The genes encoding the hydroxylase
Journal of Steroid Biochemistry &Molecular Biology 92(2004)
143–154
The genes encoding the hydroxylase of
3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione in steroid
degradation in Comamonas testosteroni TA441
Masae Horinouchi a ,?,Toshiaki Hayashi a ,Toshiaki Kudo a ,b ,c
a
RIKEN,2-1Hirosawa,Wako-shi,Saitama 351-0198,Japan b JST,2-1Hirosawa,Wako-shi,Saitama 351-0198,Japan
c
Science of Biological Supermolecular Systems,Graduate School of Integrated Science,Yokohama City University,
Suehiro,Tsurumi-ku,Yokohama 230-0045,Japan
Abstract
Steroid degradation genes of Comamonas testosteroni TA441are encoded in at least two gene clusters:one containing the meta-cleavage enzyme gene tesB ;and another consisting of ORF18,17,tesI ,H ,ORF11,12,and tesDEFG .TesH and I are,respectively,the 1-and 4(5?)-dehydrogenase of the 3-ketosteroid,TesD is the hydrolase for the product of meta-cleavage reaction,and TesEFG degrade one of the product of TesD.In this report,we describe the identi?cation of the function of ORF11(tesA2)and 12(tesA1).The TesA1-and TesA2-disrupted mutant accumulated two characteristic intermediate compounds,which were identi?ed as 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA)and its hydroxylated derivative,3,17-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione by MS and NMR analysis.A complementation experiment using a broad-host range plasmid showed that both TesA1and A2are necessary for hydroxylation of 3-HSA to 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA).?2004Elsevier Ltd.All rights reserved.
Keywords:Comamonas testosteroni ;Steroid;Testosterone;Meta -cleavage;Hydroxylase
1.Introduction
Comamonas testosteroni (formerly Pseudomonas testos-teroni )is known for its ability to utilize testosterone and var-ious steroids as sole carbon and energy sources.The mecha-nism by which testosterone is degraded in C.testosteroni was eagerly studied and the main intermediate compounds in the degradation pathway were determined in 1960s [1–9].The main pathway of testosterone degradation in C.testosteroni was predicted by these results,even though most of the genes involved in the degradation have not been determined,the exception being for genes for initial 17?-dehydrogenation [10–12]and subsequent 1-dehydrogenation [13],which were reported in 1990s.C.testosteroni strain TA441uti-lizes certain steroids as well as a number of aromatic com-?
Corresponding author.Tel.:+81484679545;fax:+81484624672.E-mail address:masae@riken.jp (M.Horinouchi).
pounds.For the purpose of isolating the steroid degrada-tion genes in TA441,we simultaneously identi?ed genes and intermediate compounds that are accumulated by the gene-disrupted mutants and revealed the testosterone degra-dation pathway in TA441as presented in Fig.1[14–16].The process is initiated by dehydrogenation of the 17?-hydroxyl group on testosterone to 4-androstene-3,17-dione (4-AD)(reaction (1)in Fig.1),which then undergoes 1-dehydrogenation to 1,4-androstadiene-3,17-dione (ADD)by TesH (reaction (2))[16],followed by 9?-hydroxylation to produce 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA)(reaction (3)and the following spon-taneous cleavage).ORF17,which is located in the region downstream of tesH ,is probably the reductase component of 9?-hydroxylase [16].C-4of 3-HSA is hydroxylated to yield 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA)(reaction (4)),which is cleaved between C-4and C-5via a meta-cleavage reaction by
0960-0760/$–see front matter ?2004Elsevier Ltd.All rights reserved.doi:10.1016/j.jsbmb.2004.09.002
144M.Horinouchi et al./Journal of Steroid Biochemistry&Molecular Biology92(2004)143–154
Fig.1.Proposed testosterone degradation pathway in C.testosteroni TA441.Closed arrows indicate the main pathway and broken lines indicate minor reactions.
M.Horinouchi et al./Journal of Steroid Biochemistry &Molecular Biology 92(2004)143–154145
TesB (reaction (5))[14].The product of the meta-cleavage reaction of 3,4-DHSA,4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandorosta-1(10),2-dien-4-oic acid (4,9-DSHA),is de-graded into 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 2-hydroxyhexa-2,4-dienoic acid by TesD (reac-tion (6))[15].One of the products,probably 2-hydroxyhexa-2,4-dienoic acid,is degraded by TesEFG [15].TesB and Tes-DEFG show homology to corresponding enzymes in bac-terial aromatic compound degradation,such as BphC and BphDHIJ,respectively.Identities are about 40%,between TesB and BphC,and between TesD and BphD,and about 60,80,80%between TesEFG and BphHIJ,respectively.Although they showed signi?cant homology to the corre-sponding aromatic compound degradation enzymes,northern hybridization analysis showed that these enzymes were in-duced in steroid-grown TA441cells (testosterone and cholic acid)but not in aromatic compound-grown TA441cells (phe-nol,biphenyl,and 3-(3-hydroxyphenyl)propionic acid)[16].Steroid degradation genes of TA441are encoded in at least two gene clusters:one containing the meta-cleavage enzyme gene tesB and the following ORF1,2,and 3,all of which are necessary in steroid degradation;and another consisting of ORF18,17,tesI ,H ,ORF11,12,and tesDEFG (Fig.2).TesI,is the 3-ketosteroid 4(5?)-dehydrogenase,which is not necessary for degradation of testosterone ( 4(5?)-bond is double),but is indispensable for degradation of
andros-
Fig.2.Partial restriction map of a steroid degradation gene cluster of C.testosteroni TA441.The genes and putative ORFs are indicated by large open arrows.Open arrowheads below these ORFs indicate putative promoters,and the putative terminators locate just downstream of ORF18and tesG .Deletion plasmids are indicated below the restriction map;the remaining gene segments (lines)are also shown.The closed arrowheads indicate the direction of transcription regulated by the lac promoter of the pUC19vector.Abbreviations are as follows:Bb,Bgl II;Bm,Bam HI;EI,Eco RI;EV ,Eco RV;Hc,Hin cII;Hd,Hin dIII;Mn,Mun I;Ms,Mst I;Nr,Nru I;Nt,Not I;Ps,Pst I;Sc,Sac I;Sl,Sal I;Sm,Sma I;Sn,Sna BI;Sp,Sph I;Xb,Xba I;Xh,Xho I.
terone ( 4(5?)-bond is single)[16].Putative terminators are found just downstream of ORF18and tesG ,and the genes in the downstream region of these terminators are not necessary for steroid degradation.In the present study,we describe the function of ORF11and ORF12.2.Materials and methods 2.1.Culture conditions
C.testosteroni TA441and the mutant strains were grown at 30?C in LB medium,C medium,or mixed LB +C medium (mixture of equal volume of LB and C media)[14,15].Testos-terone was added as a ?ltered DMSO solution at a ?nal con-centration of 0.1%(w/v).
2.2.Construction of plasmids and mutant strains
Bacterial strains and plasmids used in this study are listed in Table 1.ORF11,ORF12,and tesD were disrupted by dele-tion and insertion of a Km-resistance gene into the two Nru I sites of p12-26HB1(Fig.2and Table 1).About 1.8kb gene region between the two Nru I sites was deleted in the resul-tant plasmid,p1112D-Km r .Plasmid p1112D-Km r was used for inactivation of the ORF11,ORF12,and tesD gene in
146M.Horinouchi et al./Journal of Steroid Biochemistry&Molecular Biology92(2004)143–154
Table1
Bacterial strains and plasmids
Strain or plasmid Characteristics Source or reference Bacterial strains
C.testosteroni TA441Wild-type,Tes+*[27]
ORF11?ORF11::Km r mutant of TA441[15]
ORF12?ORF12::Km r mutant of TA441[15]
ORF1112D?ORF11,ORF12,and tesD::Km r mutant of TA441This work Plasmids
pUC19Ap r,lac Z[28]
pUC118Ap r,lac Z[28] pSuperCosI Ap r,Cosmid vector[29]
pMFY42Tc r,Km r,RSF1010-based broad-host range plasmid[21]
pC26Ap r,pSuperCosI with Bam HI insert of TA441[15]
p12-26HB1Ap r,pUC19with8.8kb Hin dIII-Bgl II insert of pC26This work pUCORF11Ap r,pUC19with1.2kb Stu I-Eco RI insert of pC26This work pUCORF12Ap r,pUC19with1.4kb Sph I-Stu I insert of pC26This work pUCORF(11)12Ap r,pUC19with2.7kb Sph I-Eco RI insert of pC26This work pUCORF1211Ap r,pUCORF(11)12based plasmid(1.4kb Nae I-Stu I fragment of the
insert of pUCORF(11)12is inverted)
This work
pUCORF1718Ap r,pUC118with5.2kb Bgl II-Hin dIII insert of pC26This work pUCORF12111718Ap r,pUCORF1718with2.7kb Sph I-Eco RI insert of pUCORF1211This work pUCORF11-18Ap r,pUC19with8.8kb Avr II-Hin dIII insert of pC26This work pMFYORF11Ap r,pMFY42with the same insert as pUCORF11This work pMFYORF12Ap r,pMFY42with the same insert as pUCORF12This work pMFYORF(11)12Ap r,pMFY42with the same insert as pUCORF(11)12This work pMFYORF1211Ap r,pMFY42with the same insert as pUCORF1211This work
Tes+*:grow on testosterone as the sole carbon source,Ap:ampicillin,Km:kanamycin,Tc:tetracycline.
TA441by homologous recombination.The plasmid was in-troduced into TA441by electroporation,and a Km-resistant and carbenicillin-sensitive TA441mutant was selected.Inser-tion of the Km-resistance gene was con?rmed by southern hybridization using the Km-resistance gene and ORF12as probes.The ORF11-disrupted mutant and ORF12-disrupted mutant were constructed in the same manner in the previous study[15].
2.3.Three-dimensional HPLC
Twice the volume of methanol was added to the cul-ture,which was then centrifuged and the supernatant was directly injected into the HPLC.A Waters2690HPLC (Nihon Waters,Tokyo,Japan)was utilized with an Inert-sil ODS-3column(2.1mm×150mm),and elution car-ried out using a linear gradient from20%solution A (CH3CN:CH3OH:TFA=95:5:0.05)and80%solution B (H2O:CH3OH:TFA=95:5:0.05)to65%solution A and35% solution B over10min,then maintained for3min,and then changed to20%solution A for5min.The?ow rate was 0.21mL/min.
2.4.Isolation of the intermediate products X1and X2 accumulated by ORF12-disrupted mutant
For the isolation of the intermediate products accumu-lated by ORF12-disrupted mutant(compounds X1and X2), a300ml culture of ORF12-disrupted mutant incubated in mixed LB+C medium with0.1%testosterone was twice ex-tracted with the same volume of ethyl acetate.The ethyl acetate layer was concentrated in vacuo,and the residue was dissolved in a small amount of methanol and loaded onto the Waters650HPLC(Nihon Waters,Tokyo,Japan) with an Inertsil ODS-3column(20mm×250mm,GL Science,Tokyo,Japan)and a solvent with the composi-tion CH3CN:MeOH:H2O:TFA of50:40:10:0.05,?ow rate 8mL/min,at room temperature.X1and X2were detected at 210nm.The fractions containing X1and X2were collected and dried.X1and X2were dissolved in a small amount of methanol and puri?ed by HPLC by elution with the compo-sition CH3CN:H2O of1:1at40?C.Other conditions are the same as described above.
2.5.General experimental procedures
For gas chromatography–mass spectrometry(GC–MS), a GCMS-QP5050A(Shimadzu Corporation,Kyoto,Japan)?tted with a capillary column HP-5MS(0.25mm internal diameter by30m,0.25?m?lm thickness,Agilent Tech-nologies,CA,USA)was used.UV spectra were recorded with an Ultrospec3300(Amersham Biosciences Corp.,NJ, USA).NMR spectra(1D,1H and13C,distortionless en-hancement by polarization transfer[DEPT],pulsed?eld gradients-correlated spectroscopy[PFG-DQFCOSY],pulsed ?eld gradients-heteronuclear multiple quantum coherence [PFG-HMQC],pulsed?eld gradients-heteronuclear multi-ple bond coherence[PFG-HMBC])were taken on a JNM-
M.Horinouchi et al./Journal of Steroid Biochemistry&Molecular Biology92(2004)143–154147
ECP500spectrometer(JEOL,Tokyo,Japan)in CDCl3solu-tion with tetramethylsilane(TMS)at0ppm as the internal standard for1H and13C chemical shifts.
3.Results
3.1.HPLC analysis of the culture media of
ORF11-disrupted mutant and ORF12-disrupted mutant incubated with testosterone
C.testosteroni TA441utilizes certain steroids,such as testosterone,using enzymes that are induced when TA441is incubated with steroids.In previous studies, we identi?ed two steroid degradation gene clusters in TA441.One contains meta-cleavage enzyme gene tesB and three following ORFs[14],all of which were neces-sary for steroid degradation in TA441,and the other con-sists of ORF18,17,tesI,H,ORF11,12,and tesDEFG (Fig.2)[15,16].TesD degrades4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid(4,9-DSHA) into9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and2-hydroxyhexa-2,4-dienoic acid.The latter compound is probably degraded by TesEFG[15].TesH and I are,re-spectively,the 1-and 4(5?)-dehydrogenase of the3-ketosteroid.The putative amino acid sequences of ORF11and 12,encoded in the center of this gene cluster,showed homol-ogy to some oxygenases,such as the phenol hydroxylase of Bacillus stearothermophilus BR219[17](ORF11)and com-ponent B of nitrilotriacetate monooxygenase of Chelatobac-ter strain ATCC29600[18](ORF12),though the identity was about30%at maximum.The ORF11-disrupted mutant and ORF12-disrupted mutant showed limited growth on testos-terone,indicating that both are necessary for steroid degra-dation in TA441[15].To analyze the function of these ORFs, the mutants were incubated in the mixed LB and C medium with testosterone for2days.The mixed LB and C medium is a mixture of equal volumes of both media,and gene-disrupted mutants cultured in the mixed medium accumulate a larger amount of intermediate compounds than in normal C medium.Fig.3shows the results of three-dimensional HPLC analysis of the culture of both mutants.The ORF12-disrupted mutant converted most of the testosterone into the interme-diate compound X1,showing a characteristic UV-spectrum, and a smaller amount of compound X2,which showed a sim-ilar UV-spectrum to that of X1.The ORF11-disrupted mu-tant accumulated a smaller amount of X1and X2than the ORF12-disrupted mutant together with androst-4-en-3,17-dione(4-AD),androsta-1,4-diene-3,17-dione(ADD),and 17-hydroxy-1,4-androstadiene-3-one,which are intermedi-ates in the early steps in testosterone degradation(Fig.1).This is probably because the disruption of ORF11affected the ex-pression of genes in the region downstream of ORF11(tesH, tesI,ORF17,and ORF18:TesH is the 1-dehydrogenase of 4-AD to produce ADD and ORF17is probably the reduc-tase component of9?-hydroxylase of ADD).The
estimated Fig.3.Three-dimensional HPLC analysis of the culture of the ORF11-disrupted mutant and the ORF12-disrupted mutant incubated with testos-terone.The vertical axis indicates wavelength,the horizontal axis indicates the RT,and the UV absorbance of each compound is represented in con-tour.For the analysis,a Waters2690HPLC was used with an Inertsil ODS-3 column(2.1mm×150mm;GL Science,Tokyo,Japan),and elution was car-ried out using a linear gradient from20%solution A(CH3CN:CH3OH:TFA [95:5:0.05])and80%solution B(H2O:CH3OH:TFA[95:5:0.05])to65% solution A and35%solution B over10min,maintained for3min,and then changed to20%solution A for5min.The?ow rate was0.21ml/min.Ab-breviations are as follows:Ts,testosterone;4-AD,4-androstene-3,17-dione; ADD,1,4-androstadiene-3,17-dione;and17OH-ADD,17?-hydroxy-1,4-androstadiene-3-one.
amount of X1in the culture of the ORF12-disrupted mu-tant presented in Fig.3was around90%of the predicted amount,and that of X2was only a few percent.In the case of ORF11-disrupted mutant,the rates were about15%and a few https://www.sodocs.net/doc/037677424.html,ually,ORF12-disrupted mutant accumulates around70–90%of X1and ORF11-disrupted mutant accu-mulates around20%.
3.2.Isolation and puri?cation of the intermediate compounds X1and X2
The ORF12-disrupted mutant was incubated with0.1% testosterone at30?C for about2days,and the resultant cul-ture was centrifuged to remove cells and other precipitation. The supernatant of the culture was extracted twice with the same volume of ethyl acetate,and the ethyl acetate frac-tion was dried and concentrated in vacuo.The remnant was dissolved in a small amount of methanol and separated by HPLC(Waters600with ODS-3column)eluted with ace-tonitrile:methanol:water:TFA(50:10:40:0.05).The fractions containing X1and X2were collected from the eluent.Since these fractions were not suf?ciently pure,the fractions were concentrated,dissolved in a small amount of methanol,and separated by HPLC again with acetonitrile:water(1:1)as the mobile phase.The purity of the isolated X1and X2was con-?rmed by HPLC and analyzed further.
3.3.Characterization of compounds X1and X2
Maximum UV absorbance of X1and X2are280.6(2325) and280.0(1918).The molecular formula and weight of X1 are C19H24O3and300,and of X2are C19H26O3and302.The
148M.Horinouchi et al./Journal of Steroid Biochemistry&Molecular Biology92(2004)143–154
Table2
NMR data for X1and X2a,b
Number X1X2
13C NMR,δ(ppm)1H NMR,δ(ppm),J(Hz)13C NMR,δ(ppm)1H NMR,δ(ppm),J(Hz) 1131.2 6.99d(8.2)131.1 6.98d(8.2)
2112.8 6.60dd(8.2,2.7)112.6 6.58dd(8.2,2.8)
3153.8153.6
4115.7 6.67d(2.7)115.7 6.65d(2.8)
5141.8142.3
631.0 2.72ddd(13.3,11.5,4.6)31.0 2.67ddd(13.3,11.9,5.0)
2.46ddd(1
3.3,11.9,5.5) 2.43m
726.7 1.88m,1.69m27.2 1.76m,1.57m
849.7 2.52m50.4 2.41m
9211.3212.4
10127.9128.1
1137.4 2.55–2.45m37.9 2.52ddd(15.6,14.2,7.3),2.38m 1230.4 2.02m,1.70m35.5 2.02ddd(12.8,6.4,1.8),1.53m 1347.643.3
1449.2 1.89m49.7 1.62m
1522.5 2.06m24.1 1.72m
1.73m 1.50m
1636.1 2.58br dd(19.0,7.8),2.26ddd(19.0,9.6,9.6)31.1 2.19m,1.64m
17218.880.5 3.77dd(8.7,8.7)
1813.5 1.17s10.7 1.08s
1918.4 2.26s18.3 2.25s
Abbreviations for NMR signals are as follows:s,singlet;d,doublet;m,multiplet;br,broad.
a The molecular formula and weight of X1and X2are C19H24O3and300,and C19H26O3and302,respectively.
b Maximum UV absorbance of X1and X2are280.6(2325)and280.0(1918).
UV-spectra and MS data of X1and X2corresponded to those
of3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione
(3-HSA)and3,17-dihydroxy-9,10-secoandrosta-1,3,5(10)-
triene-9-one(3,17-DHSA)presented by Dodson et al.in1958
and1961[1–3].Table2shows the1H NMR,13C NMR,
and2D-NMR(FGDQCOSY,FGHMQC,HMBC,and DEPT)
data of compounds X1and X2.NMR data of X1agreed,on
the whole,with the partial13C NMR data of3-HSA presented
by Furukawa et al.in1979[19],and with the partial13C NMR
data of Hamato et al.in1988[20],the exceptions being for
C1(131.2ppm in our data and127.3ppm in theirs),C10
(127.9ppm and130.9ppm,respectively),C8(49.7ppm and
49.0ppm,respectively),and C14(49.2ppm and49.6ppm,re-
spectively).From these results,X1and X2were identi?ed as
3-HSA and3,17-DHSA,respectively(Fig.4).Our data
pre-
Fig. 4.Structure of compounds X1and X2.R O:3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione and R OH:3,17-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9-one.sented in Table2is the?rst complete13C and1H NMR data of3-HSA,together with complete NMR data of3,17-DHSA. NMR data of3,17-DHSA has not been reported before. 3.4.Conversion of3-HSA by ORF11and ORF12
As the ORF11-and ORF12-disrupted mutants accumu-lated3-HSA and both of the ORFs showed about30%iden-tity to some oxygenases,ORF11and12were expected to en-code the hydroxylase for converting3-HSA to3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione(3,4-DHSA). We constructed pUC19-derivative plasmids encoding ORF11 (pUCORF11in Fig.2)and ORF12(pUCORF12)and intro-duced the plasmids into E.coli.The E.coli cells harboring each of the plasmids were incubated with induction by IPTG, and cell free extract was prepared as described in our previous study of TesD[15].To prepare3-HSA,the ORF12-disrupted mutant was incubated with testosterone until all the added testosterone was converted to3-HSA and3,17-DHSA.The cells were removed by centrifugation and the supernatant was used as reaction solution.The reaction solution was treated with E.coli harboring each of the plasmids encoding ORF11 and ORF12,and the cell free extract.As3,4-DHSA was not detected by HPLC,the remainder of the3-HSA was ana-lyzed by HPLC at suitable intervals.However,neither the E.coli cells nor their cell free extracts showed conversion of3-HSA(data not shown).Then we constructed pUC19-derivative plasmids encoding ORF11and12in the opposite direction(pUCORF(11)12),ORF12and11in the same di-rection(pUCORF1211),ORF11–18(pUCORF11–18),and
M.Horinouchi et al./Journal of Steroid Biochemistry&Molecular Biology92(2004)143–154
149
Fig.5.HPLC charts of the culture of ORF11-disrupted mutant carrying a broad-host range plasmid pMFY42incubated with testosterone(a),ORF11-disrupted mutant carrying pMFYORF11(pMFY42based plasmid expressing ORF11)(b),ORF12-disrupted mutant carrying pMFY42incubated with testosterone(c), and ORF12-disrupted mutant carrying pMFYORF12(pMFY42based plasmid expressing ORF12)(d).The analysis was carried out under conditions identical to those described in the legend of Fig.3.Abbreviations are as follows:Ts,testosterone;4-AD,4-androstene-3,17-dione;ADD,1,4-androstadiene-3,17-dione;17OH-ADD,17?-hydroxy-1,4-androstadiene-3-one;3-HAS,3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione-1,4-androstadiene-3,17-dione; and3,17-DHSA,3,17-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione-1,4-androstadiene-3,17-dione.Identi?cation of these compounds except for 3-HSA and3,17-DHSA was performed in the previous study[15].
ORF11–12,17,and18(pUCORF12111718)(Fig.2),and introduced each of them into E.coli.The conversion exper-iment was performed in the same manner as above using E. coli cells and the cell free extract with these plasmids,but reduction of3-HSA was not observed.As this might be due to proteins encoded by ORF11and ORF12not having activ-ity when expressed in E.coli cells,the gene region encoding each ORF was transferred into the broad-host range plasmid, pMFY42[21],which can be maintained in Pseudomonas and its relatives(pMFYORF11and pMFYORF12).Plasmids pMFYORF11and pMFY42(negative control)were individ-ually introduced into the ORF11-disrupted mutant of TA441, and pMFYORF12and pMFY42(negative control)were indi-vidually introduced into the ORF12-disrupted mutant.These mutants were incubated with testosterone and the remaining 3-HSA was analyzed by HPLC at suitable intervals.HPLC chart of the culture about38h after start of the incubation is presented in Fig.5.3-HSA accumulated in the culture of the mutants with pMFY42(negative control),while3-HSA was not detected in the culture of the ORF11-disrupted mu-tant with pMFYORF11or the ORF12-disrupted mutant with pMFYORF12.This indicates that both ORFs are involved in testosterone degradation in TA441.Then we considered a similar complementation experiment using a mutant gene-disrupted for both ORF11and12to con?rm that both ORFs are necessary for conversion of3-HSA.ORF activity may be determined by measuring the amount of unreacted3-HSA, but detection of the produced3,4-DHSA would be a more direct means of determining ORF activity.The method for con?rming presence of3,4-DSHA in culture is described in the next section.
3.5.Detection of3,4-DSHA
The complementation experiment described in the previ-ous section indicated that both ORF11and ORF12are in-volved in the conversion of3-HSA in testosterone degrada-tion by TA441.The product of the hydroxylation of3-HSA is thought to be3,4-DHSA,but we were not successful in de-tecting it in our previous study.3,4-DHSA was reported to be one of the intermediate compounds in steroid degradation by Nocardia restrictis[6],which degrades steroids by a degra-dation pathway as almost identical to that of C.testosteroni [1–9].In testosterone degradation in TA441,a meta-cleavage enzyme TesB,encoded in another steroid degradation clus-ter of TA441,converts3,4-DHSA to4,9-DSHA[14].Fig.6a shows the HPLC chart of the culture of a TesB-disrupted mu-tant incubated with testosterone in the mixed LB+C media. This mutant is assumed to accumulate3,4-DHSA;however, only4-AD and ADD,the?rst and the second intermediates in the testosterone degradation of TA441,were detected as intermediate compounds.As we could not detect3,4-DHSA
150M.Horinouchi et al./Journal of Steroid Biochemistry &Molecular Biology 92(2004)
143–154
Fig.6.HPLC charts of the culture of TesB-disrupted mutant incubated with testosterone (a),with ADD for three days (b),for one day (c),and the culture of (c)treated with the cell free extract of E.coli expressing TesB (d).The analysis was carried out under conditions identical to those described in the legend of Figs.3and 4,9-DSHA:4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid.Other abbreviations are the same as those described in the legend of Fig.5.
in the culture of the TesB-disrupted mutant incubated with testosterone,we incubated the TesB-disrupted mutant with several steroids,with the expectation of detecting the sub-strate of the meta-cleavage reaction.To our surprise,cul-tures of TesB-disrupted mutant incubated with cholic acid and those incubated with ADD became dark red within a few days,while the same color change was not observed following incubation with testosterone.A dark red color is often observed when catechol derivatives accumulate dur-ing bacterial degradation of aromatic compounds.Catechol is a compound comprising a benzene ring with two adja-cent hydroxyl moieties and automatically oxidizes to produce quinone derivatives,which gives dark reddish color.As ADD is an intermediate compound in testosterone degradation by TA441,the accumulated compound should be 3,4-DHSA.HPLC analysis of this dark red culture showed a number of peaks between RT 8and 12,which were not detected in the culture of any other gene-disrupted mutant incubated with testosterone (Fig.6b)[15,16].These compounds would be produced by automatic oxidation of 3,4-DHSA and would color the culture medium dark red.The isolation and charac-terization of these compounds was dif?cult,and therefore we con?rmed the accumulation of 3,4-DHSA by conversion of 3,4-DHSA to 4,9-DSHA by TesB,followed by detection of 4,9-DSHA by HPLC.As a preliminary experiment showed that accumulation of red compounds prevented the TesB re-action from proceeding,we interrupted the incubation of the TesB-disrupted mutant with ADD before the culture showed pigmentation.Following incubation for about 1day,fewer peaks were detected between RT 8and 12than in the 3-day
culture,and a small amount of 3-HSA was detected (Fig.6c).The cells of the TesB-disrupted mutant were separated from the culture by centrifugation,and the supernatant was trans-ferred to a new test tube.About 2h after the addition of the E.coli ?cells expressing TesB,the reaction solution was yellow (4,9-DSHA is brilliant yellow under neutral and ba-sic conditions [15]).HPLC analysis of this yellow solution showed the accumulation of 4,9-DSHA (Fig.6d),indicating that we could detect 3,4-DHSA by conversion to 4,9-DSHA.When we treated the culture of TesB-disrupted mutant in-cubated with testosterone in the same way with E.coli ?cells expressing TesB,smaller amount of 4,9-DSHA was detected.
3.6.Conversion of 3-HSA by ORF11and ORF12in ORF11,12,and tesD-disrupted mutant
For the complementation experiment for both ORF11and 12,we constructed the gene-disrupted mutant 1112D ?(an ORF11,ORF12,and tesD -disrupted mutant).As TesB is en-coded in a different gene cluster from that of ORF11and 12,when 3,4-DHSA is produced in this mutant,it is converted to 4,9-DSHA by TesB,and the resultant 4,9-DSHA accumu-lates because TesD,the hydrolase for 4,9-DSHA,is disrupted (Fig.7).We constructed pMFY42-based broad-host range plasmids encoding ORF11and 12in the opposite direction (pMFYORF(11)12)and ORF12and 11in the same direction (pMFYORF1211).Plasmids pMFYORF11,pMFYORF12,pMFYORF(11)12,pMFYORF1211,and pMFY42were indi-vidually introduced into the gene-disrupted mutant 1112D ?.
M.Horinouchi et al./Journal of Steroid Biochemistry&Molecular Biology92(2004)143–154
151
https://www.sodocs.net/doc/037677424.html,plementation experiment of ORF11and ORF12.HPLC chart of the culture of the1112D?(ORF11,12and TesD-disrupted mutant)carrying pMFY42incubated with testosterone(a),1112D?carrying pMFYORF11(b),1112D?carrying pMFYORF12(c)1112D?carrying pMFYORF(11)12(d),and 1112D?carrying pMFYORF1211.pMFYORF(11)12is a broad-host range plasmid encoding ORF11and ORF12in the opposite direction and pMFYORF1211 is a broad-host range plasmid encoding ORF12and ORF11in the same direction.The analysis was carried out under the same conditions stated in the legend of Fig.3.4,9-DSHA-N:4-aza-9,17-dioxo-9,10-secoandrosta-1,3,5(10)-triene-3-carboxylic acid.4,9-DSHA-N was produced from4,9-DSHA non-enzymatically by ammonium ion in the culture media added as a nitrogen source[15].Other abbreviations are the same as those described in the legend of Figs.5and6.
The mutants were incubated with ADD,and the culture was analyzed by HPLC at suitable intervals.The amount of4,9-DSHA detected by HPLC peaked at38h after the start of the incubation(Fig.7).In the culture of1112D?with pMFY42 (negative control),1112D?with pMFYORF11,and1112D?with pMFYORF12,3-HSA accumulated and4,9-DSHA was only detected in a very small amount in the culture of1112D?with pMFYORF11(Fig.7a–c).In the culture of1112D?with pMFYORF(11)12and1112D?with pMFYORF1211, 4,9-DSHA was detected with little amount of3-HSA.The amount of4,9-DSHA was larger in the culture of1112D?with pMFYORF1211than in that of1112D?with pMFY-ORF(11)12.These results indicate that both ORF11and12 are necessary for effective hydroxylation of3-HSA at the C4-position.We therefore named ORF11and ORF12tesA2and tesA1,respectively.
4.Discussion
In C.testosteroni TA441,steroids are degraded via aromatization of the A-ring,then meta-cleaved and further degraded by hydrolysis.In our previous study,we reported two steroid degradation gene clusters in TA441;one con-taining the meta-cleavage enzyme gene tesB,which converts 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dio-ne(3,4-DHSA)to4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid(4,9-DSHA),and the
152M.Horinouchi et al./Journal of Steroid Biochemistry &Molecular Biology 92(2004)
143–154
Fig.8.Alignment of the TesA1and proteins showing similarity to TesA1:(*)amino acids identical among all the proteins;(:)and (.)amino acids of high and low similarity,respectively.Abbreviations are U39411:nitrilotriacetate monooxygenase component B of Chelatobacter heintzii (Aminobacter aminovorans )ATCC 29600[18,23],AE008260:?avoprotein oxidoreductase Atu4239of Agrobacterium tumefaciens strain C58[24],AF380367:putative NADH:FMN oxidoreductase of Burkholderia sp.DBT1(available only on database under accession number AF380367),AP005957:nitrilotriacetate monooxygenase of Bradyrhizobium japonicum USDA110[25],BX640449:putative monooxygenase component of Bordetella bronchiseptica RB50[26],BX640420:putative monooxygenase component of Bordetella pertussis Tohama I [26],and BX640434:putative monooxygenase component of Bordetella parapertussis 12822[26].
other containing tesD ,a gene encoding the hydrolase for 4,9-DSHA,and consisting of ORF18,17,tesI ,H ,A2,A1,and tesDEFG .Of these,TesA1and TesA2were considered to be oxygenases because they showed homology (maxi-mum about 30%)to some oxygenases,such as the phenol hydroxylase of B.stearothermophilus BR219[17](TesA2)and component B of nitrilotriacetate monooxygenase of Chelatobacter strain ATCC 29600[18](TesA1),but the functions were not clari?ed in the previous study.In the present study,two characteristic intermediate compounds accumulated by the gene-disrupted mutants of TesA1and TesA2grown on testosterone were isolated,puri?ed,and identi?ed by MS and NMR analysis as 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA)and its hydroxylated derivative,3,17-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,17-DHSA).Several sets of NMR data were available for 3-HSA,but they were not complete [19,20].Our data in the present study are the ?rst complete set of NMR data for 3-HSA and 3,17-DHSA,as well as the ?rst report identifying the enzyme genes for conversion of 3-HSA in bacterial steroid degradation.Hydroxylation and dehydroxylation at the C17-position is probably a reversible reaction,as both 3-HSA and 3,17-DHSA accumulated when either testosterone,which has a hydroxy group at position C17,or ADD,which has a ketone group at position C17,was used as the substrate.3-HSA is
M.Horinouchi et al./Journal of Steroid Biochemistry&Molecular Biology92(2004)143–154153
the major intermediate compound,and3,17-DHSA is minor. Although we could not detect hydroxylation of3-HSA in E.coli transformants expressing TesA1and/or TesA2,or in the cell free extract,complementation experiments using broad-host plasmids carrying the same gene region showed that both TesA1and TesA2are necessary for the effective hydroxylation of3-HSA at the C4-position.One possible reason for the absence of the activity in E.coli expressed TesA1and TesA2is overexpression.TesA2may have low hydroxylation activity without TesA1,as a weak conversion of3-HSA was detected from TesA2in the complementation experiment.
For more information,we analyzed the putative amino acid sequences of tesA1and tesA2using the sequence motif search of GenomeNet service(Kyoto University Bioinfor-matics Center,http://motif.genome.ad.jp/).GenomeNet ser-vice search provides motif searches against PROSITE Pat-tern(Swiss Institute of Bioinformatics),PROSITE Pro?le (Swiss Institute of Bioinformatics),BLOCKS(Fred Hutchin-son Cancer Research Center),ProDom(Insitut National de la Recherche Agronomique),PRINTS(University of Manch-ester),and Pfam(Washington University,St.Louis and Sanger Centre)at a time.Although TesA2showed weak hydroxylation activity,no putative functional domains were found in TesA2.Prodom presented a group of enzymes with ID008747as similar to TesA2.Most of the enzymes in-cluded in this group are thought to be hydroxylases from their amino acid sequences,but most of them were only pu-tative hydroxylases.Of these,an oxygenase of Rhodococ-cus erythropolis TA421[22]showed the maximum iden-tity,33%,with TesA2.E.coli expressed TA421oxygenase showed oxidization of indole,which resulted in pigmenta-tion on the E.coli colony,but E.coli with the plasmid ex-pressing TesA2did not show the pigmentation.The search results for TesA1by BLOCKS,ProDom,and Pfam indicated a?avin reductase-like domain in TesA1.The region contain-ing the?avin reductase-like domain in TesA1proposed by the search was not identical,but all the amino acids proposed as the?avin reductase-like domain are contained in the re-gion from the aa number10–160.Fig.8shows the alignment of TesA1and proteins showing homology to it.Most of the proteins which have homology to TesA1consist of170–180 aa,and a few consist of about320aa,which is almost the same length as TesA1.Though they have the similar length, they showed little homology with each other after about170 aa.NtaB,component B of nitrilotriacetate monooxygenase of Chelatobacter strain ATCC29600[18],showed the high-est homology—about33%—to TesA1.The nitrilotriac-etate monooxygenase of ATCC29600is a two-component monooxygenase encoded by ntaA and ntaB,which are di-vergently oriented.NtaB is a?avin mononucleotide(FMN)-containing protein with nitrilotriacetate-stimulated NADH-oxidizing activity.The role of NtaA remains unclear,but its presence was absolutely necessary for oxygenation of nitrilo-triacetate.NtaA shows about40%homology to dibenzothio-phene monooxygenase SoxA.The mixture of puri?ed NtaA and NtaB showed oxygenation of nitrilotriacetate,while ad-dition of puri?ed NtaA to NtaB overexpressed by E.coli did not show oxygenation.As some of these characteristics of NtaA and NtaB are similar to those of TesA2and TesA1, TesA1and TesA2are thought to be a two-component oxyge-nase.
Our study clari?ed the functions of all genes,except for ORF17and ORF18,in the gene cluster consisting of ORF18, 17,tesI,H,A2,A1,and tesDEFG.However,the function of most of the genes in the other cluster containing tesB is unclear.Further studies are required to con?rm the na-ture of the substrates and products of the enzymes derived from these genes.Another steroid degradation gene cluster is likely present in TA441because some of the predicted enzyme genes such as an oxygenase component of the hy-droxylase of ADD at the C9-position were not found in the analyzed gene region.Characterization of the predicted but not yet isolated steroid degradation genes will also be impor-tant.
Acknowledgments
We thank Dr.Hiroyuki Koshino in RIKEN for his prac-tical advice on identi?cation of puri?ed intermediate com-pounds.This work was partly supported by a grant from the Eco Molecular Sciences Research Program from RIKEN. MH was supported by a grant from the Special Postdoctoral Researchers Program from RIKEN.
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中英文对照版合同翻译样本
1.Sales Agreement The agreement, (is) made in Beijing this eighth day of August 1993 by ABC Trading Co., Ltd., a Chinese Corporation having its registered office at Beijing, the People’ Repubic of China (hereinafter called “Seller”) and International Tradi ng Co., Ltd., a New York Corporation having its registered office at New York, N.Y., U.S.A. (hereinafter called “Buyer”). 2.WITNESSETH WHEREAS, Seller is engaged in dealing of (product) and desires to sell (product)to Buyer, and WHEREAS, Buyer desires to purchase(product) from Sellers, Now, THEREFORE, it is agreed as follows: 3.Export Contract This Contract is entered into this 5th day of August 1993 between ABC and Trading Co., Ltd. (hereinafter called “Seller”) who agrees to sell, and XYZ Trading Co., Ltd. (hereinafter called “Buyer”) who agrees to buy the following goods on the following terms and condition. 4.Non-Governmental Trading Agreement No. ＿＿This Agreement was made on the＿day of＿19＿, BETWEEN ＿ (hereinafter referred to as the Seller) as the one Side and ＿ (hereinafter referred to as the Buyer) as the one other Side. WHEREAS, the Seller has agreed to sell and the buyer has agreed to buy ＿ (hereinafter referred to as the Goods ) the quantity, specification, and price of which are provided in Schedule A. IT IS HEREBY AGREED AS FOLLOWS: 5.Contract For Joint-Operation Enterprise ＿＿ COMPANY LTD., a company duly organized under the Law of ＿＿ and having its registered office at (hereinafter called “Party A”) AND ＿＿ COMPANY LTD., a company duly organized under the Law of ＿＿ and having its registere d office at (hereinafter called “Party B”) Party A and Party B (hereinafter referred to as the “Parties”) agree to jointly form a Co-operation Venture Company (hereinafter referred to as the “CVC”) in accordance with “the Laws of the People’s Republic of C hina on Joint Ventures Using Chinese and Foreign Investment” and the “Regulations for the Implementation of the Laws of the People’s Republic of China on Joint Ventures Using Chinese and Foreign Investment” and other applicable laws and regulations. 6.MODEL CONTRACT Contract No. Date: Seller: Signed at: Address: Cable Address: Buyer: Address: Cable Address: The Seller and the Buyer have agreed to conclude the following transactions according to the terms and conditions stipulated below: https://www.sodocs.net/doc/037677424.html, of Commodity: 2.Specifications: 3.Quantity: 4.Unit Price: 5.Total Price: U.S.$: 6.Packing: 7.Time of Shipment: days after receipt of L/C. 8.Loading Port & Destination Port: From via to . 9.Insurance:
对翻译中异化法与归化法的正确认识
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专业合同系列，下载即可用 英文购销合同_中英文对照购销合同 范本(2021版) 导语：合同目的具有确定性。一般情况下，在订立合同时，当事人的合同目的应该是确定的。从严格意义而言，任何人的行为都是有目的的。合同的订立和履行也属于人的行为，而且是比较正式的行为，所以更应该具有一定的具体目的。因此，对于特定的合同当事人，其合同目的是确定的。 英文购销合同_中英文对照购销合同范本(一) Buyer: 买方： Add.: 地址： Seller: 卖方： Add.: 地址： This purchase contract (hereafter abbreviated “contract”) is signed by and between the Buyer and the Seller upon equal negotiations based on the Contract Law of P..R .China and other relevant laws and regulations.. Both parties agree to sell and buy goods on following terms and conditions. 此销售合同(以下简称“合同”)根据 >及相关法律法规并经由买卖双方经平等协商后共同签定，买方与卖方均同意以下条款和条件购买和出售货物。 1. COMMODITY NAME品名：Work glves 劳保手套
中英文对照合同11
采购合同 (Purchasing Contract) 合同编号(Contract NO.)： 合同签订地点(Signed At)：东营 签订时间(Date)：年月日出卖人(Seller)： 买受人(Buyer)： 鉴于：根据【中华人民共和国合同法】及相关法律法规，本着平等自愿、等价有偿、诚实诚信的原则，经双方协商一致，订立本合同。 According to the Contract law of the People's Republic of China and relevant laws and regulations,we conclude the contract on the basis of equality, voluntariness,compensation of equal value and sincerity. 第一条标的、数量、价款及交（提）货时间 Item 1 (object)Quantity Price and Delivery time 物资编码名称及规格型号Commodity code and type 生产厂家 Manufacture r 计量单位 Unit 数量 Quantity 单价（元） Unit Price 总金额（元） Total Amount 备注 Note 不含税总金额（人民币） Total Amount (V AT excluded) 交提货时间、数量：年月日至年月日全部交货。 Delivery time: From to 第二条质量标准：标准代号、编号和标准名称：GB150-98. Item 2 Quality standard: standard code,serial number and standard name:GB150-98. 第三条出卖人对质量负责的条件及期限 Item 3 Conditions and time limits that Sellers shall be held responsibility for 1. 质量负责条件和期限按正常运行12个月止执行。 1. Quality responsible conditions and time limits shall not be valid until the product has been functioning properly for 12 months 2. 出卖人对标的物的质量负责，产品均附质检报告单，质量保证书。如果出卖人知道或 者应当知道所处卖产品存在质量缺陷，所承担的质量保证期限不受前款质量负责期限的约束，应依法承担相应责任。 2. Sellers shall be responsible for the quality of the marks.The products should include the Quality Inspection Report and Quality Guarantee.If the sellers know or should know the products they sell with defects in quality,the quality assurance time limits should not be subject to the previous quality responsibility deadline and they should be held due responsibility for it. 第四条包装标准：标准包装，产品所需包装由出卖人合理包装，适合水运和长途内陆运输，防潮，防湿，防锈，防震。 Item 4 Packaging standard: Standard packaging, the sellers should package the products properly, it should be suitable for shipment and long distance inland transportation. The packaging of the products should be resistant of dampness, moisture, rust and shock. 第五条随机的必备品、配件、技术资料、工具数量及供应方法：随设备交货一起交齐。 Item 5 The accompanied necessary materia l, accessories, technical information ,quantity of
翻译的归化与异化
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翻译的归化与异化 作者：熊启煦 作者单位：西南民族大学,四川,成都,610041 刊名： 西南民族大学学报（人文社科版） 英文刊名：JOURNAL OF SOUTHWEST UNIVERSITY FOR NATIONALITIES（HUMANITIES AND SOCIAL SCIENCE） 年，卷(期)：2005,26(8) 被引用次数：14次 参考文献(3条) 1.鲁迅且介亭杂文二集·题未定草 2.刘英凯归化--翻译的歧路 3.钱钟书林纾的翻译 引证文献(15条) 1.郭锋一小议英语翻译当中的信达雅[期刊论文]-青春岁月 2011(4) 2.许丽红论汉英语言中的文化差异与翻译策略[期刊论文]-考试周刊 2010(7) 3.王笑东浅谈汉英语言中的差异与翻译方法[期刊论文]-中国校外教育（理论） 2010(6) 4.王宁中西语言中的文化差异与翻译[期刊论文]-中国科技纵横 2010(12) 5.鲍勤.陈利平英语隐喻类型及翻译策略[期刊论文]-云南农业大学学报(社会科学版) 2010(2) 6.罗琴.宋海林浅谈汉英语言中的文化差异及翻译策略[期刊论文]-内江师范学院学报 2010(z2) 7.白蓝跨文化视野下文学作品的英译策略[期刊论文]-湖南社会科学 2009(5) 8.王梦颖探析汉英语言中的文化差异与翻译策略[期刊论文]-中国校外教育（理论） 2009(8) 9.常晖英汉成语跨文化翻译策略[期刊论文]-河北理工大学学报（社会科学版） 2009(1) 10.常晖对翻译文化建构的几点思考[期刊论文]-牡丹江师范学院学报（哲学社会科学版） 2009(4) 11.常晖认知——功能视角下隐喻的汉译策略[期刊论文]-外语与外语教学 2008(11) 12.赵勇刚汉英语言中的文化差异与翻译策略[期刊论文]-时代文学 2008(6) 13.常晖.胡渝镛从文化角度看文学作品的翻译[期刊论文]-重庆工学院学报(社会科学版) 2008(7) 14.曾凤英从文化认知的视角谈英语隐喻的翻译[期刊论文]-各界 2007(6) 15.罗琴.宋海林浅谈汉英语言中的文化差异及翻译策略[期刊论文]-内江师范学院学报 2010(z2) 本文链接：https://www.sodocs.net/doc/037677424.html,/Periodical_xnmzxyxb-zxshkxb200508090.aspx
房屋租赁合同中英文对照范本
房屋租赁合同LEASE CONTRACT-中英文对照 发布时间：2010-12-19 11:18:55 http: 本合同双方当事人 出租方（甲方）： 承租方（乙方）： 根据国家有关法律、法规和本市有关规定，甲、乙双方在平等自愿的基础上，经友好协商一致，就甲方将其合法拥有的房屋出租给乙方使用，乙方承租使用甲方房屋事宜，订立本合同。 一、建物地址 甲方将其所有的位于上海市区的房屋及其附属设施在良好状态下出租给乙方使用。 LEASE CONTRACT Lessor （hereinafter referred to as Party A）： Lessee （hereinafter referred to as Party B）： Party A and B have reached an agreement through friendly consultation to conclude thefollowing contract. 1.Location of the premises Party A will lease to Party B the premises and attached facilities owned by itself which is located at and in good condition for .
二、房屋面积 出租房屋的登记面积为平方米（建筑面积）。 三、租赁期限 租赁期限自____年__月__日起至____年__月__日止，为期年，甲方应于年月日将房屋腾空并交付乙方使用。 2.Size of the premises The registered size of the leased premises is __squaremeters （Gross size）。 3.Lease term The lease termwill be from（month）（day）（year） to_（month）___（day）___（year）。Party A will clear the premises and provide it to Party B for use before ___（month） ___（day）___（year）。 四、租金 1.数额： 双方商定租金为每月___元整（含管理费）。乙方以___形式支付给甲方. 2.租金按___月为壹期支付；第一期租金于___年___月___日以前付清；以后每期租金于每月的___日以前缴纳，先付后住（若乙方以汇款形式支付租金，则以汇出日为支付日，汇费由汇出方承担）；甲方收到租金后予书面签收。 3.如乙方逾期支付租金超过七天，则每天以月租金的 0.3％支付滞纳金；如乙方逾期支付租金超过十天，则视为乙方自动退租，构成违约，甲方有权收回房屋，并追究乙方违约责任。 4.Rental