Pseudomonas perfectomarinus was found to grow anaerobically at the expense of nitrate, nitrite, or nitrous oxide but not chlorate or nitric oxide. In several repetitive experiments, anaerobic incubation in culture media containing nitrate revealed that an average of 82% of the cells in aerobically grown populations were converted to the capacity for respiration of nitrate. Although they did not form colonies under these conditions, the bacteria synthesized the denitrifying enzymes within 3 hr in the absence of oxygen or another acceptable inorganic oxidant. This was demonstrated by the ability, after anaerobic incubation, of cells and of extracts to reduce nitrite, nitric oxide, and nitrous oxide to nitrogen. From crude extracts of cells grown on nitrate, nitrite, or nitrous oxide, separate complex fractions were obtained that utilized reduced nicotinamide adenine dinucleotide as the source of electrons for the reduction of (i) nitrite to nitric oxide, (ii) nitric oxide to nitrous oxide, and (iii) nitrous oxide to nitrogen. Gas chromatographic analyses revealed that each of these fractions reduced only one of the nitrogenous oxides.

Barbaree and Payne ( 1 ) found gas chromatographic methods, known to be reliable for determining the components of specifically prepared mixtures of gases ( 2 ) , useful in the analysis of products of denitrification carried out by Pseudomoms perjectomorinuscells and extracts. Employing these procedures, we can now demonstrate routinely that nitric and nitrous oxides (as well as nitrogen) are products of nitrate dissimilation by enzymes in cell-free extracts of this bacterium. I t has been generally known for some time, however, that in growing cultures of denitrifiers the flow of reduction is delayed. Before the oxides and nitrogen are released, nitrite accumulates nearly stoichiometrically with the quantity of nitrate reduced ( 3 ) . Only after nitrate is depleted does dissimilative nitrate reduction go rapidly to completion (e.g., release of gaseous nitrogen). This blockage occurs in P. perfectomarinus as in other denitrifiers and might be explained either (i) by a delay in induction of enzymes for reduotion of nitrate and the intermediate nitrogenous oxides until nitrate is removed, or (ii) by suppression by nitrate of the functioning of the reductive enzymes further along the pathway. The purpose of this paper is to present results supporting the latter explanation.Materials and Methods. We cultured P. perfectomarinus as described by Rhodes et al. (4) in tryptone-yeast extract-sea salt medium containing nitrate (TYSN) . Cells were harvested by centrifugation after 20 hr and washed twice in 0.052 M MgClP, pH 7.0.From these we prepared cell-free extracts as before ( 1 ) and fractionated with ammonium sulfate. The precipitate from 40 to 70% saturation of the extract contained all the dentrifying enzymes. This crude fraction was dialyzed against 0.02 M phosphate buffer, pH Isupported by ONR Grant No. 3677(01) and NIGMS Training Grant 1 TO1 GM01968-01.7.0, for 12 hr with three evenly spaced changes of buffer and then assayed. For certain experiments, the dialyzed fraction was loaded onto a diethylaminoethyl cellulose (DEAE, Whatman DE-52) column, 2.5 X 3.5 cm, and discontinuous elution gradients were employed further to fractionate the protein.We concentrated the column eluates with an Amicon model 50 ultrafiltration cell (UM-10 membrane) before assaying for activity. Protein contents of the extracts were measured by the biuret method (5) and by absorption a t 280 nm. Gas chromatographic analyses were performed as described by Barbaree and Payne ( 1 ) .Release of nitrogen and the nitrogenous gases detectable by gas chromatography was accomplished by supplying the following components: dialyzed extract or eluted fraction as source of enzymes, reduced nicotinamide adenine dinucleotide (NADH) as the electron donor, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) as cofactors, and the appropriate oxide of nitrogen as the terminal electron acceptor (6). Before initiation of reactions by the addition of NADH and the appropriate terminal electron acceptor, we sparged the reaction mixtures with helium t...

Pseudomonas pickettii strains were studied to determine the characteristics essential for their identification in the clinical microbiology laboratory. Preliminary investigations indicated that these glucose-oxidizing, denitrifying, gram-negative rods were quite similar to an unclassified group of clinical isolates designated VA-2. Gas liquid chromatography of trimethylsilyl derivatives of whole cell hydrolysates of P. pickettii and VA-2 strains yielded nearly identical elution profiles. The VA-2 cultures were concluded to be probable strains of P. pickettii. A method is presented for differentiating cultures of P. pickettii from other similar bacteria encountered in clinical specimens.

The intracellular and constitutive arylamidase from Pseudomonas aeruginosa was purified 528-fold by salt fractionation, ion-exchange chromatography, gel filtration, and adsorption chromatography. This enzyme hydrolyzed basic and neutral N -terminal amino acid residues from amino-β-naphthylamides, dipeptide-β-naphthylamides, and a variety of polypeptides. Only those substrates having an l -amino acid with an unsubstituted α-amino group as the N -terminal residue were susceptible to enzymatic hydrolysis. The molecular weight was estimated to be 71,000 daltons. The lowest K m values were associated with substrates having neutral or basic amino acid residues with large side chains with no substitution or branching on the β carbon atom.

Pseudomonas vesicularis CDC group WO-1 Alcaligenes faecalis I Alcaligenes faecalis II Alcaligenes odorans Alcaligenes denitrificans CDC group IVc-2 CDC group IVe Bordetella bronchiseptica Flavobacterium odoratum CDC group M-5 CDC group M-6 Acinetobacter calcoaceticus subsp Iwoffi Moraxella urethralis Moraxella osloensis Moraxella nonliquefaciens Moraxella phenylpyruvica Moraxella atlantae No. of strains 39