scholarly journals Biosynthetic Intermediate Analysis and Functional Homology Reveal a Saxitoxin Gene Cluster in Cyanobacteria

2008 ◽  
Vol 74 (13) ◽  
pp. 4044-4053 ◽  
Author(s):  
Ralf Kellmann ◽  
Troco Kaan Mihali ◽  
Young Jae Jeon ◽  
Russell Pickford ◽  
Francesco Pomati ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Condensation Reaction ◽  
Comparative Sequence Analysis ◽  
Mass Spectrometry Analysis ◽  
Cylindrospermopsis Raciborskii ◽  
Shellfish Poisoning ◽  
Biosynthesis Gene Cluster ◽  
Spectrometry Analysis ◽  
Psp Toxins

ABSTRACT Saxitoxin (STX) and its analogues cause the paralytic shellfish poisoning (PSP) syndrome, which afflicts human health and impacts coastal shellfish economies worldwide. PSP toxins are unique alkaloids, being produced by both prokaryotes and eukaryotes. Here we describe a candidate PSP toxin biosynthesis gene cluster (sxt) from Cylindrospermopsis raciborskii T3. The saxitoxin biosynthetic pathway is encoded by more than 35 kb, and comparative sequence analysis assigns 30 catalytic functions to 26 proteins. STX biosynthesis is initiated with arginine, S-adenosylmethionine, and acetate by a new type of polyketide synthase, which can putatively perform a methylation of acetate, and a Claisen condensation reaction between propionate and arginine. Further steps involve enzymes catalyzing three heterocyclizations and various tailoring reactions that result in the numerous isoforms of saxitoxin. In the absence of a gene transfer system in these microorganisms, we have revised the description of the known STX biosynthetic pathway, with in silico functional inferences based on sxt open reading frames combined with liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates. Our results indicate the evolutionary origin for the production of PSP toxins in an ancestral cyanobacterium with genetic contributions from diverse phylogenetic lineages of bacteria and provide a quantum addition to the catalytic collective available for future combinatorial biosyntheses. The distribution of these genes also supports the idea of the involvement of this gene cluster in STX production in various cyanobacteria.

scholarly journals Characterization of the Ergosterol Biosynthesis Pathway in Ceratocystidaceae

2021 ◽  
Vol 7 (3) ◽  
pp. 237
Author(s):  
Mohammad Sayari ◽  
Magrieta A. van der Nest ◽  
Emma T. Steenkamp ◽  
Saleh Rahimlou ◽  
Almuth Hammerbacher ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Ergosterol Biosynthesis ◽  
Terpene Biosynthesis ◽  
Biosynthesis Gene Cluster ◽  
Spectrometry Analysis ◽  
Genes Encoding ◽  
Ergosterol Synthesis

Terpenes represent the biggest group of natural compounds on earth. This large class of organic hydrocarbons is distributed among all cellular organisms, including fungi. The different classes of terpenes produced by fungi are mono, sesqui, di- and triterpenes, although triterpene ergosterol is the main sterol identified in cell membranes of these organisms. The availability of genomic data from members in the Ceratocystidaceae enabled the detection and characterization of the genes encoding the enzymes in the mevalonate and ergosterol biosynthetic pathways. Using a bioinformatics approach, fungal orthologs of sterol biosynthesis genes in nine different species of the Ceratocystidaceae were identified. Ergosterol and some of the intermediates in the pathway were also detected in seven species (Ceratocystis manginecans, C. adiposa, Huntiella moniliformis, Thielaviopsis punctulata, Bretziella fagacearum, Endoconidiophora polonica and Davidsoniella virescens), using gas chromatography-mass spectrometry analysis. The average ergosterol content differed among different genera of Ceratocystidaceae. We also identified all possible terpene related genes and possible biosynthetic clusters in the genomes used in this study. We found a highly conserved terpene biosynthesis gene cluster containing some genes encoding ergosterol biosynthesis enzymes in the analysed genomes. An additional possible terpene gene cluster was also identified in all of the Ceratocystidaceae. We also evaluated the sensitivity of the Ceratocystidaceae to a triazole fungicide that inhibits ergosterol synthesis. The results showed that different members of this family behave differently when exposed to different concentrations of triazole tebuconazole.

scholarly journals vanM, a New Glycopeptide Resistance Gene Cluster Found in Enterococcus faecium

2010 ◽  
Vol 54 (11) ◽  
pp. 4643-4647 ◽  
Author(s):  
Xiaogang Xu ◽  
Dongfang Lin ◽  
Guoquan Yan ◽  
Xinyu Ye ◽  
Shi Wu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Amino Acid Identity ◽  
Mass Spectrometry Analysis ◽  
Clinical Strain ◽  
Glycopeptide Resistance ◽  
Spectrometry Analysis ◽  
Acid Protein ◽  
Inducible Synthesis ◽  
Intra Abdominal Infection

ABSTRACT Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcus faecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 μg/ml) and teicoplanin (MIC, 96 μg/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated vanM. The vanM ligase gene was 1,032-bp in length and encoded a 343-amino-acid protein that shared 79.9, 70.8, 66.3, and 78.8% amino acid identity with VanA, VanB, VanD, and VanF, respectively. Although the vanM DNA sequence was closest to vanA, the organization of the vanM gene cluster was most similar to that of vanD. Upstream from the vanM cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Liquid chromatography-mass spectrometry analysis of peptidoglycan precursors extracted from the VanM-type strain Efm-HS0661 treated with vancomycin or teicoplanin revealed a modified precursor (UDP-N-acetylmuramic acid [MurNAc]-tetrapeptide-d-Lac), indicating that VanM, like VanA, confers glycopeptide resistance by the inducible synthesis of precursor ending in d-Ala-d-Lac.

scholarly journals Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes

2019 ◽  
Vol 7 (11) ◽  
pp. 560 ◽  
Author(s):  
Emma J. Ludlow ◽  
Simone Vassiliadis ◽  
Piyumi N. Ekanayake ◽  
Inoka K. Hettiarachchige ◽  
Priyanka Reddy ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Gene Cluster ◽  
Perennial Ryegrass ◽  
Biosynthetic Pathway ◽  
Whole Genome Sequence ◽  
In Planta ◽  
Biosynthesis Gene Cluster ◽  
Biosynthesis Gene ◽  
Lolitrem B ◽  
Epichloë Endophytes

Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3; e.g., NEA12) and LpTG-4 (e.g., E1). Epichloë epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichloë. A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I–IV in Epichloë endophytes is proposed.

scholarly journals Molecular Basis and Phylogenetic Implications of Deoxycylindrospermopsin Biosynthesis in the Cyanobacterium Raphidiopsis curvata

2012 ◽  
Vol 78 (7) ◽  
pp. 2256-2263 ◽  
Author(s):  
Yongguang Jiang ◽  
Peng Xiao ◽  
Gongliang Yu ◽  
Tomoharu Sano ◽  
Qianqian Pan ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Phylogenetic Trees ◽  
Purifying Selection ◽  
Neutral Evolution ◽  
Cylindrospermopsis Raciborskii ◽  
Insertion Mutation ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Cell Extracts ◽  
Phylogenetic Implications

ABSTRACTNew insights into the distribution and biochemistry of the cyanotoxin cylindrospermopsin (CYN) have been provided by the recent determination of its biosynthesis gene cluster (cyr) in several cyanobacterial species.Raphidiopsis curvataCHAB1150 isolated from China was analyzed for CYN analogues. Only 7-deoxy-CYN was detected in the cell extracts. Thecyrgene cluster ofR. curvataCHAB1150 was sequenced, and thecyrgenes of this strain were found to have extremely high similarities (96% to 100%) to those from other nostocalean species. These species includeCylindrospermopsis raciborskiiAWT205,Aphanizomenonsp. strain 10E6, andAphanizomenon ovalisporumILC-146. Insertion mutation was identified within thecyrIgene, and transcripts ofcyrIand another functional genecyrJwere detected inR. curvataCHAB1150. General congruence between the phylogenetic trees based on bothcyrand 16Srrnwas displayed. Neutral evolution was found on the whole sequences of thecyrgenes, and 0 to 89 negative selected codons were detected in each gene. Therefore, the function of CyrI is to catalyze the oxygenation of 7-deoxy-CYN in CYN biosynthesis. The transcripts of the mutatedcyrIgene may result from polycistronic transcription. The high conservation of thecyrgenes may be ascribed to purifying selection and horizontal gene transfer.

scholarly journals Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters

2008 ◽  
Vol 190 (18) ◽  
pp. 6060-6069 ◽  
Author(s):  
Erin L. Westman ◽  
Andrew Preston ◽  
Robert A. Field ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bordetella Pertussis ◽  
Biosynthetic Pathway ◽  
Gene Clusters ◽  
Mass Spectrometry Analysis ◽  
Genomic Context ◽  
Spectrometry Analysis ◽  
Mannuronic Acid ◽  
Knockout Mutants ◽  
Dehydrogenase Gene

ABSTRACT Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). A five-enzyme biosynthetic pathway that requires WbpA, WbpB, WbpE, WbpD, and WbpI has been proposed for the production of this sugar in P. aeruginosa, based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB to wbpI were present, but a putative dehydrogenase gene was missing; therefore, the biosynthetic mechanism for UDP-d-ManNAc3NAcA was unclear. Nonpolar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans. Thus, the genes that encode the putative oxidase, transaminase, N-acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa. Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO 1629 and wbpO 3150, were shown to complement a wbpA knockout of P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO1629 and WbpO3150, and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP-d-GlcNAc, UDP-d-GalNAc, to a lesser extent, and UDP-d-Glc, to a much lesser extent. Together, these results suggest that B. pertussis produces UDP-d-ManNAc3NAcA through the same pathway proposed for P. aeruginosa, despite differences in the genomic context of the genes involved.

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