Molecular relationships between Pseudomonas INC P-9 degradative plasmids TOL, NAH, and SAL

PLASMID

10, 164-174 (1983)

Molecular Relationships between Pseudomonas INC P-9 Degradative Plasmids TOL, NAH, and SAL PHILIP R. LEHRBACH,’

IAN MCGREGOR,

JOHN M. WARD, AND PAUL BRODA

Department of Biochemistry, University of Manchester Institute qf Science and Technology, P.O. Box 88, Manchester A460 IQD. United Kingdom

Received January 26, 1983 We have examined the extent to which the degradative plasmids SAL, NAH, and TOL of the Inc P-9 incompatibility group share common DNA sequences.The homology we observe using “P-labeled SAL and NAH DNA probes can be assigned to six regions of the TOL (pWW0) restriction endonuclease cleavage map. At least three of these regions are probably related to transfer and replication functions, whereas a fourth region is related to the common metacleavage pathway. Restriction endonuclease maps of the SAL and NAH plasmids are derived and the relationships between these plasmids discussed.

The Pseudomonas Inc P-9 incompatibility group contains the plasmids NAH, SAL, and TOL (pWW0) (Bayley et al., 1979) which, respectively, encode all the enzymes necessary for the bacterial utilization of naphthalene (and salicylate) (Dunn and Gun&us, 1973) salicylate (Chakrabarty, 1972), and toluene (and related compounds) (Worsey and Williams, 1975; Kunz and Chapman, 1981) via a common pathway involving the meca-cleavage of catechol. Previous DNA:DNA hybridization studies with these plasmids (Heinaru et al., 1978; Bayley et al., 1979) have shown that they sharesignificant homology; however, the locations of thesehomologous regions were not determined. To study these relationships in more detail we first compared plasmid DNA endonuclease restriction patterns and then tested the relatedness of cleavage fragments by DNA:DNA hybridization techniques using whole plasmids or particular restriction fragments as 32P-labeled DNA probes. We identify six regions of the pWW0 plasmid which show homology with 32P-labeledSAL or NAH plasmid DNA. We propose that at least three of these regions

(regions 1, 2, and 3) specify the transfer and replication functions, and that a common m&a-cleavage pathway is specified by region 5. A preliminary account of some of these results was presented at a Society for General Microbiology symposium in Cambridge, England in April 1981. MATERIALS

AND

METHODS

Bacterial Strains and Plasmids

Theseare listed in Table 1. Plasmid pBR322 and its recombinant derivatives were maintained in Escherichia coli K-12 ED8654, which is met- supE supF hsdR- hsdM+ (Borck et al., 1976). Chemicals, Media, Plasmid Isolation, Digestion, and Electrophoresis

Theseare as previously described(Lehrbach et al., 1983). DNA endonuclease restriction

fragments were isolated by the method of Thuring et al. ( 1975). Transfer of DNA to Nitrocellulose

’ present address:Department of Medical Biochemistry, University Medical Centre, University of Geneva, Geneva 12I I, Switzerland. 0147-619X/83 $3.00 Copyngbt C 1983 by Academic Press. Inc. All nghfs of reproductmn in any form reserved.

Filters

DNA restriction fragments separated by agarosegel electrophoresis were transferred to 164

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TABLE I BACTERIALSTFMNSANDPLAMIDS Strain”

Plasmid*

Source or reference

P. putida mt-2 (PAWl) ToI+ P. aeruginosa PAC (AC165) Sal’ P. putida (4G7) Nab+ P. putida (PgG1064) Nab+ P. putida (PpG2100) Sal+ E. coli K12 (ED8654) ApRTp

PWWG SAL NAH NAH7 SAL1 pBR322 pMT057 (A) pMTIO5 (B) pMTl06 (C) pED3306 (D) pED33 10 (E) pED3307 (P) pED33 I I (G) pED3312 (I) pED3313 (J) pED33 14 (K)

Williams and Murray (1974) Chakmbarty (1972), Heinaru et al., (1978) Dunn and Gunsalus (1973) Yen and Gunsalus (1982) Gunsalus and Yen (I 98 I) Bohvar et al. (1977) Lehrbach ef al. (1983) This study This study Meulien et al. (I 98 I) Lehrbach et al. (1983) Meuhen et al. (198 I) Meulien and Broda (1982) Meulien and Broda (1982) Meulien and Broda (1982) Meulien and Broda (1982)

’ Abbreviations: Degradative abilities: Tol, toluene; m and pxylene (Kunz and Chapman, 1981); Sal: salicylate; Nab: naphthalene. Resistances:Ap: ampicillin; Tc: tetracycline. b Letters in parenthesesrefer to the Hind111fragments of pWW0 present in the ApRTcs derivatives of pBR322.

nitrocellulose filters (Schleicher and Schuell, BA85) by the method of Southern (1975). Preparation of 32P-labeled Plasmid DNA and DNA:DNA Hybridization Conditions

Plasmid DNA or purified restriction fragments (approx 1 pg) were ‘*P-labeled by nick translation essentially as described by Bigby et al. (1977) using “P-labeled dATP as the labeled precursor. After 4 h incubation the unreacted triphosphates were removed by passing the mixture through a Sephadex G50 column. Nitrocellulose filters, containing the denatured DNA fragments, were wetted with 2 X SSC (1 X SSC = 0.15 M NaCL, 15 mM Na citrate, pH 7.0)-O. 1%sodium dodecyi sulfate (SDS) and 50% formamide for 10 min at 37°C in confined plastic bags. Excessfluid was then removed. “P-labeled probe DNA, denatured by boiling for 10 min, was added to the filters. The plastic bagswere then sealed and hybridizations were carried out at 37°C for 16 h. After hybridization the filters were washed twice in 2 X SSC-0.1% SDS at 37°C and then twice in 2 X SSC (37°C). For au-

toradiography dried filters were exposed to sensitized photographic film next to an intensification screen at -70°C. Autoradiography was carried out for 24 h and also for a further 3-6 days to reveal weakly hybridizing bands. RESULTS Comparison of EcoRI and HindIII Restriction Patterns of p WWO, SAL, and NAH Plasmid DNA

The molecular sizesof restriction fragments of pWW0, SAL, and NAH, determined by agarosegel electrophoresis, are summarised in Table 2. We calculated that the plasmid sizes are: pWW0 115 kb,2 SAL 68 kb, and NAH 81 kb. Based on the assumption that related plasmids will yield similar cleavagefragments several conclusions can be drawn from these data. First, the SAL and NAH plasmids are closely related. Fragments with the sameelectrophoretic mobility as Hind111 fragments D ’ Abbreviations: kb, kilobase; Ap’, ampicillin resistance; Tc’: tetracycline resistance.

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and E and also G to 0 of NAH are present in the Hind111digests of SAL. For the EcoRI patterns, fragments with the same electrophoretic mobility as EcoRI fragments C to F, H to Q, and T to X of NAH are present in the EcoRl digests of SAL. However, second, the SAL and NAH plasmids have several fragments that do not cor-

ET AL.

respond in size to any from the other plasmid. The number of fragment size differences suggeststhat the relationships between these plasmids cannot be explained by the simple loss of a single DNA segment from the larger plasmid (NAH) to form SAL (or an addition to the SAL plasmid) (as previously pointed out by Heinaru et al. ( 1978)). It seemsthat a com-

TABLE Hind111 AND EcoRI RESTRICTION FRAGMENT SIZES OF TOL, SAL, AND NAH PLASMID DNA” Restriction

fragment sizes (kb)

Hind111 Fragment No. A B C D E F G H I J K L M N 0 i R S T u V W X Y Z Z’

pwwo

EcoRI

SAL

NAH

23.5 22.4 18.2 9.8 8.4 6.9 6.1 5.1 4.4 3.6 2.1 1.4 1.1 0.6 ost 0.5 0.4 0.3 0.2

20* 16* 1.5 5.5 3.4 3.1 2.8* 2.2 2.2 2.0 1.5 1.2 0.9 0.5t

18* 17* 12* 1.5 5.5 4.0* 3.4 3.1 2.2 2.2 2.0 1.5 1.2 0.9 0.5t 0.4*

115.5

68.8

79.44

zii

Z’” Z” Total (kb)

pwwo 15.6 10.1 7.8 7.8 6.8 5.9-f 5.5 5.5 5.1 4.9 4.9 4.3t 3.7 3.2 2.8t 2.5t 2.5 2.4 1.9 1.8 I .6t 1.5t 1.35 1.35 0.95t 0.95t 0.80 0.63 0.q 0.3 114.9

SAL

NAH

14* 6.5* 5.9 5.9t 5.5* 4.3t 3.4 2.8t 2.5t 2.45 2.0 2.0 1.7 1.6t 1.55 I.57 I.2 1.1t 1.1t 0.55 0.5t

24* 7.3* 5.9 5.9t 4.3t 3.4 3.0* 2.8t 2.5-f 2.45 2.45* 2.0 2.0 1.7 I .6t 1.55 1.5t 1.4s 1.35* 1.2 1.l.t 1.1t 0.55 0.5t 0.4*

68.05

81.95

a Fragment sizes were calculated from their electrophoretic mobility in 0.5, 0.7, and 1% agarose gels. Fragments co.35 kb were not detected by this method. Bacteriophage lambda digested with Hind111 and pWW0 DNA digested with Hind111 or X/z01 (Downing and Broda, 1979) were used as DNA size markers. * Fragment size differences between the SAL and NAH plasmids are indicated. t Apparent size analogues found in the three plasmids are indicated.

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bination of additions and deletions at different points on the plasmid molecules is involved (see later section). Third, the SAL and NAH plasmids have only a few fragment sizes in common with pWW0 suggesting that they are not closely related to it. 6.

DNA SequenceHomology between EcoRI and HindIII Restriction Fragments of pWW0, SAL, and NAH Previous DNA:DNA hybridization data obtained from experiments using 32P-labeled SAL and NAH DNA with Southern blots of EcoRI fragments of pWW0 (Heinaru et al., 1978; Bayley et al., 1979) also suggestedthat SAL and NAH are essentially homologous (Heinaru et al., 1978), whereasthe NAH plasmid (and also SAL) only have about 9 kb that are homologous with pWW0. Using our recently-completed EcoRI restriction map of pWW0 (Lehrbach et al., 1982) we can locate these homologous regions on this map (Fig. 1). The homologies between pWW0 and SAL or NAH plasmid DNA are distributed over the two large pWW0 Hind111 fragments B and C (and J) (regions 1, 2, and 3) with three other homologous regionsbeing presentwithin Hind111fragments A (region 5) E and G (region 6), and I (region 4). The regions shown in Fig. 1 must be regarded as a maximum estimate of the homology between these plasmids since this type of analysis does not provide information on the degree of homology (i.e., percentage homology) within individual EcoRI fragments. Homologous regions identified from autoradiographs exposed for a period of 3-6 days were assigned on the basis of whether (i) an individual EcoRI band showed homology and (ii) whether identical EcoRI fragment sizes were present in the digestsof the two plasmids being compared. For instance 32P-labeledSAL and NAH DNA both hybridize to pWW0 EcoRI fragments F (5.9 kb) and J or K (4.9 kb) which make up a doublet band within the pWW0 EcoRI digests. Therefore (i) with respect to F, since a 5.9-kb fragment is present

FIG. 1. Regions of homology between pWW0, SAL, and NAH. The EcoRI, HindIII, and XhoI restriction maps of pWW0 are shown and fragments are labeled alphabetically in order of decreasing size. Smaller fragments are not labeled (see earlier publications; Downing and Broda, 1979;Inouye et al.. 1981a,b;Lehrbach et al., 1982). Regions (I to 6) showing relatively strong (I) or weak (IX) hybridization to pWW0 DNA with “P-labeled NAH (inner circle) or SAL DNA (outer circle) are shown. Results are based on stringent hybridization conditions (see Materials and Methods) and the intensity of hybridizing fmgments was assessedafter 3-6 days autoradiography (Heinaru et al., 1978; Bayley ef al., 1979). Regions of pWW0 specifying degradative functions (taken from Inouye et al., 1981a,b, Lehrbach et al.. 1983) and the autonomous replication and conjugal transfer functions Tm/Rep (taken from Franklin et al., 1981) are shown.

in the three plasmids (Table 2) and EcoRI-F from pWW0 gives a relatively strong hybridization signal we may conclude that it is closely related to DNA from SAL and NAH. On the other hand (ii) since the 4.9-kb fragments J and/or K are unique to pWW0 DNA (Table 2) and give a relatively weak hybridization signal, they are unlikely to be completely homologous with SAL or NAH DNA. Moreover, (iii) pWW0 EcoRI fragments J and K (4.9 kb) appear as a doublet and it is unknown whether the observed homology with the SAL or NAH DNA probes is due to hybridization to one or both fragments. To accommodate theseobservations the regions of homology within the pWW0 Hind111 B and C fragments are represented as in Fig. 1. Similar considerations were used to assign homology in other regions (4-6). From these

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A

a

bc

a

def

b

E

c

d

e

f

HG

RG. 2. Hybridization of HindUI-treated pWW0 (lanes c and d), SAL (lanes b and e), and NAH (lanes a and f) DNA, fractionated by agarosegel electrophoresis and transferred to nitrocellulose. The filters were then hybridised with 32P-labeledpWWOderived fragments: A, HindIII-B (HB); B, Hi&III-D (HD); C, HindII1-G (HG); D, Hi&II-I (HI); and E, HindIII-J (I-H). Autoradiographs developed aher 24 h are shown. Arrows point to weak hybridization observed more clearly on overexposed autoradiographs. Note that HindIII-D shares I .4 kb complete homology with pWW0 HindI&F (Meulien ef al.. 1981).

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DEGRADATIVE

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PLASMIDS

has been suggestedto represent a DNA fragment carrying a common “core sequence” (Bayley et al., 1979). EcoRI fragments of SAL and NAH homologous with the pWW0 HindIII-C probe were also identical with two exceptions. The intensity of hybridization was much less than observed with the control pWW0 HindIII-C digests (data not shown) and there were no fragment sizescommon to the three plasmids. This would suggest that this region is less closely related.

experiments it appearsthat the SAL and NAH plasmids are very similar in their regions of homology with TOL. The approximate sizes of these regions are: 1, 8.0 kb; 2, 7.2 kb; 3, 11.0 kb; 4, 3.0 kb; 5, 7.0 kb; and 6, 4.0 kb. To further investigate these relationships, 32P-labeledDNA pBR322-based recombinant plasmids containing individual pWW0 Hind111 fragments A to K (in the case of pWW0 HindIII-H, the isolated fragments from HindIII-digested pWW0 DNA were used) were hybridized to EcoRI and Hind111 restriction fragments of SAL and NAH DNA transferred to nitrocellulose filters. Control experiments with 32P-labeledpBR322 DNA showed no hybridization with SAL or NAH DNA under the hybridization conditions used. Homologous fragments were detected with probes made from pWW0 Hind111fragments A, B. C, E, G, I, and J (Fig. 2 and Table 3) whereas Hind111 fragments D (Fig. 2), F, H, and K showedno homology with SAL or NAH DNA (data not shown). The pattern of hybridization detected with pWW0 Hind111 B and C probes (Table 3) shows that a similar set of EcoRI fragments of SAL and NAH hybridized with a particular probe. These Z&RI fragments are therefore part of an identical region of the SAL and NAH plasmids. One region identified with the TOL HindIII-B probe includes the 5.9-kb fragment present in all three plasmids which

HomologJj between the Meta-Cleavage Regions We wished to determine the extent of homology between the DNA sequences determining meta-cleavage pathways of the three plasmids. It was known that the pWW0 HindIII-A fragment encodes an intact regulated me&r-cleavagepathway (Inouye et al., 1981b; Lehrbach et al., 1983) so this DNA fragment was used to probe for homologous sequencesin the SAL and NAH plasmids. We also used plasmid DNA as “P-labeled probe in the reciprocal hybridization experiment. As shown in Figs. 1 and 3 the NAH probe hybridizes with two EcoRI subfragments of pWW0 HindIII-A, fragments E and D, which are known to encode structural genes of the meta-cleavagepathway (Inouye et al., 198la,b; Franklin et al., 1983). The pWW0 HindIII-

TABLE 3 RESTRICTION

FRAGMENTS

OF SAL AND NAH PLASMID DNA HYBRIDIZING “P-LABELED TOL Hind111 FRAGMENTS

Homologous pWW0 Hind111 fragments as labeled DNA probes (kb)

WITH DENATURED

Hind111 (H) and EcoRI (E) restriction fragments’

Of SAL plasmid

Of NAH ptasmid

A (23.5)

HC; EA. EC

HD; EA, EC

B (22.4)

HA; ED, EG, EJ

HB; ED. EF, EJ

C (18.2) E (8.4) G (6.1) I (4.4) J (3.6)

HB, HB, HB, HA, HB;

HC, HB, HB, HE, HC,

a After 6 days of autoradiography;

HN; HD; HD; HD, n.d.

EB, EH, EL, EO n.d. n.d. HF; n.d.

n.d.. not determined.

HO; HE; HE; HH; HP;

EB, EH, EM, EP, ES n.d. n.d. n.d. n.d.

a

b

c

+-TOLEcoRI-E

+-TOLfroRI-0

Bd

ef

.

EcoRI-A

wNAH/SAL EcoRI -C

-SALEcoRI-A

-NAH

FIG. 3. Extent of homology between SAL and NAH DNA and the pWW0 mern-cleavage pathway. ‘2P-labeled hybridization probes were (A) NAH and (B) pMT057 (Table 1). Hybridizations were to filters containing EcoRl restriction digests of NAH (lanes a and f); SAL (lanes b and e), and pMT057 (lanes c and d).

A

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FIG. 4. Derivation and comparison of XhoI and SrnaI restriction maps of NAH, NAH7, SAL, and SAL1 plasmids. The points of DNA insertions in SAL1 and SAL DNA and the extent of deleted DNA in SAL DNA are shown. Restriction fragments are labeled alphabetically in order of size (seeTable 4). Scaledivisions (inside circle) are 10 kb. The position of upper pathway, sahcylate hydroxylase, and mera-pathway genes are shown (taken from Yen and Gunsalus, 1982; Lehrbach unpublished data).

A probe hybridizes with two EcoRI fragments of both SAL and NAH; these are EcoRI-A ( 14 kb) and EcoRI-C (5.9 kb) of SAL and EcoRI-A (24 kb) and EcoRI-C (5.9 kb) of NAH (Fig. 4, Table 2).

It appears that the structural genes of the m&a-cleavage pathway of the SAL and NAH plasmids are encoded on at least two EcoRI fragments and a single EiindIII fragment NAH HindIII-D or SAL HindIII-C (7.5 kb).

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ET AL.

Physical Variation in SAL and NAH Plasmid DNA Isolated@om D$erent Host Strains

terns and the relationships of the plasmids could be explained on the following basis(Fig. 4): (i) the SAL 1 plasmid (83 kb) results from a 2.3-kb insertion in the SnzaI-B/XhoI-E rePrevious studies (Farrell et al., 1978; Heigion of NAH; (ii) the SAL plasmid (68 kb) naru et al., 1978; Bayley et al., 1979; Farrell results from a 22-kb deletion which includes and Chakrabarty, 1979; Gunsalus and Yen, the %naI-A and-B fragments (XhoI-A, D, and 198I) that have assessedthe relationships between the SAL and NAH plasmids pointed E) and also an 8- to 9-kb insertion within the XhoI-A (SmaI-A) fragment of NAH. to considerable structural variation between these plasmids. Comparison of SAL1 and DISCUSSION NAH7 plasmid DNA isolated from PseudoIn this study we have determined the exmonas putida strains (Farrell et al., 1978, Farrell and Chakrabarty, 1979) indicated that the tent of DNA sequence homology, using 83-kb NAH7 plasmid gave rise to the larger DNA:DNA hybridization techniques, between 85-kb SAL 1 plasmid by a single site addition. the IncP-9 degradative plasmids pWW0, SAL, Heinaru et al. (1978) concluded that the SAL and NAH. By a comparison of the restriction plasmid isolated from P. aeruginosa PAC endonuclease (HindIII, EcoRI) patterns of (AC165) was smaller in size than the NAH7 these plasmids and then a seriesof hybridizaplasmid and exhibited complex restriction tion experiments using 32P-labeledSAL and NAH DNA, six regions of varying hybridizapattern differences. We compared the SmaI and XhoI restriction tion strength and therefore amounts of hofragments from the plasmids from strains mology were identified on pWW0 (Fig. 1). PpG1064 (NAH7) and PpG2100 (SALl) (ob- The three largest regions ( 1,2, and 3) and two tained from I. C. Gunsalus) with those of the smaller ones (regions 4 and 6) are in portions NAH and SAL plasmids discussedabove (see of pWW0 to which functions have not yet Table 1). From this analysis (Table 4) NAH7 been assigned. While it is apparent that these and NAH plasmids are indistinguishable. Re- sequencesdo not determine catabolic funcstriction digests with HindIII, EcoRI, PstI, tions (Nakazawa et al., 1980; Lehrbach et al., SalI, and BamHI also support this conclusion 1982) they may encode common replication (McGregor and Lehrbach, data not shown). and transfer functions. Consistent with this is However, the SAL plasmids differ in both mo- the finding that region 3 includes the 5.9-kb lecular weights and restriction fragment pat- EcoRI fragment present in four IncP-9 plasTABLE 4 COMPARISON OF XhoI AND SmaI RESTRICTION FRAGMENTS FOR NAH, NAH7, SAL AND SAL1 PLASMID DNA’ Restriction

fragment sizes (kb)

Xhd Fragment A B C D E F

NAH, NAH7 >40 13 8.8 5.4 2.3 1.3

’ See Table 2 for details.

SmaI

SAL

SAL1

NAH, NAH7

SAL

SAL1

>40 13 8.8 1.3

>40 13 8.8 5.4 4.5 1.3

>40 17.6 12.7 7.0 3.7

>35 12.7 7.0 3.7 1.9

>40 17.6 12.7 7.0 3.7 2.6

Pseudomonas DEGRADATIVE

mids, two R plasmids, and NAH and pWW0, so far examined (Bayley et al. ( 1979) Table 2). This fragment may therefore contain a “core sequence” involved in replication functions. Also, a comparison of the pWW0 DNA still present in various deletion derivatives showed that all retained XhuI fragments A and H (Franklin et al., 1981) which include the regions 1, 2, and at least part of region 3 defined here. It was concluded that this segment of TOL DNA encodesautonomous replication and conjugal transfer functions. Two smaller homologous regions (regions 4 and 6) that show relatively strong hybridization to SAL and NAH “P-labeled DNA were also found within TOL XhoI fragments A and E, but the possible significance of these regions is not clear. The six regions represent 34% of TOL plasmid DNA showing possible homology with SAL or NAH DNA. This figure is higher than the estimate of common sequencesbetween TOL and NAH (8%) based on a quantitative method (Bayley et al., 1979), and the disparity may suggestthat the regions shown in Fig. 1 give a maximum estimate of homology between theseplasmids. However, now that these regions have been located, further experiments can be undertaken to define them more closely and accesstheir functional significance. DNA hybridization experiments using particular pWW0 Hind111 fragments as 32P-labeled probes confirmed the location of the six homologous regions; hybridization to SAL or NAH DNA was detected with those Hind111 fragments included in regions 1 to 6 (i.e., pWW0 Hind111 fragments A, B, C, E, G, I, and J); but no hybridization wasdetected with pWW0 Hind111 fragments outside the six regions (i.e., pWW0 Hind111 fragments D, F, H, and K). These experiments also identified various homologous Hind111and EcoRI fragments of the SAL and NAH plasmids. For instance NAH Hind111 fragments D, B, and C plus 0 sharesomeDNA sequencehomology with pWW0 Hind111 A, B, and C fragments respectively (Table 3). The completion of a more detailed restriction map (including Hind111 and Z&RI fragment maps) of SAL

PLASMIDS

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and NAH will enable us to locate these homologous fragments on the circular plasmid DNA molecule and to further study the relationships between these regions. SAL and NAH 32P-labeledDNA hybridized to pWW0 EcoRI fragments E and D of region 5 (Fig. l), which encode structural genes of the me&-cleavage pathway. From the same experiments no homology was detected with pWW0 restriction fragments (Xho -F, -G, or -C) encoding pWW0 upper pathway genes (i.e., xylA, xylB, and xylc) (Lehrbach et al., 1982; Franklin et al., 1981). By restriction analysis we have derived XhoI and SmaI physical DNA maps of NAH/ NAH7, SAL, and SAL1 (Fig. 4). The SmaI map of NAH/NAH7 is in agreement with that of Yen and Gunsalus (1982). These physical maps provide an explanation of the reported size difference between these plasmids (Farrell and Chakrabarty, 1979). In one cast (Fig. 4, right branch) a single DNA insertion with the NAH plasmid has presumably rendered the upper pathway, necessaryfor the conversion of naphthalene to salicylate (Yen and Gunsalus, 1982), inactive. Consequently the slightly larger plasmid (SAL 1) that is produced by the insertion now only determines active genes for the utilization of salicylate as sole carbon source. The relationships between the plasmids SAL and NAH can also be inferred by restriction analysis; however, in this case (Fig. 4, left branch) both a deletion of the DNA region encoding the upper pathway and a separate DNA insertion must have occurred. The combination of these events explains the complex restriction pattern differences between these plasmids with several DNA restriction fragments either totally absent or altered in size (Tables 1 and 4). ACKNOWLEDGMENTS

Wearegrateful to Professor I. C. Gunsalus for providing bacterial strains. We thank the Medical Research Council and the Science and Engineering Research Council support.

for

REFERENCES BAYLEY, S. A., MORRIS, D. W., AND BRODA, P. (1979). The relationship of degradative and resistance plasmids

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