Pseudomonas aeruginosa contains an inducible methylcitrate synthase

Assaying for enzymes of propionyl-CoA catabolism in crude extracts ofPseudomonas aeruginosa was complicated by the presence of a short-chain acyl-CoA hydrolase. Interference by the hydrolase was avoided by using permeabilized whole cells. Cells treated this way exhibited low or undetectable levels of propionyl-CoA carboxylase, propionyl-CoA dehydrogenase, and α-hydroxyglutarate synthase, enzymes initiating propionyl-CoA catabolism in many prokaryotes, but did contain high levels of methylcitrate synthase, an enzyme of the methylcitrate cycle for propionyl-CoA catabolism. Gel filtration experiments revealed a peak of methylcitrate synthase distinct from the citrate synthase of the bacterium. The enzyme was induced by growth on propionate and on propanol and heptanoate, precursors of propionate. These results suggest thatP. aeruginosa may utilize the methylcitrate cycle to metabolize propionate, a pathway heretofore only described in the yeastCandida lipolytica.     

Evidence for induced synthesis of an active transport factor for mandelate in Pseudomonas putida

1. At low concentrations of mandelate there is a lag before induction of the mandelate regulon begins at a sub-maximum rate. Cells preinduced with a saturating concentration of inducer do not exhibit this lag when they are transferred to sub-maximum inducer concentrations and are able to maintain a high rate of induction under these conditions. 2. Chloramphenicol was used to show that a protein is synthesized during the lag period that is probably responsible for the maintenance effects observed in batch and continuous cultures. 3. Direct measurements appear to show that induced cells possess an active transport factor for mandelate, sensitive to dinitrophenol, and this is presumably responsible for the maintenance effect. Mandelate is accumulated unchanged against a concentration gradient from media containing low concentrations. At higher external concentrations no accumulation occurs and only a rapid equilibration is noted. Uninduced cells merely equilibrate mandelate.     

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 an inducible glucose transport system in Kluyveromyces lactis.

To find the cause of delayed glucose oxidation in succinate-grown Kluyveromyces lactis, glucose transport was studied in glucose- and in succinate-grown cells. The initial rate of 2-deoxyglucose (2-dGlc) accumulation, as well as the appearance of 2-deoxyglucose 6-phosphate, was higher in the glucose-grown cells. In both cell types, 2-dGlc was apparently transported in the free form to be phosphorylated intracellularly. In glucose-grown cells the level of free 2-dGlc in the pool was always less than the external concentration. Exchange transport in starved, poisoned cells loaded with unlabeled 2-dGlc was 140-fold greater in glucose- than in succinate-grown cells, probably beacuse of the presence of an inducible transport component. The development of the increased rate of transport in a succinate-grown uracil-requiring auxotroph after transfer to glucose depends on the presence of uracil.     

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.     

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.     

Evidence for an inducible glucose transport system in Kluyveromyces lactis

To find the cause of delayed glucose oxidation in succinate-grown Kluyveromyces lactis, glucose transport was studied in glucose- and in succinate-grown cells. The initial rate of 2-deoxyglucose (2-dGlc) accumulation, as well as the appearance of 2-deoxyglucose 6-phosphate, was higher in the glucose-grown cells. In both cell types, 2-dGlc was apparently transported in the free form to be phosphorylated intracellularly . In glucose-grown cells the level of free 2-dGlc in the pool was always less than the external concentration. Exchange transport in starved, poisoned cells loaded with unlabeled 2-dGlc was 140-fold greater in glucose- than in succinate-grown cells, probably because of the presence of an inducible transport component. The development of the increased rate of transport in a succinate-grown uracil-requiring auxotroph after transfer to glucose depends on the presence of uracil.     

Methionine transport in Pseudomonas aeruginosa.

A high-affinity (Km = 2.7 x 10(-7) M) energy-requiring methionine-transport system has been characterized in RM 46 and RM 48, two different PAO methionine auxotrophs of Pseudomonas aeruginosa. After 8 s of transport 40--60% of the methionine label in the alcohol extract appears in S-adenosyl-L-methionine (SAM) with the remaining activity in free methionine. Methionine transport required a high degree of structural specificity for transport. Stimulation of transport occurred by addition of glucose or organic acids. The ability of a given substrate to stimulate transport was related to the type of carbon source used for growth. Transport was sensitive to sulfhydryl reagents and required oxidative phosphorylation, as indicated by the inhibitory effects of anaerobiosis, cyanide, and arsenate. The degree of inhibition by arsenate correlated with the level of ATP in the cell. Rapid transport in a SAM-deficient mutant (TM 1) and inhibition by arsenate of transport in this mutant suggested that SAM formation was not directly linked to transport and that ATP supplied energy for transport. Inhibition by arsenate was more severe in glucose- compared to citrate-stimulated cells. This result was also observed with proline transport indicating that this was not a peculiarity of the methionine-transport system. These data emphasize the close link between glucose metabolism, ATP levels, and transport. This ATP level is not so critical for transport in cells metabolizing citrate.     

Constitutive choline transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the presence of succinate and ammonium chloride as the carbon and nitrogen source, respectively. This system obeys Michaelis-Menten kinetics with an apparent K_m value of 53 μM; its activity is not inhibited by high osmolarities in the medium but is partially inhibited by choline metabolites such as betaine and dimethylglycine.     

Isolation and properties of an inducible and a constitutive beta-lactamase from Pseudomonas aeruginosa

The inducible beta-lactamase from Pseudomonas aeruginosa NCTC 8203 and the constitutive beta-lactamase from strain 1822 S/H have been isolated and compared. These two enzymes are apparently periplasmic since they are released by freezing and thawing. They resemble each other closely in their molecular weights, amino acid composition, isoelectric points and electrophoretic mobility as well as in their catalytic properties, and they may be identical. Neither enzyme contains a free thiol group.     

Inositol polyphosphate-mediated iron transport in Pseudomonas aeruginosa

It has previously been shown that myo-inositol hexakisphosphate (myo-InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron-dependent growth inhibition. In this study, the iron transport properties of myo-inositol trisphosphate and tetrakisphosphate regio-isomers were studied. Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo-Ins(1,2,3)P3 or myo-InsP6. Iron accumulation from other compounds, notably D/L myo-Ins(1,2,4,5)P4 and another inositol trisphosphate regio-isomer, D-myo-Ins(1,4,5)P3, was dramatically increased. Iron transport profiles from myo-InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild-type, indicating that these porins are not involved in the transport process. The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps. aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate-determining step.     

Enterobactin-mediated iron transport in Pseudomonas aeruginosa.

A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin. Apparently, at least two uptake systems for ferrienterobactin exist in P. aeruginosa: one of higher affinity which is specifically inducible by enterobactin under iron-limiting conditions and the second, of lower affinity, which is also inducible under iron-limiting conditions but is independent of enterobactin for induction.     

Pyocin R1 inhibits active transport in Pseudomonas aeruginosa and depolarizes membrane potential.

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, inhibited active transport of proline in the presence of high concentrations of malate and magnesium salt. Pyocin R1 did not affect the respiration of sensitive cells nor induce cell lysis, but it caused a decrease in the intracellular ATP level. In addition, a passive inflow of [14C]thiocyanate anion, a probe of membrane potential, was induced by pyocin R1, showing a depolarization of the cytoplasmic membrane. It is considered that membrane depolarization is a primary action of pyocin R1.