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Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C
Abstract:
An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.
Insights
Researchers created a Bacillus licheniformis mutant with blocked alkaline phosphatase secretion, revealing defects in phoP and phoR genes. This mutant also showed altered malate utilization and cell wall structure, providing insights into gene regulation.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Alkaline phosphatase is a crucial enzyme in Bacillus licheniformis.
- Understanding the regulation of enzyme secretion is vital for biotechnological applications.
- Genetic mutations can significantly alter cellular functions and protein secretion pathways.
Purpose of the Study:
- To isolate and characterize a mutant of Bacillus licheniformis 749/C with a complete block in alkaline phosphatase secretion.
- To investigate the genetic basis of the secretion defect, focusing on the phoP and phoR genes.
- To analyze the biochemical and structural changes associated with the blocked secretion.
Main Methods:
- Utilizing N-methyl-N'-nitro-N-nitrosoguanidine for mutagenesis to induce mutations in closely linked genes.
- Employing a two-step selection process involving cephalosporin C for malate utilization defects and dye medium for alkaline phosphatase secretion defects.
- Characterizing the mutant through biochemical analysis of alkaline phosphatase extraction and SDS-PAGE, and electron microscopy of cell wall structure.
Main Results:
- A stable mutant (B. licheniformis 749/cNM 105) was obtained with a total block in alkaline phosphatase secretion.
- The mutation affected the phoP and phoR gene regions, and the malate gene, indicating their linkage.
- The mutant exhibited membrane-bound, non-extractable alkaline phosphatase, altered protein profiles, impaired malate utilization, and an additional electron-dense layer on the cell wall.
Conclusions:
- The phoP and phoR genes are essential for the secretion of alkaline phosphatase in Bacillus licheniformis.
- The mutation impacts multiple cellular processes, including enzyme secretion, nutrient utilization, and cell wall integrity.
- This mutant provides a valuable tool for studying the complex mechanisms of protein secretion and gene regulation in Gram-positive bacteria.