snr-1 gene is required for nitrate reduction in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is able to use nitrate for both assimilation and anaerobic respiration. One set of genes, designated snr (for "shared nitrate reduction"), have been recently cloned and partially characterized. In this study, we demonstrate that the snr-1 gene encodes a predicted 52.5-kDa protein that is 82% similar to a unique cytochrome c of Desulfomonile tiedjei DCB-1. Importantly, the Snr-1 protein sequence of P. aeruginosa differed from that of the cytochrome c of D. tiedjei primarily in the first 25 amino acids, which are required for membrane attachment in D. tiedjei. In P. aeruginosa, the Snr-1 protein hydropathy profile indicates that it is a soluble protein. An isogenic snr-1::Gm insertional mutant was unable to grow aerobically with nitrate as a sole nitrogen source or anaerobically with nitrate as an electron acceptor. Complementation of the snr-1::Gm mutant with the snr-1 gene restored the wild-type phenotypes. Interestingly, anaerobic growth rates were significantly higher in the snr-1 mutant harboring a multicopy plasmid containing snr-1. In contrast, aerobic growth rates of the restored mutant using nitrate as the sole nitrogen source were similar to those of the wild type. Transcriptional lacZ fusions demonstrated that snr-1 was not regulated by molybdate, oxygen, or nitrate.

Snr, new genetic loci common to the nitrate reduction systems of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is able to both assimilate and dissimilate nitrate. On the basis of the characteristics of mutants unable to dissimilate or assimilate nitrate to nitrite, it was revealed that two different sets of genes (represented by Class I and Class II mutants) were shared between the nitrate-to-nitrite reduction steps of both pathways. The genes represented by Class I and II mutants have been separated into distinct genetic loci using two cosmids, pAD1695/96. The two different genetic loci have been designated snr (shared nitrate reduction) and mol (MoCo processing genes) based on the phenotypic characteristics of the mutants complemented. Restriction analyses of pAD1695/96 followed by subcloning confirmed the complementation results. The snr loci, which represent a unique and hitherto uninvestigated set of genes for nitrate reduction, were mapped on the P. aeruginosa chromosome by linkage analysis with sex factor FP2.

NarK is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia coli.

Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest-lived radioactive isotope of nitrogen, 13N-nitrate (half-life = 9.96 min) and the nitrite-sensitive fluorophore N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide have now been used to define the function of NarK. At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange. This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase.

Nitrate transport and its regulation by O2 in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an obligate respirer which can utilize nitrate as a terminal electron acceptor under anaerobic conditions (denitrification). Immediate, transient regulation of nitrate respiration is mediated by oxygen through the inhibition of nitrate uptake. In order to gain an understanding of the bioenergetics of nitrate transport and its regulation by oxygen, the effects of various metabolic inhibitors on the uptake process and on oxygen regulation were investigated. Nitrate uptake was stimulated by the protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, indicating that nitrate uptake is not strictly energized by, but may be affected by the proton motive force. Oxygen regulation of nitrate uptake might in part be through redox-sensitive thiol groups since N-ethylmaleimide at high concentrations decreased the rate of nitrate transport. Cells grown with tungstate (deficient in nitrate reductase activity) and azide-treated cells transported nitrate at significantly lower rates than untreated cells, indicating that physiological rates of nitrate transport are dependent on nitrate reduction. Furthermore, tungstate grown cells transported nitrate only in the presence of nitrite, lending support to the nitrate/nitrite antiport model for transport. Oxygen regulation of nitrate transport was relieved (10% that of typical anaerobic rates) by the cytochrome oxygen reductase inhibitors carbon monoxide and cyanide.

Oxygen regulation of nitrate transport by diversion of electron flow in Escherichia coli.

Anaerobic nitrate respiration is regulated by oxygen at the level of nitrate transport; however, the mechanism of O2 inhibition is unknown. Potentially, oxygen could inhibit directly by causing conformational changes in the porter system or indirectly through diversion of electron flow from the nitrate reductase complex to oxygen reduction. Inhibition due to electron diversion implies that nitrate reduction is required for nitrate transport. In this regard, nitrate uptake and its regulation by oxygen were studied in mutants of Escherichia coli (strain AN386) deficient in cytochrome d (RG98), cytochrome o (RG101), and a mutant deficient in both cytochrome d and cytochrome o (RG99). Respiratory nitrate uptake in RG99 was highly resistant to the effects of oxygen supporting the indirect mechanism of electron diversion in oxygen regulation. Nitrate transport in RG98 and RG101 is highly sensitive to oxygen; these mutants exhibited 81 and 85% inhibition, respectively, which is similar to inhibition in the wild type. These results indicate that during nitrate respiration, O2 inhibits transport by limiting the supply of electrons to the nitrate reductase complex.

Regulation and energization of nitrate transport in a halophilic Pseudomonas stutzeri.

Nitrate transport and its regulation by oxygen was studied in denitrifying halophilic Pseudomonas stutzeri, strain Zobell, and a Tn-5 transposon nitrite reductase mutant of this organism. The rate of nitrate transport was found to be 130 nanomoles nitrate min-1 mg protein-1 and 150 nanomoles nitrate min-1 mg protein-1 in the wildtype and the nitrite reductase mutant respectively as compared to 26.4 nanomoles nitrate min-1 mg protein-1 in a non-halophilic Pseudomonas stutzeri. Asparagine was found to be the best energy source for nitrate uptake. The ratio of nitrate import to nitrite export was established by measuring extracellular nitrate and nitrite concentrations using HPLC/UV analysis. There was a 1.3:1 (NO3-/NO2-) exchange. High concentrations of nitrate during growth was found to have a negative effect on nitrite metabolism. Oxygen exerted an inhibitory effect on nitrate uptake which was reversible and more pronounced in cells grown on low concentrations of nitrate compared to cells grown at high concentrations of nitrate.

Oxygen inhibition of nitrate uptake is a general regulatory mechanism in nitrate respiration.

It has recently been demonstrated that oxygen inhibits nitrate uptake by denitrifying Pseudomonas aeruginosa. The purpose of the present investigation was to determine if this novel mechanism of regulation is universal for the regulation of nitrate respiration in other widely divergent species of bacteria. Nitrate transport by whole cell suspensions was completely and reversibly inhibited in 11 out of 12 species tested, whereas nitrate reduction by cell-free extracts was not affected by oxygen or was only partially inhibited in some cases. These results indicate that oxygen inhibition of nitrate uptake is a general regulatory phenomenon.

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.

Nicotinic acid transport in Escherichia coli.

The uptake of nicotinic acid by Escherichia coli is dependent on the presence of the enzyme nicotinic acid phosphoribosyl transferase and a source of energy. Glucose concentrations between 0.1 and 0.5%, a temperature of 46 degrees C and an external concentration of 2.5 X 10(-5) were optimal conditions for nicotinic acid uptake. Saturation kinetics occur with a Km of 1.75 microM and a Vmax of 0.116 nmoles/min/mg dry weight. The intracellular molarity of the accumulated pyridine compounds is 44-fold that of the initial concentration. Inhibitors of respiration and anaerobiosis do not significantly inhibit uptake rate. However, an inhibitor of glycolysis, uncouplers of ATP production and sodium arsenate reduce vitamin transport. A mutant defective in ATPase does not accumulate exogenously supplied nicotinic acid when lactate is used as an energy source, although L-proline, the transport of which is independent of ATP production, is accumulated.

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.

Evidence for gene sharing in the nitrate reduction systems of Pseudomonas aeruginosa.

Pseudomonas aeruginosa mutants unable to assimilate or dissimilate nitrate were isolated. Transduction and reversion analyses of these mutants revealed that single genetic lesions are responsible for the double phenotypes. The mutants were divided into two classes based on the ability to utilize hypoxanthine. It can be concluded from this study that at least two genes are shared between the two nitrate reduction systems.

Antibiotic action of pyocyanin.

Biologically produced pyocyanin was purified, and the nature of its antibacterial action was determined for several bacteria. The pigment was shown to be bactericidal for all susceptible organisms. The bactericidal effect was dependent upon pyocyanin concentration and resulted in decreases in viability ranging from 1 to 8 log viable cells ml-1. The gram-positive bacteria were more susceptible as a group to the antibiotic action than were the gram-negative bacteria. All apyocyanogenic pseudomonads tested were totally resistant to the pigment, suggesting that resistance may be a characteristic of the genus. Pseudomonas aeruginosa, the producer organism, was also essentially unaffected by high concentrations of pyocyanin. Facultative anaerobes were twofold or more times resistant to the action of the pigment under fermentative conditions; however, the antibiotic action did not require oxygen since denitrifying bacteria were more susceptible during anaerobic respiration than during aerobic respiration.

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.

A cytochrome b-like pigment with a peak at 567 nm in Pseudomonas aeruginosa.

A cytochrome b - like pigment with an absorption peak at 567 nm was detected in Pseudomonas aeruginosa irrespective of whether the organism was grown aerobically or anaerobically under denitrifying conditions. This pigment has not been reported previously for P. aeruginosa but it has been detected in other denitrifying bacteria including closely related Pseudomonas species.

Determination of 30 elements in coal and fly ash by thermal and epithermal neutron-activation analysis.

Thirty elements are determined in coal and fly ash by instrumental neutron-activation analysis using both thermal and epithermal irradiation. Gamma-ray spectra were recorded 7 and 20 days after the irradiations. The procedure is applicable to the routine analysis of coals and fly ash. Epithermal irradiation was found preferable for the determination of Ni, Zn, As, Se, Br, Rb, Sr, Mo, Sb, Cs, Ba, Sm, Tb, Hf, Ta, W, Th and U, whereas thermal irradiation was best for Sc, Cr, Fe, Co, La, Ce, Nd, Eu, Yb and Lu. Results for SRM 1632 (coal) and SRM 1633 (fly ash) agree with those of other investigators.

Decrease in nitrate reductase activity in extracts of Trichoderma viride Incubated with chlorides.

Reduced nicotinamide adenine dinucleotide phosphate-dependent nitrate reductase activity in crude extracts of Trichoderma virde was significantly inhibited by physiological concentrations of ammonium chloride, sodium chloride, and potassium chloride, but not by ammonium or sodium sulfate. The chloride inhibition of nitrate reductase activity increased in a linear manner with chloride concentration.

The inhibitory effect of the artificial electron donor system, phenazine methosulfate-ascorbate, on bacterial transport mechanisms.

The artificial electron donor system, phenazine methosulfate (PMS)-ascorbate, inhibited active transort of solutes in Pseudomonas aeruginosa irrespective of whether the active transport systems were shock sensitive or shock resistant. N,N,N',N'-tetramethylphenylenediamine could be substituted for PMS but a higher concentration was required. PMS-ascorbate also inhibited active transport in several other bacterial species with the exception of Escherichia coli and of a nonpigmented strain of Serratia marcescens. PMS-ascorbate previously has been shown to energize active transport in isolated membrane vesicles, even those prepared from the same bacterial species in whose intact cells active transport was inhibited. The apparent Km of glucose active transport in untreated cells of P. aeruginosa was 40 micron while the Km of glucose transport in cells incubated with PMS-ascorbate was 25 mM, and PMS-ascorbate had no effect on efflux of accumulated glucose. These results strongly suggested that facilitated diffusion resulted upon exposure of the cells to PMS- ascorbate. Thus, PMS-ascorbate appeared to have an uncoupler-like effect on cells of P. aeruginosa. The experimental data also pointed out that there are fundamental differences between the response of intact cells and membrane vesicles to exogenous electron donors.

Studies on the modes of action of azaserine in Escherichia coli. Mechanism of resistance to azaserine.

Growth of wildtype Escherichia coli was inhibited by azaserine. There was an inverse relationship between the initial rate of uptake of phenylalanine and the azaserine concentration. Moderately azaserine-resistant mutants exhibited an initial rate that was similar to that of an aroP mutant, but highly azaserine-resistant mutants exhibited little, if any, uptake of phenylalanine. All of the azaserine-resistant organisms tested harboured a mutation in the aroP+ gene. However, resistance to the antibiotic was not due solely to this lesion.