scholarly journals Novel Pathway of Salicylate Degradation by Streptomyces sp. Strain WA46

2004 ◽  
Vol 70 (3) ◽  
pp. 1297-1306 ◽  
Author(s):  
Daisuke Ishiyama ◽  
Dusica Vujaklija ◽  
Julian Davies
Keyword(s):  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Pcr Amplification ◽  
Amino Acid Sequence Similarity ◽  
Chromosomal Dna ◽  
Degenerate Primers ◽  
Putative Gene ◽  
Cell Extracts ◽  
Plasmid Library ◽  
Coa Ligase

ABSTRACT A novel salicylate-degrading Streptomyces sp., strain WA46, was identified by UV fluorescence on solid minimal medium containing salicylate; trace amounts of gentisate were detected by high-pressure liquid chromatography when strain WA46 was grown with salicylate. PCR amplification of WA46 DNA with degenerate primers for gentisate 1,2-dioxygenase (GDO) genes produced an amplicon of the expected size. Sequential PCR with nested GDO primers was then used to identify a salicylate degradation gene cluster in a plasmid library of WA46 chromosomal DNA. The nucleotide sequence of a 13.5-kb insert in recombinant plasmid pWD1 (which was sufficient for the complete degradation of salicylate) showed that nine putative open reading frames (ORFs) (sdgABCDEFGHR) were involved. Plasmid pWD1 derivatives disrupted in each putative gene were transformed into Streptomyces lividans TK64. Disruption of either sdgA or sdgC blocked salicylate degradation; constructs lacking sdgD accumulated gentisate. Cell extracts from Escherichia coli DH5α transformants harboring pUC19 that expressed each of the sdg ORFs showed that conversions of salicylate to salicylyl-coenzyme A (CoA) and salicylyl-CoA to gentisyl-CoA required SdgA and SdgC, respectively. SdgA required CoA and ATP as cofactors, while NADH was required for SdgC activity; SdgC was identified as salicylyl-CoA 5-hydroxylase. Gentisyl-CoA underwent spontaneous cleavage to gentisate and CoA. SdgA behaved as a salicylyl-CoA ligase despite showing amino acid sequence similarity to an AMP-ligase. SdgD was identified as a GDO. These results suggest that Streptomyces sp. strain WA46 degrades salicylate by a novel pathway via a CoA derivative. Two-dimensional polyacrylamide gel electrophoresis and reverse transcriptase-PCR studies indicated that salicylate induced expression of the sdg cluster.

scholarly journals Distribution and Diversity of Geminiviruses in Trinidad and Tobago

1998 ◽  
Vol 88 (12) ◽  
pp. 1262-1268 ◽  
Author(s):  
Pathmanathan Umaharan ◽  
Malla Padidam ◽  
Ralph H. Phelps ◽  
Roger N. Beachy ◽  
Claude M. Fauquet
Keyword(s):  
Trinidad And Tobago ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Sweet Pepper ◽  
Yellow Mosaic Virus ◽  
Restriction Enzyme Digestion ◽  
Weed Species ◽  
Dot Blot ◽  
Degenerate Primers

Seven crop and eight weed species from 12 agricultural locations in Trinidad and Tobago were assayed for the presence of whitefly-transmitted geminiviruses (WTGs) by using dot blot hybridization and polymerase chain reaction (PCR) amplification of the N-terminal coat protein sequence with degenerate primers. The amplified fragments were cloned and analyzed by restriction enzyme digestion to determine fragment length polymorphism among the cloned fragments. Representative clones were then sequenced and subjected to phylogenetic analysis to determine the sequence similarity to known WTGs. WTGs were found in every location sampled and in 10 of the 15 species investigated: Lycopersicon esculentum(tomato), Capsicum annuum (pepper), Capsicum frutescens (sweet pepper), Abelmoschus esculentus (okra), Phaseolus vulgaris (beans), Alternanthera tenella, Desmodium frutescens, Euphorbia heterophylla, Malva alceifolia, and Sida acuta. The geminiviruses infecting these plants were closely related to potato yellow mosaic virus from Venezuela (PYMV-VE) and tomato leaf curl virus from Panama (ToLCV-PA). However, in pepper, sweet pepper, okra, Alternanthera tenella, Euphorbia heterophylla, Des-modium frutescens, and in one sample of tomato, a PYMV-VE-related virus was found in mixed infections with a virus related to pepper huasteco virus. Full-length infectious DNA-A and DNA-B of a tomato-infecting geminivirus from Trinidad and Tobago were cloned and sequenced. DNA-A appears to be a recombinant derived from PYMV-VE or ToLCV-PA, and Sida golden mosaic from Honduras. The implications of these findings in the control of WTGs are discussed.

scholarly journals Indole-Diterpene Gene Cluster from Aspergillus flavus

2004 ◽  
Vol 70 (11) ◽  
pp. 6875-6883 ◽  
Author(s):  
Shuguang Zhang ◽  
Brendon J. Monahan ◽  
Jan S. Tkacz ◽  
Barry Scott
Keyword(s):  
Aspergillus Flavus ◽  
Biosynthetic Pathway ◽  
Sequence Similarity ◽  
Soil Fungus ◽  
Amino Acid Sequence Similarity ◽  
Degenerate Primers ◽  
Functional Homolog ◽  
Penicillium Paxilli ◽  
Diterpene Biosynthesis ◽  
Tremorgenic Mycotoxin

ABSTRACT Aflatrem is a potent tremorgenic mycotoxin produced by the soil fungus Aspergillus flavus and is a member of a large structurally diverse group of secondary metabolites known as indole-diterpenes. By using degenerate primers for conserved domains of fungal geranylgeranyl diphosphate synthases, we cloned two genes, atmG and ggsA (an apparent pseudogene), from A. flavus. Adjacent to atmG are two other genes, atmC and atmM. These three genes have 64 to 70% amino acid sequence similarity and conserved synteny with a cluster of orthologous genes, paxG, paxC, and paxM, from Penicillium paxilli which are required for indole-diterpene biosynthesis. atmG, atmC, and atmM are coordinately expressed, with transcript levels dramatically increasing at the onset of aflatrem biosynthesis. A genomic copy of atmM can complement a paxM deletion mutant of P. paxilli, demonstrating that atmM is a functional homolog of paxM. Thus, atmG, atmC, and atmM are necessary, but not sufficient, for aflatrem biosynthesis by A. flavus. This provides the first genetic evidence for the biosynthetic pathway of aflatrem in A. flavus.

scholarly journals Fungal gene expression during ectomycorrhiza formation

1995 ◽  
Vol 73 (S1) ◽  
pp. 541-547 ◽  
Author(s):  
F. Martin ◽  
P. Laurent ◽  
D. de Carvalho ◽  
T. Burgess ◽  
P. Murphy ◽  
...  
Keyword(s):  
Cell Walls ◽  
Molecular Mechanisms ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Pisolithus Tinctorius ◽  
Amino Acid Sequence Similarity ◽  
Cell Fractionation ◽  
Fungal Cell ◽  
Temporal Expressions ◽  
Fungal Genes

Ectomycorrhiza development involves the differentiation of structurally specialized fungal tissues (e.g., mantle and Hartig net) and an interface between symbionts. Polypeptides presenting a preferential, up-, or down-regulated synthesis have been characterized in several developing ectomycorrhizal associations. Their spatial and temporal expressions have been characterized by cell fractionation, two-dimensional polyacrylamide gel electrophoresis, and immunochemical assays in the Eucalyptus spp. – Pisolithus tinctorius mycorrhizas. These studies have emphasized the importance of fungal cell wall polypeptides during the early stages of the ectomycorrhizal interaction. The increased synthesis of 30- to 32-kDa acidic polypeptides, together with the decreased accumulation of a prominent 95-kDa mannoprotein provided evidence for major alterations of Pisolithus tinctorius cell walls during mycorrhiza formation. Differential cDNA library screening and shotgun cDNA sequencing were used to clone symbiosis-regulated fungal genes. Several abundant transcripts showed a significant amino acid sequence similarity to a family of secreted morphogenetic fungal proteins, the so-called hydrophobic. In P. tinctorius, the content of hydrophobin transcripts is high in aerial hyphae and during the ectomycorrhizal sheath formation. Alteration of cell walls and the extracellular matrix is therefore a key event in the ectomycorrhiza development. An understanding of the molecular mechanisms that underlies the temporal and spatial control of genes and proteins involved in the development of the symbiotic interface is now within reach, as more sophisticated techniques of molecular and genetic analysis are applied to the mycorrhizal interactions. Key words: cell walls, ectomycorrhiza, ectomycorrhizins, fungal development, hydrophobins, symbiosis-regulated polypeptides.

Molecular Characterization of a New Lectin from the Marine Alga Ulva pertusa

2004 ◽  
Vol 36 (2) ◽  
pp. 111-117 ◽  
Author(s):  
Sheng Wang ◽  
Fu-Di Zhong ◽  
Yong-Jiang Zhang ◽  
Zu-Jian Wu ◽  
Qi-Ying Lin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Divalent Cations ◽  
Sequence Similarity ◽  
Ulva Pertusa ◽  
Amino Acid Sequence Similarity ◽  
Degenerate Primers ◽  
Heat Stable ◽  
Rapid Amplification

Abstract A new lectin, named UPL1, was purified from a green alga Ulva pertusa by an affinity chromatography on the bovine-thyroglobulin-Sepharose 4B column. The molecular mass of the algal lectin was about 23 kD by SDS-PAGE, and it specifically agglutinated rabbit erythrocytes. The hemagglutinating activity for rabbit erythrocytes could be inhibited by bovine thyroglobulin and N-acetyl-D-glucosamine. The lectin UPL1 required divalent cations for maintenance of its biological activity, and was heat-stable, and had higher activity within pH 6–8. The N-terminal amino acid sequence of the purified lectin was determined (P83209) and a set of degenerate primers were designed. The full-length cDNA of the lectin was cloned by rapid amplification of cDNA ends (RACE) method (AY433960). Sequence analysis of upl1 indicated it was 1084 bp long, and encoded a premature protein of 203 amino acids. The N-terminal sequence of the mature UPL1 polypeptide started at amino acid 54 of the deduced sequence from the cDNA, indicating 53 amino acids lost due to posttranslational modification. The primary structure of the Ulva pertusa lectin did not show amino acid sequence similarity with known plant and animal lectins. Hence, this protein may be the paradigm of a novel lectin family.

scholarly journals Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6

2006 ◽  
Vol 72 (3) ◽  
pp. 1825-1832 ◽  
Author(s):  
Mikio Sasoh ◽  
Eiji Masai ◽  
Satoko Ishibashi ◽  
Hirofumi Hara ◽  
Naofumi Kamimura ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Large Subunit ◽  
Transcriptional Regulator ◽  
Gene Clusters ◽  
Small Subunit ◽  
Amino Acid Sequence Similarity ◽  
Comamonas Testosteroni ◽  
Cell Extracts ◽  
Two Component

ABSTRACT We isolated Comamonas sp. strain E6, which utilizes terephthalate (TPA) as the sole carbon and energy source via the protocatechuate (PCA) 4,5-cleavage pathway. Two almost identical TPA degradation gene clusters, tphR I C I A2 I A3 I B I A1 I and tphR II C II A2 II A3 II B II A1 II, were isolated from this strain. Based on amino acid sequence similarity, the genes tphR, tphC, tphA2, tphA3, tphB, and tphA1 were predicted to code, respectively, for an IclR-type transcriptional regulator, a periplasmic TPA binding receptor, the large subunit of the oxygenase component of TPA 1,2-dioxygenase (TPADO), the small subunit of the oxygenase component of TPADO, a 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) dehydrogenase, and a reductase component of TPADO. The growth of E6 on TPA was not affected by disruption of either tphA2 I or tphA2 II singly; however, the tphA2 I tphA2 II double mutant no longer grew on TPA, suggesting that both TPADO genes are involved in TPA degradation. Introduction of a plasmid carrying tphR II C II A2 II A3 II B II A1 II conferred the TPA utilization phenotype on Comamonas testosteroni IAM 1152, which is able to grow on PCA but not on TPA. Disruption of either tphR II or tphC II on this plasmid resulted in the loss of the growth of IAM 1152 on TPA, suggesting that these genes are essential to convert TPA to PCA in E6. The genes tphA1 II, tphA2 II, tphA3 II, and tphB II were expressed in Escherichia coli, and the resultant cell extracts containing TphA1II, TphA2II, and TphA3II converted TPA in the presence of NADPH into a product which was transformed to PCA by TphBII. On the basis of these results, TPADO was strongly suggested to be a two-component dioxygenase which consists of the terminal oxygenase component (TphA2 and TphA3) and the reductase (TphA1), and tphB codes for the DCD dehydrogenase.

scholarly journals Characterization of the Operon Encoding the Alternative ςB Factor from Bacillus anthracis and Its Role in Virulence

2000 ◽  
Vol 182 (18) ◽  
pp. 5036-5045 ◽  
Author(s):  
Agnès Fouet ◽  
Olivier Namy ◽  
Guillaume Lambert
Keyword(s):  
Transcription Factor ◽  
Bacillus Anthracis ◽  
Pcr Amplification ◽  
Promoter Sequence ◽  
Inverse Pcr ◽  
Chromosomal Dna ◽  
Degenerate Primers ◽  
Reading Frame ◽  
A Minor

ABSTRACT The operon encoding the general stress transcription factor ςB and two proteins of its regulatory network, RsbV and RsbW, was cloned from the gram-positive bacterium Bacillus anthracis by PCR amplification of chromosomal DNA with degenerate primers, by inverse PCR, and by direct cloning. The gene cluster was very similar to the Bacillus subtilis sigB operon both in the primary sequences of the gene products and in the order of its three genes. However, the deduced products of sequences upstream and downstream from this operon showed no similarity to other proteins encoded by the B. subtilis sigB operon. Therefore, the B. anthracis sigB operon contains three genes rather than eight as in B. subtilis. TheB. anthracis operon is preceded by a ςB-like promoter sequence, the expression of which depends on an intact ςB transcription factor in B. subtilis. It is followed by another open reading frame that is also preceded by a promoter sequence similarly dependent on B. subtilis ςB. We found that in B. anthracis, both these promoters were induced during the stationary phase and induction required an intact sigBgene. The sigB operon was induced by heat shock. Mutants from which sigB was deleted were constructed in a toxinogenic and a plasmidless strain. These mutants differed from the parental strains in terms of morphology. The toxinogenicsigB mutant strain was also less virulent than the parental strain in the mouse model. B. anthracis ςBmay therefore be a minor virulence factor.

scholarly journals A Begomovirus Isolated from Chlorotic and Stunted Soybean Plants in Mexico is a New Strain of Rhynchosia golden mosaic virus

Plant Disease ◽  
2006 ◽  
Vol 90 (7) ◽  
pp. 972-972 ◽  
Author(s):  
J. Méndez-Lozano ◽  
L. L. Perea-Araujo ◽  
R. D. Ruelas-Ayala ◽  
N. E. Leyva-López ◽  
J. A. Mauricio-Castillo ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Dna Sequences ◽  
Sequence Similarity ◽  
Pcr Amplification ◽  
Nucleotide Identity ◽  
Degenerate Primers ◽  
Viral Dna ◽  
Field Samples ◽  
Pcr Products ◽  
Soybean Plants

Soybean (Glycine max Merr.) is an alternative crop during the summer in Sinaloa, a northern state of Mexico. During the last 4 years, symptoms of yellowing, curled leaves, and stunting have been observed on soybean plantings, and a scrutiny of field samples collected in 2003 identified a begomovirus related to Pepper golden mosaic virus in symptomatic plants (4). A new survey was conducted during the summer of 2004 when the soybean disease was prevalent in the region. Affected plants appeared as patches displaying symptoms ranging from mild to severe yellow mosaic with leaf deformation and stunted growth in several parcels of commercial fields of northern Sinaloa. More than 100 samples from symptomatic soybean plants and weeds growing within the same fields were collected and analyzed for the presence of begomoviruses using DNA hybridization with the coat protein gene of Pepper huasteco yellow vein virus as a probe. Thirty-eight soybean, 12 Rhynchosia sp., and 14 sunflower hybridization-positive samples were subsequently used for polymerase chain reaction (PCR) amplification with the degenerate primers pRep-DGR and pCP70-Mot (1). PCR products were cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced. The amplified viral DNA (915 nt) from two soybean plants, Sb1 and Sb2 (GenBank Accession Nos. AY955101 and AY957561, respectively), one isolate from Rhynchosia minima (GenBank Accession No. AY955102), and one from Heliantus annum (GenBank Accession No. AY957560) were sequenced and compared with DNA sequences available at NCBI database using BLAST. The highest sequence similarity was obtained with the two known isolates of Rhynchosia golden mosaic virus, RhGMV [Honduras] (GenBank Accession No. AF239671), and RhGMV [Chiapas] (GenBank Accession No. AF408199), displaying a nucleotide identity of approximately 89% with the Sinaloa isolates. Sequence comparisons of the latter isolates showed that viruses in the weeds were 97% identical to one of the soybean isolates, RhGMV-Sb1, but differed significantly (88% of nucleotide identity) from the second soybean isolate, RhGMV-Sb2. The complete genome A sequence of RhGMV-Sb1 was determined using PCR amplification of viral DNA with four degenerate primers recently described (2), cloning of overlapping PCR products into pGEM-T Easy vector (Promega) and sequencing. The 2,604-bp DNA-A of RhGMV-Sb1 (GenBank Accession No. DQ347950) was compared with the homologous genome of RhGMV [Chiapas] and RhGMV [Honduras] using the CLUSTAL alignment method (MegAlign, DNASTAR software, London) and an overall nucleotide identity of 89.2 and 88.6%, respectively, was determined. Current taxonomic criteria for begomoviruses establish that a DNA-A sequence identity lower than 93% with other isolates of a virus is indicative of a separate strain (3). Therefore, the virus identified in this study is a new strain of RhGMV that is provisionally named Rhynchosia golden mosaic virus-Soybean [Mexico:Sinaloa:2004]. This is the first soybean-infecting begomovirus from the American continent whose genome A has been completely characterized as of today. References: (1) J. T. Ascencio-Ibañez et al. Plant Dis. 86:692, 2002. (2) R. De La Torre-Almaraz et al. Plant Dis. 90:378, 2006. (3) C. Fauquet et al. Arch. Virol. 150:2151, 2005. (4) J. Mendez-Lozano et al. Plant Dis. 90:109, 2006.

scholarly journals Identification and Characterization of Lactobacillus helveticus PepO2, an Endopeptidase with Post-Proline Specificity

2003 ◽  
Vol 69 (2) ◽  
pp. 1276-1282 ◽  
Author(s):  
Yo-Shen Chen ◽  
Jeffrey E. Christensen ◽  
Jeffery R. Broadbent ◽  
James L. Steele
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Sequence Similarity ◽  
Lactobacillus Helveticus ◽  
Peptide Sequence ◽  
Amino Acid Sequence Similarity ◽  
Nucleotide Sequence Analysis ◽  
Northern Hybridization ◽  
Bitter Peptides ◽  
Cell Extracts

ABSTRACT A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-β-casein-(f203-209)-ρ-nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that L. helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L. helveticus PepO, Lactococcus lactis PepO2, L. lactis PepO, and Lactobacillus rhamnosus PepO, respectively. Northern hybridization results indicated that the transcript containing pepO2 was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L. helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as β-casein(f193-209).

scholarly journals Overexpression of the pitaya phosphoethanolamine N-methyltransferase gene (HpPEAMT) enhanced simulated drought stress in tobacco

2021 ◽  
Author(s):  
Ai-Hua Wang ◽  
Lan Yang ◽  
Xin-Zhuan Yao ◽  
Xiao-Peng Wen
Keyword(s):  
Drought Stress ◽  
Stress Response ◽  
Transgenic Plants ◽  
Drought Tolerance ◽  
Transgenic Tobacco ◽  
Sequence Similarity ◽  
Amino Acid Sequence Similarity ◽  
Transgenic Tobacco Plants ◽  
Wild Type ◽  
Tobacco Plants

AbstractPhosphoethanolamine N-methyltransferase (PEAMTase) catalyzes the methylation of phosphoethanolamine to produce phosphocholine and plays an important role in the abiotic stress response. Although the PEAMT genes has been isolated from many species other than pitaya, its role in the drought stress response has not yet been fully elucidated. In the present study, we isolated a 1485 bp cDNA fragment of HpPEAMT from pitaya (Hylocereus polyrhizus). Phylogenetic analysis showed that, during its evolution, HpPEAMT has shown a high degree of amino acid sequence similarity with the orthologous genes in Chenopodiaceae species. To further investigate the function of HpPEAMT, we generated transgenic tobacco plants overexpressing HpPEAMT, and the transgenic plants accumulated significantly more glycine betaine (GB) than did the wild type (WT). Drought tolerance trials indicated that, compared with those of the wild-type (WT) plants, the roots of the transgenic plants showed higher drought tolerance ability and exhibited improved drought tolerance. Further analysis revealed that overexpression of HpPEAM in Nicotiana tabacum resulted in upregulation of transcript levels of GB biosynthesis-related genes (NiBADH, NiCMO and NiSDC) in the leaves. Furthermore, compared with the wild-type plants, the transgenic tobacco plants displayed a significantly lower malondialdehyde (MDA) accumulation and higher activities of the superoxide dismutase (SOD) and peroxidase (POD) antioxidant enzymes under drought stress. Taken together, our results suggested that HpPEAMT enhanced the drought tolerance of transgenic tobacco.

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