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6.2.1 -
Detection of AFP active isolates in DGGE community profiles
Fourteen AFP active isolates were compared against 7 community profiles taken
from the lakes from which the cultivated strains were isolated. Two DGGE gels were run.
firstly a gel which comprised the 10 isolates which demonstrated an AFP activity of 4-is
(SPLAT scoring, section 2.10.2) with 3 community profiles (Fig 6.1); secondly a gel
comprising 9 isolates, which represented the taxonomic groups demonstrated by the
amplified ribosomal DNA restriction analysis (ARDRA, refer to Chapter >) with 4
community profiles (Fig 6.2). The seven community profiles were not taken from the
depths and times from which the majority of the AFP active strains were isolated,
because community samples were only isolated during September and November, 2000
and eight of the AFP active bacteria were isolated from dates other than these. Five of the
remaining isolates were taken from depths at which due to the lack of community profile
DNA, it was not possible to include the appropriate community profile in this analysis.
Only one isolate, 213, was compared against a community profile from the lake, depth
and time from which it was isolated. However, it was assumed that the other 13 isolates
may have been represented in the communities throughout the depth of their respective
lakes.
In DGGE gel 1 (Fig 6.3a), lanes 4,5 and 8 each showed a single band match with
bands in the community profiles. However, none of the lanes show complete matching
which indicates that these isolates were either not present within the selected community
profiles (Fig 6.3a, Lanes 1,2 & 3) or were not represented by the DGGE community
analysis due to biases inherent in the protocol (see section 6.3.2 for a discussion of bias
within DGGE analysis). DGGE gel 2 (Fig 6.2/Fig 6.3b) indicated that 5 of the AFP active
isolates were present within the community profiles. It was observed that the majority of
the isolates showed more than one putative 16S rDNA copy (putative because they were
not confirmed through 16S rDNA sequencing). Only isolates 54 (Fig 6.1, Lane 10). 794
(Fig 6.1, Lane 13) and 583 (Fig 6.2, Lane 9) showed a single copy of the 16S rRNA gene.
Isolate 302 (Fig. 6.3b. Lane 3) showed 2 bands that were also present in the
community profile for Ace Lake, 4m from September (Fig. 6.3b, Lane 10), even though
this isolate was isolated from Triple Lake. Om during January (Table 5.2). Isolate 732
(Fig. 6.3b, Lane 6) was isolated from Ace Lake at 2m in March (Table 5.2). but matched
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