beta-agarases I and II from Pseudomonas atlantica. Purifications and some properties

The agarose-degrading system of Pseudomonas atlantica has been re-examined. In addition to the previously reported extracellular endo-beta-agarase [Yaphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333-335] a second, membrane-bound endo-enzyme activity, beta-agarase II has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose] and also to degrade partially 6-O-methylated agarose to neoagarobiose and 6(1)-O-methyl-neoagarbiose. Novel assays were devised for beta-agarase II and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of beta-agarase I and II. beta-Agarase I was purified 670-fold from the bacterial medium by a new method using ammonium sulphate precipitation and gel filtration on Sephadex G-100. The enzyme was resolved from the small amount of extracellular beta-agarase II. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32000. Activity was observed against agar over the pH range 3.0-9.0 and optimally at pH 7.0. The enzyme could be used indefinitely at 30 degrees C but only for up to 2 h at 40 degrees C. beta-Agarase II was partially purified (5-fold) from the soluble fraction of disrupted cells by chromatography on Sephadex G-100, hydroxyapatite and DEAE-Sepharose CL-6B. This preparation was free of beta-agarase I and disaccharidase. beta-Agarase II was stimulated by NaCl, optimally in the range 0.10-0.20 mol dm-3 (2.4-fold the activity at 0.010 mol dm-3 NaCl). Alkali earth metal (0.002 mol dm-3 CaCl2 or 0.005 mol dm-3 MgCl2) gave 1.2-fold the normal activity. Optimum activity was over pH 6.5-7.5.     

Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107.

An agarase gene (agaA) was cloned from genomic DNA of Vibrio sp. strain JT0107. An open reading frame of 2,985 nucleotides gave a primary translation product composed of the mature protein, agarase 0107 (975 amino acid residues, with a molecular weight of 105,271) and a signal peptide of 20 amino acid residues at the N terminus. Comparison of the deduced amino acid sequence of agarase 0107 with those of Streptomyces coelicolor and Pseudomonas atlantica suggests that these enzymes share two regions in common. The AgaA protein which was expressed in Escherichia coli had the agarase activity. Agarase 0107 hydrolyzes not only agarose but also neoagarotetraose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galact opy ranosyl (1-->3)-D-galactose] to yield neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose]. This is a quite unique characteristic for a beta-agarase.     

Enzymatic hydrolysis of agar: purification and characterization of beta-neoagarotetraose hydrolase from Pseudomonas atlantica.

Agarose is degraded by a beta-agarase from Pseudomonas atlantica to neoagarooligosaccharides of degree of polymerization (DP), 4, 6, 8, and 10. A beta-neoagarotetraose hydrolase cleaves the central beta-linkage in neoagarotetraose and the beta-linkage near the nonreducing end in neoagarohexaose and -octaose to yield neoagarobiose. The beta-neoagarotetraose hydrolase was localized on or outside the cytoplasmic membrane, in the cell wall region. The enzyme was activated by NaCl, KCl, CaCl2, MnCl2, and MgSO4, has a Km of 3.4 X 10(-3) M for neoagarotetraose, was free from beta-agarase and alpha-neoagarobiose hydrolase activity, and showed no transglycosidic activity.     

Nucleotide sequence of IS492, a novel insertion sequence causing variation in extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica.

The complete nucleotide sequence of insertion element IS492, which causes reversible inactivation of extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica, is presented. Insertion of IS492 results in the EPS- phenotype, and excision results in restoration of EPS+. DNA sequencing of the site of insertion in the eps locus showed that insertion of IS492 generates a 5-base-pair repeat and that its excision is precise. IS492 is 1,202 nucleotides in length and contains one large open reading frame encoding a protein of 318 amino acids, a candidate for transposition function. No similarity between IS492 and other transposable elements has been found. Unlike the situation with other insertion sequences, no direct or inverted repeats exist at the termini of IS492.     

Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94

A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T). The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 degrees C. The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).     

Excision of IS492 Requires Flanking Target Sequences and Results in Circle Formation in Pseudoalteromonas atlantica

The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492 insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced into Escherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 in E. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.     

Cloning of the novel gene encoding beta-agarase C from a marine bacterium, Vibrio sp. strain PO-303, and characterization of the gene product

The beta-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. beta-Agarase C was identified as the first beta-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.     

Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8

The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented. IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication. There are at least 6 copies of IS1000 in the genome of T. thermophilus HB8. A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica.     

Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp.

The nucleotide sequence of a 2.1-kilobase-pair fragment containing the Streptomyces choA gene, which codes a secreted cholesterol oxidase, was determined. A single open reading frame encodes a mature cholesterol oxidase of 504 amino acids, with a calculated Mr of 54,913. The leader peptides extend over 42 amino acids and have the characteristics of a signal sequence, including basic amino acids near the amino terminus and a hydrophobic core near the signal cleavage site. Analyses of the total amino acid composition and amino acid sequencing of the first 21 amino acids from the N terminus of the purified extracellular enzyme agree with the values deduced from nucleotide sequencing data.     

Nucleotide sequence of the maltohexaose-producing amylase gene from an alkalophilic Bacillus sp. #707 and structural similarity to liquefying type alpha-amylases

The nucleotide sequence of the gene for maltohexaose-producing amylase from an alkalophilic Bacillus sp. #707 was determined. Starting at an ATG initiation codon, an open reading frame was composed of 1554 bp (518 amino acids). The NH2-terminal portion encoded a 33 amino acid-long signal peptide. The deduced amino acid sequence of the extracellular mature enzyme was more than 60% homologous to those of the liquefying type alpha-amylases but not to those of the saccharifying type alpha-amylases. The sequence of its signal peptide was completely different from those of other alpha-amylases.     

The complete amino acid sequence of diphtheria toxin fragment B. Correlation with its lipid-binding properties

The complete amino acid sequence of fragment B from diphtheria toxin has been determined. The polypeptide chain was split with cyanogen bromide, o-iodosobenzoic acid, clostripain and trypsin; all amino acid sequence analyses were made by automated Edman degradation. Fragment B, which corresponds to the carboxy terminus of the toxin molecule, contains 342 amino acids and has an Mr of 37240. The proposed amino acid sequence fully confirms the structure recently deduced from the nucleotide sequence of the structural gene. The complete sequence is analyzed in relationship with the role of fragment B in the transfer of diphtheria toxin fragment A from the extracellular medium into the cell cytoplasm.     

Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica.

The XPR2 gene encoding an alkaline extracellular protease (AEP) from Yarrowia lipolytica was cloned, and its complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AEP consists of 297 amino acids with a relative molecular weight of 30,559. The gene codes for a putative 22-amino-acid prepeptide (signal sequence) followed by an additional 135-amino-acid propeptide containing a possible N-linked glycosylation site and two Lys-Arg peptidase-processing sites. The final Lys-Arg site occurs at the junction with the mature, extracellular form. The mature protease contains two potential glycosylation sites. AEP is a member of the subtilisin family of serine proteases, with 42.6% homology to the fungal proteinase K. The functional promoter is more than 700 base pairs long, allowing for the observed complex regulation of this gene. The 5' and 3' flanking regions of the XPR2 gene have structural features in common with other yeast genes.     

Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene.

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.     

Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli.

A DNA fragment of Serratia marcescens directing an extracellular serine protease (Mr, 41,000) was cloned in Escherichia coli. The cloned fragment caused specific excretion of the protease into the extracellular medium through the outer membrane of E. coli host cells in parallel with their growth. No excretion of the periplasmic enzymes of host cells occurred. The cloned fragment contained a single open reading frame of 3,135 base pairs coding a protein of 1,045 amino acids (Mr 112,000). Comparison of the 5' nucleotide sequence with the N-terminal amino acid sequence of the protease indicated the presence of a typical signal sequence. The C-terminal amino acid of the enzyme was found at position 408, as deduced from the nucleotide sequence. Artificial frameshift mutations introduced into the coding sequence for the assumed distal polypeptide after the C terminus of the protease caused complete loss of the enzyme production. It was concluded that the Serratia serine protease is produced as a 112-kilodalton proenzyme and that its N-terminal signal peptide and a large C-terminal part are processed to cause excretion of the mature protease through the outer membrane of E. coli cells.     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis

The extracellular acid protease of Candida tropicalis was purified from the supernatant fraction of culture medium containing bovine serum albumin as nitrogen source and the NH2-terminal amino acid (aa) sequence of the protein was determined. The gene for the acid protease (ACP) was isolated using a pool of synthetic oligonucleotides as a probe and a segment of the deduced aa sequence was found to be in agreement with the NH2-terminal aa sequence of the protein. The deduced aa sequence of ACP is similar to the aa sequence of proteases of the pepsin family. The nucleotide sequence of the 5' portion of this gene revealed a coding sequence for a 60 residue propeptide containing two Lys-Arg amino acid pairs that have been identified as sites for peptidase processing of several exported peptides and proteins. The final Lys-Arg site occurs at the junction with the mature extracellular form of the acid protease.     

Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation.     

Characterization of the agarase system of a multiple carbohydrate degrading marine bacterium

A marine bacterium strain 2-40 (2-40) degraded numerous complex carbohydrates, such as agar, chitin and alginate. It may play an important role in altering carbon fluxes in marine environments. End-product analyses revealed that 2-40 synthesized an agarase system that consisted of at least three enzymes, beta-agarase I, beta-agarase II and alpha-agarase, which acted in concert to degrade polymeric agar to D-galactose and 3,6-anhydro-L-galactose. The agarase system was shown to be both cell envelope-associated and extracellular, with the relative concentrations depending on the growth phase. The principal depolymerase, a beta-agarase I, hydrolysed agar to both neoagarotetrose and neoagarobiose, as identified by thin layer chromatography. This agarase had a mass of 98 kD and a Pi of 4.3. The agarase system was repressed by D-glucose and D-galactose and induced by agar, agarose, neoagarobiose, neoagarotetrose and neoagarohexose.     

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.