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A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell
1Department of Molecular and Cell Biology, University of California at Berkeley, 94720-3206, USA.
Abstract:
A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes.
Insights
A mutant Mycobacterium smegmatis strain (LIMP7) exhibits altered cell envelope properties and increased susceptibility to certain antibiotics due to a defect in inositol metabolism. This impacts bacteriophage interactions and cell wall component synthesis.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mycobacterium smegmatis is a model organism for studying mycobacterial cell envelope biogenesis.
- The cell envelope is crucial for mycobacterial survival and pathogenicity.
- Inositol plays a vital role in mycobacterial cell wall structure and function.
Purpose of the Study:
- To characterize a novel mutant of Mycobacterium smegmatis with altered cell envelope properties.
- To identify the genetic basis for the observed phenotypic changes.
- To elucidate the role of inositol metabolism in mycobacterial cell envelope integrity and phage susceptibility.
Main Methods:
- Transposon mutagenesis and mutant screening.
- Phenotypic characterization including growth rate, clumping, antibiotic resistance/sensitivity, and permeability assays.
- DNA cloning and sequencing to identify the mutated gene.
- Analysis of inositol-containing cell wall components.
Main Results:
- Isolation of mutant strain LIMP7 with increased bacteriophage plaque size, altered growth, and modified antibiotic resistance.
- LIMP7 displays altered cell envelope permeability to lipophilic and hydrophilic molecules.
- The mutation in LIMP7 affects the impA gene, homologous to inositol monophosphate phosphatases, leading to reduced phosphatidylinositol dimannoside levels.
Conclusions:
- The impA gene is essential for inositol metabolism and cell envelope integrity in Mycobacterium smegmatis.
- Alterations in inositol metabolism impact mycobacterial cell wall synthesis, affecting lipoarabinomannan precursor levels.
- The identified mutation provides insights into the regulation of cell envelope biogenesis and its influence on bacteriophage-host interactions.
