Characterization of a phenanthrene degradation plasmid from Alcaligenes faecalis AFK2

JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 69, NO. 1, 54-56. 1990

Characterization of a Phenanthrene Degradation Plasmid from Alcaligenes faecalis AFK2 H O H Z O H KIYOHARA, 1. NOBORU TAKIZAWA, 1 HITOSHI D A T E ] SHIN TORIGOE, l AND KEIJI YANO 2

Biotechnology Laboratory, Department of Applied Chemistry, Faculty of Engineering, Okayama University of Science, 1-1 Ridai-cho, Okayama 700,1 and Radiation Microbiology Laboratory, Department of Agricultural Chemistry, Faculty of Agriculture, Tokyo University, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113,2 Japan Received 30 June 1989/Accepted 6 November 1989 A plasmid pHK2 involved in phenanthrene degradation was isolated from Alcaligenesfaecalis AFK2. Some mitomycin C treated cells lost the pHK2 plasmid. The plasmid could be transformed into Pseudomonasputida and A. faecalis. They gained the ability to utilize phenanthrene as well as o-phthalate, an intermediate of phenanthrene degradation. Phenanthrene dioxygenase could be detected from the transformants after induction with phenanthrene. The molecular size of pHK2 D N A was determined to be 42.5 kilobase (kb), and its physical map was constructed.

from the wild type strain AFK2, was tested for the loss of the phenotype and the presence of plasmids after being culture for 1 or 2 d in LB medium containing a curing reagent such as 5/.tg-ml i mitomycin C (MC), 30¢tg-ml acrydine orange (AO), 1,000/lg.ml i ethidium bromide (EB) or 500 gg. ml- 1 sodium sulfate (SDS), MC, AO and SDS resulted in the loss of the phenotype at frequencies of 2.0, 2.3 and 1.1 °0/, respectively, while EB did not show any curing effect. One strain (AFK2111) of the 7 isolates cured with MC, which was unable to utilize PH as well as PCA, lost both of the plasmids (Fig. 1). At that time, clones forming black colonies on the PH sprayed MM2 plates (15) were found at a low frequency. The strain AFK217, one of such clones, utilized PH and carried only pHK2. These findings strongly suggested the involvement of the pHK2 plasmid in PH degradation. The pHK2 DNA and a DNA mixture of pHK1 and pHK2 were isolated from the cells of AFK217 and AFK2, respectively, and purified by centrifugation at 318,000 × g for 4 h in an ethidium bromide-cesium chloride buoyant gradient. According to the procedure described by Bagdasalian et al. (16), transformation experiments were carried out with the purified DNA, and the Phn ~ clones were selected as clear zone forming colonies on PH sprayed MM2 plates containing 0.01% yeast extract (MM2Y). Consequently, pHK2 DNA transformed the cured strain AFK2111 (tet) at a frequency of 3.65 × 102 cells./lg D NA ~; Pseudomonas putida ACI0 (met) at 1.02x 104cells.ztg DNA 1; p. putida TN1032 (trp, leu) at 1.05 × 103 cells-/~g DNA 1; P. putida PpG1064 (trp/NAH) at 7.90x 102 cells./~g D N A - l ; and P. putida AC31 (rnet/SAL) at 5.50 x 104 cells./~g DNA 1. All of the transformants were found to carry pHK2 plasmid by agarose gel electrophoresis (Fig. 1). When a DNA mixture of pHK1 and pHK2 was used for the transformation of PpG1064 and AC31, transformants occurred at frequencies of 1.35 × 103 and 1.00x 104 cells ./~g DNA 1, respectively. These transformants, too, only had pHK2 other than NAH or SAL plasmid. Other bacteria tested such as Escherichia coli C600, Pseudomonas aeruginosa AC59, and Bacillus subtilis IAM1145 were not transformed. In liquid MM2Y containing 0.4% (wt.vol ~) solid PH

The bacterial utilization of polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, phenanthrene and anthracene has been well documented. The major features of the catabolic pathways by soil bacteria were first elucidated by Evans and his colleagues (I, 2) in 1964. Later, the initial oxidative steps were extensively investigated by Gibson and his group (3). We have found that Aeromonas sp. $45P1 and Alcaligenes faecalis AFK2 catabolize phenanthrene (PH) through 1-hydroxy-2-naphthoate (1H2NA), 2-carboxybenzaldehyde (2CBAL), o-phthalate (OPA) and protocatechuate (PCA) (4-6). Thus, the catabolic pathway we proposed does not converge to the naphthalene degrading pathway shown by Evans et al. (2). The PH degrading pathway of Aeromonas sp. $45P1 has been demonstrated in other PH degrading bacteria such as Pseudomonas sp., Vibrio sp. (7), Micrococcus sp. (8), Mycobacterium sp. (9), as well as others (10). The involvement of plasmids in the degradation of many aromatic compounds such as toluene (11), salicylate (12), and naphthalene (13) has been reported. Some catabolic genes, including regulatory genes, have been extensively analyzed. Little, however, is known about the genetic information of bacterial catabolism of low volatile, water insoluble, solid PAHs such as phenanthrene. We have previously reported the loss of the PH degrading phenotype (Plan +) ofA. faecalis AFK2 after successive subculturing on OPA, and also suggested the involvement of a plasmid in its phenotype (5). We present here definite evidence that a plasmid carries the genes responsible for the catabolism. The isolation of plasmid DNA from A. faecalis AFK2 was attempted by a slight modification of Hansen and Olsens method (14). Two plasmids were isolated, and their molecular weights estimated to be 66.9 and 33.4 kb from the relative mobilities on electrophoresis at 50 volts for 15 h on an 0.8% agarose slab gel in TBE buffer (89mM Tris, 2.5 mM EDTA, 89 mM boric acid, pH 8.2) (Fig. 1). The plasmids were denoted pHK1 and pHK2, respectively. Then, to test whether the plasmids are involved in the Phn ~ phenotype, AFK21, a tetracycline resistant derivative * Corresponding author. 54

VOL. 69, 1990

NOTES

TABLE 1.

55

Oxygen uptake rates with possible intermediates by resting cell suspensions of strains carrying the pHK2 plasmid grown on phenanthrene Induction with pH

Strain

Plasmid

AFK2

pHKI~pHK2

+

AFK217

pHK2

-

AFK2111

--

+

PpA 13

pHK2

-

ACI0

--

+

Oxygen consumption (nmol. min ~.mg dry cell ') with the following possible intermediates PH l H2NA 2CBAL OPA PCA NH SD SA CAT 361 2 61 8 0 0 60 7 3 3

1510 93 139 29 10 0 690 32 0 8

167 20 33 3 0 0 95 0 0 0

50 6 74 3 4 0 51 1 0 8

151 13 28 9 0 0 72 0 0 1

25 0 11 1 0 0 0 0 7 7

31 19 0 0 0 0 4 0 17 17

0 0 0 6 5 0 2 3 0 0

0 7 0 0 5 0 8 12 0 0

The suspensions of cells (5 mg dry cells) which had been grown for 24 h in MM2Y with (--) or without ( ) phenanthrene (PH) were measured for oxygen uptake rates with possible intermediates (10/~mol) of protocatechuate and catechol converging pathways on phenanthrene degradation as follows: 1H2NA, 1-hydroxy-2-naphthoate; 2CBAL, 2-carboxybenzaldehyde; OPA, o-phthalate: PCA, protocatechuate; NH, naphthalene; SD, salicylaldehyde; SA, salicylate; CAT, catechol.

as a sole c a r b o n s o u r c e , t h e s e t r a n s f o r m a n t s grew well, b u t n o t as well as t h e wild A F K 2 . T h e r e f o r e , in o r d e r to e l u c i d a t e t h e c a t a b o l i c steps w h i c h p H K 2 e n c o d e s , A F K 2 (pHK1 and pHK2), AFK217 (pHK2), AFK2111 (cured strain) and a transformant, P p A 1 3 ( p H K 2 ) , o f P. putida A C 1 0 were t e s t e d f o r t h e i r o x i d a t i v e abilities a g a i n s t p o s s i b l e i n t e r m e d i a t e s (10 m M ) u s i n g a YSI o x y g e n e l e c t r o d e . A s s h o w n in T a b l e 1, A F K 2 , A F K 2 1 7 a n d P p A 1 3 c o n s u m e d o x y g e n w i t h P H as well as all i n t e r m e d i a t e s , b u t d i d n o t w i t h n a p h t h a l e n e a n d its degr a d a t i v e i n t e r m e d i a t e s , s a l i c y l a l d e h y d e (SD), salicylate (SA) a n d c a t e c h o l ( C A T ) . T w o s t r a i n s , A F K 2 1 1 1 a n d P. putida A C 1 0 , w h i c h d i d n o t c a r r y p H K 2 s h o w e d n o o x i d a t i o n a g a i n s t t h e s e c o m p o u n d s . T h e s e r e s u l t s suggest t h a t t h e p H K 2 p l a s m i d e n c o d e s e n z y m e s i n v o l v e d in t h e c a t a b o l i c steps f r o m P H u p t o P C A . E n s l e y et al. (17) f o u n d t h a t E. coli c o n t a i n i n g a r e c o m b i nant DNA carrying the naphthalene dioxygenase gene (nahA) f o r m s i n d i g o , to w h i c h i n d o l e c o n v e r t e d f r o m t r y p t o p h a n b y t r y p t o p h a n a s e in t h e h o s t cells is o x i d i z e d w i t h t h e nahA g e n e p r o d u c t . T o o b t a i n f u r t h e r e v i d e n c e f o r t h e i n v o l v e m e n t o f p H K 2 in t h e c a t a b o l i s m o f P H , t h e synthesis o f p h e n a n t h r e n e d i o x y g e n a s e was j u d g e d a c c o r d i n g to t h e f o r m a t i o n o f i n d i g o f r o m i n d o l e . A s s h o w n in T a b l e

A

B

C

D

E

F

G

2, t h e wild s t r a i n A F K 2 p r o d u c e d a blue dye i n d i g o o n l y w h e n 2 m M i n d o l e was a d d e d a n d c u l t u r e d o v e r n i g h t a f t e r b e i n g c u l t u r e d o v e r n i g h t in M M 2 Y S ( M M 2 Y s u p p l e m e n t e d w i t h 10 m M d i s o d i u m s u c c i n a t e as a g r o w t h s u b s t r a t e ) c o n t a i n i n g 0.40/00 ( w t . v o l ') P H ( T a b l e 2, D c o l u m n ) . T h i s p r o d u c t i o n suggests t h a t p h e n a n t h r e n e dioxy g e n a s e c a n oxidize i n d o l e a d d e d to give i n d i g o . A F K 2 1 7 a n d P p A 1 3 c a r r y i n g p H K 2 f o r m e d t h e dye, b u t A F K 2 1 11, A C 1 0 a n d T N 1 0 3 2 w i t h o u t t h e p l a s m i d did n o t . T h a t is, the indigo formation apparently suggested the expression o f a p h e n a n t h r e n e d i o x y g e n a s e g e n e (phnA) o n p H K 2 in

P. putida. T o test t h e m o l e c u l a r p r o p e r t i e s o f t h e p l a s m i d , t h e p u r i f i e d D N A was p h o t o g r a p h e d b y e l e c t r o n m i c r o s c o p y a c c o r d i n g t o a p r o c e d u r e p r e v i o u s l y d e s c r i b e d (18). T h e p h o t o g r a p h p r o d u c e d a 42. l - k b circle. T h e D N A was t h e n d i g e s t e d w i t h a single species a n d t h e d o u b l e c o m i n a t i o n s

TABLE 2.

Expression of phenanthrene dioxygenase gene (phnA) encoded by pHK2

Strain AFK2(pHK1, pHK2) AFK217(pHK2) AFK2111 (pHK2) AFK2111 PpAI3(pHK2) AC10 TNI032(pHK2) TN1032

H

4.,,

FIG. 1. Plasmid DNAs in A. faecalis AFK2, its cured strains, AFK217 and AFK2111, and P. putida AC10 and its Phn + transformant, PpA13. Lanes, A to C, show marker plasmid DNA [A, F plasmid (96Kbp); B, T P l l 6 plasmid (218Kb); C, RP4 plasmid (55 Kbp)], and lanes, D, E, F, G and H, plasmids in AFK2, AFK217, AFK2111, ACI0 and PpAl3, respectively. An arrow indicates the band of chromosomal DNA.

Formation of indigo from indole (Abs. 598 nm) A B C D E F" 0.042 0.000 0.010 0.000 0.026 0.000 0.017 0.000

0.072 0.122 0.178 0.000 0.152 0.001 0.107 0.001

0.013 0.005 0.013 0.000 0.031 0.000 0.142 0.035

0.317 0.232 0.378 0.005 0.260 0.002 0.420 0.002

0.010 0.005 0.016 0.000 0.059 0.001 0.039 0.000

0.182 0.078 0.094 0.002 0.222 0.002 0.241 0.001

a Formation of indigo was determined by using the cells (OD6~0 =0.5) which had been grown for 2 d in MM2YS~phenanthrene (0.4°/00, w/v) by the following procedures: A, the cells were cultured for 2 d in MM2YS + 2 mM indole; B, after the cells were cultured overnight in MM2YS, 2 mM indole was added and then the culture was shaken overnight; C, the cells were cultured for 2 d in MM2YS + indole+phenanthrene; D, after the cells were cultured overnight in MM2YS+phenanthrene, indole was added and the culture shaken overnight; E, after the cells were cultured overnight in MM2YS + indole, phenanthrene was added and the culture shaken overnight; F, the cells were cultured for overnight in MM2YS, indole and phenanthrene were added and the culture shaken overnight. Indigo formed was extracted in a hot bath from 5 ml of the cultures with 5 ml of ethyl acetate. The extracts were filled up to 5 ml and the absorbance measured at 598 nm.

56

KIYOHARA ET AL.

J. FERMENT. BIOENG.,

REFERENCES ,,...,

L,O

0

pra

pHK2 30

~0

42.5

kbp 20

\

FIG. 2.

Restriction map of pHK2 DNA.

o f 30 r e s t r i c t i o n e n d o n u c l e a s e s . Aatl, AatII, A valI, BanII,

BglII, EcoRV, HpaI, HindIII, KpnI, NcoI, NruI, Pstl, SalI, SmaI a n d XrnaI g a v e a single f r a g m e n t ; DraI, Pvul a n d StuI, 2 f r a g m e n t s ; NarI a n d ScaI, 3 f r a g m e n t s ; Bali, 6 f r a g m e n t s ; SphI, 7 f r a g m e n t s ; EcoRI a n d AvaI, II f r a g m e n t s ; Banl, BanIII, HaeII, HinfI a n d NciI, numerous fragments. From the average of the sums of the size o f single digests w i t h t h e s e e n z y m e s a n d digests w i t h t h e d o u b l e c o m b i n a t i o n s a m o n g NarI, DraI, PvuI a n d StuI, t h e m o l e c u l a r size o f t h e D N A was e s t i m a t e d to be 42.5 k b . T h e v a l u e a g r e e d well w i t h t h a t o b t a i n e d b y elect r o n m i c r o s c o p y . F r o m t h e results, t h e r e s t r i c t i o n sites o f NarI, Dra.I, PvuI a n d StuI were m a p p e d o n t o t h e p l a s m i d D N A (Fig. 2). T h e p r e s e n c e o f p l a s m i d s in P h n ~ b a c t e r i a s u c h as Beijerinckia sp. (18) a n d Mycobacterium sp. (10) h a v e a l r e a d y b e e n r e p o r t e d . H o w e v e r , t h e s e results d i d n o t p r o v i d e c r u c i a l e v i d e n c e f o r t h e i n v o l v e m e n t o f t h e p l a s m i d s in t h e P h n ~ p h e n o t y p e . T h e p r e s e n t r e p o r t r e p r e s e n t s t h e first d e s c r i p t i o n o f a P h n ~ p l a s m i d w i t h definitive e v i d e n c e supp o r t e d a n d s t r e n g t h e n e d b y t h e t r a n s f o r m a t i o n experiments. Our correct studies are directed towards cloning a n d a n a l y z i n g genes e n c o d i n g p h e n a n t h r e n e dioxy g e n a s e , a n e n z y m e i n v o l v e d in t h e initial step o f b a c t e r i a l PH degradation. We express our appreciation to Mr. K. Akiyama, Miss 1. Nozawa, Mr. T. Kotani, and Mr. A. Fujimori for their excellent experimental assistance. A part of the study was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.

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