Common Ancestry and Novel Genetic Traits of Francisella novicida-Like Isolates from North ...

13 Pages·2011·2.05 MB·English

APPLIEDANDENVIRONMENTALMICROBIOLOGY,Aug.2011,p.5110–5122 Vol.77,No.15
0099-2240/11/$12.00 doi:10.1128/AEM.00337-11
Copyright©2011,AmericanSocietyforMicrobiology.AllRightsReserved.
Common Ancestry and Novel Genetic Traits of Francisella novicida-Like
Isolates from North America and Australia as Revealed
(cid:1)
by Comparative Genomic Analyses †
Shivakumara Siddaramappa,1 Jean F. Challacombe,1 Jeannine M. Petersen,2 Segaran Pillai,3
Geoff Hogg,4 and Cheryl R. Kuske1*
BioscienceDivision,LosAlamosNationalLaboratory,LosAlamos,NewMexico875451;DivisionofVector-BorneInfectiousDiseases,
CentersforDiseaseControlandPrevention,FortCollins,Colorado805212;ChemicalandBiologicalDivision,Scienceand
TechnologyDirectorate,DepartmentofHomelandSecurity,Washington,D.C.3;andMicrobiologicalDiagnosticUnit,
D
Public Health Laboratory, Department of Microbiology and Immunology, o
University of Melbourne, Parkville, Victoria 3010, Australia4 w
n
Received15February2011/Accepted31May2011 lo
a
d
FrancisellanovicidaisacloserelativeofFrancisellatularensis,thecausativeagentoftularemia.Thegenomes e
d
of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and
f
comparedtoF.novicidastrainU112andF.tularensisstrainSchuS4.Thestrain3523chromosomeis1,945,310 ro
bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 m
protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly h
colinear,whereasthegenomeofstrain3523hasgaps,translocations,and/orinversionscomparedtogenomes tt
p
ofstrainsFx1andU112.Usingthegenomesequencedataandcomparativeanalyseswithothermembersofthe :
/
genusFrancisella,severalstrain-specificgenesthatencodeputativeproteinsinvolvedinRTXtoxinproduction, /a
polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine bio- e
m
synthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open
.
readingframes(ORFs)andwasnamedrtxCABD.Basedonthealignmentofconservedsequencesupstreamof a
s
operonsinvolvedinthiaminebiosynthesisfromvariousbacteria,aputativeTHIboxwasidentifiedinstrain m
3523.Theglucuronatecatabolismlociofstrains3523andFx1containaclusterofnineORFsorientedinthe .
o
samedirectionthatappeartoconstituteanoperon.StrainsU112andSchuS4appearedtohavelosttheloci r
g
for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host /

adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common o
n
ancestryandnovelgenetictraitsofthesestrains.
D
e
c
Francisellatularensisisanintracellularpathogenthatcauses oratory studies, primarily due to its pathogenicity for rodents e
m
tularemiainhumans,andthepublichealthimportanceofthis andamenabilitytogeneticmanipulation(24).
b
bacterium has been well documented in recent history (81). F.novicidawasformallyincludedinthegenusFrancisellain e
r
The genome sequences of several F. tularensis isolates from 1959,andstrainU112wasthesolememberofthespeciesuntil 2
disparategeographicoriginshavebeensequencedtodate(4,6, 1989(62).Sincethen,therehavebeenatleastsevenreportsof 0
,
12,13,41).Comparativegenomesequenceanalyseshavepro- F. novicida or F. novicida-like bacteria isolated from humans. 2
0
vided insights into the taxonomy, physiology, and pathogenic These include the earliest human isolates described by the 1
evolutionofdifferentsubspeciesofF.tularensis(33,91).Fran- Centers for Disease Control and Prevention from Louisiana 8

cisella novicida strain U112, which is (cid:1)-galactosidase negative andCalifornia(30),twoisolatesfromTexas(15),andaniso- by
butcitrullineureidaseandglycerolpositive,originallywasiso- lateeachfromAustralia(95),Thailand(42),andArizona(8). g
lated from water collected in the Ogden Bay Bird Refuge in Someofthesereportswerefromimmunocompromisedpa- ue
Utah(39).EarlycomparativestudiesindicatedthatF.novicida tientswhomanifestedalocalized,relativelymildillness,which s
t
strain U112 is less fastidious than F. tularensis, and it differs
isconsistentwithF.novicidabeinganopportunisticpathogen.
from the latter in antigenic composition as well as virulence
Despite these reports, F. novicida is thought to constitute an
(64).Thelipopolysaccharide(LPS)ofF.novicidastrainU112
environmental lineage along with Francisella philomiragia,
alsoisstructurallydistinctandbiologicallymoreactivethanF.
whichalsohasbeenassociatedwithhumandisease(30,94).
tularensis LPS (87, 92). Nevertheless, F. novicida strain U112
ThegenomeofF.novicidastrainU112hasbeensequenced
has been used as a surrogate of F. tularensis in scores of lab-
andcomparedtothegenomesequencesofF.tularensispatho-
genictohumans(75).ThedraftgenomesequencesofFranci-
sella isolates from Louisiana and California (GA99-3548 and
*Correspondingauthor.Mailingaddress:BioscienceDivision,Mail GA99-3549,respectively)alsohavebeencomparedtothege-
StopM888,LosAlamosNationalLaboratory,LosAlamos,NM87545. nomesequencesofF.tularensisstrains(12).Thedraftgenome
Phone:[phone].Fax:[phone].E-mail:[email protected].
sequences of F. novicida strains FTE (a mouse passage of
†Supplementalmaterialforthisarticlemaybefoundathttp://aem
strain U112; GenBank project number 30717) and FTG (a
.asm.org/.
(cid:1)Publishedaheadofprinton10June2011. humanisolate;GenBankprojectnumber55313)areavailable.
5110
VOL.77,2011 COMPARATIVE GENOMICS OF F. NOVICIDA-LIKE STRAINS 5111
TABLE 1. CharacteristicsofthegenomesofstrainsU112,Fx1,and3523
No.of No.ofrRNAortRNAgenesb
Chromosome No.of GenBank
Strain protein-coding GCcontent(%)
size(bp) subsystemsa accessionno.
genesa 5S 23S 16S tRNA
U112 1,910,031 1,821 250 32.48 3(cid:4)1 3 3 38 CP000439
Fx1 1,913,619 1,819 253 32.54 3(cid:4)1 3 3 38 CP002557
3523 1,945,310 1,854 253 32.32 3(cid:4)1 3 3 37 CP002558
aGenesandsubsystemspredictedbyRASTserver.
bEachstraincontainsthreerRNAoperons(5S-23S-16S)andanorphan5SrRNAfragment.
WhereasF.tularensisisahighlyinfectiouszoonoticagent,the annotationservice(3).Proteinsdeemedtobespecifictoeachstrainwerecom-
mechanismoftransmissionofF.novicidaamongvertebrateor paredagainsttheNCBInonredundantproteindatabasetodeterminewhether
D
they were hypothetical or conserved hypothetical. If there was no adequate
invertebrate species is unknown. Despite the presence of o
(cid:1)97.7% average nucleotide identities among the genome se- alignmentwithanyprotein(lessthan25%identityorthealignedregionisless w
than25%ofthepredictedproteinlength),thetranslatedopenreadingframe n
quencesofF.novicidaandF.tularensisstrains,theyhavebeen (ORF)wasdesignatedahypotheticalprotein. lo
proposedtoconstituteseparatespeciesbasedonevolutionary Multiple genome comparisons were performed using the progressive align- a
analyses (40). However, amending the genus Francisella and mentoptionavailableintheprogramMAUVE,version2.3.0.Defaultscoring de
thereclassificationofF.novicidaasasubspeciesofF.tularensis and parameters were used for generating the alignment. A synteny plot was d
generatedusingtheprogramNUCmer.Theprogramusesexactmatching,clus- f
havebeenformallyproposed(31). tering, and alignment extension strategies to create a dot plot based on the ro
Francisella isolate Fx1, which is (cid:1)-galactosidase, (cid:1)-lacta- numberofidenticalalignmentsbetweentwogenomes.Prophageregions(PRs) m
mase,andcitrullineureidasepositivebutglycerolnegative,was wereidentifiedusingProphinder(http://aclame.ulb.ac.be/Tools/Prophinder/),an h
culturedfromthebloodofadiabeticpatientintheGalveston algorithmthatcombinessimilaritysearches,statisticaldetectionofphagegene- tt
enriched regions, and genomic context for prophage prediction. Insertion se- p
Bay area of Texas (15). The patient was thought to have dis- quences(ISs)wereidentifiedbywhole-genomeBLASTanalysisofstrainsFx1, ://
seminatedinfectionduetoisolateFx1manifestingwithbacte- 3523,andU112usingtheISfinder(http://www-is.biotoul.fr/).Geneacquisition a
e
remia, pneumonia, and brain abscesses. Virulence studies in and loss among the three strains were determined by comparing gene order, m
laboratoryanimalsperformedbystandardmethodsshowedno orientation of genes (forward/reverse), GC content of genes (the percentage .
a
aboveorbelowthewhole-genomeaverage),featuresofintergenicregions(e.g.,
difference between F. tularensis subsp. tularensis and isolate s
remnantsofISelements,integrationsites,etc.),andthesimilarityofproteins m
Fx1 (15). Subsequent studies have designated this isolate F. encodedbygenesatalocusofinterest((cid:2)90%identityatthepredictedprotein .
o
tularensissubsp.novicidastrainFx1andF.novicidastrainFx1 level). r
(22,66).Francisellaisolate3523,thefirstreportedFrancisella DNAandproteinsequenceswerealignedusingtheClustalW(http://www.ebi g/
from the Southern Hemisphere, was cultured from a patient .ac.uk/Tools/clustalw2/index.html)andBOXSHADE(http://www.ch.embnet.org o
/software/BOX_form.html)programsasdescribedpreviously(80).Multiple-se- n
whohadcutatoeinbrackishwaterintheNorthernTerritory
quence alignments for phylogenetic analyses of strains 3523 and Fx1 were D
ofAustralia(95).Thepatientwasafebrile,hadnootherclin- performedusingtheprogramMUSCLE,whichisavailableatthewebsitehttp: e
icalsymptoms,andrecoveredafterantibiotictreatment.Stud- //www.phylogeny.fr/(20,21).Thealignmentwasfollowedbyabootstrapped(n(cid:3) c
e
iesusingSwiss-Webstermiceshowedthatisolate3523wasless 100)neighbor-joiningmethodforinferringthephylogenies(85).Onlyfull-length m
virulent than F. tularensis subsp. tularensis (95). The true na- 16S rRNA and succinate dehydrogenase (sdhA) gene sequences from high- b
quality,finishedFrancisellagenomeswereincludedinthesecomparisons. e
ture of this isolate, which is glycerol and (cid:1)-galactosidase pos- r

2
itive, is unknown and has been tentatively designated a novi-
RESULTSANDDISCUSSION 0
cida-like subspecies of F. tularensis (95). Since these isolates ,
2
were from different continents/hemispheres, they were desir- Generalfeaturesofthechromosomes.Thechromosomeof 0
1
ablecandidatesforgenomesequencingandcomparativeanal- strain 3523 was 31,692 bp larger than that of strain Fx1. Al-
8
yses.Theobjectivesofthepresentstudyweretodecipherthe though the chromosomes of strains 3523, Fx1, and U112 dif- b
genomesofF.novicida-likestrainsFx1and3523andtoiden- feredinsize,theiraverageGCcontentandthepercentageof y

g
tifythegeneticdifferencesbetweenthesestrainsandF.tular- sequence that encodes proteins were similar (Table 1). u
ensissubsp.novicidastrainU112bycomparativegenomeanal- Genomic islands (GIs) are clusters of genes in prokaryotic e
s
yses. Since genetic relationships and evolutionary contexts genomes of probable horizontal origin (38). Comparative t
couldbebetterunderstoodbywhole-genomeanalysesofcon- genomicanalysisindicatedthatstrain3523containedtwoGIs
served operons, it was envisaged to include the genomes of (GI1, 4,352 to 15,978 bp, 28.8% GC; GI2, 1,012,013 to
different Francisella species and strains in the comparisons 1,056,964 bp, 34.2% GC). Strain U112 also contained two
whenavailable. genomic islands (GI1, 4,244 to 15,027 bp, 29.2% GC; GI2,
369,322 to 376,086 bp, 30.7% GC). However, strain Fx1 con-
MATERIALSANDMETHODS tainedasinglegenomicisland(GI1,4,244to13,232bp,27.9%
GC). Whereas GI2 of strain 3523 was unrelated to GI2 of
BacterialcultivationandchromosomalDNAextractionwereperformedatthe
CentersforDiseaseControlandPrevention,FortCollins,CO,usingstandard strainU112,GI1ofstrains3523,Fx1,andU112wererelated
protocols(57,66).Genomiclibraryconstruction,sequencing,andfinishingwere to each other, suggesting a common lateral origin. Interest-
performedattheGenomeScienceFacilitiesofLosAlamosNationalLaboratory, ingly, none of these GIs contained genes that have a role in
LosAlamos,NewMexico,asdescribedpreviously(4,6,98).Thepredictionof
pathogenicity.
the number of subsystems and pairwise BLAST comparisons of protein sets
Short sequence repeats, which include insertion sequences
within strains Fx1 and 3523 were performed using Rapid Annotation using
SubsystemsTechnology(RAST),whichisafullyautomated,prokaryoticgenome (IS), are the hallmark of F. tularensis genomes, and the IS ele-
5112 SIDDARAMAPPA ET AL. APPL.ENVIRON.MICROBIOL.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
e
m
FIG. 1. (A) Alignment of the chromosomes of strains Fx1, U112, and 3523 using MAUVE 2. Identically colored boxes, known as locally .a
colinearblocks(LCBs),depicthomologousregionsinthethreechromosomes.TheedgesofLCBsindicatechromosomerearrangementsdueto s
m
recombination,insertions,and/orinversions.SequencesofstrainsFx1andU112invertedinrelationtothoseofstrain3523areshownasblocks
.
belowthehorizontalline.TheverticallinesconnectingtheLCBspointtoregionsofhomologyamongthethreechromosomes.Numbersabove o
themapsindicatenucleotidepositionswithintherespectivechromosomes.(B)SyntenyplotsofthechromosomesofstrainsFx1,U112,and3523 rg
generatedbyNUCmer.Eachplotshowsregionsofidentitybetweenthechromosomesbasedonpairwisealignments.Plus-strandmatchesare /
o
slantedfromthebottomlefttotheupperrightcornerandareshowninred.Minus-strandmatchesareslantedfromtheupperlefttothelower
n
rightandareshowninblue.Thenumberofdots/linesshownintheplotisthesameasthenumberofexactmatchesfoundbyNUCmer.Numbers
D
indicatenucleotidepositionswithintherespectivechromosomes.
e
c
e
m
mentsarethoughttobegenerallystableamongdifferentisolates ISelementsmentionedabove.TheoccurrenceofISelements b
e
despitetheirdiversegeographicalorigins(75,86).Whole-genome atgenomicbreakpointsalsohasbeenobservedincomparisons r

2
BLASTN and BLASTX analyses using IS finder showed that ofthegenomesofdifferentsubspeciesofF.tularensis(75). 0
strain 3523 contained a single copy of an IS481 family element BLASTcomparisonofproteinsets.Athree-waycomparison ,
2
(FN3523_0714, 348 amino acids [aa]) that had no homologs in of strains 3523, Fx1, and U112 revealed that they contained 0
1
strainsFx1andU112.StrainFx1containedsixcopiesoftheIS110 1,583 orthologous protein-coding genes (bidirectional best
8
familyelement(FNFX1_0028,FNFX1_1255,FNFX1_1555,and hits). A similar comparison indicated that strain 3523 con- b
FNFX1_1557,315aaeach;FNFX1_0715andFNFX1_0718,290 tained 149 protein-coding genes with no homologs in strains y

g
aaeach)thathadnohomologsinstrains3523andU112.Strains Fx1 and U112. Strain Fx1 contained 70 protein-coding genes u
3523 and Fx1 contained IS982 family elements (FN3523_1458, withnohomologsinstrains3523andU112.StrainU112con- e
s
258 aa; FNFX1_0226, 187 aa) that had no homologs in strain tained 69 protein-coding genes with no homologs in strains t
U112. Whereas strain 3523 lacked ISFtu1, ISFtu3, and ISFtu5 3523andFx1.Atwo-waycomparisonofprotein-codinggenes
sequences, strain Fx1 lacked only ISFtu5 sequences. Neverthe- of strains 3523, Fx1, and U112 is shown in Table S1 in the
less, both strains contained full-length or partial homologs of supplementalmaterial.Instrain3523,494genescouldnotbe
otherISFtuelementsinvariouscopynumbers(datanotshown). assigned a function based on BLAST analysis and therefore
Whole-genomealignmentusingMAUVEshowedthepres- had been annotated as encoding hypothetical or conserved
enceofextensiveblocksofhomologousregionsamongstrains hypotheticalproteins.InstrainFx1,447geneshadbeenanno-
3523, Fx1, and U112 (Fig. 1A). NUCmer analyses revealed tatedasencodinghypotheticalorconservedhypotheticalpro-
that the genomes of strains Fx1 and U112 were mostly colin- teins.
ear, whereas the genome of strain 3523 had gaps, transloca- IronmetabolismandFPIgenes.Aclusterof17to19genes
tions, and/or inversions compared to the genomes of strains has been proposed to constitute the Francisella pathogenicity
Fx1andU112(Fig.1B).In-depthsequenceexaminationindi- island (FPI), and it is found in a single copy in F. tularensis
cated that some of these gaps and/or inversions were associ- subsp.novicidastrainU112(FTN_1309toFTN_1325),butitis
ated with integrative and conjugative elements, including the duplicatedinthegenomesofF.tularensissubsp.tularensis(75,
VOL.77,2011 COMPARATIVE GENOMICS OF F. NOVICIDA-LIKE STRAINS 5113
FIG. 2. (A) RTX toxin locus of strain 3523. Arrow 1, glutamate-1-semialdehyde aminotransferase; 2, glutathione synthetase; 3, methionyl-
tRNA formyltransferase; 4, hypothetical protein; 5, methyltransferase; 6, probable fatty acid hydroxylase; 7, tRNA-Val-GAC; 8, hypothetical
protein;9,recombinationassociatedprotein;10,Rhodanese-likedomainprotein;11,hypotheticalprotein;P1,hypotheticalprotein;P2,phage-
relatedtailprotein;P3(FN3523_1033),site-specificrecombinase,phageintegrasefamily;rtxD(FN3523_1034),membranefusionprotein(toxin
D
secretionprotein);rtxB(FN3523_1035),ABC-typetoxinexporter;rtxA(FN3523_1036),RTXtoxin;rtxC(FN3523_1037),RTXtoxinactivation
o
protein;rtxA(FN3523_1038),vestigialRTXtoxingene.(B)RTXtoxinlocusofstrainFx1.Arrows1to11areasdescribedforpanelA.Arrow w
rtxA (FNFX1_0797), vestigial RTX toxin gene; rtxC (FNFX1_0799), RTX toxin activation protein; rtxA1 and rtxA2 (FNFX1_0800 and n
FNFX1_0801), RTX toxins; rtxB (FNFX1_0802), ABC-type toxin exporter; rtxD (FNFX1_0803 and FNFX1_0805), membrane fusion protein lo
(toxin secretion protein, fragmented due to transposon insertion); hipBA (FNFX1_0806 and FNFX1_0807), persister cell locus; X1 and X2, a
hypotheticalproteins;T1,IS1016familytransposase.(C)ArsenicresistancelocusofstrainU112.Arrows1to11areasdescribedforpanelA. d
e
Arrow rtxA (FTN_0793), vestigial RTX toxin gene; hipB (FTN_0795), repressor of hipA (absent from this strain); U1 (FTN_0792) and U2, d
hypotheticalproteins;T2,ISFtu2transposase;emrE(FTN_0799),multidrugresistanceantiporter;arsRB(FTN_0800andFTN_0801),arsenite
f
resistanceproteins. ro
m

h
t
91). Genome comparisons revealed that strains 3523 and Fx1 onitslocationwithintheoperonandpredictedproteinmolec- t
p
also contained a single copy of the FPI (FN3523_1373 to ular mass (356 aa, 41.81 kDa), rtxC may encode a protein :/
/
FN3523_1389 and FNFX1_1347 to FNFX1_1363, respec- involvedinfattyacylationduringtheactivationofprotoxinto a
e
tively). The order and orientation of genes within the FPI of cytotoxin. The second ORF (FN3523_1036, rtxA) encoded a m
strains 3523, Fx1, and U112 were identical. The predicted pro- putativeprotein(1,829aa,195kDa)thathad30%identityto .a
teinswithintheputativeFPIsofstrains3523andFx1hadaverage theRTXcytotoxin-relatedFrpCprotein(1,829aa,197kDa)of s
m
identitiesof87and97%,respectively,tothosefromstrainU112. Neisseria meningitidis FAM20 (88) and (cid:5)-hemolysin HlyA
.
o
Furthermore, strains 3523, Fx1, and U112 contained the ferric (1,024 aa, 110 kDa) of Escherichia coli (82). The third ORF r
g
uptake regulator gene (fur) as well as the fslABCDEF operon (FN3523_1035,rtxB)encodedaputativeABCtransporter(703 /

(FN3523_1749toFN3523_1755,FNFX1_1722toFNFX1_1728, aa,79.83kDa)thathad50%identitytothe(cid:5)-hemolysintrans- o
n
andFTN_1681toFTN_1687,respectively),whichisimplicatedin locator ATP-binding protein HlyB (706 aa, 79.83 kDa) of E.
D
thebiosynthesisofapolycarboxylatesiderophoreinF.tularensis coli (29). The fourth ORF (FN3523_1034, rtxD) encoded a e
c
subsp. tularensis strain Schu S4 and F. tularensis subsp. novicida putativemembranefusionprotein(490aa,56.41kDa)thathad e
strain U112 (37, 71). An ortholog of strain Schu S4 fupA 32%identitytothetype1translocatorproteinHlyD(478aa, m
(FTT0918),whoseproductisrequiredfortheefficientutilization 54.48 kDa) of E. coli (67). The toxin-encoding ORF b
e
of siderophore-bound iron (47), also was found in strains 3523, (FN3523_1036, 5,490 bp) was the largest among all protein- r

2
Fx1, and U112 (FN3523_0408, FNFX1_0437, and FTN_0444, codinggenespredictedinstrain3523.SeveralotherFrancisella 0
respectively).ThepresenceofFPI,fur,fslABCDEF,andfupAin genomes contained truncated genes related to rtxA of strain ,
2
almost all Francisella genomes suggests that these functions are 3523(e.g.,FTM_1222,FTL_1124,FTT_1077c,FTH_1098,and 0
1
essentialfortheirsurvivalintheenvironmentand/orhost. FTA_1184;76to94%identityatthepredictedproteinlevel).
8
RTXtoxin-relatedgenes.SeveralpathogenicGram-negative However, homologs of rtxB, rtxC, and rtxD were not found in b
bacteria produce potent pore-forming cytotoxins that contain anyofthesegenomes. y

g
calcium-bindingglycineandaspartate-richrepeatregions(93). ThegenomicanalysisofstrainFx1revealedalocus(12,973 u
ThegeneticdeterminantsofRTXtoxinsynthesisandtransport bp,32.6%GC)encodingproteinsrelatedtoRTXtoxins(Fig. e
s
usuallyconsistofasingleoperon(rtxCABD)andanunlinked 2B). The first ORF (FNFX1_0799, rtxC) had no homologs in t
gene (tolC) encoding an outer membrane channel (46). Al- the databases. Based on its location within the operon and
thoughsomeresearchershavealludedtothepresenceoftoxins predicted protein molecular mass (337 aa, 39 kDa), rtxC may
in cellular preparations of F. tularensis, there is no consensus encodeaproteininvolvedinfattyacylationduringtheactiva-
opinion on the synthesis of toxins, and genes encoding toxins tion of protoxin to cytotoxin. The second and third ORFs
have not been found in this species (69, 90). However, ho- (FNFX1_0800andFNFX1_0801,rtxA1andrtxA2,respectively)
mologsoftolC(FTT_1095candFTT_1724c)havebeenchar- encoded putative proteins (1,234 and 1292 aa, 134.7 and 140
acterized in F. tularensis (25) and also are found in strains kDa, respectively) that are unrelated to (cid:5)-hemolysin HlyA of
U112 (FTN_0779 and FTN_1703), 3523 (FN3523_1096 and E.colibuthad28to30%identitytotheRTXcytotoxin-related
FN3523_1775),andFx1(FNFX1_0783andFNFX1_1744). proteins FrpA and FrpC of Neisseria meningitidis and the pu-
Thepredictedprophageregionofstrain3523containedan tativeproteinencodedbyrtxAofstrain3523.Furthermore,the
operon (10,160 bp, 35.5% GC) related to those involved in putativeproteinsencodedbyrtxA1andrtxA2had30%identity
RTX toxin synthesis and secretion (Fig. 2A). The first ORF to each other. Interestingly, the rtx locus of strain Fx1 also
(FN3523_1037,rtxC)hadnohomologsinthedatabases.Based contained rtxB and rtxD ORFs found in strain 3523. Whereas
5114 SIDDARAMAPPA ET AL. APPL.ENVIRON.MICROBIOL.
the rtxB ORFs (FNFX1_0802 and FN3523_1035) were 96% regulator and an arsenite exporter (arsRB) (Fig. 2C). At the
identicalamongthetwostrains,thertxDORFofstrainFx1was proteinlevel,strainU112ArsB(FTN_0800,342aa)was61%
fragmented due to a transposition event (FNFX1_0803 and identical to the arsenite efflux transporter of Bacillus subtilis
FNFX1_0805).Withinthisregion,strainFx1containedagene (BSU25790,346aa),andArsR(FTN_0801,116aa)was38%
encoding a hypothetical protein (FNFX1_0797, 141 aa) that identical to the ArsR repressor of B. subtilis (BSU25810, 105
had86%identitytotheputativeRTXtoxin(1,829aa,encoded aa).AgeneencodingaputativeIS4familytransposase(247aa)
byrtxA)ofstrain3523.Anoperon(1,487bp;31%GC)related wasfoundadjacenttothearsRBoperonofstrainU112.Within
to the hipBA locus of multidrug-tolerant bacteria also was thisregion,strainU112alsocontainedageneencodingaputative
found in this region (Fig. 2B), and putative HipA protein(FTN_0799,109aa)thatwas44%identicaltothesmall
(FNFX1_0806,418aa)ofstrainFx1was30%identicaltothe multidrug resistance antiporter EmrE of E. coli. The arsRB
HipA persistence factor (2WIU_C, 446 aa) of E. coli. Ge- operonandhomologsofemrEwerepresentinF.tularensissubsp.
nomes of several other F. tularensis subsp. tularensis strains novicida strain GA99-3548 and F. philomiragia strains ATCC
contained a similar hipBA operon, but strain U112 contained 25015and25017,butnotinstrains3523andFx1.InstrainU112, D
only a hipB ortholog (FTN_0795) and strain 3523 lacked thetransposasegeneassociatedwiththearsRBoperonwassep- o
w
hipBA. arated from an identical gene upstream by genes encoding a
n
Althoughthe(cid:5)-hemolysinofE.coliistheprototypeofRTX putative fatty acid hydroxylase (FTN_0797, 182 aa), an rRNA lo
toxins,genesencodingRTXtoxinsappeartobemorecommon methyltransferase (FTN_0798, 718 aa), and a hipB gene. Adja- a
d
in pathogenic members of the Pasteurellaceae and are almost centtothistransposasegene,strainU112containedtwoORFs e
d
always associated with genes encoding a type I secretion sys- encoding hypothetical proteins (FTN_0792 and FTN_0793, 140

f
tem. The horizontal transfer of genes encoding RTX toxins and189aa,respectively)(Fig.2C).HomologsofFTN_0792were ro
acrossdifferentbacterialfamilieshasbeensuggestedbasedon present in F. tularensis subsp. novicida strains GA99-3548 and m
phylogeneticanalyses(23).Theidentificationofalocusencod- GA99-3549 but not in strains 3523 and Fx1. Furthermore, h
t
ing putative RTX toxins in strains 3523 and Fx1 is especially FTN_0793had91%identitytotheputativeRTXtoxin(1,829aa, t
p
intriguing,sincesuchgeneshavenotbeenreportedthusfarin encodedbyrtxA)ofstrain3523.Theoccurrenceofdifferentloci :/
/
any of the francisellae genomes. The bacteriocin ABC trans- (prophageregioncontainingrtxCABDinstrain3523,transposon a
e
portersofsomebacteriahavesimilaritiestoABCtransporters associatedwithrtxCABDinstrainFx1,andtransposonassociated m
oftypeIsecretionsystems(99).AlthoughbacteriocinsofFran- with arsenite resistance genes in strain U112) flanked by con- .a
cisella have been investigated previously and designated tu- served genes implies that this variable region is a genomic hot s
m
larecins(1,2,5),verylittleisknownaboutthegeneticbasisof spotamongdifferentFrancisellastrains.
.
o
their synthesis/secretion. The putative RTX toxin of strain Polysaccharidebiosynthesisgeneclusters.TheLPSofFran- r
g
3523 is also related to the bacteriocins of Rhizobium legumi- cisella spp. has several unique features and has been demon- /

nosarum and Nitrococcus mobilis Nb-231 (data not shown). strated to undergo antigenic variation (28). In contrast to F. o
n
Since the bacteriocin of R. leguminosarum has similarities to tularensis subsp. tularensis, F. tularensis subsp. novicida strain
D
RTXtoxins(63)andsomecytolysinscanfunctionasbacterio- U112 expresses a single chemotype of LPS which has been e
c
cins (11), the putative RTX toxin of strain 3523 also may proposed to contribute to virulence in a mouse model of in- e
possess properties of bacteriocins and afford environmental fection(34).Immunobiologicalstudieshavedemonstratedthe m
fitness. The biochemical characterization of rtxCABD loci of activationofcomplementbypathogenicstrainsofF.tularensis b
e
strains 3523 and Fx1 is required to verify these hypotheses. subsp. tularensis as well as F. tularensis subsp. novicida strain r

2
Furthermore, the presence of a vestigial RTX toxin/bacterio- U112 and implicated their LPS O-antigen in mediating resis- 0
cin-encoding ORF in several Francisella genomes, including tance to complement-mediated lysis (16). It also has been ,
2
strains 3523 (FN3523_1038), Fx1 (FNFX1_0797), and U112 shown that mice immunized with LPS from strain U112 were 0
1
(FTN_0793),indicatesthatthecommonancestorofthesebac- protected against strain U112 but not against F. tularensis
8
teria was toxigenic/bacteriocinogenic. It is possible that the subsp.holarctica,andimmunizationwithLPSfromF.tularen- b
ancestrallocuswastruncatedduringhabitatrestrictionand/or sis subsp. tularensis protected against F. tularensis subsp. hol- y

g
reductive evolution of these bacteria, and that it was reac- arcticabutnotagainststrainU112(87).Thewbtgenecluster u
quiredbystrains3523andFx1throughhorizontaltransfer. ofF.tularensissubsp.tularensisstrainSchuS4(17,378bp,31% e
s
Arsenic resistance genes. Arsenic is an environmental pol- GC) is involved in LPS biosynthesis and contained 15 ORFs t
lutant,andsomemicroorganismshaveevolvedmechanismsof (87). In contrast, a similar gene cluster of F. tularensis subsp.
resistance to this cytotoxic agent. Arsenic exists in two oxida- novicidastrainU112(13,880bp,30.6%GC)containedonly12
tionstates,arsenite[As(III)]andarsenate[As(V)],inbiolog- ORFs(87).TheLPSOantigensofF.tularensissubsp.novicida
icalsystems.Inmostbacteria,theminimalarsenicalresistance andF.tularensissubsp.tularensishavebeenshowntobestruc-
operoncontainsthreeORFs(arsRBC),whereintheconversion turallyandimmunologicallydistinct,dueinparttothediffer-
ofarsenatetoarseniteisaccomplishedbyareductase(product encesinwbtgenesinvolvedintheirbiosynthesis(73,87).
ofarsC),arseniteistransportedoutofthecellbyamembrane- Comparativegenomicanalysesrevealedthatstrains3523and
bound efflux pump (product of arsB), and arsR encodes an Fx1containedaclusterof21(23,452bp,31%GC)and16(15,841
arsenic resistance regulatory protein. The plasmid- or trans- bp,30.6%GC)ORFs,respectively,thatwererelatedtothewbt
poson-mediatedhorizontaltransferofgenesthatconferarse- gene cluster of F. tularensis subsp. tularensis strain Schu S4. In
nic resistance has been well documented (58). F. tularensis addition,thesestrainscontainedconservedORFsencodingpro-
subsp. novicida strain U112 contained a two-gene operon teinsputativelyinvolvedinmannosemodificationadjacenttothe
(1,218bp,30.4%GC)thatencodedaputativetranscriptional wbt gene cluster (FN3523_1475 to FN3523_1476 and
VOL.77,2011 COMPARATIVE GENOMICS OF F. NOVICIDA-LIKE STRAINS 5115
FNFX1_1451 to FNFX1_1452). Table S2 in the supplemental an activated donor to an acceptor molecule and are usually
material contains a comprehensive list of the annotated ORFs specific for the glycosidic linkages created (48). The genomes
found in the wbt gene clusters of these bacteria. The 10 strain- ofstrainsU112,Fx1,3523,andSchuS4containedyetanother
specificORFsfoundinthewbtgeneclusterofstrain3523were cluster of 10 to 17 ORFs oriented in the same direction that
organized into groups of five (FN3523_1480 to FN3523_1484, encoded putative GTs and other proteins related to enzymes
4,575 bp, 29.6% GC), two (FN3523_1486 and FN3523_1487, involvedinpolysaccharidebiosynthesisorcellwall/membrane
2,328bp,29.8%GC),andthree(FN3523_1492toFN3523_1494, biogenesis. This cluster has been tentatively designated psl
3,232bp,28.9%GC)contiguousORFs.Ofthesixstrain-specific (polysaccharidesynthesislocus).TableS3inthesupplemental
ORFs found in the wbt gene cluster of strain Fx1, four were material contains a list of the annotated ORFs found within
contiguous(FNFX1_1457toFNFX1_1460,3,888bp,28.6%GC). thisgenecluster.Strain3523had11contiguousstrain-specific
Ofthefivestrain-specificORFsfoundinthewbtgeneclusterof ORFs(FN3523_1278toFN3523_1288,10,580bp,30.4%GC)
strainSchuS4,fourwerecontiguous(FTT_1452ctoFTT_1455c, within the psl cluster. Strain Fx1 had two contiguous strain-
4,150bp,30%GC).ThewbtgeneclusterofF.tularensissubsp. specific ORFs (FNFX1_1260 and FNFX1_1261, 1,897 bp, D
novicidastrainU112containedthreenoncontiguousORFsthat 26.8% GC) within the psl cluster. Strain Schu S4 had three o
w
werestrainspecific(FTN_1422,FTN_1424,andFTN_1428).The contiguous strain-specific ORFs (FTT_0794 to FTT_0796,
n
wbt gene clusters of F. novicida-like strains 3523 and Fx1 con- 2,675 bp, 27% GC) within the psl cluster. The psl clusters of lo
tained four contiguous orthologous ORFs (FN3523_1488 to strainsSchuS4andFx1containedtwoorthologousORFsthat a
d
FN3523_1491, 4,175 bp, 30.7% GC, and FNFX1_1461 to were not found in strain 3523 (FTT_0797/FNFX1_1259 and e
d
FNFX1_1464, 4,204 bp, 30.6% GC, respectively) that had no FTT_0793/FNFX1_1262). These strain-specific ORFs were

f
homologsinstrainsU112andSchuS4.Thewbtgeneclustersof flanked by three contiguous orthologous ORFs at both ends. ro
strainsU112andSchuS4containedthreecontiguousorthologous Furthermore,strainFx1containedacopyoftheIS110family m
ORFs (FTN_1425 to FTN_1427, 3,380 bp, 32.5% GC, and transposase(FNFX1_1255;315aa)adjacenttotheORFthat h
t
FTT_1459ctoFTT_1461c,3,391bp,32.5%GC,respectively)that encodedaputativeHADfamilyhydrolase(FNFX1_1256;see t
p
hadnohomologsinstrains3523andFx1. TableS3inthesupplementalmaterial).Basedongenecontent :/
/
The wbt gene clusters of strain Fx1 and F. tularensis subsp. andorganization,itmaybesurmisedthatthepslgenecluster a
e
tularensis Schu S4 contained two contiguous orthologous was involved in LPS and/or exopolysaccharide (EPS) biosyn- m
ORFs(FNFX1_1466andFNFX1_1467,1,225bp,32.7%GC, thesis.Sincethepslgeneclusterofstrain3523containedmore .a
and FTT_1462c and FTT_1463c, 1,411 bp, 31% GC, respec- genes than strains Fx1, U112, and Schu S4, it is possible that s
m
tively)thathadnohomologsinstrains3523andU112.Thewbt theLPS/EPSofthisstrainismorecomplex.
.
o
gene clusters of strains 3523 and U112 contained two contig- Thiamine biosynthesis genes. Vitamin B1 (thiamine pyro- rg
uous orthologous ORFs (FN3523_1495 and FN3523_1496, phosphate)isinvolvedinseveralmicrobialmetabolicfunctions /

2,209 bp, 34% GC, and FTN_1429 and FTN_1430, 1,735 bp, (74).Prokaryoteshaveevolvedelaboratemechanismstoeither o
n
34.3% GC, respectively) that had no homologs in strains Fx1 synthesize this important cofactor de novo or acquire it from
D
and Schu S4. The strain-specific ORFs in these strains were their niche (7). Thiamine biosynthesis in most bacteria is ac- e
c
flanked by highly conserved orthologous ORFs putatively en- complishedbytwomajorpathways;oneinvolvestheformation e
codingWbtM(FTT_1450c,FN3523_1477,andFNFX1_1453) of hydroxymethylpyrimidine pyrophospate (HMP-PP) from m
and WbtA (FTT_1464c, FN3523_1497, FNFX1_1468, and aminoimidazoleribotideusingThiCandThiD,andtheother b
e
FTN_1431).Furthermore,thewbtgeneclusterofstrainSchu involves the formation of hydroxyethylthiazole phosphate r

2
S4 was flanked by genes encoding ISFtu1/IS630 transposases (HET-P)usingThiS,ThiF,ThiG,andThiO.Theenzymethi- 0
(126 aa each), and the wbt gene cluster of strain U112 con- amine phosphate synthase (ThiE) combines HMP-PP and ,
2
tained a copy of ISFtu3/IS1016 (233 aa) that appears to have HET-P to produce thiamine phosphate, which is phosphory- 0
1
truncated the ORF encoding a putative dTDP-D-glucose 4,6- lated by thiamine monophosphate kinase (ThiL) to produce 8
dehydratase (FTN_1420c; WbtM). However, the wbt gene thiaminepyrophosphate(7).TheinvitrogrowthofmostFran- b
clustersofstrains3523andFx1lackedtransposasegenes(see cisellaspeciesisachievedbysupplementingculturemediawith y

g
TableS2inthesupplementalmaterial). thiaminehydrochlorideorthiaminepyrophosphate,indicating u
Since the wbt gene clusters of strains U112, Fx1, 3523, and the absence of the thiamine biosynthesis (TBS) pathway in e
s
SchuS4displayacassette/mosaicstructurewithanoutercon- thesebacteria(59). t
served region and an inner variable region, it can be hypoth- Strain 3523 and F. philomiragia strain ATCC 25017 con-
esizedthatgenesintheouterregionencodefunctionscommon tained an operon with six ORFs (thiCOSGDF; FN3523_1212
to all strains, whereas genes in the inner region encode sero- toFN3523_1217,6,035bp,32%GC)encodingproteinsrelated
group-specific functions. If the number of genes in the inner to enzymes involved in thiamine biosynthesis in several pro-
variable region is an indicator of the complexity of LPS, then karyotes (Table 2). This gene cluster was not found in other
the LPS of strains 3523 and Fx1 likely is more different from Francisella genomes and was associated with a transposable
thatofstrainsU112andSchuS4.Asimilarchimericarrange- elementinF.philomiragiastrainATCC25017(Fig.3A).The
menthasbeenobservedinthegeneclustersencodingpolysac- geneticorganizationofthestrain3523thiCOSGDFlocuswas
charide antigens in Salmonella enterica, and it has been pro- similartothatoftheplasmid-encodedthiCOGElocusinvolved
posed that genes in the outer conserved region mediate the in thiamine biosynthesis in Rhizobium etli (56). An analogous
conspecificexchangeofgenesintheinnervariableregion(44). cluster (thiOGF) is found within plasmid pEA29 of the plant
Biosynthesis of polysaccharides requires several glycosyl- pathogen Erwinia amylovora strain Ea88 (50). The chromo-
transferases(GTs),whichcatalyzethetransferofsugarsfrom some of lithoautotrophic bacterium Ralstonia eutropha H16
5116 SIDDARAMAPPA ET AL. APPL.ENVIRON.MICROBIOL.
TABLE 2. Strain3523genesinvolvedinthiaminebiosynthesis,glucuronatemetabolism,andspermidinebiosynthesis
ClosesthomologoutsideFrancisella
Protein
Locustagandfunction Annotation
size(aa) Protein
Locustag %Identity Evalue
size(aa)
Thiaminebiosynthesis
FN3523_1212 251 Thiazolebiosynthesisadenylyltransferase(ThiF) Mrub_1727 266 37 3e(cid:6)39
FN3523_1213 494 Fusedproteinphosphomethylpyrimidinekinase lpg1568 495 36 9e(cid:6)76
(ThiD)/thiamine-phosphatepyrophosphorylase(ThiE)
FN3523_1214 259 Thiazolesynthase(ThiG) lpg1567 263 59 2e(cid:6)87
FN3523_1215 66 Thiaminebiosynthesisprotein(ThiS) IL-0768 66 34 5e(cid:6)05
FN3523_1216 339 Thiaminebiosynthesisoxidoreductase(ThiO) Kkor_0127 351 33 6e(cid:6)44
FN3523_1217 592 Thiaminebiosynthesisprotein(ThiC) CV_0235 632 72 0.0
Glucuronatemetabolism D
FN3523_0892 325 Inositoloxygenase 56727Miox 285 38 7e(cid:6)48 o
FN3523_0893 462 D-Xylose-protonsymporter(XylT) CBUD_1731 463 43 2e(cid:6)91 w
FN3523_0894 473 Glucuronateisomerase(UxaC) Sde_1272 471 51 2e(cid:6)140 n
FN3523_0895 182 KDPGaldolase(KdgA) BC1003_2949 213 37 5e(cid:6)36 lo
FN3523_0896 306 2-Keto-3-deoxygluconatekinase(KdgK) Sde_1269 296 43 1e(cid:6)59 ad
FN3523_0897 396 Mannonatedehydratase(UxuA) PROSTU_04181 396 58 2e(cid:6)134 e
FN3523_0898 490 D-Mannonateoxidoreductase(UxuB) CJA_0180 492 43 2e(cid:6)106 d
FN3523_0899 774 Alpha-glucosidase BL00280 802 53 0.0 fr
FN3523_0900 486 Rhamnogalacturonidetransporter(RhiT) KPK_1307 502 45 8e(cid:6)116 o
m

Spermidinebiosynthesis h
FN3523_0489 163 S-Adenosylmethioninedecarboxylase(SpeD) Tcr_0272 167 66 3e(cid:6)47 ttp
FN3523_0490 289 Spermidinesynthase(SpeE) Pfl01_1732 291 57 7e(cid:6)97 :
/
FN3523_0491 550 Argininedecarboxylase(SpeA) Avin_07050 636 31 8e(cid:6)64 /a
FN3523_0492 328 Agmatinedeiminase(AguA) Kkor_1127 327 51 5e(cid:6)90 e
FN3523_0493 286 N-Carbamoylputrescineamidase(AguaB) VP1774 288 57 4e(cid:6)92 m
.
a
s
m
.
alsocontainsathiCOSGElocusthatisproposedtobeinvolved was (cid:7)50% identical to ThiG of R. etli (AAC45974; E value, o
r
inthedenovosynthesisofthiamine(68).Attheproteinlevel, 4e(cid:6)66)andR.eutropha(H16_A0238;Evalue,8e(cid:6)77). g
/

strain 3523 ThiC was 68% identical to ThiC of R. etli Instrain3523,FN3523_1213appearedtoencodeaputative o
n
(AAC45972) and R. eutropha (H16_A0235), whereas strain fused protein containing hydroxy-phosphomethylpyrimidine
D
3523 ThiF was 35% identical to ThiF of E. amylovora kinase and thiamine-phosphate pyrophosphorylase domains.
e
(NP_981993;Evalue,3e(cid:6)24).Furthermore,strain3523ThiO In some bacteria, these functions are encoded by two different c
e
andThiSwere27and31%identicaltoThiO(H16_A0236)and ORFs(thiDandthiE,respectively).HomologsofFN3523_1213 m
ThiS (H16_A0237) of R. eutropha, respectively (E values, werefoundinseveralbacteria(e.g.,Legionellapneumophila,Cox- b
9e(cid:6)27to0.001).PutativethiazolesynthaseThiGofstrain3523 iellaburnetii,Geobactersulfurreducens,andColwelliapsychreryth- er

2
0
,

2
0
1
8

b
y

g
u
e
s
t
FIG. 3. (A)Thiaminebiosynthesislocus.Fn,F.novicida-likestrain3523;Fp,F.philomiragiaATCC25017.Arrow1,metabolite:H(cid:4)symporter
(MHS) family protein; 2, hypothetical protein; 3, exodeoxyribonuclease VII large subunit; 4, transglutaminase-like superfamily protein; 5,
conservedhypotheticalprotein;6,conservedhypotheticalprotein;7,hypotheticalprotein;8,majorfacilitatorsuperfamilytransporter;9,glutamate
racemase;10,hypotheticalprotein;11,excinucleaseBsubunit;12,exodeoxyribonuclease;COSGDF(FN3523_1212toFN3523_1217),thiamine
biosynthesis proteins; T, transposase; X, hypothetical protein; Y, hypothetical protein; Z, major facilitator transporter. (B) Putative thiamine
regulatoryelement.Aconserved36-bpsequenceupstreamofthiCfromeightspeciesisboxed.Nucleotidesidenticalinalleightsequencesare
showninboldface.ThelengthofsequencebetweentheTHIboxandthestartcodonofthiCineachspeciesisindicated(bptoATG).Thelocus
tagandthenumberofaminoacidsencodedbythiCineachspeciesareshowninparentheses.
VOL.77,2011 COMPARATIVE GENOMICS OF F. NOVICIDA-LIKE STRAINS 5117
raea;(cid:7)30% protein identity) and plants (e.g., Arabidopsis thali- otherFrancisellagenomes,withtheexceptionofF.philomira-
ana,Zeamays,andBrassicanapus;(cid:7)29%proteinidentity).Ithas gia strain ATCC 25015, which appeared to contain the entire
beenproposedthatthesebifunctionalenzymesareinvolvedinthe cluster(79to89%identityattheproteinlevel).
synthesisofHMP-PPaswellasthecondensationofHMP-PPand The predicted mannonate dehydratase, 2-keto-3-deoxy-
HET-Ptoproducethiaminemonophosphate(36,72,74).The5(cid:8) gluconokinase, KDPG aldolase, and glucuronate isomerase
untranslatedregionsofoperonsinvolvedinthiaminebiosynthesis proteinsfromstrain3523had57,37,37,and28%identitiesto
andtransporthavebeenshowntocontainaregulatoryelement E.coliUxuA(ECs5281),KdgK(ECs4406),KdgA(ECs2560),
called the THI box sequence (55). Based on the alignment of and UxaC (ECs3974), respectively (E values, 5e(cid:6)132 to
conserved sequences upstream of operons involved in thiamine 9e(cid:6)26). These enzymes catalyze the dehydration of D-man-
biosynthesisfromvariousbacteria,aputativeTHIboxsequence nonatetoKDG,phosphorylationofKDG,cleavageofKDPG
wasidentifiedupstreamofthiCinstrain3523andF.philomiragia to pyruvate and G3P, and the conversion of D-glucuronate to
strainATCC25017(Fig.3B).TheidentificationofaputativeTHI D-fructuronate,respectively(65).TheGlcUAutilizationgene
box upstream of thiC in strain 3523 suggests a thiamine-depen- clustersofstrains3523andFx1hadsomesimilaritiestothatof D
dentregulationofthisgene,similarlytootherbacteriathathave Bacillus stearothermophilus T-6, which has been predicted to o
w
TBSgenes(74). metabolize GlcUA akin to E. coli and Bacillus subtilis (79).
n
In bacteria that lack a TBS pathway, thiamine kinases may Furthermore,oneendoftheGlcUAutilizationgeneclusterof lo
facilitatethesalvageofdephosphorylatedthiamineintermedi- B. stearothermophilus T-6 also contains an ORF encoding a a
d
atesfromtheenvironmentorgrowthmedium(32).Agenethat proteinthathassimilaritiestotransposasesoftheIS481family e
d
encodes a putative thiamine pyrophosphokinase (TPK) was (49),indicatingthepossiblehorizontaltransferofthislocus.

f
found in most members of Francisella, including strains 3523, TheORFsencodingaputativeinositoloxygenaseinstrains ro
Fx1, and U112 (FN3523_0611, FNFX1_0669, and FTN_0662, 3523,Fx1,andATCC25015hadnobacterialhomologsinthe m
respectively). F. novicida strain U112 TPK was 27% identical publicdatabasesoutsidethegenusFrancisella.However,strain h
to Bacillus subtilis TPK (THIN_BACSU; E value, 3e(cid:6)10), 3523 inositol oxygenase had 38% identity to Mus musculus tt
p
whichcatalyzesthedirectconversionofthiaminetothiamine myo-inositol oxygenase (56727 Miox; E value, 7e(cid:6)48), which :/
/
pyrophosphate (52). In Listeria monocytogenes, which has an catalyzes the conversion of myo-inositol to GlcUA (9). myo- a
e
incomplete thiamine biosynthesis pathway, it has been shown Inositol and its derivatives are ubiquitous among eukaryotes m
thatproliferationwithinhostcellsisreduceduponthedeletion andarchaea,buttheirsynthesisandmetabolismisbelievedto .a
of thiD and thiT, encoding an HMP salvage protein and a be less common among bacteria (54). Although none of the s
m
thiamine transporter, respectively (77). In addition, strains of francisellae genomes sequenced to date contained ORFs en-
.
o
E.amylovorathatlackthethiOSGFoperon-containingplasmid coding proteins putatively involved in the transport and/or r
g
pEA29 have been shown to be altered in exopolysaccharide metabolism of myo-inositol, most of them, including strains /

biosynthesis and less virulent (51). Aspergillus nidulans, the 3523 and Fx1, had a suhB homolog (FN3523_1410 and o
n
filamentousascomycetewhichinducesafatalsystemicmycosis FNFX1_1384,respectively).Thisevolutionarilyconservedgene
D
in mice, has been shown to be less pathogenic when its thia- encodedinositol-1-monophosphatase,whichhydrolyzesmyo-ino- e
c
mine biosynthetic pathway was blocked by mutation (70). It sitol-1-phosphatetoyieldfreemyo-inositol(60,61).Thus,itap- e
alsohasbeensuggestedthatBrucellaabortusandR.etli,which pearsthatmostmembersofFrancisellacanconvertmyo-inositol- m
havesimilarintracellularlifestyles,requireincreasedthiamine 1-phosphatetofreemyo-inositol.However,onlystrains3523and b
e
inthestationaryphase(56,76).Inviewoftheseobservations, Fx1 as well as F. philomiragia strain ATCC 25015 are able to r

2
theTBSgeneclusterofstrain3523couldservetoenhanceits utilizemyo-inositoltosynthesizeGlcUA,whichthenismetabo- 0
survivalintheenvironment/hostandalsomayhaveanindirect lizedusingtheEntner-Doudoroffpathway. ,
2
role in pathogenesis. Furthermore, the TBS genes of strain Polyamine biosynthesis and transport genes. Polycations 0
1
3523 could be useful in accelerating the growth of thiamine suchasputrescineandspermidineareprecursorsforseveralcel-
8
auxotrophsofF.tularensisinthelaboratory. lular components, and pathways for the biosynthesis of these b
Glucuronatemetabolismgenes.Somebacteriahaveevolved polyamines have been described in a number of species (97). y

g
mechanisms for the metabolism of uronic acids and uronates Polyaminesalsohavebeenimplicatedinbacterialoxidativestress u
using the Entner-Doudoroff pathway (65). In this pathway, response and virulence (78). The ubiquitous presence of genes e
s
(cid:5)-D-glucuronicacid(GlcUA)isconvertedinto2-keto-3-deoxy- encodingputrescinetransportsystems(potFGHI)inbacterialge- t
gluconate (KDG) by a three-step process. The subsequent nomes suggests that these small molecules play crucial roles in
phosphorylationofKDGyields2-keto-3-deoxy-6-phosphoglu- cellular physiology. Strains 3523, Fx1, and U112 as well as F.
conate (KDPG), which is finally cleaved to produce glyceral- tularensis subsp. tularensis strains Schu S4, WY96-3418, and
dehyde 3-phosphate (G3P) and pyruvate. Comparative OSU18 contained a cadA gene (FN3523_0462, FNFX1_0489,
genomicanalysesindicatedthatstrains3523andFx1contained FTN_0504,FTT_0406,FTW_1667,andFTH_0474,respectively).
aclusterofnineORFsthatappeartoconstituteapolycistronic Thisgeneencodedaputativeproteinthathad55%identitytoE.
operon (FN3523_0892 to FN3523_0900 and FNFX1_0904 to coli lysine decarboxylase (NP_418555; E value, 0), which is in-
FNFX1_0912, respectively, 11,784 bp, 31.7% GC). These volved in cadaverine biosynthesis (53). The strains mentioned
ORFs encoded putative proteins related to enzymes involved above also contained a potGHI operon (e.g., FN3523_1141 to
inGlcUAcatabolism(Table2).Furthermore,genesencoding FN3523_1143)andanunlinkedpotFgene(e.g.,FN3523_0514).
putative transposases of IS1106 and IS1016 families were Furthermore, strain 3523 contained a cluster of five ORFs
foundneartheGlcUAutilizationgeneclusterofstrainFx1but (FN3523_0489to_0493,4,873bp,32.6%GC)encodingputa-
notinstrain3523(Fig.4A).Thisgeneclusterwasnotfoundin tiveproteinsrelatedtoenzymesinvolvedinthebiosynthesisof
5118 SIDDARAMAPPA ET AL. APPL.ENVIRON.MICROBIOL.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/

FIG. 4. (A)Glucuronatemetabolismlocus.Fn1,F.novicida-likestrainFx1;Fn3,F.novicida-likestrain3523.Arrow1,hypotheticalprotein;2, o
n
zinc-ironpermeasefamilyprotein;3,cobalaminsynthesisprotein(putativeGTPase);4,zinc-ironuptakeregulatoryprotein;5,RNaseT2family
D
protein;6,hypotheticalprotein;7,phenylalanyl-tRNAsynthetasebetachain;8,phenylalanyl-tRNAsynthetasealphachain;9,drug/metabolite
e
transportersuperfamilyprotein;10,putativetransporter;11,sugartransporter;12,conservedhypotheticalprotein;13,hypotheticalprotein;14, c
transcriptionalregulator;15,hypotheticalprotein;16,HollidayjunctionDNAhelicaseRuvB;17,short-chaindehydrogenasefamilyproteinFabG; e
18,CBSdomainpairprotein;19,GDSL-likelipolyticenzyme;20,hypotheticalprotein;21,1-deoxy-D-xylulose-5-phosphatesynthase;22,GMP m
synthase; 23, amino acid permease; ABCDEFGHI (FNFX1_0904 to FNFX1_0912, FN3523_0892 to FN3523_0900), glucuronate metabolism b
e
genes;J,hypotheticalprotein;T1,IS1106transposase;T2,IS1016transposase.(B)Lactoseandpolyaminemetabolismloci.Fp,F.philomiragia r
ATCC 25017; Ft, Francisella tularensis subsp. tularensis WY96-3418; Fn, F. novicida-like strain 3523. Arrow 1, glutaminyl-tRNA synthetase; 2, 2
peptide transporter; 3, ABC transporter ATP-binding protein; 4, tetracycline efflux protein TetA; 5, conserved domain protein; 6, drug:H(cid:4) 0
antiporter-1 (DHA1) family protein; 7, 23S rRNA (guanosine-2(cid:8)-O)-methyltransferase (RlmB); 8, UDP-N-acetylmuramate:L-alanyl-gamma-D- , 2
glutamyl-meso-diaminopimelateligase;9,orotatephosphoribosyltransferase;10,UDP-2;3-diacylglucosaminehydrolase;11,threoninesynthase; 0
12, homoserine kinase; 13, hypothetical protein; 14, arginyl-tRNA synthetase; 15, organic solvent tolerance protein; Y (FN3523_0487), sugar 1
8
permease;Z(FN3523_0488),beta-galactosidase;K(FN3523_0489),S-adenosylmethioninedecarboxylase;L(FN3523_0490),spermidinesynthase;
M (FN3523_0491), biosynthetic arginine decarboxylase; N (FN3523_0492), agmatine deiminase; O (FN3523_0493), N-carbamoylputrescine b
y
amidase; P, hypothetical protein; T1, transposase/integrase-like protein; T2, ISFtu1 transposase fragments. (C) Presumptive lac promoter of
g
Francisella.Aconserved61-bpsequenceupstreamofthelacZORFfromninestrainsofFrancisellaisdepicted.Nucleotidesidenticalinallnine u
sequencesareshowninboldface.The(cid:6)10(TATAAT),(cid:6)35(TTGAACorTTGAAG),and17-bpspacerregionsareindicated.Anarrowpoints e
to the putative transcription start site ((cid:4)1). The predicted start codon of lacZ is underlined. Sequences 4 through 9 are identical. Row 1, F. s
t
philomiragiaATCC25017;2,F.novicida-likestrainFx1;3,F.novicida-likestrain3523;4,F.tularensisFSC198;5,F.tularensisWY96-3418;6,F.
tularensisFSC147;7,F.tularensisOSU18;8,F.tularensisFTNF002-00;9,F.tularensisLVS.
spermidineandputrescine(Table2,Fig.4B).Thisgenecluster ninedecarboxylasehad55and28%identitiestoE.coliSpeE
wasnotfoundinstrainsFx1andU112butwaspresentinother (3O4F_A; E value, 1e(cid:6)92) and SpeA (3NZQ_B; E value,
F. tularensis subsp. tularensis genomes (e.g., strains Schu S4, 3e(cid:6)60), respectively. Strain 3523 agmatine deiminase and N-
OSU18, and FSC147). In F. tularensis subsp. tularensis strain carbamoylputrescine amidohydrolase had 37 and 57% identi-
WY96-3418,fragmentedISFtu1elementswerefoundnearthis ties to Pseudomonas aeruginosa PAO1 AguA (PA0292; E
gene cluster (Fig. 4B). Strain 3523 S-adenosylmethionine de- value, 1e(cid:6)58) and AguB (PA0293; E value, 8e(cid:6)94), respec-
carboxylase had 45% identity to SpeD (MJ_0315; E value, tively. These proteins are key components in the biosynthesis
9e(cid:6)27) of Methanocaldococcus jannaschii, but it is unrelated of polyamines in M. jannaschii, E. coli, and P. aeruginosa (14,
to SpeD of E. coli. Strain 3523 spermidine synthase and argi- 35, 45). The acquisition of genes encoding polyamine biosyn-
VOL.77,2011 COMPARATIVE GENOMICS OF F. NOVICIDA-LIKE STRAINS 5119
thesis/metabolism functions may enhance the adaptive capa- geneanditspromoteridentifiedinthisstudyshouldbeuseful
bilities of a bacterium (78, 97). The presence of spermidine ingeneticstudiesrequiringareporter/markerwithinthisgroup
biosynthesisgenesinstrain3523aswellasF.tularensissubsp. ofbacteria.
tularensisstrains,buttheirabsenceinstrainsFx1,U112,andF. Summaryofimportantgenetictraitsandphylogeneticanal-
philomiragia strain ATCC 25017, is a notable feature from a yses. Based on whole-genome comparisons of different Fran-
pathogenicandevolutionarystandpoint. cisella species and strains, the following genetic acquisitions
Lactose metabolism genes. The lac operon, which contains and losses are evident. While strain Fx1 contained the lac
the structural genes encoding proteins that facilitate lactose operoninadditiontogenesforGlcUAutilization,strain3523
metabolism, is found in a variety of bacteria. Lactose is im- contained the lac operon along with GlcUA utilization, thia-
portedintothecellasafreesugarbymeansofapermease,and mine biosynthesis, and spermidine biosynthesis genes.
the enzyme (cid:1)-galactosidase hydrolyzes this disaccharide into WhereasmostF.tularensissubsp.tularensisstrainshadlostloci
galactoseandglucose(96).Genomecomparisonsrevealedthat for GlcUA utilization and thiamine biosynthesis, they con-
strain 3523 contained a cluster of two ORFs (3,165 bp, 32% tained a vestigial lacZ gene and a complete speDE system. D
GC)adjacenttothespermidinebiosynthesislocusthatappear Furthermore,strains3523andFx1weretheonlyonescontain- o
w
toconstituteanoperon(FN3523_0487toFN3523_0488)(Fig. ing the loci for RTX toxin production. Notably, strain U112
n
4B).ThepredictedLacZofstrain3523had29%identitytothe lackedalloftheselocibutcontainedanarsRBoperon,which lo
(cid:1)-galactosidase(AAF16519,BgaB;Evalue,2e(cid:6)79)ofCarno- was absent from strains 3523 and Fx1 as well as F. tularensis a
d
bacteriummaltaromaticum(17)and26%identitytothe(cid:1)-ga- subsp.tularensisstrains.Asummaryofthepresence/absenceof e
lactosidase(O07012.2,GanA;Evalue,8e(cid:6)76)ofBacillussub- these loci is presented in Fig. 5A. It is possible that strain d

tilis (19), but it is unrelated to the (cid:1)-galactosidase of E. coli. U112,anenvironmentalisolateofF.tularensissubsp.novicida, fro
Strain3523LacYhad25%identitytotheputativeoligogalac- hadretainedanarsRBoperonbuthadlostlociforlactoseand m
turonide transporter (NP_752266; E value, 3e(cid:6)07) of E. coli GlcUA utilization as well as genes for thiamine biosynthesis h
t
CFT073 and 22% identity to the putative sugar transporter because of niche selection. Strains 3523 and Fx1 appeared to t
p
(YP_081973;Evalue,1e(cid:6)08)ofBacilluscereus.Asimilarlac beintransitionfrombeingenvironmentaltopathogenic,since :/
/
operon was found in strain Fx1 and F. philomiragia strains theyhavelostthearsRBoperonbutcontainlociforlactoseand a
e
ATCC 25015 and 25017. However, genes encoding the galac- GlcUAutilization,inadditiontogenesforRTXtoxinproduc- m
toside O-acetyltransferase (lacA) and the regulatory protein tion.ExtantstrainsofF.tularensissubsp.tularensisappearedto .a
(lacI) were not found in any of these bacteria. In F. philomi- have lost the arsRB operon and loci for lactose and GlcUA s
m
ragia strain ATCC 25017, an ORF encoding a putative trans- utilization,inadditiontogenesforthiaminebiosynthesis,asa
.
o
posase/integrase-like protein was found near the lac operon consequenceofhostadaptationandreductiveevolution.Since r
g
(Fig. 4B). Furthermore, other F. tularensis subsp. tularensis strain3523andmostF.tularensissubsp.tularensisstrainscon- /

genomes (e.g., strains Schu S4, WY96-3418, OSU18, and tain a complete speDE system, the role of spermidine in the o
n
FSC147)containedatruncatedlacZ.Basedonthealignment pathogenesisoftularemianeedstobecarefullystudied.Thisis
D
of conserved sequences upstream of lacZ from nine different especially important in light of recent reports implicating e
c
Francisella genomes, a putative lac promoter also was identi- spermineandspermidineinthestimulationofhostcellsbyF. e
fied(Fig.4C).ThispromoterhadhighATcontent(78to86%) tularensissubsp.tularensis(10). m
andwassimilartothelacpromotersofseveralGram-negative Based on these observations, it may be surmised that the b
e
bacteria. genes/loci for arsenite resistance, GlcUA utilization, lactose r

2
Although the classic lac operon of E. coli consists of three utilization, and thiamine biosynthesis are more ancient. It is 0
ORFs (lacZYA), which are regulated by the product of the possiblethatgenesforRTXtoxinproductionandspermidine ,
2
adjacentlacIrepressorgene,bacteriacontainingonlylacZYor biosynthesis were acquired later by an F. tularensis/novicida- 0
1
lacZhavebeenidentified(27,43,83).Whiletheevolutionary like clade. Although strains 3523 and Fx1 were isolated in
8
originoftheE.colilacoperonisuncertain(83),theoccurrence differentpartsoftheglobe,bothwereassociatedwithmarine b
oflacgenesnearintegrativeandconjugativeelementsandthe environments, and their recent common ancestry cannot be y

g
identificationofE.coli-likelacoperonsinsomeGram-positive ruledout.Deletion-basedphylogeneticanalyseshaveindicated u
bacteria suggest their lateral mobility (18, 26, 89). From ge- F.tularensissubsp.novicidatobetheoldestandthatboththe e
s
nomecomparisons,itappearedthatthelastcommonlactose- acquisition and loss of genes have occurred during the evolu- t
utilizingancestorofstrainsFx1and3523,F.philomiragiastrain tion of F. tularensis (84). Phylogenetic analyses based on full-
ATCC 25017, and F. tularensis subsp. tularensis strains have length16SrRNAandsdhAgenessupportthepropositionthat
acquiredthelacZYoperonbytransposon-mediatedhorizontal F. tularensis subsp. novicida is very closely related to F. tular-
transfer. The loss of this operon in strain U112 and most F. ensissubsp.tularensisandindicatethatstrain3523represents
tularensissubsp.tularensisstrainsmaybeduetonicheselection anewlineageparentaltotheotherF.tularensissubsp.novicida
or through genetic drift, and a similar mechanism for some andF.tularensissubsp.tularensisstrains(Fig.5BandC).
members of Enterobacteriaceae has been proposed (83). The Inconclusion,previousgenomecomparisonshaveindicated
ability to metabolize lactose probably affords strains Fx1 and that subspecies of F. tularensis have independently and inter-
3523 a growth advantage in environments where the sugar is mittently acquired and lost genes, and that the net loss in F.
present,andthesemayrepresentasubsetofFrancisellaspecies tularensissubsp.tularensisismorethanthenetlossinF.tula-
that have retained an ancestral copy of the lac operon. Al- rensis subsp. novicida (4, 75). Based on the analyses of unidi-
thoughdetailedphylogeneticanalysesarerequiredtoestablish rectionalgenomicdeletioneventsandsingle-nucleotidevaria-
theevolutionaryoriginoftheFrancisellalacoperon,thelacZ tions,ithasbeensuggestedthatthedifferentsubspeciesofF.