incomplete combustion of organic material, motor vehicle emissions, coal liquefaction
and gasification processes, cigarette smoke, and industrial wastes (Blumer, 1976).
Using a replica plate method, it has been observed that hydrocarbon-polluted sediments
in central and northern Chesapeake Bay are enriched in phenanthrene-degrading microorganisms (Cooney and Shiaris, 1982, West et al., 1984). Phenanthrene is not acutely
toxic, carcinogenic, or mutagenic ; however, it has been used as a model substrate in
studies of environmental degradation of polycyclic aromatic hydrocarbons, since its
structure is found in the nucleus of carcinogenic PAHs, such as benzo(a) anthracene and
3-methylcholanthrene (Cerniglia and Yang, 1984). Hydrocarbon degrading bacteria play
an important role in the clean-up of oil pollution (Colwell and Walker, 1977 ; Atlas, 1981)
and in the cycling of carbon in the environment (Gibson, 1977). Plasmid mediation of key
steps in the degradation of hydrocarbon molecules is now well established (Chakrabarty,
1980 ; Yen and Gunsalus, 1982). In general, degradation requires an interaction between
chromosomal and plasmid genes (Fennewald et al, 1978 ; Zuniga et al., 1981).
It has been suggested that phenanthrene degradation may be under plasmid control in
Alcaligenes faecalis (Kiyohara et al, 1982). Although they did not definitively associate
plasmids with oil degradation, Hada and Sizemore (1981) found that plasmid incidence
was greater in Vibrio spp. isolated from an oil field in the northwestern Gulf of Mexico
than in isolates from pristine control sites. It was, therefore, the aim of our study to
examine the plasmids in phenanthrene-degrading estuarine bacteria for their ability to
mediate the catabolism of this recalcitrant environmental pollutant.
MATERIALS AND METHODS
Bacterial strains
Bacterial strains used in this study are listed in Table 1. Details of the methods employed
in isolating and identifying Vibrio sp. W10, V. fluvialis W20 and Klebsiella/Enterobacter/Hafnia/Serratia group strains W32, W33, S53 and S54 have been reported
elsewhere (West et al, 1984). Flavobacterium sp. strains SB23 and W45, as well as the
unidentified Gram negative rods, were isolated on estuarine salts agar (ESWA) plates
(Sherga et al, 1979). The surface of each plate was uniformly spread with 0.2 ml of a
filtered ethanol solution of phenanthrene (containing 0.5 g phenanthrene per 100 ml),
using the method of Shiaris and Cooney (1983). All isolates were maintained on ESWA
slants onto which crystals of phenanthrene were sprinkled.
Preparation of Deoxyribonucleic Acid (DNA)
One and one-half milliliters of late logarithmic phase cultures grown in Luria broth
(casein Hydrolysate, 10 g ; NaCl, 10 g ; yeast extract, 5 g ; deionized water, 1 liter) at 30°C
were introduced into 1.5 ml microcentrifuge tubes and centrifuged in a microcentrifuge
(Eppendorf model 5412, Brinkman Instruments, Inc., Westbury, N.Y.). The cell pellet
was lysed and DN A preparation obtained using the procedure described by Kado and Liu
(1981).
Agarose gel electrophoresis
The agarose gel electrophoresis technique described by Meyers et al (1976) was used for
bacterial plasmid screening. Following electrophoresis, plasmids were detected by staining the gel slab in ethidium bromide (Sigma), 0.5 µg/ml, for 2 hr at room temperature.
Photographic records were prepared using a Polaroid MP4 land camera.
602
and gasification processes, cigarette smoke, and industrial wastes (Blumer, 1976).
Using a replica plate method, it has been observed that hydrocarbon-polluted sediments
in central and northern Chesapeake Bay are enriched in phenanthrene-degrading microorganisms (Cooney and Shiaris, 1982, West et al., 1984). Phenanthrene is not acutely
toxic, carcinogenic, or mutagenic ; however, it has been used as a model substrate in
studies of environmental degradation of polycyclic aromatic hydrocarbons, since its
structure is found in the nucleus of carcinogenic PAHs, such as benzo(a) anthracene and
3-methylcholanthrene (Cerniglia and Yang, 1984). Hydrocarbon degrading bacteria play
an important role in the clean-up of oil pollution (Colwell and Walker, 1977 ; Atlas, 1981)
and in the cycling of carbon in the environment (Gibson, 1977). Plasmid mediation of key
steps in the degradation of hydrocarbon molecules is now well established (Chakrabarty,
1980 ; Yen and Gunsalus, 1982). In general, degradation requires an interaction between
chromosomal and plasmid genes (Fennewald et al, 1978 ; Zuniga et al., 1981).
It has been suggested that phenanthrene degradation may be under plasmid control in
Alcaligenes faecalis (Kiyohara et al, 1982). Although they did not definitively associate
plasmids with oil degradation, Hada and Sizemore (1981) found that plasmid incidence
was greater in Vibrio spp. isolated from an oil field in the northwestern Gulf of Mexico
than in isolates from pristine control sites. It was, therefore, the aim of our study to
examine the plasmids in phenanthrene-degrading estuarine bacteria for their ability to
mediate the catabolism of this recalcitrant environmental pollutant.
MATERIALS AND METHODS
Bacterial strains
Bacterial strains used in this study are listed in Table 1. Details of the methods employed
in isolating and identifying Vibrio sp. W10, V. fluvialis W20 and Klebsiella/Enterobacter/Hafnia/Serratia group strains W32, W33, S53 and S54 have been reported
elsewhere (West et al, 1984). Flavobacterium sp. strains SB23 and W45, as well as the
unidentified Gram negative rods, were isolated on estuarine salts agar (ESWA) plates
(Sherga et al, 1979). The surface of each plate was uniformly spread with 0.2 ml of a
filtered ethanol solution of phenanthrene (containing 0.5 g phenanthrene per 100 ml),
using the method of Shiaris and Cooney (1983). All isolates were maintained on ESWA
slants onto which crystals of phenanthrene were sprinkled.
Preparation of Deoxyribonucleic Acid (DNA)
One and one-half milliliters of late logarithmic phase cultures grown in Luria broth
(casein Hydrolysate, 10 g ; NaCl, 10 g ; yeast extract, 5 g ; deionized water, 1 liter) at 30°C
were introduced into 1.5 ml microcentrifuge tubes and centrifuged in a microcentrifuge
(Eppendorf model 5412, Brinkman Instruments, Inc., Westbury, N.Y.). The cell pellet
was lysed and DN A preparation obtained using the procedure described by Kado and Liu
(1981).
Agarose gel electrophoresis
The agarose gel electrophoresis technique described by Meyers et al (1976) was used for
bacterial plasmid screening. Following electrophoresis, plasmids were detected by staining the gel slab in ethidium bromide (Sigma), 0.5 µg/ml, for 2 hr at room temperature.
Photographic records were prepared using a Polaroid MP4 land camera.
602