[Transcriptional regulation of aco gene cluster in Bacillus thuringiensis].
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We analyzed the transcriptional regulation of aco gene cluster and the phenotype of acoR mutant, to determine the effect of acoR deletion on sporulation efficiency and Cry protein production.Sequence of aco gene cluster in Bacillus thuringiensis was analyzed by sequence alignment. RT-PCR was carried out to reveal the transcriptional units of the aco gene cluster. acoR insertion mutant was constructed by homologous recombination. Transcriptional activity was analyzed by promoter fusions with lacZ gene. Comparison of the Cry1Ac protein production was determined by protein quantitation.The aco gene cluster was composed of four genes. The acoABCL formed one transcriptional unit. The transcriptional activity of acoA promoter sharply decreased in sigL and acoR mutants, respectively. Deletion of acoR had no effect on growth and Cry protein production, but decreased the motility of cells and sporulation efficiency.The aco gene cluster is controlled by Sigma 54 and activated by AcoR. Deletion of acoR has no effect on Cry protein production, but decreased the motility of the cells.Keywords:
Gene cluster
Bacillus thuringiensis
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ABSTRACT Pseudomonas azelaica HBP1 degrades the toxic substance 2-hydroxybiphenyl (2-HBP) by means of three enzymes that are encoded by structural genes hbpC , hbpA , and hbpD . These three genes form a small noncontiguous cluster. Their expression is activated by the product of regulatory gene hbpR , which is located directly upstream of the hbpCAD genes. The HbpR protein is a transcription activator and belongs to the so-called XylR/DmpR subclass within the NtrC family of transcriptional activators. Transcriptional fusions between the different hbp intergenic regions and the luxAB genes of Vibrio harveyi in P. azelaica and in Escherichia coli revealed the existence of two HbpR-regulated promoters; one is located in front of hbpC , and the other one is located in front of hbpD . Northern analysis confirmed that the hbpC and hbpA genes are cotranscribed, whereas the hbpD gene is transcribed separately. No transcripts comprising the entire hbpCAD cluster were detected, indicating that transcription from P hbpC is terminated after the hbpA gene. E. coli mutant strains lacking the structural genes for the RNA polymerase ς 54 subunit or for the integration host factor failed to express bioluminescence from P hbpC - and P hbpD -luxAB fusions when a functional hbpR gene was provided in trans . This pointed to the active role of ς 54 and integration host factor in transcriptional activation from these promoters. Primer extension analysis revealed that both P hbpC and P hbpD contain the typical motifs at position −24 (GG) and −12 (GC) found in ς 54 -dependent promoters. Analysis of changes in the synthesis of the hbp mRNAs, in activities of the 2-HBP pathway enzymes, and in concentrations of 2-HBP intermediates during the first 4 h after induction of continuously grown P. azelaica cells with 2-HBP demonstrated that the specific transcriptional organization of the hbp genes ensured smooth pathway expression.
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Sigma factor
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The Bacillus subtilis chromosomal locus that contains the genes encoding the menaquinone biosynthetic enzymes (the men genes) was cloned by using an integrable plasmid vector. The men cluster was reconstituted on three overlapping recombinant plasmids, and a tentative gene order was derived. Evaluations of the direction of transcription and of transcriptional boundaries suggested that the men genes are expressed in the form of at least one polycistronic message. In addition, a spectrum of Men phenotypes resulting from the integration of different internal fragments of the cluster indicate transcriptional complexities, possibly including an internal promoter. The size of cloned DNA fragments required to encompass the transcription unit, as well as the locations of known men mutations within these fragments, suggests that a gene(s) not previously identified with the men system is also located within the cluster. The cloned men sequences make available probes to examine the patterns of transcription from the men locus in response to changing environmental conditions and during the developmental sequence represented by endospore formation.
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Cloning (programming)
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In a screen for morphological mutants from the T1 generation of approximately 50 000 activation-tagging lines, we isolated four dominant mutants that showed hyponastic leaves, downward-pointing flowers and decreased apical dominance. We designated them isoginchaku (iso). The iso-1D and iso-2D are allelic mutants caused by activation of the AS2 gene. The T-DNAs were inserted in the 3' downstream region of AS2. Iso-3D and iso-4D are the other allelic mutants caused by activation of the ASL1/LBD36 gene. These two genes belong to the AS2 family that is composed of 42 genes in Arabidopsis. The only recessive mutation isolated from this gene family was of AS2, which resulted in a leaf morphology mutant. Applying reverse genetics using a database of activation-tagged T-DNA flanking sequences, we found a dominant mutant that we designated peacock1-D (pck1-D) in which the ASL5/LBD12 gene was activated by a T-DNA. The pck1-D mutants have lost apical dominance, have epinastic leaves and are sterile. These results strongly suggest that activation tagging is a powerful mutant-mining tool especially for genes that make up a gene family.
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We analyzed the transcriptional regulation of aco gene cluster and the phenotype of acoR mutant, to determine the effect of acoR deletion on sporulation efficiency and Cry protein production.Sequence of aco gene cluster in Bacillus thuringiensis was analyzed by sequence alignment. RT-PCR was carried out to reveal the transcriptional units of the aco gene cluster. acoR insertion mutant was constructed by homologous recombination. Transcriptional activity was analyzed by promoter fusions with lacZ gene. Comparison of the Cry1Ac protein production was determined by protein quantitation.The aco gene cluster was composed of four genes. The acoABCL formed one transcriptional unit. The transcriptional activity of acoA promoter sharply decreased in sigL and acoR mutants, respectively. Deletion of acoR had no effect on growth and Cry protein production, but decreased the motility of cells and sporulation efficiency.The aco gene cluster is controlled by Sigma 54 and activated by AcoR. Deletion of acoR has no effect on Cry protein production, but decreased the motility of the cells.
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Bacillus thuringiensis
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The transcriptional organization and regulation of region 1 expression of the Escherichia coli K5 capsule gene cluster were studied. Region 1 was transcribed as an 8.0-kb polycistronic mRNA which was processed to form a separate 1.3-kb transcript encoding the 3'-most gene kpsS. Transcription of region 1 of the E. coli K5 capsule gene cluster was directed from a single promoter 225 bp upstream of a previously unidentified gene, kpsF. The promoter had -35 and -10 consensus sequences typical of an E. coli sigma 70 promoter, with no similarities to binding sites for other sigma factors. Two integration host factor (IHF) binding site consensus sequences were identified 110 bp upstream and 130 bp downstream of the transcription start site. In addition, two AT-rich regions separated by 16 bp identified upstream of the region 1 promoter were conserved upstream of the region 3 promoter. The kpsF gene was 98.8% identical with the kpsF gene identified in the E. coli K1 antigen gene cluster and confirms that the kpsF gene is conserved among group II capsule gene clusters. An intragenic Rho-dependent transcriptional terminator was discovered within the kpsF gene. No essential role for KpsF in the expression of the K5 antigen could be established. The temperature regulation of region 1 expression was at the level of transcription, with no transcription detectable in cells grown at 18 degrees C. Mutations in regulatory genes known to control temperature-dependent expression of a number of virulence genes had no effect on the temperature regulation of region 1 expression. Likewise, RfaH, which is known to regulate expression of E. coli group II capsules had no effect on the expression of region 1. Mutations in the himA and himD genes which encode the subunits of the IHF led to a fivefold reduction in the expression of KpsE at 37 degrees C, confirming a regulatory role for IHF in the expression of region 1 genes.
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Terminator (solar)
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Bacterial alginate genes are chromosomal and fairly widespread among rRNA homology group I Pseudomonads and Azotobacter. In both genera, the genetic pathway of alginate biosynthesis is mostly similar and the identified genes are identically organized into biosynthetic, regulatory and genetic switching clusters. In spite of these similarities, still there are transcriptional and functional variations between P. aeruginosa and A. vinelandii. In P. aeruginosa all biosynthetic genes except algC transcribe in polycistronic manner under the control of algD promoter while in A. vinelandii, these are organized into many transcriptional units. Of these, algA and algC are transcribed each from two different and algD from three different promoters. Unlike P. aeruginosa, the promoters of these transcriptional units except one of algC and algD are algT-independent. Both bacterial species carry homologous algG gene for Ca2+-independent epimerization. But besides algG, A. vinelandii also has algE1-7 genes which encode C-5-epimerases involved in the complex steps of Ca2+-dependent epimerization. A hierarchy of alginate genes expression under 22(algT) control exists in P. aeruginosa where algT is required for transcription of the response regulators algB and algR, which in turn are necessary for expression of algD and its downstream biosynthetic genes. Although algTmucABCD genes cluster play similar regulatory roles in both P. aeruginosa and A. vinelandii but unlike, transcription of A. vinelandii, algR is independent of 22. These differences could be due to the fact that in A. vinelandii alginate plays a role as an integrated part in desiccation-resistant cyst which is not found in P. aeruginosa. Keywords: Pseudomonads, Azotobacter, alginate genes, regulation, biosynthesis
Azotobacter vinelandii
Sigma factor
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Gene cluster
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Sigma factor
Salmonella enterica
Transcription
Specificity factor
SOS response
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Abstract To understand the basis of broad-spectrum disease resistance in rice, we isolated a gamma-ray-induced IR64 mutant G978 that showed enhanced resistance to blast and bacterial blight. The resistance is quantitative and non-race specific against the bacterial and fungal pathogens. The mutation is inherited as a single recessive gene, designated as Bsdr 1 and causes shorter stature relative to the wild type; however, it does not show lesion mimics phenotype under the conditions tested. The mutation was mapped as a quantitative trait locus to a 3.8-Mb region on chromosome 12. By comparing the gene expression profiles of the mutant and wild type, we identified a candidate gene encoding a U-box domain-containing protein. The disrupted gene showed a loss of expression in the mutant and co-segregated with mutant phenotype. The mutant provides a useful tool for investigating the important genes responsible for non-race specific resistance to two distinct diseases.
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Bacillus thuringiensis
Transcription
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Bacillus thuringiensis subspecies morrisoni strain HD12, whose genome harbors multiple insecticidal protein-encoding genes, includes eight cry genes, as indicated by genome sequencing. This strain produces crystals that are toxic to lepidopteran species. These crystal inclusions were purified by sucrose gradients and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by liquid chromatography-mass spectrometry, and found to contain five proteins (Cry1Da, Cry1Ae, Cry1Bb, Cry1Fb, and Cry1Ja). The transcriptional activities of the cry1Da, cry1Ae, cry1Bb, cry1Fb, and cry1Ja promoters indicated that transcription of cry1Da is controlled by SigE; however, the other four cry genes were found to be controlled by both SigE and SigK. The activities of the cry1Ja and cry1Fb promoters were the strongest among the five genes studied. These promoters were therefore used to direct the expression of the Cry1Ac- and Cry2Ab-encoding genes concurrently in a single strain. Our findings indicate that promoters with the same transcriptional profile may be used to direct the expression of different cry genes in one strain. Our results are expected to be valuable for the construction of strains with efficient expression of multiple cry genes in order to overcome current limitations associated with the application of B. thuringiensis-based insecticides.
Bacillus thuringiensis
Cry1Ac
Strain (injury)
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