Physiological and Comparative Genomic Analysis of Arthrobacter sp. SRS-W-1-2016 Provides

19 Pages·2017·4.48 MB·English

G C A T genes
T A C G
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BriefReport
Physiological and Comparative Genomic Analysis of
Arthrobacter sp. SRS-W-1-2016 Provides Insights on
Niche Adaptation for Survival in Uraniferous Soils
AshviniChauhan1,*,AshishPathak1,RajneeshJaswal1,BobbyEdwardsIII1,
DemarioChappell2,ChristopherBall3,ReynaGarcia-Sillas4,PaulStothard5andJohnSeaman6
1 EnvironmentalBiotechnologyandGenomicsLaboratory,SchooloftheEnvironment,1515S.MartinLuther
KingJr.Blvd.,Suite305B,FSHScienceResearchCenter,FloridaA&MUniversity,Tallahassee,FL32307,USA;
[email protected](A.P.);[email protected](R.J.);[email protected](B.E.)
2 DepartmentofBiology,CollegeofScienceandTechnology,1610S.MartinLutherKingBlvd.,
FloridaA&MUniversity,Tallahassee,FL32307,USA;[email protected]
3 DepartmentofBiologicalSciences,LifeScienceBuilding,AlabamaStateUniversity,915SJacksonStreet,
Montgomery,AL36101,USA;[email protected]
4 SchoolofLifeSciences,CollegeofLiberalArtsandSciences,ArizonaStateUniversity,723E6thSt,
Tempe,AZ85281,USA;[email protected]
5 DepartmentofAgricultural,FoodandNutritionalScience,UniversityofAlberta,
Edmonton,ABT6G2P5,Canada;[email protected]
6 SavannahRiverEcologyLaboratory,UniversityofGeorgia,Aiken,SC29802,USA;[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
Received:21November2017;Accepted:5January2018;Published:11January2018
Abstract: Arthrobactersp. strainSRS-W-1-2016wasisolatedonhighconcentrationsofuranium(U)
fromtheSavannahRiverSite(SRS)thatremainsco-contaminatedbyradionuclides,heavymetals,and
organics. SRSislocatedonthenortheastbankoftheSavannahRiver(SouthCarolina,USA),whichis
aU.S.DepartmentofEnergy(DOE)managedecosystemlefthistoricallycontaminatedfromdecades
of nuclear weapons production activities. Predominant contaminants within the impacted SRS
environmentincludeUandNickel(Ni),bothofwhichcanbetransformedmicrobiallyintolesstoxic
formsviametalcomplexationmechanisms. StrainSRS-W-1-2016wasisolatedfromtheuraniferous
SRSsoilsonhighconcentrationsofU(4200µM)andNi(8500µM),butrapidgrowthwasobservedat
muchlowerconcentrationsof500µMUand1000µMNi,respectively. Microcosmstudiesestablished
withstrainSRS-W-1-2016revealedarapiddeclineintheconcentrationofspikedUsuchthatitwas
almostundetectableinthesupernatantby72hofincubation.Conversely,Niconcentrationsremained
unchanged,suggestingthatthestrainremovedUbutnotNiunderthetestedconditions. Toobtaina
deeperunderstandingofthemetabolicpotential,adraftgenomesequenceofstrainSRS-W-1-2016was
obtainedatacoverageof90×,assemblinginto93contigswithanN50contiglengthof92,788bases.
ThegenomicsizeofstrainSRS-W-1-2016wasfoundtobe4,564,701baseswithatotalnumberof
4327putativegenes. Anin-depth,genome-widecomparisonbetweenstrainSRS-W-1-2016andits
fourclosesttaxonomicrelativesrevealed1159distinctgenes,representing26.7%ofitstotalgenome;
manyassociatingwithmetalresistanceproteins(e.g.,forcadmium,cobalt,andzinc),transporter
proteins, stress proteins, cytochromes, and drug resistancefunctions. Additionally, several gene
homologuescodingforresistancetometalswereidentifiedinthestrain,suchasoutermembrane
effluxpumpproteins,peptide/nickeltransportsubstrateandATP-bindingproteins,ahigh-affinity
nickel-transportprotein,andthespoT gene,whichwasrecentlyimplicatedinbacterialresistance
towardsU.DetailedgenomemininganalysisofstrainSRS-W-1-2016alsorevealedthepresenceofa
plethoraofsecondarymetabolitebiosyntheticgeneclusterslikelyfacilitatingresistancetoantibiotics,
biocides,andmetals. Additionally,severalgenehomologousforthewell-knownoxygenaseenzyme
systemwerealsoidentified,potentiallyfunctioningtogenerateenergyviathebreakdownoforganic
Genes 2018,9,31;doi:10.3390/genes9010031 www.mdpi.com/journal/genes
Genes 2018,9,31 2of19
compoundsandthusenablingthesuccessfulcolonizationandnaturalattenuationofcontaminants
byArthrobactersp. SRS-W-1-2016attheSRSsite.
Keywords: bioremediation; uranium; nickel; whole genome sequencing; resistome; comparative
genomics;Arthrobacter
1. Introduction
SavannahRiverSite(SRS)locatedinAiken,SouthCarolina,functionedasanuclearmaterials
productionfacilityfortheU.S.DepartmentofEnergy(DOE),wheremetal-claduranium(U)targets
wereusedintheproductionofplutonium[1]. From1954to1982,SRSwastewateroriginatingfrom
themetalplatingandfabricationprocesseswasreleaseddirectlyintoTimsBranch,asecond-order
stream. Becauseoftheseactivities,largequantitiesofbothU(depletedaswellasnaturally-occurring)
andNickel(Ni)werereleasedanddepositedintostreamsedimentsandanabandonedfarmpond-the
SteedsPond,thatservedasanaturalsettlingbasinalongTimsBranch[1,2]. Althoughcontaminant
distribution is heterogeneous in the Steeds Pond sediments, U and Ni concentrations are found
upwards of 1000 mg/kg [2]. To gauge in-situ remediation (natural attenuation) and recommend
appropriate strategies forthe decontaminationof SRS contaminated ecosystems, it is necessaryto
obtainadeeperunderstandingofthemechanismsexhibitedbythenativesoilmicrobiotathathave
evolvedtoresistmetalsandradionuclides.Infact,microorganismsexposedtothecontaminantstresses
notonlyhavetheunparalleledabilitytosurviveinradionuclidecontaminatedenvironments, but
also potentially reduce the toxicity of both U [3–5] and Ni [6], respectively. Microbially-mediated
mechanisms for the reduction of U toxicity include bioreduction, biosorption, biomineralization,
and bioaccumulation [4]. For Ni, most commonly known resistance mechanisms are mediated
by efflux pumps such as cnr CBA (cobalt-nickel resistance) from Cupriavidus metallidurans CH34,
NccCBA(Nickel-cobalt-cadmium)andNreB(nickelresistance)fromAchromobacterxylosoxidans31A,
andCznABC(Cadmium-zinc-nickel)fromHelicobacterpylori[6]. However,thegenomicmechanisms
underpinningUandNiconversionsintothelessmobileandlesstoxicformscontinuetoremainunclear.
Soilsamplesforthisstudywerecollectedfromsite101locatedwithintheTimsBranchsystem.
Atthissite,Uconcentrationsaretypicallypresentbetween700and800ppm[5],whichareconsidered
high Uconcentrationsinvoking thecriteria ofMumtaz etal. [7]. Therefore, thissite representsan
opportunity to study genome-enabled mechanisms recruited by the SRS-native microorganisms,
facilitatingtheirsurvivalinco-contaminatedenvironments. Infact,thestressposedbyenvironmental
contaminantsfacilitatestherecruitmentofgenesbyhorizontalgenetransfermechanismsthatenable
themicrobialcellstonotonlyresist,butalsobioremediate,thecontaminants,mostlybythesynthesisof
proteinsforcellularsurvival[8]. Someexamplesofsuchgenomic-mechanismsincludeeffluxsystems,
thepresenceofmetalresistantgenes,detoxificationgenes,andbiosorption/bioaccumulationofthe
contaminantatorwithinthebacterialcellmembrane[9].
Furthermore, a growing body of research is demonstrating that metal contaminants in the
environmenthavethepropensitytoco-selectforantibioticresistancewithinthenaturally-occurring
microbiota[10]. Co-selectionoccursthroughthefollowingmechanisms: (a)co-resistance-whenthere
isphysicalproximityoftheresistomeencodingantibioticsand/ormetalresistance,e.g.,onthesame
geneticelement(plasmid)orinthesamecell(e.g.,merAandKPCbeta-lactamase);(b)cross-resistance-
when a single resistome functions to provide efflux and antibiotic resistance (e.g., mdrL confers
resistancetometals,suchaszinc,cobalt,andchromium,alongwithantibiotics,suchaserythromycin,
josamycin,andclindamycin)[11];and(c)co-regulatoryresistance-inthissituation,multipleresistance
genesconferringresistancetodifferenttoxiccompounds,includingantibiotics,biocides,andmetals,
arecontrolledbyasingleregulatorygeneelement(e.g.,czcRregulatingtheexpressionoftheCzcCBA
Genes 2018,9,31 3of19
efflux pump, resulting in the resistance to zinc, cadmium, and cobalt, along with co-regulating
resistancetoantibioticcarbapenems)[12].
Tofurtherunderstandenvironmentally-relevantgenomicmechanismsthatunderpinmicrobial
survivalinradionuclideandheavymetal-richecosystems,werecentlyisolatedseveralbacterialstrains
inthepresenceofhighconcentrationsofbothUandNi[13]. 16S-genebasedanalysisrevealedthat
theisolatedstrainsmainlybelongedtoBurkholderiaspp. andArthrobacterspp.—generademonstrated
to serve as bioindicators of environmental contamination, as well as agents of bioremediation,
especiallyU[14]. In fact, Arthrobacter spp., were found to be predominant in the U-contaminated
Hanfordsite[15],whichisanotherDOEsiteinWashingtonStatehavingasimilarUcontamination
history as the SRS site. Arthrobacter spp., have also been isolated from ecosystems under extreme
environmental stresses, such as a nuclear waste plume amd U mined site, as well as heavy metal
contaminatedhabitats[14,16–20]. Therefore,itappearsthatArthrobacterspp.,haverecruitedstrong
genomictraitstobeabletocolonizeandsurviveinradionuclideandmetalcontaminatedhabitats.
Understandingthebasisofmetal-microbialinteractions,therefore,isaprerequisiteforthesuccessful
managementand/orrehabilitationofnuclear-legacycontaminatedenvironments.
WerecentlycharacterizedthegenomictraitsofBurkholderiasp. strainSRS-W-2-2016,abacterial
strainisolatedfromthesamecohortasArthrobactersp. SRS-W-1-2016[13];whichrevealedthepresence
ofseveralgenehomologuespreviouslydemonstratedtocodefortheresistanceofheavymetalsand
radionuclides,formingthebasisofthisstudy. Specifically,inthisstudy,wereportseveralecologically
relevant genomic traits present in Arthrobacter sp. SRS-W-1-2016, including a suite of substrate
bindingproteins, permeases, transportregulators, andeffluxpumps-likelyworkinginconcertto
potentiallydetoxifytoxicmetalsandthusfacilitatingthenaturalattenuationofcontaminantswithin
theSRSimpactedecosystem. Suchgenome-enabledstudieswillfacilitateadeeperunderstanding
of heavy metal and antibiotic resistance and hydrocarbon degradative mechanisms in complex,
mixed contaminant habitats and provide recommendations for the environmental stewardship of
anthropogenically-impactedenvironments.
2. ResultsandDiscussion
2.1. UraniumandNickelDepletionPotentialofArthrobactersp. SRS-1-W-2016
TheabilityofbacterialstrainstoresistUtoxicityhasbeenpreviouslydemonstrated. Infact,90%
ofbacteriarepresentativeoftaxaFirmicutes, Gammaproteobacteria, Actinobacteria, Bacteroidetes,
andBetaproteobacteriaidentifiedfromtheUraniumminingsiteofDomiastat,India,werefoundto
be resistant to a high U concentration of 4 mM [21]. Kulkarni et al. [22] isolated two phosphatase
producing bacteria- Escherichia coli and Deinococcus radiodurans, which were found to grow in as
high as 20 mM U. Burkholderia fungorum isolated from Rifle, Colorado, was found to grow on
10 µM U [23]. Similarly, several strains resistant to particularly high concentrations of Ni have
beenisolatedfromvariouscontaminatedsources. Alboghobeishetal.[24]isolatedseveralstrains,
namelyMethylobacteriumsp. ATHA7andKlebsiellaoxytocaATHA6resistantto24mMand16mM
Ni, respectively, fromindustrialsewage. Cupriavidustaiwanensisisolatedfromtherootnodulesof
Mimosapudica was found to be resistant to 1.5 mM Ni, along with some other heavy metals [25].
TodeterminetheresistancepotentialofstrainSRS-1-W-2016againstUandNi,differentconcentrations
ofthesemetalsweresupplementedin4MmediaandthegrowthresultsarepresentedinFigure1a.
Strain SRS-1-W-2016 could resist up to 4.2 mM of U and 8.5 mM of Ni, which are the typical
concentrationsofthesemetalsinthesamplinglocation[13],albeit,rapidgrowthwasonlyobservedat
muchlowerconcentrationsof500µMUand1000µMNi,respectively;astationaryphasewasreached
in48h. Asimilartrendwasshowninthepresenceof1000µMNi. Whengrowninthecombinationof
1000µMNiand500µMU,thestationaryphasewasonlyachievedafter60hofincubation,potentially
duetothecombinedstressofbothUandNi. Inthenativesoilenvironmentfromwherethestrainwas
Genes 2018,9,31 4of19
isolated,bothUandNiarethemajorco-contaminantsandthereforethegrowthprofileobtainedfor
theseco-contaminantsprovidesamoreaccuraterepresentationofthestrain’smetalresistanceability.
TofurtherevaluatethebioremediationpotentialofstrainSRS-1-W-2016forUandNiseparately,
aswellasincombination,microcosmswereestablishedforquantificationoftheamendedmetalsby
Genes 2018, 9, 31 4 of 19
inductivelycoupledplasma-massspectrometry(ICP-MS)andtheseresultsareshowninFigure1b.
ItisclearthTaot ftuhrethienr ietviaallucaoten tcheen btiroaretimoendioaftiUon ipnottehnetiaml oicf rsotrcaoins SmRsS-d1-eWcl-i2n0e1d6 froar pUi danlyd Nini smepearrealtyely2,4 hand
mostofatsh weeslpl aisk eind coUmdbeincaltiinoend, mciocmrocpolsemtesl yweinre tehsetasbulispheerdn faotra qnutabnytif7ic2athio,ns torfo thneg alymseundgegde msteintaglst bhya tstrain
inductively coupled plasma-mass spectrometry (ICP-MS) and these results are shown in Figure 1b.
SRS-1-W-2016hastheabilitytoquicklybiotransformand/orbioremediateU.Severalmechanismsare
It is clear that the initial concentration of U in the microcosms declined rapidly in merely 24 h and
recognizedbywhichmicrobesareabletotransformandresisttoxicityposedbyextracellularU.Some
most of the spiked U declined completely in the supernatant by 72 h, strongly suggesting that strain
ofthesemechanismsincludereductiveprecipitationbyoutermembranecytochromes,conductivepili,
SRS-1-W-2016 has the ability to quickly biotransform and/or bioremediate U. Several mechanisms are
orsporerse,cosgunrifzaecde bayd wsohircpht mioincrbobyees xaroep aobllye stoa ctrcahnasrfoidrme (aEnPd Sre)soisrt tSo-xlaicyiteyr ps,osoerdp brye ecxiptriatcaetlilounlarw Ui.t Shopmheo sphate
compouonf dthse,sree mspeechctainviesmlys. iTnocleuvdae lrueadtuecttihvee pmreeccihpiatantiisomn buyt ioluizteerd mbeymsbtrraanien cSyRtoSc-h1r-oWm-e2s,0 c1o6ntdoucgtrivoew inthe
presencpeiloi,f oUr ,swpoereuss, esdurtfhacee haidstsoorcphteiomn icbayl egxroopwoltyhsaacscshaayridoen (EtrPySp) toors eS-plahyoerssp, hoar teprmeceiptihtaytliognr eweinth( TPMG)
phosphate compounds, respectively. To evaluate the mechanism utilized by strain SRS-1-W-2016 to
media. WeobservedanintensegreencolorationformedbystrainSRS-W-1-2016duringgrowthon
grow in the presence of U, we used the histochemical growth assay on tryptose phosphate methyl
anLuriaBertani(LB)platesupplementedwithU(Figure1c),indicatingtheactivityofphosphatase
green (TPMG) media. We observed an intense green coloration formed by strain SRS-W-1-2016
enzymed-ubrainseg dgrbowiotrhe omn eadn iLautiroian BeorftaUni. (SLuB)r pplraitsei nsugplpy,lenmoendteedcl winiteh iUn (tFhigeucreo 1ncc)e, inntdriacatitoinngs thoef atchtieviatym ended
Niwasoofb psheorsvpehdatausne denezrytmhee-bteassetedd bicoorenmdeidtiioatniosn( Fofi gUu. rSeur1pbr)is.inItgliys, hniog dhelcylinliek ienl ytheth caotnceefnflturaxtiomnes cohf anisms
wereemthpel oaymeedndbeyd tNhie wstarsa oinbsteorvperde uvnednetrt thhee cteesltleudl acronudpittiaoknes o(FfigNuirea n1bd).h Iet nisc heignholyb liiokreelym tehdati aetfiflounx activity
mechanisms were employed by the strain to prevent the cellular uptake of Ni and hence no
againstNiwasdocumented.
bioremediation activity against Ni was documented.
No Amendment 500 µM U
1000 µM Ni 500 µM U + 1000 µM Ni
1
0.8
)
m
n
0 0.6
0
6
(
D 0.4
O
0.2
0
0 12 24 36 48 60 72 84 96
Time (h)
(a)
Nickel Nickel in U + Ni
Uranium Uranium in U + Ni
900 180
800 160
700 140
kel 600 120um
c500 100ni
Ni a
M 400 80 Ur
µ300 60 M
μ
200 40
100 20
0 0
0 24 48 72
Time (h)
(b)
Figure1.Cont.
Genes 2018,9,31 5of19
Genes 2018, 9, 31 5 of 19
(c)
Figure 1. Shown are (a) growth experiments to determine the resistance abilities of Arthrobacter sp.
Figure1. Shownare(a)growthexperimentstodeterminetheresistanceabilitiesofArthrobactersp.
strain SRS-W-1-2016 to Uranium (U) and Nickel (Ni), respectively. Growth of strain is shown in 4M
strainSRS-W-1-2016toUranium(U)andNickel(Ni),respectively.Growthofstrainisshownin4M
media without amendments, as well as supplemented with 500 μM U, 1000 μM Ni and, and in the
mediawcoitmhboiunattaiomn eonf 5d0m0 μenMt sU, aansdw 1e00ll0 aμsMs uNpi,p rleesmpeectnivteeldy; w(bi)t hsh5ow00n µarMe thUe, m1e0a0s0urµeMd coNniceanntrda,tiaonnsd inthe
combinaotfi Uon anodf N50i 0ovµeMr a tUhraeen-dda1y0 p0e0riµodM thNati w,reersep speickteivd einly m;(icbr)ocsohsomws ncoanrtaeinthineg mstreaains uSrReSd-Wc-o1n-2c0e1n6;t rations
ofUand(cN) hiiostvoechreamtihcarle ep-ldataey-bpaseerdio gdrotwhatht wasesraey stpo iksheodwi npmhoiscprhoactoassem esnczoymntea-ibnaisnedg sUt-rparienciSpRitaSt-iWon- 1-2016;
potential of strain SRS-W-1-2016. Bacteria that display a phosphatase-positive phenotype appear
(c)histochemical plate-based growth assay to show phosphatase enzyme-based U-precipitation
bright green on Tryptose Phosphate Methyl GreenTPMG agar plate.
potential of strain SRS-W-1-2016. Bacteria that display a phosphatase-positive phenotype appear
brig2h.2t. gGreeneonmoe-nCTenrytrpict oEsvealPuhatoisonp hoaf tSetrMaine tShRySl-GWr-e1e-n20T1P6M Gagarplate.
The genome size of strain SRS-W-1-2016 was predicted to be approximately 4,564,701 bases with
2.2. Genome-CentricEvaluationofStrainSRS-W-1-2016
a G+C content of 64.1. Moreover, the strain contained a total of 4327 coding sequences, with 58 total
RNA genes, 57 tRNA genes, and two copies of the 16S rRNA genes, respectively. A circular genomic
ThegenomesizeofstrainSRS-W-1-2016waspredictedtobeapproximately4,564,701baseswith
map of strain SRS-W-1-2016 using the 93 contigs with an N50 contig length of 92,788 bases is shown
aG+Ccontentof64.1. Moreover,thestraincontainedatotalof4327codingsequences,with58total
in Figure 2.
RNAgenes,57tRNAgenes,andtwocopiesofthe16SrRNAgenes,respectively. Acirculargenomic
Further genome-centric evaluation of strain SRS-W-1-2016 revealed that out of 4318 total protein
mapofscotrdaining SgRenSe-sW, 6-14.-7250%1 6geunseisn gastshoceia9t3edc ownitthig csluwstiethrs aonf Nor5th0ocloognotuisg gleronugptsh ooff p9r2o,t7e8in8s b(aCsOeGssi)s; shown
inFigur7e42.3.4% genes annotated to protein coding function predictions; 24.36% genes annotated as protein
Furctohdeinrgg geennoems we-icthe nentrziycmeev aplruodauticotniono;f asntdra 2i5n.5S9R%S g-eWne-s1 -w2e0r1e6 corenvneecatleedd toth Kaytootou tEoncfy4c3lo1p8etdoiata olf protein
Genes and Genomes (KEGG) pathways. Note that COGs represent proteins that are an ortholog or
codinggenes,64.75%genesassociatedwithclustersoforthologousgroupsofproteins(COGs);74.34%
direct evolutionary counterpart among bacterial genomes as they evolve over time. When the whole
genesannotatedtoproteincodingfunctionpredictions;24.36%genesannotatedasproteincoding
genome sequence of strain SRS-W-1-2016 was taxonomically analyzed using the One Codex RefSeq
geneswdiathtabeanszey, 5m2.e5%p roof dcluascstiifoiend; raeandds (2r5e.a5d9c%oungte 4n2e) sclwuseterreedc ownitnhe ActrethdrotboacKtery aoutroesEcennsc yTCcl1o, pfoeldloiwaeodf Genes
andGenboy m25e%s ((rKeaEdGcoGu)ntp 2a0t)h wwitahy As.rtNhroobtaecttehr agtloCbifOorGmiss rNeBpRreCs 1e2n,1t3p7r, oretsepinecstitvhealyt (aFrieguarne 3o)r. tThwoolo-wgaoyr direct
evolutioanvaerryagceo nuunctleeorptidaert idaemntoitnyg (AbaNcIt)e breiatwlegeenn sotmraiens SaRsSt-hWe-y1-e2v01o6l vaendo vtheer ctliomsees.t Wrelhateinvet Aherthwrohboaclteerg enome
aurescens TC1 was estimated to be 80.28%, (SD: 5.24%; Supplementary Figure S1), from 3101
sequenceofstrainSRS-W-1-2016wastaxonomicallyanalyzedusingtheOneCodexRefSeqdatabase,
fragments that were included in this analysis, indicating that SRS-W-1-2016 may be a new species
52.5%ofclassifiedreads(readcount42)clusteredwithArthrobacteraurescensTC1,followedby25%
that is distinctly different than Arthrobacter aurescens TC1. Similar to the ANI, when digital DNA-
(readcount20)withArthrobacterglobiformisNBRC12,137,respectively(Figure3). Two-wayaverage
DNA hybridization (dDDH) was estimated using the Genome-to-Genome Distance web service, it
nucleotiwdaesi desetinmtiattyed( AthNatI )stbraeitnw SeReSn-Wst-1ra-2i0n16S RwSa-sW 21-.12-02%0 1s6imainladr ttoh Aerctlhorosbeasctterre alaurteivsceenAs rTtChr1o (bdaacttae rnoatu rescens
TC1wassheoswtinm). aNtoetde tthoatb aecc8o0r.d2i8n%g t,o( SthDe :p5ip.2e4lin%e; uSsuedp, pDlDemH e>n 7t0a%ry (vFiaig luogriestSic1 r)e,gfrreosmsion3)1 w01ouflrda gmmeaenn tsthat
wereincthluatd tehde tiwnot gheinsoamneasl ybesliosn,gin tod tihcea tsianmge tshpaectiSesR. TSo-Wge-th1e-2r,0 th1i6s mis satyrobnge eavindeewnces ptheact iSeRsSt-hWa-t1-i2s0d16is tinctly
has a unique taxonomic position relative to other members of Arthrobacter species for which whole
differentthanArthrobacteraurescensTC1. SimilartotheANI,whendigitalDNA-DNAhybridization
genome sequence data is available.
(dDDH)wasestimatedusingtheGenome-to-GenomeDistancewebservice, itwasestimatedthat
strainSRS-W-1-2016was21.20%similartoArthrobacteraurescensTC1(datanotshown). Notethat
accordingtothepipelineused,DDH>70%(vialogisticregression)wouldmeanthatthetwogenomes

belongtothesamespecies. Together,thisisstrongevidencethatSRS-W-1-2016hasauniquetaxonomic
position relative to other members of Arthrobacter species for which whole genome sequence data
isavailable.
GeGneenses2 021081,89, ,93, 131 66o fo1f 919
Genes 2018, 9, 31 6 of 19

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fourth rings depicting clusters of orthologous groups of proteins (COG) categories of protein coding
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genes on the forward and reverse strands, respectively. The second and third rings show the locations
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of protein coding, tRNA, and rRNA genes on the forward and reverse strands, respectively. The black
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deopf ipccrtosontGeteiCnnt cc aoobndotiveneng tt,h wteR igtNehnAtoh,m eaenp daev arekRrsaNgeAex,t wegnehdneirenesag ost ntoh wtohsaeer fdeoxsrtwethnaedriodnug at tnsoiddw eraerovdfest rthsheee csciterrncatlneerdr mse,p arrreekss pseeengctmitnivegnetGlsy Cw. Ticthohen btelnactk
abpolovteG dCteh peciocgntetsen nGotC ml oecwoanevrt eetrnhatag nwe ,itthwhe h thgeeerne paoesmatekh soa veseexrtaeegxnetd.e innTdhgie nt oginwntaoerwrdmasor tsdht sep tolhouet tscdieednpeti ceotrsf mtGhaCer kcsikrsceewlge. m rBeepontrhtes sbewansitteihn gG GCC
cocnotnectneotnmltop awobseoitrviotenh t aphnleo ttghse ewngoeermen ego emanveeeraraavtegeder a,u gwsienh.geT rahe sealsiid ntihnnoegrs wme ienoxdsttoepwnld ooitfn d5g0e ,tp0o0iwc0t nasrtG.d Cs tshkee cwe.nBteort hmbaarske sceogmmpeonstsit iwonith
plGoCts wcoenretegnetn elorwateerd uthsainng tahsel idgeinngomwein davoweraogfe5. 0,T0h0e0 nint.nermost plot depicts GC skew. Both base
composition plots were generated using a sliding window of 50,000 nt.

Figure 3. Whole genome sequence-based phylotree of Arthrobacter sp. strain SRS-W-2-2016 relative to
a cohort of 24 Arthrobacter spp., for which genome sequences are available. The highest taxonomic
affiliation of strain was found with Arthrobacter aurescens TC1 based on the highest number of whole
genome sequence reads (42; shown in parenthesis) that clustered with this group. The tree was
FiFgiugrugereen3 e.3r.Wa Wtehdho oblealesg egeden ononom mtheee s pseerqequsueenennccece eo--fb btahasese ePddfa ppmhh yfyullonotctrrteieoeen o ocffa tAAegrrttohhrrryoo abbmaaccottenerrg ssstpp A.. srsttthrraraoibnina cSStReRrS sS-pW-Wp.- 2-a2-v2-a20i0l1a16b6 lreer ielnal atitvivee to
toaa ccotohhheoo rintr ttoeogff r2a24t4e AdA rmrtthihcrrrooobbbaaiccattle egrr essnppoppm..,,e ffeooxrrp wewrhth riicechvhi eggween n(oIoMmmGee/ sesere)q qduuaetenanbccaeesssea . arDreiesa pavlvaayaileialdab bnleloe.d.T eThs heaerhe hitghihgohesese tswtt aittxhao xanot onmomicic
afafiflfiiallietaiaotsinto onon foesf rtsretaaridan i(n<w1 wa%sa osffo fcuolanusdnsidwfi ewidthi rtheAa Adrtsrh)t.rh orboabcatcetrera uaruersecsecnesnTs CTC11b absaesdedo nonth teheh ihgihgehsetsnt unmumbebreor fowf whohloele
gegneonmomees esqeuqeunecnecer eraedasds( 4(24;2;s hsohwownni ninp apraernetnhtehseissi)s)t htahtact luclsutsetreerdedw withithth tihsisg rgoruopu.p.T Thheet rtereeew wasas
Arthrobacter species, which are members of the soil actinobacteria, are ubiquitously distributed
gegneenreartaetdedb absaesdedo nonth tehep rperseesnecneceo fotfh teheP fPafmamfu fnucntciotinonc actaetgeogroyrya mamonognsgtsAt Artrhtrhorboabcatcetresrp spp.pa. vaavialailbalbelein in
in diverse ecological habitats, and are known for their ability to survive in extreme conditions such
thtehein itnetgergartaetdedm micircorboibailagl egneonmomeeex epxepretrrte rveivewiew(I M(IMG/Ge/re)r)d dataatbaabsaes.eD. Disipsplalyaeydedn nododesesa raereth tohsoesew withitha tat
laeslae sUatsVot n oiernrreae draiedaatd(i<o (n1<% 1a%nod fo cfrl aacdlsasiosifisaiecfditeivdrie trayed a[s2d)6.s]).. Efficient molecular adaptation mechanisms in Arthrobacter
enhance the stability of nucleic acids, proteins, and lipids, allowing bacteria to survive in extreme
conditions [27], such as the SRS co-contaminated soils. Some species of Arthrobacter such as
Arthrobacter species, which are members of the soil actinobacteria, are ubiquitously distributed
A rthrobacterspecies,whicharemembersofthesoilactinobacteria,areubiquitouslydistributed
in diverse ecological habitats, and are known for their ability to survive in extreme conditions such
in diverse ecological habitats, and are known for their ability to survive in extreme conditions
as UV irradiation and radioactivity [26]. Efficient molecular adaptation mechanisms in Arthrobacter
such as UV irradiation and radioactivity [26]. Efficient molecular adaptation mechanisms in
enhance the stability of nucleic acids, proteins, and lipids, allowing bacteria to survive in extreme
conditions [27], such as the SRS co-contaminated soils. Some species of Arthrobacter such as
Genes 2018,9,31 7of19
Arthrobacterenhancethestabilityofnucleicacids,proteins,andlipids,allowingbacteriatosurvive
inextremeconditions[27],suchastheSRSco-contaminatedsoils. SomespeciesofArthrobactersuch
as Arthrobacter crystallopoietes [28] and Arthrobacter chlorophenolicus [29] have been utilized for the
bioremediation of chromium and 4-chlorophenol in contaminated soils. Generally, Arthrobacter is
knowntogrowoncomplexaromaticsubstratessuchashydroxybenzoates,pyridine,andpicoline[30],
which is a unique biodegradative ability of this group of soil bacteria. In fact, as stated earlier
inthisstudy,Arthrobacterspp.,havebeenshowntosuccessfullycolonizehabitatscharacterizedby
extreme environmental stresses, including nuclear waste plume heavy metal contaminated sites,
as well as uraniferous niches, such as the U-impacted DOE Hanford site [15], which points to the
genomic-mechanismspossessedbyArthrobacterspp.,forthecolonizationofmetal-richecosystems.
To further analyze the genome of strain SRS-1-W-2016, a KEGG -based hierarchical functional
gene clustering analysis was performed (Table 1), which revealed the presence of a total of 1124
KEGG-associatinggenes;thetopfivecategoriesbelongedtocarbohydratemetabolism(284genes;25.27%);
amino acid metabolism (256 genes; 22.78%); energy metabolism (135 genes; 12.01%); vitamin and
cofactormetabolism(128genes;11.39%);andmembranetransport(105genes;9.34%),respectively.
Additionally, a total of 2844 protein coding genes associated with COGs in strain SRS-W-1-2016
werefoundwithfurtherclassificationinto25categories,withthemostabundantCOGsubsystems
as follows: carbohydrate transport and metabolism; and amino acid transport and metabolism.
Thetranscriptionofmanyofthesefeaturessuggeststhepropensityofthisstraintoengageinhigh
metabolicactivityinitsnativecontaminatedenvironment. Theseanalysesprovideastrongfoundation
forthepresenceofseveralgenome-enabledmetabolicandcatabolicprocessesinArthrobactersp. strain
SRS-W-1-2016whichlikelyplayasignificantroleinitssuccessfulcolonizationandsurvivalwithinthe
SRSco-contaminatedsoilhabitat.
Table1.FunctionaltraitsconnectedwithKyotoEncyclopediaofGenesandGenomes(KEGG)pathways
identifiedfromthewholegenomesequenceofArthrobactersp.strainSRS-W-1-2016.Thisfigurewas
prepared by analysis of the whole genome using the integrated microbial genome expert review
(IMG/er)andvalueswereplottedusingMicrosoftExcel.
KEGGCategory Percentage(%)
GlobalandOverviewMaps 63.08
CarbohydrateMetabolism 25.27
AminoAcidMetabolism 22.78
EnergyMetabolism 12.01
MetabolismofCofactorsandVitamins 11.39
MembraneTransport 9.34
NucleotideMetabolism 8.72
LipidMetabolism 7.56
Translation 7.21
XenobioticsBiodegradationandMetabolism 6.32
SignalTransduction 5.87
MetabolismofotherAminoAcids 4.8
ReplicationandRepair 4.54
Folding,SortingandDegradation 4.18
MetabolismofTerpenoidsandPolyketides 3.91
BiosynthesisofotherSecondaryMetabolites 3.2
GlycanBiosynthesisandMetabolism 2.85
CellMotility 2.67
CellGrowthandDeath 1.42
DrugResistance 1.33
TransportandCatabolism 0.98
Transcription 0.44
EnvironmentalAdaptation 0.18
ExcretorySystem 0.18
Genes 2018,9,31 8of19
Genes 2018, 9, 31 8 of 19
2.23..3 G. Geneense PsoPtoetnetniatilalyll yCConofnefrerrirnign gMMeteatla RlReseissitsatnacnec eini nStSrtariani nSSRRSS-W-W-1--12-021061 6
OOnen enontoatbalbel eatattrtirbiubtuet eofo tfhteh eAArtrhtrhorboabcatcetre rgegneunus sisi sthtehieri rabaibliitliyt ytot odedgergardaed exexneonboiboitoitci ccocmomppououndnds s
anadn drerseissitsatnacnec etot ohheaevavy ymmeteatlasl s[2[62,63,13–13–43]4,] ,ininclculuddiningg UU aanndd NNi i[2[277].] .GGenenoomme-ec-ecnentrtirci casassessessmsmenetn tofo f
strain SRS-W-1-2016 revealed the presence of several gene homologues previously demonstrated to
strain SRS-W-1-2016 revealed the presence of several gene homologues previously demonstrated
play a part in the resistance against heavy metal/radionuclides, such as several efflux and MFS
toplayapartintheresistanceagainstheavymetal/radionuclides,suchasseveraleffluxandMFS
transporters, P-type ATPase translocators, and heavy metal-responsive transcriptional regulators.
transporters,P-typeATPasetranslocators,andheavymetal-responsivetranscriptionalregulators.
Specifically, for metals resistance, the genome of strain SRS-W-1-2016 contains the high affinity
Specifically, for metals resistance, the genome of strain SRS-W-1-2016 contains the high
traafnfisnpiotyrt trsaynsstpemor tfsoyrs tenmickfeol/rconbicaklte l/(NcoiCbaolTt (gNeinCeo;T HgoexnNe;/HHuopxNN//NHixuAp Nf/aNmiixlAy), faams ilyw),elal saws etllhea s
cothbealtc/ozibnacl/tc/azdinmci/ucmad rmesiiustmantr epsriosttaenint (pCrzocDte)in. Th(Cez NcDiC)o.TT shyestNemiC, osThoswynst reemla,tisvheo two fnourre lhaotimveolotogofuosu r
gene systems (Figure 4a), was found to occur as a 1056 bp fragment (contig41300;
homologous gene systems (Figure 4a), was found to occur as a 1056 bp fragment (contig41300;
fifgi|g6|666666666666.62.3223720780.8p.epge.g20.28048)4, )w,whihleil ethteh eCzCczDcD susbusbyssytsetmem, (,F(iFgiugruer e4b4)b w)wasa sa a1018098 9bpb pgegneen efrfargamgmenetn t
(contig51456; fig|6666666.232708.peg.2658), respectively. In addition to these genes, bacterial
(contig51456;fig|6666666.232708.peg.2658),respectively. Inadditiontothesegenes,bacterialresistance
resistance to metals is also governed by genes for the resistance, nodulation, and cell division (RND)
to metals is also governed by genes for the resistance, nodulation, and cell division (RND) family
family proteins- all part of transenvelope protein complexes which detoxify the cellular environment
proteins- all part of transenvelope protein complexes which detoxify the cellular environment by
by exporting toxic metal cations from the periplasm to the outside. Several RND-type efflux gene
exporting toxic metal cations from the periplasm to the outside. Several RND-type efflux gene
homologues were found interspersed within the genome of strain SRS-W-1-2016 (data not shown).
homologues were found interspersed within the genome of strain SRS-W-1-2016 (data not shown).
Additionally, the ABC-type transporters and heavy-metal transport/detoxification proteins were
Additionally, the ABC-type transporters and heavy-metal transport/detoxification proteins were
abundantly present within the genome of strain SRS-W-1-2016, potentially maintaining metal
abundantly present within the genome of strain SRS-W-1-2016, potentially maintaining metal
homeostasis and survival in the SRS co-contaminated soil habitat.
homeostasisandsurvivalintheSRSco-contaminatedsoilhabitat.
(a)
(b)
Figure 4. Shown are (a) the chromosomal region of the cobalt/zinc/cadmium subsystem gene
Figure4.Shownare(a)thechromosomalregionofthecobalt/zinc/cadmiumsubsystemgene(1056bp)
(1056 bp) in Arthrobacter sp. strain SRS-W-2-2016 relative to four other organisms and (b) the
inArthrobactersp.strainSRS-W-2-2016relativetofourotherorganismsand(b)theHoxN/HupN/NixA
HoxN/HupN/NixA family nickel/cobalt transporter gene (1089 bp) in Arthrobacter sp. strain SRS-W-2-
familynickel/cobalttransportergene(1089bp)inArthrobactersp.strainSRS-W-2-2016relativetofour
2016 relative to four other organisms. The graphic is centered on the focus gene, which is red and
otherorganisms.Thegraphiciscenteredonthefocusgene,whichisredandnumbered1.Setsofgenes
numbered 1. Sets of genes with a similar sequence are grouped with the same number and color.
withasimilarsequencearegroupedwiththesamenumberandcolor.Geneswhoserelativepositionis
Genes whose relative position is conserved in at least four other species are functionally coupled and
conservedinatleastfourotherspeciesarefunctionallycoupledandsharegraybackgroundboxes.
share gray background boxes.
There are several mechanisms by which bacteria can reduce the mobility and hence toxicity
There are several mechanisms by which bacteria can reduce the mobility and hence toxicity of
of U and one process that shows bioremediation potential is by a phosphatase enzyme mediated
U and one process that shows bioremediation potential is by a phosphatase enzyme mediated
biotransformation of U (VI) into phosphate-minerals [27,29]. It has been demonstrated that
biotransformation of U (VI) into phosphate-minerals [27,29]. It has been demonstrated that
microbially-mediatedalkalineoracidphosphataseactivitiescanresultintheprecipitationofmore
microbially-mediated alkaline or acid phosphatase activities can result in the precipitation of more
than90%ofsolubleU,resultingintheformationofawidearrayofuranylphosphateminerals[30].
than 90% of soluble U, resulting in the formation of a wide array of uranyl phosphate minerals [30].
Therefore, the microbially-mediated formation of uranyl phosphate from U (VI) warrants further
Therefore, the microbially-mediated formation of uranyl phosphate from U (VI) warrants further
researchforthecontrolofUmobilityinU.S.DOEcontaminatedenvironments.Toidentifythepresence
research for the control of U mobility in U.S. DOE contaminated environments. To identify the
presence of the phosphatase enzyme within the genome of strain SRS-W-1-2016, we manually
Genes 2018,9,31 9of19
of the phosphatase enzyme within the genome of strain SRS-W-1-2016, we manually queried the
annotatedgenome,revealingthepresenceoftwoacidphosphatases(NCBIgeneaccession#OOP63314
andOOP61946),aswellasanalkalinephosphatase(NCBIgeneaccession#OOP64358). Moreover,
wealsoidentifiedthespoTgene(contig50361;fig|6666666.232708.peg.2556)instrainSRS-W-1-2016
(datanotshown);thespoTgeneencodesaguanosinetetraphosphate(ppGpp)hydrolase/synthetase
whichwasrecentlyimplicatedintheresistanceofUbyCaulobactercrescentus,butonlyundercarbon
starvationconditions[35]. Collectively,thesemechanismslikelyrenderthemetabolicabilitytostrain
SRS-W-1-2016tonotonlysurviveintheSRSsoils,butalsoperformcriticalbiodegradativefunctions.
Moreover,inadditiontothemetal-relatedgenes,severalgenesthatpotentiallyconfertheability
todegradeorganiccontaminantswerealsoidentifiedinstrainSRS-W-1-2016;especiallyoxygenase
enzymes(datanotshown). Thisfindingisrelevantbecauseoxygenaseshavebeenpreviouslyshownto
co-metabolizethedegradationoftrichloroethylene(TCE),oneofthewidespreadorganiccontaminants
identifiedinSRS-impactedhabitats[36]. Therefore,itislikelythatstrainSRS-W-1-2016ismetabolically
activeinthebiodegradationandbiomineralizationofnotonlymetals,butalsohydrocarbonsinthe
SRSsoils.
2.4. ResistomeofArthrobactersp. StrainSRS-W-1-2016
WhenthegenomeofstrainSRS-W-1-2016wasminedforthepresenceofantibioticresistancegenes
(theresistome),genedeterminantsforthefollowingdrugresistancewerenoted(percentidentitywith
matchingregionareshownintheparenthesis): fluoroquinolone(36%),isoniazid(46%),fosfomycin
(43%), aminocoumarin (66%), mupirocin (54%), and rifamycin (33%), respectively. Several other
genesthatpotentiallyconferantibioticresistancewerealsoidentified,includingtheantibiotictarget
protectionprotein(36%),geneinvolvedinself-resistancetoantibiotic(66%),effluxpumpcomplexor
subunitconferringantibioticresistances[(39%withImrBand40%withImrD—bothconferresistance
tolincosamides)and55%withnovA(resistancetonovobiocin)],antibioticresistantgenevariantsor
mutants,andtheantibioticinactivationenzyme(SupplementaryFigureS2). Becausealowerdegreeof
homology,rangingbetween36%to66%,wasobservedbetweentheresistomeofstrainSRS-W-1-2016
withthepreviouslycharacterizedantibioticresistancegenes,thereisastrongpossibilityofanovel
resistomethathasdevelopedinthisstrain,likelyduetotheselectivepressureofheavymetals,U,
andorganiccompounds.
Moreover,whenthegenomeofstrainSRS-W-1-2016wasevaluatedforthepresenceofbiosynthetic
gene clusters (BCs) responsible for the synthesis of secondary metabolites (SMs), seven BCs with
150associatedgenes were identified (Supplementary Figure S3). The predominant BC identified
fromstrainSRS-W-1-2016affiliatedwiththewell-knownnonribosomalpeptidesynthetase(NRPS)-
type III polyketide synthase (T3PKS), with a total of 69 pfams; other BCs were also identified for
bacteriocin(27pfams)andbutyrolactone(11pfams). Interestingly,manypfamsthatwerefoundtobe
overrepresentedacrossthesevenBCsidentifiedinstrainSRS-W-1-2016havebeenshowntoplayarole
inmetals-microbeinteractions(appearasredboxesintheheatmap;SupplementaryFigureS3).
Notably,atotalof46secondarymetaboliteclusterswereidentifiedusingtheantiSMASHpipeline
withgeneshomologoustothebiosynthesisof(similarityshowninparenthesis): Amphotericin(23%);
Streptomycin(15%);Arsenopolyketides(12%);Echosides(11%);Guadinomine(11%);Thiolutin(8%);
Kanamycin (7%); Chlorizidine (7%); Pristinamycin (5%); Meilingmycin (2%); Lobosamide (4%);
Enduracidin (4%); and Herboxidiene (2%), respectively. Overall, this resistome analysis depicts
thebroadmetabolicandbiosyntheticpotentialofArthrobactersp. strainSRS-W-1-2016.
2.5. PredictionofPhenotypicTraitsinArthrobactersp. StrainSRS-W-1-2016
Tofurtherprobethegenotypetophenotypetraits,arecentlydevelopedpipelinewasutilizedfor
strainSRS-W-1-2016relativetoseveralotherArthrobacterspp.,whichrevealedtheabilityforsugar
hydrolysissuchasmaltose,trehalose,mannitol,rhamnose,andmannosebyboththeclassifiers,while
glucose,xylose,andcellobiosewereconfirmedbyonlyoneoftheclassifiersduringTraitaranalysis
Genes 2018,9,31 10of19
Genes 2018, 9, 31 10 of 19
ofArthrobactersp. strainSRS-W-1-2016(Figure5). However,certaintraitssuchastheutilizationof
arabinose,fermentationofglucose,andutilizationofacetate,whichwereobservedinotherArthrobacter
arabinose, fermentation of glucose, and utilization of acetate, which were observed in other
strains,wereabsentinstrainSRS-W-1-2016.
Arthrobacter strains, were absent in strain SRS-W-1-2016.

FFiigguurree 55.. IInn--ssiilliiccoo pphheennoottyyppiicc ccoommppaarriissoonn ooff AArrtthhrroobbaacctteerr sspp.. SSRRSS WW--11--22001166 wwiitthh ssiixx cclloosseellyy rreellaatteedd
AArrtthhrroobbaacctteerrs spppp.. uussiinngg tthhee TTrraaitiatarrp pipipeelilninee..
Using the phypate + PGL predictor built in the pipeline also confirmed the presence of the
Using the phypate + PGL predictor built in the pipeline also confirmed the presence of the
alkaline phosphatase enzyme in Arthrobacter sp. strain SRS-W-1-2016, which may be associated with
alkaline phosphatase enzyme in Arthrobacter sp. strain SRS-W-1-2016, which may be associated
U precipitation and could be one of the factors to confer resistance to the bacterium at a toxic U
withUprecipitationandcouldbeoneofthefactorstoconferresistancetothebacteriumatatoxicU
concentration. Interestingly, Arthrobacter sp. strain SRS-W-1-2016 was the only motile bacterium relative
concentration.Interestingly,Arthrobactersp.strainSRS-W-1-2016wastheonlymotilebacteriumrelative
to the other selected Arthrobacter spp. group of bacteria, according to the phypat predictor; motility is a
totheotherselectedArthrobacterspp. groupofbacteria,accordingtothephypatpredictor;motilityisa
useful trait that can facilitate bacterial chemotaxis and enhance biodegradative functions [37].
usefultraitthatcanfacilitatebacterialchemotaxisandenhancebiodegradativefunctions[37].
22..66.. GGeennoommiicc IIssllaannddss iinn AArrtthhrroobbaacctteerr sspp.. SSttrraaiinn SSRRSS--WW--11--22001166
AAnnootthheerr iinntteerreessttiinngg ggeennoommiicc ttrraaiitt ooff ssttrraaiinn SSRRSS--WW--11--22001166 iiss tthhee pprreesseennccee ooff sseevveerraall ggeennoommiicc
iissllaannddss (G(GEEIsIs).). BBaacctteerriiaall ggeennoommeess ccoonnssiisstt ooff aa sseett ooff ccoorree ggeenneess eennccooddiinngg ffoorr eesssseennttiiaall mmeettaabboolliicc
ffuunnccttiioonnssw whihchichar easruep psulepmpelenmteednwteidth gweinthes agceqnueisr edacvqiuaihreodri zvoinat alhgoernizeotnratanls fgeren(He GtTra)nmsfeecrh a(nHisGmTs)
tmhaetchmaanyisrmensd tehraatd mdiatiyo nraelnmdeert aabdodliictifounnaclt imonestatobotlhice rfeucnructiitoinngs btoac ttehrei arseucrcuhiatisnagd bapactitvereiatr asiutscha nads
gaednaopmtivice ptrlaasittsic aitnyd, fgaecinloitmatiicn gpleavsotilcuittyio, nfaacriylitsautrinvgiv eavlo[3lu8]t.ioGnEarIys hsuavrveivbaele n[3m8].o GreEcIos mhamvoen blyeeknn moworne
tcoomremndoenrlyv ikrnuolewnnce too rreanndteibr ivoitriculreensciset oanr caenttiobitohteich roessitsbtaanccteer tioa ,thbeu thomsot rbeacrteecreina,t lbyu,tw mhoorlee rgeecneonmtlye,
whole genome sequencing studies have also revealed other GEI-encoded functional traits classified
into the following four broad categories - pathogenicity islands (PAIs), that code for virulence genes;
metabolic islands (MIs), genes for the biosynthesis of secondary metabolites; resistance islands (RIs),