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Transcriptional Fusion (transcriptional + fusion)
Selected AbstractsA new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxesENVIRONMENTAL MICROBIOLOGY, Issue 4 2006Robin Tecon Summary The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor ,Burkholderia sp. strain RP037 , produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed. [source] Antibiotics, arsenate and H2O2 induce the promoter of Staphylococcus aureus cspC gene more strongly than coldJOURNAL OF BASIC MICROBIOLOGY, Issue 2 2009Palas Kumar Chanda Abstract Proteins expressed by the bacterial cold shock genes are highly conserved at sequence level and perform various biological functions in both the cold-stressed and normal cells. To study the effects of various agents on the cold shock genes of Staphylococcus aureus, we have cloned the upstream region of cspC from S. aureus Newman and found that the above region possesses appreciable promoter (Pc) activity even at 37 °C. A reporter S. aureus strain CHANDA2, constructed by inserting the Pc - lacZ transcriptional fusion into S. aureus RN4220 genome, was found to express very low level of , -galactosidase after cold shock, indicating that low temperature induces Pc very weakly. Interestingly, transcription from Pc was induced very strongly by several antibiotics, hydrogen peroxide and arsenate salt. Cold shock proteins expressed by S. aureus are highly identical at sequence level and bear single-strand nucleic acid binding motifs. A 16 nt downstream box and a 13 nt upstream box were identified at the downstream of initiation codon and at the upstream of ribosome binding site of csp transcripts. Their roles in S. aureus cold shock gene expression have been discussed elaborately. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Activity of Serratia plymuthica IC1270 gene chiA promoter region in Escherichia coli mutants deficient in global regulators of transcriptionJOURNAL OF BASIC MICROBIOLOGY, Issue 6 2005I. A. Khmel To study the regulation of expression of the Serratia plymuthica gene chiA encoding a 58-kDa endochitinase, its 586-bp-long upstream regulatory region was cloned, sequenced and fused to a promoterless lac operon in phage ,RS45 to obtain a single-copy transcriptional fusion (PF1chiA - lac ) in lysogens of Escherichia coli wild-type strains or their mutants deficient in various global regulators of transcription. The level of PF1chiA - lac expression increased about 20- and 90-fold, respectively, in E. coli K12 ,hns and double ,hns stpA mutants deficient in H-NS, and in both H-NS and StpA DNA-binding histone-like proteins, as compared to levels in the wild-type strain. In a ,lrp mutant deficient in the leucine-responsive transcriptional regulator Lrp, the level of PF1chiA - lac expression increased only up to threefold, whereas even smaller differences relative to the wild-type strain were observed in rpoS and ,crp mutants deficient in the ,S subunit of RNA polymerase and catabolite-repression protein (CRP), respectively. Deletion of the inverted-repeat sequences and curved DNA regions located in the upstream region of chiA essentially did not influence strain IC1270's chiA promoter activity in E. coli . (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] The tetraspanin BcPls1 is required for appressorium-mediated penetration of Botrytis cinerea into host plant leavesMOLECULAR MICROBIOLOGY, Issue 3 2004M. Gourgues Summary Animal tetraspanins are membrane proteins controlling cell adhesion, morphology and motility. In fungi, the tetraspanin MgPls1 controls an appressorial function required for the penetration of Magnaporthe grisea into host plants. An orthologue of MgPLS1, BcPLS1, was identified in the necrotrophic fungal plant pathogen Botrytis cinerea. We constructed a Bcpls1::bar null mutant by targeted gene replacement. Bcpls1::bar is not pathogenic on intact plant tissues of bean, tomato or rose, but it infects wounded plant tissues. Both wild type and Bcpls1::bar differentiate appressoria on plant and artificial surfaces, a process involving an arrest of polarized growth, apex swelling and its cell wall reinforcement. Although wild-type appressoria allowed the penetration of the fungus into the host plant within 6,12 h, no successful penetration events were observed with Bcpls1::bar, suggesting that its appressoria are not functional. An eGFP transcriptional fusion showed that BcPLS1 was specifically expressed in conidia, germ tubes and appressoria during host penetration. Our results indicate that BcPLS1 is required for the penetration of B. cinerea into intact host plants. The defect in pathogenicity of Bcpls1::bar also demonstrates that functional B. cinerea appressoria are required for a successful penetration process. As Bcpls1::bar and Mgpls1,::hph penetration defects are similar, fungal tetraspanins are likely to be required for an essential appressorial function widespread among fungi. [source] Environmental regulation of recA gene expression in Porphyromonas gingivalisMOLECULAR ORAL MICROBIOLOGY, Issue 3 2001Y. Liu The recA gene product in Porphyromonas gingivalis is involved in DNA repair. Further, disruption of this gene can affect the proteolytic activity and expression of other virulence factors in this organism. Since several known environmental factors can influence virulence gene expression in P. gingivalis, we investigated the influence of these signals on the expression of the recA gene in this organism. A heterodiploid strain of P. gingivalis (designated FLL118) containing a transcriptional fusion of the recA promoter region and the promoterless tetracycline-resistant gene [tetA(Q)2] and xylosidase/arabinosidase (xa) gene cassette was constructed. The recA promoter activity was assessed by measurement of xylosidase activity in FLL118. The expression remained relatively constant during different growth phases, at different pH levels and in the presence of DNA-damaging agents. In response to hemin limitation and in the presence of calcium there was a moderate increase in recA promoter activity. Temperature also affected the expression. The highest level of xylosidase activity was observed in cultures at 32°C with a decline of approximately 46% as growth temperature increased to 41°C. Reverse transcriptase polymerase chain reaction analysis revealed that this regulation may be occurring at the transcriptional level. These results suggest that expression of the recA gene in P. gingivalis W83 is responsive to several environmental signals but is not regulated by a DNA damage,inducible SOS-like regulatory system. [source] Use of a whole-cell biosensor to assess the bioavailability enhancement of aromatic hydrocarbon compounds by nonionic surfactantsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2008Angela Keane Abstract The whole-cell bioluminescent biosensor Pseudomonas putida F1G4 (PpF1G4), which contains a chromosomally-based sep-lux transcriptional fusion, was used as a tool for direct measurement of the bioavailability of hydrophobic organic compounds (HOCs) partitioned into surfactant micelles. The increased bioluminescent response of PpF1G4 in micellar solutions (up to 10 times the critical micellar concentration) of Triton X-100 and Brij 35 indicated higher intracellular concentrations of the test compounds, toluene, naphthalene, and phenanthrene, compared to control systems with no surfactants present. In contrast, Brij 30 caused a decrease in the bioluminescent response to the test compounds in single-solute systems, without adversely affecting cell growth. The decrease in bioluminescent response in the presence of Brij 30 did not occur in the presence of multiple HOCs extracted into the surfactant solutions from crude oil and creosote. The effect of the micellar solutions on the toluene biodegradation rate was consistent with the bioluminescent response in single-solute systems. None of the surfactants were toxic to PpF1G4 at the doses employed in this study, and PpF1G4 did not produce a bioluminescent response to the surfactants nor utilize them as growth substrates. TEM images suggest that the surfactants did not rupture the cell membranes. The results demonstrate that for Pseudomonas putida F1, nonionic surfactants such as Triton X-100 and Brij 35, at doses between 2 and 10 CMC, may increase the bioavailability and direct uptake of micellar phase HOCs that are common pollutants at contaminated sites. Biotechnol. Bioeng. 2008;99: 86,98. © 2007 Wiley Periodicals, Inc. [source] A novel sensor kinase,response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosaMOLECULAR MICROBIOLOGY, Issue 4 2004Michelle A. Laskowski Summary The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced by contact with eukaryotic cells and by growth in low-calcium media. We have identified a protein, RtsM, that is necessary for expression of the TTSS genes in P. aeruginosa. RtsM possesses both histidine kinase and response regulator domains common to two-component signalling proteins, as well as a large predicted periplasmic domain and seven transmembrane domains. Deletion of rtsM resulted in a defect in production and secretion of the type III effectors. Northern blot analysis revealed that mRNAs encoding the effectors ExoT and ExoU are absent in the ,rtsM strain under TTSS-inducing conditions. Using transcriptional fusions, we demonstrated that RtsM is required for transcription of the operons encoding the TTSS effectors and apparatus in response to calcium limitation or to host cell contact. The operon encoding the TTSS regulator ExsA does not respond to calcium limitation, but the basal transcription rate of this operon was lower in ,rtsM than in the wild-type parent, PA103. The defect in TTSS effector production and secretion of ,rtsM could be complemented by overexpressing ExsA or Vfr, two transcriptional activators involved in TTSS regulation. ,rtsM was markedly less virulent than PA103 in a murine model of acute pneumonia, demonstrating that RtsM is required in vivo. We propose that RtsM is a sensor protein at the start of a signalling cascade that induces expression of the TTSS in response to environmental signals. [source] DNA damage induction of recA in Mycobacterium tuberculosis independently of RecA and LexAMOLECULAR MICROBIOLOGY, Issue 3 2002Elaine O. Davis Summary The ubiquitous and highly conserved RecA protein is generally expressed from a single promoter, which is regulated by LexA in conjunction with RecA. We show here using transcriptional fusions to a reporter gene that the Mycobacterium tuberculosis recA gene is expressed from two promoters. Although one promoter is clearly regulated in the classical way, the other remains DNA damage inducible in the absence of RecA or when LexA binding is prevented. These observations demonstrate convincingly for the first time that there is a novel mechanism of DNA damage induction in M. tuberculosis that is independent of LexA and RecA. [source] A novel NO-responding regulator controls the reduction of nitric oxide in Ralstonia eutrophaMOLECULAR MICROBIOLOGY, Issue 3 2000Anne Pohlmann Ralstonia eutropha H16 mediates the reduction of nitric oxide (NO) to nitrous oxide (N2O) with two isofunctional single component membrane-bound NO reductases (NorB1 and NorB2). This reaction is integrated into the denitrification pathway that involves the successive reduction of nitrate to dinitrogen. The norB1 gene is co-transcribed with norA1 from a ,54 (RpoN)-dependent promoter, located upstream of norA1. With the aid of norA1,,lacZ transcriptional fusions and the generation of regulatory mutants, it was shown that norB1 gene transcription requires a functional rpoN gene and the regulator NorR, a novel member of the NtrC family of response regulators. The regulator gene maps adjacent to norAB, is divergently transcribed and present in two copies on the megaplasmid pHG1 (norR1) and the chromosome (norR2). Transcription activation by NorR responds to the availability of NO. A nitrite reductase-deficient mutant that is incapable of producing NO endogenously, showed a 70% decrease of norA1 expression. Addition of the NO-donating agent sodium nitroprusside caused induction of norA1,,lacZ transcription. Truncation of the N-terminal receiver domain of NorR1 interrupted the NO signal transduction and led to a constitutive expression of norA1,,lacZ. The results indicate that NorR controls the reductive conversion of NO in R. eutropha. This reaction is not strictly co-ordinated on the regulatory level with the other nitrogen oxide-reducing steps of the denitrification chain that are independent of NorR. [source] CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenesMOLECULAR MICROBIOLOGY, Issue 4 2000Shamila Nair Stress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens. In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth. The first gene of the clpC operon of L. monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes. An L. monocytogenes ctsR -deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42°C), but its level of virulence in the mouse was not affected. The virulence of a wild-type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response. Regulation of the L. monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B. subtilis as a host. The L. monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B. subtilis. The purified CtsR protein of L. monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting. Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B. subtilis. This is the first description of a stress response regulatory gene in a pathogen. [source] Graft transmission of induced and spontaneous post-transcriptional silencing of chitinase genesTHE PLANT JOURNAL, Issue 5 2001Patrice Crété Summary Sense and antisense tobacco chitinase (CHN) transgenes, Luciferase-CHN transcriptional fusions, and promoterless CHN cDNAs were introduced biolistically into CHN transformants of tobacco that never exhibit spontaneous gene silencing. All of the constructs tested induced systemic silencing of the resident CHN transgene and endogenes. Nuclear run-on transcription assays showed that local introduction of additional gene copies triggers systemic post-transcriptional gene silencing (PTGS). Together, this provides evidence that additional transgene copies need not be either highly transcribed or produce sense transcripts to evoke production of systemic PTGS signals. CHN PTGS was transmitted by top grafting, but not by reciprocal grafting of mature stems or the exchange of tissue plugs. Thus, the commonly encountered difficulties in achieving graft-transmission could reflect the method used. Silencing in sense but not antisense transformants was transmitted by grafting to a high-expressing sense CHN scion suggesting that the elaboration of mobile signals may not be an essential feature of antisense-mediated gene silencing. [source] | ||||||||||