Involvement of nitrate reductase and pyoverdine in competitiveness of Pseudomonas fluorescens strain C7R12 in soil

Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain Pseudomonas fluorescens C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (-1 and -10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar(-)), the pyoverdine (Pvd(-)), or both (Nar(-) Pvd(-)) were used. The Nar(-) and Nar(-) Pvd(-) mutants were obtained by site-directed mutagenesis of the wild-type strain and of the Pvd(-) mutant, respectively. The selective advantage given by nitrate reductase and pyoverdine to the wild-type strain was assessed by measuring the dynamic of each mutant-to-total-inoculant (wild-type strain plus mutant) ratio. All three mutants showed a lower competitiveness than the wild-type strain, indicating that both nitrate reductase and pyoverdine are involved in the fitness of P. fluorescens C7R12. The double mutant presented the lowest competitiveness. Overall, the competitive advantages given to C7R12 by nitrate reductase and pyoverdine were similar. However, the selective advantage given by nitrate reductase was more strongly expressed under conditions of lower aeration (-1 kPa). In contrast, the selective advantage given by nitrate reductase and pyoverdine did not differ in bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in the saprophytic ability of C7R12 in soil environments.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

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

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 denitrifying Pseudomonas 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.     

Inactivation of gacS Does Not Affect the Competitiveness of Pseudomonas chlororaphis in the Arabidopsis thaliana Rhizosphere

Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac⁻ phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by Pseudomonas chlororaphis, a gnotobiotic system based on Arabidopsis thaliana seedlings in soil was investigated. Transposon insertion mutants of P. chlororaphis isolate SPR044 carrying insertions in different genes required for the production of N-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative gacS mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the gacS mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the gacS mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in populations containing the wild type and Gac⁻ mutants.     

Fitness in soil and rhizosphere of Pseudomonas fluorescens C7R12 compared with a C7R12 mutant affected in pyoverdine synthesis and uptake

Fluorescent pseudomonads have evolved an efficient strategy of iron uptake based on the synthesis of the siderophore pyoverdine and its relevant outer membrane receptor. The possible implication of pyoverdine synthesis and uptake on the ecological competence of a model strain (Pseudomonas fluorescens C7R12) in soil habitats was evaluated using a pyoverdine minus mutant (PL1) obtained by random insertion of the transposon Tn5. The Tn5 flanking DNA was amplified by inverse PCR and sequenced. The nucleotide sequence was found to show a high level of identity with pvsB, a pyoverdine synthetase. As expected, the mutant PL1 was significantly more susceptible to iron starvation than the wild-type strain despite its ability to produce another unknown siderophore. As with the wild-type strain, the mutant PL1 was able to incorporate the wild-type pyoverdine and five pyoverdines of foreign origin, but at a significantly lower rate despite the similarity of the outer membrane protein patterns of the two strains. The survival kinetics of the wild-type and of the pyoverdine minus mutant, in bulk and rhizosphere soil, were compared under gnotobiotic and non-gnotobiotic conditions. In gnotobiotic model systems, both strains, when inoculated separately, showed a similar survival in soil and rhizosphere, suggesting that iron was not a limiting factor. In contrast, when inoculated together, the bacterial competition was favorable to the pyoverdine producer C7R12. The efficient fitness of PL1 in the presence of the indigenous microflora, even when coinoculated with C7R12, is assumed to be related to its ability to uptake heterologous pyoverdines. Altogether, these results suggest that pyoverdine-mediated iron uptake is involved in the ecological competence of the strain P. fluorescens C7R12.     

Nodulating Competitiveness of a Nonmotile Tn7 Mutant of Bradyrhizobium japonicum in Nonsterile Soil

A nonmotile mutant of Bradyrhizobium japonicum serogroup 127 was generated by Tn7 mutagenesis and matched with the wild type against a common competitor in studies of soybean nodulation in nonsterile soil. The Tn7 mutant was very similar to the wild type in growth rate in culture, soybean lectin-binding ability, flagellar morphology, and nodulating capability, but it had a longer lag phase. Competing strains were distributed uniformly in soil in various ratios and at different population densities prior to planting. Mutant and wild type were equally prevalent in the seedling rhizosphere at about the time of nodule initiation, suggesting that motility conferred no advantage in rhizosphere colonization. Nodulation success of the Tn7 mutant was lower than that of the wild type under all test conditions. Differences were greatest at low soil populations of competitors and much less pronounced at initial populations of 10⁷ g⁻¹. The longer lag phase of the Tn7 mutant may have contributed to its decreased competitiveness, especially at the higher inoculation levels. The antibiotic and motility markers were stable, and the rifampin resistance derived from the parent did not affect adversely the competitiveness of the Tn7 mutant. We found motility to be of limited importance to the competitiveness of a strain in normal nonsterile soil, where the significance, if any, of this ability may be in migration at the immediate root surface in soils sparsely populated with rhizobial symbionts.     

Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA⁺ (toluene o-monooxygenase) genes from Burkholderia cepacia PR1₂₃(TOM_23C). A transposon integration vector was used to insert tomA⁺ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR1₂₃(TOM_23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h⁻¹) and colonized wheat (3 × 10⁶ CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h⁻¹; level of colonization, 4 × 10⁶ CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.     

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g⁻¹) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ~2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.     

Fusaric Acid-Producing Strains of Fusarium oxysporum Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in Pseudomonas fluorescens CHA0 In Vitro and in the Rhizosphere of Wheat

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA′-′lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA′-′lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA⁺) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA⁻) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.     

Pseudomonas fluorescens Pirates both Ferrioxamine and Ferricoelichelin Siderophores from Streptomyces ambofaciens

Iron is essential in many biological processes. However, its bioavailability is reduced in aerobic environments, such as soil. To overcome this limitation, microorganisms have developed different strategies, such as iron chelation by siderophores. Some bacteria have even gained the ability to detect and utilize xenosiderophores, i.e., siderophores produced by other organisms. We illustrate an example of such an interaction between two soil bacteria, Pseudomonas fluorescens strain BBc6R8 and Streptomyces ambofaciens ATCC 23877, which produce the siderophores pyoverdine and enantiopyochelin and the siderophores desferrioxamines B and E and coelichelin, respectively. During pairwise cultures on iron-limiting agar medium, no induction of siderophore synthesis by P. fluorescens BBc6R8 was observed in the presence of S. ambofaciens ATCC 23877. Cocultures with a Streptomyces mutant strain that produced either coelichelin or desferrioxamines, as well as culture in a medium supplemented with desferrioxamine B, resulted in the absence of pyoverdine production; however, culture with a double mutant deficient in desferrioxamines and coelichelin production did not. This strongly suggests that P. fluorescens BBbc6R8 utilizes the ferrioxamines and ferricoelichelin produced by S. ambofaciens as xenosiderophores and therefore no longer activates the production of its own siderophores. A screening of a library of P. fluorescens BBc6R8 mutants highlighted the involvement of the TonB-dependent receptor FoxA in this process: the expression of foxA and genes involved in the regulation of its biosynthesis was induced in the presence of S. ambofaciens. In a competitive environment, such as soil, siderophore piracy could well be one of the driving forces that determine the outcome of microbial competition.     

Role of Chemotaxis in the Ecology of Denitrifiers

A modification of the Adler capillary assay was used to evaluate the chemotactic responses of several denitrifiers to nitrate and nitrite. Strong positive chemotaxis was observed to NO₃⁻ and NO₂⁻ by soil isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri, with the peak response occurring at 10⁻³ M for both attractants. In addition, a strong chemoattraction to serine (peak response at 10⁻² M), tryptone, and a soil extract, but not to NH₄⁺, was observed for all denitrifiers tested. Chemotaxis was not dependent on a previous growth on NO₃⁻, NO₂⁻, or a soil extract, and the chemoattraction to NO₃⁻ occurred when the bacteria were grown aerobically or anaerobically. However, the best response to NO₃⁻ was usually observed when the cells were grown aerobically with 10 mM NO₃⁻ in the growth medium. Capillary tubes containing 10⁻3 M NO₃⁻ submerged into soil-water mixtures elicited a significant chemotactic response to NO₃⁻ by the indigenous soil microflora, the majority of which were Pseudomonas spp. A chemotactic strain of P. fluorescens also was shown to survive significantly better in aerobic and anaerobic soils than was a nonmotile strain of the same species. Both strains had equal growth rates in liquid cultures. Thus, chemotaxis may be one mechanism by which denitrifiers successfully compete for available NO₃⁻ and NO₂⁻, and which may facilitate the survival of naturally occurring populations of some denitrifiers.     

Quinolobactin, a New Siderophore of Pseudomonas fluorescens ATCC 17400, the Production of Which Is Repressed by the Cognate Pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of ⁵⁹Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Thiamine-Auxotrophic Mutants of Pseudomonas fluorescens CHA0 Are Defective in Cell-Cell Signaling and Biocontrol Factor Expression

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 × 10⁻⁸ M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Biochemical, Genetic, and Zoosporicidal Properties of Cyclic Lipopeptide Surfactants Produced by Pseudomonas fluorescens

Zoospores play an important role in the infection of plant and animal hosts by oomycetes and other zoosporic fungi. In this study, six fluorescent Pseudomonas isolates with zoosporicidal activities were obtained from the wheat rhizosphere. Zoospores of multiple oomycetes, including Pythium species, Albugo candida, and Phytophthora infestans, were rendered immotile within 30 s of exposure to cell suspensions or cell culture supernatants of the six isolates, and subsequent lysis occurred within 60 s. The representative strain SS101, identified as Pseudomonas fluorescens biovar II, reduced the surface tension of water from 73 to 30 mN m⁻¹. The application of cell suspensions of strain SS101 to soil or hyacinth bulbs provided significant protection against root rot caused by Pythium intermedium. Five Tn5 mutants of strain SS101lacked the abilities to reduce the surface tension of water and to cause lysis of zoospores. Genetic characterization of two surfactant-deficient mutants showed that the transposons had integrated into condensation domains of peptide synthetases. A partially purified extract from strain SS101 reduced the surface tension of water to 30 mN m⁻¹ and reached the critical micelle concentration at 25 μg ml⁻¹. Reverse-phase high-performance liquid chromatography yielded eight different fractions, five of which had surface activity and caused lysis of zoospores. Mass spectrometry and nuclear magnetic resonance analyses allowed the identification of the main constituent as a cyclic lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon hydroxy fatty acid. The other four zoosporicidal fractions were closely related to the main constituent, with molecular massesranging from 1,111 to 1,169 Da.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana

After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO₃⁻ as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH₄⁺-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein.     

Rapid Colony Screening Method for Identifying Hydrogenase Activity in Rhizobium japonicum

A method has been developed for the rapid screening of Rhizobium japonicum colonies for hydrogenase activity based on their ability to reduce methylene blue in the presence of respiratory inhibitors and hydrogen. Hydrogen uptake-positive (Hup⁺) colonies derepressed for hydrogenase activity were visualized by their localized decolorization of filter paper disks impregnated with the dye. Appropriate responses were seen with a number of Hup⁺ and Hup⁻ wild-type strains of R. japonicum as well as Hup⁻ mutants. Its specificity was further confirmed in selected strains on the basis of comparisons with chemolithotrophic growth and the presence of other genetic markers. Utilization of the method in identifying Hup⁺ colonies among 16,000 merodiploid derivatives of the Hup⁻ mutant strain PJ17nal containing cloned DNA fragments of the Hup⁺ strain 122 DES has demonstrated its applicability as a screening procedure in the genetic analysis of the R. japonicum hydrogen uptake system.     

Nitrogen Control of Atrazine Utilization in Pseudomonas sp. Strain ADP

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas⁻ mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas⁻ mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.