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F M Hulett

Publications and source records attributed to F M Hulett.

At least 37 records · Page 2Linked to original sources
PubMed1996

Two-component regulatory proteins ResD-ResE are required for transcriptional activation of fnr upon oxygen limitation in Bacillus subtilis.

Bacillus subtilis can grow anaerobically in the presence of nitrate as a terminal electron acceptor. The two component regulatory proteins, ResD and ResE, and an anaerobic gene regulator, FNR, were previously shown to be indispensable for nitrate respiration in B. subtilis. Unlike Escherichia coli fnr, B. subtilis fnr transcription was shown to be highly induced by oxygen limitation. fnr is transcribed from its own promoter as well as from a promoter located upstream of narK, the first gene in the narK-fnr dicistronic operon. DNA fragments containing the narK promoter, the fnr promoter, and both of the promoters were used to construct three lacZ fusions to examine the transcriptional regulation of the narK-fnr operon. ResDE was found to be required for transcriptional activation of fnr from the fnr-specific promoter, and FNR was required for activation of narK-fnr transcription from the FNR-dependent narK operon promoter under anaerobiosis. In order to determine if the requirement for ResDE in nitrate respiration is solely to activate fnr transcription, fnr was placed under control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter, Pspac. The observed defect in anaerobic growth of a Pspac-fnr delta resDE mutant in the presence of IPTG indicated that resDE has an additional role in B. subtilis anaerobic gene regulation.

Anaerobiosis↗
PubMed1996

Regulators of aerobic and anaerobic respiration in Bacillus subtilis.

Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration. The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD. They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially. ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex). The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins. resD null mutations are more deleterious to the cell than those of resE. resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate. A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation. The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis.

Aerobiosis↗
PubMed1994

A pho regulon promoter induced under sporulation conditions.

Sporulation-induced alkaline phosphatases (APases) of Bacillus subtilis require the products of the sporulation stage-0 genes and certain stage-II genes, including the spoIIA operon, for induction. Mutations in either sapA or sapB bypass this requirement [Piggot and Taylor, J. Gen. Microbiol. 10 (1977) 69-80], resulting in APase production in a spoIIA sapA or spoIIA sapB strain, under sporulation conditions. B. subtilis has multiple structural genes encoding APases, which are induced either by phosphate starvation or during sporulation, or under both conditions. We report studies designed to determine which APase(s) were being expressed in the sap mutants, and from which promoters. phoB (formerly phoAIII), one of the structural genes encoding an APase in B. subtilis, is expressed under both sporulation and phosphate starvation conditions, but from separate promoters [Chesnut et al., Mol. Microbiol. 5 (1991) 2181-2190]. The spoIIA sapA and spoIIA sapB strains express phoB under sporulation conditions. Interestingly, the expression of phoB during sporulation was from Pv, the phosphate starvation-inducible promoter of phoB, rather than from Ps, the sporulation-specific promoter. Since the induction of phosphate starvation-inducible promoters during phosphate limitation requires the phoPR operon [Miki et al., Genetics 52 (1965) 1093-1100], we asked if the phoPR products were involved in regulating Pv expression under sporulation conditions. The phoPR genes are transcribed under sporulation conditions, regulated by sapA and sapB under sporulation conditions, and required for expression from Pv under sporulation conditions.

Alkaline Phosphatase↗
PubMed1994

Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis.

Bacillus subtilis has an alkaline phosphatase (APase) gene family composed of at least four genes. All members of this gene family are expressed postexponentially, either in response to phosphate starvation or sporulation induction or, in some cases, in response to both. The phoA gene (formerly called phoAIV) and the phoB gene (formerly called phoAIII) products have both been isolated from phosphate-starved cells, and a mutation in either gene decreased the total APase expressed under phosphate starvation conditions. Data presented here show that a phoA phoB double mutant reduced APase production during phosphate starvation by 98%, indicating that these two genes are responsible for most of the APase activity during phosphate-limited growth. The promoter for phoA was cloned and used, with the phoB promoter, to examine phosphate regulation in B. subtilis. phoA-lacZ reporter gene assays showed that the expression of the phoA gene commences as the culture enters stationary phase as a result of limiting phosphate concentrations in the growth medium, thereby mimicking the pattern of total APase expression. Induction persists for approximately 2 h and is then turned off. phoA is transcribed from a single promoter which initiates transcription 19 bp before the translation initiation codon. PhoP and PhoR are members of the two-component signal transduction system believed to regulate gene expression in response to limiting phosphate. The expression of phoA or phoB in response to phosphate starvation was equally dependent on PhoP and PhoR for induction. lacZ-promoter fusions showed that both phoA and phoB were hyperinduced, or failed to turn off induction after 2 h, in a spo0A strain of B. subtilis. Mutations in genes which are required for phosphorylation of Spo0A, spo0B and spo0F, also resulted in phoA and phoB hyperinduction, suggesting that phosphorylation of Spo0A is required for the repression of both APases in wild-type strains. The hyperinduction of either APase gene in a spo0A strain was dependent on PhoP and PhoR. Analysis of a phoP-lacZ promoter fusion showed that the phoPR operon is hyperinduced in a spo0A mutant strain, suggesting that Spo0A approximately P represses APases by repressing phoPR transcription. We propose a model for PHO regulation in B. subtilis whereby the phoPR operon is transcribed in response to limiting phosphate concentration, resulting in activation of the PHO regulon transcription, including transcription of phoA and phoB. When the phosphate response fails to overcome the nutrient deficiency, signals for phosphorylation of Spo0A result in production of Spo0A approximately P, which represses transcription of phoPR, thereby repressing synthesis of the PHO regulon.

Alkaline Phosphatase↗
PubMed1993

Bacillus subtilis transcription regulator, Spo0A, decreases alkaline phosphatase levels induced by phosphate starvation.

Alkaline phosphatase (APase) is induced as a culture enters stationary phase because of limiting phosphate. The results presented here show that expression of APase is regulated both negatively and positively. PhoP, a homolog of a family of bacterial transcription factors, and PhoR, a homolog of bacterial histidine protein kinases, are required for induction of APases when phosphate becomes limiting. The induction period lasts 2 to 3 h, after which the rate of APase accumulation is decreased. Mutant strains defective in the Spo0A transcription factor failed to decrease APase production. The consequent hyperinduction of APase in a spo0A strain was dependent on phoP and phoR. spo0B and spo0F strains also overexpressed APase, suggesting that phosphorylated Spo0A is required for repression of APase. An abrB mutant allele in the presence of the mutant spo0A allele in these strains did not significantly change the APase hyperinduction phenotype, demonstrating that Spo0A repression of abrB expression is not the mechanism by which Spo0A-P regulates APase expression. Our previous report that spo0A mutants do not express APases is in conflict with the present data. We show here that the previously used mutants and a number of commonly used spo0 strains, all of which have an APase deficiency phenotype, contain a previously unrecognized mutation in phoR.

Alkaline Phosphatase↗
PubMed1991

Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure.

Bacillus subtilis has an alkaline phosphatase multigene family. Two members of this gene family, phoAIII and phoAIV, were cloned, taking advantage of in vitro constructed strains containing a plasmid insertion within one or the other of the structural genes. The DNA sequences of the two genes showed approximately 64% identity at the DNA level and 63% identity in the deduced primary amino acid sequences. The phoAIII and phoAIV genes code for predicted proteins of 47,149 and 45,935 Da, respectively. Comparison of the deduced primary amino acid sequence of the mature proteins with other sequenced alkaline phosphatases from Escherichia coli, yeast, and humans shows 25-30% identity. Based on the refined crystal structure of E. coli alkaline phosphatase, it appears that the active site and the core of the structure are retained in both Bacillus alkaline phosphatases. However, both proteins are truncated at the amino terminus compared with other mature alkaline phosphatases, three sizable surface loops of E. coli are deleted, and a minidomain is replaced with a larger domain in the model. Neither Bacillus alkaline phosphatase sequenced contains any cysteine residues, an amino acid implicated in intrachain disulfide bond formation in other alkaline phosphatases.

Alkaline Phosphatase↗
PubMed1991

Identification of four unique clones encoding 10 kDa proteins from Bacillus that cause phenotypic complementation of a phoA mutant strain of Escherichia coli.

A number of clones have been isolated from two Bacillus species which complement the PhoA- phenotype of Escherichia coli mutants under conditions that induce the expression of alkaline phosphatase (APase). These clones were initially thought to carry XPases because the transformed host could hydrolyse a common APase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate). The sequences of the open reading frames responsible for the phenotypic complementation showed no sequence similarity to ATPases of E. coli, human (bone-liver-kidney, intestinal or placental) or Bacillus. Therefore, these clones were designated as XPA (for X Phosphatase Activity) clones. Four of the clones encoded small (10 kDa), basic, hydrophobic proteins. Two of these, xpaB from B. subtilis 168 and xpaL2 from B. licheniformis MC14, shared 62% identity at both the DNA and the predicted amino acid sequence level. The fact that homologues from two Bacillus strains were cloned indicated that the screen was specific, but not for APase genes. It is clear that phenotypic complementation with cloned DNA from another genus does not ensure the identification of an APase gene. Possible mechanisms for the abnormal phenotypic complementation are discussed.

Alkaline Phosphatase↗
PubMed1991

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis.

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.

Alkaline Phosphatase↗
PubMed1991

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

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.

Alkaline Phosphatase↗
PubMed1990

The Bacillus subtilis phoAIV gene: effects of in vitro inactivation on total alkaline phosphatase production.

A degenerative oligodeoxyribonucleotide probe deduced from the first 19 amino acids of the mature alkaline phosphatase IV (APase IV) protein was used to clone a DNA fragment internal to the coding region of the phoAIV gene of Bacillus subtilis. An insertional mutation was constructed in the phoAIV locus using the integrative plasmid, pJM103, containing the cloned DNA fragment. The strain with the interrupted phoAIV gene showed no detectable APase IV product on Western-blot analysis. The impact of the phoAIV interruption on total APase production in B. subtilis 168 was analyzed under both phosphate starvation and sporulation culturing conditions. The mutation in phoAIV reduced total APase-specific activity by 75% in phosphate-starved cells, and resulted in the elimination of a salt-extractable membrane APase, as well as the secreted APase IV. Analysis of this membrane APase indicated that it is a phoAIV gene product which is localized within the membrane fraction of the lysed cell and not secreted. There was no effect on the production of sporulation APase. The phoAIV::pJM103 insertion was mapped and determined to be located at approx. 73 degrees on the B. subtilis 360 degrees chromosome.

Alkaline Phosphatase↗
PubMed1990

Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis.

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. Southern analysis of genomic DNA with degenerative oligomers based on the amino acid sequences suggest that there are two structural genes for alkaline phosphatase in the genome of B. subtilis.

Alkaline Phosphatase↗
PubMed1990

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

The first alkaline phosphatase (APase) structural gene mutant of Bacillus 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 vegetative, phosphate-starved cells. DNA sequencing of the clone revealed the first 80 amino acids of the APase III protein, including a typical procaryotic signal sequence of 32 amino acids preceding the start of the mature protein. The 29 amino acids encoded by the predicted open reading frame immediately following the signal sequence are identical to the first 29 amino acids of the sequenced mature protein. This region shows 80% identity to strand A of the beta sheet that is very well conserved in Escherichia coli and mammalian APases. A phoAIII structural mutant was constructed by insertional mutagenesis with a fragment internal to the coding region. The effects of this mutation on APase production in B. subtilis 168 were analyzed under both phosphate starvation and sporulation conditions. The mutation in APase III reduced the total vegetative APase specific activity by approximately 40% and sporulation APase specific activity by approximately 45%. An APase protein was isolated from sporulating cells at stage III and was identified as APase III by protein sequencing of the amino terminus and by its absence in the phoAIII mutant. The APase III gene has been mapped to approximately 50 degrees on the B. subtilis chromosome.

Alkaline Phosphatase↗
PubMed1989

Protoplast transformation of Bacillus licheniformis MC14.

A protoplast transformation system has been developed for Bacillus licheniformis MC14. Optimum regeneration conditions were achieved by raising the incubation temperature of the regeneration plates to 46 degrees C. Regenerated transformed colonies could be isolated in 3 to 5 d under these conditions. Plasmids introduced by this method were stably maintained by B. licheniformis MC14 and could be recovered and used to transform Bacillus subtilis.

Bacillus↗
PubMed1988

Critical roles of spo0A and spo0H in vegetative alkaline phosphatase production in Bacillus subtilis.

Growth conditions established to optimize vegetative alkaline phosphatase production and stability in Bacillus subtilis were used to compare alkaline phosphatase synthesis and secretion in isogenic strains JH646 (spo0A12) and JH646MS (spo0A12 abrB15). A mutation in spo0A blocked vegetative alkaline phosphatase production, and a second mutation at the abrB locus resulted in hyperinduction of vegetative alkaline phosphatase. Phosphate regulation of vegetative alkaline phosphatase synthesis was unaffected in the double mutant. spo0H, on a multicopy plasmid, partially overcame the spo0A effect.

Alkaline Phosphatase↗
PubMed1986

Purification and characterization of the secreted alkaline phosphatase of Bacillus licheniformis MC14: identification of a possible precursor.

The most abundantly secreted protein from Bacillus licheniformis MC14 is alkaline phosphatase (APase). A twofold purification yields a homogeneous enzyme. No discernible chemical-physical differences in the secreted APase distinguish this enzyme from the cell-bound APase(s) except a 10-fold higher specific activity. During the growth phase in which the bacterium was secreting APase into the medium an inactive cytosol protein antigenically similar but 3000 Da heavier than the subunit of the mature secreted APase was immunoprecipitated from the cytosol. A pulse-chase experiment showed the kinetics of disappearance of this protein from the cytosol to be correlated with the appearance of the secreted APase in the medium, suggesting that it may be a precursor to the secreted APase.

Alkaline Phosphatase↗
PubMed1985

Two alkaline phosphatase genes positioned in tandem in Bacillus licheniformis MC14 require different RNA polymerase holoenzymes for transcription.

Southern transfer analysis of Bacillus licheniformis MC14 DNA, using as probe a DNA fragment from within the coding region of a previously cloned alkaline phosphatase (APase) gene, revealed a second area of hybridization adjacent to the cloned APase gene. A second APase gene (APase II) was subcloned from the same plasmid clone, pMH8, from which the first APase gene (APase I) had been subcloned. The two genes are arranged in tandem with several hundred base pairs separating them. Immunoblot analysis showed that both code for Mr 60,000 proteins that crossreact with anti-APase. Both proteins enzymatically cleave 5-bromo-4-chloro-3-indolyl phosphate. In vitro transcription showed that APase I and APase II are transcribed in the same direction but that the two genes require different forms of Bacillus RNA polymerase: sigma 55- and sigma 37-containing RNA polymerase holoenzymes, respectively.

Alkaline Phosphatase↗
PubMed1984

Cloning and characterization of the Bacillus licheniformis gene coding for alkaline phosphatase.

The structural gene for alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1) of Bacillus licheniformis MC14 was cloned into the Pst1 site of pMK2004 from chromosomal DNA. The gene was cloned on an 8.5-kilobase DNA fragment. A restriction map was developed, and the gene was subcloned on a 4.2-kilobase DNA fragment. The minimum coding region of the gene was localized to a 1.3-kilobase region. Western blot analysis was used to show that the gene coded for a 60,000-molecular-weight protein which cross-reacts with anti-alkaline phosphatase prepared against the salt-extractable membrane alkaline phosphatase of B. licheniformis MC14 .

Alkaline Phosphatase↗