Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity. (9/1206)

Melittin, the 26-residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C-terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5-7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been transpositioned to the N-terminal region from the C-terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.  (+info)

In vitro transcriptional studies of the bkd operon of Pseudomonas putida: L-branched-chain amino acids and D-leucine are the inducers. (10/1206)

BkdR is the transcriptional activator of the bkd operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of Pseudomonas putida. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5' region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the bkd operon was obtained by using a vector containing the bkdR-bkdA1 intergenic region plus the putative rho-independent terminator of the bkd operon. Substrate DNA, BkdR, and any of the L-branched-chain amino acids or D-leucine was required for transcription. Branched-chain keto acids, D-valine, and D-isoleucine did not promote transcription. Therefore, the L-branched-chain amino acids and D-leucine are the inducers of the bkd operon. The concentration of L-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the bkd operon by BkdR during enzyme induction which incorporates these results is presented.  (+info)

NahY, a catabolic plasmid-encoded receptor required for chemotaxis of Pseudomonas putida to the aromatic hydrocarbon naphthalene. (11/1206)

Pseudomonas putida G7 exhibits chemotaxis to naphthalene, but the molecular basis for this was not known. A new gene, nahY, was found to be cotranscribed with meta cleavage pathway genes on the NAH7 catabolic plasmid for naphthalene degradation. The nahY gene encodes a 538-amino-acid protein with a membrane topology and a C-terminal region that resemble those of chemotaxis transducer proteins. A P. putida G7 nahY mutant grew on naphthalene but was not chemotactic to this aromatic hydrocarbon. The protein NahY thus appears to function as a chemoreceptor for naphthalene or a related compound. The presence of nahY on a catabolic plasmid implies that chemotaxis may facilitate biodegradation.  (+info)

The IIANtr (PtsN) protein of Pseudomonas putida mediates the C source inhibition of the sigma54-dependent Pu promoter of the TOL plasmid. (12/1206)

The gene cluster adjacent to the sequence of rpoN (encoding sigma factor sigma54) of Pseudomonas putida has been studied with respect to the C source regulation of the Pu promoter of the upper TOL (toluene catabolism) operon. The region includes four open reading frames (ORFs), two of which (named ptsN and ptsO genes) encode proteins similar to components of the phosphoenolpyruvate:sugar phosphotransferase system. Each of the four genes was disrupted with a nonpolar insertion, and the effects in the inhibition caused by glucose on Pu activity were inspected with a lacZ reporter system. Although cells lacking ORF102, ORF284, and ptsO did not display any evident phenotype under the conditions tested, the loss of ptsN, which encodes the IIANtr protein, made Pu unresponsive to repression by glucose. The ptsN mutant had rates of glucose/gluconate consumption identical to those of the wild type, thus ruling out indirect effects mediated by the transport of the carbohydrate. A site-directed ptsN mutant in which the conserved phospho-acceptor site His68 of IIANtr was replaced by an aspartic acid residue made Pu blind to the presence or absence of glucose, thus supporting the notion that phosphorylation of IIANtr mediates the C source inhibition of the promoter. These data substantiate the existence of a molecular pathway for co-regulation of some sigma54 promoters in which IIANtr is a key protein intermediate.  (+info)

Characterization of a Pseudomonas putida allylic alcohol dehydrogenase induced by growth on 2-methyl-3-buten-2-ol. (13/1206)

We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium Pseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of Acinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.  (+info)

Effect of organic solvents on the yield of solvent-tolerant Pseudomonas putida S12. (14/1206)

Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h-1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.  (+info)

cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type. (15/1206)

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.  (+info)

Cloning, sequence analysis, and expression of the Pseudomonas putida 33/1 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase gene, encoding a carbon monoxide forming dioxygenase. (16/1206)

1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from the 1H-4-oxoquinoline utilizing Pseudomonas putida strain 33/1, which catalyzes the cleavage of 1H-3-hydroxy-4-oxoquinoline to carbon monoxide and N-formylanthranilate, is devoid of any transition metal ion or other cofactor and thus represents a novel type of ring-cleavage dioxygenase. Gene qdo was cloned and sequenced. Its overexpression in Escherichia coli yielded recombinant His-tagged Qdo which was catalytically active. Qdo exhibited 36% and 16% amino acid identity to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) and atropinesterase (a serine hydrolase), respectively. Qdo as well as Hod possesses a SXSHG motif, resembling the motif GXSXG of the serine hydrolases which comprises the active-site nucleophile (X=arbitrary residue).  (+info)