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Characterization of a glycosyl transferase inactivating macrolides, encoded by gimA from Streptomyces ambofaciens
A Gourmelen1, M H Blondelet-Rouault, J L Pernodet
1Institut de Génétique et Microbiologie, UMR 2225, Université Paris-Sud XI, Orsay, France.
Abstract:
In Streptomyces ambofaciens, the producer of the macrolide antibiotic spiramycin, an open reading frame (ORF) was found downstream of srmA, a gene conferring resistance to spiramycin. The deduced product of this ORF had high degrees of similarity to Streptomyces lividans glycosyl transferase, which inactivates macrolides, and this ORF was called gimA. The cloned gimA gene was expressed in a susceptible host mutant of S. lividans devoid of any background macrolide-inactivating glycosyl transferase activity. In the presence of UDP-glucose, cell extracts from this strain could inactivate various macrolides by glycosylation. Spiramycin was not inactivated but forocidin, a spiramycin precursor, was modified. In vivo studies showed that gimA could confer low levels of resistance to some macrolides. The spectrum of this resistance differs from the one conferred by a rRNA monomethylase, such as SrmA. In S. ambofaciens, gimA was inactivated by gene replacement, without any deleterious effect on the survival of the strain, even under spiramycin-producing conditions. But the overexpression of gimA led to a marked decrease in spiramycin production. Studies with extracts from wild-type and gimA-null mutant strains revealed the existence of another macrolide-inactivating glycosyl transferase activity with a different substrate specificity. This activity might compensate for the effect of gimA inactivation.
Insights
A novel glycosyltransferase gene, gimA, was identified in Streptomyces ambofaciens, conferring low-level macrolide resistance through glycosylation. Its inactivation had no adverse effects, but overexpression reduced spiramycin production.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Streptomyces ambofaciens produces the macrolide antibiotic spiramycin.
- Genes conferring resistance, like srmA (rRNA monomethylase), are known.
- Glycosyltransferases are implicated in macrolide inactivation.
Purpose of the Study:
- To identify and characterize novel genes involved in macrolide resistance in S. ambofaciens.
- To investigate the function and substrate specificity of the newly identified gimA gene.
- To assess the impact of gimA expression on spiramycin production and bacterial survival.
Main Methods:
- Gene cloning and expression in a susceptible host (S. lividans).
- Enzymatic assays using cell extracts to detect glycosylation activity.
- Gene replacement and overexpression studies in S. ambofaciens.
- Analysis of macrolide resistance profiles and antibiotic production.
Main Results:
- A novel gene, gimA, encoding a putative glycosyltransferase, was identified.
- Expression of gimA conferred low-level resistance to certain macrolides via glycosylation, modifying precursors like forocidin but not spiramycin.
- Inactivation of gimA in S. ambofaciens had no negative impact on survival.
- Overexpression of gimA significantly decreased spiramycin production.
- Evidence for an alternative macrolide-inactivating glycosyltransferase activity was found.
Conclusions:
- GimA is a novel macrolide-inactivating glycosyltransferase with specific substrate preferences.
- GimA plays a role in modulating macrolide resistance and antibiotic production.
- The presence of compensatory glycosyltransferase activities suggests complex regulation of macrolide metabolism.