Alkaline Phosphatase Mutants of Bacillus subtilis

RGlenn, A

doi:10.1071/bi9750323

Cited by 13 publications

(4 citation statements)

References 5 publications

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“…Both mother cell and developing forespore are permeable to phosphates until the appearance of refractility (11,21). Previously isolated Bacillus "phoA" mutants (now known to be mutations in regulatory genes) that expressed very low levels of all APases under phosphate starvation conditions had wild-type sporulation APases (4,5,19). Similarly, many sporulation-regulatory mutants that were sporulation APase and engulfment negative (blocked at stage II) induced normal amounts of vegetative APases when grown in lowphosphate medium (25) …”

Section: Discussionmentioning

confidence: 99%

“…When covalently closed circular plasmid DNA containing homologous DNA is transformed into B. subtilis, a Campbell-type integration can occur, resulting in a duplication of the homologous region flanking heterologous DNA. If the homology is entirely within a transcriptional unit, recombination results in the production of two truncated, nonfunctional units flanking the 3.7-kilobase vector, as shown in part 4. In this case, verification that the desired integration had taken place used two different probes (part 5).…”

Section: Methodsmentioning

confidence: 99%

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The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis

et al. 1990

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The first alkaline phosphatase (APase) structural gene mutant of Bacilus subtilis 168 was constructed by using a clone identified by hybridization to a synthetic degenerative oligonucleotide. The design of the probe was based on the first 29 amino acids of the sequenced mature APase III protein, which had been isolated from the secreted fraction of Bacillus species produce alkaline phosphatase (APase) when phosphate becomes growth limiting (termed vegetative) as well as during sporulation, when phosphate supplies are abundant. We have shown that there are at least two different APase proteins responsible for vegetative APase activity (9) and present evidence here that at least two are produced during sporulation. When the vegetative APases are derepressed in culture medium which does not support sporulation, the two secreted proteins that are responsible for the major APase activity are APase III and APase IV. APase III has a native molecular weight of 80,000 and is active as a dimer. APase IV is active as a monomer and has a native molecular weight of 45,000 (9).During the process that leads to formation of a mature spore, APase synthesis is derepressed as a developmental stage-specific event unrelated to the supply of phosphate in the medium (4-6, 11, 33). The function of the sporulation APase proteins in producing a mature exospore or later in the germination process is unknown because previously the only mutations that eliminated sporulation APase were in regulatory genes that block all developmental events past stage II.In order to understand the function and regulation of the multiple alkaline phosphatase proteins that are synthesized in Bacillus subtilis, we have isolated, purified, and sequenced the amino termini of two of these proteins (9). These sequences allowed us to synthesize oligomers that were used to probe genomic libraries. We report here the cloning, partial sequence, and insertional inactivation of one of those APases, APase III. We also present evidence that the APase III protein, unlike APase IV, is synthesized during both vegetative and sporulation-derepressing conditions. Analysis of the phoAIII strain, the first structural APase mutant in Bacillus species, indicates that this gene * Corresponding author.responds to phosphate starvation as well as to the developmental signals of sporulation. MATERIALS AND METHODSBacterial strains and plasmids. B. subtilis JH646MS (trpC2 pheAl spoOA12 abrB15) (from J. Hoch) was used to isolate the APase proteins. B. subtilis 168 (trpC2) was the wild-type strain from which the gene was cloned and in which the structural mutations were made. The plasmid pJM103 (J. Hoch), derived from a ligation between pUC19 and the gram-positive chloramphenicol resistance gene, was used in cloning and construction of the structural mutant. The recipient Escherichia coli strain used for cloning, subcloning, and culturing the plasmid was DH5a

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis

et al. 1990

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“…vegetative APase) was dependent on pho-regulon genes, phoP and phoR (Miki etaf., 1965), while total sporulation APase induction required stage II sporulation genes spollA. spollG and spollE (Grant, 1974;Glenn, 1975;Piggott and Taylor, 1977). In order to understand the mechanisms by which these various genes regulate APase expression, it was necessary to determine their effects on the individual APase genes that contribute to total APase activity.…”

Section: Introductionmentioning

confidence: 99%

Separate promoters direct expression of phoAIII, a member of the Bacillus subtilis alkaline phosphatase multigene family, during phosphate starvation and sporulation

Chesnut

Bookstein

Hulett

1991

Molecular Microbiology

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Alkaline phosphatase (APase) expression can be induced in Bacillus subtilis by phosphate starvation or by sporulation. We have recently shown that there are multiple APase structural genes contributing to the total alkaline phosphatase expression in B. subtilis. The expression of the alkaline phosphatase III gene (phoAIII) was analysed under both phosphate-starvation induction and sporulation induction conditions. phoAII is transcribed from two promoter regions, PV and PS. The PV promoter initiated transcription 37 bp before the translation initiation codon and was used to transcribe phoAIII during phosphate-starvation induction in vegetative cells. The PS promoter initiated transcription 119 bp before the translation initiation codon and was used during sporulation induction. Genes which have previously been shown to affect total vegatative APase, pho regulon genes phoP, phoR and phoS, affected expression of phoAIII during phosphate starvation. Genes known to affect expression of total sporulation APase, i.e. spoIIA, spoIIG and spoIIE, affected phoAIII expression during sporulation induction. Our data show that one member of the APase multigene family, phoAIII, contributes to the total APase expression both during phosphate-starvation induction and sporulation induction, and that the mechanism of regulation includes two promoters, each requiring different regulatory genes.

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“…The pho regulon genes phoP, phoR, and phoT (14) are involved in the regulation of vegetative APase but not sporulation APase. However, we have recently shown that two early sporulation genes, spoOA and spoOH, also are involved in the regulation of vegetative APase (9), suggesting that overlapping control systems (sporulation and the pho regulon) function to regulate the synthesis of vegetative APase. The fact that the biochemically indistinguishable APase proteins were isolated from the two cell types led to the hypothesis that a single gene under complex regulatory control is responsible for all APase activity in B. subtilis (5). In contrast, despite an extensive search by a number of groups, including ours (5)(6)(7)12), no phosphatase mutant which is the result of a structural-gene mutation has yet been isolated.…”

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confidence: 99%

Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis

1990

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Two secreted alkaline phosphatase proteins were purified from cultures of Bacillus subtilis JH646MS. The two proteins showed slight differences in subunit molecular weight, substrate specificity, and charge characteristics. A total of 62% of the first 22 amino-terminal amino acids were identical. Both sequences showed conservation of structural features identified in Escherichia coli and human alkaline phosphatases. One alkaline phosphatase was a monomer and the other was a dimer. Culture conditions for APase production. The culture conditions used to maximize vegetative APase production in B. subtilis were a modification of the procedure developed for B. licheniformis (17). The liquid defined medium in which each culture was grown and assayed was composed of ammonium sulfate (3.03 mM), sodium citrate (0.68 mM), ferric chloride (3.04 mM), manganese sulfate (1.00 mM), magnesium sulfate (35 mM), potassium phosphate (dibasic) (0.42 mM), Trizma (50 mM), and zinc chloride (0.01 mM). Before the medium was sterilized, the pH was adjusted to 7.12 by the addition of glacial acetic acid. Before inoculation, the following sterile solutions were added to the medium to achieve the indicated concentrations: fructose (135 mM) and amino acids to supplement auxotrophic requirements (50 ,ug/ml

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Alkaline Phosphatase Mutants of Bacillus subtilis

Cited by 13 publications

References 5 publications

The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis

The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis

Separate promoters direct expression of phoAIII, a member of the Bacillus subtilis alkaline phosphatase multigene family, during phosphate starvation and sporulation

Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis

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