DTIC ADA574806: Analysis of Redox Responses During TNT Transformation by Clostridium acetobutylicum ATCC 824 and Mutants Exhibiting Altered Metabolism

0.57 MB·English

by  Defense Technical Information Center

REPORT DOCUMENTATION PAGE Form Approved OMB NO. 0704-0188
The public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions,
searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments
regarding this burden estimate or any other aspect of this collection of information, including suggesstions for reducing this burden, to Washington
Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA, 22202-4302.
Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any oenalty for failing to comply with a collection of
information if it does not display a currently valid OMB control number.
PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS.
1. REPORT DATE (DD-MM-YYYY) 2. REPORT TYPE 3. DATES COVERED (From - To)
New Reprint -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Analysis of redox responses during TNT transformation by W911NF-09-1-0119
Clostridium acetobutylicum ATCC 824 and mutants exhibiting
5b. GRANT NUMBER
altered metabolism
5c. PROGRAM ELEMENT NUMBER
611103
6. AUTHORS 5d. PROJECT NUMBER
Matthew Servinsky, Xianpeng Cai, James Kiel, Christian Sund, George
N. Bennett 5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAMES AND ADDRESSES 8. PERFORMING ORGANIZATION REPORT
NUMBER
William Marsh Rice University
Office of Sponsored Research
William Marsh Rice University
Houston, TX 77005 -
9. SPONSORING/MONITORING AGENCY NAME(S) AND 10. SPONSOR/MONITOR'S ACRONYM(S)
ADDRESS(ES) ARO
U.S. Army Research Office 11. SPONSOR/MONITOR'S REPORT
P.O. Box 12211 NUMBER(S)
Research Triangle Park, NC 27709-2211 55744-LS.1
12. DISTRIBUTION AVAILIBILITY STATEMENT
Approved for public release; distribution is unlimited.
13. SUPPLEMENTARY NOTES
The views, opinions and/or findings contained in this report are those of the author(s) and should not contrued as an official Department
of the Army position, policy or decision, unless so designated by other documentation.
14. ABSTRACT
The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been
examined to analyze the maximal rate of initial transformation, determine the effects of metabolic mutations of the
host on transformation rate and to assess the cell metabolic changes brought about during TNT transfromation.
Little difference in the maximal rate of TNT degradation in early acid phase cultures was found between the
parental ATCC824 strain and strains altered in the acid forming pathways (phosphotransacetylase, or butyrate
15. SUBJECT TERMS
electron mediators, metabolism, redox, anaerobe, hydrogenase
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 15. NUMBER 19a. NAME OF RESPONSIBLE PERSON
a. REPORT b. ABSTRACT c. THIS PAGE ABSTRACT OF PAGES George Bennett
UU UU UU UU 19b. TELEPHONE NUMBER
713-348-4920
Standard Form 298 (Rev 8/98)
Prescribed by ANSI Std. Z39.18
Report Title
Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants
exhibiting altered metabolism
ABSTRACT
The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been
examined to analyze the maximal rate of initial transformation, determine the effects of metabolic mutations of the
host on transformation rate and to assess the cell metabolic changes brought about during TNT transfromation. Little
difference in the maximal rate of TNT degradation in early acid phase cultures was found between the parental
ATCC824 strain and strains altered in the acid forming pathways (phosphotransacetylase, or butyrate kinase) or in a
high solvent producing strain (mutant B). This result is in agreement with the previous findings of a similar
degradation rate in a degenerate strain, (M5) that had lost the ability to produce solvent. A series of antisense
constructs were made that reduced the expression of hydA, encoding the Fe-hydrogenase, or hydE and hydF, genes
encoding hydrogenase maturating proteins. While the antisense hydA strain had only ~30% of the activity of wild
type, the antisense hydE strain exhibited a TNT degradation rate around 70% that of the parent. Over expression of
hydA modestly increased the TNT degradation rate in acid phase cells, suggesting the amount of reductant flowing
into hydrogenase rather than the hydrogenase level itself was a limiting factor in many situations. The redox
potential, hydrogen evolution and organic acid metabolites produced during rapid TNT transformation in early log
phase cultures were measured. The redox potential of the acid producing culture decreased from -370 to -200 mV
immediately after addition of TNT and the hydrogen evolution rate decreased, lowering the hydrogen to carbon
dioxide ratio from 1.4 to around 1.1 for 15 minutes. During the time of TNT transformation, the treated acidogenic
cells produced less acetate and more butyrate. The results show that during TNT transformation the cells shift
metabolism away from hydrogen formation to reduction of TNT and the resulting effects on cell redox cofactors
generate a higher proportion of butyrate.
REPORT DOCUMENTATION PAGE (SF298)
(Continuation Sheet)
Continuation for Block 13
ARO Report Number 55744.1-LS
Analysis of redox responses during TNT transfo ...
Block 13: Supplementary Note
© 2012 . Published in Applied Microbiology and Biotechnology, Vol. Ed. 0 (2012), (Ed. ). DoD Components reserve a
royalty-free, nonexclusive and irrevocable right to reproduce, publish, or otherwise use the work for Federal purposes, and to
authroize others to do so (DODGARS §32.36). The views, opinions and/or findings contained in this report are those of the
author(s) and should not be construed as an official Department of the Army position, policy or decision, unless so designated by
other documentation.
Approved for public release; distribution is unlimited.
ApplMicrobiolBiotechnol
DOI10.1007/s00253-012-4253-3
ENVIRONMENTAL BIOTECHNOLOGY
Analysis of redox responses during TNT transformation
Clostridium acetobutylicum
by ATCC 824 and mutants
exhibiting altered metabolism
Xianpeng Cai&Matthew Servinsky&James Kiel&
Christian Sund&George N.Bennett
Received:24April2012/Revised:14June2012/Accepted:15June2012
#Springer-Verlag2012
Abstract The transformation of trinitrotoluene (TNT) by ratearound70%thatoftheparent.OverexpressionofhydA
several mutant strains of Clostridium acetobutylicum has modestly increased the TNT degradation rate in acid phase
been examined to analyze the maximal rate of initial cells,suggestingtheamountofreductantflowingintohydrog-
transformation, determine the effects of metabolic muta- enase rather than the hydrogenase level itself was a limiting
tions of the host on transformation rate, and to assess factorinmanysituations.Theredoxpotential,hydrogenevo-
the cell metabolic changes brought about during TNT lution, and organic acid metabolites produced during rapid
transformation. Little difference in the maximal rate of TNT transformation in early log phase cultures were mea-
TNT degradation in early acid phase cultures was found sured. The redox potential of the acid-producing culture de-
between the parental ATCC 824 strain and strains al- creasedfrom−370to−200mVimmediatelyafteradditionof
tered in the acid forming pathways (phosphotransacety- TNTandthehydrogenevolutionratedecreased,loweringthe
lase, or butyrate kinase) or in a high-solvent-producing hydrogen to carbon dioxide ratio from 1.4 to around 1.1 for
strain (mutant B). This result is in agreement with the 15 min. During the time of TNT transformation, the treated
previous findings of a similar degradation rate in a acidogeniccellsproducedlessacetateandmorebutyrate.The
degenerate strain (M5) that had lost the ability to pro- results show that during TNT transformation, the cells shift
duce solvent. A series of antisense constructs were metabolism away from hydrogen formation to reduction of
made that reduced the expression of hydA, encoding TNTandtheresultingeffectsoncellredoxcofactorsgenerate
the Fe-hydrogenase, or hydE and hydF, genes encoding ahigherproportionofbutyrate.
hydrogenase maturating proteins. While the antisense
hydA strain had only ∼30 % of the activity of wild Keywords Electronmediators .Metabolism .Redox .
type, the antisense hydE strain exhibited a TNT degradation Anaerobe .Hydrogenase
G.N.Bennett(*) Introduction
DepartmentofBiochemistry&CellBiology,RiceUniversity,
MS140,6100MainStreet, Trinitrotoluene(TNT)isacommonnitroaromaticcompound
Houston,TX77005-1892,USA
contaminating the surface and subsurface soil of many mili-
e-mail:[email protected]
tary sites, as a result of the accumulation of residues from
: :
M.Servinsky J.Kiel C.Sund explosives (Spain 1995). Data describing TNT presence in
USArmyResearchLaboratory, militarysiteshavebeencompiledinseveralreviews(Ahmad
2800PowderMillRoad,
andHughes2000;Spain1995).Thetoxicityandmutagenicity
Adelphi,MD20783,USA
ofTNThasbeenwidelyknownfordecades(Wonetal.1976),
PresentAddress: andinformationonthetoxicityandmutagenicityofsynthetic
X.Cai andbiodegradativeintermediatesalsohasbeenconsideredin
ConagenInc,
efforts to remove toxicity and study the transformation of
Suite238,1005NorthWarsonRoad,
these compounds (Siciliano et al. 2000; Padda et al. 2000).
St.Louis,MO63132,USA
[email protected] Duetothehighcostofincinerationtreatmentsforsuchsoils,
ApplMicrobiolBiotechnol
bioremediation, especially by strains of anaerobic bacteria All medium components for clostridium culture were
such as Clostridium, has become a promising and cost- obtained from Difco (Detroit, MI) or Sigma-Aldrich. All
effective decontamination measure (Tan et al. 1992; Shin RestrictionenzymeswereobtainedfromNewEnglandBiol-
andCrawford1995;Daunetal.1998).Theabilitytomonitor abs, Inc. (Beverly, MA). pCR2.1-TOPO vector from Invi-
thesoilcommunitiesandtheongoingmetabolismisimportant trogen Corporation (Carlsbad, CA) was used for PCR
in estimating the course of progress in decontamination of product cloning. Automated DNA sequencing was per-
hazardouscompounds(FrischeandHoper2003;Eyersetal. formedbyLoneStarautomatedDNAsequencing(LoneStar
2006;Wuetal.2008). Laboratories Inc., Houston, TX).
TheprevalenceofClostridiuminsoilmakesthestudyof
these organisms relevant for bioremediation studies, and
Bacterialstrains, plasmids, andgrowthconditions
various Clostridium species have been reported to degrade
TNT through alternative routes (Ahmad and Hughes 2000;
All bacteria strains andplasmids are listed in Table 1.
Spain1995;ZhangandBennett2005).Thiswidelyisolated Escherichiacoliculturesweregrownaerobicallyat37°C
species and related anaerobes are important contributors to in Luria-Bertani medium, C. acetobutylicum was grown
the degradation of a variety of compounds in the oxygen-
anaerobically at 37 °C in buffered clostridium growth me-
depleted environment of contaminated subsurface soil.
dium (CGM) in a Forma Scientific anaerobic chamber
Clostridium acetobutylicum is known to enzymatically re-
(Thermo Forma, Marietta, OH) as described previously
duce TNT primarily through the activity of a Fe-only hy- (Cai and Bennett 2011; Zhao et al. 2005). For E. coli
drogenase, which transfers electrons to TNT through an
recombinant strains, the medium was supplemented with
iron–sulfur center (Watrous et al. 2003; Kutty and Bennett ampicillin (100 μg/mL), chloramphenicol (35 μg/mL),
2006;AhmadandHughes2000;Hughesetal.1998b;Khan kanamycin (50 μg/mL), or erythromycin (200 μg/mL) as
et al. 1997). A more thorough understanding of the regula- appropriate. For C. acetobutylicum strains, erythromycin
tionofhydrogenaseactivityandtheredoxinputstohydrog- (40 μg/mL for solidified agar plate and 100 μg/mL for
enaseduringTNTreductionmayyieldbettertechniquesfor liquid medium) and thiamphenicol (25 μg/mL) were used
accentuating the degradation ofTNT. when necessary. For long-term storage, E. coli strains were
In this study, we compared TNT reduction in C. aceto- cultivated and stored as glycerol stocks at −80 °C. C. ace-
butylicum strains containing different genetic backgrounds. tobutylicum strains were stored as lyophilized stocks at
Since hydrogenase was found to be important in TNT deg- room temperature or glycerol stocks at −80 °C (Scotcher
radation, we investigated if strains that produced an altered
and Bennett 2005).
pattern of acids and solvents would have different abilities
to degrade TNTat various stages of growth. The measure-
Analysisoffermentation products
ment of culture parameters and metabolic products during
TNTdegradationledtothefindingthatatmaximalratesthe
Cell growth was monitored by A with a Beckman DU-
TNTreductionwasverycompetitivewithcellprocessesand 600
800 spectrophotometer. In a fermentor, cell growth was
accounted for a significant fraction of the available reduc-
monitored by a cell density sensor. The concentrations of
tant produced by glucose metabolism. The cell physiology
butanol, acetone, ethanol, butyrate, and acetate were deter-
interactions accompanying TNT degradation are discussed
mined using gas chromatography with a Hewlett-Packard
in relation to the distribution of the reduced metabolites
produced by Clostridium cultures. 5890 Series IIinstrument (Hewlett-Packard Company, Palo
Alto, CA) as described previously (Scotcher and Bennett
2005; Zhao et al. 2003, 2005). Glucose was measured by
HPLC as described (Wang etal. 2011).
Materials and methods
Chemicals and reagents TNTassay inC. acetobutylicum culture
2,4,6-trinitrotoluene, 2,6-dinitrotoluene, 2-amino-4,6- TNTconcentrationintheculturemediumwasfollowedbya
dinitrotoluene, and 4-amino-2,6-dinitrotoluene were pur- spectrometricprotocolmodifiedfromJenkinsetal.(Jenkins
chased from ChemService (Westechester, PA); p-nitro- andWalsh1992;Watrousetal.2003).Freshlymadesodium
toluene and 2,4-dinitrotoluene were purchased from sulfite solution (0.2 g/mL) in 50 mM at pH 8.0 Tris–HCl
Sigma-Aldrich (St. Louis, MO); 2-hydroxylamino-4,6- buffer reacts with TNTsolution to form a colored complex
dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, and with a maximum absorption at A that remains stable for
414
2,4-diamino-6-nitrotoluene were from SRI International hoursunderassayconditions.A readingsincreaselinear-
414
(Menlo Park, CA). lyup to a500-μMTNTconcentration.
ApplMicrobiolBiotechnol
Table1 Bacterialstrainsand
plasmids Strain/plasmid Description Reference/source
Strains
Clostridiumacetobutylicum
ATCC824 Wildtype ATCC
M5 pSOL1¯ Clarketal.(1989)
824(buk) Disruptionofbutyratekinasegenein824 Greenetal.(1996)
824(pta) Disruptionofphosphotransacetylasegenein824 Greenetal.(1996)
824(mutantB) DisruptionofSolRbypO1X Nairetal.(1999)
amcrAΔmcrBCmethylcytosine-
Escherichiacoli
specificrestrictionsystemabol-
ished,recA1homologousre- DH10β mcrAΔmcrBCrecA1Strr NEB
combinationabolished,Strr
Plasmids
streptomycinresistant,MLSr
pSOS94 ptbpromoter,AprMLSrColEl,andrepL Tummalaetal.
macrolide-lincosamide-
streptograminBresistant,repL (2003)
pIM13gram-positiveoriginof pSC12 Thlr,shuttlevector,ColEI,andrepL Zhaoetal.(2003)
replication,ColElgram- pDHKM Kmr,Φ3TImethyltransferaseinpDHK29 Zhaoetal.(2003)
negativeoriginofreplication,
pSOS-del ControlplasmidbasedonpSOS94bydeletion Thisstudy
Kmrkanamycinresistant,Apr
ofclostridiumstructuralgenes
ampicillinresistant,Strrstrepto- pSOS-as-hydA Antisensedown-regulationofhydAgene Thisstudy
mycinresistant,Thlrthiamphe-
nicolresistant,ATCCAmerican pSOS-as-hydE Antisensedown-regulationofhydEgene Thisstudy
TypeCultureCollection(Mana- pSOS-as-hydF Antisensedown-regulationofhydFgene Thisstudy
ssas,VA),NEBNewEngland pSOS-hydA OverexpressionofhydAgene Thisstudy
Biolabs(Beverly,MA)
Single C. acetobutylicum colonies from freshly grown chamber,andallsolutionswerepre-equilibratedinthecham-
plates were inoculated into buffered CGM medium with berfor24h.Oncethethreetimesampleswerecollectedand
appropriateantibioticsandincubatedovernightasseedcul- centrifugedat13,000rpmfor10min,theA absorbanceof
414
tures.Anappropriateamountfromseedculturewasusedfor eachsupernatantwasmeasured,andthedilutionfactorswere
thesubculturetoyieldastartingA of0.1.Theculturewas considered to calculate the specific TNT degradation rates.
600
incubatedat37°Candgrowth(A )measuredperiodically. Sincethetimeoftheinitialratemeasurementwasshortafter
600
TNTratewasassayedduringearly-exponentialphase(A , the dilution compared with a typical lag phase recovery of
600
0.3),mid-exponentialphase(A ,1.0),andstationaryphase growth rate, the measured rates are considered indicative of
600
(24-h culture with A , varied) by diluting the culture at themetabolismofthecellatthetimepointsused.
600
differenttimesintofreshmediumcontaining400μMTNT.
At different time points, 250 μL samples were taken and Plasmidconstructions andtransformation
immediatelyreactedwith750μLsodiumsulfitesolutionby
vortex; the samples were further centrifuged at 14,000 rpm PlasmidsfromE.coliwerepurifiedusingQIAprepMiniprep
for 10 min, and the supernatant was used for the TNT protocols (Qiagen Inc., Valencia, CA). Plasmids from C.
spectrophotometric assay. The TNT degradation rate μ acetobutylicumwerepurifiedaccordingtotheprotocol(Harris
max
fordifferentstrainswerecomparedbycalculatingtherateof etal.2002).GenomicDNAwaspurifiedfromC.acetobuty-
TNT decrease during thefirst40 min. licum using the Genomic DNA Purification Kit from Pure-
InordertodeterminetheeffectofculturedilutiononTNT gene (Gentra Systems, Minneapolis, MN). All commercial
degradationandconfirmthelinearrangeofthemeasurements, enzymesusedinthisstudy(Taqpolymerase,restrictionendo-
five C. acetobutylicum cultures were prepared to ultimately nucleases, calf intestinal phosphatase, T4 DNA ligase, and
yield×10,×20,×30,×40,and×50dilutions,respectively.For KlenowfragmentofDNApolymeraseI)wereusedaccording
each dilution sample, three subcultures were made. The ap- tothemanufacturers’recommendations.
propriateamountsofC.acetobutylicum(A ,0.3)andCGM/ E. coli transformation followed the Sambrook protocol
600
TNT mixture,for a total volumeof10mL, weretransferred (Sambrook et al. 1989). As to C. acetobutylicum transfor-
anaerobically into 15 mL culture tubes. The tubes were im- mation,plasmidDNAwasmethylatedbytheΦ3TImethyl-
mediatelyvortexed,anda0.25mLsampleofeachtubewas transferase to prevent restriction by the clostridial
addedto0.75mLassaybuffer.Thetubeswerelefttoincubate endonuclease Cac824I (Mermelstein and Papoutsakis
at 37 °C, and 0.25 mL samples were again taken at 20 and 1993).Thiswasachievedbytransformation oftherequired
40 min. All operations were conducted in the anaerobic plasmid into E. coli DH10β harboring vector pDHKM
ApplMicrobiolBiotechnol
(Zhao et al. 2003), carrying an active copy of the Φ3TI were centrifuged at 10,000 rpm for 10 min at 4 °C. The
methyltransferase gene. Electrotransformation of C. aceto- supernatant was decanted and the cell pellets were re-
butylicumwascarriedoutaccordingtoamodificationofthe suspended in 750 μL of a solution containing a 2:1 ratio of
protocol (Mermelstein and Papoutsakis 1993); positive RNAProtect(QiagenInc.Valencia,CA)toPBS.Afterincu-
transformantswereisolatedonagar-solidifiedCGMsupple- bation for 5 min at room temperature, the cell suspensions
mented with the appropriate antibiotic; and transformations were pelleted by centrifugation and stored at −80 °C. Cell
were also confirmed byplasmidDNA purification. pelletswerethawedonice,andthetotalRNAwasextracted
Oligonucleotide sequences used are listed inTable 2. For and purified using an RNeasy mini kit (Qiagen) with on
antisenseconstructs,pSOS94wasusedasthebackboneplas- column DNase I treatment according to the manufacturer’s
mid.ItwasdigestedwithBamHIandAvaIandligatedwith instructions.
thedigestedtargetantisensefragments(as-hydA,175bp;as- First-strand cDNA was synthesized from 0.15 μg total
hydE,244bp;andas-hydF,244bp)toformpSOS-as-hydA, RNAusinganIscriptcDNAsynthesiskit(Bio-Rad,Hercules,
pSOS-as-hydE, and pSOS-as-hydF. pSOS 94 digested by CA) per the manufacturer’s instructions. Real-timePCR pri-
BamH I and Ava I was treated and self-ligated to form the mers were designed using Primer3 software to amplify spe-
control plasmid pSOS-del. In order to overexpress the hydA cificproductsthatwerebetween100and170bpinsize.Each
gene,primershydA-FandhydA-Rwereusedtoamplifythe real-timePCRcontained12.5μLiQSYBRGreenSupermix
1,989 bp fragment from C. acetobutylicum 824 genomic (Bio-Rad), 200 nM of each primer, 1 μL diluted cDNA
DNA.Thisnewconstructwasfurtherconfirmedbysequenc- template, and water to bring the final reaction volume to
ingbeforebeingligatedtopSOS94digestedbyBamHIand 25 μL. Real-time PCR was performed in a CFX96 PCR
AvaItoyieldtheplasmidpSOS-hydA. machine (Bio-Rad) using the standard program: 98 °C for
2 min (1 cycle) then 98 °C for 2 s and 55 °C for 5 s (40
Real-time PCR quantification ofgene expression cycles). Fluorescence data were collected and melting-curve
inC. acetobutylicum analysiswasperformedfollowingamplificationtoensurethat
a single product was formed. The PCR efficiency for each
Cultures were grown to an A of 0.3, at which point the primersetwasdeterminedbyanalyzingthreereplicatesof2-
600
cultures were treated with rifampicin, chloramphenicol, and fold serial dilutions of cDNA. To determine relative expres-
phenylmethanesulfonyl fluoride at final concentrations of sionlevels,datawereanalyzedasdescribedbyPfaffl(2001)
30mg/L,50mg/L,and0.5mM,respectively.Cultureswere byusingCAC0905andGapCasinternalstandards.
immediately placed on ice for 15 min, after which 7.5 mL
Measurementof redox potential
Table2 Oligonucleotidesequences
C. acetobutylicum ATCC 824 was grown anaerobically in a
Primers Sequencea(5′-3′) four-vesselbioreactor(DASGIP)onCGMwithconstantag-
itation at 37 °C. The redox and growth of the culture was
hydA-as-F CTCGGGTAATGTAATTACTTTTAGT
recorded every 30 s, and the headspace was flushed with a
hydA-as-R GGATCCGAGTGTTGGGATATCTAC
constantstreamofN withaflowrateof0.1L/min.Theredox
hydE-as-F CTCGGGATAGGAGAAATTATGG 2
potential was measured using Broadly-James F-995 Redox
hydE-as-R GGATCCGTCTTAAACCACAATACATAC
FermProbe which measures the difference in potential be-
hydF-as-F CTCGGGACGAGGTGATTTTAATG
tweenaplatinumelectrodeinthefermentationmediaandan
hydF-as-R GGATCCTCATCAAGACCAGCAGTATC
Ag/AgClreferenceelectrode.Theelectrodesarenotcalibrated
hydA-F GGATCCTTGGGAGGATAAACATGAAAAC
because the zero points and slope of the half-cells do not
hydA-R CTCGGGTAAAATAAATGTGCCTCAACTT
change (Broadley-James FermProbe ORP Electrodes Manu-
Cac0905-rt-F TAGTGATTGTGGGTGGCGGT al).On-linegrowthreadingwascalibratedwithaspectropho-
Cac0905-rt-R GCCACCTAGGCTATCCTCTGCT tometer.OncetheculturesreachedanA of0.25thenTNT
600
GapC-rt-F GCTCACGTAAGAGCAGGCGC was added inanacetonitrilesolution through a septumport.
GapC-rt-R AGCCATTGGAGCTAAGCAGTTAGT As a control, the same amount of acetonitrile without TNT
hydA-rt-F AGCAGCAACAGGACAAGCGAT wasaddedtoothervesselsinasimilarmanner.
hydA-rt-R TGCGGTAGATGCGTAGCTGC
hydE-rt-F ACCGGCAACTACGGCAATGGAAT
Measurementof carbon dioxide andhydrogen gas
hydE-rt-R GTCTGCAGTGCCCAGGAGTGT
production
hydF-rt-F AGCATGCAGCCAGCACAATGAAC
hydF-rt-R AGAAGCCTGCACCCACCACC
The fermentation vessels received a constant flow of nitro-
Restrictionsitesaresetinitalics gengaswhichmixedwiththeheadspacegasesproducedby
ApplMicrobiolBiotechnol
the metabolizing organism. These gases were exhausted example, the difference in acetate vs. butyrate production
through a water-cooled off-gas condenser which was can result in a different level of hydrogen formation and
plumbed directly to an Agilent MicroGC 3000A that was potentially alter the availability of reductant for TNT trans-
usedtomeasurenitrogen,carbondioxide,andhydrogengas formation. Also, since some of these strains, the buk and
levels.Thegasanalyzerwassettotakesamplesevery5min mutant H strains, have previously been shown to enter the
for approximately 1 h before the addition of TNT and solvent-producingphaseearlierthanthewild-typeparent,it
continuing for about 4 h after theaddition. was anticipated they may have lower ability to transform
TNTintheearlyormid-logphasesamplesastheywouldbe
physiologicallymoreliketheearly-solvent-producing state.
Results Samples from cultures of each C. acetobutylicum strain
(Table 1) were diluted in order to measure the initial rate
The colorimetric TNTassay protocol of the TNT transformation of the specific culture. To mea-
sure the TNT degradation rate during different growth
AsimplifiedJenkinsTNTassayprotocolwasadaptedinour phases, samples were analyzed from cultures at A of 0.3
600
experiments. Major nitro compounds and TNT metabolites (for early exponential phase), A of 1.0 (for mid-
600
donotinterferewithourTNTassay,inwhichTNTshowed exponential phase), and 24-h culture (for stationary phase
an absorption peak at 414 nm while other partially reduced culture, solventgenic phase). The initial rate of TNT trans-
speciesdidnot.Theothernitrocompoundsshowedabsorp- formationofeachcultureisshowninFig.1.Theexperiment
tionpeaksaround350nmwithamuchlowerintensitythan showsthemaximalrateofthereactionwithdifferentstrains
thepeakat414nmforTNT(datanotshown).Themeasured and cell preparations. It was found that all strains tested
TNTconcentration in the culture declined with time during showed similar initial TNT degradation rate, ranging from
the assay, and the rate of decrease had diminished consid- 10,000to12,000μMh−1A −1duringtheearlyexponential
600
erably after 1 h. It was found that sample dilution affected phase.Allstrainstestedshowedthesametrendofadecrease
the calculated specific TNT degradation rate. Samples of in the specific TNT degradation rate with the advancement
clostridial cultures diluted in higher volumes of the CGM/ of growth phase, with those entering or in the stationary-
TNT mixture exhibited increasing levels of specific TNT solvent-producing phase having the lowest specific rate of
degradation rate up to a dilution factor of 40, and further TNT transformation. The maximal specific TNT degrada-
dilution incurred no additional effect on the specific rate of tion rate may be limited by hydrogenase levels or by other
transformation. Therefore, the maximal specific TNT deg- factors such as the glucose uptake rate, growth rate, supply
radation rate as measured by this method was used in the of reductant ferredoxin (Fd) reduced (Fd ), or other
reduced
figures and tables. components.Toinvestigatetheeffectsoftheseotherfactors,
TNT degradation rate remains relatively stable among
different C. acetobutylicum strains
StrainsofC.acetobutylicumwerecomparedfortheirrateof
TNT transformation. The strains analyzed were the wild-
typeATCC824;strainM5(Clarketal.1989;Stim-Herndon
etal.1996),astrainthathaslostthepSOLplasmidanddoes
notproducebutanol;strain824(pta),withadisruptedphos-
photransacetylase gene, pta, which produces a lower level
ofacetateduringtheearlyphaseofculture;strain824(buk)
with a disrupted butyrate kinase gene,buk, whichproduces
butyrate at a lower level during the early phase of culture
(Green et al. 1996); and 824(mutant B), a strain that has a
disruption in the gene upstream of the solvent operon and
has increased butanol formation (Nair et al. 1999). These
strains could reveal differences in the TNT transformation Fig. 1 Comparison of TNT degradation rates among C. acetobutyli-
rate correlated with the metabolite pattern of the cells, cumstrainswithdifferentgeneticbackgrounds(differentsolventpro-
especiallyintheearlylogphase.Sincethemetabolicmuta- files) revealed no major differences. The rate of TNT transformation
was assayed during the early exponential phase (A , 0.3), mid-
tionsaffectedtheproductsofearlymetabolism,thischange 600
exponentialphase(A ,1.0),andstationaryphase(24-hculturewith
inmetabolicpatterncouldinturnaffecttherelativepropor- A ,varied)bydilut6in00gthecultureatdifferenttimesintofreshmedi-
600
tion of hydrogen produced during the early log phase. For umcontaining400μMTNT.Standarddeviationsareshown
ApplMicrobiolBiotechnol
analysesweredoneonthemediabeforeandduringthetime would place TNT among the group of dyes that can be
theTNTwas being degraded. reduced by Clostridium (Rao and Mutharasan 1987), and
In order to quantitate the glucose metabolism during the thus this work could have broader implications in the gen-
TNT transformation, the metabolites were analyzed from eralcontrol/understandingofredoxregulationandresponse
parentalC.acetobutylicumATCC824culturestransforming in these anaerobes. In the case of TNT, the reduction is
TNTatthehighratespresentedinFig.1.Sincetheamount irreversible and continues beyond an initial reduction step
ofglucoseutilization duringtheperiodofTNTtransforma- (Hughesetal.1998a,b)whileformanydyesthereactionis
tionwaslow,weassessedthemetabolismduringthisperiod reversible and the reduced dye can transfer electrons to
by two independent measurements. We measured glucose another carrier or redox reaction and affect the metabolite
directly and also measured the metabolic products of glu- pattern. This feature is the well-known observation that
cose metabolism (acetate and butyrate) independently. The violgen addition results in higher butanol levels in late
TNT transformation rate measured in this series of five growth cultures (Rao and Mutharasan 1987).
culture experiments gave a TNT transformation rate of
15.2 mM h−1A −1.
600 Redox and metabolite changesare observedduring TNT
Glucose uptake rates in three of the culture experiments
transformation as shown by measurements of theproducts
were found by measurement of the acetate and butyrate
of thecell cultureduring theTNTreduction period
concentrations before and after the TNT transformation
period. The correlation to glucose utilization used the stoi-
We found TNT addition alters the redox potential of the
chiometric amount of acetate and butyrate produced by
culture. Addition of TNT to cultures in exponential growth
Clostridiacultures(A 00.3)withinanhourofTNTaddi-
600 resulted in a dose dependent spike in the redox potential,
tion. Since the stoichiometric ratio of metabolites produced
whichisshowninFig.2.Thisinitialspikewasfollowedby
toglucoseconsumedis2:1foracetateand1:1forbutyrate,
a persistent increase in the redox potential of the cultures
themolarincreaseobservedinthesetwoproductsduringthe
receiving TNTwhen compared with the redox level of the
first hour was used to determine the amount of glucose
control culture. The relative increase in redox potential
metabolism to acids during the period of TNT transforma-
lasted for the remainder of exponential growth indicating
tion. Ethanol and butanol were measured and discounted
from the analysis as negligible. These measurements gave
glucoseuptakeratesof22,26,and34mMh−1A −1.Intwo
600
oftheexperiments,theglucoseuptakebyClostridiaculture
(A 00.3)wasanalyzedbyHPLCdirectly.Foreachexper-
600
iment,theglucoseconcentrationvaluesweremeasuredboth
before and after the hour-long run with TNT. The rate of
glucoseconsumptionwasdirectlydeterminedfromthecon-
centration difference showing values of 17 and 14 mM h−1
A −1. The ratios of TNT to glucose metabolism in these
600
TNTstudiesrangedfrom0.45to1.0.Soitappeared,amole
of TNTwas reduced for about every 1 to 2 mol of metab-
olized glucose.
These relatively high ratios suggest that TNT can be
reduced competitively against regular cellular metabolism
andthatwhenoperatingatthemaximalrateTNTreduction
isnotaminorfactorinthecellsmetabolicactivitiesandcan
represent a significant distortion and sink for the cell’s
reducing power. Thus, rather than being a minor side reac-
tionofthecellsmetabolism,theTNTisaneffectiveinvivo
reactant with the redox carriers/enzymes such as Fd -
reduced
hydrogenase, and it can affect the cells metabolism during
Fig. 2 The effect of TNT addition on redox potential. The redox
this time. The reactionwith TNT notonly canuse asignif- potentialofC.acetobutylicumculturesincreasedinadose-dependent
icant proportion of the cells reducing power, but in doing matter after the addition of TNT. Acetonitrile or an acetonitrile/TNT
thisitaffectstheoverallpatternofmetabolismandisinturn solution was added to C. acetobutylicum cultures in the exponential
phaseofgrowth (A of ∼0.25)tofinalconcentrations of0,100,or
affectedbycompetitorswhichinfluencetheredoxsituation, 600
500μMTNT.Theculturesweregrowninabioreactor,andtheredox
e.g., the more effective diversion of redox to solvent pro-
potentialoftheculturewasrecordedevery30s.Detailsaregiveninthe
ducingredoxpathwayslaterintheculture.Thisperspective “Materialsandmethods”
ApplMicrobiolBiotechnol
that TNTaddition resultsin a change to the culture’s redox Regulationof hydrogenase affectsTNT degradation rate
status for 3 h. and solvent production
We observed addition of TNT resulted in a decrease of
hydrogen production by cultures of C. acetobutylicum Thematurationofactivehydrogenasebyotherproteinshas
ATCC 824. TNTcan act as an alternative electron acceptor beeninvestigatedrecently(Kingetal.2006;McGlynnetal.
for the hydrogenase enzyme, so the effect of TNTaddition 2007),andwesoughttoassessifreductionofexpressionof
onH productioninculturesduringexponentialgrowthwas genes encoding these proteins by using antisense technolo-
2
determinedbymeasuringtheH /CO ratio. Figure3shows gy would generate a greater effect on reduction of hydrog-
2 2
thattherewasasharpreductionintheH /CO ratiofollow- enaseactivitythananantisensedirectedtowardhydAitself.
2 2
ingtheadditionofTNT,whichwasduetoadecreaseinH If so and if these low-concentration maturation proteins
2
production since CO production remained relatively con- were limiting, we might thereby observe a much lower
2
stant.ThisprovidesfurtherevidencethatTNTcancompete TNT transformation rate in strains with lowered expression
with the natural electron acceptors of the hydrogenase en- of the maturation proteins. We chose hydE and hydF as
zyme in actively growing cells and indicates that TNT genes to manipulate in this way and compared the results
reductioncould alter themetabolic output of thecells. with the analogous reduction of hydA expression by a sim-
We noted that when TNT was added, and the reaction ilar antisense construct. The previous work using hydA
proceedsoveraperiodoftime,adifferentmetabolicpattern antisense constructs demonstrated the feasibility of the
was observed. More butyrate and less acetate are observed. methodandhadshownacorrelationofhydrogenaseexpres-
A culture grown under the conditions where the TNT deg- sion and activity with TNT degradation rate (Watrous etal.
radation rate was analyzed (Fig. 1) was processed and sub- 2003).Theconstructionofthehomologousplasmidseriesis
jected to gas chromatography to determine the metabolites presented in Fig. 4a. Small fragments containing the up-
presentinthepre-TNTaddedcultureandinthecultureafter streamandstartsectionofthecodingregionsofhydA,hydE,
1 h when the starting TNT had been depleted. The culture and hydF were targeted for antisense down-regulation.
continued to grow during this time and the metabolism of Meanwhile, the hydA gene was overexpressed based on
glucose continued during the 1-h period. Analysis of the the same backbone plasmid to increase hydrogenase activ-
metabolites formed during the period when TNT is being ity. The scheme for making the constructs is shown in
transformedshowsasignificantchangeintheproportionsof Fig. 4a. Figure 4b shows that down-regulation of hydA
acetate and butyrate. The metabolites formed during the significantly reduced the specific TNT degradation rate
periodofTNTtransformationweredeterminedbysubtract- while overexpression of hydA improved the specific TNT
ing the concentrations of metabolites at the time of TNT degradation rate to only a very small degree but not to the
addition from those determined after 1 h of TNT level of theextra dosage ofthe hydrogenase genebased on
transformation. thehighercopynumberoftheplasmid.Down-regulationof
AsshownintheexperimentsofFig.2showingtheredox hydEreducedTNTdegradationrateslightlywhileantisense
potential after TNTaddition and in Fig. 3 where measure- against hydF did not show a decrease in TNT degradation
ments of CO and hydrogen are included, we noted that activity. This result might indicate that hydF is present in
2
when TNTwas added the hydrogen production decreased. excess under normal physiological conditions. Antisense
ThiswouldbeexpectedifTNTisactingatthehydrogenase down-regulation of hydA caused hydrogenase activity to
andtakingsomeoftheelectronsthat wouldnormally goto decrease significantly. However, overexpression of hydA
hydrogen formation to instead reduce the TNT. We mea- caused only a slight increase in hydrogenase activity under
suredtheacidmetabolitesformedduringthetimeafterTNT the assay conditions. All antisense constructs showed
addition. A culture grown under the conditions where the growth patterns similar to the control strain. A test for
TNT degradation rate was analyzed was processed and solvent production showed that all antisense strains pro-
subjected to gas chromatography to determine the metabo- duced slightly more butanol (as well as total solvent, data
litespresentinthepre-TNTaddedcultureandintheculture not shown) than the strain bearing the control plasmid.
1 h after the TNT addition, a time when the starting TNT Reverse transcription polymerase chain reaction (RT-PCR)
had been depleted. The culture continued to grow during confirmed anti-sense down-regulation of target genes in all
this time, and the metabolism of glucose continued during three strains (Fig. 4c). mRNA for hydA, hydE, and hydF in
the 1-h period and seemed unchanged in rate as judged by theantisenseconstructswasreducedbyaround2-to5-fold
thetotalproductionofacidsduringthistime.Analysisofthe relative to the 824(pSOS-del) control. hydA and hydE un-
metabolitesformedovertheperiodduringTNTtransforma- derwentsimilarlevelsofreduction,butthedown-regulation
tion showed a significant change in the proportions of of hydF showed the largest expression decrease. RT-PCR
acetate and butyrate, with a large amount of butyrate being also showed that hydA overexpression generated a large
produced incomparisonto verylittle acetate (Table 3). (∼50-fold) increase in hydA mRNA. Since those proteins,