Agric. Biol Chem., 49 (6), 1703-1709, 1985 1703 Identification of Nitrogen-fixing Hydrogen Bacterium Strain N34 and Its Oxygen-resistant Segregant Strain, Y38 Yoshihiro Nakamura, Takashi Yamanobeand Jiro Ooyama Fermentation Research Institute, Tsukuba Science City, Ibaraki 305, Japan Received October 29, 1984 A hydrogen bacterium strain, N34, and its oxygen-resistant segregant strain, Y38, were subjected to a taxonomical study. Since both strains were capable of N2-fixation, N2-fixing facultative hydrogen autotrophs listed in "Bergey's Manual of Systematic Bacteriology" were used for comparison. Both strains produced a water-insoluble carotenoid pigment, zeaxanthin di- rhamnoside, indicating that both should be classified into the genus Xanthobacter. Then, the differential characteristics of the two species of the genus Xanthobacter, X. autotrophicus and X. flavus, were investigated as to both strains. The vitamin requirement, the sensitivity to oxygen under autotrophic conditions, the inducibility of hydrogenase, the substrate range of carbohydrates and N2-fixing growth characteristics of both strains were almost completely opposite to those of X. flavus. Moreover, both strains coincided exactly with X. autotrophicus in morphological and other physiological characteristics. From these results both strains were identified as Xanthobacter auto trophicus. In 1971, Ooyama isolated a nitrogen-fixing strain N34 and its nitrogen-fixing growth on hydrogen bacterium from oily soil in a nor- manyorganic compounds,culture conditions thern district of Japan.1} This was the first and the effect of oxygen tension on nitrogenase isolation of a nitrogen-fixing hydrogen bac- have been studied.9~n) Recently, an oxygen- terium. After that, in 1974, Gogotov and resistant strain was isolated during plate culti- Schlegel also isolated some nitrogen-fixing hy- vation of strain N34 under high oxygen ten- drogen bacteria,2) followed by the isolation by sion using combined nitrogen as a nitrogen various authors of manyof the samekind of source.12) bacteria.3'40 All of these strains belonged to In the present study, we identified both Corynebacterium autotrophicum5) but the ob- strain N34 and strain Y38, the newly isolated servations on the Gramstain reaction of these oxygen-resistant segregant, as Xanthobacter strains were contradictory. After extensive autotrophicus.8) The identification was pre- taxonomical studies, Corynebacterium auto- liminarily oriented according to the tests for trophicum Baumgartenet ah was transferred a rapid and primary identification proposed to a newgenus, Xanthobacter, as Xanthobac- by Aragno and Schlegel.13) Then, the iden- ter autotrophicus (Baumgarten et ah).6) In tification of both strains was completed by 1980, the proposal of the new genus ofXantho- referring to the descriptions of species listed bacter was approved by the International in "Bergey's Manual of Systematic Bacteri- Committee on Systematic Bacteriology ology."^ This paper reports the results of this (ICSB),7) and the genus Xanthobacter Wiegel study. et ah appeared in the newly published "Bergey's Manual of Systematic Bacteri- MATERIALS AND METHODS ology. "8) The first isolated unidentified nitrogen- Organisms. Strain N341} isolated from oily soil in a fixing hydrogen bacterium was designated as northern district of Japan and strain Y38,12) an oxygen- 1704 Y. Nakamura, T. Yamanobe and J. Ooyama resistant segregant of strain N34, were used. termined by the Laybourn's modification of the staining Growth media. Both strains were grown for taxonomical method.16) Accumulation of poly-/?-hydroxybutyrate tests on nutrient agar at 30°C. For autotrophic growth, the (PHB) was detected with Sudan Black B.18) mineral mediumpreviously reported12) was used and the culture were incubated under an atmosphere ofH2, O2and Qualitative determination of carotenoid pigment. The CO2 (8:1:1, 7:2:1 or 5:4:1, by volume). For the carotenoid pigment of both strains was extracted with nitrogen fixation test, the N-source in the mineral medium acetone-methanol (3 : 1, v/v). The absorption spectrum wasomitted and the cultures were incubated as described was compared with that of zeaxanthin dirhamnoside.19) by Wiegel et al.6) For the test on N2-fixing growth characteristics, 0.2% K-gluconate was used as the C- Isolation ofDNAand determination of the G + C content. source and the cultures were incubated under gas mixtures Isolation of DNAwas carried out by Marmur's method.20) with various oxygen tensions balanced by N2. The DNAbase composition was determined from the thermal denaturation temperature (Tm) in accordance Tests for a rapid andprimary identification. As a means with the procedure of Marmur and Doty.21) The DNA of orientation for the identification of both strains, the from Escherichia coli strain Bwas used as a control. tests for a rapid and primary identification of hydrogen- oxidizing bacteria proposed by Aragno and Schlegel13) werecarried out. The test were on key characteristics such RESULTS as cell morphology, motility, Gramstaining, acid fast-test response, pigmentation, denitrification, nitrogen-fixation, Morphological characteristics tetrathionate reductase and utilization of carbon sources; sucrose, trehalose, D-fructose, D-xylose and benzoate. Cell The morphological characteristics of both form and growth on nutrient agar were observed after 1 to strains were as follows. Rod-shaped cells, 0.6 7 days' incubation at 30°C. Cell shape was also observed in to 1.3 by 1.4 to 6.2jum; branched cells were the mineral medium previously reported12) containing produced on succinate-containing medium. 0.2% Na-succinate. Motility was examined both by the Polyphosphate and poly-jS-hydroxybutyrate hanging drop method and stab culture on semisolid were accumulated in the cells. Non-motile. nutrient agar. Gramstain was determined by Hucker's modified method.14) The acid-fast test was carried out Gram-negative. Acid-fastness negative. Agar by the method of Ziehl-Neelsen.14) Other tests were colonies were circular, entire, convex, slimy carried out according to the methods of Aragno and and yellow pigmented. Figure 1 shows cells of Schlegel. 1 3) both strains. Other physiological characteristics. Nitrate reduction was determined in nitrate and succinate-nitrate broth15) Physiological and biochemical characteristics after 2, 4 and 7 days' incubation at 30°C. The Voges- The physiological and biochemical charac- Proskauer test response, indole production, catalase ac- teristics of both strains were as follows: Nitrate tivity, oxidase activity and urease activity were determined reduction positive. Denitrification negative. using the methods of Skerman.16) Hydrogen sulfide pro- Voges-Proskauer reaction negative. Indole duction was detected using triple-sugar iron (TSI) agar production negative. Hydrogen sulfide pro- after 10 days' incubation. Hydrolysis of starch was de- tected by the iodo-starch reaction after incubation on duction negative. Hydrolysis of starch neg- nutrient agar containing 0.2% soluble starch for 4 days. ative. Gelatin liquefaction negative. Catalase Liquefaction of gelatin was determined according to positive. Oxidase positive. Tetrathionate re- Smith's modification of the method of Frazier.14) ductase positive. Urease positive. Optimum Production of acid and gas from carbohydrates was temperature: 25~35°C. Optimum pH: observed for 2 weeks using peptone water15) supplemented with 1 %carbohydrate and 0.001% B.C.P. A Durham tube 6.0~9.5. Grew as chemolithoautotrophs in was inverted in a test tube. The carbohydrates tested were mineral mediumunder an atmosphere of H2, D-arabinose, D-cellobiose, D-fructbse, D-galactose, glucose, O2and CO2. Grew also as chemoorganotrophs inositol, lactose, maltose, mannitol, mannose, raffinose, D- on methanol, ethanol, w-propanol, ^-butanol, ribose, sucrose, trehalose and D-xylose. Utilization of sole carbon sources was investigated by the method of Stanier formate, acetate, citrate, gluconate, malonate, etal.17) propionate and succinate as sole carbon sources, Glucose, fructose, galactose, cello- Determination of polyphosphate and poly-$-hydroxy- biose, arabinose, maltose, sucrose, xylose, tre- butyrate. Formation of polyphosphate granules was de- halose, ribose, benzoate, phenylalanine and Identification of Nitrogen-fixing Hydrogen Bacteria 1705 Table I. Comparison of the Key Characteristics of Strains N34 and Y38 with Those of Xanthobacter autotrophicusa Characteristics N34, Y38 _ ^ _L. bXanthobacterautotrophicush Cell morphology M otility Gram stain A cid-fastness Pigm entation D enitrification N2 -fixation Tetrathionate reductase Growth on D-Fructose D-X ylose Sucrose Trehalose Benzoate a Symbols: cor, coryneform; r, rods; v, variable; y, yellow to orange. b According to Aragno and Schlegel.13) c Somestrains mayappear gram variable. the keys between both strains and X. auto- trophicus is shown in Table I. Amongthe key Fig. 1. Photomicrographs of Cells Grown Auto- characteristics in Table I yellow pigmentation, trophically. nitrogen-fixation and the presence of tetra- A, strain N34; B, strain Y38. thionate reductase seemed to be the diagnostic characters of X. autotrophicus. histidine were not utilized as sole carbon sources. Neither acid nor gas was produced Salient characteristics of nitrogen-fixing hy- from some of the carbohydrates listed in Ma- drogen autotrophs terials and Methods. Growth factors were Originally two main characters were con- not required. Atmospheric nitrogen was fixed sidered to be typical for Xanthobacter: