scholarly journals Transcriptional Regulation of the Equol Biosynthesis Gene Cluster in Adlercreutzia equolifaciens DSM19450T

Nutrients ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 993 ◽  
Author(s):  
Flórez ◽  
Vázquez ◽  
Rodríguez ◽  
Redruello ◽  
Mayo
Keyword(s):  
Gene Cluster ◽  
Expression Patterns ◽  
Transcriptional Analysis ◽  
Whole Genome Sequence ◽  
Intestinal Bacterium ◽  
Rt Pcr ◽  
Biotechnological Production ◽  
Biosynthesis Gene Cluster ◽  
Intergenic Regions ◽  
Metabolic Transformation

Given the emerging evidence of equol’s benefit to human health, understanding its synthesis and regulation in equol-producing bacteria is of paramount importance. Adlercreutzia equolifaciens DSM19450T is a human intestinal bacterium —for which the whole genome sequence is publicly available— that produces equol from the daidzein isoflavone. In the present work, daidzein (between 50 to 200 μM) was completely metabolized by cultures of A. equolifaciens DSM19450T after 10 h of incubation. However, only about one third of the added isoflavone was transformed into dihydrodaidzein and then into equol. Transcriptional analysis of the ORFs and intergenic regions of the bacterium’s equol gene cluster was therefore undertaken using RT-PCR and RT-qPCR techniques with the aim of identifying the genetic elements of equol biosynthesis and its regulation mechanisms. Compared to controls cultured without daidzein, the expression of all 13 contiguous genes in the equol cluster was enhanced in the presence of the isoflavone. Depending on the gene and the amount of daidzein in the medium, overexpression varied from 0.5- to about 4-log10 units. Four expression patterns of transcription were identified involving genes within the cluster. The genes dzr, ddr and tdr, which code for daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase respectively, and which have been shown involved in equol biosynthesis, were among the most strongly expressed genes in the cluster. These expression patterns correlated with the location of four putative ρ-independent terminator sequences in the cluster. All the intergenic regions were amplified by RT-PCR, indicating the operon to be transcribed as a single RNA molecule. These findings provide new knowledge on the metabolic transformation of daidzein into equol by A. equolifaciens DSM19450T, which might help in efforts to increase the endogenous formation of this compound and/or its biotechnological production.

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 Fimbrial ghf Gene Cluster of Genital Strains of Haemophilus spp.

2002 ◽  
Vol 70 (10) ◽  
pp. 5438-5445 ◽  
Author(s):  
Guillaume Bruant ◽  
Nathalie Gousset ◽  
Roland Quentin ◽  
Agnes Rosenau
Keyword(s):  
Gene Cluster ◽  
Chromosome Analysis ◽  
Gene Clusters ◽  
Large Deletion ◽  
Gene Promoters ◽  
Rt Pcr ◽  
Divergent Promoters ◽  
Intergenic Regions ◽  
Flanking Regions ◽  
Palindromic Sequences

ABSTRACT We analyzed the LKP fimbrial gene clusters of six piliated strains of a cryptic genospecies of Haemophilus isolated from the genital tracts of adult patients (five strains) and from an infected neonate. In a group of 19 genital strains, LKP-like genes have been found in only these 6 strains. In addition to the ghfA, ghfD, and ghfE genes previously described, we characterized two genes, designated ghfB and ghfC, encoding the putative chaperone and assembly platform proteins. All six strains had a complete and unique LKP-like gene cluster consisting of the five genes ghfA to ghfE, homologous to genes hifA to hifE of Haemophilus influenzae. The sequences of the coding and intergenic regions of the ghf clusters of the six strains were remarkably homologous. Unlike hif clusters, which are inserted between purE and pepN, the ghf cluster was inserted between purK and pepN on the chromosome. Analysis of the flanking regions of the ghf cluster identified a large deletion, identical in the 5′ end regions of all strains, including the whole purE gene and much of the purK gene. Ultrastructural observations, an attempt at enriching LKP fimbriae, and hemagglutination experiments demonstrated that none of the strains had LKP-type fimbriae. Nevertheless, reverse transcription (RT)-PCR showed that ghf genes were transcribed in four of the six strains. Sequencing of the intergenic ghfA-ghfB regions, including the ghf gene promoters, showed that the absence of transcripts in the remaining two strains was due to a decrease in the number of TA repeats (4 or 9 repeats rather than 10) between the −10 and −35 boxes of the two overlapping and divergent promoters. The other four strains, which had ghf transcripts, had the optimal 10 TA repeats (one strain) or 5 repeats associated with putative alternative −35 boxes (three strains). The absence of 10 repeated palindromic sequences of 44 or 45 nucleotides upstream of ghfB induces an increased instability of mRNA, as quantified by real-time RT-PCR, and may explain why the LKP fimbrial gene cluster is not expressed in these strains.

scholarly journals Analysis of a DtxR-Regulated Iron Transport and Siderophore Biosynthesis Gene Cluster in Corynebacterium diphtheriae

2005 ◽  
Vol 187 (2) ◽  
pp. 422-433 ◽  
Author(s):  
Carey A. Kunkle ◽  
Michael P. Schmitt
Keyword(s):  
Gene Cluster ◽  
Iron Uptake ◽  
Iron Transport ◽  
Genetic Locus ◽  
Corynebacterium Diphtheriae ◽  
Transcriptional Analysis ◽  
Biosynthesis Gene Cluster ◽  
Iron Source ◽  
Siderophore Biosynthesis ◽  
Siderophore Transport

ABSTRACT This report describes a genetic locus associated with siderophore biosynthesis and transport in Corynebacterium diphtheriae. A BLAST search of the C. diphtheriae genome identified a seven-gene cluster that included four genes, designated ciuA, ciuB, ciuC, and ciuD, whose predicted products are related to ABC-type iron transporters. Downstream from ciuD is the ciuE gene, whose predicted product is similar to the aerobactin biosynthetic enzymes IucA and IucC. The CiuE protein, which has a predicted mass of 121,582 Da and is approximately twice the size of either IucC or IucA, is homologous to each of these proteins in both its N- and C-terminal regions. C. diphtheriae ciuE deletion mutants exhibited a defect in siderophore production, iron uptake, and growth in low-iron medium. Mutations in the ciuA gene, whose predicted product is a lipoprotein component of an iron transport system, resulted in a severe defect in iron uptake and reduced ability to use the C. diphtheriae siderophore as an iron source. Site-directed mutations in irp6A, a gene previously reported to be associated with siderophore transport, had no effect on iron uptake or the utilization of the C. diphtheriae siderophore as an iron source. Transcriptional analysis demonstrated that expression of ciuA and ciuE is DtxR and iron regulated, and DNase I protection experiments confirmed the presence of DtxR binding sites upstream from each of these genes. Thus, this iron- and DtxR-regulated gene cluster is involved in the synthesis and transport of the C. diphtheriae siderophore.

scholarly journals The Hox genes of the direct-type developing sea urchin Peronella japonica

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S73-S73
Author(s):  
Shoutaro Yamaguchi ◽  
Yuko Hano ◽  
Akane Hayashi ◽  
Masaaki Yamaguchi
Keyword(s):  
Gene Expression ◽  
Gene Cluster ◽  
Sea Urchin ◽  
Hox Genes ◽  
Expression Patterns ◽  
Spatial Patterning ◽  
Rt Pcr ◽  
Good System ◽  
Adult Rudiment ◽  
Anterior Posterior

The Hox gene cluster controls spatial patterning mechanisms along the anterior/posterior axis of bilateral metazoans. There exists a co-linearity between the order of the Hox genes in the genome and the spatial order of their domains of expression during development. Echinoderms have pentamerous radial body plans that are different from those of other deuterostomes. Thus expression patterns of echinoderm Hox genes in the adult rudiment might illuminate their evolutionary transformations from bilateral to radial structures. Since Peronella japonica is a direct-type developer that metamorphoses in 3 days without feeding, it provides a good system in which to analyse Hox gene expression in the rudiment.We PCR-amplified the Hox-type sequences of P. japonica using genome DNA and cDNA prepared from larval RNAs as a template. We used two pairs of degenerated primers that corresponded to the first and third helices of the homeodomain of the Hox genes. As a result we obtained 13 Hox-type sequences, named Pj1–Pj13 in order of their determination. All the sequences were detected by RT-PCR, suggesting that they are being expressed in larvae (Table 1).

scholarly journals Transcriptional Control by A-Factor of strR, the Pathway-Specific Transcriptional Activator for Streptomycin Biosynthesis in Streptomyces griseus

2005 ◽  
Vol 187 (16) ◽  
pp. 5595-5604 ◽  
Author(s):  
Ayami Tomono ◽  
Yisan Tsai ◽  
Haruka Yamazaki ◽  
Yasuo Ohnishi ◽  
Sueharu Horinouchi
Keyword(s):  
Gene Cluster ◽  
Transcriptional Activation ◽  
Transcriptional Control ◽  
Streptomyces Griseus ◽  
Transcriptional Activator ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Biosynthesis Gene Cluster ◽  
Regulatory Cascade ◽  
Streptomycin Biosynthesis

ABSTRACT A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) triggers streptomycin production by inducing the transcription of strR, encoding the pathway-specific transcriptional activator, through signal transduction in the A-factor regulatory cascade in Streptomyces griseus. AdpA, one of the key transcriptional activators in the cascade, bound two upstream activation sites, approximately at nucleotide positions −270 and −50 with respect to the transcriptional start point of strR, as determined by gel mobility shift assays and DNase I footprinting. Transcriptional analysis of the strR promoter with mutated AdpA-binding sites showed that both sites were required for full transcriptional activation of strR by AdpA. Potassium permanganate footprinting showed that AdpA assisted RNA polymerase in forming an open complex at an appropriate position for transcriptional initiation of strR. Nine transcriptional units within the streptomycin biosynthesis gene cluster, including the strR-aphD operon, depended on StrR, indicating that StrR is the pathway-specific transcriptional activator for the whole gene cluster. Consistent with this, expression of strR under the control of a constitutively expressed promoter in an adpA null mutant caused the host to produce streptomycin.

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.

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