N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

An experiment in enzyme evolution studies withPseudomonas aeruginosa amidase

The regulation of amidase synthesis inP. aeruginosa is under positive control. This review describes the experimental evolution of amidase and its regulator protein for the hydrolysis of novel substrates and experiments to elucidate the mechanism of the control system.     

Bacteriophage lambda DNA packaging: A mutant terminase that is independent of integration host factor

Summary ⁺ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF⁺ cells. In vitro, however, we find that the DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of . Variants of cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.     

A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae

Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.     

Initial Studies on the Regulation of Toluene Degradation by Pseudomonas Putida F1

Pseudomonas putida Fl oxidizes toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is the substrate for “meta” fission of the aromatic nucleus. Kinetic and induction experiments indicate that the genes encoding enzymes for these reactions are part of an operon, designated the tod operon, that is coordinately induced and regulated. Strains unable to utilize toluene as a growth substrate were isolated at high frequencies by using screening procedures that utilize the redox dye, 2,3,5-triphenyl-2H-tetrazolium chloride. Biochemical characterization of strains with mutations in the structural genes of the tod operon showed that toluene induces the first four enzymes in toluene degradation by P. putida Fl. The isolation and characterization of pleiotropicnegative mutants together with mutants altered in terms of their expression of tod genes suggests that the tod operon may be under the control of a positive regulatory element.     

Environmentally directed mutations in the dehalogenase system of Pseudomonas putida strain PP3

Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids — HAAs) which were toxic to P. putida; and/or the presence of a potential growth substrate. Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs. the mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH. Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P. putida from toxic compounds in its growth environment. Other mutations partially restored P. putida's dehalogenating capability under conditions where toxic substrates were absent. Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment.     

Influence of soil type on the transfer of plasmid RP4p from Pseudomonas fluorescens to introduced recipient and to indigenous bacteria

Abstract Transfer of plasmid RP4p from introduced Pseudomonas fluorescens to a co-introduced recipient strain or to members of the indigenous bacterial population was studied in four different soils of varying texture planted with wheat. Donor and recipient strains showed good survival in the four soils throughout the experiment. The numbers of transconjugants found in donor and recipient experiments in two soils, Ede loamy sand and Löss silt loam were significantly higher in the rhizosphere than in corresponding bulk soil. In the remaining two soils, Montrond and Flevo silt loam, transconjugant numbers were not significantly higher in the rhizosphere than in the bulk soil.
The combined utilization of a specific bacteriophage eliminate the donor strain and the pat sequence as a specific marker to detect RP4p was found to be very efficient in detecting indigenous transconjugants under various environmental conditions. The numbers of indigenous transconjugants were consistently higher in rhizosphere than bull soil. A significant rhizosphere effect on transconjugant numbers of transconjugants were recovered from Flevo and Montrond silt loam; these soils possess characteristics such as clay or organic matter contents which may be favorable to conjugation.     

Initial reactions in the mineralization of 2-sulfobenzoate by Pseudomonas sp. RW611

Abstract Pseudomonas sp. strain RW611 utilized the ammonium salt of 2-sulfobenzoate as sole source of carbon, sulfur, nitrogen, and energy. The xenobiotic sulfo substituent was dioxygenolytically eliminated as sulfite, which was then slowly oxidized to sulfate. 2,3-Dihydroxybenzoate, which resulted from desulfonation underwent meta -cleavage, mediated by 2,3-dihydroxybenzoate 3,4-dioxygenase activity. This enzyme was inhibited by 3-chlorocatechol and 2,3,4-trihydroxybenzoate.     

Development of a detection system for ferric pseudobactin using monoclonal antibodies

Certain root-colonizing fluorescent pseudomonads have been shown to promote plant growth and prevent plant disease in part through the production of siderophores. However, these favorable results have not been reproduced consistently from the laboratory to the greenhouse or from the greenhouse to the field. In some circumstances siderophores appear to play no role in disease prevention. In order to understand the dynamics of competition for iron in the rhizosphere it is essential that the localization and concentration of siderophores produced by both biocontrol agents and plant pathogens be determined. We have produced monoclonal antibodies (MAbs) to ferric pseudobactin, the siderophore of plant growth-promoting Pseudomonas B10. Three IgG1 MAbs cross-react with certain ferric pseudobactins but not with others. A competitive ELISA has been developed to detect and quantify ferric pseudobactin.