Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ Nucleotide Sequence Databanks and have been assigned the accession number X78413     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.     

Studies on the Biosynthesis of Fosfomycin. Conversion of 2-Hydroxyethylphosphonic Acid and 2-Aminoethylphosphonic Acid to Fosfomycin

Two types of 130 kDa insecticidal protein genes isolated from large plasmid DNAs of Bacillus thuringiensis var. israelensis HDS22 were cloned in an Escherichia coli plasmid vector. Analysis of the nucleotide sequences revealed that the two genes encoded a 127,500-dalton protein (ISRH3) consisting of 1,135 amino acids and a 134,400-dalton protein (ISRH4) consisting of 1,180 amino acids. The two insecticidal proteins were identical in a region of the C-terminal 467 amino acids.     

Characterization of a 3-dehydroquinase gene from Actinobacillus pleuropneumoniae with homology to the eukaryotic genes qa-2 and QUTE

A gene was cloned from Actinobacillus pleuropneumoniae strain 4074 by complementation of an aroD strain of Escherichia coli. The E. coli gene aroD codes for a 3-dehydroquinase enzyme of type I, active in the aromatic biosynthesis pathway. The A. pleuropneumoniae gene, termed aroQ, displays no base or ami no acid sequence homoiogy to aroD of E. coli. It is instead homologous to the QUTE and qa-2 genes, respectively of Aspergillus niduians and Neurospora crassa. These genes code for 3-dehydroquinase enzymes of type ii, involved in the catabolism of quinic acid. The 1.6 kb fragment, which includes aroQ, carries four overlapping or adjacent open reading frames: a dapD gene; aroQ; one without homology to sequences in GenBank; and one with homology to the C-terminal 40% of chlN of E. coli.     

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments

The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroA‐like) from pure‐culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite‐oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite‐oxidizing environments. Geochemical analyses, including measurement of arsenite‐oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA‐like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6–3.6) Fe‐oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near‐neutral (pH 6.2–8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and β‐Proteobacteria. Modified primers designed around previously characterized and newly identified aroA‐like genes successfully amplified new lineages of aroA‐like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA‐like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences identified in the current study expand the phylogenetic distribution of known Mo‐pterin arsenite oxidase genes, and suggest the importance of three prominent genera of the order Aquificales in arsenite oxidation across geochemically distinct geothermal habitats ranging in pH from 2.6 to 8.     

Characterization of a HoxEFUYH type of [NiFe] hydrogenase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> and some EPR and IR properties of the hydrogenase module

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M _r = 52 kDa) and a small (M _r = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe–S clusters, three [2Fe–2S] clusters and five [4Fe–4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe–S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H₂-dependent reduction of NAD⁺. Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni–Fe active site and a [4Fe–4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni_a–C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.     

Ribosomal Protein S1(RpsA) of Buchnera aphidicola,the Endosymbiont of Aphids: Characterization of the Gene and Detection of the Product

Buchnera aphidicola is a prokaryotic endosymbiont found in specialized cells of the aphid Schizaphis graminum. Many of the previously cloned B. aphidicola genes are preceded by a poor ribosome-binding site. Ribosomal protein S1 (RpsA) allows the translation of messenger RNAs that lack or have a poor ribosome binding site. We have cloned and sequenced a 4.5-kilobase (kb) B. aphidicola DNA fragment containing four open reading frames corresponding to aroA–rpsA–himD–tpiA. The deduced amino acid sequence of B. aphidicola RpsA was 75% identical to that of the Escherichia coli protein. The major difference was in the number of basic amino acids, which were present in higher numbers in B. aphidicola RpsA. Antiserum to E. coli RpsA was prepared and used to detect B. aphidicola RpsA in cell-free extracts of aphids. During the first 12 days of aphid growth there is a slight decrease in the amount of RpsA per unit of aphid weight. The three additional genes found on the 4.5-kb DNA fragment encoded for proteins involved in aromatic amino acid biosynthesis (aroA), DNA bending (himD), and carbohydrate metabolism (tpiA). The presence of these genes in B. aphidicola is additional evidence of its similarity to free-living bacteria.     

Differential expression of two SOD (Superoxide dismytase) genes from small radish (Rhaphanus sativus L. var.sativus)

We have isolated two Superoxide dismutase cDNA clones (RsCu/ZnSod andRsFeSod) from small radish (Raphanus sativus L.) by cDNA library screening.RsCu/ZnSod is 563 bp long, with an open reading frame of 153 amino acids, and corresponds to a protein of predicted molecular mass 15.1 kDa and a pl of 5.44. The 823-bp RsFeSod has an ORF of 213 amino acids, corresponding to a protein of predicted molecular mass 25.4 kDa and a pl of 8.77. Their nucleotide and deduced amino acid sequences show the highest homology with those ofArabidopsis. Genomic Southern blot analysis, using each cDNA clone as probe, has revealed that the SOD genes are present as at least two copies in the small radish genome. Nondenaturing polyacrylamide gels for SOD activity has demonstrated the presence of several isozymes, depending on the organ type and developmental stage. TheseRsSod genes also have differential expression patterns in response to treatments with white light, xenobiotics, UV, osmoticums, plant hormones, and salicylic acid. Therefore, we suggest that they are involved in an antioxidative defense mechanism against stress induced by environmental change.     

Comparative genomics and phylogenetic analysis of <Emphasis Type="Italic">S. dysenteriae</Emphasis> subgroup

Genomic compositions of representatives of thirteen S. dysenteriae serotypes were investigated by performing comparative genomic hybridization (CGH) with microarray containing the whole genomic ORFs (open reading frames, ORFs) of E. coli K12 strain MG1655 and specific ORFs of S. dysenteriae A1 strain Sd51197. The CGH results indicated the genomes of the serotypes contain 2654 conserved ORFs originating from E. coli. However, 219 intrinsic genes of E. coli including those prophage genes, molecular chaperones, synthesis of specific O antigen and so on were absent. Moreover, some specific genes such as type II secretion system associated components, iron transport related genes and some others as well were acquired through horizontal transfer. According to phylogenic trees based on genetic composition, it was demonstrated that A1, A2, A8, A10 were distinct from the other S. dysenteriae serotypes. Our results in this report may provide new insights into the physiological process, pathogenicity and evolution of S. dysenteriae.     

Identification of a New Gene Encoding EPSPS with High Glyphosate Resistance from the Metagenomic Library

Glyphosate, a powerful nonselective herbicide, acts as an inhibitor of the activity of the enzyme 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by the aroA gene involved in aromatic amino acid biosynthesis. An Escherichia coli mutant AKM4188 was constructed by insertion a kanamycin cassette within the aroA coding sequence. The mutant strain is an aromatic amino acids auxotroph and fails to grow on M9 minimal media due to the inactive aroA. A DNA metagenomic library was constructed with samples from a glyphosate-polluted area and was screened by using the mutant AKM4188 as recipient. Three plasmid clones, which restored growth to the aroA mutant in M9 minimal media supplemented with chloramphenicol, kanamycin, and 50 mM glyphosate, were obtained from the DNA metagenomic library. One of them, which conferred glyphosate tolerance up to 150 mM, was further characterized. The cloned fragment encoded a polypeptide, designated RD, sharing high similarity with other Class II EPSPS proteins. A His-tagged RD fusion protein was produced into E. coli to characterize the enzymatic properties of the RD EPSP protein.     

Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene

Summary The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.     

Cloning and comparative sequence analysis of the gene coding for isopenicillin N synthase in Streptomyces

Summary The genes coding for isopenicillin N synthase (IPNS) in Streptomyces jumonjinensis and S. lipmanii were isolated from recombinant phage lambda libraries using the S. clavuligerus IPNS gene as a heterologous probe. The S. jumonjinensis IPNS gene has an open reading frame coding for 329 amino acids, identical in size to that of the previously cloned S. clavuligerus IPNS gene. A partial nucleotide sequence was also determined for the S. lipmanii IPNS gene. Comparison of the predicted amino acid sequences of all three streptomycete IPNS proteins shows that they exhibit more than 70% similarity, close to that found in comparisons among fungal IPNS proteins and significantly greater than that found, approximately 60%, between Streptomyces and fungal IPNS proteins. We conclude that procaryotic and eucaryotic IPNS genes are subgroups of a single family of microbial IPNS genes. Hybridization probes prepared from IPNS genes of the above streptomycete species were used to detect analogous genes in eight other strains that included both penicillin and cephalosporin producers and non-producers. Each producer strain responded with all three probes implying the presence of an IPNS gene. Surprisingly, several non-producer strains also responded with one or two of the probes. Our results suggest that IPNS-related genes may be more prevalent in Streptomyces than previously believed.     

Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.)

Three novel low molecular weight (LMW) glutenin subunits from cultivated einkorn (Triticum monococcum L., A^mA^m, 2n = 2x = 14) were characterized by SDS-PAGE and molecular weights determined by MALDI-TOF-MS. Their coding genes were amplified and cloned with designed AS-PCR primers, revealing three complete gene sequences. All comprised upstream, open reading frame (ORF), downstream and no introns were present. The deduced amino acid sequences showed that all three genes, named as LMW-M1, LMW-M3 and LMW-M5, respectively, belonged to the LMW-i type subunits with the predicted molecular weight between 38.5206 and 38.7028 kDa. They showed high similarity with other LMW-i type genes from hexaploid bread wheats, but also displayed unique features. Particularly, LMW-M5 subunit contained an extra cysteine residue in the C-terminus except for eight conserved cysteines, which resulted from a single-nucleotide polymorphism (SNP) of the T–C transition, namely arginine → cysteine substitution at position 242 from the N-terminal end. This is the first report that the LMW-i subunit contained nine cysteines residues that could result in a more highly cross-linked and more elastic glutenin suggesting that LMW-M5 gene may associates with good quality properties. In addition, a total of 25 SNPs and one insertions/deletions (InDels) were detected among three LMW-i genes, which could result in significant functional changes in polymer formation of gluten. It is anticipated that these SNPs could be used as reliable genetic markers during wheat quality improvement. The phylogenetic analysis indicated that LMW-i type genes apparently differed from LMW-m and LMW-s type genes and diverged early from the primitive LMW-GS gene family, at about 12.92 million years ago (MYA) while the differentiation of A^m and A genomes was estimated at 3.98 MYA.Q. Zhang had the same contribution to this work as the first author.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Synthesis of chitinase in Streptomyces thermoviolaceus is regulated by a two-component sensor-regulator system

The chiS and chiR genes located upstream of the chitinase locus (chi40) on the chromosome of Streptomyces thermoviolaceus OPC-520 were cloned and sequenced. The deduced amino acid sequences revealed that ChiS (390 amino acids, 40.9 kDa) and ChiR (213 amino acids, 22 kDa) show significant sequence similarities to histidine kinases and response regulators, respectively, of typical prokaryotic two-component regulatory systems. The extracellular chitinase activity of Streptomyces lividans 66 (pTSR2 (bearing chiS, chiR and chi40)) was significantly enhanced by a high dosage of the chiS and chiR genes.     

Cloning and Sequencing of Alginate Lyase Genes from Deep-Sea Strains of Vibrio and Agarivorans and Characterization of a New Vibrio Enzyme

Four alginate lyase genes were cloned and sequenced from the genomic DNAs of deep-sea bacteria, namely members of Vibrio and Agarivorans. Three of them were from Vibrio sp. JAM-A9m, which encoded alginate lyases, A9mT, A9mC, and A9mL. A9mT was composed of 286 amino acids and 57% homologous to AlxM of Photobacterium sp. A9mC (221 amino acids) and A9mL (522 amino acids) had the highest degree of similarity to two individual alginate lyases of Vibrio splendidus with 74% and 84% identity, respectively. The other gene for alginate lyase, A1mU, was shotgun cloned from Agarivorans sp. JAM-A1m. A1mU (286 amino acids) showed the highest homology to AlyVOA of Vibrio sp. with 76% identity. All alginate lyases belong to polysaccharide lyase family 7, although, they do not show significant similarity to one another with 14% to 58% identity. Among the above lyases, the recombinant A9mT was purified to homogeneity and characterized. The molecular mass of A9mT was around 28 kDa. The enzyme was remarkably salt activated and showed the highest thermal stability in the presence of NaCl. A9mT favorably degraded mannuronate polymer in alginate. We discussed substrate specificities of family 7 alginate lyases based on their conserved amino acid sequences.     

Utilization of the TEF1-a gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae

For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1α, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-α protein consisted of 460 amino acids possessing high identity to other fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A. oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 °C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C. Received: 28 November 1997 / Received revision: 24 February 1998 / Accepted: 6 March 1998     

Molecular cloning and nucleotide sequence of the psaA and psaB genes of the cyanobacterium Synechococcus sp. PCC 7002

The psaA and psaB genes, which encode the P700 chlorophyll a apoproteins of the Photosystem I complex, have been cloned from the unicellular, transformable cyanobacterium Synechococcus sp. PCC 7002. The nucleotide sequence of these genes and of their flanking sequences have been determined by the chain termination method. As found in the chloroplast genomes of higher plants, the psaA gene lies 5 to the psaB gene; however, the cyanobacterial genes are separated by a greater distance (173 vs. 25–26 bp). The psaA gene is predicted to encode a polypeptide of 739 amino acid residues (81.7 kDa), and the psaB gene is predicted to encode a polypeptide of 733 residues (81.4 kDa). The cyanobacterial psa gene products are 76% to 81% identical to their higher plant homologues; moreover, because of conservative amino acid replacements, the cyanobacterial sequences are more than 95% homologous to those determined for higher plants. These results provide the basis for a genetic analysis of Photosystem I, and are discussed in relationship to structural and functional aspects of the Photosystem I complexes of both cyanobacteria and higher plants.