Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSION4.2.2.5 Duration for chemical expression of bioactive metabolitesAn extended duration of actinobacterial fermentations in submerged culture were runover 288 hrs (12 days) instead of the usual average of 120 –240hrs [5 – 10 days](Iwai & Ōmura, 1982). By using antimicrobial activity as a indicator of expressivityof SM, it was shown that a cultivation period of 240 hrs (10 days) was sufficient forproduction of antimicrobial compounds in most of the media evaluated (Table 50). Asantimicrobial screening was the only parameter tested for secondary metaboliteproduction throughout the duration of the fermentation, this may have not beensensitive enough to detect minor quantities of the compound/s. Thus, further testingby TLC or HPLC-MS may have provided been better suited to detect all secondarymetabolites produced by the isolate throughout the fermentation.4.3 Correlation between genetic screening and antibioticeffectsIn certain cases the PCR screening strategy used in this study was able to detectputative SMBG’s in the actinobacterial isolates and this was able to be correlated withthe antimicrobial activities detected. However this correlation was not encounteredwith all the isolates. Interpretation of a positive PCR result must be treated withcaution as this indicates only that the strain being screened is presumed to posses thegenes necessary for the biosynthesis of that type of antibiotic. It does not indicatewhether the genes are expressed, nor does it indicate that the strain possess all thebiosynthetic genes for that class of antibiotic. Two cases were encountered that didnot adhere to this correlation. In the first case isolate A1488 produced a PCR productwith the absence of antimicrobial activity, this result may partially be explained byeither by the lack of sensitivity of the biological activity screens not been able todetect the activity or the fermentation conditions may have not been favorable topromote the production of the compound. In the second case isolate A2834 did notproduce a PCR product, however antimicrobial activity was detected. This result maybe explained by the limitation in the sensitivity of the assay as the primers may havenot been appropriate to detect the SMBG responsible for the antimicrobial activity orthat it may have been a false negative where the strain being screened may possess thebiosynthetic genes for the antibiotic, but no PCR product is seen. False negatives in_____________________________________________________________________124