Nitrogen-fixing Bacterium Azotobacter Research Articles

The Recultivation of the Soils, Contaminated with Oil, Petrol and Diesel Fuel, with the Help of Earthworms Dendrobena veneta and the Complex of Microorganisms

The study was performed of the bioremediation efficiency of the soil contaminated with oil (20 to 100 g/kg), petroleum (20 to 60 g/kg) and diesel fuel (20 to 40 g/kg) with the help of earthworms Dendrobena veneta in the presence of bacteria Pseudomonas, nitrogen fixing bacteria Azotobacter and Clostridium, yeasts Saccharomyces, fungi Aspergillus and Penicillium, as well as Actinomycetales, all being components of biopreparation Baykal-EM. It was demonstrated that in oil-contaminated soil, the content of hydrocarbons decreased by 95% after 22 weeks in the presence of worms and bacteria.

Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.

Responses of Bruguiera sexangula propagules to beneficial microbes in the nursery

Bruguiera sexangula (Lour) Poir., a threatened mangrove tree, was inoculated with beneficial microbes in a nursery to assess any improvements in growth and biomass. From soil samples from the rhizosphere of B. sexangula in a mangrove forest in Panangadu of Kerala India, nitrogen-fixing bacteria Azotobacter chroococcum and Azospirillum brasilense were isolated. The phosphate-solubilising bacterium Bacillus megaterium and potassium-mobilizing bacteria Frateruria aurantia were also isolated and cultured on suitable media. Later, ripe propagules of B. sexangula were collected from matured trees and raised in sterilized soil bags (13 × 25 cm) containing sterilized soil and sand (2:1 ratio). The cultured beneficial microbes were propagated and used to inoculate the ripe propagules of B. sexangula and maintained in the nursery for 6 months. After 6 months, growth and biomass of the inoculated propagules were greater than for the uninoculated control propagules. Shoot length, number of leaves, stem girth and root length were also significantly greater than in the controls. This study showed that the mangrove-specific beneficial microbes influenced the growth of B. sexangula in the nursery and will help in the establishment of B. sexangula in degraded mangrove forests.

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation.

Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium.IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

Biofertilizer effect of free living nitrogen fixing bacterium on the growth and water quality properties of Penaeid prawn culture system

The role of phytoplankton is significant in improving the yield of prawn culture. The growth of phytoplankton will be effected various factors viz. dissolved oxygen, production of natural food to shrimp larvae in control of ammonia. The growth of phytoplankton can be enhanced by application of nitrogeneous fertilizers to improve water quality and growth performance of Penaeus indicus. The present research work is focussed on a free-living nitrogen fixing bacterium Azotobacter chroococcum supplemented by nitrogeneous fertilizers to increase the content of proteins, carbohydrates and wet weight of P. indicus. Ten animals of juveniles of P. indicus were collected and grown in controlled conditions for fifteen days. The liquid suspension of free living nitrogen fixing bacteria A. chroococcum has been inoculated for experimental treatment purpose. The inoculum has been introduced to four treatments and for findings of water quality analysis andphytoplankton growth analysis by Strickland and Parsons method and biochemical analysis by Raymont et al., method. The research findings revealed that A. chroococcum supplemented with fertilizers will affect the growth of phytoplankton which is vital in prawn culture and enhance the growth of hormones and dry weight for improvement of the yield.

Effects of Rhamnolipid and Microbial Inoculants on the Vermicomposting of Green Waste with Eisenia fetida.

The effects of adding the biosurfactant rhamnolipid, the lignolytic and cellulolytic fungus Phanerochete chrysosporium, and the free-living nitrogen-fixing bacterium Azotobacter chrococcum on vermicomposting of green waste with Eisenia fetida was investigated. The addition of rhamnolipid and/or either microorganism alone or in all combinations significantly increased E. fetida growth rate, the number of E. fetida juveniles and cocoons, the population densities of cellulolytic fungi and Azotobacter bacteria, and cellulase and urease activities in the vermicomposts. The quality of the final vermicompost (in terms of electrical conductivity, nutrient content, C/N ratio, humic acid content, lignin and cellulose contents, and phytotoxicity to germinating seeds) was enhanced by addition of rhamnolipid and/or microorganisms. The physical characteristics of vermicomposts produced with rhamnolipid and/or microorganisms were acceptable for agricultural application. The best quality vermicompost was obtained with the combined addition of P. chrysosporium, A. chrococcum, and rhamnolipid.

Characterization of divergent pseudo-sucrose isomerase from Azotobacter vinelandii: Deciphering the absence of sucrose isomerase activity

Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6-glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_08330) encoding a putative SIase (AZOG: Azotobacterglucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the “RLDRD” motif, a SIase-specific region in 329–333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain α-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-α-glucoside (MαG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MαG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G.

Impact of a Number of Factors on Physiological and Biochemical Activity of Strains – Components of Azogran, a Complex Bacterial Preparation

The use of microbial preparations in plant-growing can be due to the correction of biological processes in agroecosystems and stimulates growth and development of plants. The efficiency of this process is dependent on biotic and abiotic factors, however their influence on introduction microorganisms in phytosphere is insufficiently studied. The article summarizes some results of recent studies, related to the impact of a number of environmental factors on physiological and biochemical activity of nitrogen-fixing bacteria Azotobacter vinelandii IMV B-7076 and phosphate-mobilizing strain Bacillus subtilis IMV B-7023 – components of Azogran, a complex bacterial preparation for plant growing. The dependence of the physiological and biochemical activity of these bacteria, including their antioxidant potential, on biotic and abiotic environmental agents was determined. The impact of a number of factors on chemotaxis, energy metabolism of these bacteria, their synthesis of substances of phenol nature, and other biologically active substances, which may influence the efficiency of using this preparation in plant growing, was studied. Azogran inhibits the spread of phytopathogens and some kinds of phytophages in agroecosystems, is capable of protecting plants from the oxidative stress and enhancing on 16–37 % their crop productivity.

Systemic effect of microbial preparations on causative microorganisms of bacterial diseases of tomato plants

Biologicals Phytohelp, Phytocide and Ekstrasol, based on the bacteria Bacillus subtilis, showed different antibacterial activity to phytopathogens, due to the peculiarities of used strains, cells titer and concentration of biologically active products of microorganisms. Biologicals Phytohelp and Phytocide showed high antibacterial activity against the agents of bacterial cancer C. michiganensis subsp. michiganensis and bacterial black spotting X. vesicatoria, and the no growth zone diameter ranged from 73 to 80 mm. Under these conditions no growth zone diameter of the studied strains C. michiganensis subsp. michiganensis and X. vesicatoria for microbiological preparation Ekstrasol did not exceed 40 mm. Among these preparations more active against bacterial speck of tomatoes P. syringae pv. tomato was microbial preparation Ekstrasol no growth zone diameter of which was 23±3.0 mm. Biological Azotofit, based on cells of nitrogen-fixing bacteria Azotobacter chroococcum, showed high antibacterial activity against the agent of bacterial cancer C. michiganensis subsp. michiganensis, no growth zone diameter was 78±2.0 mm. This preparation was middle-active against the agent of bacterial black spotting X. vesicatoria and showed no activity against the agent of bacterial speck of tomato P. syringae pv. tomato. It was revealed that the most active against the agents of bacterial black spotting and bacterial cancer are biologicals Phytocide and Phytohelp, based on the bacteria Bacillus subtilis that are recommended by us for use in the manufacture.

Effects of common oak (Quercus robur L.) defolition on the soil properties of an oak forest in Western Plain of Romania

This study was conducted to evaluate the variability of the chemical properties of the soil of an oak forest affected by defoliation and the corresponding microbial abundance. Soil samples were collected from a control zone (zone 1) without outbreaks of defoliating insects and from a sample zone (zone 2) where the trees were affected by Lymantria dispar L. defoliation. The research was conducted to determine the changed conditions for soil microorganisms produced as a consequence of defoliation. The results indicated, by means of analysis of variance (two-way ANOVA, P = 0.05), statistically significant differences (P < 0.0001) with respect to soil hydrolytic acidity, pH, ammonium nitrogen, heterotrophic bacteria, nitrogen fixing bacteria from genus Azotobacter and fungi. The data revealed a low number of heterotrophic bacteria and low pH values in samples taken from the area affected by defoliation. Soils under stands of defoliated trees showed higher values with respect to soil acidity, ammonium nitrogen, fungi and nitrogen fixing bacteria Azotobacter. Moreover, the soil moisture, nitrate nitrogen, organic matter content, organic carbon, the number of heterotrophic bacteria and the number of bacteria from genus Azotobacter exhibited statistically significant seasonal differences between the two zones studied. The correlations between the tested parameters showed that soil parameters such as moisture content, soil acidity, pH, organic matter content, organic carbon, total nitrogen and nitrate nitrogen are important factors influencing the soil populations of aerobic mesophilic heterotrophic bacteria, fungi and nitrogen fixing bacteria in the studied forest ecosystem.

The Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

BackgroundOriginally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1→4) linkages in maltodextrin/starch and the synthesis of consecutive (α1→6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD. MethodsThe purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D [1]H/[13]C NMR spectroscopy and enzymatic degradation studies. ResultsThe A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1→6) glucosidic bonds. Instead, it forms a high molecular mass and branched α-glucan with alternating (α1→4) and (α1→6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose. ConclusionsIn view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes. General significanceThis study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient.

Biological Remediation of the Petroleum and Diesel Contaminated Soil with Earthworms Eisenia Fetida

The study was performed of the bioremediation efficiency of the soil contaminated with petroleum and diesel with the help of earthworms E. fetida in the presence of bacteria Pseudomonas, nitrogen fixing bacteria Azotobacter and Clostridium, yeasts Saccharomyces, fungi Aspergillus and Penicillium, as well as Actinomycetales, all being components of biopreparation Baykal-EM. It was demonstrated that in petroleum-contaminated soil with 20-60 g/kg of petroleum, the content of hydrocarbons decreases by 99% after 22 weeks in the presence of worms and biopreparation. In diesel-contaminated soil with diesel concentration of 40 g/kg in the presence of earthworms E. fetida and microbiological preparation, 30% of the earthworm species died after 14 days because of the toxic impact of the diesel fuel.

Interrelated effects of mycorrhiza and free-living nitrogen fixers cascade up to aboveground herbivores.

Aboveground plant performance is strongly influenced by belowground microorganisms, some of which are pathogenic and have negative effects, while others, such as nitrogen-fixing bacteria and arbuscular mycorrhizal fungi, usually have positive effects. Recent research revealed that belowground interactions between plants and functionally distinct groups of microorganisms cascade up to aboveground plant associates such as herbivores and their natural enemies. However, while functionally distinct belowground microorganisms commonly co-occur in the rhizosphere, their combined effects, and relative contributions, respectively, on performance of aboveground plant-associated organisms are virtually unexplored. Here, we scrutinized and disentangled the effects of free-living nitrogen-fixing (diazotrophic) bacteria Azotobacter chroococcum (DB) and arbuscular mycorrhizal fungi Glomus mosseae (AMF) on host plant choice and reproduction of the herbivorous two-spotted spider mite Tetranychus urticae on common bean plants Phaseolus vulgaris. Additionally, we assessed plant growth, and AMF and DB occurrence and density as affected by each other. Both AMF alone and DB alone increased spider mite reproduction to similar levels, as compared to the control, and exerted additive effects under co-occurrence. These effects were similarly apparent in host plant choice, that is, the mites preferred leaves from plants with both AMF and DB to plants with AMF or DB to plants grown without AMF and DB. DB, which also act as AMF helper bacteria, enhanced root colonization by AMF, whereas AMF did not affect DB abundance. AMF but not DB increased growth of reproductive plant tissue and seed production, respectively. Both AMF and DB increased the biomass of vegetative aboveground plant tissue. Our study breaks new ground in multitrophic belowground–aboveground research by providing first insights into the fitness implications of plant-mediated interactions between interrelated belowground fungi–bacteria and aboveground herbivores.

FREE-FLOWING COMPLEX BACTERIAL PREPARATION FOR CROP AND EFFICIENCY OF ITS USE IN AGROECOSYSTEMS

The present study investigated the influence of different nanomaterials on physiological and biochemical activity of the nitrogen-fixing bacteria Azotobacter vinelandii IMV V-7076 and phosphate mobilizing bacteria Bacillus subtilis IMV V-7023 for the development of high-efficient free-flowing bacterial complex for crop production. Among the studied nanomaterials, vermiculite stimulated the most effectively bacterial growth, synthesis of amino acids and phytohormones, dehydrogenase, catalase and peroxidase activities. Based on vermiculite and highly efficient strains of bacteria Azotobacter vinelandii IMV V-7076 and Bacillus subtilis IMV V-7023, a free-flowing bacterial complex preparation for crop production was created. The preparation was stable during storage, it improved the nitrogenous and phosphorus nutrition of plants stimulated their growth by biologically active substances and protected plants from lesion by phytopathogenic micromicetes and bacteria.

Biological Remediation of the Engine Lubricant Oil-contaminated Soil with Three Kinds of Earthworms, Eisenia Fetida, Eisenia Andrei, Dendrobena Veneta, and a Mixture of Microorganisms

The survival of the earthworms in used engine oil-contaminated soil with concentration of 20-100g/kg was estimated. The investigation was made of the contaminated soil bioremediation efficiency with the use of Eisenia fetida, Eisenia andrei and Dendrobena veneta in presence of the photosynthetic bacteria Thiorhodaceae, Athiorhodaceae and Chlorobacteriaceae, nitrogen-fixing bacteria Azotobacter and Clostridium, yeast cells Saccharomyces, fungi Aspergillus and Penicillium, and Actinomycetales, composing microbiologic product Baykal EM. It is determined that the used oil concentration in the soils with earthworms decreases 4-10 times faster than in contaminated soil without earthworms. High rates of engine oil removal are registered in presence of E. andrei and D. veneta. Oil concentration in soil decreased by 60-90%. With oil concentration of 60-100g/kg and earthworms (Eisenia fetida) cultivation, the oil concentration decreased 4-6 times, with introduction of Californian earthworms (Eisenia andrei) 12 times and with Dendrobena veneta 12-23 times. At the addition of the microbiologic product along with earthworms, oil content in the soil decreased 58 times and constituted 1.1g/kg, soil remediation efficiency being 99.9%.

Melanin from the Nitrogen-Fixing Bacterium Azotobacter chroococcum: A Spectroscopic Characterization

Melanins, the ubiquitous hetero-polymer pigments found widely dispersed among various life forms, are usually dark brown/black in colour. Although melanins have variety of biological functions, including protection against ultraviolet radiation of sunlight and are used in medicine, cosmetics, extraction of melanin from the animal and plant kingdoms is not an easy task. Using complementary physicochemical techniques (i.e. MALDI-TOF, FTIR absorption and cross-polarization magic angle spinning solid-state 13C NMR), we report here the characterization of melanins extracted from the nitrogen-fixing non-virulent bacterium Azotobacter chroococcum, a safe viable source. Moreover, considering dihydroxyindole moiety as the main constituent, an effort is made to propose the putative molecular structure of the melanin hetero-polymer extracted from the bacterium. Characterization of the melanin obtained from Azotobacter chroococcum would provide an inspiration in extending research activities on these hetero-polymers and their use as protective agent against UV radiation.

English

Significant variation was recorded in various quantitative parameters of the mulberry with the application of various bio-inoculants like phosphate solubilising microorganisms, nitrogen fixing bacteria and arbuscular mycorrhizal fungi. The longest shoot length was recorded in T4 (175.43 cm) receiving 350: 140 kg/ha/year N and P along with co-inoculation followed by T6 (172.78 cm) receiving 175: 70 kg/ha/year N and P along with co-inoculation respectively. The treatment T8 receiving 175 kg nitrogen and 70 kg phosphorus/ha/year (with rock phosphate as a source of P) has indicated significantly higher number of shoots per plant (6.43) as compared to T0 control recording only (5.64) number of shoots per plant. Significantly higher number of leaves per shoot was recorded in treatment T8 (27.53) as compared to the rest of the treatments including T0 (26.37) as control. The study also clearly indicated that it is possible to curtail the application of nitrogenous and phosphatic fertilizers in mulberry cultivation to an extent of 25 to 50% without any adverse effect on leaf yield and quality by supplementing nitrogen and phosphorus through the use of nitrogen fixing bacteria Azotobacter, phosphate solubilizing bacteria, fungi and VA-mycorrhiza. The studies on economics of using bio-inoculants in mulberry also indicated that approximately Rs 2000 to 4500 /ha/year can be saved only on the input cost of nitrogen and phosphorus in mulberry cultivation. Key words: Mulberry, biofertilisers, economics.

Functional interaction of Azotobacter vinelandii cytoplasmic cyclophilin with the biotin carboxylase subunit of acetyl-CoA carboxylase

Cyclophilins (E.C. 5.1.2.8) are protein chaperones with peptidyl-prolyl cis/trans isomerase activity (PPIase). In the present study, we demonstrate a physical interaction among AvppiB, encoding the cytoplasmic cyclophilin from the soil nitrogen-fixing bacterium Azotobacter vinelandii, and AvaccC, encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. A decrease in AvppiB PPIase activity, in the presence of AvaccC, further confirms the interaction. However, PPIase activity seems not to be essential for these interactions since a PPIase active site mutant of cyclophilin does not abolish the AvaccC binding. We further show that the presence of cyclophilin largely influences the measured ATP hydrolyzing activity of AvaccA in a way that is negatively regulated by the PPIase activity. Taken together, our data support a novel role for cyclophilin in regulating biotin carboxylase activity.

Biochemical characterization of two Azotobacter vinelandii FKBPs and analysis of their interaction with the small subunit of carbamoyl phosphate synthetase

FK-506 binding proteins (FKBPs) belong to the peptidyl-prolyl cis/trans isomerase superfamily (PPIases, EC: 5.2.1.8) which catalyzes the interconversion of peptidyl-prolyl bonds while they can also act on polypeptides, as folding helper enzymes. Here, we biochemically characterize two recombinant FKBPs, AvfkbA1 and AvfkbA2, from the soil nitrogen-fixing bacterium Azotobacter vinelandii and show that both possess PPIase activity while AvfkbA2 possesses chaperone activity as well. Further, we demonstrate their physical interaction with AvcarA, the small subunit of carbamoyl phosphate synthetase. Using RT-qPCR, we show that AvfkbA1 and AvfkbA2 are co-expressed with AvcarA under the same growth conditions. A decrease in AvfkbA1 or AvfkbA2 PPIase activity, in the presence of AvcarA, further confirms each interaction. However, PPIase activity does not seem to be essential for these interactions since PPIase active site mutations of both FKBPs do not abolish the AvcarA binding. The P(358) residue of AvcarA, possibly retaining a cis configuration, is critical only for the interaction with AvfkbA1. The presence of either of the two FKBPs did not influence the measured glutamine hydrolyzing activity of AvcarA. Taken together, these data indicate that although the two FKBPs have a common biological substrate they probably have differing physiological roles.

Transcriptional and biochemical characterization of two Azotobacter vinelandii FKBP family members

Peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), a class of enzymes that catalyse the rate-limiting step of the cis/trans isomerization in protein folding, are divided into three structurally unrelated families: cyclophilins, FK506-binding proteins (FKBPs), and parvulins. Two recombinant FKBPs from the soil nitrogen-fixing bacterium Azotobacter vinelandii, designated as AvfkbX and AvfkbB, have been purified and their peptidyl-prolyl cis/trans isomerase activity against Suc-Ala-Xaa-Pro-Phe-pNA synthetic peptides characterised. The substrate specificity of both enzymes is typical for bacterial FKBPs, with Suc-Ala-Phe-Pro-Phe-pNA being the most rapidly catalysed substrate by AvfkbX and Suc-Ala-Leu-Pro-Phe-pNA by AvfkbB. Both FKBPs display chaperone activity as well in the citrate synthase thermal aggregation assay. Furthermore, using real-time RT-qPCR, we demonstrated that both genes were expressed during the exponential growth phase on glucose minimal medium, while their expression declined dramatically during the stationary growth phase as well as when the growth medium was supplied exogenously with ammonium.