Cloning, sequencing, and characterization of the iturin A operon

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.     

Detection of several strains of the bacterium-like organism of citrus greening disease by DNA probes

Greening disease of citrus is caused by a phloem-restricted, bacterium-like organism (BLO). DNA was purified from phloem tissue of periwinkle plants infected with an indian strain of the greening BLO, restricted withHindIII endonuclease, and cloned in the replicative form of bacteriophage M13mp18.By differential hybrizations involving DNA from healthy and infected periwinkle plants, we have selected three recombinant phages containing BLO DNA. The BLO DNA inserts (In-2.6, In-1.9, and In-0.6) have been purified from the viral replicative forms and used as probes. Southern and dot hybridizations have shown that In-2.6 and In-1.9 recognized all asian strains tested (strains from India, Thailand, the Philippines, Indonesia, China, and Taiwan), but not a South African strain. In-0.6 reacted only with the indian BLO strain.     

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.     

Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa3-600 quinol oxidase.

Bacillus subtilis contains two aa3-type terminal oxidases (caa3-605 and aa3-600) catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H2O (Lauraeus, M., Haltia, T., Saraste, M., and Wikstr?m, M. (1991) Eur. J. Biochem. 197, 699-705). Previous studies characterized only the structural genes of caa3-605 oxidase. We isolated the genes coding for the four subunits of a B. subtilis terminal oxidase from a genomic DNA library. These genes, named qoxA to qoxD, are organized in an operon. Examination of the deduced amino acid sequence of Qox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases. In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B. subtilis caa3-605 cytochrome c oxidase. We produced, by in vitro mutagenesis, a mutation in the qox operon. From the phenotype of the mutant strain devoid of Qox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein. Although several terminal oxidases are found in B. subtilis, Qox oxidase (aa3-600) is predominant during the vegetative growth and its absence leads to important alterations of the phenotype of B. subtilis.     

Molecular cloning,characterization, and sequencing of the hemolysin gene from Edwardsiella tarda

Hemolysis is a major symptom of diseased eels infected by Edwardsiella tarda. The hemolysin gene of E. tarda strain ET16 was cloned into plasmid pSK and expressed in Escherichia coli. The mol. mass of the functional β-hemolysin was estimated to be approximately 34 kDa by gel filtration and by SDS-PAGE followed by in situ hemolysin activity analysis. The cloned fragment containing the β-hemolysin locus from E. tarda strain ET16 expressed in E. coli was estimated to be 5.3 kb in length; the deduced gene product was identical in mol. mass and properties to the extracellular products of E. tarda strain ET16. The presence of EcoRI and XbaI sites within the β-hemolysin gene of E. tarda was determined from the loss of hemolytic activity in subclones. Analysis of the DNA sequence of a 2,436-bp HaeIII-HindIII fragment that included EcoRI and XbaI sites revealed three ORFs organized as an operon that encoded three predicted polypeptides of 15,874, 7,055, and 34,804 Da. A 34-kDa polypeptide expressing hemolytic activity in cell lysates of the clone DH5α(pETH3E) is very likely the β-hemolysin encoded by the third ORF. The observation that hemolytic activity appeared in the culture medium of E. tarda, but not in that of E. coli strain DH5α(pETH3E) indicates the existence of a mechanism for transporting the hemolysin across the cell envelope in E. tarda that is different from that of E. coli. Received: 7 July 1995 / Accepted: 24 October 1995     

Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich, and turkey

Transferrins form an important class of iron-binding proteins widely distributed in the physiological fluids of vertebrates and invertebrates. In vertebrates they are present mostly in serum as serotransferrins. In birds and reptiles transferrins are also found in eggs as ovotransferrins. However, until now only chicken and duck ovotransferrin sequences have been published. This paper presents data on the purification, biochemical analysis, cloning and sequencing of ovotransferrins from red-eared turtle, African ostrich and turkey, revealing their significant homology with other known ovotransferrin sequences. The proteins were purified by size-exclusion and anion-exchange chromatography. Isoelectric points, iron-saturated and iron-free spectra, as well as the mRNA nucleotide sequences of 2,409 nt (ORF: 2,106 nt encoding a 701-amino-acid polypeptide; ), 2,418 nt (ORF 2,118 nt encoding a 705-amino-acid polypeptide; ), and 2,397 nt (ORF: 2,118 nt encoding a 705-amino-acid polypeptide; ) were determined for ostrich (OtrF), red-eared turtle (TtrF), and turkey (MtrF) ovotransferrin, respectively.     

Phosphoglycerate kinase gene from Zymomonas mobilis: cloning, sequencing, and localization within the gap operon.

The Zymomonas mobilis gene encoding phosphoglycerate kinase (EC 2.7.3.2), pgk, has been cloned into Escherichia coli and sequenced. It consists of 336 amino acids, including the N-terminal methionine, with a molecular weight of 41,384. This promoterless gene is located 225 base pairs downstream from the gap gene and is part of the gap operon. Previous studies have shown that the specific activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase do not change coordinately in Z. mobilis, although the two enzymes appear to be under the control of a common promoter. The translated amino acid sequence for the Z. mobilis phosphoglycerate kinase is less conserved than those of eucaryotic genes. A comparison of known sequences for phosphoglycerate kinase revealed a high degree of conservation of structure with 102 amino acid positions being retained by all. In general, the amino acid positions at the boundaries of beta-sheet and alpha-helical regions and those connecting these regions were more highly conserved than the amino acid positions within regions of secondary structure.     

Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization

Pseudozyma antarctica JCM 10317 exhibits a strong degradation activity for biodegradable plastics (BPs) such as agricultural mulch films composed of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA). An enzyme named PaE was isolated and the gene encoding PaE was cloned from the strain by functional complementation in Saccharomyces cerevisiae. The deduced amino acid sequence of PaE contains 198 amino acids with a predicted molecular weight of 20,362.41. High identity was observed between this sequence and that of cutinase-like enzymes (CLEs) (61–68 %); therefore, the gene encoding PaE was named PaCLE1. The specific activity of PaE against emulsified PBSA was 54.8?±?6.3 U/mg. In addition to emulsified BPs, PaE degraded solid films of PBS, PBSA, poly(ε-caprolactone), and poly(lactic acid).     

Purification, characterization cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2

Streptomyces septatus TH-2 secretes a large amount of a protease when cultured on a medium containing K(2)HPO(4) and glucose. The enzyme was purified to homogeneity by a three-step procedure. This enzyme had a molecular mass of approximately 35kDa, and was particularly inhibited by EDTA and phosphoramidon. Its substrate specificity was investigated using novel fluorescence energy transfer combinatorial libraries. The protease was found to prefer Phe and Tyr at the P(1) position, a hydrophobic or basic residue at the P(2) position, and a basic or small residue at the P(3) position. Its gene was cloned and sequenced, and its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, confirming that it encodes metalloendopeptidase. The primary structure of the enzyme showed 40 and 69% identities with that of thermolysin from Bacillus thermoproteolyticus and that of a metalloendopeptidase from Streptomyces griseus, respectively.     

Regulation of the glyoxylate bypass operon: cloning and characterization of iclR.

In Escherichia coli, expression of the glyoxylate bypass operon appears to be controlled, in part, by the product of iclR+. Mutations in iclR have been found to yield constitutive expression of this operon, suggesting that iclR+ encodes a repressor protein. We have cloned iclR+ by taking advantage of its tight genetic linkage with the glyoxylate bypass operon. The clone complemented a mutant allele of iclR in trans, restoring an inducible phenotype for this operon. Deletion analysis identified a region of ca. 900 base pairs that was necessary and sufficient for complementation. The nucleotide sequence of the insert was then determined. Translation of this sequence revealed an open reading frame capable of encoding a protein with Mr 29,741 preceded by a potential Shine-Dalgarno ribosome-binding site. The deduced amino acid sequence includes a region at the amino terminus that may form a helix-turn-helix motif, a structure found in many DNA-binding domains.     

Twelve polymorphic microsatellite loci from the Asian citrus psyllid,Diaphorina citri Kuwayama,the vector for citrus greening disease,huanglongbing

Twelve polymorphic microsatellite markers were developed from microsatellite‐enriched DNA libraries and mined from an expressed sequence tags library of Diaphorina citri, the vector of the citrus greening disease (huanglongbing). Analysis of 288 individuals from Florida, Texas, and Brazil showed that allelic diversity ranged from three to eight alleles per locus and observed and expected heterozygosities ranged from 0.014 to 0.569 and from 0.052 to 0.653, respectively. These variable microsatellite loci can provide means for assessing overall genetic variation and migration patterns for this agriculturally important pest species. This information can be used to aid in developing successful management strategies.     

Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon.

Cytochromes P-450 are extremely important in the oxidative metabolism of a variety of endogenous and exogenous compounds in pro- and eukaryotic organisms. Progress in understanding the structure and mechanism of action of this superfamily of enzymes has been hampered by the properties of the eukaryotic enzymes and the availability of only one well-characterized prokaryotic enzyme as a model. We report here the isolation of a Pseudomonas species which will utilize a monoterpene natural product, alpha-terpineol, as its sole source of carbon and energy. Approximately 1% of the soluble protein in the cell-free extract is a novel cytochrome P-450 (P-450terp). This enzyme and its associated iron sulfur protein electron carrier (terpredoxin) have been purified to homogeneity and their NH2-terminal amino acid sequences determined. The amino acid sequences of six tryptic peptide fragments of cytochrome P-450terp have also been determined. This sequence information was used to clone the gene encoding cytochrome P-450terp. Three clones representing approximately 8 kilobase pairs of unique sequences were selected and sequenced. Five non-overlapping open reading frames (ORFs) were found in the sequences, and the translated sequences were used to search the Protein Identification Resource for comparable proteins. The ORFs were identified as: 1) an alcohol dehydrogenase, 2) an aldehyde dehydrogenase, 3) cytochrome P-450terp, 4) terpredoxin reductase, and 5) terpredoxin. The identification of both the cytochrome P-450terp and terpredoxin DNA sequence was confirmed by the presence of each of the corresponding amino acid sequences found in the purified proteins. The five ORFs were bounded on both the 5' and 3' ends by consensus factor-independent terminator sequences. A consensus promoter sequence was found immediately 5' to the first ORF. These results indicate that we have sequenced the complete terp operon. Comparison of the amino acid sequence of cytochrome P-450terp to that of all other cytochromes P-450 has shown that it is the first member of the gene family CYP108. Preliminary characterization of the chemical and physical properties and the preparation of crystals of this new cytochrome P-450, suitable for x-ray diffraction analysis, indicate that it will be useful in comparison studies with other members of this class of proteins.     

ABO genotyping by PCR-RFLP and cloning and sequencing

A refined PCR-RFLP based method was established to genotype ABO blood groups. The main objective of this study was to make the techniques also suitable for working with degraded DNA. Specific primer design was carried out to choose fragments shorter than 200 bp as necessary in forensic and archaeological applications. Four fragments of exon 6 and 7 of the ABO gene were amplified and digested by in total 7 restriction endonucleases. Particular attention was paid to the base changes at nucleotide positions 261(delG), 297, 526, 703, 721, 771, 796 and 1060(delC) in order to distinguish the six common alleles A101, A201, B, O01, O02 and O03. Furthermore, this method also enables determination of seven of the less frequent alleles: A104, A204, Ax02, Ax03, O05, O06 and O07. The method was applied successfully to a series of blood samples with known phenotypes and genotypes as well as DNA extracted from a thirty year old blood stain and an ancient tooth sample. However, working with ancient DNA requires additional cloning and sequencing of the RFLP-typing results due to DNA post mortem damages such as deaminations, which could lead to false typing results.     

Glycinamide ribonucleotide synthetase from Escherichia coli: cloning, overproduction, sequencing, isolation, and characterization

The purD gene of Escherichia coli encoding the enzyme glycinamide ribonucleotide (GAR) synthetase, which catalyzes the conversion of phosphoribosylamine (PRA), glycine, and MgATP to glycinamide ribonucleotide, MgADP, and Pi, has been cloned and sequenced. The protein, as deduced by the structural gene sequence, contains 430 amino acids and has a calculated Mr of 45,945. Construction of an overproducing strain behind a lambda pL promoter allowed a 4-fold purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of the sequence with those of other GAR synthetases confirm the amino acid sequence deduced from the gene sequence. Initial velocity studies and product and dead-end inhibition studies are most consistent with a sequential ordered mechanism of substrate binding and product release in which PRA binds first followed by MgATP and then glycine; Pi leaves first, followed by loss of MgADP and finally GAR. Incubation of [18O]glycine, ATP, and PRA results in quantitative transfer of the 18O to Pi. GAR synthetase is very specific for its substrate glycine.     

Gene cloning, sequencing, and biochemical characterization of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

The gene encoding endoxylanase (xynA) from Thermoanaerobacterium saccharolyticum B6A-RI was cloned and expressed in Escherichia coli. A putative 33-amino-acid signal peptide, which corresponded to the N-terminal amino acids, was encoded by xynA. An open reading frame of 3,471 bp, corresponding to 1,157 amino acid residues, was found, giving the xynA gene product a molecular mass of 130 kDa. xynA from T. saccharolyticum B6A-RI had strong similarity to genes from family F beta-glycanases. The temperature and pH optimum for the activity of the cloned endoxylanase were 70 degrees C and 5.5, respectively. The cloned endoxylanase A was stable at 75 degrees C for 60 min and displayed a specific activity of 227.4 U/mg of protein on oat spelt xylan. The cloned xylanase was an endo-acting enzyme.     

Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A.

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 37 degrees C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.