Related Experiment Videos
A 7.6kb DNA region from Streptomyces kasugaensis M338-M1 includes some genes responsible for kasugamycin biosynthesis
S Ikeno1, T Tsuji, K Higashide
1Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Abstract:
A 7.6kb PstI-KpnI DNA fragment including a sequence highly similar to kasugamycin acetyltransferase gene (kac) was isolated from Streptomyces kasugaensis M338-M1 and sequenced. Nine open reading frames (ORFs), designated as ORF A, B, C, D, E, F, G, H and I, were recognized in this region, although ORF A was incomplete. ORF G runs in the opposite direction to the others. The amino acid sequence deduced from ORF H showed 98% similarity to that of the kasugamycin acetyltransferase from S. kasugaensis MB273-C4, another kasugamycin (KSM) producer. Transformation of E. coli JM109 with ORF H made the strain highly resistant to KSM. The deduced amino acid sequences of the ORF A, C and D products were similar, respectively, to glucosyltransferase I from E. coli (26%), beta-alanine: pyruvate transaminase from Pseudomonas putida (32%) and dTDP-D-glucose 4,6-dehydratase (StrE) from Streptomyces griseus (37%). The strE-like ORF (ORF D) seems to be the gene responsible for formation of the 6-deoxy structure of the kasugamine moiety. ORF A and ORF C are also likely to have roles in KSM biosynthesis. Taken together, our analyses strongly suggest that this DNA region includes at least a part of the gene cluster of KSM biosynthesis.
Insights
Researchers identified a DNA region in Streptomyces kasugaensis containing genes potentially involved in kasugamycin (KSM) biosynthesis. One gene confers high resistance to KSM when expressed in E. coli.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Kasugamycin (KSM) is an important antibiotic produced by Streptomyces species.
- Understanding the genetic basis of KSM biosynthesis is crucial for its efficient production.
Purpose of the Study:
- To identify and characterize genes involved in kasugamycin biosynthesis in Streptomyces kasugaensis.
- To investigate the function of specific open reading frames (ORFs) within a cloned DNA fragment.
Main Methods:
- DNA isolation and sequencing of a 7.6kb PstI-KpnI fragment from Streptomyces kasugaensis M338-M1.
- Bioinformatic analysis to identify open reading frames (ORFs).
- Functional characterization of ORF H through transformation of E. coli JM109.
Main Results:
- A DNA fragment containing nine ORFs (A-I) was sequenced, with ORF H showing 98% similarity to kasugamycin acetyltransferase.
- Expression of ORF H in E. coli conferred high resistance to kasugamycin.
- ORFs A, C, and D showed similarities to known enzymes involved in glycosylation and sugar metabolism, suggesting roles in KSM biosynthesis.
Conclusions:
- The analyzed DNA region likely contains a portion of the kasugamycin biosynthesis gene cluster.
- ORF H encodes a kasugamycin acetyltransferase, contributing to KSM resistance.
- Other identified ORFs may be involved in the formation of the kasugamine moiety and overall KSM production.