During Stationary Phase, Beijerinckia Derxii Shows Nitrogenase Activity Concomitant with the Release and Accumulation of Nitrogenated Substances
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Microbiol. Res. (2003) 158, 309–315 http://www.urbanfischer.de/journals/microbiolres During stationary phase, Beijerinckia derxii shows nitrogenase activity concomitant with the release and accumulation of nitrogenated substances Natália Reiko Sato Miyasaka1, Daniela Strauss Thuler1, Eny Iochevet Segal Floh2, Walter Handro2, Mariana Braga Duarte Toledo1, Sônia Maria Gagioti3 and Heloiza Ramos Barbosa1* 1 Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo. Av. Prof. Lineu Prestes, 1374, CEP- 05508-900, São Paulo, Brasil 2 Plant Cell Biology Laboratory, Department of Botany, Institute of Biosciences, University of São Paulo. Rua do Matão, 277- CEP. 05422-970, São Paulo, Brasil 3 Department of Histology and Embriology, Institute of Biomedical Sciences, University of São Paulo. Av. Prof. Lineu Prestes, 1524, 05508-900, São Paulo, Brasil Accepted: July 22, 2003 Abstract Beijerinckia derxii, a free-living nitrogen-fixing bacterium, the activity of their nitrogenase enzyme. Metabolic and maintained an increasing nitrogenase specific activity during genetic adaptations ensure that the nitrogen fixed by the stationary growth phase. To verify the destination of the the bacteria may be used by the microorganism itself, nitrogen fixed during this phase, intra and extracellular nitro- as free-living bacteria or by the host plant in symbio- genated contents were analyzed. Organic nitrogen and amino tic associations (Schubert, 1995). The very low NH + acids were detected in the supernatant of the cultures. An 4 increase in intracellular content of both nitrogen and protein assimilation by bacteroids (Espín et al., 1994) and the occurred. Cytoplasmic granules indicated the presence of inhibition of microbial cell division (Postgate, 1998) + arginine. The ability of a non-diazotrophic bacterium (E. coli) are features that promote NH4 excretion. Free-living to use B. derxii proteins as a source of nitrogen was observed bacteria, on the other hand, being fully able to engage in concomitantly with E. coli growth. There is a suggestion that cell division, need to incorporate fixed nitrogen into B. derxii contributes to the environment by both releasing their own structures and proteins. These microorga- nitrogenated substances and accumulating substances capable + nisms have an efficient system for NH4 assimilation. of being consumed after its death. Since low intracellular nitrogen levels are necessary for diazotrophic microorganisms to perform nitrogen fixa- Key words: Beijerinckia – N fixation – nitrogenated sub- 2 tion (Pati et al., 1994), nitrogenated substances not stances incorporated into the cell structures and enzymes must be excreted or stored as insoluble material. In general, the excretion of ammonium by free-living diazotrophs is observed in mutants that suffered physiological Introduction suppression or genetic manipulation of the enzymes involved in ammonium assimilation (Bali et al., 1992). However, Narula et al. (1981) showed that several The ability of N2-fixing bacteria to survive as free-living diazotrophs or as symbionts associated to plants, deter- strains of Azotobacter chroococcum and two strains of mines the destination of the nitrogen fixed as a result of A. vinelandii, obtained from laboratories or isolated from the soil, were able to release ammonium into the culture medium (in concentrations varying from traces –1 Corresponding author: Heloiza Ramos Barbosa to 46 µg.ml ). There is evidence that free-living N2- e-mail: [email protected] fixing bacteria release substances that may be utilized 0944-5013/03/158/04-309 $15.00/0 Microbiol. Res. 158 (2003) 4 309 by other organisms. Pati et al. (1994) showed that phyl- Cultures preparation. B. derxii inoculum was carried lospheric isolates identified as Beijerinckia indica, out in N-free LGa medium. Culture I preparation: A. chroococcum and Corynebacterium sp were able to an Erlenmeyer flask (500 mL) containing 130 mL of the release several amino acids. LGb medium was inoculated with 20 mL of a 48h One way of keeping the product of nitrogen fixation B. derxii culture (about 108 CFU.mL–1), incubated for inside the cell is to accumulate a nitrogen reserve. 74 h at 30°C in a rotary shaker (200 rpm), followed by Such nitrogen reserves were only observed in some a still incubation at 30°C, for a maximum period of cyanobacteria. During the stationary phase, and when in 550 h. Periodically, samples of the culture were taken to the presence of an excessive large source of nitrogen determine: a) Viable Cells Counts (Colony Forming (Suarez et al., 1999) and a shortage of other essential Units-CFU) using the drop method (Barbosa et al., nutrients (Newton and Tyler, 1987), these organisms 1995). Six replicates of the dilutions of the culture were produced cyanophicin, a peptide consisting of co-poly- plated on solid LGa medium. The B. derxii CFU number mers of aspartic acid and arginine. in stationary phase was confirmed by direct counting This paper deals with the destination of the products (Koch, 1994); b) Cell Protein Content, according to of N2 fixation by Beijerinckia derxii, not used for cell Lowry (1951) and Bradford (1976) methods and c) growth, for the purpose of gaining a better understand- Nitrogenase Activity, using the acetylene reduction ing of the ecological role of this free-living N2-fixing assay (Turner and Gibson, 1980). In a 384 h B. derxii bacterium. Using bacteria grown in a N-free medium, culture, arginine was tested in cell granules by the Saka- the authors seeked to establish the correlation between guchi reaction (Pearse, 1968). nitrogenase activity, i.e., the enzyme responsible for Extracellular determinations in B. derxii cultures providing fixed nitrogen to the cells, and the factors were performed in two supernatants called A and B. listed as follows: growth phase, liberation of nitro- Supernatant A, was obtained by centrifugation of the genated substances and accumulation of both N/protein culture at 12,100 x g for 30 min and filtered through inside the cells. Moreover, a model using a non-diazo- Millipore membranes (0.22 µm). Supernatant B was trophic microorganism (E. coli) was constructed in obtained by initial centrifugation of the culture at order to observe the possible consumption by this 12,100 x g for 30 min. After that, several re-suspensions bacterium of nitrogenated material, measured as pro- of cells in water and centrifugations followed, until the tein, proceeding from disrupted B. derxii cells. cells were free from a mucous layer which detached itself from the cells as a gel. The supernatant B, a pool of all centrifugations, was filtered through a 0.22 µm Millipore membrane to discard the remaining cells. Materials and methods Supernatant A was used to determine ammonium con- tent, using the method of Chaney and Marbach (1962), glucose using the method of glucose oxidase (Henry Bacterial strains. Beijerinckia derxii, a free-living, N - 2 et al., 1974), extracellular protein using the method of fixing, bacterium, was isolated by our group from acid Bradford (1976) with bovine serum albumin (Fluka) as soil, which is also poor in nitrogen and organic matter, standard, and amino acids (only in the supernatant of a in Pirassununga, Brazil. The bacterium was identified 250 h grown culture) by the method described below. To and catalogued as ATCC 33962. Escherichia coli ICB19 determine total extracellular nitrogen (converted to was isolated from human faeces in our laboratory and NH +) in supernatant B by the method of micro-Kjeldahl identified by biochemical and morphological tests 4 (Daniels et al., 1994; Eaton et al., 1995), this prepara- (Farmer III, 1995). This bacterium was chosen to indi- tion was concentrated to a volume compatible with the cate if B. derxii nitrogenated substances might be method used (8–80 µg.ml–1). consumed as a N source. – Amino acid analysis – Fifteen ml samples from super- Culture media. LGa medium, containing (mM): natant A, previously filtered through Millipore mem- K HPO , 0,57; CaCl .2H O, 0,14; MgSO .7H O, 0,81; 2 4 2 2 4 2 branes (0.22 µm), were centrifuged (14,000 x g, for Na MoO .2H O, 0,008; KH PO , 2,2; FeCl . 6H O, 2 4 2 2 4 3 2 0.5 h) to detect amino acids (Astarita et al. 2003) by 0,037; CoCl .2H O, 0,0054 and glucose, 55,0 (pH 5.7). 2 2 High-Performance Liquid Chromatography (HPLC). For solid medium, 12 g.l–1 agar were added. An LGb medium, similar to LGa except for the phosphate Culture II preparation: another culture, called the concentration being raised to 9,6mM K2HPO4 and culture II was prepared, in which E. coli cells were cul- 40 mM KH2PO4 (pH 6.2), was also employed. Media tivated in a LGb medium to which disrupted components were supplied by Merck AG (Darmstadt) B. derxii cells were added. A sonicated diazotroph or by Difco: Nutrient broth (NB) and nutrient agar suspension (8 ml) and 125 µl of a diluted E. coli suspen- (NA). sion (about 4.107 CFU.ml–1) were added to 42 ml of an 310 Microbiol. Res. 158 (2003) 4 LGb medium. The sonicated suspension was obtained plained by the following. The idea that bacteria are as follows. A 504 h B. derxii culture was centrifuged at capable of regu-lating their metabolic reactions to 12,100 x g for 30 min at 4°C. The cells were washed achieve maximum economy and efficiency, in order to with sterile distilled water. The cells were re-suspended obtain yield of cells proportional to the amount of ATP in LGb medium and sonicated in a Branson Sonifier produced, is contradicted by the observation that 450, in an ice bath, for about 5 min. The confirmation “resting-cells suspensions” can utilize energy sources in of cell burst was done by obser-vation under an optical the complete absence of growth (Russell and Cook, microscope.