene. Unlike the PR ORF theAA transporter is fungal in origin. Most CTG species con-tain a single neutral AA transporter; however C. parapsilosisand D. hansenii have four.

We located tRNA genes for nearly all CTG species besidethe large conserved syntenic block (Figure 4). It has beenshown that tRNA genes are associated with genomicbreakpoints [46]. We hypothesize that a genomic rear-rangement has occurred at this site in the LCA of C. parap-silosis and L. elongisporus. We cannot determine if thebacterial PR homolog was inserted into the LCA of L.elongisporus/C. parapsilosis and subsequently lost in L.elongisporus, or gained by C. parapsilosis after speciation.

We also investigated the gene order around the Pezizomy-cotina PR homologs [see additional file 3]. Gene syntenyaround the PR homologs found in clade-D (Figures 2 &3)is not conserved (not shown). Interestingly however, bothA. niger and G. zeae in clade-D (Figures 2 &3) have genescontaining a FAD dependent oxidoreductase domain inclose proximity to their PR homologs (not shown).According to Pfam [47], FAD dependent oxidases includeD-amino acid oxidases, that catalyze the oxidation of neu-tral and basic D-amino acids into their correspondingketo acids. The presence of these oxidases may be anotherexample of an adaptive translocation to enhance the activ-ity of the PR gene in these Pezizomycotina species.

A. oryzae has three PR homologs (Figures 2 &3 clade-C).All of these have orthologs in its close relative A. flavus(Figure 3 clade-C), and synteny around these is conserved[see additional file 3 clade-D]. The remaining two speciesin clade-C are A. niger and G. zeae. There is no evidence ofconserved gene order within these species, or with A.oryzae or A. flavus. Gene order around the A. flavus and A.terreus PR homologs found in the Metazoan/Pezizomy-cotina clade (Figures 2 &3) is also conserved [see addi-tional file 3], as is the order between A. fumigatus and N.fishceri [see additional file 3]. We could not locate aminoacid transporters or FAD dependent oxidases beside anyof the PR homologs found in clades B or C (Figure 2).

Proline racemase codon usageIt has been shown that recently acquired genes often dis-play an atypical codon preference when compared toother genes in the genome [48,49]. However, the trans-ferred PR homologs have a codon usage consistent withthe rest of their genomes [see additional file 4]. We under-took an analysis of variation in synonymous codon usageon all PR genes shown in Figure 2. Homologs from relatedspecies cluster together [see additional file 5]. For exam-ple, the Actinobactria, the Firmicutes and the Burkholderiaspecies all inhabit unique areas in two dimensional corre-spondence analysis space [see additional file 5].

The majority of fungal and Metazoan PRs are clusteredtogether [see additional file 5]. The C. parapsilosis PRhomolog has a codon usage distinct from the other Pezi-zomycotina fungal PR homologs [see additional file 5],



which is unsurprising as C. parapsilosis belongs to the Sac-charomycotina subphylum. The C. parapsilosis homolog isalso separate from the Burkholderia (β-proteobacteria)genes with which it forms a closely related phylogeneticgroup (Figures 2 &3). This suggests that the gene may have

originated from a genome with no other close relativesamong the species analyzed here.

Proline racemase activityThe PR active site from Trypanosoma cruzi, Clostridium stick-landii, Agrobacterium tumefaciens, Brucella melitensis and

Gene order around C. parapsilosis proline racemase geneFigure 4Gene order around C. parapsilosis proline racemase gene. Species names and identifiers are shown in each box. Gene identifiers relate to annotations from the Broad Institute [66]. On the left hand side orthologous genes are stacked under one another in pillars. Relative positions of t-RNA genes are shown and may indicate a breakpoint. After the breakpoint, synteny is conserved between C. albicans, C. dubliniensis, C. tropicalis, D. hansenii and Cl. lusitaniae. Synteny between C. parapsilosis and L. elongisporus is conserved but differs to the other CTG species. C. parapsilosis has a proline racemase (PR CPAG_02038) and a neutral amino acid transporter (AA cpar5437) insertion in this region. cpar5437 is absent from the Broad gene list but present in our manual gene call.

C. albicansorf19.1136

C. parapsilosisCPAG_02042

L. elongisporusLELG_00344

CLUG_03490

P. guillermondiiPGUG_01868

C. tropicalisCTRG_03467

C. dubliniensis

D. hansenii

F11187g




CLUG_03488



C. dubliniensis

D. hansenii

F11231g




CLUG_03489



C. dubliniensis

D. hansenii

F11209g

tRNA




CLUG_03491



C. dubliniensis

D. hansenii

F11165g tRNA

tRNA

tRNA

tRNA

AAPR

C. parapsilosis

CPAG_02037

C. parapsilosis

CPAG_02036

L. elongisporus

LELG_00342

L. elongisporus

LELG_00341

C. albicansorf15282

CLUG_03492


C. dubliniensis

D. hanseniiF11121g

C. albicansorf15281


C. dubliniensis

Cl. lusitaniae Cl. lusitaniaeCl. lusitaniaeCl. lusitaniae

Cl. lusitaniae

Cd11070 Cd11090

Cd11110

Cd11080Cd11060

Cd11100

C. parapsilosis

C. parapsilosis CPAG_02038

cpar5437



Pseudomonas aeruginosa all contain cysteine at amino acidposition 330 [43,50]. This amino acid is essential forenzymatic function, because substitution with serineabolishes activity [41]. However, PR homologs fromhuman, mouse, Rhizobium and Brucella contain a threo-nine instead of a cysteine at position 330 [41]. Weobserved that cysteine is found in the equivalent positionin many of the bacterial proteins. The Pezizomycotina PRgenes found in clade-B and clade-D contain a cysteine atthe active site (Figure 3). The PR homologs found in theMetazoan/Pezizomycotina clade (clade-B) have a threo-nine at position 330. Similarly, the C. parapsilosis PRhomolog, together with its relatives from Burkholderia allcontain a threonine (Figure 3). However, Burkholderia spe-cies have multiple PR homologs [see additional file 2]with a cysteine as the active site (not shown). It is not clearwhat effect the substitution has on enzyme activity. It hasbeen suggested that homologs containing threonine at theactive site are not true PRs [41], but may instead belong toa superfamily. We cannot detect any difference in the abil-ity of C. parapsilosis, the other CTG species or any of thePezizomycotina species to utilize D-proline as growthmedia (data not shown). We therefore cannot confidentlyinfer the function of the PR homologs in the fungi ana-lyzed here.

Phenazine F phylogeny and characterisationThe C. parapsilosis gene (designated CPAG_03462) is mostsimilar to a Photorhabdus luminescens phenazine F (PhzF)protein with 61% pairwise identity (Figure 1B). Phena-zines are biologically active compounds, all of which havea characteristic tricyclic ring system and have been shownto confer a selective growth advantage to organisms whichsecrete them, as they possess broad-spectrum antibioticactivity towards bacteria, fungi and higher eukaryotes[51]. In Pseudomonas, the best studied phenazine pro-ducer, PhzF is part of an operon required for the conver-sion of chorismic acid to phenazine-1-carboxylate (PCA)[52]. PhzF homologs were identified in most of the CTGspecies tested as well as several other fungal species. How-ever, we could not identify a PhzF homolog in the L. elong-isporus genome, even when multiple TBlastN and BlastNsearches were used.

PhzF homologs were extracted from GenBank for subse-quent phylogenetic analysis. In total 181 representativeprotein coding sequences distributed amongst 154 organ-isms were used. These taxa were distributed amongst α, β,γ and δ-proteobacteria, Actinobacteria, Fungi, Firmicutes awell as other bacterial groups.

We aligned all sequences and reconstructed a PhzF MLphylogeny (Figure 5). The C. parapsilosis PhzF homolog isfound in a clade with members of the β-proteobacteria(Burkholderia multiovorans, Burkholderia cepacia, Burkholde-

ria ambifaria), α-proteobacteria (Roseovarius) and the γ-proteobacteria (Azotobacter vinelandii, Acinetobacter bau-mannii, Shewanella baltica and Photorhabdus luminescens)(81% BP). In contrast, all other PhzF homologs from CTGspecies are in a completely separate clade (Figure 5). Theseform a sister group (63% BP) to PhzF homologs fromother Saccharomycotina species (C. glabrata, Saccharomy-ces cerevisiae, Kluyveromyces lactis and Vanderwaltozymapolyspora). All three clades are grouped together in a largerclade with high support (75% BP).

The sister group relationship between the PhzF homologsfrom the Ascomycota and the proteobacteria clade isintriguing (Figure 5), as it suggests that an ancestral Sac-charomycotina species gained the PhzF homolog from aproteobacteria. The bacterial PhzF gene has subsequentlybeen retained after multiple speciation events, but lost inC. parapsilosis. We hypothesize that C. parapsilosis hasrecently reacquired a bacterial PhzF homolog from a pro-teobacterial source, as it is grouped (81% BP) within aproteobacterial subclade. To test this hypothesis we recon-structed constrained trees that placed C. parapsilosistogether with the remaining Ascomycota species [seeadditional file 6 C-H]. The AU test of phylogenetic treeselection [53], showed that the original unconstrainedtree (groups C. parapsilosis with proteobacteria) receivesthe optimal likelihood tree score, and the differences inlikelihood scores when compared to the constrained trees[see additional file 6], are significant (P < 0.05). This isalso supported by spectral analysis [see additional file 7].

Our phylogeny shows that the Schizosaccharomyces pombePhzF homolog is found in a clade containing all CTGPhzF homologs (Figure 5 99% BP). Furthermore it isgrouped beside D. hansenii (66% BP). S. pombe is not amember of the Saccharomycotina, it belongs to theTaphrinomycotina subphylum. The genome sequences ofSchizosaccharomyces japonicus and Schizosaccharomyces oct-osporus have recently been completed [54]. We could notlocate a PhzF homolog in S. japonicus but did locate ahomolog in S. octosporus using a TBlastN search strategy.Phylogenetic analysis has shown that S. pombe and S. oct-osporus are more closely related to one another than to S.japonicus [55]. Therefore we hypothesize that the LCAancestor of S. pombe and S. octosporus gained the PhzF genefrom an ancestral D. hansenii-like species after speciationfrom S. japonicus. We reconstructed a constrained tree thatplaced S. pombe outside the Saccharomycotina clade [seeadditional file 6B]. The approximately unbiased test ofphylogenetic tree selection (AU test) [53], showed that thephylogenetic inferences of the unconstrained tree are sig-nificantly better (P < 0.05) than the constrained tree [seeadditional file 6]. This infers that S. pombe has obtained aPhzF homolog from a member of the CTG clade.



A small basidiomycete clade is evident amongst prokary-ote species (Figure 5). Both Ustilago maydis and Malasseziaglobosa belong to the Ustilaginomycotina subphylum.Therefore our phylogeny infers that an ancestral Ustilagi-nomycotina species gained a PhzF gene from an unknown

bacterial source, and both species have retained this afterspeciation.

A correspondence analysis of synonymous codon usagefor all PhzF homologs was also performed and is shown

PhzF maximum likelihood phylogenyFigure 5PhzF maximum likelihood phylogeny. The optimum model of protein substitution was found to be WAG+G. The number of gamma rate categories was 4 (alpha = 0.873). Bootstrap resampling (100 iterations) was undertaken and are dis-played. For display purposes branches with less than 50% support were collapsed. Branches are colored according to their tax-onomy. Fungal branches are shown in green. The S. pombe PhzF homolog is highlighted with a red rectangle.

Saccharopolyspora erythraea

Mycobacterium ulcerans Agy99

Salinispora tropica CNB440


Corynebacterium glutamicum

Corynebacterium glutamicum

Congregibacter litoralis KT71

Methylibium

petroleiphilum PM

1

Zymom

onas mobilis ZM

4

Gluconacetobacter diazotrophicus PAL5

Kineococcus radiotolerans SRS30216

Arthrobacter sp FB24

Arthrobacter chlorophenolicus A6

Arthrobacter aurescens TC1

Acidovorax sp JS42

Acidovorax avenae A

AC

00

Nocardioides sp JS

614

Janibacter sp HTC

C2649

Pse

udom

onas

syr

inga

e st

r D

C30

00P

seud

omon

as s

yrin

gae

1448

A

Pse

udom

onas

syr

inga

e B

728aC

hromobacterium

violaceumP

seudomonas fluorescens P

f5

Bradyrhizobium

sp BTA

i1

Bradyrhizobium

sp OR

S278 P

seud

omon

as p

utid

a W

619

Pse

udom

onas

aer

ugin

osa

PA

O1

Pse

udom

onas

aer

ugin

osa

PA

7

Pse

udom

onas

aer

ugin

osa

PA

CS

2

Pse

udom

onas

aer

ugin

osa

C37

19

Pse

udom

onas

aer

ugin

osa

UC

BP

PP

A14

Frankia sp E

AN

1pec

Frankia alni A

CN

14aF

rankia sp CcI3

Malassezia globosa C

BS

7966

Ustilago m

aydis 521A

cidiphilium cryptum

JF5

Agrobacterium

tumefaciens str C

58B

urkholderia cenocepacia AU

1054D

elftia acidovorans SP

H1

Rho

dofe

rax

ferr

iredu

cens

T11

8

Pol

arom

onas

nap

htha

leni

vora

ns C

J2

Pol

arom

onas

sp

JS66

6

Del

ftia

acid

ovor

ans

SP

H1

Com

amon

as te

stos

tero

ni K

F1

Burkholderia phymatum STM815

Burkholderia xenovorans LB400

Burkholderia phytofirmans PsJN

Burkholderia oklahomensis C6786

Burkholderia oklahomensis EO

147

Burkholderia thailandensis MSM

B43

Burkholderia pseudomallei 91

Burkholderia pseudomallei B7210

Burkholderia pseudomallei 9

Burkholderia pseudom

allei 7894


allei BC

C215

Burkholderia thailandensis E

264

Burkholderia thailandensis T

XD

OH

Burkholderia m

allei ATC

C 23344


allei 668


allei 305


allei K96243


allei 14

Burkholderia cenocepacia AU 1054

Burkholderia cenocepacia MC03

Burkholderia cenocepacia PC184

Burkholderia sp 383

Burkholderia ubonensisBurkholderia dolosa AUO158

Burkholderia multivorans ATCC1761


Burkholderia cepacia AMM

D

Burkholderia vietnamiensis G4

Erwinia carotovora SCRI1043

Erwinia chrysanthemi str 3937

Sodalis glossinidius str morsitans

Yersinia frederiksenii ATCC 33641

Roseova

rius s

p 217


Azotobacter vinelandii AvOP


Burkholderia cepacia AMMD

Burkholderia multivorans ATCC 17616

Photorhabdus luminescens TTO1

Shewanella baltica OS155


Acinetobacter baumannii ATCC 17978


Candida glabrata

Saccharomyces cerevisiae YJM789

Kluyveromyces lactis

Vanderwaltozyma polyspora

Clavispora lusitaniae

Debaryomyces hansenii CBS767

Schizosaccharomyces pombe 972hPichia guilliermondii ATCC 6260Pichia stipitis CBS 6054Candida tropicalisCandida dubliniensisCandida albicans SC5314

Candida albicans

Steno

troph

omon

as m

alto

philia

R55

13

Xanthomonas oryz

ae MAFF 311018

Xanthomonas oryz

ae KACC10331

Xanthomonas oryz

ae BLS256

Xanth

omon

as a

xono

podis

str 3

06

Xantho

monas

campe

stris

str 85

Xanth

omon

as c

ampe

stris

ATC

C3391

3

Xanth

omon

as ca

mpe

stris

pv ca

mpe

stris

Ste

notro

phom

onas

mal

toph

ilia

R55

13

Xan

thom

onas

cam

pest

ris A

TCC

3391

3

Xan

thom

onas

cam

pest

ris s

tr 85

Xant

hom

onas

axo

nopo

dis

str 3

06

Xant

hom

onas

ory

zaeB

LS25

6

Xant

hom

onas

ory

zae

KAC

C10

331

Xanth

omon

as o

ryza

e M

AFF31

1018

Paracoccus denitrificans P

D1222

Zym

omonas m

obilis ZM

4

Alkaliphilus m

etalliredigenes QY

MF

Clostridium

difficile QC

D32g58

Clostridium

difficile 630

Natronom

onas pharaonis DS

M 2160

Halobacterium

sp NR

C1

Cam

pylo

bact

er fe

tus

subs

p fe

tus

8240

Clo

strid

ium

diff

icile

QC

D-6

3q42

Clo

strid

ium

ace

tobu

tylic

um A

TC

C 8

24

Clo

strid

ium

bei

jerin

cki N

CIM

B 8

052

Des

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obac

teriu

m h

afni

ense

Y51

Clo

strid

ium

bol

teae

AT

CC

BA

A

Bac

illus

sp

B14

905

Clo

strid

ium

sp

OhI

LAs

Bac

illus

pum

ilus H

erpetosiphon aurantiacus

Cal

dice

llulo

siru

ptor

sac

char

olyt

icus

Bla

stop

irellu

la m

arin

a D

SM

364

5

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la b

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H1

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dopi

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la b

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a S

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onas

ace

toxi

dans

DS

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84

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noth

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sp C

CY

0110

Syn

echo

cyst

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p P

CC

680

3

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eoba

cter

sp

CC

S2

Rho

doba

cter

ales

bac

teriu

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TCC

2654

Salin

ispo

ra a

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S205

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440

Reinekea sp MED297Bradyrhizobium japonicum USDA 110Hoeflea phototrophica DFL4343


Rhizobium leguminosarum 3841


Rhizobium etli CFN 42

Sinorhizobium meliloti 1021Sinorhizobium medicae WSM419

Rhodopseudomonas palustris BisB18

Rhodopseudomonas palustris CGA009

Rhodopseudomonas palustris TIE1




Haloba

cteriu

m sp

NRC1

Lyng

bya

sp P

CC 810

6

Trichodesm

ium erythra

eum IMS101

Chloroflexu

s aurantia

cus

Chrom

obac

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ceum

Ralst

onia

eut

roph

a JM

P134

Burkh

olde

ria c

enoc

epac

ia M

C03

Jannaschia sp CCS1

Dinoroseobacter shibae

Sagittula stellata

Methylobacterium populi BJ001

Xanthobacter autotrophicus Py2

Stappia aggregata IAM 12614

Ochrobactrum anthropiBrucella suis 1330

Sinorhizobium meliloti 1021

Sinorhizobium medicae WSM419

Pseudomonas a

eruginosa P

AO1

Erwinia ca

rotovora SCRI1043

Burkholderia sp 383

Burkholderia sp 383

Fungiα-proteobacteria

γ-proteobacteriaActinobacteria

β-proteobacteria

miscellaneous bacteriaArchaea



in additional information [see additional file 8]. The S.pombe PhzF homolog has a codon usage pattern very sim-ilar to the D. hansenii protein.

Gene order around PhzFAnalysis of the genes adjacent to the PhzF homolog in C.parapsilosis shows that there is a high conservation of gene

synteny and supports our hypothesis that PhzF wasrecently acquired in this species (Figure 6). Homologs inthe other CTG species are located in completely differentregions of the genome relative to C. parapsilosis (notshown). For example, the C. albicans PhzF homolog islocated between orf19.5619 and orf19.5621, whereas theC. parapsilosis homolog is found between orf19.6689 &

Gene order around C. parapsilosis PhzF geneFigure 6Gene order around C. parapsilosis PhzF gene. Species names and gene identifiers are shown in each box. Orthologous genes are stacked under one another in pillars. The C. parapsilosis PhzF homolog (CPAG_03462) is highlighted with a red box. Synteny relative to the C. parapsilosis PhzF homolog is conserved in all species. Other CTG PhzF homologs are found in com-pletely different regions of the genome relative to C. parapsilosis. L. elongisporus is the only CTG species missing a PhzF gene, and there is no evidence for a pseudogene in the genome.




CLUG_01660



C. dubliniensis

D. hanseniiC14586




CLUG_01662



C. dubliniensis

D. hanseniiC14630g




CLUG_01661



C. dubliniensis

D. hanseniiC14608




CLUG_01659



C. dubliniensis

D. hanseniiC14564




Cl. lusitaniaeCLUG_01663



C. dubliniensis

D. hanseniiC146740g


Cl. lusitaniaeCl. lusitaniaeCl. lusitaniaeCl. lusitaniae

Cd73180Cd73190Cd73200Cd73320 Cd73170



orf19.6687 relative to C. albicans SC5314 (Figure 6).However, the L. elongisporus genome contains no PhzFhomolog, either at a position equivalent to the C. parapsi-losis copy or elsewhere in the genome.

We propose that the LCA of L. elongisporus and C. parapsi-losis lost the PhzF gene present in the other CTG species,and a second (new) copy was subsequently gained by C.parapsilosis after speciation. We have partial sequence data(unpublished) from Candida orthopsilosis, a species soclosely related to C. parapsilosis that it was once designatedC. parapsilosis group II [56]. We located a C. orthopsilosis PRhomolog that is 83% identical (at the amino acid level) tothe C. parapsilosis copy. This implies that the commonancestor of C. parapsilosis and C. orthopsilosis acquired thebacterial PhzF homolog after speciation from L. elongis-porus.

Mechanisms of gene transfer into fungi are poorly under-stood. To date no DNA uptake mechanism has been iden-tified in CTG species. Interkingdom conjugation betweenbacteria and yeast has been observed however [57-59].Similarly, Saccharomyces cerevisiae has been shown to betransformant competent under certain conditions [60].CTG species are known interact with bacteria in vivo [61],and it is therefore possible that interkingdom conjugationand transformation may facilitate DNA transfer in C. par-apsilosis. These mechanisms may also be applicable to thePezizomycotina species examined in this analysis.

ConclusionWe investigated the frequency of recent interkingdomgene transfer between CTG and bacterial species. Welocated two strongly supported incidences of HGT, bothwithin the C. parapsilosis genome. We also located inde-pendent transfers into the Pezizomycotina, Basidiomy-cotina and Protozoan lineages.

We cannot determine the exact origin of the PR homolog(CPAG_02038) found in the C. parapsilosis genome. How-ever, based on its phylogenetic position it either origi-nated from a Burkholderia source, or more likely anorganism not yet represented in the sequence databases.Our PR phylogenetic analysis also suggests there were twoindependent transfers into Pezizomycotina species, onefrom an Actinobacterial source, and the second is from anunknown proteobacterial source. There is also evidencethat T. cruzi has obtained its PR homolog from a Firmi-cutes species. The transferred PR genes analyzed herebelong to a superfamily of proline racemases, althoughwe cannot determine their exact function in the fungalspecies examined. Their proximity to an amino acid trans-porter (in C. parapsilosis) and a FAD dependent oxidore-ductase (in A. niger and G. zeae) suggests they do have arole in amino acid metabolism. Furthermore, evidence of

multiple independent transfers into fungi suggests theprotein does confer a biological advantage, although wecannot determine what is. The bacteria-derived PR genehas the potential to be a novel antifungal drug target asthere would be no undesired host protein-drug interac-tions.

The bacterial PhzF homolog (CPAG_03462) found in C.parapsilosis most likely originated from a proteobacterialsource. Most CTG species examined contained PhzFhomologs, with the exception of L. elongisporus. The crys-tal structure the PhzF homolog in S. cerevisiae has beendetermined and while its function remains unknown, it isnot thought to be involved in phenazine production [62].We postulate that the PhzF homolog present in other CTGspecies was initially lost by the ancestor of C. parapsilosisand L. elongisporus, but subsequently regained by C. parap-silosis through HGT. The loss of eukaryote genes and sub-sequent reacquisition of a prokaryotic copy has previouslybeen described in yeast, and can confer specific metaboliccapabilities. An analysis of the biotin biosynthesis path-way discovered that the ancestor of Candida, Debaryomy-ces, Kluyveromyces and Saccharomyces lost the majority ofthe pathway after the divergence from the ancestor of Y.lipolytica. However, Saccharomyces species have rebuilt thebiotin pathway through gene duplication/neofunctional-ization after horizontal gene transfer from α and γ proteo-bacterial sources [20]. The acquisition of the URA1 gene(encoding dihydroorotate dehydrogenase) from Lactoba-cillus and replacement of the endogenous gene in S. cere-visiae, allowed growth under anaerobic conditions [19].Similarly, acquisition of BDS1 (alkyl-aryl-sulfatase) fromproteobacteria may have enabled the survival of S. cerevi-siae in a harsh soil environment [19]. Our PhzF phylogenysuggests that the PhzF homolog found in most CTG spe-cies originated from an ancient HGT event, from a mem-ber of the proteobacteria. Our analysis also shows that S.pombe has obtained a PhzF homolog from a CTG species,most likely one closely related to D. hansenii. There is alsophylogenetic evidence showing that an ancestral Ustilagi-nomycotina species gained a PhzF gene from an unknownbacterial source. We cannot however, determine the bio-logical advantage to the organisms.

Although it was not the major goal of this study, we didlocate HGT from bacteria into fungal genomes outside theCTG clade, and also inter-fungal transfers. In a previousanalysis of HGT in diplomonads, fifteen genes were foundto have undergone HGT [18]. There is phylogenetic evi-dence that these genes have undergone independenttransfers into other eukaryotic lineages including Fungi.Therfore, in eukaryotes just as HGT has affected some spe-cies more than others [63], there may be groups of genesthat are more likely to be taken up through HGT than oth-ers. We cannot test this directly however, as we have not



identified all cases of HGT from bacteria to fungi outsidethe CTG clade.

Our analysis indicates that recent interkingdom genetransfer into extant CTG species is negligible. This sup-ports a previous hypothesis that genetic code alterationsblocks horizontal gene transfer [64]. It should be notedhowever that we searched for recent bacterial gene trans-fers into individual CTG species, and not for more ancienttransfers. We took this approach because the presence ofrecently gained bacterial genes in a eukaryote genomeshould be readily detected compared to older transfers.Similarly, we have not investigated eukaryote-to-eukary-ote transfers. It is therefore possible that we have underes-timated the overall rate of HGT into the CTG lineage. Thediscovery of HGT in other fungal lineages implies thatHGT plays an important role in fungal evolution anddeserves further analysis. In particular a strategy which candetect ancient gene transfers would be meaningful.

MethodsSequence dataThe complete C. albicans (SC5314) genome (Assembly19) was obtained from the Candida genome database[65]. The Broad institute have sequenced and annotatedfive CTG species (C. albicans (WO-1), C. tropicalis, L. elong-isporus, P. guilliermondii, and Cl. lusitaniae). Thesegenomes were obtained directly from the Broad Institute[66]. Gene sets for the C. dubliniensis were downloadedfrom GeneDB [44].

The incomplete C. parapsilosis geneome was downloadedfrom the Sanger Institute [67]. Gene annotations wereperformed using two separate approaches. The firstinvolved a reciprocal best BLAST [31] search with a cutoffE- value of 10-7 of Candida albicans SC5314 protein codinggenes against the unannotated C. parapsilosis genome. TopBLAST hits longer than 300 nucleotides were retained asputative open reading frames. The second approachinvolved a pipeline of analysis that combined several dif-ferent gene prediction programs, including ab initio pro-grams SNAP [68], Genezilla [69], and AUGUSTUS [69],with gene models from Exonerate [70] and Genewise [71]based on alignments of proteins and Expressed SequenceTags. Putative gene sets from both approaches wereimported into Artemis [72] and cross corroborated manu-ally. The resultant gene sets contained 5,823 protein-cod-ing genes. The C. parapsilosis genome was also annotatedby the Broad Institute, and where possible we have usedthe gene names they assigned.

The UniProt database (v11.1) was downloaded [73].Database searches against GenBank refer to release 164.0.

Blast based approach to detect potential horizontally transferred genesTaking one CTG species at a time, we located gene familiesof interest by comparing individual protein coding genesagainst the UniProt database (v11.1) using the BlastPalgorithm [31] with a cutoff expectation (E) value of 10-20.To use all available sequence data, CTG proteins with atop database hit to a bacterial protein in UniProt wereextracted for a second round of database searching againstGenBank (E value of 10-20). Proteins which also had a topdatabase hit to a bacterial protein in GenBank were con-sidered as possible incidences of horizontal gene transfer.All putative homologs were extracted from GenBank andsearched against the relevant CTG genome to ensure areciprocal best Blast hit. For completeness, CTG proteinsnot yet deposited in GenBank were added to gene familiesof interest where appropriate.

Accession numbers for all sequences used in this analysiscan be found in additional material [see additional file 2].

Phylogenetic methodsGene families were aligned using MUSCLE (v3.6) [74]using the default settings. Obvious alignment ambiguitieswere corrected manually.

Phylogenetic relationships were inferred using maximumlikelihood methods. Appropriate protein models of sub-stitution were selected for each gene family using Model-Generator [75]. One hundred bootstrap replicates werethen carried out with the appropriate protein model usingthe software program PHYML [76] and summarized usingthe majority-rule consensus method.

We performed the approximately unbiased test of phylo-genetic tree selection [53], to assess whether differences intopology between constrained and unconstrained genetrees are no greater than expected by chance.

Codon usage analysis and spectral analysisTo determine if the putative HGT genes had a differentcodon usage pattern to the host genome an analysis ofvariation in synonymous codon usage was undertakenusing the GCUA software [77]. Individual correspondenceanalyses of raw codon counts for the Candida parapsilosis,Ustilago maydis, Malassezia globosa, Aspergillus flavus,Aspergillus niger, Gibberella zeae, Aspergillus oryzae, Phae-osphaeria nodorum, and Schizosaccharomyces pombegenomes were performed, with the first four principal axesbeing used to evaluate synonymous codon usage patterns.Similar analyses were also carried out on members of theproline racemase and phenazine F gene families displayedin Figures 2 and 4. We used spectrum [78] to perform aspectral analysis on a subset of the phenazine data.



Authors' contributionsDAF, MEL and GB were involved in the design phase. MELpredicted genes in unannotated genomes. DAF sourcedhomologs, examined synteny and performed phyloge-netic analyses. DAF and GB drafted the manuscript. Allauthors read and approved the final manuscript.

Additional material

AcknowledgementsThe authors wish to acknowledge the Wellcome Trust Sanger Institute and Broad institute of MIT & Harvard for releasing data ahead of publication. We would like to acknowledge the financial support of the Irish Research Council for Science, Engineering and Technology (IRCSET), the Irish Health Research Board (HRB) and Science Foundation Ireland (SFI). We wish to acknowledge the SFI/HEA Irish Centre for High-End Computing (ICHEC) for the provision of computational facilities and support. We thank Mike Lorenz and Paul Dyer for fungal strains. We also thank Jason Stajich for help with gene annotations.

Additional file 1Examples of reported incidences of interkingdom gene transfer between prokaryotes and fungi. One Kluyveromyces lactis gene (KLLA0D19949g) previously highlighted [22], been omitted as it is no longer recognized as an ORF. Y. lipolytica genes denoted with a * and ^ indicate possible gene duplications after HGT.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-8-181-S1.doc]

Additional file 2GenBank accession numbers for PR (A) and PhzF (B) sequences used in this analysis. Species identified with an * use the accession numbers created by the Broad Institute [66] or the Wellcome Trust Sanger Institute [67].Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-8-181-S2.doc]

Additional file 3Gene order around Pezizomycotina proline racemase genes. Species names and identifiers are shown in each box. PR genes are labeled. Gene identifiers relate to annotations from the Broad Institute. Clade letters in parentheses correspond to those in Figure 2. There is evidence for con-served gene synteny between some species such as A. oryzae and A. flavus (clade-C). A. flavus/A. terreus and N. fischeri/A. fumigatus in the Metazoan/Pezizomycotina clade (B). The A. flavus gene denoted by a * is absent from the Broad gene set but we were able to locate it with a BlastX search.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-8-181-S3.eps]

Additional file 4Correspondence analysis of codon usage. Correspondence analysis of codon usage in the C. parapsilosis (1), U. maydis (2), M. globosa (3), A. flavus (4), A. niger (5), G. zeae (6), A. oryzae (7), P. nodorum (8), and S. pombe (9) genomes. Transferred genes are highlighted. All have a codon usage similar to the rest of their genomes which is unsurpris-ing as transferred genes have been shown to ameliorate their codon usage to their hosts [79].Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-8-181-S4.pdf]

Additional file 5Correspondence analysis of codon usage in the proline racemase gene family analyzed in this study. Major groups are color-coded. The C. par-apsilosis PR gene has a codon usage pattern distinct from other fungal species in this analysis. It is also quite distinct from the Burkholderia (β-proteobacteria) species, which were found to be its phylogenetic neighbors.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-8-181-S5.eps]

Additional file 6Trees for approximately unbiased test for PhzF homologs. Tree A is the original unconstrained topology, which groups C. parapsilosis with pro-teobacteria. Topology B is a constrained tree that places S. pombe outside the Saccharomycotina clade. Topologies C-H are constrained and place C. parapsilosis amongst the other Saccharomycotina species. Log likelihood scores for each tree are given. To assess the likelihood that any differences in topology between the inferred trees is no more significant that that expected by chance, we performed the approximately unbiased test. The AU test shows that the unconstrained tree receives the optimal likelihood tree score. Furthermore, the differences in likelihood scores when com-pared to the constrained trees are significant (P < 0.05). Therefore based on these results the placement of the C. parapsilosis homolog in the pro-teobacterial clade to the exclusion of the Saccharomycotina and S. pombe within the Saccharomycotina clade is significant.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-8-181-S6.eps]

Additional file 7PhzF spectral analysis. Analysis was performed on the Saccharomycotina and selected proteobacterial clade. Bars above the x-axis represent fre-quency of support for each split. Bars below the x-axis represent the sum of all corresponding conflicts. Clad grams above columns represent the cor-responding splits in the data. There is no support for the placement of C. parapsilosis with the other Saccharomycotina species.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-8-181-S7.eps]

Additional file 8Correspondence analysis of codon usage in the PhzF gene family ana-lyzed in this study. Major groups are color-coded. The C. parapsilosis PhzF gene has a codon usage pattern similar to other CTG species ana-lyzed. It is quite distinct from the proteobacterial species that were found to be its phylogenetic neighbors.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-8-181-S8.eps]


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evidence of recent inter kingdom horizontal gene transfer between bacteria and candida is

Documents

hgt gene

horizontal gene transfer

ctg species

lateral gene transfer

process of gene transfer

important role gene

cases of interdomain

ctg homolog