Transfer of Bacillus mucilaginosus and Bacillus edaphicus to the genus Paenibacillus as Paenibacillus mucilaginosus comb. nov. and Paenibacillus edaphicus comb. nov.

Bacillus mucilaginosus, which can release nutritional ions from soils and minerals, is widely used as a biofertilizer in agricultural applications (Glick, 1995; Sheng et al., 2000; Liu et al., 2006). It was first described phenotypically in 1967 (Aleksandrov et al., 1967) and phylogenetically characterized in 1986 (Avakyan et al., 1986). However, the name was not validly published until 1998, which coincided with the identification of Bacillus edaphicus, a novel species with similar functions (Shelobolina et al., 1997). Recently, B. mucilaginosus and B. edaphicus were found to be more closely related to the genus Paenibacillus than to the genus Bacillus on the basis of phylogenetic analysis (Hu et al., 2006), but this finding requires further clarification.

Paenibacillus, a genus separated from the genus Bacillus, was first proposed by Ash et al. (1993) and later emended by Shida et al. (1997). Many species in this genus possess useful properties, such as nitrogen fixation (Achouak et al., 1999) and macromolecule degradation (Ash et al., 1993), and play an economically important role in agriculture. The typical characteristics of species in the genus Paenibacillus are as follows (Shida et al., 1997): rod-shaped with a flagellum; producing ellipsoidal spores with swollen sporangia; positive for catalase; negative for H₂S production; variable for oxidase, nitrate reduction and Voges–Proskauer reaction; DNA G+C content ranging between 45 % and 54 mol%, with anteiso-C_15 : 0 as the major cellular fatty acid, MK-7 as the major isoprenoid quinone, meso-diaminopimelic acid as the diagnostic diamino acid, and more than 89.6 % intra-genus similarity in 16S rRNA gene sequences. These distinctive characteristics distinguish the genus Paenibacillus from other members of the family Bacillaceae. In this study, we present phenotypic and phylogenetic data that support the transfer of B. mucilaginosus and B. edaphicus to the genus Paenibacillus.

The following strains were used in this study: B. edaphicus VKPM B-7517^T, B. mucilaginosus VKPM B-7519^T, CGMCC 1.231, CGMCC 1.2326, CGMCC 1.3714, CGMCC 1.153, KNP413 and KNP414 (Hu et al., 2006), Bacillus circulans ACCC 10228^T, Bacillus megaterium ACCC 10245^T, Bacillus subtilis ACCC 92068^T, Bacillus azotoformans ACCC 10226^T, Paenibacillus apiarius ACCC 10193^T, Paenibacillus azotofixans ACCC 10251^T, Paenibacillus validus CCTCC 95016^T and Paenibacillus polymyxa CCTCC 92076^T. The B. mucilaginosus and B. edaphicus strains were cultured on Aleksandrov agar (Avakyan et al., 1986) and the other strains were grown on nutrient agar medium. Aleksandrov medium consisted of (w/v) 1.0 % sucrose, 0.2 % K₂HPO₄ . 3H₂O, 0.05 % MgSO₄ . 7H₂O, 0.1 % CaCO₃ and 2.0 % agar, pH 7.5. All strains were incubated for 24–48 h at 30 °C.

Genomic DNA extraction, amplification and sequencing of the 16S rRNA gene were performed as described previously (Hu et al., 2006). The 16S rRNA gene sequences were aligned with representatives from the genera Bacillus and Paenibacillus that had been deposited in the GenBank, EMBL and DDBJ databases. Phylogenetic trees were constructed with three methods: neighbour-joining (mega version 3.1), maximum-parsimony and maximum-likelihood (phylip version 3.65) (Saitou & Nei, 1987). Bootstrap resampling analysis was performed to estimate the confidence of the tree topologies.

The almost-complete 16S rRNA gene sequence (1502 bases) was determined for B. mucilaginosus strains CGMCC 1.231, CGMCC 1.2326, CGMCC 1.3714 and CGMCC 1.153. The 16S rRNA gene sequence-based phylogenetic analysis revealed that the intraspecies similarity values for B. mucilaginosus were 99.1–99.9 %, with the exception of strain VKM B-1480D^T, which shared only 94.3–96.9 % similarity with the other strains. However, strain HSCC 1605^T, deposited in Japan, presented a high intraspecies similarity of 99.1–99.9 %. Thus, the 16S rRNA gene sequence of strain VKM B-1480D^T, which is similar to that of VKPM B-7519^T was analysed, and it found that the sequence submitted as GenBank accession no. AF006077 might contain various errors. Resequencing of the 16S rRNA gene proved that the sequence of strain VKPM B-7519^T differed significantly from that of strain VKM B-1480D^T. In addition, there was only one base discrepancy between the sequences of strains VKPM B-7519^T (FJ039528) and HSCC 1605^T(AB045091), indicating the similarity of these two strains. Therefore, the strains of Bacillus mucilaginosus tested were members of the same species with 99.0–99.9 % intraspecies similarity.

Strains of B. mucilaginosus and B. edaphicus strain VKPM B-7517^T showed 90.4–96.4 % and 90.3–95.1 % similarity to the representatives of the Paenibacillus cluster, respectively. However, both species shared less than 88.5 % similarity with representatives of the other clusters of the family Bacillaceae. The >90.4 % 16S rRNA gene sequence similarity to strains of the genus Paenibacillus places B. mucilaginosus and B. edaphicus within the realm of the genus Paenibacillus rather than in that of the genus Bacillus (Shida et al., 1997). The phylogenetic trees constructed with all three treeing methods indicated that the B. mucilaginosus and B. edaphicus clustered with the genus Paenibacillus (Fig. 1⇓). Within this cluster, B. mucilaginosus strains and B. edaphicus VKPM B-7517^T were found to be closely related to each other and to the species P. validus, which was consistent with an earlier result (Shelobolina et al., 1997).

The gyrB gene, a single-copy gene encoding the B-subunit protein of DNA gyrase, is increasingly used as a phylogenetic marker for bacterial systematics and for bacterial taxonomic and phylogenetic studies (Yamada et al., 1999). The gyrB gene was used in this study to confirm the taxonomic and phylogenetic placements of the species tested. The amplification of the gyrB gene was performed with universal primers (UP-1, CAYGCNGGNGGNAARTTYG; UP-2r, CCRTCNACRTCNGCRTCNGTCAT; Yamamoto & Harayama, 1995) and the sequencing and phylogenetic analysis were carried out as described above.

The 1210 bp gyrB genes of the strains examined were sequenced and their GenBank accession numbers are given in Fig. 2⇓. The intraspecies similarity values for the strains of B. mucilaginosus were 98.2–99.9 %. All B. mucilaginosus strains showed 70.4–78.0 % gene sequence similarity to the representatives of Paenibacillus cluster and to B. edaphicus VKPM B-7517^T. B. edaphicus VKPM B-7517^T shared 70.3–79.2 % gene sequence similarity with strains of the genus Paenibacillus. However, B. mucilaginosus and B. edaphicus shared only 61.2–68.9 % similarity with representatives of the other clusters of the family Bacillaceae. It would appear that there are threshold values of 98 % for a species delineation and 70 % for a genus delineation for the gyrB gene, which require further substantiation. The high gyrB gene sequence similarity between B. mucilaginosus and B. edaphicus and members of the genus Paenibacillus suggested that these species should be placed within the genus Paenibacillus rather than the genus Bacillus.

The phylogenetic trees constructed with the three treeing methods (Fig. 2⇑) also indicated that B. mucilaginosus and B. edaphicus clustered within the genus Paenibacillus, and showed that the two species were closely related to each other and to P. validus. However, B. mucilaginosus strains were divided into two groups, Group I (KNP413, CGMCC 1.3714 and CGMCC 1.2326) and Group II (VKPM B-7519, CGMCC 1.231, CGMCC 1.153 and KNP414). Strains within each group shared 99.6–99.9 % gene sequence similarites for the gyrB gene, but strains in the different groups showed only 98.2–98.6 % sequence similarity. Strains in Group II, which possessed plasmids (about 7 kb) and large capsules (data not shown), differed at 17 positions when compared with those in Group I. Although differences existed between the two groups, which are of interest for further study, such differences were not significant enough to prevent the two groups from clustering into the same species. The results based on the gyrB gene sequence coincided closely with those of the 16S rRNA gene sequence comparisons and further confirmed the powerful discriminative ability of the gyrB gene.

The DNA base ratio was determined from the thermal denaturation temperature, as described by Marmur & Doty (1962). The DNA G+C contents of strains of B. mucilaginosus and B. edaphicus ranged from 54.5 to 56.8 mol% (Table 1⇓). These values are similar to those previously determined by Shelobolina et al. (1997); however, they exceeded the DNA G+C content level expected for members of the genus Paenibacillus (Shida et al., 1997). Most strains of the genus Paenibacillus have a DNA G+C content of 45–54 mol% (Shida et al., 1997), with the exception of strains of Paenibacillus dendritiformis (Tcherpakov et al., 1999) and Paenibacillus koleovorans (Takeda et al., 2002), which have a higher DNA G+C content of 54–55 mol%. Thus, B. mucilaginosus and B. edaphicus provide further evidence that the DNA G+C content of the genus Paenibacillus can exceed 54 %.

DNA–DNA hybridization was performed using a thermal denaturation technique (De Ley et al., 1970). The DNA–DNA hybridization values confirmed that B. mucilaginosus strains VKPM B-7519^T, KNP414 and CGMCC 1.2326 were members of the same species. Strain VKPM B-7519^T exhibited 89.2 % and 88.7 % relatedness values with strains KNP414 and CGMCC 1.2326, respectively. Organisms that have <97.0 % 16S rRNA gene sequence similarity will not recombine with DNA relatedness values >60 %, regardless of the hybridization method employed (Stackebrandt & Goebel, 1994). We did not determine the DNA–DNA relatedness values between B. mucilaginosus and other strains of the genus Paenibacillus because the 16S rRNA gene sequence similarities between them were <96.4 %, which demonstrated that B. mucilaginosus and B. edaphicus are not related to any of the previously described Paenibacillus taxa at the species level.

Cell-wall peptidoglycan was purified and the cell-wall amino acids and the peptides in the cell-wall hydrolysates were analysed by two dimensional ascending TLC on cellulose plates using the solvent systems of Schleifer & Kandler (1972). The isomer of diaminopimelic acid was identified using the method of Rhuland et al. (1955). Analyses of polar lipids and menaquinones were performed according to published procedures (Groth et al., 1999). The peptidoglycan type of the two species was A1γ, based on meso-diaminopimelic acid. The major polar lipids were phosphatidylglycerol and diphosphatidylglycerol, while the major isoprenoid quinone was MK-7 (94.1–95.7 %). These data were identical to and consistent with the chemotaxonomic properties characteristic of the genus Paenibacillus (Shida et al., 1997; Kämpfer et al., 2006; Chou et al., 2009).

The cellular fatty acid compositions (FAME) of B. edaphicus VKPM B-7517^T and the B. mucilaginosus strains were analysed with the Sherlock Microbial Identification System (MIS Inc.), with P. validus strain CCTCC 95016^T as the control. All aspects of the procedure were conducted according to the instructions for the MIDI System (Microbial ID; Sasser, 1990). The resulting FAME profiles were compared with those of known species in the MIDI database, TSBA version 4.10. The major cellular fatty acids of B. edaphicus and the B. mucilaginosus strains were anteiso-C_15 : 0 (43.19–48.45 %), C_16 : 1ω11c (10.78–14.20 %) and C_16 : 0 (8.72–19.83 %). The complete cellular fatty acid profiles are presented in Table 2⇓. Based on the maximum similarity of 1.2–20.2 %, the MIDI identification program indicated that B. edaphicus and B. mucilaginosus were related to P. validus. The profiles were similar to the previously determined profile of B. mucilaginosus VKPM B-7519^T, but different from that of B. edaphicus VKPM B-7517^T as determined by Shelobolina et al. (1997). The FAME profile difference observed for strain VKPM B-7517^T could be attributed to the different methods utilized in the determination of the FAME profiles. The standard method was used in this study, but Shelobolina et al. (1997) developed their own method. The differences between the two methods may be a result of the media utilized; TSBA medium containing nitrogen was used in the standard method instead of Aleksandrov medium. It has already been reported that B. edaphicus strains can be significantly influenced by the composition of the medium, especially by the presence of nitrogen (Shelobolina et al., 1997). We also observed that colonies of strain VKPM B-7517^T grown on TSBA medium were small and opaque and were clearly different from those cultured on Aleksandrov medium. Therefore, an observed difference in the FAME profiles of the cultures grown on the two different media is understandable. The predominant anteiso-C_15 : 0 fatty acids observed in the profiles of the B. edaphicus and B. mucilaginosus strains confirmed their association with the genus Paenibacillus.

Other characteristics, including Gram stain, Voges–Proskauer reaction, nitrate reduction, gelatin liquefaction, enzymic activities (catalase, oxidase) and NaCl tolerance, were determined according to the methods of Claus & Berkeley (1986). All strains of B. mucilaginosus and B. edaphicus tested (Table 2⇑) shared similar morphologies with P. validus CCTCC 95016^T, except for the presence of a flagellum. Although the presence of a flagellum is a characteristic shared by most species in the genus Paenibacillus, there are some species without flagella (Smerda et al., 2005; Sanchez et al., 2005). B. mucilaginosus and B. edaphicus were variable in tests for oxidase and nitrate reactions, positive for catalase activity, and negative for the Gram and Voges–Proskauer reactions, for gelatin liquefaction and for the production of H₂S. They could not grow at 50 °C or in media containing 2.5 % (w/v) NaCl. From these observations, it is obvious that B. mucilaginosus and B. edaphicus have phenotypic characteristics in common with strains of the genus Paenibacillus (Shida et al., 1997).

The phylogenetic and phenotypic results demonstrate that B. edaphicus VKPM B-7517^T and B. mucilaginosus, as represented by strain VKPM B-7519^T, are phylogenetically closer to the genus Paenibacillus than to the genus Bacillus. However, they are independent of any recognized species of the genus Paenibacillus. Therefore, it is proposed that B. edaphicus and B. mucilaginosus be transferred to the genus Paenibacillus as Paenibacillus mucilaginosus comb. nov. and Paenibacillus edaphicus comb. nov., respectively.