Patrick Potier

Role of Respiratory Nitrate Reductase in Ability of Pseudomonas fluorescens YT101 To Colonize the Rhizosphere of Maize

ABSTRACT Selection of the denitrifying community by plant roots (i.e., increase in the denitrifier/total heterotroph ratio in the rhizosphere) has been reported by several authors. However, very few studies to evaluate the role of the denitrifying function itself in the selection of microorganisms in the rhizosphere have been performed. In the present study, we compared the rhizosphere survival of the denitrifyingPseudomonas fluorescens YT101 strain with that of its isogenic mutant deficient in the ability to synthesize the respiratory nitrate reductase, coinoculated in nonplanted or planted soil. We demonstrated that under nonlimiting nitrate conditions, the denitrifying wild-type strain had an advantage in the ability to colonize the rhizosphere of maize. Investigations of the effect of the inoculum characteristics (density of the total inoculum and relative proportions of mutant and wild-type strains) on the outcome of the selection demonstrated that the selective effect of the plant was expressed only during the phase of bacterial multiplication and that the intensity of selection was dependent on the magnitude of this phase. Moreover, application of the de Wit replacement series technique to our results suggests that the advantage of the wild-type strain was maximal when the ratio between the two strains in the inoculum was close to 1:1. This work constitutes the first direct demonstration that the presence of a functional structural gene encoding the respiratory nitrate reductase confers higher rhizosphere competence to a microorganism.

PURIFICATION OF THE DISSIMILATIVE NITRATE REDUCTASE OF PSEUDOMONAS FLUORESCENS AND THE CLONING AND SEQUENCING OF ITS CORRESPONDING GENES

The dissimilative membrane-bound nitrate reductase from Pseudomonas fluorescens strain AK15 was purified and the alpha subunit of the enzyme partially sequenced. On the basis of this partial amino acid sequence and of conserved stretches of amino acids between Escherichia coli and Bacillus subtilis, degenerate primers were design to amplify the narG gene and part of the narH gene in a PCR approach. The deduced amino acid sequence of narG shows 72% and 52% and narH 78% and 62% identity to the homologous subunit of E. coli and B. subtilis, respectively.

Temperature-dependent changes in proteolytic activities and protein composition in the psychrotrophic bacterium Arthrobacter globiformis S155

SUMMARY: Proteolytic activities, active against casein and insulin are present in cell-free extracts of the psychrotrophic bacterium Arthrobacter globiformis S155. These activities were compared (i) following growth of the bacterium at different temperatures (10, 20 and 32°C) and (ii) after a temperature shift from 10 to 32°C. Both activities (measured at 20°C) were greater in cells grown at 32°C and after a temperature shift. Chloramphenicol prevented the increases in activities. The proteolysis of casein was stimulated by ATP; the stimulation was greater following a temperature shift. In addition, proteolysis of casein (measured at 20°C) was activated by pre-incubation at temperatures between 45 and 60°C; the proportion of proteolytic activity activated by such treatment also increased with growth temperature. Comparison by two-dimensional electrophoresis of the polypeptides synthesized at 10 and 32°C suggests that A. globiformis S155 produces proteins specific to both temperatures (13 species were either exclusive to or present in increased amounts during growth at 10°C, while at least 21 polypeptides were preferentially synthesized at 32°C). A temperature shift from 10 to 32°C promoted the synthesis of at least 16 polypeptides, many with M r values similar to those of heat shock proteins synthesized at higher temperatures in mesophiles. A temperature up-shift of A. globiformis S155 from 10 to 20°C did not provoke the synthesis of new proteins; heat shock proteins were produced only at 28°C and higher. The similarity in kinetics of the appearance of heat shock proteins and the increase in proteolytic activities suggest that in A. globiformis S155 some heat shock proteins might also possess protein catabolic function.

Polyclonal Antibodies Recognizing the AmoB Protein of Ammonia Oxidizers of the β-Subclass of the Class Proteobacteria

ABSTRACT A 41-kDa protein of Nitrosomonas eutropha was purified, and the N-terminal amino acid sequence was found to be nearly identical with the sequence of AmoB, a subunit of ammonia monooxygenase. This protein was used to develop polyclonal antibodies, which were highly specific for the detection of the four genera of ammonia oxidizers of the β-subclass of Proteobacteria(Nitrosomonas, including Nitrosococcus mobilis, which belongs phylogenetically to Nitrosomonas;Nitrosospira; Nitrosolobus; andNitrosovibrio). In contrast, the antibodies did not react with ammonia oxidizers affiliated with the γ-subclass ofProteobacteria (Nitrosococcus oceani andNitrosococcus halophilus). Moreover, methane oxidizers (Methylococcus capsulatus, Methylocystis parvus, and Methylomonas methanica) containing the related particulate methane monooxygenase were not detected. Quantitative immunoblot analysis revealed that total cell protein ofN. eutropha consisted of approximately 6% AmoB, when cells were grown using standard conditions (mineral medium containing 10 mM ammonium). This AmoB amount was shown to depend on the ammonium concentration in the medium. About 14% AmoB of total protein was found when N. eutropha was grown with 1 mM ammonium, whereas 4% AmoB was detected when 100 mM ammonium were used. In addition, the cellular amount of AmoB was influenced by the absence of the substrate. Cells starved for more than 2 months contained nearly twice as much AmoB as actively growing cells, although these cells possessed low ammonia-oxidizing activity. AmoB was always present and could even be detected in cells of Nitrosomonas after 1 year of ammonia starvation.

Use of polyclonal antibodies to detect and quantify the NOR protein of nitrite oxidizers in complex environments

In the approaches or models which aim to understand and/or predict how the functioning of ecosystems may be affected by perturbations or disturbances, little attention is generally given to microorganisms. Even when they are taken into account as indicators, variables which are poorly informative about the changes in the microbial functioning (microbial biomass or diversity or total number of microorganisms) are often used. To be able to estimate, in complex environments, the quantity of enzymes involved in key ecosystem processes may constitute a useful complementary tool. Here, we describe an immunological method for detecting and quantifying, in complex environments, the nitrite oxidoreductase (NOR), responsible for the oxidation of nitrite to nitrate. The alpha-catalytic subunit of the enzyme was purified from Nitrobacter hamburgensis and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NOR by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. They recognized the alpha-NOR of all the Nitrobacter species described to date, but no reaction was observed with members of other nitrite-oxidizing genera. The detection threshold and reproducibility of the proposed method were evaluated. The feasibility of its use to quantify NOR in a soil was tested.

ATP-dependent and -independent Protein Degradation in Extracts of the Psychrotrophic Bacterium Authrobacter sp. S1 55

SUMMARY: Proteolysis of endogenous and exogenous substrates in cell-free extracts of the psychrotrophic bacterium Arthrobacter sp. S155 has been compared. Endogenous proteins were degraded only after treatment with cyanogen bromide. The hydrolysis of exogenous proteins of high Mr, (i.e. casein) was optimum at alkaline pH and was stimulated by Ca2+, Mg2+, Mn2+ and ATP. The serine protease inhibitor phenylmethylsulphonyl fluoride had no effect on ATP stimulation. Small peptides (i.e. insulin) were degraded at very high rates. This activity was optimum at slightly acidic pH and was stimulated by Ca2+, strongly inhibited by Mn2+, but not affected by ATP. Degradation of cyanogen bromide-treated cellular proteins displayed two pH optima which corresponded to the optimum pH for the degradation of insulin and casein. The characteristics of these acidic and alkaline activities were identical to those active against insulin and casein respectively. The proteases which degraded casein were much more heat resistant than those which degraded insulin.