From a cosmid library of Streptomyces cyanogenus S136, DNA fragments encompassing approximately 35 kb of the presumed landomycin biosynthetic gene cluster were identified and sequenced, revealing 32 open reading frames most of which could be assigned through data base comparison.
On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.
The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.
These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.
The phage growth limitation (Pgl) system of Streptomyces coelicolor confers resistance to C31 and its homoimmune phages. The positions of the pgl genes within a 16-kb clone of S. coelicolor DNA were defined by subcloning, insertional inactivation, and deletion mapping. Nucleotide sequencing and functional analysis identified two genes, pglY and pglZ, required for the Pgl ؉ (phage-resistant) phenotype. pglY and pglZ, which may be translationally coupled, are predicted to encode proteins with M r s of 141,000 and 104,000, respectively. Neither protein shows significant similarity to other known proteins, but PglY has a putative ATP/GTP binding motif. The pglY and pglZ genes are cotranscribed from a single promoter which appears to be constitutive and is not induced by phage infection.The C31 resistance mechanism of Streptomyces coelicolor (termed Pgl, for phage growth limitation) prevents plaque formation on lawns of growing bacteria. Pgl Ϫ strains (sensitive to plaque formation by C31) arise spontaneously from S. coelicolor Pgl ϩ strains at high frequency (10 Ϫ3 to 10
Ϫ4) and revert to Pgl ϩ at a similar frequency (9), a situation reminiscent of various kinds of phase variation phenomena such as Salmonella typhimurium flagellar antigen variation (reference 17 and references therein), Neisseria gonorrhoeae pilin variation (18), expression of the pyelonephritis-associated pili of Escherichia coli (4), and expression of outer membrane lipopolysaccharides in Haemophilus influenzae (39).In addition to its phase variation, the Pgl system is interesting because of its novel mechanism (9). C31 propagated on a Pgl Ϫ strain (or the stably C31-sensitive strain Streptomyces lividans 66) can adsorb to and lysogenize Pgl ϩ strains of S. coelicolor at the same efficiency as Pgl Ϫ strains. Measurements of the phage lytic cycle using one-step growth curve experiments demonstrated that a single complete cycle of lytic phage development occurs in Pgl ϩ strains, resulting in a normal burst size of progeny phage. However, the progeny phage are attenuated in their ability to infect further Pgl ϩ hyphae (although they can infect Pgl Ϫ hyphae). Chinenova et al. (9) proposed that the Pgl system involves at least two components: one that modifies C31 during the initial round of infection and a second that recognizes and attenuates the lytic development of modified C31. The attenuation of modified C31 particles by Pgl ϩ hyphae does not involve adsorption-inhibition since the frequency of adsorption is the same in Pgl ϩ and Pgl Ϫ strains (9). Although there is no direct evidence of DNA modification, these observations are consistent with the Pgl system encoding a restriction-modification system in which the restriction endonuclease specifically degrades modified DNA. Restriction systems that act on foreign DNA only when it is methylated occur in several genera (19,31,35), including Streptomyces (23). However, these systems do not include a modification function, whereas Pgl ϩ strains appear to modify C31 and then restrict it in subsequent r...
A mutation in actVI-ORF1, which controls C-3 reduction in actinorhodin biosynthesis by Streptomyces coelicolor, was complemented by gra-ORF5 and -ORF6 from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22. It is hypothesized that, while gra-ORF5 alone is a ketoreductase for C-9, gra-ORF6 gives the enzyme regiospecificity also for C-3.
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