Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp

PLASMID

18,

173-177

(1987)

Physical Comparison

of Parathion Hydrolase Plasmids from Pseudomonas diminuta and flavobacterium sp.

WALTER W. MULBRY,*

PHILIPC.KEARNEY,*JUDDO.NELSON,~

AND

*Pesticide Maryland

Degradation Laboratory. 20705. and fEntomology Received Restriction

pPDL2 70-kb

of

two

KARNS**’

Agricultural Research Service, U.S. Department Department, University oj Maryland, CoIleKe May plasmids

28,

1987;

revised

encoding

parathion

is a 39-kb plasmid harbored by Havohacferium plasmid found in Pseudomonas diminuta (strain

been shown degradation) study, ments

maps

S.

JEFFREY

to share homologous parathion hydrolase as judged by DNA-DNA hybridization

we conducted from pCMSl

DNA hybridization as probes against

experiments

Flavobacterium

August

ojAgricuhure. Park, Maryland

Beltsville, 20742

6. I987

hydrolase

have

been

determined.

sp. (ATCC MC). Both

2755 1). while pCMSI plasmids previously

genes (termed and restriction

opd for organophosphate mapping.

In the

using each of nine Psll restriction plasmid DNA. The opd genes

plasmids are located within a highly conserved region of approximately homology extends approximately 2.6 kb upstream and 1.7 kb downstream No homology between the two plasmids is evident outside ofthis region.

5. I kb. from

is a have present fragof both

This region of the opd genes.

o 1987 Academic

hen.

IN

Microbial enzymes such as parathion hy- (termed opd for organophosphate degradadrolase (EC 3.1.3) are thought to play a sig- tion) from P. diminuta MG has been cloned nificant role in the environmental fate of into other bacterial hosts (Serdar and Gibparathion (O,Odiethyl-O-nitrophenyl phos- son, 1985; Mulbry el al., 1986). A 1.3-kb PstI phorothioate) and related organophosphate restriction fragment containing the P. diinsecticides. Indeed, the spread of such hy- minuta opd gene was used to identify a hodrolase genes in the microbial community mologous genetic region on one of the four may explain why some soils show increased plasmids within Flavobacterium sp. ATCC 27551 (Mulbry et al., 1986). Restriction metabolism of organophosphates (Read, experiments re1983). The growing prevalence of these so- analysis and hybridization vealed that the DNA regions within and imcalled “problem soils” poses a significant mediately surrounding the two opd genes threat to the continued efficacy of many peswere highly conserved, while no such conticides and has stimulated interest in identiservation of plasmid sequence appeared to fying the responsible genes and in understanding the dispersal of these genes among exist outside of this region (Mulbry et al.. members of the soil microflora. 1986). A more comprehensive comparison A parathion hydrolase producing Ffuvo- of the tyo opd plasmids was precluded by bacterium sp. was collected in 1972 from a the lack of restriction maps for them. The Philippine rice field (Sethunathan and Yo- present study was undertaken to characterize shida, 1973). A decade later, in Texas, Yseu- physically the two parathion hydrolase plasdomonas diminuta MG was isolated from an mids by restriction mapping and to probe the enrichment culture that utilized parathion as full extent of their homology by DNA-DNA a carbon source (Serdar er al., 1982). A plas- hybridization. We previously demonstrated that only one mid-borne gene for a parathion hydrolase plasmid (pPDL2) out of the four plasmids within Flavobacterium sp. ATCC 27551 I To whom all correspondence should be addressed. 173

0 147-6

19X/87

Copyright All ri&u

Q 1987 by Academic Press, inc. of reproduction in any form rcurd.

$3.00

SHORT

174 Plasmid

34 I P 1

pPDL2

O/39I

kb

EPP 1, I c vd

COMMUKICA’I‘IONS

5I P I

pm17

10 E

15

EP P E P II I 0

P I

30 ,

34 J

F I

e J

1

J

II

Pminl9

L-l I

Pm2

I

phWM44

LL-L--

pm6

11

I

1

1

II

1

PM7

,I

II

p-5

II

1

plili?l1019

A

LU

pvm1001

U -

pwnllO59 pi%?41036

1

u

puw1002

u

pwMlO31

UI

puw41021

I

phWlloBl

t

1

1

h II

J

IIIIJ

I-l

pu!mlOl4

I

phlmloo7 pm1135

25

-

PM

p!a?llO79

20 I

II

,

LI I

)I

I

pliWl1160

FIG. I. Restriction maps of recombinant fragments from Flawbacterium sp. plasmid

, I

I

plasmids pPDL2.

contained the opdgene (Mulbry et al., 1986). Plasmid-curing experiments were unsuccessful in isolating a derivative of Flavobacterium sp. ATCC 27551 that contained only pPDL2 (Mulbry et al., 1986). In order to construct a restriction map of pPDL2, a recombinant DNA library containing plasmid DNA from Flavobacterium ATCC 27551 was created. Plasmid DNA from the Flavobacterium was subjected to partial digestion with EcoRI and ligated into pBR325 as described previously (Mulbry et al.. 1986). To create a library of PstI fragments, Flavobacterizcm plasmid DNA was subjected to partial digestion with PstI (160 pg DNA/ml, 4.5 U YstI/pg DNA, 0.5 min at 18°C) ligated to

II

J

,

containing

overlapping

&I

(P) and EcoRl

(E)

pUCl9 (Vanisch-Perron et al., 1985) (300 &ml DNA, I:9 ratio of vector to insert, 600 U T4 DNA ligase/ml, I h at lS”C), and used to transform competent DH-Sa cells (BRL. Gaithersburg, MD). Representative recombinant plasmids containing pPDL2 DNA are shown in Fig. 1. Use of the 1.3-kb PstI fragment containing the opd gene as a hybridization probe identified three recombinant plasmids (pWWM6, pWWM44, and pWWM I 135) that contained fragments contiguous with opd. Additional clones containing restriction fragments that had significant overlap with the EcoRI and PstI fragments in pWWM6, pWWM44, and pWWMl135 allowed determination of the complete pPDL2

SHORT COMMUNICATIONS

map. Further restriction mapping of these recombinant plasmids yielded the pPDL2 restriction map shown in Fig. 2. Addition of pPDL2 restriction fragments yielded a total molecular size of 39 kb. PstI fragments from pCMS1 were cloned into pBR322 as described previously (Mulbry et al., 1986). Using the same protocol, PstI fragments also were cloned into pUC19. In order to clone the two largest pCMS1 PstI fragments (20.0 and 17.8 kb) they were purified by electrophoresis, recovered by electroelution into dialysis bags (Maniatis et al., 1982), and concentrated using Elutip-d columns (Schleicher & Schuell, Keene, NH), according to the manufacturer’s instructions. The purified fragments were ligated to PstI-digested pUC19 DNA and used to transform competent Escherichia coli DHS-a cells. DNA from pCMS1 and from recombinant clones containing pCMS1 DNA were subjected to digestion with pairs of restriction enzymes in order to construct the restriction map of pCMS1 shown in Fig. 2. Our molecular size estimate of 70 kb for pCMS 1 agrees well with

175

an estimate of 66 kb by previous investigators (Serdar et al., 1982). To determine the outer limits of homology between pCMS 1 and pPDL2, PstI fragments from recombinant plasmids containing pCMS1 DNA were nick-translated and used as probes in Southern hybridization experiments against restriction enzyme digested Flavobacterium plasmid DNA. pPDL2 restriction fragments showing hybridization to pCMS 1 fragments are listed in Table 1. Figure 3 shows the restriction maps of the opd regions delineated. The area of homology extends approximately 2.6 kb upstream and 1.7 kb downstream of opd. Within the 5. I-kb area of plasmid homology there is a 2. I-kb region in which the restriction sites of the two plasmids are highly conserved. No such conservation of plasmid restriction sites is evident outside of this region. There is one PstI fragment from pCMS1 (fragment D) that hybridized weakly to a region of pPDL2 that is well within the region of homology, even though fragment D is from outside this region on pCMS1 (Table 1). We cannot satisfactorily explain this observation except to

FIG. 2. Restriction maps of the opd-containing plasmids pCMS 1 from Pseudomonas diminuta MG and pPDL2 from Flavobacterium sp. ATCC 2755 1. The locations and orientations of the opd genes on these plasmids are marked by arrows. The orientation of the opd gene was determined previously by directional cloning into Ml3 (Mulbry et al., 1986) and has been confirmed by directional cloning into pUC19 (data not shown).

176

SHORT COMMUNIC’ATIONS TABLE I

HYBRIDIZATION OF CLONED pCMSl WI RESTRICTION FRAGMENTS TO pPDL2 RESIRICTIO!G FRAGMENTS pCMSl Afl probe (size)

pPDL2 fragment bands showing hybridization EcoRI

A (20.0 kb) B(17.8 kb) C (17.6 kb)

&zmH I

D (5.2 kb)

14.7 (A) 7.3 (C) 7.3 (C)

E (4.1 kb)

7.3 (C)

F(1.8 kb) G (1.5 kb) H (1.3 kb)

7.3 (C)

30 (A)

No hybridization No hybridization 5.4 (D)

2.5 (B) 1.5 (E) 2.5 (B) 1.5 (E)

30 (A) I .5 (E)

I(0.7 kb)

sun

I’.trl

Smal

12.5 (B)

2.0 (D)

2.4 (E)

1.8 (F)

NT

2.4 (E)

2.7 (D) 1.8 (F)

NT

12.5 (B) 1.8 (F)

NT

No hybridization No hybridization 1.3 (H) No hybridization

NOW.Cloned Psrl restriction endonuclease fragments from pCMS I were purified from vector DNA as described in the text and used as hybridization probes against restriction endonuclease digested plasmid DNA from Fluvobacrerium sp. ATCC 2755 I (which contains four distinct plasmids). pPDL2 restriction fragments which hybridized to the pCMSl DNA probes are listed. Fragment letters in the table correspond to the lettered fragments in Fig. 2. Restriction fragments from Fhvobucferium plasmids other than pPDL2 which hybridized to pCMSl probes are not shown. NT, not tested.

note that this fragment was not homologous to any pPDL2 fragment a corresponding distance upstream from the opdgene. Thus, this anomaly appears to be unique to pCMS1 and does not alter the limits determined for the homologous regions from both plasmids. The observation of homologous catabolic genes such as opd within structurally unrelated plasmids from soil bacteria is certainly not without precedent. Williams and coworkers (Keil et al., 1985) have shown that

PPOLP

Ii 1

BP XS 1 I II .--------------------

8X

the TOL (toluene) degradation plasmids pWW0 and pWW53 contain homologous catabolic genes. However, hybridization experiments between deletion derivatives of pWW0 and deletion derivatives of pWW53 showed that these TOL plasmids shared homology only within the regions where the TOL catabolic genes were located. In another study, comparison of five independently isolated TOL plasmids with different sizes and restriction fragment profiles

PSSmBPHE I I Ir Ill

Sm

San ,

P I

OPd I 1 kb

FIG. 3. Comparison of the opdregions from pCMSl and pPDL2. Restriction maps of pCMS1 and pPDL2 DNA surrounding their opdgenes are shown. The underlined region of pPDL2 delineates the outer limits of plasmid homology which were demonstrated by hybridization experiments. Restriction endonuclease sites shown are B, BumHI; E, EcoRI; H, HindIll; P, &I; S, SalI; Sm. Smal; and X, Xhol.

SHORT

177

COMMUNICATIONS

has shown that each of these apparently dissimilar plasmids contains identical pairs of catechol 2,3-oxygenase genes (Chatfield and Williams, 1986). Other types of degradation plasmids containing homology only within their catabolic gene regions have also been documented. The 2,4-D (2,4dichlorophenoxyacetic acid) degradation plasmid pJP4 has been shown to share homology with the chlorobenzoate degradation plasmid pAC27 only in the region containing its structural genes for chlorocatechol catabolism (Ghosal et al., 1985). We have been unable to determine whether or not the opdgene is part of a transposon. Experiments designed to detect repeated sequences at or near the termini of the conserved regions of pPDL2 and pCMS I by DNA-DNA hybridization did not detect any repeated sequences. However, repeated sequences of less than 200 bp may not have been detected by our technique. Moreover, we have observed no evidence of physical transposition throughout the course of our experimentation. The nonselectable nature of the opdgene product, as well as the lack of a transposon detection system that will work well in Flavobactrrium or P. diminuta has limited our ability to detect transposition of the opd gene.

REFERENCES CHATFIELD, L. K., AND WILLIAMS, P. A. (1986). Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes. J. Bacterial. 168, 878-885.

GHOSAL, D., You, L-S., CHATTERJEE, D. K., AND CHAKRABARTY, A. M. (1985). Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4. Proc. Nut. Acad. Sci. USA 82, 1638-1642. KEIL, H., KEIL, S., PICKUP, R. W., AND WILLIAMS, P. A. ( 1985). Evolutionary conservation of genes coding for meta-pathway enzymes within TOL plasmids pWW0 and pWW53. J. Bacterial. 164, 887-895. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring-Harbor, NY. MULBRY, W. W.. KARNS, J. S., KEARNEY, P. C., NELSON,J. 0.. MCDANIEL, C. S.. AND WILD, J. R. (1986). Identification of a plasmid-borne parathion hydrolase gene from Flavohacterium sp. by Southern hybridization with opd from Pseudomonas diminuta. Appl. Environ.

Microhiol.

51, 926-930.

READ, D. C. (1983). Enhanced microbial degradation of carbofuran and fensulfothion after repeated applications to acid mineral soil. ARric. Ecosyst. Environ. 10, 37-46.

SERDAR, C. M., AND GB.XON, D. T. (1985). Enzymatic hydrolysis of organophosphates: cloning and expression of a parathion hydrolase gene from Pseudomonas diminuta. Biotechnology 3, 567-57 I. SERDAR,~. M.,GIBSON, D. T., MUNNECKE, D. M.,AND LANCASTER, J. H. (1982). Plasmid involvement in parathion hydrolysis by Pseudomonas diminuta. Appl. Environ.

Microbial.

44, 246-249.

SETHUNATHAN, N., AND YOSHIDA, T. (1973). A Flavobacterium that degrades diazinon and parathion.

ACKNOWLEDGMENT This paper is scientific article No. A-4686, contribution No. 7682 of the Maryland Agricultural Experiment Station.

Canud.

J. Microbial.

19, 873-875.

VANISCH-PERRON, C., VIEIRA, J., AND MESSING, J. W. ( 1985). Improved M I3 phage cloning vehicles and host stains: Nucleotide sequences of Ml3mpl8 and pUCl9 vectors. Gene33, 103-l 19. Communicated

by Francis

L. Macrina