scholarly journals Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

2006 ◽  
Vol 282 (7) ◽  
pp. 4681-4692 ◽  
Author(s):  
Leanne A. Pearson ◽  
Kevin D. Barrow ◽  
Brett A. Neilan
Keyword(s):  
Gene Cluster ◽  
Microcystis Aeruginosa ◽  
Hydroxy Acid ◽  
Biosynthesis Gene Cluster ◽  
Glutamate Mutase ◽  
Acid Dehydrogenase ◽  
Methyl Aspartate ◽  
Catalyzed Reactions

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce α-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.

Fur from Microcystis aeruginosa binds in vitro promoter regions of the microcystin biosynthesis gene cluster

2006 ◽  
Vol 67 (9) ◽  
pp. 876-881 ◽  
Author(s):  
Beatriz Martin-Luna ◽  
Emma Sevilla ◽  
José A. Hernandez ◽  
M. Teresa Bes ◽  
Maria F. Fillat ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Microcystis Aeruginosa ◽  
Promoter Regions ◽  
Biosynthesis Gene Cluster ◽  
Biosynthesis Gene

scholarly journals Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) inStreptomyces chartreusisNRRL 3882

2010 ◽  
Vol 55 (3) ◽  
pp. 974-982 ◽  
Author(s):  
Qiulin Wu ◽  
Jingdan Liang ◽  
Shuangjun Lin ◽  
Xiufen Zhou ◽  
Linquan Bai ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mutational Analysis ◽  
Divalent Cations ◽  
Biological Effects ◽  
Biosynthetic Gene Cluster ◽  
Type I ◽  
Biosynthesis Gene Cluster ◽  
Biosynthesis Gene ◽  
Hydroxyanthranilic Acid

ABSTRACTThe pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster fromStreptomyces chartreusisNRRL 3882. Gene replacements confirmed the identity of the gene cluster, andin silicoanalysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, anN-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.

Identification of a gene cluster for cell-surface genes of the SRS superfamily inNeospora caninumand characterization of the novelSRS9gene

Parasitology ◽  
2011 ◽  
Vol 138 (14) ◽  
pp. 1832-1842 ◽  
Author(s):  
V. RISCO-CASTILLO ◽  
V. MARUGÁN-HERNÁNDEZ ◽  
A. FERNÁNDEZ-GARCÍA ◽  
A. AGUADO-MARTÍNEZ ◽  
E. JIMÉNEZ-RUIZ ◽  
...  
Keyword(s):  
Western Blot ◽  
Gene Cluster ◽  
Neospora Caninum ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Specific Expression ◽  
Initial Characterization ◽  
Hydrophilic Region

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

scholarly journals Light-inducible carotenoid production controlled by a MarR-type regulator in Corynebacterium glutamicum

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Satoru Sumi ◽  
Yuto Suzuki ◽  
Tetsuro Matsuki ◽  
Takahiro Yamamoto ◽  
Yudai Tsuruta ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Gene Cluster ◽  
Transcriptional Start Site ◽  
Transcriptional Regulator ◽  
Carotenoid Biosynthesis ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Biosynthesis Gene Cluster ◽  
Carotenoid Production

Abstract Carotenoid production in some non-phototropic bacteria occurs in a light-dependent manner to protect cells from photo-oxidants. Knowledge regarding the transcriptional regulator involved in the light-dependent production of carotenoids of non-phototrophic bacteria has been mainly confined to coenzyme B12-based photo-sensitive regulator CarH/LitR family proteins belonging to a MerR family transcriptional regulator. In this study, we found that bacteria belonging to Micrococcales and Corynebacteriales exhibit light-dependent carotenoid-like pigment production including an amino acid-producer Corynebacterium glutamicum AJ1511. CrtR is a putative MarR family transcriptional regulator located in the divergent region of a carotenoid biosynthesis gene cluster in the genome of those bacteria. A null mutant for crtR of C. glutamicum AJ1511 exhibited constitutive production of carotenoids independent of light. A complemented strain of the crtR mutant produced carotenoids in a light-dependent manner. Transcriptional analysis revealed that the expression of carotenoid biosynthesis genes is regulated in a light-dependent manner in the wild type, while the transcription was upregulated in the crtR mutant irrespective of light. In vitro experiments demonstrated that a recombinant CrtR protein binds to the specific sequences within the intergenic region of crtR and crtE, which corresponds to −58 to −7 for crtE, and +26 to −28 for crtR with respect to the transcriptional start site, and serves as a repressor for crtE transcription directed by RNA polymerase containing SigA. Taken together, the results indicate that CrtR light-dependently controls the expression of the carotenoid gene cluster in C. glutamicum and probably closely related Actinobacteria.

scholarly journals Isolation and Characterization of Canthaxanthin Biosynthesis Genes from the Photosynthetic Bacterium Bradyrhizobiumsp. Strain ORS278

2000 ◽  
Vol 182 (13) ◽  
pp. 3850-3853 ◽  
Author(s):  
Laure Hannibal ◽  
Jean Lorquin ◽  
Nicolas Angles D'Ortoli ◽  
Nelly Garcia ◽  
Clemence Chaintreuil ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Photosynthetic Bacterium ◽  
Carotenoid Biosynthesis ◽  
Open Reading Frame ◽  
Biosynthesis Gene Cluster ◽  
Reading Frame ◽  
Functional Assignment ◽  
Isolation And Characterization ◽  
Biosynthesis Gene

ABSTRACT A carotenoid biosynthesis gene cluster involved in canthaxanthin production was isolated from the photosyntheticBradyrhizobium sp. strain ORS278. This cluster includes five genes identified as crtE, crtY,crtI, crtB, and crtW that are organized in at least two operons. The functional assignment of each open reading frame was confirmed by complementation studies.

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