Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONtesting for primer design (chapter 2 section 2.2). Application of the act04f and act06rprimers in PCR screening experiments using actinobacterial pure cultures amplifiedthe predicted 0.47kb product, in two of the three streptomyces tested. However in thethird streptomycete S. argillaceus a faint band in the expected area was observed,which may indicate that only partial sequence similarity was able to be achieved. Thepresence of unexpected low molecular weight products in the molecular weight rangeof 0.2 – 0.3 kb was also detected (Figure 22). These products may be due to thepresence of similar sequences as actinobacteria are known to contain multiple copiesof PKS genes or could be non-specific products.Partial sequencing of the amplified products from the actinobacterial Cerylid isolatesA1488 (Modestobacter) and A3023 (Actinoplanete) showed that the DNA sequenceobtained from the isolates showed a high degree of DNA sequence similarity (73-77% and 66-80 %, respectively) with aromatic KS α genes from different actinobacterialspecies (Table 35). Alignment of the deduced protein sequences translated from thetwo nucleotide sequences A1488 and A3023 Cerlylid cultures, to known KS α domainpeptide sequences from different actinobacteria genera indicated that approximately39 - 47 % similarity from the respective isolates were similar to that of aromatic KS αgenes (Figure 23). The translated sequence obtained from isolate A1488 showed thatit contains characteristic conserved domains of the protein sequence for the KS α gene(highlighted in blue in Figure 23), and is in accordance with the consensus sequencefor the KS α protein (Fernández-Moreno et al., 1997). Comparison of both the aminoacid sequences of A1488 and A3023 showed that they share 57 % similarity to eachother.The presence of the KS α gene in two environmental non-streptomycete actinobacteriais significant in that, the primer sequence chosen was designed from mainlystreptomycete KS α sequences indicating that the primer set act04f/act06r is capable todetect similar KS α sequences in different actinobacterial species. Further cloning andexpression studies of these genes in a suitable host could be used to determine thefunction and metabolic products of these putative KS α genes. Indications fromantimicrobial testing showed that isolate A3023, contained bioactivity however noactivity was detected for A1488. As only antimicrobial activity was tested providing_____________________________________________________________________114