RESULTS AND DISCUSSION
Plasmids in phenanthrene-degrading bacteria from Chesapeake Bay varied greatly in
molecular weight (Table 1) and were randomly distributed between 1 and 110 Mdal. The
number of plasmids harbored by each strain differed substantially. Some strains possessed as many as nine distinct plasmids, e.g., strains W1, W10, and W11, while others
harbored but a single plasmid, i.e., strains W7, W29, W42, W45 and SB23. There was a
higher incidence of plasmid carriage in strains isolated from the water (W) column (4.6
plasmids per plasmid-bearing strain) than in sediment isolates (3.0 plasmids per plasmidbearing strain). Only 30 % of the plasmid-containing strains were sediment isolates. The
size distributions of plasmids carried by phenanthrene degrading bacteria in water and
sediment samples also differed. Water column isolates tended to have small plasmids.
More than one half (54.3 %) of the plasmids detected in water column isolates fell within
the 1-10 Mdal size range, compared to 38.5 % of plasmids for sediment strains. Conversely, sediment isolates had large plasmids. Almost half (46.1 %) of the plasmids borne by
the sediment isolates were in the 31-50 Mdal mw class, compared to 12.9 % of plasmids in
water column strains. Carriage of plasmids in other size ranges was low. No sediment
bacteria possessed plasmid bands in the 11-30 Mdal mw range. Poor detection of
plasmids in this size class may be due to the fact that chromosomal DNA fragments band
in this area, thereby obscuring plasmid DNA bands.
Generally, the frequency of plasmid carriage was higher in strains obtained under
selective conditions than in natural, randomly selected heterotrophic populations. The
moderately high percentage (36 %) of plasmid carriage observed in our study was not
unexpected, when compared to similar findings reported by other investigators. Hada
and Sizemor (1981) have reported that 35 % of 440 Vibrio strains isolated from an oil field
in the northwestern Gulf of Mexico, without selection, carried plasmids, compared to 23
% of Vibrio strains from non-oil- impacted control sites.
In a closely related study in our laboratory of plasmid incidence in bacteria isolated from
polluted and non-polluted Atlantic Ocean samples, it was found that bacteria associated
with toxic chemical waste more frequently contained plasmid DNA and demonstrated
antibiotic resistance than bacteria isolated from domestic sewage-impacted waters or
from uncontaminated open ocean sites (Baya et al, 1986). In that study, it was observed
that plasmid molecular weights ranged from 1.4 to 151 Mdal. Plasmids in the 2.5 - 6.3
Mdal mw were most common, while those between 12.6 and 20.0 Mdal mw were
uncommon. While plasmids were detected in bacteria from all sites, the largest number of
plasmids was found in sewage effluent strains. The number of plasmids possessed by each
strain also varied, for the whole data set and between sites. Sewage effluent (SE) strains
possessed an average of 2.6 plasmids per strain, as opposed to 1.2 plasmids per strain for
sewage outfall (SO). The Ocean City (OC) and clean water (CW) strains each showed an
average of less than one plasmid for every two strains (Baya et al., 1986).
Novobiocin proved effective in curing the single 34 Mdal plasmid in Flavobacterium sp.
SB23 (Fig. 1 ). Loss of phenanthrene clearing ability by the cured derivative strain of SB 23
is concluded to be a consequence of plasmid elimination. Some investigators have
suggested that the phenanthrene degrading phenotype of a soil isolate identified as
Alcaligenes faecalis strain AFK2 may be under plasmid control. The phenanthrenedegrading (Phn+) phenotype of this organism disappeared after twenty successive subcultures in a minimal salts medium containing o-phthalate or after subculture in nutrient
broth containing mitomycin C (Kiyohara et ai, 1982). However, no actual screening for
plasmids was reported by these workers. We report here the first physical evidence of
plasmid loss correlated with loss of phenanthrene-degrading phenotype in Flavobacterium sp. SB23.
605
Plasmids in phenanthrene-degrading bacteria from Chesapeake Bay varied greatly in
molecular weight (Table 1) and were randomly distributed between 1 and 110 Mdal. The
number of plasmids harbored by each strain differed substantially. Some strains possessed as many as nine distinct plasmids, e.g., strains W1, W10, and W11, while others
harbored but a single plasmid, i.e., strains W7, W29, W42, W45 and SB23. There was a
higher incidence of plasmid carriage in strains isolated from the water (W) column (4.6
plasmids per plasmid-bearing strain) than in sediment isolates (3.0 plasmids per plasmidbearing strain). Only 30 % of the plasmid-containing strains were sediment isolates. The
size distributions of plasmids carried by phenanthrene degrading bacteria in water and
sediment samples also differed. Water column isolates tended to have small plasmids.
More than one half (54.3 %) of the plasmids detected in water column isolates fell within
the 1-10 Mdal size range, compared to 38.5 % of plasmids for sediment strains. Conversely, sediment isolates had large plasmids. Almost half (46.1 %) of the plasmids borne by
the sediment isolates were in the 31-50 Mdal mw class, compared to 12.9 % of plasmids in
water column strains. Carriage of plasmids in other size ranges was low. No sediment
bacteria possessed plasmid bands in the 11-30 Mdal mw range. Poor detection of
plasmids in this size class may be due to the fact that chromosomal DNA fragments band
in this area, thereby obscuring plasmid DNA bands.
Generally, the frequency of plasmid carriage was higher in strains obtained under
selective conditions than in natural, randomly selected heterotrophic populations. The
moderately high percentage (36 %) of plasmid carriage observed in our study was not
unexpected, when compared to similar findings reported by other investigators. Hada
and Sizemor (1981) have reported that 35 % of 440 Vibrio strains isolated from an oil field
in the northwestern Gulf of Mexico, without selection, carried plasmids, compared to 23
% of Vibrio strains from non-oil- impacted control sites.
In a closely related study in our laboratory of plasmid incidence in bacteria isolated from
polluted and non-polluted Atlantic Ocean samples, it was found that bacteria associated
with toxic chemical waste more frequently contained plasmid DNA and demonstrated
antibiotic resistance than bacteria isolated from domestic sewage-impacted waters or
from uncontaminated open ocean sites (Baya et al, 1986). In that study, it was observed
that plasmid molecular weights ranged from 1.4 to 151 Mdal. Plasmids in the 2.5 - 6.3
Mdal mw were most common, while those between 12.6 and 20.0 Mdal mw were
uncommon. While plasmids were detected in bacteria from all sites, the largest number of
plasmids was found in sewage effluent strains. The number of plasmids possessed by each
strain also varied, for the whole data set and between sites. Sewage effluent (SE) strains
possessed an average of 2.6 plasmids per strain, as opposed to 1.2 plasmids per strain for
sewage outfall (SO). The Ocean City (OC) and clean water (CW) strains each showed an
average of less than one plasmid for every two strains (Baya et al., 1986).
Novobiocin proved effective in curing the single 34 Mdal plasmid in Flavobacterium sp.
SB23 (Fig. 1 ). Loss of phenanthrene clearing ability by the cured derivative strain of SB 23
is concluded to be a consequence of plasmid elimination. Some investigators have
suggested that the phenanthrene degrading phenotype of a soil isolate identified as
Alcaligenes faecalis strain AFK2 may be under plasmid control. The phenanthrenedegrading (Phn+) phenotype of this organism disappeared after twenty successive subcultures in a minimal salts medium containing o-phthalate or after subculture in nutrient
broth containing mitomycin C (Kiyohara et ai, 1982). However, no actual screening for
plasmids was reported by these workers. We report here the first physical evidence of
plasmid loss correlated with loss of phenanthrene-degrading phenotype in Flavobacterium sp. SB23.
605