Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONThe success of PCR amplification is dependant on a suitable target sequence withavailable nucleotide sequences for the design of primers and PCR conditions. In thisstudy the majority of nucleotide sequences of SMBG retrieved from theGenBank/EMBL database were derived from Streptomyces species (Table 32,37,40and 44), and the deduced consensus sequence, on which the design of the primers wasbased, may have restricted the detection of SMBG in non-Streptomyceteactinobacteria which have different codon preferences. The designed primers mayhave displayed codon bias in amplifying only streptomycete SMBG. Strategies whichhave been used to isolate SMBG having unknown codon preferences include usingdegenerate primers (Seow et al., 1997; Nicholson et al., 2001) or heterologous probes(Sosio et al., 2000a). It was demonstrated in this study that degenerate-PCR primersappear better suited to environmental screening for SMBG, as was shown in the caseof the KS gene when non-degenerate primers were substituted for degenerate primers.Codon usage (CU) patterns in Streptomyces genes have been established (Wright &Bibb, 1992), but further analysis of the CU patterns of non-streptomyceteactinobacterial genes needs to be further evaluated. Evidence for differences in codonusages in actinobacterial genera is exemplified in the case of the aminoglycosideresistance aph (aminoglycoside phosphotransferase) gene. It has been shown that theMicromonospora aph gene has a lower G or C in the third position in codon triplets,which is significantly lower than in their corresponding Streptomyces aph gene(Salauze & Davies, 1991).Twenty two uncharacterized environmental actinobacterial isolates obtained from theCerylid culture collection (Melbourne, Australia) were individually screened with thefour sets of primers to assess the SMBG diversity amongst the isolates (Table 48).The distribution of the SMBG in the environmental isolates tested were as follows;the ketosynthase gene (KS), representive of aliphatic polyketides, was detected in 8of the 22 isolates; the β-ketoacyl synthase gene (KS α ), representative of aromaticpolyketides, was detected in 8 of the 22 isolates; the dTDP-glucose synthase generepresentative of deoxysugar aminoglycoside compounds was detected in 6 of the 22isolates; the least abundant SMBG detected amongst the isolates was the isopenicillinN synthase gene (pcbC), representative of the β-lactam antibiotics, which was onlydetected in 1 of the 22 isolates. In addition, 8 from the 22 isolates contained the_____________________________________________________________________119