Gene disruption mutants of B. The plasmids were transformed into Escherichia coli Top 10 Invitrogen and verified by sequencing. Plasmids harboring emrB or emrB-bif R were transferred to the B. Overnight cultures of the donor E. The mixed culture was centrifuged to remove LB and residual antibiotics.
The pellet was washed four times with 1. After overnight incubation, all cells were scraped off and resuspended in 1 mL of LB. The concentrations of H 2 O 2 and CuCl 2 were chosen based on a plate sensitivity assay in which these concentrations did not visibly impede growth data not shown. To determine the effect of redox inactive metal on gene expression, cells were exposed to ZnCl 2 and MgCl 2 at final concentrations of 1 mM and 30 mM, respectively. Cells were grown for 30 min and harvested by centrifugation.
The reference gene was selected based on its use as a reference in analysis of the transcriptional response of B. Overnight cultures were diluted in LB broth, and absorbance of all the strains was recorded at 1 h intervals. Data are representative of three replicates. Absorbance data were plotted on log 10 scale. Ten microliters of cells were spotted on an LB agar plate. Ten microliters of cells were spotted on preheated LB agar plates containing 0.
To assess if biofilms pellicles formed at the air—liquid interface, overnight cultures were diluted in 3 mL of LB medium. Conjugative plasmids were maintained using selective antibiotics.
Culture tubes were kept stationary for 72—96 h at room temperature. Pellicles were assayed by visual inspection of the air—liquid interface of culture tubes. Quantification of biofilm adherent to the surface of culture tubes was performed as described. To quantify the biofilm, absorbance was recorded at nm.
To compare growth of the stationary cultures, the absorbance of cells in liquid culture was measured, following which the pellicle was resuspended in the culture; the absorbance was again measured, and cells were plated for determination of CFU. The gene encoding BifR was amplified from B.
To create the CA substitution, an overhanging primer technique was used to amplify the whole plasmid using primers CA Fw and Rev Supplemental Table 1. The cell pellets were thawed for 1 h on ice and resuspended in chilled buffer containing 50 mM Tris-Cl pH 7. To 5 mL cell suspension, lysozyme 1.
After 1. Protein was concentrated using Amicon centrifugal filter device Millipore. Concentration was calculated using the BCA protein assay kit Pierce. For oligomeric state determination, the protein was cross-linked with 0. The protein was incubated for 30 min, and an equal volume of Laemmli sample buffer was added to terminate the reaction. To determine if oxidation of BifR occurs in vivo, Western Blots were performed.
After 30 min, 1 mM H 2 O 2 was added and cells were incubated an additional 30 min; control cultures received no H 2 O 2. The cell pellets were thawed and resuspended in 50 mM sodium phosphate buffer pH 7.
To 5 mL of cell suspension, lysozyme 1. Reactions were incubated for 2 h for lysis. Supernatants were collected after centrifugation at g for 1 h. The apparent dissociation constant K d was determined using electrophoretic mobility shift assays EMSA.
Synthetic oligonucleotides representing 57 bp emrB-bif R promoter region with the two identified palindromes at the center were purchased and purified by denaturing polyacrylamide gel electrophoresis.
DNA 0. Densitometric data were obtained with Image-Quant 5. Reactions were incubated at room temperature for 30 min and processed as described above. To assess the effect of oxidant on protein—DNA binding, protein was mixed with CuCl 2 in a ratio of and incubated on ice for 15 min. EMSA was performed with the oxidized protein, and the apparent dissociation constant was determined as described above.
Reactions were incubated for 30 min and electrophoresed as described above. The B. Many EcsC homologues are annotated as putative LasA protease Ensembl genomes family: protease domain. In Pseudomonas , disruption of quorum sensing has been correlated with reduced expression of virulence genes, including LasA protease. The entire locus is conserved in other Burkholderia species, including the pathogenic species, and Ortholuge predicts that the encoded proteins are orthologs.
Two imperfect palindromes in the intergenic region are shown bold and underlined. Inverted triangles indicate positions of transposon insertion. Inspection of the intergenic region between ecsC and emrB-bif R revealed two conserved, imperfect palindromes consisting of 8 bp half-sites, with the two palindromes separated by 3 bp Figure 1A. These palindromes are putative binding sites for BifR. As shown in Figure 1B , a product of the expected size was obtained, confirming the annotated operon.
We obtained and verified disruptant strains in which a transposon was inserted at position 15 of bif R or at position of the emrB open reading frames, respectively. Complementation with emrB only was not sufficient to restore the growth rate to wild-type levels, whereas complementation with emrB-bif R resulted in a growth rate equivalent to that of wild-type Figure 1C. Transcript levels of ecsC , emrB , and bif R were 0.
This shows that BifR functioned as a repressor of the divergent genes. Regulation of gene expression. Error bars represent standard deviation of three separate experiments each with three technical replicates. Plates contain insoluble elastin and clearing zone reflects elastin cleavage.
Having verified the role of BifR in repressing expression of ecsC and emrB-bif R , we examined its predicted properties in vitro. The model reflects the obligate MarR dimer with DNA recognition helices Figure 3A ; green positioned for interaction in consecutive DNA major grooves; all MarR homologues share this general fold, suggesting that the model is a reasonable approximation of the BifR structure despite the relatively low sequence conservation. Cross-linking of BifR with glutaraldehyde which cross-links lysine residues resulted in formation of a cross-linked species with M w corresponding to a dimer, suggesting that reduced BifR exists as a dimer Figure 3B , lane 2.
Dimer formation by BifR. A BifR model based on the structure of MarR family regulator pa 2fbh. Both monomers are colored blue to red amino-terminus to carboxy-terminus.
The cysteines in each monomer are in yellow sphere representation, with the cysteine in the left monomer toward the front of the protein black arrow and the cysteine in the right monomer facing the back gray arrow. Monomer M and dimer D identified at the right. C BifR titrated with increasing concentration of hydrogen peroxide.
D Elution of reduced BifR from size exclusion column. E Size-exclusion chromatogram of BifR previously incubated with CuCl 2 , resulting in a mixture of reduced dimer; circle and oxidized dimer-of-dimers; arrow species. F Elution of reduced and oxidized BifR from size exclusion column indicated by circle and arrow, respectively. M w of standards are indicated in kDa. To determine if protein oxidation alters physicochemical properties, reduced BifR was incubated with increasing concentrations of H 2 O 2.
That the dimeric species observed by protein oxidation migrated slightly slower than dimer obtained by glutaraldehyde cross-linking suggests a more extended conformation.
On the basis of the location of cysteines in dimeric BifR, we infer that oxidation resulted in formation of a trans -dimer between two monomers in separate BifR cis -dimers. Size exclusion chromatography was used to determine the oligomeric state of reduced and oxidized BifR. Reduced protein eluted as a single species with M w A significant presence of higher-order oligomers was not detected.
To assess the inference that dimer formation was due to disulfide bond formation, a mutant was created in which Cys was replaced with Ala BifR-CA , and the protein was purified to apparent homogeneity. To determine if BifR forms a dimer-of-dimers in vivo , expression of the His 6 -tagged protein was induced in E. BifR oxidation by copper. Yearly improvement in education projects in each region. Yearly improvements in food waste projects in each region.
Company Company. Download the Annual Report. Download the Corporate Governance Statement. Brambles Speak Up hotline Risk Management.
Download the Sustainability Review Contact us. Brambles Sustainability Framework. Strategy Promote and contribute to reforestation projects through partnership alliances and volunteering opportunities. Benefits Reforestation programs have habitat and biodiversity benefits and engage and educate our employees on the importance of sustainable forestry.
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Some forms of randomness concern hash or seach algorithms. Another task that is random concerns selecting music tracks. In statistical theory, randomization is an important principle with one possible application involving survey sampling. Many applications of randomization have caused several methods to exist for generating random data. Lottery games is one current application. Slot machine odds are another use of random number generators.
Toggle navigation Go back. Random string generator Allowed chars. Number of strings. About Random string generator tool. Random number generator. They once ruled the market with the ambassador. They again got significant numbers with the lancer. And then they sough of just gave up. Never really bothered to do anything significant towards improving the automobile industry. And their doom was inevitable.
Even if the Indian government would step in and revive HM i dont think it would do them any good. And i hope after the demise of HM , Mitsubishi can club with a good partner who would at least let us exploit the the amazing potential of Mitsubishi and its products. Originally Posted by Durango Dude. Motorbikes have an emotional quotient that a standard family car cannot achieve. Bullets were never a mass commuter vehicle but always a niche product, therefore what was essentially drawbacks of the motorcycle became classic Bullet characteristics.
A car like the Amby was never a 'niche' product. It was simply a big underpowered car that in todays age provides absolutely no benefit over its more modern competition The owners knew that HM is heading towards doom and were willing to let it sink. Never in its history can you see a serious attempt at reviving the company or creating a great new product.
I believe that the HM management simply want the company to close down and liquidate any valuable assets, especially the land where it stands. Interesting comparison. The only reason i can think of is that Bullet had absolutely no competition in the same category while Amby has only competetion.
BTW - Royal Enfield should be credited to improve the bike somewhat although not so refined, still way better than the 80's bikes. Amby too tried it - with the Ambassador Nova, but it was far too cosmetic to take effect. They introduced new engines like AVL ones. The recent Classic series is also done tastefully. Nothing of that sort was done by Ambassador. Last edited by Technocrat : 8th December at Originally Posted by arjab. Amby goes to Bangladesh!
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