Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs.

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by  Csorgo, Balint

Csörgőetal.MicrobialCellFactories2012,11:11
http://www.microbialcellfactories.com/content/11/1/11
RESEARCH Open Access
Escherichia
Low-mutation-rate, reduced-genome
coli
: an improved host for faithful maintenance of
engineered genetic constructs
Bálint Csörgő1, Tamás Fehér1, Edit Tímár1, Frederick R Blattner2,3 and György Pósfai1*
Abstract
Background: Molecular mechanisms generating genetic variation provide the basis for evolution and long-term
survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating
mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly
emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic
biological tasks.
Results: By constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-
genome E. coli strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of
diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant
stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in
overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free
host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental
strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the
culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The
surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-
induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/
outgrowth of mutants no longer producing an active, toxic enzyme.
Conclusions: By eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic
element-free E. coli strain MDS42 was further stabilized. The resulting strain represents an improved host in various
synthetic and molecular biological applications, allowing more stable production of growth-inhibiting
biomolecules.
Keywords: Escherichia coli, mutation rate, evolvability, reduced genome, synthetic biology, chassis
Background controlled, as in laboratory and industrial settings. In
Intrinsicmechanismsforgeneratingdiversityareimpor- fact,novel,evolvedfeaturesarisinginacarefullydesigned
tant for survival of bacterial populations in dynamically and fabricated system of biological parts can lead to
changing environmental conditions. The ability of a unwanted genotypic andphenotypicalterations,andthe
populationtoadapttovarioussituationsislargelydepen- spontaneous genetic modification of an established pro-
dent upon a constant fine-tuning of mutation rate [1]. ductionstrainoraclonelibraryisusuallyhighlyundesir-
However, what is beneficial in a natural environment is able [2]. Consequently, whether used as a production
not necessary when conditions are relatively stable and strain,acloninghost,orasasyntheticbiologicalchassis,
abacterialcellwithincreasedgenetic stabilityisofgreat
importance[3-5].
*Correspondence:[email protected] Inadditiontobeingauniversalcloninghost,Escherichia
1InstituteofBiochemistry,BiologicalResearchCenteroftheHungarian
coliisthemostcommonorganismusedintheproduction
AcademyofSciences,62Temesvárikrt,H6726Szeged,Hungary
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Csörgőetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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of proteins, metabolites, and secondary metabolites, for defective forms of toxic genes can be so effective, that it
bothresearchand industrialpurposes[6-12].Inaneffort canactuallybeuseddeliberatelytoobtainpointmutants,
toimprovethe performance ofthese ‘workhorse’ strains, asdemonstratedbyisolationofmutantsofhumanimmu-
several large-scale modifications have been made to var- nodeficiency virus protease [27] or of the PvuII DNA
ious E. coli strains using genome engineering methods methyltransferase[28].UnlikeinsertionmutagenesisbyIS
[13-17]. These efforts all follow the basic principle of elements, point mutations as a whole cannot be totally
streamliningmetabolicpathwaysfortheincreasedproduc- eliminated. Nevertheless, any reduction inmutationrate
tion of a given biomaterial coupled with reduction of expands the cloning potential of the host cell and
unwanted byproducts. Along these lines, a reduced-gen- improvesitsfunctionasasyntheticbiologicalchassis.
omeE.colistrain(MDS42)wasconstructedinourlabora- OurstrategyforreducingpointmutationrateinE.coli
tories.Mostgenesirrelevantforlaboratoryapplications,as involved disabling the effective mutation generating
wellasallknownmobileDNAsequencesandcrypticviru- enzymesoftheSOSresponse.Understressfulconditions
lence genes were precisely deleted, resulting in a geneti- (e.g.toxicclonesharboredinthecell),DNAdamagemay
cally stabilized strain that displays several advantageous occur, activating the SOS response, inducing approxi-
properties[18].TheadvantagesofusinganMDS42back- mately40membersoftheSOSregulon [29-32].Threeof
ground for industrial purposes was demonstrated by the genes induced during the SOS response of E. coli
increasedL-threonineproductioninamodifiedversionof encode DNA polymerases(Pol II, Pol IV, Pol V) thatare
the multi-deletion strain [19]. In a recent work, we have able to bypass replication barriers at damaged sites and
alsoshownthattheISelement-freeMDS42hostimproves stalledreplicationforks[33-36].AllthreeoftheSOS-indu-
maintenance of unstable genetic constructs,allowing for ciblepolymeraseshavebeenimplicatedininducedmuta-
stablecloningofcertaintoxicgenes[20]. genesis [37], with Pol IV and Pol V having error-rates
Evidence exists that some genes, in their functional approximately2to3ordersofmagnitudehigherthanthe
forms, are unusually difficult to clone in bacterial plas- high-fidelity replicative polymerase (Pol III) [38]. Pol II,
mids, and aberrant clones frequently arise [21-24]. whileshowinghighfidelityonundamagedtemplates,was
Normally, the general mutation rateofthe host cellisso shown to take part in certain types of stress-induced
low (~10-7 mutation/gene/generation) [25], that sponta- mutagenesis [37,39,40]. The SOS-regulated polymerases
neous changes in a cloned DNA fragment are extremely aredispensable;theirprimaryroleseemstobethegenera-
rare and therefore cannot solely account for the cloning tion of genetic diversity under stressful conditions [41].
artifacts.However,whentheclonedDNAfragmentinter- DNArepairhasalternativepathways,mostnotablyrecom-
fereswithnormalcellphysiologyandreducesgrowth,the bination-mediatedrepair,whichcanrescuestalledreplica-
rare mutants of the clone can be positively selected for tioninalesserror-pronemanner[42].
and can rapidly become dominant in the culture. This Weshowherethatthedisablingofstress-inducedmuta-
phenomenonbecameapparenttousinanearlierattempt genesismechanismsfurtherincreasesthegeneticstability
toclone theVP60gene ofrabbithemorrhagic virus[18]. of MDS42, a reduced-genome E. coli strain lacking all
VP60 fused to a cholera toxin component proved to be mobilegeneticelements. Weofferproofofthebeneficial
toxictothecell.Inactivationoftherecombinantgenedue effectsoftheresultingstrainasacloninghostinthestable
to IS element-transposition and insertion into the toxic maintenance ofa toxic gene.The improved strainshows
gene,followedbyrapidselectionofthemutants,resulted promisingpotentialasacellularchassisformolecularand
in only aberrant clones in normal E. coli cloning hosts. syntheticbiologicalapplications.
Using the MDS42 host cell free of all IS elements, the
recombinantgenecouldbeclonedinitsintactform. Results
Here we wish to expand this work by making further The absence of error-prone polymerasesreduces the
improvementsonthegeneticstabilityofMDS42.Beyond spontaneous mutation rate
thepreviouseliminationofallISinsertionevents,wedis- TheSOS-inducedminor(error-prone)DNA-polymerases
abled other mutation-generatingpathwaysof the host in aremajorfactorsingeneratingpointmutations.Inapub-
ordertoimprovetoleranceandfidelity.RemovalofISele- lishedattempt,preventionofderepressionofSOSregulon
ments from the host genome eliminated a major, some- geneswasaccomplishedbyintroducingamutationinthe
times dominant [26] form of mutation generation. primary regulator LexA, disabling the autocatalytic clea-
However, in many cases, toxic clones are inactivated by vage process involved in the regulation [43]. The muta-
pointmutationsordeletions.Adetailedanalysisofclones tion-decreasing effect ofsuch a LexA variant was shown
ofhepatitisCvirusgenesshowedthatthecloningproce- topreventE.colicellsfromdevelopingantibioticresistant
dure in E. coli resulted exclusively in defective, non- topoisomerase mutations [44]. Alternatively, direct dele-
expressing clones due to the selection of point mutants tion of all three SOS-inducible DNA polymerase genes
(either frameshifts or stop codons) [24]. Selection of could reduce the mutation rate. Since these polymerases
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might contribute to mutation-generation even in the MDS42polBdinB (MDS42pd) and the triple deletion
absenceofstress,and,moreover,theycanbeactivatedby strainMDS42polBdinBumuDC(MDS42pdu).Compared
stress-inducedpathwaysotherthantheSOSresponse[45] to their parent MDS42, these strains showed a close to
(Figure1),weoptedforthescarlessremovalofthegenes 50% decrease in spontaneous mutation rate (8.2*10-8
encoding PolII(polB), PolIV (dinB), and PolV (umuDC). mutation/cell/generation decreased to 4.34*10-8 and
Forcomparison, LexAandRecAmutants [46], unable to 4.45*10-8,respectively).Thedifferenceobservedbetween
inducetheSOSpathway,werealsoanalyzed. wild-type MG1655 and MDS42 is due to the absence of
The genescodingforthethreeerror-proneDNApoly- insertionevents[18].
merases(polB,dinB,umuDC)weredeletedfromthegen- To verify that the absence of the error-prone DNA
ome of MDS42 in a scarless manner using a suicide polymerases has no adverse effect on fitness, growth
plasmid-based method [47]. Gene deletions were made rates of the different strains were measured in MOPS
individually and also joinedin all possible combinations. minimal medium. 14 parallel cultures originating from
The spontaneous mutation rate of each strain was then 14 individual colonies for each strain were picked and
determinedusingaD-cycloserineresistanceassay,detect- growninaBioscreenCinstrument(Figure3).Wefound
ingalltypesofmutationsinthecycAgene[48]. that none ofthe deletions had a significant effect on fit-
The deletion of each error-prone polymerase gene by nessinMOPSminimalmedium,evenwhencombinedin
itself results in at least a 20% decrease in mutation rate the triple deletion strain MDS42pdu. This strain was
(Figure2)measuredwiththismethod.Whencombining thenchosenforfurtheranalysis.
the different deletions, the mutation rate decreased Asanadditionalmeasureoffitness,weanalyzedthesur-
further, with the lowest mutation rates being that of vivalrateofMDS42pduandMDS42inlong-termstationary
Stringent response
(p)ppGpp, DskA
Polyphosphate-mediated
nutrient-limitation response
General stress response
PolyP, Ppk, Ppx RpoS
Mutagenesis in aging
colonies
SOS response polB, dinB, umuDC
RecA,LexA
Antibiotic-induced
mutagenesis Heat-shock response
quinolones, β-lactams
GroE
Figure1Inducersoftheerror-pronepolymerasegenes.Besidesthewell-documentedSOSresponse-mediatedpathway,error-proneDNA
polymerasegenescanbeactivatedbyseveraldifferentroutes.Mainpathwaysareindicatedintheboxes,withthemaininteractingpartners
indicatedbeloweach.(Adaptedfrom[45]).
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1.6E-07
1.4E-07 ***
)
n
o
ati 1.2E-07
r
ee
aten 1.0E-07
rg
on ne/ 8.0E-08 ** * ** *** *** *** ***
atige
utn/ 6.0E-08
Mo
ati 4.0E-08
ut
m
( 2.0E-08
0.0E+00
5 2 B B C d u u u
M G165 M DS4 DS42din DS42pol 42um uD M DS42p M DS42p M DS42d M DS42pd
M M S
D
M
Figure2Spontaneousmutationratesoferror-pronepolymerasedeletionmutantandcontrolstrains.Mutationratesweredetermined
bythefluctuationanalysisofD-cycloserine-resistantmutants[48].Genenamesareindicatedforthesingle-deletionstrains,whileMDS42pd,
MDS42pu,MDS42du,andMDS42pdudenoteMDS42polBdinB,MDS42polBumuDC,MDS42dinBumuDC,andMDS42polBdinBumuDC,respectively.
Errorbarsrepresent95%confidenceintervalsfortheaverageof4independentmeasurements.ANOVArevealedasignificanceofp<0.005.
Pairwiset-testswereconductedforeachstraincomparedtotheMDS42strain,*indicatesasignificanceofp<0.05,**indicatesasignificance
ofp<0.01,***indicatesasignificanceofp<0.001.
phase (Additional file 1). No significant difference was Inactivation of the regulators of the SOS response does
observedbetweenthetwostrainsoveraperiodof7days. not lower the spontaneous mutation rate
Furthermore, when the two strains were additionally To see whether the inactivation of the whole SOS
stressedbyexpressingamoderatelytoxicproteinfromthe response via regulator mutants would have the same
pSin32plasmid(discussedlater),thesurvivalratesinsta- effects on the spontaneous mutation rate as elimination
tionaryphasewerenotsignificantlydifferenteither. of the error-prone DNA polymerases, MDS42recA and
n) 80
mi 70
( 60

e 50
m
40
ti 30
g
n 20
li 10
b
u 0
o
d M G1655 M D S42M D S42polBM D S42diMnDBS42u m uD C M D S42pd M D S42pu M D S42duM D S42pduM D S42r4e2cleAxA(S119A)
S
D
M
Figure3Doublingtimesofstrainsusedinthestudy.DoublingtimesweremeasuredinMOPSminimalmediumat37°Cinmicrotiterplates
(seemethods).Errorbarsrepresent95%confidenceintervalsfortheaverageof14independentmeasurements.ANOVArevealedasignificance
ofp<0.005.Pairwiset-testswereconductedforeachstraincomparedtotheMDS42strain,noneofthestrainsshowedasignificantdifference.
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MDS42lexA(S119A) were constructed. None of these effect of overproduction on mutation rates, genes for
modifications had an adverse effect on the overall fitness either non-toxic GFP, or the toxic small, leucine-rich
of the strains (Figure 3). Spontaneous mutation rates of hydrophobic protein ORF238 [53] were cloned on plas-
these SOS-disabled strains were analyzed by the D- midsasinducibleconstructscontrolledbyaT7promoter.
cycloserine resistance fluctuation assay. Neither strain Toexpressthem, T7 RNApolymerase encoding variants
showed a significant decrease in spontaneous mutation ofthestudiedstrainswereconstructed.Fitnessmeasure-
rate when compared to MDS42 (Figure 4). ments of these modified strains revealed no significant
decreasecomparedtoMDS42(Additionalfile2).Inaddi-
MDS42pdu is genetically stable under stress conditions tiontoMDS42pduanditsparentMDS42,thewidelyused
Duetothestress-inducednatureoftheerror-proneDNA proteinproductionstrainBL21(DE3), thewild-typeK-12
polymerases,itwasexpectedthatthedifferenceinmuta- MG1655,andalsothetwodifferentSOS-inactivatedvar-
tion rates of the polymerase-free and the parent strain iants of MDS42 (MDS42recA and MDS42lexA(S119A))
would be even more pronounced under stressful condi- weretested(Figure4).
tions. Mutation rates were therefore measured under Resultsshowedthat,withtheexception ofMDS42recA
stressful conditions, includingthe applicationofan anti- and MDS42pdu, the various stresses generally increased
biotic agent (mitomycin-C), overproduction of benign the mutation rate. Overproduction of the toxic ORF238
GreenFluorescentProtein(GFP)[49,50],andoverproduc- proteinhadthelargesteffect:a>5-foldincreaseinmuta-
tionofatoxicprotein(ORF238)[20]. tion rate was measured. Sub-inhibitory concentration of
Mitomycin-C, a DNA cross-linking agent that causes mitomycin-Ccauseda>2-3-foldincreaseinthemutation
lesionsindouble-strandedDNA[51],directlyactivatesthe rate (BL21(DE3) and MDS42recA were unable to grow
SOSresponse,leadingtotheup-regulationoferror-prone undertheseconditions).TheoverproductionofGFPhada
DNApolymeraseenzymes.Asub-inhibitoryconcentration minoreffect,a1.5to2-foldincreaseinmutationrate.
(0.1 μg/ml) of mitomycin-C was used to stress the cells In contrast, no significant increase in mutation rate in
andanalyzetheeffectonmutationrates.Proteinoverpro- thepresenceofanyofthestressorscouldbeseenineither
ductionimposesstressonthehostcell[52,53].Totestthe MDS42recA or MDS42pdu. (Interestingly, MDS42lexA
***
unstressed
7.0E-06
pET-GFP
6.0E-06
)
on 5.0E-06 * * pSG-ORF238
rati ** mitomycin
ee 4.0E-06
ation ratene/gen 11..20EE--0066 ***
Muton/g 8.0E-07 ***
utati 6.0E-07 ** ** ** **
m **
( 4.0E-07 ***
*
2.0E-07 ** *
0.0E+00
BL21(DE3) M G1655 M DS42 M DS42recA exA(S119A) M DS42pdu
2l
4
S
D
M
Figure4Mutationratesofvariousstrainsunderunstressedandstressfulconditions.StressconditionsincludeoverproductionofGFP,
overproductionofatoxicpeptidefrompSG-ORF238,andtreatmentwithmitomycin-C.AllmeasurementsweremadeusingthecycAfluctuation
assay,errorbarsrepresent95%confidenceintervalsfortheaverageof3independentmeasurements.BL21(DE3)andMDS42recAfailedtogrow
inthepresenceof0.1μg/mlmitomycin-C.ANOVArevealedasignificanceofp<0.0001.Pairwiset-testswereconductedforeachstrainundera
givenconditioncomparedtothecorrespondingMDS42pdustrain,*indicatesasignificanceofp<0.05,**indicatesasignificanceofp<0.01,
***indicatesasignificanceofp<0.001.
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(S119A)didnotfollowthisbehavior,thestrainshowedan rifampicinresistance assay (detecting point mutations in
increaseinmutationrateinresponsetoallofthestresses.) theessentialrpoBgene[54])wereconsistentwiththecycA
MDS42pdu can be characterized as the genetically most fluctuationassaydata(Additionalfile3).MDS42pduhada
stablestrain,displayingthelowestspontaneousmutation 2-fold lower spontaneous mutation frequency compared
rate and showing negligible response to stressful toMDS42.Inresponsetotheoverproductionofthetoxic
conditions. ORF238protein,aswellasinthepresenceofmitomycin-
It is also noteworthy that the commonly used protein C,the mutationrate of MDS42became significantly ele-
production strain BL21(DE3) showed a mutation rate vated, while the response of MDS42pdu was much less
almost twoordersofmagnitudehigherthanMDS42pdu. substantial.
To study what this difference could be attributed to, the
mutational spectra of BL21(DE3),MG1655, MDS42,and The error-prone polymerase-free strain provides
MDS42pduwerestudiedbyPCRanalysisofcycAincyclo- improved stability to a toxic protein-expressing plasmid
serine-resistant mutants (Figure 5). In MG1655, 74% of clone
themutationsprovedtobepointmutations,24%wereIS Inordertodemonstratethepracticaladvantageofwork-
insertions, and 2% were deletions. In contrast, in BL21 ing with a strain of higher genome stability, a plasmid-
(DE3),77%ofcycAmutationswereISinsertions.Although basedmutationscreenwasdesigned.PlasmidpSin32car-
the proportion of point mutations in BL21(DE3) was riesaninduciblecopyofsinI,codingfortheSinImethyl-
muchsmaller(74%inMG1655versus23%inBL21(DE3)), transferase of Salmonella enterica serovar Infantis. SinI
the actual rate of point mutations was also over 2-fold methylates the inner cytosines in DNA at GG(A/T)CC
higher in BL21(DE3) (2.28*10-7 compared to 9.2*10-8 in sites,producing5-methylcytosine,therebycreatingtargets
MG1655).NodeletionswerefoundamongthecycAalleles fortheMcrBCendonuclease,whichcleavesDNAcontain-
inBL21(DE3). ingmethylcytosine.AplasmidthatcarriesmethylatedSinI
ToconfirmthedataobtainedusingthecycAfluctuation sites(e.g.,pSin32,self-methylatedatits8SinIsites),there-
assay, mutation rates of MDS42 and MDS42pdu under fore cannot establish itself in a mcrBC+ host. To check
eachofthedifferentstressconditionswerealsomeasured what ratio of an induced pSin32 sample carries mutated
using a second assay. The data obtained using the sinI(notexpressingafunctionalSinI),theplasmidsample
1.0E-06
9.0E-07 IS elements
)
n
o 8.0E-07 Point mutations
ti
a
r 7.0E-07 Deletions
e e
t n
a e 6.0E-07
r g

n /
o e 5.0E-07
n
ati e
t g 4.0E-07
u /
M n
o
ti 3.0E-07
a
t
u 2.0E-07
m
( 1.0E-07
0.0E+00
) 5 2 u
3 5 4 d
E 6 S p
D 1 D 2
1( G M S4
L2 M D
B M
Figure5Comparisonofthemutationalspectraofvariousstrains.ThebargraphshowsthedistributionofcycAmutationtypes,detected
byPCRanalysis.Theshareofdeletions(toolowtobevisible)is1.6,2.4,and4.3%ofthetotalmutationsinMG1655,MDS42,andMDS42pdu,
respectively.NodeletionsweredetectedinBL21(DE3).
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is transformed in both MDS42 (McrBC-) and MG1655 mutation rate of MDS42-T7, the effect is weaker (at a
(McrBC+), and colony numbers are compared (see marginalsignificance)inMDS42pdu-T7(Figure6B),sup-
methods). portingthefindingsofthepreviousexperiments.
BL21(DE3)mcrBC, MDS42-T7, and MDS42pdu-T7 FollowingIPTG-induction,plasmidsamplesweretaken
were transformed with pSin32. (While MDS42-T7 and at various intervals. The fraction of the plasmid sample
MDS42pdu-T7areMcrBC-,BL21(DE3)hadtobespeci- thatcarriedsinI-disablingmutations(unmethylatedplas-
ficallydeletedformcrBC tobeabletohosttheplasmid.) mids) wasdetected by transforming theplasmidsamples
Incidentally,itwasfoundthat,uponinductionby IPTG, back intoMG1655(mcrBC+). Thetotalplasmidnumber
production of the SinI enzyme had a moderate growth- persamplewasdeterminedbysimultaneouslytransform-
inhibiting effect even in McrBC- strains (Figure 6A). ingthesamplesintoMDS42.Aftercorrectingeachvalue
Whilethismoderatetoxicityleadstoanelevationinthe withthetransformantnumberfromacontrolplasmidfor
A
1.6
1.4
1.2
0 1
4
5 IPTG induction uninduced
. 0.8
D
O. 0.6 induced
0.4
0.2
0
0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 6 4 2 0 8 6 4 2 0 8 6 4
1 2 3 4 4 5 6 7 8 8 9 0
1
minutes
B
1.2E-06
)
n
o uninduced
ati 1.0E-06
e er induced
at en 8.0E-07
r g
n e/
o n
ati ge 6.0E-07
ut n/
M o 4.0E-07
ati
ut
m 2.0E-07
(
0.0E+00
MDS42-T7 MDS42pdu-T7
Figure6ToxiceffectoftheproductionofSinI.(A)GrowthcurvesofMDS42-T7(anMcrBC-host)carryingpSin32withandwithoutIPTG
induction.DataareaveragesoftheO.D. valuesof25independentcolonieseach,measuredevery5minutesusingtheBioscreenC
540
automatedinstrument.(B)Mutationratesofuninducedandinduced(SinI-expressing)cells.MutationratesweremeasuredusingthecycA
fluctuationassay.Errorbarsrepresent95%confidenceintervalsfortheaverageof3independentmeasurements.ANOVArevealedasignificance
ofp<0.01.Pairwiset-testswereconductedfortheuninducedandinducedpairsresultinginasignificanceofp=0.077andp=0.067,
respectively.
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each set of electrocompetent cells, the ratio of plasmids time,SinI-inactivatingmutationsarose,whichthen,hav-
codingforfunctional/non-functional SinIwas calculated ingresumedtheir normal growthrate,quickly overgrew
(Figure7). the rest of the culture. In the low-mutation-rate
Surprisingly,96.7%ofthestarting(0-hour)plasmidsam- MDS42pdu-T7, SinI-inactivating mutations developed,
ple,originatingfromMDS42,couldnotbeestablishedin on average, over a longer time period. Growth curve
MG1655. This indicated that, even in a host lacking T7 measurementsof50independentcoloniesofMDS42-T7
polymerase,spurioustranscriptionofsinIhadresultedin andMDS42pdu-T7,allcarryingthepSin32plasmid,sup-
SinI expression, and consequently, methylation of SinI port this notion. An O.D. value of 0.7 was used as a
540
sites.ThemethylatedstatusoftheSinIsitesintheoriginal cutoff to indicate that a culture had overcome the
plasmidsample wasconfirmed by their uncleavability by growth-hindering effect of the induced plasmid. The
SinI(datanotshown). averagetimetaken forMDS42pdu-T7toreachthislevel
Differences regarding clone stability in the different of density was significantly longer than for MDS42-T7
strains became evident after IPTG-induction of SinI (727.8 and 571.8 minutes, respectively; P < 0.005, two-
expression. Thirty-six hours after transformation (28 tailed,unpairedttest).
hoursafterIPTG-induction),51.7%ofpSin32harboredin To verify that mutations had indeed takenplace in the
BL21(DE3)mcrBC cellscarried mutations preventing the plasmidsthatallowedforgrowthinMcrBC+cells,thesinI
production of active SinI. This value was significantly regionof 8different plasmid samples (taken from viable,
lower in MDS42-T7 (25.8%, p < 0.005 with a two-tailed, pSin32-transformed MG1655 colonies) were sequenced
unpaired t-test). In MDS42pdu-T7, the fraction of (Additionalfile4).Insevenoutoftheeightcases,aframe-
mutated pSin32 plasmids was even lower (8.2%, p < shiftmutationhadoccurredinsinI,resultinginanewstop
0.005).Thenon-methylatedstatusoftheSinIsitesonthe codonwithinthegene.TheeighthcasedisplayedanAto
plasmids carrying a mutated sinI gene was confirmed by C transversion, resulting in the N255T mutation of the
theircleavabilitybySinI(datanotshown). protein. Six out of the seven new stop codons caused by
It seemed evident, that accumulation of mutant plas- theframeshiftswerelocatedwithinthefirst125bpofthe
mids in BL21(DE3)mcrBC and MDS42-T7 was due to a gene.
combined effect of stress-induced mutagenesis and
growth inhibition by the SinI-expressing plasmid. Pro- Discussion
duction of the enzyme elevated mutation rates and One of the major challenges that synthetic biology must
reduced growth. In these slow-growing cultures, over face is the intrinsic variability and genetic instability of
800
s
d 700
BL21(DE3)mcrBC
mi
s 600
a
MDS42-T7
pl
500
0
0 MDS42pdu-T7
0 IPTG induction
1 400

r
e
p 300

s
nt 200
a
t
u
m 100
0
0 10 20 30 40
hours post transformation
Figure7AccumulationofplasmidswithmutatedsinIinvarioushosts.SinImethyltransferasewasexpressedfrompSin32.Plasmidswere
isolatedatvariousintervalsandscreened(bytransformationinMcrBC+andMcrBC-hosts)formutationsresultinginlossoffunctionofthe
enzyme.Errorbarsrepresent95%confidenceintervalsfortheaverageof3independentmeasurementsofmutantplasmidratios.ANOVA
revealedasignificanceofp<0.005.Pairwiset-testsofeachMDS42pdu-T7sampleweredonewiththecorrespondingMDS42-T7andBL21(DE3)
mcrBCsample,respectively.Startingfrom10hours,allMDS42pdu-T7samplesdifferedsignificantlyfromtheMDS42-T7(p<0.01)orBL21(DE3)
mcrBC(p<0.005)samples.
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living organisms [55]. As the complexity of synthetic effect of individual and combined error-prone polymer-
systems increase, the emergence and selection of new ase deletions on the mutation rate, under either
features will become a significant impediment in achiev- unstressed or stressed conditions. We determined that,
ing robust and stable performance. Improving the under unstressed conditions, elimination of each error-
genetic stability of the host organism, or synthetic biolo- prone polymerase by itself significantly decreased the
gical chassis is therefore a validated goal. spontaneousmutationrate.Theeffectofcombiningdele-
Previously,wehavedemonstratedthatgenomestabiliza- tionsΔpolBand ΔdinBwasadditive,indicatinganinde-
tionbyeliminationofmobilegeneticelementshasadvan- pendentmodeofactionforthesepolymerases.However,
tages in certain cloning applications [18,20]. To achieve ΔumuDC generatednoadditionaldecrease ofthe muta-
additionalgeneticstabilizationofthehost,wetargetedand tion rate when any of the other two error prone poly-
eliminated error-proneDNA polymerases(PolII,PolIV, merases was missing, possibly marking an interaction
Pol V), major sources of frameshift and point mutations. among the genes or their products. This phenomenon
Possiblealternativeapproachestolowerthemutationrate has been described previously regarding ΔdinB and
include the introductionof a so-called antimutator dnaE ΔumuDC, the nature of their putative interaction, how-
allele or upstream inactivation of the SOS response by everisnotyetknown[61].
introducinga recA orlexAmutation.Previousstudieson Asexpected,themostdramaticdifferencesinmutation
theeffectofantimutatorsfounda5to30foldreductionin rate between MDS42 and MDS42pdu were observed
mutation rate [56]. It was later shown that the mode of undervariousstressconditions.Asub-inhibitoryconcen-
action of these DnaE antimutators was a more effective trationoftheSOSresponse-activatingmitomycin-C,over-
abilitytoexcludeerror-proneDNApolymerasesatsitesof production ofeitherthenon-toxicGFP proteinorof the
DNA synthesis during DSB-repair associated stress- highlytoxicORF238hydrophobicproteinallsignificantly
inducedmutagenesis[57],suggestingthattheelimination increased the mutation rate of MDS42. The values for
oftheseenzymeswouldreproducetheantimutatoreffect. MDS42pduremainedstableunderthesameconditions.It
Testing the other alternative approaches, MDS42recA, isalsonoteworthy,thatamongthestrainstested,thecom-
comparedtoMDS42pdu,showedamuchlesspronounced monlyusedproductionstrainBL21(DE3)showednotjust
reduction in spontaneous mutation rate. Furthermore, thehighestspontaneousmutationrate,butalsothehigh-
owingtothecentralroleoftheRecAenzymeincellphy- est increase in mutation rates in response to the various
siology [58], unwanted pleiotropic effects might arise stresses. A difference of almost two orders of magnitude
withinthecell,manifested,amongothers,insensitivityto was observed between the mutation rate of BL21(DE3)
mitomycin-C,presumablyduetoinsufficientrepairactivity. and MDS42pdu when overproducing the toxic ORF238
MDS42lexA(S119A),carryinganon-cleavableformofthe protein.ThiselevatedrateofmutationinBL21(DE3)can
LexA repressor, did not significantly lower the mutation bemostlyattributedtoanincreasedrateofISinsertions.
rate undertheconditionsapplied. The smalleffectofthe AclearpracticaladvantageofworkingwithMDS42pdu
recAand lexAmutationscanbe explainedby therelative was demonstrated in a protein production experiment,
SOS-independence of the error-prone polymerases: the where the SinI methyltransferase was expressed from an
enzymes are present even inunstressed cells, andcan be inducibleplasmidconstruct.SinI,producing5-methylcy-
up-regulated by a number of (not just SOS) stress tosines is toxic to cells that carry the McrBC endonu-
responses(Figure1)[45]. clease. Even in cells lacking McrBC, we observed a
Several studieshave beenmadeonthe effectsoferror- negative effect on cell fitness. When SinI was produced,
pronepolymerasesonmutationratesusingvariousstrains wefoundthatthesinIgene,carriedonaplasmid,acquired
ofE.coliandvariousmethodsofmeasurement.Support- loss-of-functionmutationsapproximatelythreetimesless
ing our findings is the observation that deletion of dinB frequently in MDS42pdu than in MDS42, and over five
significantlydecreasesthemutationratesforbothframe- timeslessfrequently thaninBL21(DE3)mcrBC.Remark-
shift andbase substitution mutations ina Lac+ reversion ably, after only 16 hours of production in BL21(DE3)
system, aswellasinarifampicinresistanceassay[59].In mcrBC,almosthalfofallsinI genesencodedontheplas-
another study, the lackofPolVcaused a decrease inthe midshadsufferedadisablingmutation.
numberofArg+ growthdependentrevertants[60]. Later Clearly,theunexpectedlyhighratioofmutatedclonesin
studiesalsoshowedthatpost-exposuremutationratesin theSinI-expressingculturecannotbeexplained solelyby
the presence of ciprofloxacin were markedly reduced thestress-inducedmutagenesis,theoverallmutationrate
when all three inducible polymerases were separately ofwhichbeingtoolowinabsolutevalues(intheorderof
eliminated[44]. 10-6mutations/gene/generation)tocausesuchadramatic
Here, using a D-cycloserine resistance-based fluctua- effect.Rather,thephenomenonisinlargepartduetothe
tionanalysis[48],confirmedinsomecasesby arifampi- growthinhibitoryeffectoftheplasmid carryingthetoxic
cin resistance assay as well, we carefully quantified the gene.Thechainofeventsisthefollowing:Uponinduction
Csörgőetal.MicrobialCellFactories2012,11:11 Page10of13
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of expressionof the toxic gene, growthrate ofthe cell is shown in Table 1. All deletions and modifications were
reduced. Atthesame time, mutationrate isincreasedby verified by PCR and sequencing using flanking primers.
thestress.Onceamutant,notproducingthetoxicprotein, T7polymerase-expressingstrainvariants(MDS42-T7and
arisesintheplasmidpopulation,thecellharboringitcan MDS42pdu-T7)wereconstructedbyreplacingtheyahA-
resume normalgrowthandbecomedominantinthecul- yaiLgenomicregionwithanIPTG-induciblelacoperator/
ture. In low-mutation-rate MDS42pdu, appearance of T7polymerasecassette.PlasmidpSG-ORF238isanIPTG-
such mutants is delayed, and the cells can produce the inducible,pSG1144-basedconstructcapableofoverprodu-
functionaltoxicproteinforanextendedperiodoftime. cingthehydrophobicORF238toxicprotein[20].Plasmid
Calculating the precise advantage of MDS42pdu over pET-GFPisanIPTG-induciblepET-basedconstructcar-
the parental MDS42 or the commonly used production rying the gfp gene. Plasmid pSin32 is a pET3-His based
strainBL21(DE3)canbechallenging,duetothestochastic construct [64] carrying an inducible, N-terminal His-
natureofmutagenesis,aswellasthelackofexactdataon tagged sinI methyltransferase gene cloned into the XhoI
thefitnesscostofoverproduction.Nevertheless,itisclear site of the original vector [65]. Plasmids were prepared
thatthemoreseverethestressofoverproducingaproduct using IS-free MDS42 host. LB and LB-agar plates were
is(resultinginanelevatedmutationrateandgrowthinhi- usedforroutinecultivation[66].Thefollowingfinalanti-
bition),thegreatertheadvantageofthestabilizedhost. bioticconcentrationswereused:50μg/mlampicillin(Ap),
25 μg/ml chloramphenicol (Cm), 25 μg/ml kanamycin
Conclusions (Kan),100μg/mlrifampicin(Rp),and4μg/mlD-cycloser-
Themutationandinactivationofengineeredgeneticcon- ine (Cyc). For the cycA fluctuation assays, minimal salt
structs within a host cell is an overlooked problem that (MS)medium,supplementedwith0.2%glucose,wasused
mayhaveseriousdetrimentaleffectsonthesuccessofany [26].GrowthmeasurementsweremadeinMOPSminimal
syntheticbiological,molecularbiologicalorbiotechnologi- definedmedium(ScarabGenomics,Madison,Wisconsin,
calprocess.Ageneproductimposingametabolicburden USA). Additional file 5 lists the oligonucleotides used in
orbeingtoxictothehostdrivesanevolutionaryforcethat theexperiments.
selectsforanymutantsthatalleviatethegrowth-inhibiting
effect.Ahostcellorchassiswithenhancedgeneticstabi- Growth measurements
lity is advantageous in the stable maintenance of these Growth properties were evaluated in liquid medium in
constructs.Byeliminatingtheerror-proneDNApolymer- 100-well Honeycomb 2 plates (Oy Growth Curves Ab,
ase enzymes from the reduced-genome MDS42 strain Helsinki,Finland). Growthcurves were measured by fol-
lackingallgenomicISelements,wehavefurtherstabilized lowingtheopticaldensities(O.D.)at540nmineachwell
a strain that already showed clear advantages in cloning using the Bioscreen C Automated Microbiology Growth
applications.TheresultingMDS42pdustrainhadasignifi- Analysis System (Oy Growth Curves Ab, Helsinki, Fin-
cantstabilizingeffectonatoxicproteinexpressionclone. land). Fourteenindividual colonies fromeach straintype
Thishigh-fidelitystrain,producingdecreasedgeneticvar- were resuspended and grown in parallel to saturation at
iation in the culture, might also prove useful in applica- 37°CinMOPSmedium.Fromthesaturatedcultures,2μl
tionsrangingfromtheproductionofDNAtherapeuticsto wastransferredto198μlfreshMOPSmediumandgrown
long-termcontinuousfermentationprocesses. tosaturationinindividualwellsat37°Cusingcontinuous
shaking. The median O.D. value of the fourteen parallel
Methods culturescorresponding toeach strainwascalculatedand
Strains, plasmids, media, and oligonucleotide primers plotted for each time point. Doubling times were calcu-
Most of the strains used in this study were constructed latedfromthesegrowthcurvesusingpreviouslydescribed
fromE.coliMDS42[18],areducedgenomestrainderived methods [67]. For analysis of growth in the presence of
from K-12 MG1655 [62]. Individual, scarless deletions the SinI methyltransferase, 50 resuspended individual
(polB,dinB,umuDC,mcrBC)orallelereplacements(lexA)
were constructed by a suicide plasmid-based method.
Table 1Genomiccoordinates ofeach scarless deletion
Standard steps and plasmids (pST76-A, pSTKST) of the
procedurehavebeendescribed[47].DeletionofrecAwas Strain Coordinatesofdeletion
carriedoutwithasimilarstrategy,usingplasmidpSG2857 MDS42recA 2820783-2821861
(ScarabGenomics,Madison,Wisconsin,USA).Individual MDS42polB 63505-65764
deletionswerecombinedbyP1phagetransduction[63]of MDS42dinB 250904-251966
themarked(with integratedsuicide-plasmids)intermedi- MDS42umuDC 1230261-1231298
atesofthedeletionconstructs, followedbyendonuclease
BL21(DE3)mcrBC 4408095-4515388
cleavage-stimulated out-recombination and loss of the
ThedataregardingtheMDS42-basedstrainsaregivenusingMG1655
plasmid. Co-ordinates of the individual deletions are coordinates.