Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate reduction

An electron capture gas-chromatographic technique was developed to continuously measure nitrate (NO3-) reduction during in vitro complementation tests with extracts from Pseudomonas aeruginosa mutants deficient in both assimilatory and dissimilatory nitrate reduction as a result of a single genetic mutation. The procedure involves coupling nitrate reduction to nitrous oxide (N2O) evolution via a series of reactions specific to the denitrification pathway. The assay was dependent on nitrate concentration, and optimal activity was obtained with a final concentration of 0.2% potassium nitrate. The reduction exhibited a stoichiometry of 2:1 (NO3-/N2O), and succinate was the best electron source for the reaction, followed by glucose, pyruvate, and malate. The technique can also be used for continuously monitoring nitrate reduction. The optimal nitrite concentration in the nitrite reductase assay was 0.025%. The initial complementation studies of mutant extracts demonstrated that at least two genes are shared between the two nitrate reduction pathways in P. aeruginosa.     

Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate reduction.

An electron capture gas-chromatographic technique was developed to continuously measure nitrate (NO3-) reduction during in vitro complementation tests with extracts from Pseudomonas aeruginosa mutants deficient in both assimilatory and dissimilatory nitrate reduction as a result of a single genetic mutation. The procedure involves coupling nitrate reduction to nitrous oxide (N2O) evolution via a series of reactions specific to the denitrification pathway. The assay was dependent on nitrate concentration, and optimal activity was obtained with a final concentration of 0.2% potassium nitrate. The reduction exhibited a stoichiometry of 2:1 (NO3-/N2O), and succinate was the best electron source for the reaction, followed by glucose, pyruvate, and malate. The technique can also be used for continuously monitoring nitrate reduction. The optimal nitrite concentration in the nitrite reductase assay was 0.025%. The initial complementation studies of mutant extracts demonstrated that at least two genes are shared between the two nitrate reduction pathways in P. aeruginosa.     

CRISPR-Cas systems restrict horizontal gene transfer in Pseudomonas aeruginosa

CRISPR-Cas systems provide bacteria and archaea with an adaptive immune system that targets foreign DNA. However, the xenogenic nature of immunity provided by CRISPR-Cas raises the possibility that these systems may constrain horizontal gene transfer. Here we test this hypothesis in the opportunistic pathogen Pseudomonas aeruginosa, which has emerged as an important model system for understanding CRISPR-Cas function. Across the diversity of P. aeruginosa, active CRISPR-Cas systems are associated with smaller genomes and higher GC content, suggesting that CRISPR-Cas inhibits the acquisition of foreign DNA. Although phage is the major target of CRISPR-Cas spacers, more than 80% of isolates with an active CRISPR-Cas system have spacers that target integrative conjugative elements (ICE) or the conserved conjugative transfer machinery used by plasmids and ICE. Consistent with these results, genomes containing active CRISPR-Cas systems harbour a lower abundance of both prophage and ICE. Crucially, spacers in genomes with active CRISPR-Cas systems map to ICE and phage that are integrated into the chromosomes of closely related genomes lacking CRISPR-Cas immunity. We propose that CRISPR-Cas acts as an important constraint to horizontal gene transfer, and the evolutionary mechanisms that ensure its maintenance or drive its loss are key to the ability of this pathogen to adapt to new niches and stressors.Subject terms: Microbiology, Microbial genetics, Evolution     

Evidence for polyadenylated mRNA in Pseudomonas aeruginosa

In this paper, we report the synthesis of Pseudomonas aeruginosa cDNA in the presence of oligo(dT) primers. Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from P. aeruginosa PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.     

Evidence for phage-mediated gene transfer among Pseudomonas aeruginosa strains on the phylloplane.

As the use of genetically engineered microorganisms for agricultural tasks becomes more frequent, the ability of bacteria to exchange genetic material in the agricultural setting must be assessed. Transduction (bacterial virus-mediated horizontal gene transfer) is a potentially important mechanism of gene transfer in natural environments. This study investigated the potential of plant leaves to act as surfaces on which transduction can take place among microorganisms. Pseudomonas aeruginosa and its generalized transducing bacteriophage F116 were used as a model system. The application of P. aeruginosa lysogens of F116 to plant leaves resulted in genetic exchange among donor and recipient organisms resident on the same plant. Transduction was also observed when these bacterial strains were inoculated onto adjacent plants and contact was made possible through high-density planting.     

Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa

Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa. Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen. The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition. The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation. The inhibition appeared to be specific for nitrate uptake. Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen. The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P. aeruginosa.     

Evidence for small pores in the outer membrane of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa NCTC6750 and Escherichia coli K12 were used to study permeability of whole, intact cells to a series of labelled oligosaccharides. Stationary phase, oxygen depleted simple salts batch cultures were used. An efflux method was used to compare diffusion from cells of various ³H-labelled sugars (an homologous series based on isomaltitol) with diffusion of [¹⁴C]sucrose. Both plasmolysed and unplasmolysed cell suspensions were used. The data are consistent with an E. coli pore exclusion limit of approx. 833 Da for unplasmolysed cells and of about 670 Da for plasmolysed cells. For P. aeruginosa the data indicated a relatively small pore exclusion limit about the same size as sucrose with plasmolysis having little effect. These findings were confirmed with P. aeruginosa PAO1 grown in nutrient broth.     

Evidence for the location of OprM in the Pseudomonas aeruginosa outer membrane

Abstract OprM with a M _r of 49 K is associated with the multidrug resistance of Pseudomonas aeruginosa . Detergent fractionation of bacterial cells has demonstrated that OprM is located in the outer membrane from which it sediments with the other major outer membrane proteins. In this study we have determined the location of OprM as the P. aeruginosa outer membrane. Western immunoblots of cell fractions, obtained by sucrose density gradient centrifugation of whole cell lysates, were probed with an OprM-specific murine polyclonal antiserum.     

Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa.

Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa. Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen. The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition. The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation. The inhibition appeared to be specific for nitrate uptake. Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen. The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P. aeruginosa.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R', previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.     

Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa

Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM. Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM). Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.