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Other Gram-Positive Bacteria

Paenibacillus forsythiae sp. nov., a nitrogen-fixing species isolated from rhizosphere soil of Forsythia mira

Yu-Chao Ma, San-Feng Chen

  • 1National Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, PR China
  • 2Key Laboratory of Agro-Microbial and Application, China Agricultural University, Beijing 100094, PR China
  • 3College of Biological Sciences, China Agricultural University, Beijing 100094, PR China
  • Correspondence
    San-Feng Chen
    chensf{at}cau.edu.cn

    • International Journal of Systematic and Evolutionary Microbiology 2008; 58(2):319–323 · https://doi.org/10.1099/ijs.0.65238-0

    View at publisher PubMed

    Abstract

    A nitrogen-fixing bacterium, designated strain T98T, was isolated from rhizosphere soil of Forsythia mira. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain T98T was a member of the genus Paenibacillus. High levels of 16S rRNA gene similarity were found between strain T98T and Paenibacillus durus ATCC 35681T (97.0 %), Paenibacillus sabinae DSM 17841T (98.3 %) and Paenibacillus zanthoxyli DSM 18202T (96.8 %). Levels of 16S rRNA gene sequence similarity between strain T98T and the type strains of other recognized members of the genus Paenibacillus were below 97.0 %. Levels of DNA–DNA relatedness between strain T98T and P. durus ATCC 35681T, P. sabinae DSM 17841T and P. zanthoxyli DSM 18202T were 27.6, 30.0 and 32.1 %, respectively. The DNA G+C content of strain T98T was 50.4 mol%. The major fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. On the basis of its phenotypic characteristics and levels of DNA–DNA hybridization, strain T98T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus forsythiae sp. nov. is proposed. The type strain is T98T (=CCBAU 10203T =DSM 17842T).

    • The GenBank/EMBL/DDBJ accession numbers for the partial nifH gene sequence and 16S rRNA gene sequence of strain T98T are respectively DQ349124 and DQ338443.

    • A neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic position of strain T98T compared with all recognized species of the genus Paenibacillus is available as supplementary material with the online version of this paper.

    The genus Paenibacillus was erected by Ash et al. (1993) on the basis of analysis of the 16S rRNA gene sequences of group 3 bacilli. Members of the genus are widely distributed in nature and have physiologically diverse characteristics. Some species consistently show a great capacity to fix atmospheric nitrogen in vitro (Berge et al., 2002; Ma et al., 2007a, b; Ding et al., 2005; Elo et al., 2001; Grau & Wilson, 1962; Rodríguez-Díaz et al., 2005; Seldin et al., 1984). Here we show that a bacterial strain, designated T98T, isolated from rhizosphere soil of Forsythia mira represents a novel nitrogen-fixing species of the genus Paenibacillus.

    Strain T98T was isolated from rhizosphere soil of F. mira by screening on nitrogen-free medium (Ma et al., 2007a). The nitrogen-free medium contained 20 g sucrose, 0.1 g K2HPO4, 0.4 g KH2PO4, 0.2 g MgSO4 . 7H2O, 0.1 g NaCl, 0.01 g FeCl3 and 0.002 g Na2MoO4 per litre of water. One gram of soil was placed in 9 ml sterile water and stirred for 50 min. Aqueous volumes (100 μl of the mixture) were heated at 80 °C for 10 min and then spread on nitrogen-free medium in triplicate and incubated at 30 °C. After 3 days incubation, strain T98T was selected for further study.

    To confirm the nitrogen-fixing capacity of strain T98T, an assay for nitrogenase activity and PCR amplification of the nifH gene were carried out. For measurement of nitrogenase activity, strain T98T and several reference strains were grown on solid RCV mineral medium containing 0.5 % glucose, malate, starch and mannitol (Weaver et al., 1975). After 48 h at 30 °C, strains were incubated under 2 % (v/v) acetylene in air for 2 h and then analysed for ethylene production by GC (Berge et al., 2002). As shown in Table 1⇓, strain T98T exhibited high nitrogenase activity. Whole-cell DNA for PCR amplification was extracted according to standard methods (Sambrook et al., 1989). A 324-bp fragment of the nifH gene was amplified by using two degenerate primers for the nitrogenase Fe protein gene and then sequenced as described by Ding et al. (2005). A phylogenetic tree was generated by the neighbour-joining method by using the software package treecon for Windows (Van de Peer & de Wachter, 1994). Phylogenetic analysis based on nifH gene sequences revealed that strain T98T clustered together with species of the genus Paenibacillus (Fig. 1⇓). Levels of nifH gene sequence similarity between strain T98T and Paenibacillus zanthoxyli DSM 18202T, Paenibacillus sabinae DSM 17841T, Paenibacillus durus ATCC 35681T, Paenibacillus polymyxa DSM 36T, Paenibacillus odorifer TOD45T, Paenibacillus graminis RSA19T, Paenibacillus macerans ATCC 8244T and Paenibacillus wynnii LMG 22176T were 98.15, 97.0, 96.30, 89.0, 88.07, 80.73, 81.65 and 84.0 %, respectively.

    Figure image not available in archive
    Fig. 1.

    Phylogenetic tree based on partial nifH gene sequences, showing the position of strain T98T in relation to other reference bacterial species. Bootstrap analyses were made based on 1000 cycles; only values >50 % are shown at branch points. Bar, 0.1 substitutions per nucleotide position.

    Table 1.

    Nitrogenase activity of strain T98T compared with some nitrogen-fixing species of the genus Paenibacillus

    Results are means±sd of three determinations.

    A full-length sequence of the 16S rRNA gene (1500 bp) was obtained from a PCR product amplified from strain T98T by using the universal forward primer P1 and the universal reverse primer P6. Primer P1 (5′-AGAGTTTGATCCTGGTCAGAACGCT-3′) corresponds to positions 8–37 and primer P6 (5′-TACGGCTACCTTGTTACGACTTCACCCC-3′) corresponds to positions 1479–1506 in the Escherichia coli 16S rRNA gene (Yanagi & Yamasato, 1993). The amplified 16S rRNA gene was sequenced by using an ABI377 automatic sequencer (Applied Biosystems). The 16S rRNA gene sequence of strain T98T was aligned with sequences of recognized species of the genus Paenibacillus by using the clustal_x program (Thompson et al., 1997). Phylogenetic analysis revealed that strain T98T clustered together with Paenibacillus species (Fig. 2⇓; an extended version of this tree is available as Supplementary Fig. S1 in IJSEM Online). Strain T98T showed highest 16S rRNA gene sequence similarity with P. sabinae DSM 17841T, P. durus ATCC 35681T, P. zanthoxyli DSM 18202T and Paenibacillus stellifer DSM 14472T (98.3, 97, 96.8 and 96.5 %, respectively). Levels of 16S rRNA gene sequence similarity between strain T98T and other recognized members of the genus Paenibacillus were below 96 %.

    Figure image not available in archive
    Fig. 2.

    Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain T98T among species of the genus Paenibacillus. Bacillus subtilis NCDO 1769T was used as an outgroup. Bootstrap analyses were made based on 1000 cycles; only values >50 % are shown at branch points. Bar, 0.1 substitutions per nucleotide position. An extended version of this tree including all species of Paenibacillus with validly published names is available as Supplementary Fig. S1 in IJSEM Online.

    Strain T98T was tested for a range of phenotypic, physiological and biochemical characteristics together with the type strains of closely related Paenibacillus species, including P. durus ATCC 35681T, P. sabinae DSM 17841T, P. zanthoxyli DSM 18202T, P. polymyxa ATCC 842T, P. macerans ATCC 8244T, P. odorifer TOD45T and P. graminis RSA19T, as reference strains. For observation of cell morphology, strain T98T was grown on endospore-forming medium [0.07 % yeast extract, 0.1 % tryptone, 0.1 % glucose, 0.02 % (NH4)2SO4, 0.02 % MgSO4 . 7H2O and 0.1 % K2HPO4, pH 7.2] for 72 h, and cells were then examined by light microscopy. Strain T98T formed ellipsoidal spores, located centrally in swollen sporangia. Colonies on RCV mineral medium agar were circular, convex and glossy with entire margins. Gram-staining demonstrated that cells of strain T98T were Gram-positive or Gram-variable rods (0.4–0.5×2.0–2.6 μm). Cell motility was determined by using two methods, i.e. directly by light microscopy and observation of growth spread of cells in test tubes containing semisolid RCV medium after 3 days incubation. Cells of strain T98T were motile. To determine aerotactic ability, bacterial cells were inoculated by mixing them with semisolid medium at 40–50 °C in test tubes followed by growth at 30 °C for 3 days. Although cells of strain T98T grew throughout the medium in test tubes, cells near the surface grew better than those near the bottom, suggesting that strain T98T was facultatively aerobic.

    Most physiological and biochemical tests, including Gram staining, nitrate reduction, production of dextrin, temperature and pH optima for growth, activities of catalase, oxidase and the Voges–Proskauer reaction, growth inhibition by NaCl and lysozyme, were performed according to Gordon et al. (1973), Priest et al. (1981) and Rhodes-Roberts (1981). Hydrolysis of casein, gelatin and starch was determined as described by Cowan & Steel (1965). Utilization of various substrates as carbon and energy sources was determined as described by Shirling & Gottlieb (1966). The physiological and biochemical characteristics of strain T98T compared with those of the type strains of closely related Paenibacillus species are presented in Table 2⇓. As shown, strain T98T has physiological properties that allowed its distinction from these recognized Paenibacillus species.

    Table 2.

    Differential phenotypic characteristics between strain T98T and the type strains of selected Paenibacillus

    Strains: 1, strain T98T; 2, P. durus ATCC 35681T; 3, P. sabinae DSM 17841T; 4, P. zanthoxyli DSM 18202T; 5, P. polymyxa ATCC 842T; 6, P. macerans ATCC 8244T; 7, P. odorifer TOD45T; 8, P. graminis RSA19T. +, Positive; −, negative; nd, not determined. Data for physiological and biochemical characteristics of strain T98T and reference strains are from the present study. Data for G+C contents of strain T98T, P. sabinae DSM 17841T and P. zanthoxyli DSM 18202T are from the present study. Data for G+C contents of P. durus ATCC 35681T, P. polymyxa ATCC 842T, P. macerans ATCC 8244T, P. odorifer TOD45T and P. graminis RSA19T are from Seldin et al. (1984), Nakamura (1987) and Berge et al. (2002).

    The DNA G+C content was determined by using the thermal denaturation protocol (De Ley et al., 1970). The G+C content of strain T98T was 50.4 mol% (Table 1⇑), which is within the range of values for recognized Paenibacillus species (Shida et al., 1997).

    For DNA–DNA hybridization experiments, total genomic DNA was extracted and purified according to the method described by Marmur (1961) with some modifications. DNA–DNA hybridization was determined by the initial renaturation rate method (De Ley et al., 1970). Levels of DNA–DNA relatedness between strain T98T and P. sabinae DSM 18741T, P. durus ATCC 35681T and P. zanthoxyli DSM 18202T were 30.0, 27.6 and 32.1 %, respectively, indicating that strain T98T represented a species distinct from these three species.

    For determination of its cellular fatty acid composition, strain T98T was grown in RVC mineral medium at 30 °C for 3 days. Analysis was carried out as described by Komagata & Suzuki (1987) by using the Sherlock Identification System (MIDI) (Sasser et al., 2005). Profiles of the major fatty acids of strain T98T and of closely related Paenibacillus type strains are shown in Table 3⇓. Anteiso-C15 : 0, the major fatty acid in recognized members of the genus Paenibacillus, was also the major fatty acid component of strain T98T (29.87 %).

    Table 3.

    Cellular fatty acid profiles of strain T98T and closely related Paenibacillus type strains

    Strains: 1, strain T98T; 2, P. durus ATCC 35681T (data from Yoon et al., 2003); 3, P. sabinae DSM 17841T (Ma et al., 2007a); 4, P. zanthoxyli DSM 18202T (Ma et al., 2007b); 5, P. stellifer DSM 14472T (Suominen et al., 2003); 6, P. polymyxa DSM 36T (Yoon et al., 2003); 7, P. macerans ATCC 8244T (Elo et al., 2001). Data are percentages of the total fatty acids. nr, Not reported. For unsaturated fatty acids, the position of the double bond can be located by counting from the methyl (ω) end of the carbon chain; cis isomers are indicated by the suffix c.

    In summary, phylogenetic analysis based on the full-length 16S rRNA gene sequence and on a fragment of the nifH gene sequence, DNA G+C content, and chemotaxonomic properties revealed that strain T98T was a member of the genus Paenibacillus. Several phenotypic characteristics and levels of DNA–DNA hybridization further demonstrated that strain T98T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus forsythiae sp. nov. is proposed.

    Description of Paenibacillus forsythiae sp. nov.

    Paenibacillus forsythiae (for.sy′thi.ae. N.L. gen. n. forsythiae of Forsythia, referring to the plant Forsythia mira, the source of the rhizosphere soil from which the type strain was isolated).

    Gram-positive or Gram-variable, facultatively aerobic, motile, straight rods (0.4–0.5×2.0–2.6 μm). Ellipsoidal spores are located centrally in swollen sporangia. Colonies on RVC mineral medium are circular, convex and glossy with entire margins and measure 2.0–2.5 mm in diameter after 72 h at 30 °C. The temperature range for growth is 10–40 °C, with optimum growth at 30 °C. Grows at pH 4.0–10.0, with optimum growth at pH 7.0–7.2. Grows in the absence of NaCl and in 3 % (w/v) NaCl, but is unable to tolerate 5 % NaCl. Growth is inhibited by 0.001 % (w/v) lysozyme. Catalase-positive and oxidase-negative. Positive for the Voges–Proskauer reaction. Nitrate is reduced to nitrite. Nitrogen is fixed. Able to utilize succinate to produce acid, but not glucose, sucrose, lactose, fructose, glycerol, xylose, maltose, d-sorbitol, sodium citrate, glycine or l-aspartate. Casein is hydrolysed but gelatin and starch are not. The G+C content of the DNA is 50.4 mol% (thermal denaturation method).

    The type strain, T98T (=CCBAU 10203T =DSM 17842T), was isolated from rhizosphere soil of the plant Forsythia mira in the Beijing region, China.

    Acknowledgments

    We thank Professor Tian-Shen Tao, Chinese Academy of Agricultural Sciences, for technical help. This work was supported by the Chinese National ‘973’ Project (grant no. 001CB108904).

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