Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 1: INTRODUCTIONbeen shown to contain multiple forms of RNA polymerases. RNA polymerases playintegral roles in determining which genes to transcribe and at what rates (Ishihama,1997). Promoter recognition in bacteria requires that RNA polymerase associates witha sigma (σ) factor to form a holoenzyme (Buttner et al., 1990; Kang & Roe, 1998).Most bacteria contain multiple forms of σ factor, activation of these factors is elicitedby specific signals or stress conditions (Helmann, 1999).Several Streptomycete promoters have been identified from a variety of sources,which have high A + T content compared with coding sequences. Actinobacteriacontain several classes of promoters, some of which resemble E.coli promoters inspecific nucleotide regions 10 to 35 bp before the start point of transcription. Adistinctive feature of actinobacteria is that they contain two or more transcription startsites and associated promoter sequences. This feature is believed to aid in thedifferential expression of genes in different phases of the growth cycle when themajor RNA polymerase may have different promoter specificities (Seno & Baltz,1989). Additionally altered gene expression patterns are achieved by the regulatorysystem known as the stringent response (Bascarán et al., 1991). The system isactivated in the event of nutritional limitation in which guanosine 3’-diphosphate-5’-diphosphate (ppGpp) and guanosine 3’-triphosphate-5’-diphosphate (pppGpp) aresynthesised causing an intracellular accumulation of these compounds. Theaccumulation of these compounds causes instable initiation complexes at promotersfor stable RNA-synthesis, causing a reduction of the total rate of RNA synthesis andother cellular metabolites (Strauch et al., 1991; Takano & Bibb, 1994; Pfefferle et al.,1995).Certain compounds have been shown to regulate secondary metabolite biosynthesis atthe level of transcription, such as A-factor (2-(6’methylheptanoyl)-3Rhydroxymethyl-4-butanolide)which alleviates repressor proteins and allowsbiosynthetic genes to be transcribed (Miyake et al., 1990). The two compounds ML-236B and phenobarbitol have been shown to act synergistically, effecting thetranscriptional regulation of the cytochrome P450 sca gene and influencing theproduction of pravastatin, a cholesterol lowering drug (Watanabe & Serizawa, 1998)._____________________________________________________________________26