Volum~ =JO, number ;L 35|=~:t~ FEB$ 09S21! I ~ | Feder~ll=n at l~urop~¢+ Bio~hentkai ~ N t k ~ oOi4~r~e]/'Olt$~l,~ ADONI$ t~1145"/~191~ , V / t /
Mar~h I991
Anaerobically induced expression of the nitrite reductase cytochrome c-551 operon from Pseudomonas aeruginosa H i r o y u k i Arui, Y~tsuo Illarashi and~ T o h r u K o d a m a
Deparlm¢~t ~[ dgrt¢~dmeal Chemistry. UMwP~tIy¢+fTokya. Bunk.v,.k++.T~ky, 113. Japan Received ? D~¢embcr 1990; revised v~rsion received 25 January 1991 "The nit rite redt~¢tascilene idea+4) alld the eyto+;hrome ¢-$$1 liene (draB) arc I¢~aled only, :S0bp ~part from each other in the P~¢mlamr~l+t.~ueruxhl~a ¢hrom~.,~me, We r~p~rt evidence the'+these tw¢~=t:nes are +;o.transwibc,d .s an operon only under anaerobic (d,~nitrifyln~) conditions. The nudeo. tide ~equ,cnceo1' the prometer [rc~j[ulatg+l} rcllion or lh¢ opcru1'~Is hishl~' AT-rich and coat+tins a sequence clo,~=lyrosemblint= the consensus FNR bindinl! site in E. ¢~tL
Nitriter~luctas¢:Cylochrome ¢.~.~I,Promoter; FNR blndlnilsite:P~¢udam,nc~ ,¢ruFllnoxa
I. I N T R O D U C T I O N
#B/nil) and streptomycin (~00/41ml) for P. aeruxino~u.Transfer of plasmid~ from E. colt to P. aeruginosa was performed by the triparen.
Nitrite reductase (cytochrome cd=: EC 1.9.3,2) of Pseudomones aeruginosa ill is a soluble redox protein occurring in the pcriplasm [21. It catalyzes the reduction of nitrite to nitric oxide [3] and thus plays an important role in anaerobic respiration (denitrification). The activity of this dissimilatory enzyme has been re r~orted to be expressed only when the cells are grown an~.erobically in the presence o f nitrate [41. Coyne et at. [51 have also immunochemically shown that the enzyme protein is synthesized exclusively in denitrifying cells. It is further known that cytochrome c-551, a small redes pro, tein, acts as the direct electron donor for nitrite reductase [6]. We have recently cloned a gene cluster encoding both nitrite reductase and cytochrome c-551 and shown that the two genes are located only 50 bp apart from each other [7]. Here we report that the two genes are transcribed as an operon from an oxygen-regulated promoter. We also propose to call the nitrite reductase and cytochrome c-551 genes (lenA and denB, respectively.
tal matiall method uslnB pRK2013 as a I~elper plasmid II0l.
2. MATERIALS A N D METHODS P. aerugmosa PAOLI61 [8] and E. colt J M I 0 9 [9] were grown at 37°C in YT-broth (5 g yeast extract, 8 g trypton and 5 g NaCl/I, pH 7.3) or on YT-plate, For anaerobic cultivation, 100 mM NaNO3 or 20 mM NaNOz was added to the medium in sealed flasks and air in the heads pace was replaced by N2 gas. ~vVhennecessary, the following an. tibiotics were added to the medium: anapicillin (50/~g/ml), kanamycin (25/zg/ml) and streptomycin (10/~g/ml) for E'. colt; carbeniclllin (300 Correspondence address: T. Kodama, Department of Agricultural Chemistry, University of Tokyo, Bunkyo-ku, Tokyo 113. Japan
Published by Elsevier ,Science Publishers B, V.
Pset,domonas isolation a~ar tDifco) was used for isolation of P.
aeruainosa transtormnnts, pTSI04S (tncq, Ampicillin f, Streptomycin', :~'ylE) was used to probe the promoter activity of DNA frailments, tra functional promoter is ligated to the upstream ors.viE in this vector, the activity of catechol 2,3.dioxygenase (C230), the
.x'y/E gene product, can be expressed [i l]. Protein was determined by Bio.Rad method using bovin~ serunl albu rain as a standard, DNA sequencin~ was performed as described [71. Other DNA, manipulations were carried out as described by Sambrook et al. [91.
3. RESULTS AND DISCUSSION
3.1. Identification o f promoter region o f the nitrite reduclase gene Various restriction fragments of the upstream region of the nitrite reductase gene (denA) were ligated to the promoter probe vector pTS1045. The constructed plasmids, pHA301 through 306 (see Fig. 1), were transferred to P. aeruginosa. The promoter activities of the ligated fragments were then examined under both aerobic and anaerobic conditions by measuring the activity of C230, the xylE gene product. As shown in Table I, under aerobic conditions, no or little C230 activity was expressed in the cells harbouring a n y of the constructed plasmids even inthe presence of nitrate or nitrite. On the other hand, under anaerobic conditions the cells carrying pHA301, pHA302, pHA304 or pHA306, but not pHA303 or pHA305, expressed high C230 activities when grown in the presence of nitrate or nitrite. These results indicated that the nitrite reductase gene is transcribed only under anaerobic conditions and the promoter and regulatory region of this gene is located in the ApaI-EcoRI fragment (see Fig. 1). It was
351
Volazme ; ~ , number =
hlar~:h 1991
FI~B$ LI~TTER~
__
~_~_ =~ j~,
~
CenA-" pHA301 pHA302 L pHA303 1
I
pHA304
~
pHA305 PHA306 pHA307
~mlll
IsHA300
I
T~l~le i
_
,~
~.t~
C230 a~livtty in ¢,dl ~xtrttet of P. ~r~#l.~#. PAOLI61 ,¢o.mlni.ll pT$1045 lind pHA]0t-3011
C2]O aedvtt~, (U/roll protein) aerc,bk: ~.a,erobl~
1
VT
NO," 1OOmM
NO=° 20mM
NO~'* 100ram
NO=~ 20 mM
pTSIO4J~ pHA301 pHA302 pHA3O;I
l',61 1,47 1,67
0.99
1,91 1.42
1. t t; 2.09 2.46
1,70 20,94 29,7#
0,98 253?
1,62
l ,,~II
2.86
:L21.
217
pHA~O~
=,l~
I,t6
t.,=,~
l,=O
t,n
pHA306
1,43 I,n
1,62
2,08
20,00 to.as
~1,25
I ,
t-i
I
Fig, I. Fraltlments used for C230 a~say, "The plasmld~ pHA301.308 were constructed by lillafing the~e reslrl¢lion fragments containing for
pH^~0~ pH^30t~
28.66
i.~9
t=o
denA and./or its flanking reBIon~ to the upstream of .~'),IEof the promoter probe vector p"TSI04S, A, Apal; E, EcoRI; M, Small 5, gphl,
also suggested that the activity of this promoter (regulator) region is sensitive to molecular oxygen. 3.2. Identification o f promoter region o f the cyto.
chrome c.551 gene In the chromosome of P. aeruginosa, the cytochrome c-551 gene (denB) is located 50 bp downstream of denA and no promoter-like sequence can be found in this 50~bp region [7,12], suggesting that these two genes are transcribed as an operon. To confirm this possibility, two fragments were prepared, ligated to p'rS1045 (pHA307 and pHA308, see Fig. 1), transferred to P. aeruginosa, and their promoter activities were measured by C230 assay, pHA307 contains the (lena promoter region, in addition to the (lenA coding region, 50-bp non-coding region, and about half of the denB coding region, whereas pHA308 lacks the denA promoter region. As shown in Table 1, the cells harbouring pHA308 did not express C230 activity even under anaerobic conditions and in the presence of nitrate, indicating that the promoter for denB is not located in pHA308. On the other hand, the cells carrying pHA307 expressed C230 activity when grown anaerobically in the presence of nitrate. The growth of the latter cells was, however, very poor under anaerobic conditions, probably because of a large amount of nitrite reductase
expressed from the multicopy plasmid. The C230 activity of the cells harbouring pHA307 $rown anaerobically was also considerably lower than that expressed in the cells carrying pHA304. This low expression was probably due to the p o o r growth. In any case, it was con. eluded that denB is transcribed from the denA promoter; in other words, denA and denB are transcribed as an operon. 3.3. Nucleotide sequence o f the promoter (regulator)
region o f denAB We determined the nucleotide sequence of the (lenA promoter region (ApaI-EcoRl fragment). As shown in Fig. 2, this region is highly AT-rich as compared tothe average GC content of P. aeruginosa DNA (67-68070) [13]. This high AT content may be necessary for the binding of regulatory elements. In this region there is a sequence that is closely similar to the consensus FNR binding site (A-A-TTGAT--A-ATCAAT--.) of E. coil [14]. F N R (regulatory protein for fumarate and nitrate reduction) is a regulator for anaerobiosis of 17.. coil and activates t h e expression of proteins such as nitrate reductase, which are required for anaerobic respiration [15]. Azurin. a blue copper protein, is able to act as an alternative electron donor for nitrite reductase [6], and a putative FNR binding site is also located in the upstream region of the azurin gene of P. aeruginosa
~'~TTGA
GCAATACCGG
CAGGCCGTGC
CGCCAGGCGC
GCTCGAAGAC
TTCGATCTCA
60
TGGCCGGTGG
CCTCGTAGAA
GGGTGTCGCG
CGCTAGGGAT
120
GC~A~C~TGA
180
ATGAATTC
TAGGACCGCA
CGCTA~TCAC
AG~GGAAGG
TCCCGCATGT CCTAC?CCTG ' 0RF1 TGCCACAAGC GCAAAGCAAC
TTCCGGTCAA
GCAA~GGTAA
AGACCCTGCT
TTCTATGATC
_EcaRI CTTTCGCGCC
238
Fig. 2. Nucleotide sequence of the promoter (regulatory) region of denAB ( A p e l - E c o R l fragment). The sequence resembles to the consensus FNK binding site of E. coil is boxed, The arrow underline an open reading frame extending to the opposite direction of denA (ORFI), The putative ribosome binding site for ORFI is underlined.
352
Volume 2110, number 2
FEBS LETTEK$
[16], The occurr=nce or un FNR.like element in this orl~nism has been s~Uested by Lod~te et al. [|7]. It seems that the regulatory mechanism for anaeroblosis in P, aeru~inox# resembles thzzt in E, colt, We, also fou~d that upstream rei~ion of dana hes an an~erobic=lly inducible promoter activity in the opposite direction (dat~ not shown), This region forther contains an open reading frame (ORF! in Fig, 2), shortly after which a putative ribosome binding site is located. It is likely that ORFI encodes ~n =zctivator for the transcription o f the denAB operon, as in the case of trp/.rrpBA in P, ~en~gtnos~ [18],
Ack'~eowledfcmcnt: We Uttmk Dr T, Nak.~xawa (Y~tinagt~chi Ufliv,) for kindly providins DTSIO4S, REFERENCES [ll Y~manuke, T,. Ota, A. ~nd Okunuki, K. (1960) Biochtm. Biophys, Acts 44, 39"/-398, [2] Wood, P,M,, (t978} FEBS Left,92, 2[4-2i& [3| Henry. Y. and Besst~'.res,P. (1984) Biochimic 66, 2~g-289, [4l Yamanaka, T,, O|a, A. and Okunuki. K, (196|) Biochim. Biophys. ActP.53, 294-308,
March 1991
[:~! Coyne, M.S.. Arunakum~ri. A., Pankr#l~, H.$. ~nd Tledj~, J.M, (I~)0) J, Uacterlol. 172, 2~$#=]~62. [6) Z~mtlonl. D. (19119)B1ochlm. t~iophys,A¢[~ 19i[9,;I~=316. [7| ArA]. H,, ~=nbo~lll, Y,, tilarashl,Y, and Kodnma, T, (I~0} FEB$ L~t, 26[, 196~198. [g| Ounn, N,W, and Holl~way, B,W. (1971) G~n¢|, Bes. 18, tgS-19'~, [91 Saml)rook, J,, Pt|lsch,T. and MAni~Us, 3'. (1989) Molecular Cloning: A L~l~oratoryM,nu~l, 2rid cdn, Cold Spring Harbor Laboratory, NaveYork. [t0i Dicta, G., Stanfleld. $,. Corbtn, D, and Helinskl. D.R~ (1980) Proc, N~I{],Acid. Set. USA 77, 7347-735L [ell lno~sye,S., As~i, Y,. Nak~t~wa, A. =nd Naku~w~, T. (1986)J. Bactcriol, 166, 739-745, [121 Silvesirlni, M.¢,, Caleoui, C.L,, Qcrv~Is, M,, $chlninA, E., B~rr~, D,, Bossa, F. and Brtmorl, M. (1989) FEBS Levi, 2S4. 33=38. [13] Was(, $,E.H. and Islewski, B,H, (1988) Nucleic Acids Rex. 16. g323-933~, {14] $piro, S. and Quest, J.R, (1990) FEM$ Mtcrobiol. Bey. ?S. 399-428, [ISl Stewart, v. (1982) J, Bacterioi. iS[, i320-132S, {161 Hoitink, C,W,Q,, Woad(, L.P~, Turenbout. J.C,M., van de Kamp, .hi, and Canters, G,W, (1990) Gene 90, !$-20. {17l Lodge, J,, Williams, R., Bell, A,, Chan, B. and Busby,S. (1990) FEMS Microbiol. Let|, 67. 221-22o. [~8) Chang, M,, Fladero, A, nnd Crawford, [.P, (1989)J. Bactcriol, 171, 172-;183.
353
Mar~h I991
Anaerobically induced expression of the nitrite reductase cytochrome c-551 operon from Pseudomonas aeruginosa H i r o y u k i Arui, Y~tsuo Illarashi and~ T o h r u K o d a m a
Deparlm¢~t ~[ dgrt¢~dmeal Chemistry. UMwP~tIy¢+fTokya. Bunk.v,.k++.T~ky, 113. Japan Received ? D~¢embcr 1990; revised v~rsion received 25 January 1991 "The nit rite redt~¢tascilene idea+4) alld the eyto+;hrome ¢-$$1 liene (draB) arc I¢~aled only, :S0bp ~part from each other in the P~¢mlamr~l+t.~ueruxhl~a ¢hrom~.,~me, We r~p~rt evidence the'+these tw¢~=t:nes are +;o.transwibc,d .s an operon only under anaerobic (d,~nitrifyln~) conditions. The nudeo. tide ~equ,cnceo1' the prometer [rc~j[ulatg+l} rcllion or lh¢ opcru1'~Is hishl~' AT-rich and coat+tins a sequence clo,~=lyrosemblint= the consensus FNR bindinl! site in E. ¢~tL
Nitriter~luctas¢:Cylochrome ¢.~.~I,Promoter; FNR blndlnilsite:P~¢udam,nc~ ,¢ruFllnoxa
I. I N T R O D U C T I O N
#B/nil) and streptomycin (~00/41ml) for P. aeruxino~u.Transfer of plasmid~ from E. colt to P. aeruginosa was performed by the triparen.
Nitrite reductase (cytochrome cd=: EC 1.9.3,2) of Pseudomones aeruginosa ill is a soluble redox protein occurring in the pcriplasm [21. It catalyzes the reduction of nitrite to nitric oxide [3] and thus plays an important role in anaerobic respiration (denitrification). The activity of this dissimilatory enzyme has been re r~orted to be expressed only when the cells are grown an~.erobically in the presence o f nitrate [41. Coyne et at. [51 have also immunochemically shown that the enzyme protein is synthesized exclusively in denitrifying cells. It is further known that cytochrome c-551, a small redes pro, tein, acts as the direct electron donor for nitrite reductase [6]. We have recently cloned a gene cluster encoding both nitrite reductase and cytochrome c-551 and shown that the two genes are located only 50 bp apart from each other [7]. Here we report that the two genes are transcribed as an operon from an oxygen-regulated promoter. We also propose to call the nitrite reductase and cytochrome c-551 genes (lenA and denB, respectively.
tal matiall method uslnB pRK2013 as a I~elper plasmid II0l.
2. MATERIALS A N D METHODS P. aerugmosa PAOLI61 [8] and E. colt J M I 0 9 [9] were grown at 37°C in YT-broth (5 g yeast extract, 8 g trypton and 5 g NaCl/I, pH 7.3) or on YT-plate, For anaerobic cultivation, 100 mM NaNO3 or 20 mM NaNOz was added to the medium in sealed flasks and air in the heads pace was replaced by N2 gas. ~vVhennecessary, the following an. tibiotics were added to the medium: anapicillin (50/~g/ml), kanamycin (25/zg/ml) and streptomycin (10/~g/ml) for E'. colt; carbeniclllin (300 Correspondence address: T. Kodama, Department of Agricultural Chemistry, University of Tokyo, Bunkyo-ku, Tokyo 113. Japan
Published by Elsevier ,Science Publishers B, V.
Pset,domonas isolation a~ar tDifco) was used for isolation of P.
aeruainosa transtormnnts, pTSI04S (tncq, Ampicillin f, Streptomycin', :~'ylE) was used to probe the promoter activity of DNA frailments, tra functional promoter is ligated to the upstream ors.viE in this vector, the activity of catechol 2,3.dioxygenase (C230), the
.x'y/E gene product, can be expressed [i l]. Protein was determined by Bio.Rad method using bovin~ serunl albu rain as a standard, DNA sequencin~ was performed as described [71. Other DNA, manipulations were carried out as described by Sambrook et al. [91.
3. RESULTS AND DISCUSSION
3.1. Identification o f promoter region o f the nitrite reduclase gene Various restriction fragments of the upstream region of the nitrite reductase gene (denA) were ligated to the promoter probe vector pTS1045. The constructed plasmids, pHA301 through 306 (see Fig. 1), were transferred to P. aeruginosa. The promoter activities of the ligated fragments were then examined under both aerobic and anaerobic conditions by measuring the activity of C230, the xylE gene product. As shown in Table I, under aerobic conditions, no or little C230 activity was expressed in the cells harbouring a n y of the constructed plasmids even inthe presence of nitrate or nitrite. On the other hand, under anaerobic conditions the cells carrying pHA301, pHA302, pHA304 or pHA306, but not pHA303 or pHA305, expressed high C230 activities when grown in the presence of nitrate or nitrite. These results indicated that the nitrite reductase gene is transcribed only under anaerobic conditions and the promoter and regulatory region of this gene is located in the ApaI-EcoRI fragment (see Fig. 1). It was
351
Volazme ; ~ , number =
hlar~:h 1991
FI~B$ LI~TTER~
__
~_~_ =~ j~,
~
CenA-" pHA301 pHA302 L pHA303 1
I
pHA304
~
pHA305 PHA306 pHA307
~mlll
IsHA300
I
T~l~le i
_
,~
~.t~
C230 a~livtty in ¢,dl ~xtrttet of P. ~r~#l.~#. PAOLI61 ,¢o.mlni.ll pT$1045 lind pHA]0t-3011
C2]O aedvtt~, (U/roll protein) aerc,bk: ~.a,erobl~
1
VT
NO," 1OOmM
NO=° 20mM
NO~'* 100ram
NO=~ 20 mM
pTSIO4J~ pHA301 pHA302 pHA3O;I
l',61 1,47 1,67
0.99
1,91 1.42
1. t t; 2.09 2.46
1,70 20,94 29,7#
0,98 253?
1,62
l ,,~II
2.86
:L21.
217
pHA~O~
=,l~
I,t6
t.,=,~
l,=O
t,n
pHA306
1,43 I,n
1,62
2,08
20,00 to.as
~1,25
I ,
t-i
I
Fig, I. Fraltlments used for C230 a~say, "The plasmld~ pHA301.308 were constructed by lillafing the~e reslrl¢lion fragments containing for
pH^~0~ pH^30t~
28.66
i.~9
t=o
denA and./or its flanking reBIon~ to the upstream of .~'),IEof the promoter probe vector p"TSI04S, A, Apal; E, EcoRI; M, Small 5, gphl,
also suggested that the activity of this promoter (regulator) region is sensitive to molecular oxygen. 3.2. Identification o f promoter region o f the cyto.
chrome c.551 gene In the chromosome of P. aeruginosa, the cytochrome c-551 gene (denB) is located 50 bp downstream of denA and no promoter-like sequence can be found in this 50~bp region [7,12], suggesting that these two genes are transcribed as an operon. To confirm this possibility, two fragments were prepared, ligated to p'rS1045 (pHA307 and pHA308, see Fig. 1), transferred to P. aeruginosa, and their promoter activities were measured by C230 assay, pHA307 contains the (lena promoter region, in addition to the (lenA coding region, 50-bp non-coding region, and about half of the denB coding region, whereas pHA308 lacks the denA promoter region. As shown in Table 1, the cells harbouring pHA308 did not express C230 activity even under anaerobic conditions and in the presence of nitrate, indicating that the promoter for denB is not located in pHA308. On the other hand, the cells carrying pHA307 expressed C230 activity when grown anaerobically in the presence of nitrate. The growth of the latter cells was, however, very poor under anaerobic conditions, probably because of a large amount of nitrite reductase
expressed from the multicopy plasmid. The C230 activity of the cells harbouring pHA307 $rown anaerobically was also considerably lower than that expressed in the cells carrying pHA304. This low expression was probably due to the p o o r growth. In any case, it was con. eluded that denB is transcribed from the denA promoter; in other words, denA and denB are transcribed as an operon. 3.3. Nucleotide sequence o f the promoter (regulator)
region o f denAB We determined the nucleotide sequence of the (lenA promoter region (ApaI-EcoRl fragment). As shown in Fig. 2, this region is highly AT-rich as compared tothe average GC content of P. aeruginosa DNA (67-68070) [13]. This high AT content may be necessary for the binding of regulatory elements. In this region there is a sequence that is closely similar to the consensus FNR binding site (A-A-TTGAT--A-ATCAAT--.) of E. coil [14]. F N R (regulatory protein for fumarate and nitrate reduction) is a regulator for anaerobiosis of 17.. coil and activates t h e expression of proteins such as nitrate reductase, which are required for anaerobic respiration [15]. Azurin. a blue copper protein, is able to act as an alternative electron donor for nitrite reductase [6], and a putative FNR binding site is also located in the upstream region of the azurin gene of P. aeruginosa
~'~TTGA
GCAATACCGG
CAGGCCGTGC
CGCCAGGCGC
GCTCGAAGAC
TTCGATCTCA
60
TGGCCGGTGG
CCTCGTAGAA
GGGTGTCGCG
CGCTAGGGAT
120
GC~A~C~TGA
180
ATGAATTC
TAGGACCGCA
CGCTA~TCAC
AG~GGAAGG
TCCCGCATGT CCTAC?CCTG ' 0RF1 TGCCACAAGC GCAAAGCAAC
TTCCGGTCAA
GCAA~GGTAA
AGACCCTGCT
TTCTATGATC
_EcaRI CTTTCGCGCC
238
Fig. 2. Nucleotide sequence of the promoter (regulatory) region of denAB ( A p e l - E c o R l fragment). The sequence resembles to the consensus FNK binding site of E. coil is boxed, The arrow underline an open reading frame extending to the opposite direction of denA (ORFI), The putative ribosome binding site for ORFI is underlined.
352
Volume 2110, number 2
FEBS LETTEK$
[16], The occurr=nce or un FNR.like element in this orl~nism has been s~Uested by Lod~te et al. [|7]. It seems that the regulatory mechanism for anaeroblosis in P, aeru~inox# resembles thzzt in E, colt, We, also fou~d that upstream rei~ion of dana hes an an~erobic=lly inducible promoter activity in the opposite direction (dat~ not shown), This region forther contains an open reading frame (ORF! in Fig, 2), shortly after which a putative ribosome binding site is located. It is likely that ORFI encodes ~n =zctivator for the transcription o f the denAB operon, as in the case of trp/.rrpBA in P, ~en~gtnos~ [18],
Ack'~eowledfcmcnt: We Uttmk Dr T, Nak.~xawa (Y~tinagt~chi Ufliv,) for kindly providins DTSIO4S, REFERENCES [ll Y~manuke, T,. Ota, A. ~nd Okunuki, K. (1960) Biochtm. Biophys, Acts 44, 39"/-398, [2] Wood, P,M,, (t978} FEBS Left,92, 2[4-2i& [3| Henry. Y. and Besst~'.res,P. (1984) Biochimic 66, 2~g-289, [4l Yamanaka, T,, O|a, A. and Okunuki. K, (196|) Biochim. Biophys. ActP.53, 294-308,
March 1991
[:~! Coyne, M.S.. Arunakum~ri. A., Pankr#l~, H.$. ~nd Tledj~, J.M, (I~)0) J, Uacterlol. 172, 2~$#=]~62. [6) Z~mtlonl. D. (19119)B1ochlm. t~iophys,A¢[~ 19i[9,;I~=316. [7| ArA]. H,, ~=nbo~lll, Y,, tilarashl,Y, and Kodnma, T, (I~0} FEB$ L~t, 26[, 196~198. [g| Ounn, N,W, and Holl~way, B,W. (1971) G~n¢|, Bes. 18, tgS-19'~, [91 Saml)rook, J,, Pt|lsch,T. and MAni~Us, 3'. (1989) Molecular Cloning: A L~l~oratoryM,nu~l, 2rid cdn, Cold Spring Harbor Laboratory, NaveYork. [t0i Dicta, G., Stanfleld. $,. Corbtn, D, and Helinskl. D.R~ (1980) Proc, N~I{],Acid. Set. USA 77, 7347-735L [ell lno~sye,S., As~i, Y,. Nak~t~wa, A. =nd Naku~w~, T. (1986)J. Bactcriol, 166, 739-745, [121 Silvesirlni, M.¢,, Caleoui, C.L,, Qcrv~Is, M,, $chlninA, E., B~rr~, D,, Bossa, F. and Brtmorl, M. (1989) FEBS Levi, 2S4. 33=38. [13] Was(, $,E.H. and Islewski, B,H, (1988) Nucleic Acids Rex. 16. g323-933~, {14] $piro, S. and Quest, J.R, (1990) FEM$ Mtcrobiol. Bey. ?S. 399-428, [ISl Stewart, v. (1982) J, Bacterioi. iS[, i320-132S, {161 Hoitink, C,W,Q,, Woad(, L.P~, Turenbout. J.C,M., van de Kamp, .hi, and Canters, G,W, (1990) Gene 90, !$-20. {17l Lodge, J,, Williams, R., Bell, A,, Chan, B. and Busby,S. (1990) FEMS Microbiol. Let|, 67. 221-22o. [~8) Chang, M,, Fladero, A, nnd Crawford, [.P, (1989)J. Bactcriol, 171, 172-;183.
353
