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The two restriction enzyme digest patterns were very similar but did show some
differences. The isolates which clustered with M protea (154,124.5ice3.744.794.214
and 54), showed the same clustering for both digests (Figs. 5.2a & b) indicating they
were probably all the same species. Isolates 39 and 86 were probably also identical as
they formed a single taxon with both restriction enzymes (Figs. 5.2a & b). Isolates 302
and 33 appeared to form a single taxon with Hpa II (Fig 5.2a), however, with Alu I they
formed two separate taxa while still clustering together (Fig 5.2b), indicating that these
two isolates had a higher similarity to each other than to any of the other isolates. The
same situation was also shown by isolates 492 and 47. Isolate 494 appears as a direct
outlier to the M protea taxon in both digest patterns indicating a distant relationship.
Isolates 583 and 213 appear within the same group in both patterns, however, the degree
of relatedness differs for the different enzymes. Using Alu I (Fig 5.2b) they clustered
together with isolate 53 which when digested with Hpa II was found within the same
group but at a different degree of relatedness (Fig 5.2a). Isolate 466 moved from an
outlier of the 39/86 taxon using Hpa II (Fig 5.2a), to an outlier of the M protea taxon
when using Alu I (Fig 5.2b). Isolate 732 shows a similar jump in position from a
taxonomic grouping with the 302/33 taxon when using Hpa II (Fig 5.2a), to a grouping
with the 39/86 taxon when using Alu I (Fig 5.2b).
5.2.2 -16S rRNA nucleotide sequencing
PCR products were purified using the QlAquick PCR purification centrifugation
protocol (QIAGEN),
eluted in water and then sequenced directly (Chapter 2, section
2.9.6.4). The majority of the isolates provided approximately Ikb of nucleotide sequence
directly from the PCR product. However, some of the PCR products failed to produce
adequate sequence using this method; these sequences were cloned into the TOPO 2.1
vector (Chapter 2, section 2.9.6.3) using the TOPO TA cloning kit (Invitrogen). and
sequenced using the M 13 forward and reverse primers. This provided better sequencing
results for the remaining isolates. Isolates 494,213,492,5(ice3)
and 466 proved difficult
to clone and so these sequences have not be adequately sequenced, as time constraints did
not allow further attempts.
The available sequence data for the 16S rDNA gene was entered into the Genbank
database (http: //wNti-w. ncbi. nlm. nih. ) and assigned an accession number (Table 5.1).
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