Isolation of Anitrogen-Fixing Vibrio Species from the Roots of Eelgrass (Zostera Marina)

J. Gen. App!. Microbiol., 33, 321-330 (1987)

ISOLATION OF ANITROGEN-FIXING VIBRIO SPECIES FROM THE ROOTS OF EELGRASS (ZOSTERA MARINA)

WUNG YANG SHIER, USIO SIMIDU, ANDYOSHIHARU MARUYAMA*

Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164, Japan * Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Bunkyo, Tokyo 113, Japan

A facultative nitrogen-fixing bacterium was isolated from the roots of eelgrass (Zostera marina) growing in Aburatsubo Inlet, Kanagawa Prefecture, Japan. The isolate grew anaerobically using molecular nitrogen as the sole nitrogen source. However, NH4C1 could also serve as a nitrogen source, under either aerobic or anaerobic conditions. The nitrogenase activity of the isolate was very weak under aerobic conditions, but was easily detected within a few hours under anaerobic conditions. It was enhanced by some sugars and was Na +-dependent. Morphological, physiological, and biochemical characteristics, as well as the guanine-plus- cytosine content of its DNA (45.4 + 0.3 mol%) placed the isolate in the genus Vibrio of the family Vibrionaceae. The isolate was not identical to two previously described nitrogen-fixing species of the genus Vibrio, Vibrio diazotrophicus and Vibrio natriegens.

Zostera marina (eelgrass) develops an extensive rhizome-root system beneath the sediment surface in coastal seawater. Different views have been presented on the importance of nitrogen fixation in supplying nitrogen to eelgrass (1-3). Although heterotrophic nitrogen fixation probably supplies a part of the nitrogen requirement (4), nitrogen-fixing bacteria have not been isolated before this. In our preliminary study, we found that the nitrogenase activity of eelgrass roots was greatly enhanced by the addition of glucose under either aerobic or anaerobic conditions. The results indicate that heterotrophic nitrogen-fixing bacteria are associated with the eelgrass roots. This report describes the isolation and characterization of a halophilic, facultatively anaerobic, nitrogen-fixing bacterium from eelgrass roots.

Address reprint requests to: Dr. W. Y. Shieh, Ocean Research Institute, University of Tokyo, Minamidai 1-15-1, Nakano-ku, Tokyo 164, Japan. 321 322 SHISH, SIMIDU, and MARUYAMA VOL. 33

MATERIALS AND METHODS

Media. A nitrogen-free liquid medium (NFL medium) used for the enrichment of nitrogen-fixing bacteria contained the following components: (i) basal medium (NaCI, 28 g; MgSO4.7H2O, 2 g; KBr,100 mg; SrCl2.6H2O, 26 mg; H3B03, 20 mg; Na2MoO4.2H2O, 10 mg; Tris, 50 mmol; distilled water, 800m1) adjusted to pH 7.8; (ii) glucose, 5 g; FeCl3.6H2O, 15 mg dissolved in 100 ml of distilled water; (iii) K2HPO4, 1 g dissolved in 50 ml of distilled water; (iv) sodium L-ascorbate, 0.5 g dissolved in 50 ml of distilled water. These were mixed after autoclaving separately. The PYG broth medium contained 2.0 g Polypepton (Daigo, Tokyo, Japan), 0.5 g Yeast extract (Difco), 5.0 g glucose, and 50 mmol Tris in 1 l of 9OO seawater. The pH was adjusted to 7.6. The PY medium was identical to PYG medium except that glucose and Tris were omitted. Isolation. Eelgrass roots were collected from an eelgrass field in Aburatsubo Inlet, Kanagawa Prefecture, Japan, and were washed free of sediment by vigorous shaking in sterile seawater. To measure the nitrogenase activity of the eelgrass roots, 1-g, wet weight, samples were placed in 20-m1 test tubes. To each tube was added 2 ml of seawater buffer (20 mM Tris in 80O seawater, pH 7.6) or the buffer containing glucose at a concentration of 1 j;. The nitrogenase activity of eelgrass roots was found to be greatly enhanced by the addition of glucose under either aerobic or anaerobic conditions. One of the glucose-amended root samples that showed high nitrogenase activity under anaerobic conditions was used for the isolation of nitrogen-fixing bacteria. The roots together with the supplemented solution were homogenized and then 1 ml of the homogenate was serially diluted with 9 ml of sterile seawater containing 0.05°c sodium L-ascorbate. A 0.1 ml aliquot of each diluent was transferred to a 40-ml L-shaped tube containing 5 ml of NFL medium. After the tube was sealed with a rubber stopper, the contained air was replaced with nitrogen. The cultures were gently shaken at 25°C for about 3 days. The culture of the highest dilution that showed marked turbidity was again serially diluted, and the above procedure was repeated. For the final isolation of nitrogen- fixing bacteria, the second enrichment culture of the highest dilution positive for growth was spread on PY agar plates and the plates were incubated at 25°C for 3 days under anaerobic conditions using a GasPak Anaerobic System (BBL). Thirty colonies were picked up at random and assayed for nitrogenase activity in NFL medium. Measurement of nitrogenase activity. The nitrogenase activity of the isolates was measured by the acetylene-reduction assay method. After incubation in NFL medium under nitrogen atmosphere for 30 to 40 hr, the cells were harvested by centrifugation and then washed with artificial seawater containing 0.0500 sodium L- ascorbate. The artificial seawater consisted of 0.4 M NaCI, 0.1 M MgSO4.7H2O, 0.02 M KCI, and 0.02 M CaCl2.2H2O. The cells were resuspended in 5 ml of NFL or its modified medium, distributed in the 32-ml test tubes, and incubated at 25°C. The optical density at 600 nm of the cell suspensions was adjusted to about 0.25. To 1987 A N2-fixing Bacterium Isolated from Eelgrass Roots 323 measure aerobic nitrogenase activity, 3.2 ml of the air in the test tube was removed and then the same volume of acetylene gas was injected with a gas-tight syringe; for measuring anaerobic nitrogenase activity, all the air in the test tube was replaced with Ar/C2H2 (90% :10%, v/v). At 4-hr intervals, 0.2 ml of gas was removed with a gas-tight syringe and analyzed for ethylene and acetylene using a Shimadzu G-4CM gas chromatograph equipped with a flame ionization detector (Porapak R column 3 mm x 50 cm, 50°C, nitrogen carrier gas, 40 ml mm). Specific activities were calculated between 4 and 8 or 8 and 12 hr of incubation. No growth occurred during the period when nitrogenase activity was measured. Growth studies. The strain RSI19, which showed the highest nitrogenase activity among the isolates, was gently shaken at 25°C in NFL medium. NH4C1 when added to NFL medium served as a nitrogen source for cells growing under aerobic or anaerobic conditions. Taxonomic tests. Taxonomic tests were carried out according to the con- ventional procedures as described by SMIBERTand KRiEG(5) with exceptions and additional tests as cited below. Pigmentation, swarming, and luminescence were determined after growth for 24 to 48 hr on the PY agar plates. Luminescence was recorded after the plate had been observed for 5 min in the dark. Poly-/3-hydroxybutyrate (PHB) accumulation was tested on modified NFL media containing O.2° glucose or O.2° sodium-DL-/3- hydroxybutyrate instead of glucose as the carbon source and supplemented with 2mmol of NH4C1 per liter. The cultures were examined with a phase-contrast microscope, daily for 5 days (6). Sensitivity to 2,4-diamino-6,7-diisopropyl-pteridine (0/ 129) phosphate was determined by a disc-sensitivity testing method on PY agar plate. The discs contained 10 ug or 150 ug of 0/ 129 phosphate. Growth at different concentrations of NaCI was determined in tryptone (100, w/v) broth containing 0, 1, 3, 6, and 8 % NaCI. Ability to grow at various temperatures was tested using PY broth medium. Growth was recorded after incubation for 14 days at 4°C, and 2 days at 20, 35, and 40°C. The presence of alginase and chitinase was tested as described by BAUMANNet al. (7). The ability to produce acid from different carbohydrates was tested on stab media which differed from PY medium by containing 3 g of agar per liter, 15 ml of 0.2°c (w/v) of bromthymol blue solution per liter, and 10 g of the carbohydrates per liter. The surface of the medium was sealed with sterile liquid paraffin after inoculation. The cultures were examined for color change for a period of 7 days. NaCI was added to all tested media that did not already contain at least 0.500 NaCI to a final 100 concentration. Cells grown for 6 hr in PY broth medium at 25°C were negatively stained with potassium phosphotungstate (1.0%, w/v, pH 7.0). Photographs were taken with a JEM-100C electron microscope. Ability to utilize various compounds as sole sources of carbon and energy was determined using the modified NFL media containing 0.2°c of the tested carbon sources instead of glucose and supplemented with 2 mmol of NH4C1 per liter. Unless otherwise stated, all the tested cultures were incubated at 25°C for 7 days under aerobic conditions. Guanine-plus-cytosine content of the DNA. The guanine-plus-cytosine content 324 SHISH, SIMIDU, and MARUYAMA VOL. 33 of the bacterial DNA was determined by high-performance liquid chromatogFaphy (HPLC). Directly determining the guanine-plus-cytosine content of DNA using different models of HPLC and a wide range of column types have been found to give satisfactory results (8-10). DNA was extracted and purified by the method of MARMuR(11) with some modifications. For enzymic hydrolysis of RNA, ribonuclease A (Sigma) and ribonuclease T1 (Sigma) were used. The purified DNA was dissolved in 10 mM phosphate buffer (pH 7.0,1 mg/ml). Then the DNA solution was heated at 100°C for 15 min, and cooled rapidly in an ice bath. The denatured DNA solution (20 µl) was mixed with 20 , it of nuclease P1 solution (0.1 mg dissolved in 1 ml of 40 mM sodium acetate buffer containing 2 mM Zn5O4, pH 5.3), and incubated at 50°C for 1 hr. The hydrolysate was stored at -20 °C and 10 µl of the hydrolysate was applied on reversed-phase HPLC. The present HPLC system consisted of a Jasco Tri Rotor VI pump, a Jasco Uvidec 100-VI variable wavelength detector at 260 nm, and a Jasco DP-L220 LC data processor. An HPLC column of Senshu Pak ODS-1251-N (4.6 x 250 mm) was packed with 5 j ODS materials (M. Nagel, Germany). The nucleotides were eluted by 10 mM phosphate buffer (pH 7.0) at a flow rate of 1 ml/min at 30°C. The standard mixture of four deoxyribonu- cleotides and nuclease PI were obtained from Yamasa Shoyu (Choshi, Chiba, Japan).

RESULTS

Isolation High nitrogenase activity was observed in all the replicate samples of glucose- amended roots, although little or no nitrogenase activity appeared with unamended roots. After 48 hr incubation, all glucose-amended root samples had a nitrogenase activity higher than 1,000 nmol ethylene/g wet wt under either aerobic or anaerobic conditions. With anaerobic enrichment, bacterial growth occurred in the tubes with the nitrogen-free liquid medium (NFL medium) inoculated with decimal dilutions of homogenized roots. The 106 dilution was the highest dilution culture exhibiting positive growth. All colonies from the plating of the second enrichment culture of the highest dilution were apparently of the same type indicating a possible origin from the same cell. Six isolates selected for characterization tests proved to be identical in every respect. The strain RSI19 that showed the highest nitrogenase activity in NFL medium was chosen for further studies.

Growth and nitrogenase activity of strain RSI19 The nitrogenase activity of strain RSI19 was very weak under aerobic conditions, but was easily detected in a few hours under anaerobic conditions. As in Table 1, showing the effect of carbohydrate, glucose and sucrose were the most effective enhancers of anaerobic nitrogenase activity, while mannose was not effective. NaCI was required for the nitrogenase activity. The activity was highest at 3 % NaCI, very low at 7%, and with Nat-free medium the activity was negligible 1987 A N2- fixing Bacterium Isolated from Eelgrass Roots 325

Table 1. Effect of carbon source on nitrogenase activity of strain RSI19 incubated under anaerobic conditions.u

Table 2. Effect of NaCI on nitrogenase activity of strain RSII9 under anaerobic conditions.u

Fig. 1. Growth of strain RSI19 with Nz or NHS as the nitrogen source under different gas phase conditions. a NFL medium; b, NFL medium supplemented with 1 mM NH4CI. 326 SHIEH, SIMIDU, and MARUYAMA VOL. 33

Fig. 2. Strain RSIlQ grown for 6 hr in PY broth medium, showing a polar flagellum.

(Table 2). Although nitrogenase activity has been reported for some halophilic, facultatively anaerobic bacteria, no growth has been observed in N-free media with these bacteria (12, 13). As shown in Fig. 1, however, strain RSI 19 exhibited significant anaerobic growth in the NFL medium. The optical density at 600 nm increased from about 0.05 to 0.55 during 30 hr. No growth occurred in this medium aerobically. NH4Cl, when added to NFL medium, served as an N source for cells growing both aerobically and anaerobically under Ar (Fig. 1).

Identification Strain RSI19 was gram-negative. Cells grown in PY broth medium were straight or slightly curved rods and motile by means of a single polar flagellum (Fig. 2). The cell size was 2.0-2.5 x 0.4-0.5 ,um. Colonies produced on PY or PYG agar plates were circular, convex, and unpigmented. The biochemical and physiological characteristics of strain RSI19 are summarized in Table 3. The strain was facultatively anaerobic and oxidase-positive. It fermented glucose and other carbohydrates without producing gas, and required NaCI for its growth. Its DNA base composition was 45.4 ± 0.3 mol 0 G + C. The results of the sole carbon source utilization tests are presented in Table 4. 1987 A N2-fixing Bacterium Isolated from Eelgrass Roots 327

Table 3. Biochemical and physiological characteristics of strain RSI19.

DISCUSSION

Nitrogen fixation is a property found only among prokaryotic organisms. Cyanobacteria and bacterial species belonging to a wide variety of families and genera are able to fix molecular nitrogen. Among the nitrogen-fixing bacteria, members of Enterobacteriaceae are very common and are facultatively anaerobic, gram-negative rods. Here, strain RSI19, a facultatively anaerobic, gram-negative rod, is placed in the Vibrionaceae, not in Enterobacteriaceae, because it is oxidase- positive, halophilic, and motile by means of a single polar flagellum. The NaCI requirement for growth and the lower guanine-plus-cytosine content (45.4 + 0.3) showed that the strain belongs to the genus Vibrio or Photobacterium. Strain RSI19, like Photobacterium spp., was able to accumulate PHB as an intracellular product. However, it was excluded from the genus Photobacterium according to the following characteristics: the ability to utilize fl-hydroxybutyrate and mannitol, the production of amylase, and the inability to utilize mannose. For the above reasons, we 328 SHISH, SIMIDU, and MARUYAMA VOL. 33

Table 4. Ability of strain RSI19 to utilize compounds as sole carbon and energy sources.

placed strain RSI19 in the genus Vibrio. Strain RSI19 has some characteristics that are not common among the members of Vibrio, such as acid production from inositol, xylose, and sorbitol, the ability to accumulate PHB, the ability to utilize malonate and f3-hydroxybutyrate as the sole carbon sources, and the inability to hydrolyze casein, DNA, chitin, and lecithin. In addition, the bacterium is resistant to a disc containing 150 µg of 0/129. However, these characteristics are insufficient to exclude the bacterium from the genus Vibrio. Vibrio spp. are usually reported to be sensitive to 0/ 129. However, in a numerical taxonomy study, many strains of Vibrio species were shown to be resistant to 0/129(14). Although the current definition of Vibrio species states that no species fixes molecular nitrogen (I5), recent studies show that some strains of Vibrio natriegens isolated from a geographically diverse range of habitats can fix nitrogen (13). Vibrio diazotrophicus, a new nitrogen- fixing species was also reported recently, and this species is distributed in seawater, sediment and the gastrointestinal tracts of marine animals (12, 16). The nitrogenase activities of these bacteria, however, are quite weak compared with that of typical nitrogen-fixing bacteria: Several days of incubation are required before these activities are detectable (12, 13). Strain RSI19, which has significant nitrogenase activity in a few hours under anaerobic conditions is the first nitrogen-fixing bacterium of Vibrio species isolated from marine seagrass. Characteristics distinguishing strain RSI19 from the known nitrogen-fixing species of Vibrio are shown in Table 5. Strain RSI19, like V. natriegens, has certain characteristics that are not common among the genus Vibrio, including the ability to accumulate PHB and to utilize ,8-hydroxybutyrate and malonate. Nevertheless, this bacterium is distinguishable from V, natriegens by a combination of a variety of characteristics (Table 5). 1987 A N2- fixing Bacterium Isolated from Eelgrass Roots 329

Table 5. Characteristics distinguishing strain RSI19 from Vibrio diazotrophicus° and Vibrio natriegens!

In the present experiment, aerobic enrichment cultures of the homogenate of glucose-amended roots did not show visible growth on the enrichment medium, so aerobic nitrogen-fixing bacteria were not isolated. Facultative nitrogen-fixing bacteria, including the present Vibrio isolate, may be the most important nitrogen- fixing bacteria associated with eelgrass roots.

We are grateful to Ms. K. Kita-Tsukamoto, Ocean Research Institute, University of Tokyo, for assistance with the electron microscopy. Part of the present study was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries for "Promotion of Biotechnology Research."

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