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Deleted Mutant (deleted + mutant)
Selected AbstractsCharacterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatusFEBS JOURNAL, Issue 3 20002S] ferredoxin, Redox properties, molecular interaction with a [2Fe A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6 -tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 , Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe,2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be ,275 ± 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of , 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe,2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA -deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions. [source] Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfAMOLECULAR MICROBIOLOGY, Issue 6 2003Eliane Milohanic Summary PrfA is the major regulator of Listeria virulence gene expression. This protein is a member of the Crp/Fnr family of transcription regulators. To gain a deeper understanding of the PrfA regulon, we constructed a whole-genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild-type EGDe and a prfA -deleted mutant (EGDe ,prfA). Both strains were grown at 37°C in brain,heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA. We identified three groups of genes that are regulated differently. Group I comprises, in addition to the 10 already known genes, two new genes, lmo2219 and lmo0788, both positively regulated and preceded by a putative PrfA box. Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes (lmo0178,lmo0184) are organized in an operon. Group III comprises 53 genes, of which only two (lmo0596 and lmo2067) are preceded by a putative PrfA box. Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes. In the presence of cellobiose, all the group I genes were downregulated, whereas group III genes remained fully activated. Group II genes were repressed in all conditions tested. A comparison of the expression profiles between a second L. monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14prfA*) and its corresponding prfA -deleted mutant (P14,prfA) and the EGDe strain revealed interesting strain-specific differences. Sequences strongly similar to a sigma B-dependent promoter were identified upstream of 22 group III genes. These results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probably expressed from a sigma A-dependent promoter. In contrast, a second set of PrfA-regulated genes lack a PrfA box and are expressed from a sigma B-dependent promoter. This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors. [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] Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentationBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2005Ying Zhu Abstract Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta -deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose. © 2005 Wiley Periodicals, Inc. [source] | ||||||||||