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.     

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.     

Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium

The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 degrees C, lower than that of E. coli APase by 30 degrees C. The specific activity of SIB1 APase at 50 degrees C was 3.1 fold higher than that of E. coli APase at 80 degrees C. SIB1 APase lost activity with a half-life of 3.9 min at 70 degrees C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 degrees C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.     

Cloning,expression and characterization of a highly active thermostable alkaline phosphatase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> MTCC 1483 in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3)

Alkaline phosphatase gene of the bacterium, Bacillus licheniformis MTCC 1483 was cloned and successfully expressed in Escherichia coli BL21 (DE3). Sequence analysis revealed an open reading frame of 1662 bp encoding a 553 amino acid protein with a molecular mass of 62 kDa, as determined by SDS-PAGE. The recombinant enzyme was purified using Ni-NTA affinity column and the purified enzyme showed a specific activity of 24890 U/mg protein, which is the highest value among any other bacterial recombinant alkaline phosphatases reported so far. The enzyme exhibited optimum activity at 50°C and pH 10.0 and showed high thermostability. The recombinant alkaline phosphatase from B. licheniformis MTCC 1483 exhibited a dephosphorylation efficiency of 92.9% to dephosphorylate linear DNA fragments. The recombinant enzyme with high catalytic efficiency and thermostability has the potential for applications in clinical diagnostics which require enzyme stability against thermal deactivation during preparation or labeling procedures.     

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 possessing alkaline phosphodiesterase activity and other phosphodiesterases in Bacillus subtilis.

In Bacillus subtilis Marburg strain, single-point mutations in the phoP locus brought about simultaneous losses of the major activities of alkaline phosphatase (APase) and alkaline phosphodiesterase (APDase). Revertants recovered the two activities. APases with APDase activity were purified from the membrane fraction of B. subtilis 6160-BC6 and from the culture fluid of an APase-secreting B. subtilis mutant strain, RAN 1. In addition to these major APases with APDase activity, at least two kinds of phosphodiesterase (PDase) without phosphatase activity were found in the cytoplasmic supernatants of RAN 1 and an APase-less B. subtilis mutant strain, SP25. Another minor APase with a molecular weight of about 80,000, which had almost no PDase activity, was isolated from the membrane fraction of strain 6160-BC6. Enzyme distribution in subcellular fractions from various strains cultured in high- and low-phosphate media was analyzed. The PDases did not cross-react with rabbit antiserum against the RAN 1 APase with APDase activity. The main component of the PDases had a molecular weight of about 80,000 and was most active at pH 8.0. These results suggest that APase with APDase activity is different from PDases detected in cytoplasmic supernatants and that phoP is the structural gene for the phosphate-repressible APase with APDase activity.     

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.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Molecular cloning,sequence analysis,and characterization of a new cell wall hydrolase,CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Acid phosphatase isozymes secreted under phosphate-deficient conditions inPholiota nameko

We previously reported the purification of an acid phosphatase (APase52) secreted from the mycelia ofPholiota nameko under phosphate-deficient conditions. In the present study, two other isozymes (APase47 and APase48) were found and their structures were compared with that of APase52. Thirteen amino acid residues at theN-terminus of APase47 were completely identical with those of APase48 and had partial homology with those of APase52. The deglycosylation of proteins indicated that three APase isozymes differ in theN-linked oligosaccharide content. The protease-generated peptide maps of the APases differed from one another in the band pattern. These results suggest that the APases are the products of different genes.     

Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.     

Differential responses of oat cultivars to phosphate deprivation: plant growth and acid phosphatase activities

The effect of phosphate starvation on growth and acid phosphatases (APases) localization and activity in oat tissues was investigated. Oat cultivars (Avena sativa L.??Arab, Polar, Szakal) were grown for 1?C3?weeks in complete nutrient medium (+P) and without phosphate (?P). Pi concentration in plant tissues decreased strongly after culturing on ?P medium. Pi deficit reduced shoot growth, stimulated root elongation and increased ratio of root/shoot in all oat cultivars. Pi deficit had a greater impact on growth of oat cv. Polar than other varieties. A decrease in the internal Pi status led to an increase of acid phosphatase activities in extracts from shoots and roots, and in root exudates. The highest activity of secreted APases was observed for oat cv. Arab, during the third week of growth under Pi-deficient conditions. The activity of extracellular APase was high in young, growing zones of roots of ?P plants. Histochemical visualization indicated high activity of APases in the epidermis and vascular tissues of ?P plants. Pi deficiency increased intracellular APase activity in shoot mainly in oat cv. Polar, whereas APase activity in roots was the highest in oat cv. Szakal. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoform(s) may be affected by Pi deficiency. Three major APase isoforms were detected in all oat plants; one was strongly induced by Pi deficit. The studied oat cultivars differed in terms of acclimation to deficiency of phosphate??used various pools of APases to acquire Pi from external or internal sources.     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5αF′. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2°C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.     

Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupin

White lupin (Lupinus albus) grown under P deficiency displays a suite of highly coordinated adaptive responses. Included among these is secretion of copious amounts of acid phosphatase (APase). Although numerous reports document that plants secrete APases in response to P deficiency, little is known of the biochemical and molecular events involved in this process. Here we characterize the secreted APase protein, cDNA, and gene from white lupin. The secreted APase enzyme is a glycoprotein with broad substrate specificity. It is synthesized as a preprotein with a deduced M(r) of 52,000 containing a 31-amino acid presequence. Analysis of the presequence predicts that the protein is targeted to outside the cell. The processed protein has a predicted M(r) of 49,000 but migrates as a protein with M(r) of 70,000 on sodium dodecyl sulfate gels. This is likely due to glycosylation. Enhanced expression is fairly specific to proteoid roots of P-stressed plants and involves enhanced synthesis of both enzyme protein and mRNA. Secreted APase appears to be encoded by a single gene containing seven exons interrupted by six introns. The 5'-upstream putative promoter of the white lupin-secreted APase contains a 50-base pair region having 72% identity to an Arabidopsis APase promoter that is responsive to P deficiency. The white lupin-secreted APase promoter and targeting sequence may be useful tools for genetically engineering important proteins from plant roots.     

Induction of maize acid phosphatase activities under phosphorus starvation

Large variation in phosphorus-(P) acquisition efficiency exists among maize inbred and hybrid genotypes. Acid phosphatases are a type of enzyme that affects P acquisition and P-use efficiency in plants. The objectives of this research were (1) to characterize acid phosphatase activity in maize grown hydroponically under P starvation, and (2) to determine if there is differential induction of acid phosphatases in two maize genotypes previously characterized as P efficient (Mo17) and P inefficient (B73). B73 and Mo17 seedlings were grown hydroponically and both intracellular and secreted acid phosphatase activities were characterized. Fresh seedling weight of both genotypes decreased under P starvation, but percent fresh weight allocated to roots increased 14 days after P starvation in B73. Soluble protein concentration in shoots and roots was affected little, but secreted protein decreased by 40 and 20% in B73 and Mo17 seedlings grown without P for 14 days. Intracellular and secreted acid phosphate activity increased substantially in leaves and roots in B73 and Mo17 in response to P starvation. Secreted APase activity per unit protein increased 310 and 300% in B73 and Mo17, respectively, 7 days after P withdrawal. One of the minor isozymes identified on non-denaturing PAGE, was increased specifically in response to P starvation in both maize genotypes. The patterns and levels of change in APase activities in B73 and Mo17 were not sufficiently different to account for the diverse growth response of these genotypes in low-P conditions. The results suggest that APases may not be a major mechanism for scavenging or acquiring P and changes in APases may reflect a state of P stress in both varieties. Other factors such as root architecture, secretion of low-molecular weight carboxylates and microbial interactions might explain the difference between these two genotypes.     

Molecular characterization of a venom acid phosphatase Acph-1-like protein from the Asiatic honeybee Apis cerana

Bee venom contains a variety of peptides and enzymes, including acid phosphatases. An acid phosphatase has been identified from European honeybee (Apis mellifera) venom. However, although the amino acid sequence is known, no functional information is currently available for bee venom acid phosphatase Acph-1-like proteins. In this study, an Asiatic honeybee (Apis cerana) venom acid phosphatase Acph-1-like protein (AcAcph-1) was identified. The analysis of the predicted AcAcph-1 amino acid sequence revealed high levels of identity with other bee venom acid phosphatase Acph-1-like proteins. Recombinant AcAcph-1 was expressed as a 64-kDa protein in baculovirus-infected insect cells. The enzymatic properties of recombinant AcAcph-1, determined using p-nitrophenyl phosphate (p-NPP) as a substrate, showed the highest activity at 45 °C and pH 4.8. Northern and western blot analyses showed that AcAcph-1 was expressed in the venom gland and was present as a 64-kDa protein in bee venom. In addition, N-glycosylation of AcAcph-1 was revealed by tunicamycin treatment of recombinant virus-infected insect Sf9 cells and by glycoprotein staining of purified recombinant AcAcph-1. Our findings show that AcAcph-1 functions as a venom acid phosphatase. This paper provides the first evidence of the role of a bee venom acid phosphatase Acph-1-like protein.     

Escherichia coli signal peptidase recognizes and cleaves the signal sequence of α-amylase originating from Bacillus licheniformis

The gene encoding the α-amylase from Bacillus licheniformis was cloned, with and without the native signal sequence, and expressed in Escherichia coli, resulting in the production of the recombinant protein in the cytoplasm as insoluble but enzymatically active aggregates. Expression with a low concentration of the inducer at low temperature resulted in the production of the recombinant protein in soluble form in a significantly higher amount. The protein produced with signal sequence was exported to the extracellular medium, whereas there was no export of the protein produced from the gene without the signal sequence. Similarly, the α-amylase activity in the culture medium increased with time after induction in case of the protein produced with signal sequence. Molecular mass determinations by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing of the purified recombinant α-amylase from the extracellular medium revealed that the native signal peptide was cleaved by E. coli signal peptidase between Ala28 and Ala29. It seems possible that the signal peptide of α-amylase from B. licheniformis can be used for the secretion of other recombinant proteins produced using the E. coli expression system.