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/C/NM105) 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.

Export and secretion of proteins by bacteria

“…Included among this third group of proteins are some where genetic analyses are beginning to shed light on the possible mechanisms of secretion. Perhaps the best characterised mutants are the Sec- [237] and sacU mutants of Bacillus sp. [220,226,268a].…”

“…[220,226,268a]. The Sec-mutants of B. licheniforrnis described by Kumar et al [237] have an additional electron-dense surface layer which is not found in wild-type cells, and accumulate alkaline phosphatase in discrete sites in the plasma membrane rather than releasing it into the medium. This may provide additional evidence for the existence of specific sites for protein export in the plasma membrane, as discussed in section 5.…”

Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C

“…Bacillus licheniformis 749/C cells were grown in low-phosphate medium for alkaline phosphatase derepression. This medium contained 1% casein hydrolysate and 0.1% glucose (17). Growth continued for 4.5 h at 32°C with constant shaking.…”

Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

“…Growth,: enzyme assay, and antibody production. B. licheniformis 749/C cells were grown in low-phosphate casein hydrolysate medium containing 0.1% glucose for 4.5 h at 32°C with constant shaking (15). Alkaline phosphatase activity was assayed by monitoring the release of p-nitrophenol from p-nitrophenyl phosphate as described previously (10); protein content was estimated by the method of Lowry et al (16).…”