Appl Microbiol Biotechnol(2006)71522–532
Appl Microbiol Biotechnol(2006)71:522–532
DOI10.1007/s00253-005-0190-8
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Barbara Brezna.Ohgew Kweon.Robin L.Stingley.
James P.Freeman.
Ashraf A.Khan.Bystrik Polek.Richard C.Jones.
Carl E.Cerniglia
Molecular characterization of cytochrome P450
genes in the polycyclic aromatic hydrocarbon
degrading Mycobacterium vanbaalenii PYR-1
Received:29July2005/Revised:1September2005/Accepted:9September2005/Published online:30November2005 #Springer-Verlag2005
Abstract Mycobacterium vanbaalenii PYR-1has the abil-ity to degrade low-and high-molecular-weight polycyclic aromatic hydrocarbons(PAHs).In addition to dioxygenases, cytochrome P450monooxygenases have been implicated in PAH degradation.Three cytochrome P450genes, cyp151(pipA),cyp150,and cyp51,were detected and amplified by polymerase chain reaction from M.vanbaa-lenii PYR-1.The complete sequence of these genes was determined.The translated putative proteins were≥80% identical to other GenBank-listed mycobacterial CYP151, CYP150,and CYP51.Genes pipA and cyp150were cloned,and the proteins partially expressed in Escherchia coli as soluble heme-containing cytochrome P450s that exhibited a characteristic peak at450nm in reduced carbon monoxide difference spectra.Monooxygenation metabo-lites of pyrene,dibenzothiophene,and7-methylbenz[α] anthracene were detected in whole cell biotransformations, with E.coli expressing pipA or cyp150when analyzed by gas chromatography/mass spectrometry.The cytochrome P450inhibitor metyrapone strongly inhibited the S-oxida-tion of dibenzothiophene.Thirteen other Mycobacterium strains were screened for the presence of pipA,cyp150,and cyp51genes,as well as the initial PAH dioxygenase(nidA and nidB).The results indicated that many of the Myco-bacterium spp.surveyed contain both monooxygenases and dioxygenases to degrade PAHs.Our results provide further evidence for the diverse enzymatic capability of Mycobacterium spp.to metabolize polycylic aromatic hydrocarbons.
Introduction
Polycyclic aromatic hydrocarbons(PAHs)are ubiquitous environmental pollutants.Chemically,they constitute a class of organic compounds containing two or more fused benzene rings in linear,angular,or cluster arrangement. Because of their human and ecotoxicity,there is a con-siderable interest to determine the fate of these compounds in the environment and to consider possible use of microorganisms for remediation of polluted sites(Cerniglia and Sutherland2001;Kanaly and Harayama2000;Mueller et al.1996).
M.vanbaalenii PYR-1,isolated from a petrogenic chemical polluted site,can utilize or biotransform a wide range of PAHs(Khan et al.2002).Studies of PAH metab-olites showed that this bacterium uses both dioxygenase(s) and cytochrome P450monooxygenase(s)to metabolize PAHs(Heitkamp et al.1988;Kelley et al.1990;Khan et al. 2001;Kim et al.2004a,b,2005;Moody et al.2001,2002, 2003,2004,2005).While cis-dihydrodiols produced by this strain are typical metabolites of aromatic ring-hy-droxylating dioxygenases,trans-dihydrodiols(Heitkamp et al.1988;Kelley et al.1990;Kim et al.2005;Moody et al.2001,2003,2004,2005)are presumably formed by cytochrome P450catalyzed epoxidation of the aromatic nucleus with enzymatic hydration by epoxide hydrolase.
B.Brezna.O.Kweon.R.L.Stingley.
A.A.Khan.C.E.Cerniglia(*)
Division of Microbiology,
National Center for Toxicological Research,
US Food and Drug Administration,
3900NCTR Road,
Jefferson,AR72079,USA
e-mail:ccerniglia@https://www.wendangku.net/doc/ba6888773.html,
Tel.:+1-870-5437341
Fax:+1-870-5437307
B.Brezna.B.Polek
Institute of Molecular Biology,Slovak Academy of Sciences, 84551Bratislava,Slovakia
J.P.Freeman
Division of Biochemical Toxicology,National Center
for Toxicological Research,Food and Drug Administration, Jefferson,AR72079,USA
R.C.Jones
Division of Systems Toxicology,National Center
for Toxicological Research,Food and Drug Administration, Jefferson,AR72079,USA
Among other suspected cytochrome P450metabolites formed by M.vanbaalenii PYR-1are4-hydroxybiphenyl (Moody et al.2002),dibenzothiophene(DBT)sulfoxide(un-published results),and a7-hydroxymethyl-12-methylbenz [α]anthracene(Moody et al.2003).Despite the metabolic evidence that implicated cytochrome P450,it has not been identified in M.vanbaalenii PYR-1.However,there are genetic data on several cytochrome P450families in dif-ferent Mycobacterium spp.that were not associated with PAH degradation(Aoyama et al.1996,1998;Bellamine et al. 1999;Cole and Barrell1998;Jackson et al.2003;Kelly et al.2003;Lepesheva et al.2001;McLean et al.2002; Mowat et al.2002;Poupin et al.1999a;Trigui et al.2004). In this study,we report the detection and molecular char-acterization of three CYPs from M.vanbaalenii PYR-1, two of which were cloned and expressed in Escherchia coli and assessed for their ability to oxygenate PAHs.In addi-tion,13other Mycobacterium strains were screened for the presence of cytochrome P450and aromatic ring-hydrox-ylating dioxygenase genes.
Materials and methods
Chemicals
Pyrene,DBT,and piperidine hydrochloride were pur-chased from Sigma Chemical Company(St.Louis,MO, USA).Solvents were purchased from J.T.Baker,Inc.(Miamisburg,OH,USA).7-Methylbenz[α]anthracene(7-MBA)was synthesized by Dr.Peter Fu at the National Center for Toxicological Research,(Jefferson,AR,USA). Strains and media
Mycobacterium strains used in this study are listed in Table1.For cloning,E.coli host strains EPI300(Epicentre, Madison,WI,USA),DH5α(Promega,Madison,WI, USA),Novablue(Novagen,Madison,WI,USA),and vector pET-17b(Novagen)were used.Middlebrook7H11 medium was purchased from Remel(Lenexa,KS,USA). Mineral salts medium(MBS)with nutrients and MBS agar amended with phenanthrene by using a spray-plate tech-nique were prepared as described by Heitkamp et al. (1988).
DNA preparation
Mycobacterium strains were cultivated for7days on Middlebrook agar7H11,or on MBS amended with phenanthrene,if they were capable of phenanthrene PAH utilization.The cells were scraped from the plates,and their total genomic DNA was isolated with the DNeasy tissue kit (Qiagen,Valencia,CA,USA).Plasmid and fosmid DNA preparations from E.coli strains were made by using the Qiaprep Spin Miniprep kit(Qiagen).
Table1Mycobacterium strains used in this study that were screened for the presence of cytochrome P450genes and nidA and nidB genes Strain Substrate or other characteristic Detection of
nidA c nidB d pipA e cyp150f cyp51g M.aurum(ATCC23366)Type strain(?)(?)?++ M.austroafricanum(ATCC33464)Type strain,related to M.vanbaalenii(?)(?)?++ M.austroafricanum GTI-23a PAHs+++??M.chlorophenolicum PCP-1(ATCC49826)Polychlorinated phenols(?)(?)??+ M.flavescens PYR-GCK(ATCC700033)PAHs(?)(+)++?M.frederiksbergense FAn9T(DSM44346)PAHs(?)(+)+++ M.gilvum(ATCC43909)Type strain???+?M.gilvum BB1(DSM9487)PAHs(+)(+)++?M.petroleophilum(ATCC21497)n-Paraffins(?)(?)?++ M.smegmatis mc2155(ATCC700084)Transformation host???++ M.vaccae JOB-5(ATCC29678)Gaseous,long chain,cycloparaffinic
and monoaromatic hydrocarbons
(?)(?)?++
M.vanbaalenii PYR-1(DSM7251)PAHs(+)(+)+++ Mycobacterium sp.7E1B1W(ATCC29676)Gaseous and long chain hydrocarbons(?)(?)?+?Mycobacterium sp.PAH2.135(RJGII-135)b PAHs(+)(+)+?+“+”PCR product of expected size was present,“?”PCR product of expected size was not obtained.In brackets are the cumulative PCR and Southern hybridization results from the previous study as follows:“(+)”the studied gene is present(Brezna et al.2003),“(?)”the studied gene is not present(Brezna et al.2003)
a Obtained from Dr.B.W.Bogan at the Gas Technology Institute in Des Plaines,IL
b From Dr.D.Warshawsky at the University of Cincinnati
c PCR primers nidAf an
d nidAr wer
e used
d Primers nidBf and nidBr
e Primers RP1F1and RP1R2
f Primers FM10F1and FM10R2
g Primers Cyp51F and Cyp51R
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PCR reactions
Polymerase chain reaction(PCR)primers used in this study are listed in Table2.Regular PCR was performed with Taq DNA polymerase and supplied PCR solutions according to the manufacturer’s instructions(Qiagen).The PCR regime consisted of3min preincubation at95°C;30cycles of30-s denaturation at94°C,30-s annealing at55°C,and1-min extension at72°C;followed by a final hold at72°C for 7min.In each PCR reaction,the concentration of primers was0.5μM each,and the template DNA was added at a final concentration of10pgμl?1.
Proofreading PCR was performed with PCR Supermix High Fidelity(Invitrogen,Carlsbad,CA,USA).The conditions were the same as for regular PCR,but the primers were added at a final concentration of0.2μM and the extension step of the PCR cycle was90s. Southern hybridization
pipA-specific digoxigenin(DIG)-labeled DNA probe was prepared using PCR DIG-labeling kit(Roche Diagnostics, Indianapolis,IN,USA),primers RP1F1and RP1R2,and the total genomic DNA from M.vanbaalenii PYR-1as a PCR template.c yp150-specific DIG-labeled DNA probe was prepared in the same way,using PCR primers FM10F1 and FM10R2.DNA was transferred from colonies grown on Petri dishes to positively charged nylon membranes (Roche)using the procedures described in the Genius System User’s Guide(Roche).Hybridization and detection was performed according to the DIG DNA Labeling and Detection Kit instruction manual(Roche).The results were visualized using the chemiluminiscent substrate CSPD (Roche).Detection of cytochrome P450
Primers RP1F1and RP1R2for detection of pipA were
designed according to conserved regions in GenBank
sequences AF102510(pipA from Mycobacterium smeg-
matis mc2155)and AJ310142(morA from Mycobacterium
sp.RP1).Primers FM10F1and FM10R2for cyp150detec-
tion were designed from the conserved regions in se-
quences AF107047(probable cytochrome P450from M.
smegmatis mc2155)and AF107046(probable cytochrome
P450from Mycobacterium sp.FM10).To design primers
MSCYP51F1and MSCYP51R2for detection of sterol14α-demethylase cytochrome P450(cyp51),sequence cover-ing cyp51and surrounding regions from Mycobacterium
tuberculosis H37Rv(BX842574)was aligned with se-
quences from unfinished genome sequencing projects of
M.smegmatis mc2155and Mycobacterium avium104
available at https://www.wendangku.net/doc/ba6888773.html,,the web site of the
Institute for Genomic Research.Total genomic DNA
from M.vanbaalenii PYR-1was used as a PCR template.
PCR screening of pipA,cyp150,nidA,and nidB genes
in14Mycobacterium strains
Total genomic DNA of each strain was used as a PCR template at a final concentration50pgμl?1.For screening of pipA,the primer pairs RP1F1and RP1R2were used.For screening of cyp150,FM10F1and FM10R2were used; Cyp51F and Cyp51R were used for cyp51.In strains where genes nidA and nidB were not screened previously(Brezna et al.2003),the nidA detection using nidAF and nidAR primers and the nidB detection using nidBF and nidBR were performed.
Table2PCR primers used in this study
Primer name Primer sequence Reference microorganism Reference sequence Position
RP1F1agctggatcctcaacaag Mycobacterium sp.RP1AJ3101421,161–1,178
RP1F2tcatcgcgatcatgctc1,354–1,370
Fm10F1ccctacttcgatcacctgcgc Mycobacterium sp.FM10AF107046489–509
Fm10R2ccgaacgcgatgtgctcgcg1,504–1,485 MSCYP51F1gggccgatgttccagccg M.smegmatis mc2155contig33121,289,527–1,289,544 MSCYP51R2tcgccgagacgccgcgcg1,291,356–1,291,339 PipAclonF acgccatatg tcgtcggccactgtcggttctgtc a,b M.vanbaalenii PYR-1AY4859981–27
PipAclonR agctaagcttcaatggtgatggtgatggtgggaa
gcgggcgtgaagccga a,c
1,200–1,181
Cyp150clonF acgccatatg agcgacttcgacacgatcgactac a,b M.vanbaalenii PYR-1AY496703322–348
Cyp150clonR agctaagcttcaatggtgatggtgatggtgtcga
accggggtgaacgtga a,c
1,589–1,608
Cyp51F cgacggcctgcctgatcg M.vanbaalenii PYR-1AY575951507–524
Cyp51R tcctcggggatccggttg1,137–1,120
a Italic denotes parts of primers not aligning to target sequence
b Underlined are Nde I restriction sites
c Underline
d ar
e Hin dIII restriction sites,double underlined are6xHis-tagged codons
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Cloning and sequencing
A fosmid genomic library of M.vanbaalenii PYR-1was constructed previously(Stingley et al.2004a,b).Colonies of library clones were transferred from the petri dishes to positively charged nylon membranes.pipA-or cyp150-containing clones were identified by colony hybridization with pipA-or cyp150-specific DIG-labeled DNA probes, respectively.One positive fosmid clone was selected for each cytochrome gene.The fosmid DNA was digested with Eco RI in the case of the PipA-containing clone and with Sac I in the case of the CYP150-containing clone.The restriction fragments were subcloned into pGEM-11zf(+) (Promega),and the resulting subclones were rescreened by colony hybridization with pipA-or cyp150-specific DIG-labeled DNA probes.pipA-and cyp150-containing subclones were named pGEM-PIP and pGEM-CYP,respec-tively,and were sequenced.For subcloning into expression vector pET-17b(Novagen),the genes were amplified with proofreading PCR.In the forward PCR primers,an Nde I restriction site was incorporated;in the reverse primers,the 6-His-tag codon and a Hin dIII restriction site were incor-porated.Primers PipAclonF and PipAclonR for amplifi-cation of pipA and Cyp150clonF and Cyp150clonR for amplification of cyp150are listed in Table2.As the PCR template,the plasmid DNA of pGEM-PIP and pGEM-CYP was used.The PCR amplicons were subcloned into pET-17b,resulting in plasmids pET-17b-PIP and pET-17b-CYP. Recombinant plasmids were transformed into NovaBlue E. coli host strain and subsequently retransformed into BL21 (DE3)pLysS host strain(Novagen).
DNA sequencing was performed on an Applied Biosystems Model377DNA sequencer at the University of Arkansas for Medical Sciences,Little Rock,AR,USA. Sequences were compiled,translated,and analyzed using Lasergene software(DNASTAR,Madison,WI,USA)and compared to similar genes and proteins using online database searches(https://www.wendangku.net/doc/ba6888773.html,/BLAST/). Protein expression,purification,and identification
A single colony of BL21(DE3)pLysS E.coli cells ex-pressing cytochrome PipA or cytochrome CYP150,re-spectively,were grown overnight on L
B plates with100μg ampicillin ml?1.A single colony of each E.coli clone was transferred into10ml of liquid LB medium containing 100μg ampicillin ml?1.After overnight incubation with shaking,these starter cultures were added to250ml of LB medium with100μg ampicillin ml?1.Cultures were incubated with shaking at20°
C until the OD600reached 0.5.Subsequently,2mM of heme precursor5-aminolevu-linic acid(ALA),10μg ml?1of FeCl3,and1mM of the inducer isopropylthiogalactoside(IPTG)were added.For apo-P450synthesis analysis,ALA and FeCl3were not added.Cells were incubated overnight at20°C and then spun at4,000×g.The cells were lysed by boiling for3min or by sonication of six10-s bursts at300W,with a10-s cooling period between each burst depending on its usage. The lysates were centrifuged at8,400×g to pellet the cellular debris.
The6xHis-tagged proteins were purified from the total soluble protein fraction using the Ni-NTA resin,as described in the Qiaexpressionist handbook(Qiagen).All the buffers were adjusted to pH7.4.
PipA and CYP150containing covalently attached heme were identified by heme-staining with dimethoxybenzidine (Francis and Becker1984)after separation via denaturing polyacrylamide gel electrophoresis(PAGE).The stained bands were excised,digested robotically with trypsin,and analyzed using nano liquid chromatography(LC)–tandem mass spectrometry(MS/MS)on an LCQ Deca XP Plus ion trap mass spectrometer(Thermo,San Jose,CA,USA) (Edmondson et al.2002).Samples(40μl)were loaded using an Endurance autosampler(Micro-Tech Scientific, Vista,CA,USA)onto an IntegraFrit(New Objective, Woburn,MA,USA)vented column(Licklider et al.2002) (75μm×3cm)packed with1cm Jupiter C12material (Phenomenex,Torrance,CA)at14μl min?1and eluted with a50-min gradient(0.1–30%B in35min,30–50%B in10min,and50–80%B in5min,where A=99.8%H2O, 0.1%acetonitrile,0.1%formic acid;and B=80%acetoni-trile,19.9%H2O,0.1%formic acid)at200nl min?1 (generated with a split tee)using an UltraPlus II capillary HPLC pump(Micro-Tech Scientific)over a75μm×15cm IntegraFrit analytical column packed also with Jupiter C12 material.The column was coupled to a stainless steel emitter(30μm ID×3cm;Proxeon,Odense,Denmark). MS/MS was performed on the top four ions in each MS scan using the data-dependent acquisition mode.Normal-ized collision energy was set at35%,and three microscans were summed following AGC implementation(target values for MS and MS/MS were2×108and6×107counts, respectively).Dynamic exclusion and repeat settings ensured each ion was selected only once and excluded for30s thereafter.Product ion data were searched against the NCBInr protein database using a locally stored copy of the Mascot search engine(Matrix Science,London,UK). Spectrophotometric analysis of cytochrome P450
Total soluble protein fractions were prepared from E.coli cells expressing cytochromes PipA and Cyp150or those containing pET-17b vector without insert,as described earlier in the protein expression and purification section. Reduced CO difference spectra were measured in soluble protein extracts as described previously(Omura and Sato 1964;Schlenk et al.1994).
Biotransformation experiments
The transformed E.coli BL21(DE3)pLysS(pET-17b-PIP), BL21(DE3)pLysS(pET-17b-PIP)(pBRCD),BL21(DE3) pLysS(pET-17b-CYP150),BL21(DE3)pLysS(pET-17b-CYP150)(pBRCD),and control E.coli BL21(DE3)pLysS
525
(pET-17b)were cultivated analogously as in the protein expression experiment.The total culture volumes were 50ml.After the addition of IPTG,FeCl3,and ALA for holo-P450and IPTG only for apo-P450,the cultures were grown for8h at20°C with vigorous shaking.Afterwards, the cells were spun at4,000×g,washed with50ml of 50mM sodium–phosphate buffer(pH7.4),and resus-pended in20ml of the same buffer,with the final cell suspension adjusted to OD600=4.3.To each flask,7.5μl of the prepared substrate stock solutions,which were10% DBT,7-MBA,or pyrene in dimethylformamide,was added.For cytochrome P450inhibition studies,metyr-apone was added to a final concentration of0.27mM.After a16-h incubation with shaking at20°C,the transformation reaction was stopped by the addition of an equal volume of ethyl acetate.The cell suspensions were extracted three times with70ml ethyl https://www.wendangku.net/doc/ba6888773.html,bined ethyl acetate fractions were evaporated at25°C on a vacuum rotary evaporator,redissolved in3ml ethyl acetate,and dried in a vacuum evaporator.
Gas chromatography and mass spectrometry
After collection,the metabolites were analyzed by gas chromatography(GC)/electron ionization mass spectrom-etry(EI-MS)with a TSQ700or TSQ7,000tandem quad-rupole mass spectrometer(ThermoFinnigan,San Jose,CA, USA).The mass spectrometer was operated in the single quadrupole mode with70eV electron ionization(EI)en-ergy and150°C ion source temperature.AVarian3,400gas chromatograph was employed for the GC/EI-MS analyses. Separation was achieved with a30m×0.25mm×0.25μm DB-5ms capillary column(J&W Scientific,Folsom,CA, USA).The column was heated from70°C to280°C at 20°C min?1.The helium carrier gas flow rate was con-trolled at15psi.In order to estimate the relative amounts of the detected metabolites,extracted ion chromatograms and base peak ions were generated for the molecular ions and for the metabolites,respectively,and the resulting chromatographic peaks were integrated electronically with the chromatographic software.Ratios were calculated for the resulting peak areas and averaged for each metabolite. Results
Detection,cloning,and sequence analysis
of cytochrome P450in M.vanbaalenii PYR-1
Carbon monoxide difference spetra of cellular lysates of M. vanbaalenii PYR-1grown in the presence of PAHs indicated that the100,000-g supernatant fraction contained trace levels of cytochrome P450.As a strategy for a more sensitive detection of cytochrome(s)P450in M.vanbaa-lenii PYR-1,we used a genomic approach and considered most likely that member(s)of the CYP150or the CYP151 family would be present since they originate from fast-growing environmental Mycobacterium strains RP1(Trigui et al.2004)and FM10(AF107046).The presence of
CYP51in M.vanbaalenii PYR-1was also hypothesized
because of its conservation among several mycobacteria
and even in different biological kingdoms(Aoyama et al.
1996).
PCR screening of M.vanbaalenii PYR-1genomic DNA
with two primer pairs designed from strain RP1for pipA
gene and from strain FM10for cyp151gene(Table2)gave
expected PCR products sizes,0.25and1.0kb,respectively.
The preliminary sequencing of PCR products confirmed
that they were indeed parts of the targeted cyp isogenes.
Afterwards,the DIG-labeled versions of these PCR prod-
ucts were used as probes to screen M.vanbaalenii PYR-1
genomic https://www.wendangku.net/doc/ba6888773.html,plete sequences of both cyp isogenes
were obtained from positive library subclones.The PCR
product of expected size1.8kb of the third cyp isogene,
cyp51,in M.vanbaalenii PYR-1resulted from the primer
combination MSCYP51F1and MSCYP51R2.Since this
PCR product covered the complete cyp51gene,no sub-
sequent library screening was necessary.The1.8-kb PCR
product was sequenced by primer-walking.
The sequence of M.vanbaalenii PYR-1pipA region was
submitted to GenBank under accession number AY485998.
This sequence contains a gene for cytochrome P450
PipA,a ferredoxin,and a partial sequence of glutamine
synthetase(Fig.1).This locus organization is identical
to M.smegmatis mc2155(GenBank accession number
AF102510)and similar to Mycobacterium sp.RP1(GenBank
accession number AJ310142).However,strain RP1has a
gene for a putative ferredoxin reductase located between a
ferredoxin and a putative glutamine synthetase gene unlike
M.vanbaalenii PYR-1.The cytochrome P450and the
ferredoxin genes were identified based on≥86%and ≥69%identity,respectively,of proposed proteins to their counterparts in M.smegmatis mc2155(AF102510)and
Mycobacterium sp.RP1(AJ310142)and by using the
conserved domain database search at http://www.ncbi.nlm.
https://www.wendangku.net/doc/ba6888773.html,.Partial sequence of the putative glutamine synthe-
tase gene covers only33aminoacids at the N terminus of
the proposed protein,where no conserved domains are
present.However,this N-terminal sequence is75%
identical to putative protein of M.smegmatis
(AF102510),where beta-grasp domain of glutamine syn-
thetase(pfam03951)is present.
The sequence of the M.vanbaalenii PYR-1cyp150
region was submitted to GenBank under accession number
AY496703.In addition to the open reading frame(ORF)
for cytochrome P450(cyp150),the sequence contains an
incomplete ORF coding for a possible protein that contains
a conserved domain of bacterial regulatory TetR-family
proteins,as well as two hypothetical proteins(Fig.1).
There is a similar locus organization in Mycobacterium sp.
FM10(AF107046)and in M.smegmatis(AF107047).
CYP150from M.vanbaalenii PYR-1is96and88%
identical to its counterparts in Mycobacterium sp.FM10
and M.smegmatis,respectively,at the protein level.
The sequence of the cyp51region of M.vanbaalenii
PYR-1(GenBank accession number AY575951)contains
an incomplete ORF coding for a probable oxidoreductase,
526
cytochrome CYP51,and a ferredoxin,and another incom-plete ORF coding for a hypothetical protein of unknown function (Fig.1).This ORF organization is conserved in several other Mycobacterium spp.(Jackson et al.2003).CYP51from M.vanbaalenii is 80–92%identical to those of M.smegmatis ,M.avium M.tuberculosis ,and Myco-bacterium bovis subsp.bovis (GenBank accession numbers BX842574,AE006970,BX248336,and AE017229and unfinished genomes of M.smegmatis mc 2155and M.avium 104at https://www.wendangku.net/doc/ba6888773.html, ).
A phylogenetic tree was constructed by the neighbor –joining (NJ)approach of the collection of 29aligned cytochrome P450protein sequences including three cyto-chrome P450s from M.vanbaalenii PYR-1(Fig.2).The tree shows six distinct groups for the 29cytochrome P450s.The three cytochrome P450s from M.vanbaalenii PYR-1fall into three different groups but belong to the same class I.PipA of M.vanbaalenii PYR-1belongs to the PipA (morA)group that shows over 86%identity to each other.CYP150of M.vanbaalenii PYR-1belongs to the CYP150group displaying very high identity (≥82%)to CYP150s from other Mycobacterium spp.but with very low identity (≤39%)to the remaining CYP150s.CYP51of M.vanbaa-lenii PYR-1is placed in CYP51group and shows very high identity (≥79%)to CYP51s from Nocardia farcinica
IFM10152and other Mycobacterium spp.but with very low identity (≤36%)to other CYP51s.The pairwise distance values obtained by using Gonnet weight matrix were less than 0.700within each group,with the exception of the group CYP150.The CYP150group can be divided into two subgroups by using the pairwise distance value,0.700.Within each group,the pairwise distance values were less than 0.700.
Expression,purification,and identification of PipA and CYP150
Recombinant His-tagged protein PipA heterologously ex-pressed in E.coli was soluble.After a passage through Ni-NTA resin,partial purification was achieved,i.e.,His-tagged PipA was a predominant protein in the resin eluate (Fig.3a,lane 5).His-tagged CYP150was localized mainly in the insoluble fraction when expressed in E.coli (data not shown),probably due to the formation of inclusion bodies.However,some of the protein was also produced in the soluble form and was partially purified from the soluble fraction using the Ni-NTA resin.
The overexpressed PipA and CYP150were stained with dimethoxybenzidine,and the two stained bands were
ana-
Fig.1Physical maps and conserved sequence alignments of the cytochrome P450monooxygenases and ferredoxins from M.vanbaalenii PYR-1with those from other sources.The amino acid residues involved in binding to heme (FX 2GX 3CXG)and to the [3Fe-4S]cluster (CX 5CX n C)are indicated by highlighted char-acters .Designations:CYP151(PipA ),CYP150,and CYP51—cytochromes P450;Fdx —ferredoxins;GlnA —putative glutamine synthetase;orf2and orf3—hypothetical proteins;orf4—probable regulatory protein from TetR-family.GenBank accession numbers are as follows.PipA of M.vanbaalenii PYR-1,AY485998;PipA of M.smegmatis mc 2155,AF102510;morA of Mycobacterium tokaiense THO100,AY816211;morA of Mycobacterium sp.RP1,
AJ310142;morA of Mycobacterium sp.HE5,AY816211;CYP51of M.vanbaalenii PYR-1,AY575951;CYP51of M.avium subsp.paratuberculosis str.k10,NC_002944;CYP51of M.tuberculosis CDC1551,AE000516;CYP51of M.tuberculosis H37Rv,NC_000962;M.bovis AF2122/97NC_002945;CYP150A2of M.smegmatis mc 2155,;CYP150of M.vanbaalenii PYR-1,AY496703;CYP150of Mycobacterium sp.FM10,AF107046;CYP of Burkholderia fungorum LB400,NZ_AAAJ03000005;CYP150of Arthrobacter sp.FB24,NZ_AAHG01000018;CYP107L2of Streptomyces avermitilis MA-4680,BA000030;CYP of Pseudomonas aeruginosa PA01,NC_002516
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lyzed by LC –MS/MS.The resultant product ion data were searched against the public NCBI protein database.The 44.8-kDa band for CYP150matched to cytochrome P450from M.vanbaalenii PYR-1(AY496703)with 46unique peptides and 75%sequence coverage.The 48.7-kDa band for PipA (Fig.3a,lane 4)also matched to cytochrome P450s from the same species with accession number AY485998showing 31peptides and 78%sequence coverage.
Reduced CO-difference spectra of soluble protein cell extracts from E.coli expressing PipA and CYP150showed a typical peak at 450nm,confirming the cytochrome P450-like character of these proteins (Fig.3b).A negative con-trol,E.coli containing a vector without insert,showed no peak at 450nm.(Fig.3b).
Biotransformation of DBT,7-MBA,and pyrene by PipA and CYP150
The expression of pipA and cyp150in E.coli lacking the electron-transport components produced one metabolite
from DBT,which had a retention time (12.44min)(Fig.4a,b)and mass spectral fragmentation pattern (m /z 200and m /z 184)(Fig.4f)identical to DBT 5-oxide (Schlenk et al.1994).Moreover,supplementation of pBRCD,containing the cis-trons encoding [3Fe-4S]ferredoxin (phdC )and ferredoxin reductase (phdD )component from Nocardioides sp.KP7(Saito et al.2000)in pipA and cyp150,increased DBT 5-oxide formation twofold.
To confirm PipA DBT S-oxidase activity,the effects of PipA inhibitor metyrapone and ALA and FeCl 3were tested.As shown in Fig.4c,metyrapone markedly inhib-ited DBT-sulphoxide production in E.coli containing pET-17b-PIP (43%decrease),but was less efficient in E.coli containing pET-17b-PIP (pBRCD)(11.8%decrease)(data not shown).
To support more direct evidence of the role of PipA in DBT oxidation,apo-PipA lacking heme was expressed without the addition of ALA and FeCl 3and confirmed by heme-staining with dimethoxybenzidine (Fig.3a,lanes 8and 9).Apo-PipA and the negative control showed no DBT 5-oxidation activity (Fig.4d,e).This result indicates
that
Fig.2Phylogenetic tree obtained from the alignment of three cytochrome P450s from M.vanbaalenii PYR-1with related proteins.The protein sequences of the 29cytochrome P450s are classified.The amino acid sequences were aligned with the Clustal X package (version 1.83),and the tree was constructed by the NJ method and displayed with the program TreeView X (1.6.6).Scale bar indicates the percentage divergence.The pairwise distance matrix was obtained by using Clustal X (Gonnet 250).Class I cytochrome P450s are three-component systems comprising of a flavin adenine dinucleotide (FAD)-containing reductase,an iron –sulfur protein (ferredoxin),and a cytochrome P450.The eukaryotic class I enzymes are associated with the mitochondrial membrane.Class II cytochrome P450s are two-component systems,and both class III and class IV are a single polypeptide.In a class II system,the cytochrome P450is partnered with a diflavin (FDA/FMN)reductase,whereas in the class III systems,the diflavin (FDA/FMN)reductase is fused to the cytochrome P450.Class IV system is made up of FMN-containing reductases with a ferredoxin-like center linked to a cytochrome P450(Roberts et al.2002).GenBank accession numbers are as follows (refer to Fig.1for the remaining protein sequences):P450of Ralstonia metallidurans CH34,NZ_AAAI00000000;P450RhF of Rhodococcus .sp.NCIMB 9784,AF459424;P450of Rhodococcus rubber DSM 44319,;P450cam of Pseudomonas putida ,M12546;P450Novosphingo-bium aromaticiviorans DSM 12444,NZ_AAAV02000002;CYP505of Fusarium oxysporum MT-811,AB030037;P450BM-3of Bacil-lus megaterium Fulco PB85,J04832;P450of Bacillus cereus ATCC 14579,AE017008;P450of B.cereus E33L,NC_006274;CYP51of Sorghum bicolor SS1000,U74319;CYP51of Homo sapiens ,CH236949;CYP51of N.farcinica IFM 10152
528
PipA was responsible for S-oxygenation of DBT and needed heme as a prosthetic group for enzyme activity.As shown in Fig.3a (lanes 6and 8),the addition of ALA and FeCl 3did not increase the expression level of PipA.
When the substrate was 7-MBA,one metabolite was observed in each sample eluting at 17.7min.The mass spec-trum consisted of an apparent molecular ion at m /z 258and a major fragment ion at m /z 229.The mass spectrum and re-tention time are consistent with an authentic 7-hydroxymethyl-benz[α]anthracene (Fig.5b)(Cerniglia et al.1982).
GC/MS analysis of the pyrene extracts produced three chromatographic peaks with apparent molecular mass of 218that contained a major fragment ion at m /z 189.These peaks eluted at 16.6,16.8,and 19.5min.The mass spectral fragmentation data were identical to pyrenols (Cerniglia
et al.1986).Authentic 1-hydroxypyrene eluted at the same retention time (19.5min)and produced the same mass spectrum as the third peak.Since pyrene is a symmetrical molecule,the only isomers that could be formed are 1-hy-droxy-,2-hydroxy-,or 4-hydroxypyrene (Fig.5
c).
Fig.3Expression and purification of recombinant PipA of M.vanbaalenii PYR-1at 20°C.a Lane M molecular size marker,lane 1cell extract from E.coli (BL21)(pET-17b),lane 2cell extract from E.coli (BL21)(pET-17b-PIP)prepared by glass bead cell disruption,lane 3cell extract from E.coli (BL21)(pET-17b-PIP)prepared by boiling,lane 4heme-stain of the same cell extract as lane 2,lane 5partial purification of 6xHis-tagged PipA on Ni-NTA resin (Qiagen),lane 6Coomassie blue stained cell extract from E.coli (BL21)(pET-17b-PIP)grown in the presence of ALA and FeCl 3,lane 7heme-stain of lane 6,lane 8Coomassie-blue-stained cell extract from E.coli (BL21)(pET-17b-PIP)grown in the absence of ALA and FeCl 3,lane 9heme-stain of lane 8.b Reduced CO difference spectra of total soluble E.coli protein extracts from cultures of expressing transgenic PipA (solid line ),CYP150(dashed line ),and E.coli containing pET-17b vector without insert (dash and dotted line ).The protein concentrations were 6mg ml ?
1
Fig.4GC/EI-MS extracted ion chromatograms of DBT extracts for (a )CYP150,(b )PipA,(c )PipA+metyrapone,(d )apo-PipA,(e )negative control sample,and (f )mass spectrum of DBT
5-oxide
Detection of cytochrome P450genes pipA,cyp150,
and cyp51and of dioxygenase genes nidA and nidB
in Mycobacterium strains
The amplification of0.25-kb PCR product indicating pipA presence,1.0-kb product indicating cyp150,and0.63-kb product indicating cyp51presence varied among the strains, as documented in Table1.nidA and nidB genes coding for the large and small subunits of an aromatic ring-hy-droxylating dioxygenase were detected by PCR in Myco-bacterium austroafricanum GTI-23.Mycobacterium gilvum ATCC43909and M.smegmatis mc2155did not produce the PCR products(Table1).For the rest of the strains,the results of nidA and nidB screening from the previous study (Brezna et al.2003)are summarized in Table1. Discussion
M.vanbaalenii PYR-1was the first organism known to produce both cis-dihydrodiol and trans-dihydrodiol me-tabolites of high-molecular-weight PAHs such as pyrene (Heitkamp et al.1988;Kelley et al.1990;Kim et al.2005; Moody et al.2001),indicating that both dioxygenase(s) and cytochrome P450monooxygenase(s)can initiate PAH degradation in this bacterium.One of these enzymes,aro-matic ring-hydroxylating dioxygenase,is encoded by nidA and nidB genes and has been cloned and characterized previously(Khan et al.2001;Kim et al.2004a;Stingley et al.2004b).This study complements the previous in-formation by identifying three genes encoding alternative PAH-oxidative enzymes,cytochromes P450,in this organ-ism.To our knowledge,this is the first study proving that functional cytochrome P450genes can coexist with aro-matic ring-hydroxylating dioxygenase in a high-molecular-weight PAH utilizer.Three CYPs were detected in M. vanbaalenii PYR-1using the PCR approach that were >80%identical to other mycobacterial CYP151,CYP150, and CYP51,respectively.However,considering the high number of CYP isozymes in complete genomes of some mycobacteria,i.e.,20CYPs in M.tuberculosis(Cole and Barrell1998),18CYPs in M.bovis(Garnier et al.2003), 42CYPs in M.avium ssp.paratuberculosis(NC_002944), and approximately40CYPs in M.smegmatis(Jackson et al. 2003)and M.avium104(Kelly et al.2003),it is quite likely that M.vanbaalenii also has more than three CYPs. Only the complete genome sequence of M.vanbaalenii PYR-1will tell the total number of CYPs present in this bacterium.
Studies on the activity of bacterial CYPs towards PAHs are scarce(Carmichael and Wong2001;England et al. 1998;Harford-Cross et al.2000;Joo et al.1999;Li et al. 2001;Taylor et al.1999),and none of these previously assayed CYPs originate from a Mycobacterium or from a PAH utilizing strain.In our study,two CYPs from M. vanbaalenii PYR-1,PipA and CYP150,were heterolo-gously expressed in E.coli,and whole cell biotransforma-tion experiments were performed to prove their ability to oxygenate PAHs.
Cytochrome P450s are multicomponent enzymes con-sisting of two separated functional classes,electron transfer and oxygenation.Interaction and complementation be-tween two functional classes are necessary for the full catalytic function.The expression in E.coli of both PipA and CYP150from M.vanbaalenii PYR-1with functional activity suggests that the electron transport system for PipA and CYP150can be complemented by the unidentified electron transport systems of E.coli used as a host.This result has been observed in many cytochrome P450s and dioxygenases(Joo et al.1999;Khan et al.2001;Kurkela et al.1988;Laurie and Lloyd-Jones1999;Simon et al.1993). Moreover,the supplementation of phdCD electron-trans-port system of a nonheme dioxygenase from Nocardioides sp.KP7(Saito et al.2000)increased the transforming activity of PipA and CYP150.This suggests the relatively low specificity of the electron-transport systems toward PipA and CYP150.The PhdC protein used in our study was the first example of the[3Fe-4S]type of ferredoxin in nonheme dioxygenases(Saito et al.2000)and has38% identity to the[3Fe-4S]type of ferredoxin component of PipA from M.vanbaalenii PYR-1.The three cysteine res-idues that serve as ligands for the iron–sulfur cluster are conserved in both PhdC and ferredoxin of PipA(Fig.1). This tolerance between cytochrome P450s and other elec-tron transport systems gives clues for understanding the relationships in the number and the specificity of a redox partnership between cytochrome P450and electron trans-port systems.
In order to obtain direct evidence of the oxidation of PAHs by cytochrome P450in M.vanbaalenii PYR-1,PipA that was expressed mainly as a functionally active form in the cytosol was chosen and tested for the inhibition of cytochrome P450and the oxidation activity of apo-PipA. Based on the previous investigation(Schlenk et al.1994), DBT and metyrapone were used as a substrate and in-hibitor,respectively.S-oxidation of DBT in E.coli that can express active PipA was decreased by metyrapone.In ad-dition,apo-PipA lacking heme could not form DBT5-oxide. These conclusive results support the metabolic evidence of the involvement of cytochrome P450s in PAH metabolism in M.vanbaalenii PYR-1.According to Richardson et al. (1995),the influence of ALA on cytochrome P450synthe-sis in E.coli depends on the form of cytochrome P450.The addition of ALA and FeCl3did not increase the expression level of PipA,based on the intensity of the bands corre-sponding to holo-PipA and apo-PipA in the SDS-PAGE gel (Fig.3a,lanes6and8),indicating that heme synthesis was not a rate-limiting step in PipA production.
According to PCR results,pipA,cyp150,and cyp51 detection varied among the strains and exhibited no corre-lation with the strains’PAH-degrading ability(Table1).In contrast to this,the alternative PAH-oxygenation enzyme, the aromatic ring-hydroxylating dioxygenase encoded by nidA and nidB genes,was consistently present in PAH-utilizing mycobacteria and absent in PAH nonutilizers (Table1)(Brezna et al.2003).It seems that the genes nidA and nidB are specialized for the degradation of PAHs, whereas the primary role of pipA,cyp150,and cyp51is
530
different and the PAH monooxygenation is only a fortu-itous or nonspecific reaction.The true biological function of PipA(CYP150family)is degradation of heterocyclic amines like morpholine or piperdine(Poupin et al.1999a, b;Sielaff et al.2001;Taylor et al.1999;Trigui et al.2004), while the physiological roles of CYP150and mycobacte-rial CYP51are obscure(Cole and Barrell1998).Regard-less of the primary function of the three CYPs,the results of the PCR screening showed that it is not unusual for soil mycobacteria to have both ring-hydroxylating dioxy-genases and cytochromes P450since all of the strains that contained nidAB genes possessed at least one of the studied cyp isogenes.Thus,several mycobacteria have a potential to use both dioxygenation and monooxygenation reactions for initial biotransformation of PAHs. Acknowledgements This work was supported in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agree-ment between the US Department of Energy and the US Food and
Drug Administration.
References
Aoyama Y,Noshiro M,Gotoh O,Imaoka S,Funae Y,Kurosawa N, Horiuchi T,Yoshida Y(1996)Sterol14-demethylase P450 (P45014DM*)is one of the most ancient and conserved P450 species.J Biochem(Tokyo)119:926–933
Aoyama Y,Horiuchi T,Gotoh O,Noshiro M,Yoshida Y(1998) CYP51-like gene of Mycobacterium tuberculosis actually encodes
a P450similar to eukaryotic CYP51.J Biochem(Tokyo)124:
694–696
Bellamine A,Mangla AT,Nes WD,Waterman MR(1999) Characterization and catalytic properties of the sterol14alpha-demethylase from Mycobacterium tuberculosis.Proc Natl Acad Sci U S A96:8937–8942
Brezna B,Khan AA,Cerniglia CE(2003)Molecular characteriza-tion of dioxygenases from polycyclic aromatic hydrocarbon-degrading Mycobacterium spp.FEMS Microbiol Lett223:177–183
Carmichael AB,Wong LL(2001)Protein engineering of Bacillus megaterium CYP102.The oxidation of polycyclic aromatic hydrocarbons.Eur J Biochem268:3117–3125
Cerniglia CE,Sutherland JB(2001)Bioremediation of polycyclic aromatic hydrocarbons by ligninolytic and non-ligninolytic fungi.In:Gadd GM(ed)Fungi in bioremediation.Cambridge University Press,Cambridge,pp136–187
Cerniglia CE,Fu PP,Yang SK(1982)Metabolism of7-methylbenz
[a]anthracene and7-hydroxymethylbenz[a]anthracene by Cun-
ninghamella elegans.Appl Environ Microbiol44:682–689 Cerniglia CE,Kelly DW,Freeman JP,Miller DW(1986)Microbial metabolism of pyrene.Chem Biol Interact57:203–216
Cole ST,Barrell BG(1998)Analysis of the genome of Mycobac-terium tuberculosis H37Rv.Novartis Found Symp217:160–172;discussion172–167
Edmondson RD,V ondriska TM,Biederman KJ,Zhang J,Jones RC, Zheng Y,Allen DL,Xiu JX,Cardwell EM,Pisano MR,Ping P (2002)Protein kinase C epsilon signaling complexes include metabolism-and transcription/translation-related proteins:com-plimentary separation techniques with LC/MS/MS.Mol Cell Proteomics1:421–433
England PA,Harford-Cross CF,Stevenson JA,Rouch DA,Wong LL(1998)The oxidation of naphthalene and pyrene by cytochrome P450cam.FEBS Lett424:271–274Francis RT Jr,Becker RR(1984)Specific indication of hemopro-teins in polyacrylamide gels using a double-staining process.
Anal Biochem136:509–514
Garnier T,Eiglmeier K,Camus JC,Medina N,Mansoor H,Pryor M, Duthoy S,Grondin S,Lacroix C,Monsempe C,Simon S, Harris B,Atkin R,Doggett J,Mayes R,Keating L,Wheeler PR,Parkhill J,Barrell BG,Cole ST,Gordon SV,Hewinson RG (2003)The complete genome sequence of Mycobacterium bovis.Proc Natl Acad Sci U S A100:7877–7882
Harford-Cross CF,Carmichael AB,Allan FK,England PA,Rouch DA,Wong LL(2000)Protein engineering of cytochrome p450 (cam)(CYP101)for the oxidation of polycyclic aromatic hydrocarbons.Protein Eng13:121–128
Heitkamp MA,Freeman JP,Miller DW,Cerniglia CE(1988)Pyrene degradation by a Mycobacterium sp.;identification of ring oxidation and ring fission products.Appl Environ Microbiol 54:2556–2565
Jackson CJ,Lamb DC,Marczylo TH,Parker JE,Manning NL, Kelly DE,Kelly SL(2003)Conservation and cloning of CYP51:a sterol14alpha-demethylase from Mycobacterium smegmatis.Biochem Biophys Res Commun301:558–563 Joo H,Lin Z,Arnold FH(1999)Laboratory evolution of peroxide-mediated cytochrome P450hydroxylation.Nature399:670–673
Kanaly RA,Harayama S(2000)Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by bacteria.J Bacteriol 182:2059–2067
Kelley I,Freeman JP,Cerniglia CE(1990)Identification of metabolites from degradation of naphthalene by a Mycobacte-rium sp.Biodegradation1:283–290
Kelly SL,Lamb DC,Jackson CJ,Warrilow AG,Kelly DE(2003) The biodiversity of microbial cytochromes P450.Adv Microb Physiol47:131–186
Khan AA,Wang RF,Cao WW,Doerge DR,Wennerstrom D, Cerniglia CE(2001)Molecular cloning,nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp.strain PYR-1.Appl Environ Microbiol67:3577–3585
Khan A,Kim SJ,Paine DD,Cerniglia CE(2002)Classification of a polycyclic aromatic hydrocarbon-metabolizing bacterium,My-cobacterium sp.strain PYR-1,as Mycobacterium vanbaalenii sp.Int J Syst Evol Microbiol52:1997–2002
Kim SJ,Jones RC,Cha CJ,Kweon O,Edmondson RD,Cerniglia CE(2004a)Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods.Proteomics4:3899–3908
Kim YH,Moody JD,Freeman JP,Engesser KH,Cerniglia CE (2004b)Evidence for the existence of PAH-quinone reductase and catechol-O-methyltransferase in Mycobacterium vanbaale-nii PYR-1.J Ind Microbiol Biotechnol31:507–516
Kim YH,Freeman JP,Moody JD,Engesser KH,Cerniglia CE (2005)Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1.Appl Microbiol Biotechnol67:275–285
Kurkela S,Lehvaslaiho H,Palva ET,Teeri TH(1988)Cloning, nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816.
Gene73:355–362
Laurie AD,Lloyd-Jones G(1999)The phn genes of Burkholderia sp.strain RP007constitute a divergent gene cluster for polycyclic aromatic hydrocarbon catabolism.J Bacteriol181: 531–540
Lepesheva GI,Podust LM,Bellamine A,Waterman MR(2001) Folding requirements are different between sterol14-alpha-demethylase(CYP51)from Mycobacterium tuberculosis and human or fungal orthologs.J Biol Chem276:28413–28420
Li QS,Ogawa J,Schmid RD,Shimizu S(2001)Engineering cytochrome P450BM-3for oxidation of polycyclic aromatic hydrocarbons.Appl Environ Microbiol67:5735–5739
531
Licklider LJ,Thoreen CC,Peng J,Gygi SP(2002)Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column.Anal Chem74:3076–3083 McLean KJ,Cheesman MR,Rivers SL,Richmond A,Leys D, Chapman SK,Reid GA,Price NC,Kelly SM,Clarkson J, Smith WE,Munro AW(2002)Expression,purification and spectroscopic characterization of the cytochrome P450CYP121 from Mycobacterium tuberculosis.J Inorg Biochem91:527–541
Moody JD,Freeman JP,Doerge DR,Cerniglia CE(2001)Degra-dation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp.strain PYR-1.Appl Environ Microbiol 67:1476–1483
Moody JD,Doerge DR,Freeman JP,Cerniglia CE(2002)Degra-dation of biphenyl by Mycobacterium sp.strain PYR-1.Appl Microbiol Biotechnol58:364–369
Moody JD,Fu PP,Freeman JP,Cerniglia CE(2003)Regio-and stereoselective metabolism of7,12–dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1.Appl Environ Micro-biol69:3924–3931
Moody JD,Freeman JP,Fu PP,Cerniglia CE(2004)Degradation of benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1.Appl Environ Microbiol70:340–345
Moody JD,Freeman JP,Cerniglia CE(2005)Degradation of benz
[a]anthracene by Mycobacterium vanbaalenii strain PYR-1.
Biodegradation16:513–526
Mowat CG,Leys D,McLean KJ,Rivers SL,Richmond A,Munro AW,Ortiz Lombardia M,Alzari PM,Reid GA,Chapman SK, Walkinshaw MD(2002)Crystallization and preliminary crystallographic analysis of a novel cytochrome P450from Mycobacterium tuberculosis.Acta Crystallogr D Biol Crystal-logr58:704–705
Mueller JG,Cerniglia CE,Pritchard PH(1996)Bioremediation: principles and applications.Cambridge University Press, Cambridge
Omura T,Sato R(1964)The carbon monoxide-binding pigment of liver microsomes.I.Evidence for its hemoprotein nature.J Biol Chem239:2370–2378
Poupin P,Ducrocq V,Hallier-Soulier S,Truffaut N(1999a)Cloning and characterization of the genes encoding a cytochrome P450 (PipA)involved in piperidine and pyrrolidine utilization and its regulatory protein(PipR)in Mycobacterium smegmatis mc2155.J Bacteriol181:3419–3426Poupin P,Godon JJ,Zumstein E,Truffaut N(1999b)Degradation of morpholine,piperidine,and pyrrolidine by Mycobacteria: evidences for the involvement of a cytochrome P450.Can J Microbiol45:209–216
Richardson TH,Jung F,Griffin KJ,Wester M,Raucey JL,Kemper B,Bornheim LM,Hassett C,Omienski CJ,Johnnson EF (1995)A universal approach to the expression of human and rabbit cytochrome P450s of the2C subfamily in Escherichia coli.Arch Biochem Biophys323:87–96
Roberts GA,Gideon G,Andy G,Sabine LF,Nicholas JT(2002) Identification of a new class of cytochrome P450from a Rhodococcus sp.J Bacteriol184:3898–3908
Saito A,Iwabuchi T,Harayama S(2000)A novel phenanthrene dioxygenase from Nocardioides sp.strain KP7:expression in Escherichia coli.J Bacteriol182:2134–2141
Schlenk D,Bevers RJ,Vertino AM,Cerniglia CE(1994)P450 catalysed S-oxidation of dibenzothiophene by Cunninghamella elegans.Xenobiotica24:1077–1083
Sielaff B,Andreesen JR,Schrader T(2001)A cytochrome P450and
a ferredoxin isolated from Mycobacterium sp.strain HE5after
growth on morpholine.Appl Microbiol Biotechnol56:458–464 Simon MJ,Osslund TD,Saunders R,Ensley BD,Suggs S,Harcourt A,Suen WC,Cruden DL,Gibson DT,Zylstra GJ(1993) Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7and NCIB9816-4.Gene 127:31–37
Stingley RL,Brezna B,Khan AA,Cerniglia CE(2004a)Novel organization of genes in a phthalate degradation operon of Mycobacterium vanbaalenii PYR-1.Microbiology150:3749–3761
Stingley RL,Khan AA,Cerniglia CE(2004b)Molecular character-ization of a phenanthrene degradation pathway in Mycobacte-rium vanbaalenii PYR-1.Biochem Biophys Res Commun 322:133–146
Taylor M,Lamb DC,Cannell R,Dawson M,Kelly SL(1999) Cytochrome P450105D1(CYP105D1)from Streptomyces griseus:heterologous expression,activity,and activation effects of multiple xenobiotics.Biochem Biophys Res Commun263: 838–842
Trigui M,Pulvin S,Truffaut N,Thomas D,Poupin P(2004) Molecular cloning,nucleotide sequencing and expression of genes encoding a cytochrome P450system involved in secondary amine utilization in Mycobacterium sp.strain RP1.
Res Microbiol155:1–9
532
专业英语八级改错练习题及答案解析(30)
专业英语八级改错练习题及答案解析(30) Why does the idea of progress loom so large in the modern world? Surely because progress of particular kind is actually taking place around us and is more and more manifesting. Although mankind has undergone no general improvement in intelligence or morality, it has made extraordinary progress in the accumulation of knowledge. Knowledge begins to increase as soon as the thoughts of one individual could be communicated to another by mean of speech. With the invention of writing, knowledge could be communicated and stored. Libraries made education possible, and education in turn added libraries: the growth of knowledge followed a kind of compound-interest law, which was greatly enhanced by the invention of printing. All this was comparatively slow until, with the coming of science, the tempo was suddenly risen. Then knowledge began to be accumulated according to a systematic plan.However, as soon as new knowledge is acquired, it is now turned to practical account. What is called “modern civilization” is not the result of a balanced development of all man’s nature, but not of accumulated knowledge applied to practical life. The problem now facing humanity is: What is going to be done with all this knowledge? Like is often pointed out, knowledge is a two edged weapon which could be used equally for good or evil. It is now being frequently used indifferently for both. Could any spectacle, for instance, be more grimly whimsical than that gunners using science to shatter men’s bodies while, clo se at hand, surgeons use it to restore them. 1 ________ 2 ________ 3 ________ 4 ________ 5 ________ 6 ________ 7 ________ 8 ________ 9 ________ 10 _______
上海市重点小学排名50(最新)
【第一梯队学校评价标准】 1. 独特的教学和教育理念; 2.独树一帜的教育方法或深厚的办学积淀; 3.足够高的社会美誉度，并受到市级乃至国家级的表彰; 4.一流的软硬件; 5.较好的对口初中等。 【第二梯队学校评价标准】 1. 优异且稳定的升学成绩(或有推优); 2.区内突出的教学特色; 3.足够的师资力量; 4.发展劲头明显或受到市、区级相关单位的重点关注等。 【第三梯队学校评价标准】 1.潜力巨大的新学校或被翻牌后进步明显的普小(以及部分略有退步的原重点小学)，具备在未来成为优秀学校的潜质，但目前在成绩或生源方面还有一定的完善空间。 徐汇区 【第一梯队】世界外国语小学、盛大花园小学、爱菊小学、逸夫小学、建襄小学、高安路一小、向阳小学、汇师小学 【第二梯队】上海小学、上师大一附小、东安路第二小学、园南小学、田林第三小学、徐汇一中心、田林四小、求知小学、上海师范大学第三附属实验学校、康健外国语小学、东安路二小 【第三梯队】徐汇实验小学、康宁科技实小、交大附小、上实附小、华理附小、徐教院附小、启新小学 黄浦区 【第一梯队】上海实验小学、蓬莱二小、卢湾二中心、黄埔上外 【第二梯队】上师大附属卢湾实验小学、曹光彪小学、徽宁路三小、黄浦一中心、复兴东路三小、卢湾一中心、私立永昌学校、师专附小 【第三梯队】北京东路小学、黄教院附属中山学校、巨鹿路一小、裘锦秋实验学校 静安区 【第一梯队】一师附小、静教院、静安一中心
【第二梯队】万航渡路小学、静安三中心、上外静小 【第三梯队】静安实验、陈鹤琴小学、威海路三小、市西小学、西康路三小、静安二中心、静安小学 老闸北 【第一梯队】闸北实验小学 【第二梯队】闸北区大宁国际小学、扬波外国语小学、闸北区第三中心小学、闸北区第一中心小学、彭浦实验小学、童园小学(民办)、闸北区第二中心小学、彭浦新村第一小学 【第三梯队】静安区第四中心小学、中山北路小学、田家炳小学 浦东新区 【第一梯队】上海实验学校、明珠小学(ABC)、福山外国语小学、六师附小 【第二梯队】浦东外国语小学、浦东二中心小学、竹园小学、海桐小学、上实东校、浦明师范附属小学、进才实验小学、昌邑小学、建平实验小学、新世界实验小学、平和双语学校、华二浦东实验学校(预计)、上海科技大学附属学校(预计) 【第三梯队】上外临港外国语学校(预计)、东方小学、元培学校、张江高科实验小学、洋泾-菊园实验学校、浦东南路小学、外高桥保税区实验小学、南码头小学、上南二村小学、龚路中心小学、北蔡镇中心小学、上南实验小学、御桥小学、莲溪小学、澧溪小学、观澜小学、园西小学、工商附小、惠南小学 杨浦区 【第一梯队】二师附小、控江二小、打虎山路第一小学 【第二梯队】民办打一外国语小学、复旦大学附属小学、上理工附小、杨浦小学、齐齐哈尔路第一小学、水丰路小学、上音实验学校小学部、复旦科技园实验学校小学部 【第三梯队】六一小学、上音实验学校、上外附属双语学校、沪东外国语学校、同济小学、平凉路三小、许昌路第五小学、中原路小学、五角场小学、杨教院实验学校小学部、凤城新村小学、政立路二小、回民小学、内江路二小、二联小学、市东实验学校小学部(原市东小学)、怀德路第一小学 虹口区
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2009年中国水资源公报 2012-04-26 中华人民共和国水利部 2009年，我国气候异常，一些地区降雨之多、台风之强、旱情之重为历史 罕见。旱情来得早、去得晚、范围广、影响大，特别是冬麦主产区年初的冬春连旱，东北西部、华北北部和西北东部的夏伏旱，江南大部、华南大部和西南局部的秋冬连旱，对农业生产带来严重影响。全年有9个台风在我国沿海登陆，发生强风、暴雨、高潮同时出现的最不利形势。受多次大范围、高强度降雨过程影响，全国有210多条河流相继发生超警戒水位以上洪水。在党中央、国务院的正确领导下，水利部门坚持以人为本，超前部署，科学决策，精心调度，各级地方党委、政府全力以赴，广大军民奋力抗灾救灾，最大程度地减轻了洪涝干旱台风灾害损失。 2009年，党中央、国务院高度重视水利工作，国家把加快水利基础设施建 设作为应对金融危机、扩大国内需求的重要领域。各级水利部门深入学习实践科学发展观，坚决贯彻落实中央保增长、保民生、保稳定的各项决策部署，保持了水利又好又快发展的强劲势头。扩大内需水利基础设施建设取得显著成效，有力地促进了经济平稳较快增长；强力推进十七届三中全会明确的病险水库除险加固、农村饮水安全工程、灌区续建配套与节水改造三大任务，涉及民生的水利问题加快解决；为应对我国水资源短缺、用水效率不高、水污染严重的突出问题，明确提出实行最严格的水资源管理制度，节水型社会建设全面推进；一大批重点水利工程开工建设，创近年来新开工大型水利工程数量历史新高；水利法制建设稳步
推进，依法治水、科学管水能力不断提升。水利工作取得的显著成效，为促进经济平稳较快增长提供了有力支撑。 一、水资源量 降水量 2009年，全国平均年降水量591.1mm，折合降水总量为55965.5亿m3，比常年值偏少8.0%。从水资源分区看，松花江、辽河、海河、黄河、淮河、西北诸河6个水资源一级区（以下简称北方6区）面平均降水量为315.7mm，比常年值偏少3.8%；长江（含太湖）、东南诸河、珠江、西南诸河4个水资源一级区（以下简称南方4区）面平均降水量为1079.7mm，比常年值偏少10.0%。在31个省级行政区中，降水量比常年值偏多的有9个省（自治区、直辖市），其中海南和青海偏多程度约30%；降水量比常年值偏少的有22个省（自治区），其中北京和云南偏少约25%。 地表水资源量 2009年全国地表水资源量23125.2亿m3，折合年径流深244.2mm，比常年值偏少13.4%。从水资源分区看，北方6区地表水资源量比常年值偏少13.1%；南方4区比常年值偏少13.5%。在31个省级行政区中，地表水资源量比常年值偏多的有6个省（自治区、直辖市），比常年值偏少的有25个省（自治区、直辖市），其中黑龙江、上海、青海、海南偏多程度在23%～56%之间，江西、吉林、云南、河南、福建、内蒙古、宁夏偏少程度在25%～37%之间，山西、辽宁、河北、北京偏少程度在45%～62%之间。 2009年，从国境外流入我国境内的水量为154.9亿m3，从我国流出国境的水量为5193.3亿m3，从我国流入国际边界河流的水量为1090.7亿m3,全国入海水量为13960.9亿m3。
英语专业八级改错练习题及答案解析
英语专业八级改错练习题及答案解析 About half of the infant and maternal deaths in developing countries could be avoided if women had used family planning methods to prevent high risk ____1____ pregnancies, according to a report publishing recently by the Johns Hopking ____2____ University. The report indicates that 5.6 million infant deaths and 2,000,000 maternal Deaths could be prevented this year if women chose to have theirs children ____3____ within the safest years with adequate intervals among births and limited their ____4____ families to moderate size. This amounts to about half of the 9.8 million infant and 370.000 maternal deaths in developing countries, excluded China, estimated for this year by ____5____ the United Nation’s Children’s Fund and the US Centers for Disease Control respectably. China was excluded because very few births occur in the high ____6____
2018-2019学年上海市宝山区宝山实验学校六年级第一学期期中考试数学试卷
哈佛北大精英创立 2018学年上海市宝山区宝山实验学校六年级 第一学期期中考试数学试卷 一、填空题 1. 既是素数又是偶数的是 【答案】2 2. 分解素因数：45= 【答案】533?? 3. 8和9 的最小公倍数是 【答案】72 4. 在18、27、30、44、102中，能被5整除的数是 【答案】30 5. 已知自然数A 与自然数B 是连续的偶数，那么A 、B 的最大公因数是 【答案】2 6. 既是12的倍数又是36的因数的数是 【答案】12.36 7. 如图（1），点A 表示的数是 【答案】4 11 8. = 10 3-54计算 【答案】2 1 9. 是千克的4132 【答案】 6 1 10. 学校教学楼走廊长56米，如果在走廊上等间隔地拜访8盆花，并且走廊的两端各放一盆花，那么每两盆花之间的距离是 米 【答案】8 11. 如图（2），将正方形看做一个整体，那么阴影部分可以用最简分数 来表示
哈佛北大精英创立 【答案】10 12. 三张卡片上分别写有1、2、3三个数字，从这三张卡片中依次抽出两张，就能组成一个两位数，在所得的两位数中，素数有 个 【答案】3 13. 是 按从小到大的顺序排列、、16 5 73.08 3· 【答案】·73.08 3 165ππ 14. 的所有最简分数之和是之间，分母是和在155 4 31 【答案】15 111 15. 的值为 那么如果， ，个数，如分子、分母中较大的那表示，规定对于分数m m m A A A ,12}7 {}3{16}13 16 {3}32{}{=+== 【答案】5 二、填空题 1. 4是8和16的（） 【A 】公因数 【B 】最大公因数 【C 】最小公倍数 【D 】公倍数 【答案】A 2. 的分数有（）个且小于大于6 561 【A 】4 【B 】5 【C 】6 【D 】无数个 【答案】D 3. 如果m 是技术，那么下列数中（）也是奇数 【A 】m+1 【B 】m+2 【C 】m+3 【D 】m+m
2017年电大高等数学基础形成性考核册作业答案
高等数学基础作业 作业1 一、CCBC DCA 二、1、（3， +∞) ，2、 x 2 - x ，3、 e 1/ 2 ，4、 e ， 5、 x=0 ，6、 无穷小量 。 三、 1、f(-2) = - 2，f(0) = 0， f(1) = e 2、由 01 2>-x x 解得x<0或x>1/2，函数定义域为(-∞，0)∪(1/2，+∞) 3、如图梯形面积A=(R+b)h ，其中22h R b -= ∴ 4、 5、 6、 7、 8、 h h R R A )(2 2-+=2 3 22sin 2 33sin 3 lim 2sin 3sin lim 00==→→x x x x x x x x 2)1() 1sin(1lim )1sin(1lim 12 1-=-++=+--→-→x x x x x x x 33cos 33sin 3lim 3tan lim 00==→→x x x x x x x x x x x x x x x sin )11()11)(11(lim sin 11lim 222020++-+++=-+→→0 sin 11lim sin )11(1 )1(lim 20 220=++=++-+=→→x x x x x x x x x x x x x x x x x x x x )3 41(lim )343(lim 31(lim +-+=+-+=+-∞→∞→∞→
9、 10、 ∴函数在x=1处连续 不存在，∴函数在x=-1处不连续 作业2 一、 BDADC 二、1、f ＇(0)= 0 ，2、f ＇(lnx)= (2/x)lnx+5/x ， 3、 1/2 ， 4、 y=1 ， 5、 2x 2x (lnx+1) ， 6、 1/x 。 三、1、求y ＇ (1)、y=(x 3/2+3)e x ，y ＇=3/2x 1/2e x +(x 3/2+3)e x =(3/2x 1/2+x 3/2+3)e x (2)、y ＇=-csc 2x + 2xlnx +x (3)、y ＇=(2xlnx-x)/ln 2x (4)、y ＇=[(-sinx+2x ln2)x 3-3x 2(cosx+2x )]/x 6 4 3 4 43) 3 41(] )341[(lim ---+∞→=+-+-+=e x x x x 32)4)(1()4)(2(lim 4586lim 4224=----=+-+-→→x x x x x x x x x x 1)(lim 1)21()(lim 1 2 1 ===-=- +→→x f x f x x )1(1)(lim 1 f x f x ==→011)(lim 1)(lim 1 1=+-=≠-=-+-→-→x f x f x x )(lim 1 x f x -→x x x x x x x 22sin cos )(ln sin )21 ()5(---、
2007年中国水资源公报
2007年中国水资源公报 2008-10-13 2007年，在党中央、国务院的正确领导下，各级水利部门认真贯彻落实科学发展观，积极践行可持续发展治水思路，着力解决水利发展中的突出问题，水利事业保持了投资加大、建设加快，基础增强、管理加强，效益提高、民生改善的良好态势。 2007年，我国极端天气事件频发，水旱灾害呈现先旱后涝、旱涝急转和旱涝并发的局面。淮河发生新中国成立以来仅次于1954年的流域性大洪水，台风前少后多强度大，山洪灾害频发，城市暴雨内涝严重。北方大部及南方一些地区发生冬春连旱，江南、华南等地发生严重夏伏旱，旱情主要发生在粮食主产区和作物生长关键期，波及范围广，持续时间长，影响程度深。面对严重的水旱灾情，党中央高度重视，国务院有关部门周密部署，受灾地区干部齐心合力，军民团结一致，夺取了防汛抗旱工作的全面胜利。 一、水资源量 降水量 2007年全国平均降水量610.0mm，折合降水总量为57763亿m3，比常年值（多年平均值，下同）偏少5.1%。从水资源分区看，松花江、辽河、海河、黄河、淮河、西北诸河六个水资源一级区（简称北方六区，下同）面平均降水量317.9mm，比常年值偏少3.1%；长江（含太湖）、东南诸河、珠江、西南诸河四个水资源一级区（简称南方四区，下同）面平均降水量1128.4mm，比常年值偏少6.0%。在31个省级行政区中，降水量比常年值偏多的有14个省（自治区、直辖市），其中山东和甘肃分别偏多13.8%和11.5%；降水量比常年值偏少的有17个省（自治区、直辖市），其中内蒙古、江西、黑龙江的偏少程度超过20%，北京、广西、河北、湖南、广东、天津偏少15%～10%。
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会建设试点工作取得成效，水资源调度工作得到进一步强化。《全国水资源综合规划》、《太湖流域水功能区划》获得国务院批复，修订后的《中华人民共和国水土保持法》公布，水利事业快速发展。 一、水资源量 降水量 2010年，全国平均年降水量695.4mm，折合降水总量为65849.6亿m3，比常年值（多年平均值，下同）偏多8.2%。从水资源分区看，松花江、辽河、海河、黄河、淮河、西北诸河6个水资源一级区（简称北方6区，下同）面平均降水量为365.8mm，比常年值偏多11.5%；长江（含太湖）、东南诸河、珠江、西南诸河4个水资源一级区（简称南方4区，下同）面平均降水量为1280.2mm，比常年值偏多6.7%。在31个省级行政区中，降水量比常年值偏多的有20个省（自治区、直辖市），其中新疆、辽宁和吉林等3省（自治区）偏多程度大于30%；降水量比常年值偏少的有11个省（自治区），其中天津、北京和重庆分别偏少18.2%、12.6%和10.6%。 地表水资源量 2010年全国地表水资源量29797.6亿m3，折合年径流深314.7mm，比常年值偏多11.6%。从水资源分区看，北方6区地表水资源量比常年值偏多16.1%；南方4区比常年值偏多10.7%。在31个省级行政区中，地表水资源量比常年值偏多的有19个省（自治区、直辖市），其中辽宁和吉林偏多程度大于80%，海南、浙江、江西、福建、河南、安徽和新疆偏多程度在30%～60%之间；比常年值偏少的有12个省（自治区、直辖市），其中北京、河北、天津、山西和内蒙古偏少程度在30%～60%之间。
专八改错习题及答案解析
英语专业八级改错练习题及答案解析(一) About half of the infant and maternal deaths in developing countries could be avoided if women had used family planning methods to prevent high risk ____1____ pregnancies, according to a report publishing recently by the Johns Hopking ____2____ University. The report indicates that 5.6 million infant deaths and 2,000,000 maternal Deaths could be prevented this year if women chose to have theirs children ____3____ within the safest years with adequate intervals among births and limited their ____4____ families to moderate size. This amounts to about half of the 9.8 million infant and 370.000 maternal deaths in developing countries, excluded China, estimated for this year by ____5____ the United Nation?s Children?s Fund and the US Centers for Disease Control respectably. China was excluded because very few births occur in the high ____6____ risk categories. The report says that evidences from around the world shows the risk of ____7____ maternal or infant ill and death is the highest in four specific types of ____8_____ pregnancy; pregnancies before the mother is 18 year old; those after the ____9____ mother is 35 years old; pregnancies after four births; and those lesser than ____10____ two years apart. 参考答案及解析: 1 将had used 改为used。因为此句是虚拟语气，表示与现在事实相反，故条件从句中应使用一般过去时。例如：Many would be wise if they did not think themselves wise. 许多人原本会成为聪明人－如果他们不自以为聪明的话。 2 将publishing改为published;report和publish时逻辑动宾关系，故应使用publish的过去分词短语来修饰report。例如：Any discovery that we may make, however small, will remain acquired knowledge. 任何可能的发现，不管多么微不足道，都将成为知识宝库中的一部分。 3 将theirs改为their; 4 将among改为between;在两次怀孕期间留出足够的间隔时间，故用between。 5 将过去分词excluded改为介词excluding。excluding意为“不包括…” 6 将respectably改为respectively;respectively 意为“分别地”，符合句子的意思。而respectably 意为“可敬的，值得尊敬地”。 7将evidences改为evidence。evidence是不可数名词。 8将ill改为illness。 9将year改为years。 10将lesser改为less 英语专业八级改错练习题及答案解析(二) “Home, sweet home” is a phrase that express an essential attitude in the United States. Whether the reality of life in the family house is sweet or no sweet, the cherished ideal of home _____1_____ has great importance for many people. This ideal is a vital part of the American dream. This dream, dramatized in the history of nineteenth century European settlers of American West, was to find a piece of place, build a house _____2_____