Novel nitrogen-fixing Acetobacter nitrogenifigens sp. nov., isolated from Kombucha tea

Endophytic bacteria colonize the internal tissues of the host plant for mutual benefits. The nitrogen-fixing endophyte Gluconacetobacter diazotrophicus has been found to be associated with sugarcane, pineapple, cameroon grass, sweet potato, mango and banana (Muthukumarasamy et al., 2002), while Gluconacetobacter azotocaptans and Gluconacetobacter johannae inhabit coffee plants (Fuentes-Ramírez et al., 2001) and provide host plants with useful fixed nitrogen and growth stimulants. The first report of the occurrence and association of nitrogen-fixing Acetobacter peroxydans along with G. diazotrophicus inhabiting cultivated wetland rice varieties demonstrated the presence of nitrogen-fixing property in the genus Acetobacter (Muthukumarasamy et al., 2005). Based on the striking resemblance of the high sugar and low pH habitats of sugarcane and Kombucha tea (Blanc, 1996), we explored and isolated a nitrogen-fixing strain RG1^T belonging to the genus Acetobacter of the family Acetobacteraceae. This family has been divided into eight genera; Acetobacter, Gluconacetobacter, Gluconobacter, Acidomonas, (Yamada et al., 1997), Asaia (Yamada et al., 2000), Kozakia (Lisdiyanti et al., 2002), Saccharibacter (Jojima et al., 2004) and Swaminathania (Loganathan & Nair, 2004). The genus Acetobacter comprises, at present, 15 validly described species that were delineated mainly on the basis of DNA–DNA relatedness and phylogenetic relationships (Sokollek et al., 1998; Lisdiyanti et al., 2000, 2001; Cleenwerck et al., 2002; Silva et al., 2006). This report of the isolation of nitrogen-fixing strain RG1^T from the novel source Kombucha tea is only second in the genus Acetobacter after A. peroxydans (Muthukumarasamy et al., 2005).

We present morphological, biochemical and genetic evidence which indicates that this isolate represents a novel nitrogen-fixing species within the genus Acetobacter isolated from a novel source. We propose the name Acetobacter nitrogenifigens for the RG1^T isolate.

Aliquots of Kombucha mat suspension, after teasing the mat apart in the soup, were spread on to LGI (0.06 % KH₂PO₄, 0.02 % K₂HPO₄, 0.02 % MgSO₄, 0.002 % CaCl₂, 0.001 % FeCl₃, 0.0002 % Na₂MoO₄, 10 % sucrose, pH 4.5; Cavalcante & Döbereiner, 1988) agar plates containing 150 mg cycloheximide l⁻¹ (Jimenez-Salgado et al., 1997) and 150 mg nystatin l⁻¹. Plates were incubated at 30 °C for 5 days. The bacterial isolate was purified by repeated streaking on to LGI plates, which have no combined nitrogen source. Gas tight vials of bacteria-inoculated LGI medium (under microaerophilic environment, without shaking) were assayed for acetylene reduction activity (Stal, 1988). Nitrogenase-positive isolates were selected for further characterization. Nitrogen-fixing bacterial strain RG1^T was isolated. Other reference strains and the novel isolates were grown in mannitol broth for further study. Colony morphology was examined on LGI agar plates and on potato agar plates containing 10 % sucrose. Various phenotypic and morphological studies were performed using standard techniques described elsewhere (Cavalcante & Döbereiner, 1988; Caballero-Mellado et al., 1995; Jimenez-Salgado et al., 1997; Cleenwerck et al., 2002). Isoprenoid quinone of the isolate was extracted with chloroform/methanol [2 : 1, (v/v)], and then purified by TLC on silica gel 60 F₂₅₄ (20×20 cm; Merck) by using benzene as the developing solvent. Quinones recovered from the TLC plates were dissolved in acetone and analysed by HPLC (Lu et al., 1999). The HPLC system was equipped with a reverse-phase column [Luna 5U C18 (2) 100A, 250×4.6 mm; Phenomenex] and a mixture of methanol/2-propanol [2 : 1, (v/v)] was used as the mobile phase at a flow rate of 1 ml min⁻¹. Types of quinone were identified by absorption at 275 nm and compared with coenzymes Q-9 and Q-10 standards from Sigma-Aldrich. Ubiquinone Q-9 was present in strain RG1^T and fitted with the previous observations that showed the presence of this ubiquinone type in the genus Acetobacter (Cleenwerck et al., 2002). The type strain RG1^T deviated biochemically and morphologically from other species of the genus Acetobacter as shown in Table 1⇓.

A 1451 bp fragment of 16S rRNA gene was PCR amplified with bacteria-specific primers fD1 and rD1 (Weisburg et al., 1991) using Taq polymerase and genomic DNA from strain RG1^T as template. The nucleotide sequence was similar to the extent of 98.2 % with Acetobacter aceti, 97.7 % with Acetobacter estunensis, 97.4 % with Acetobacter indonesiensis, 97.2 % with Acetobacter tropicalis, 97.2 % with Acetobacter oeni, 97.1 % with Acetobacter cibinongensis, 97.0 % with Acetobacter cerevisiae, 97.0 % with Acetobacter malorum, 96.9 % with Acetobacter orleansis, 96.6 % with Acetobacter orientalis and 97.0 % with Acetobacter syzygii after performing a similarity search with fasta (ungapped). The phylogenetic tree (Fig. 1⇓) was deduced using mega version 3.1 (Kumar et al., 2004) software after multiple alignment of 16S rRNA gene sequences of other acetic acid bacteria with clustal w (Thompson et al., 1994). Distances (distance options according to the Kimura two-parameter model) and clustering with the neighbour-joining method was determined by using bootstrap values (Felsenstein, 1985) based on 100 replications. Two subclusters were evident in the genus Acetobacter: one composed of Acetobacter pomorum, Acetobacter pasteurianus, A. syzygii, Acetobacter lovaliensis and A. peroxydans and the other included Acetobacter aceti and other acetobacters along with the novel nitrogen-fixing strain RG1^T. The clustering of strain RG1^T with A. aceti was further confirmed by the formation of dihydroxyacetone from glycerol by both strains RG1^T and A. aceti ATCC 15973^T.

Cell extracts were prepared for multilocus enzyme electrophoresis (MLEE) and run on a starch gel as described previously (Selander et al., 1986; Caballero-Mellado & Martínez-Romero, 1994). A. cibinongensis JCM 11196^T, A. orleansis LMG 1583^T, A. malorum LMG 1746^T, A. aceti ATCC 15973 ^T, A. tropicalis LMG 19825^T, A. oeni B13^T, A. orientalis JCM 11195^T, A. indonesiensis LMG 19824^T, A. estunensis LMG 1626^T and A. cerevisiae LMG 1625^T were included as reference strains. The relationships among non-nitrogen-fixing species of Acetobacter and RG1^T are illustrated by the dendrogram (Supplementary Fig. S1 available in IJSEM Online) derived by program etmega (T. S. Whittam; ) and mega version 3.1 software based on the electrophoretic mobility of nine metabolic enzymes, indophenol oxidase, alcohol dehydrogenase, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine dehydrogenase, leucine dehydrogenase, lysine dehydrogenase, hexokinase and esterase. In MLEE, strain RG1^T exhibited highest similarity to A. aceti ATCC 15973^T, which is in agreement with its phylogenetic position (Fig. 1⇑). Analysis of the dendrogram revealed that strain RG1^T formed a unique branch with A. aceti ATCC 15973^T that deviated at a genetic distance of more than 0.5. This value has been used as a criterion to suggest species limits (Selander et al., 1985; Musser et al., 1987). Thus, MLEE results strongly support the existence of strain RG1^T as a novel Acetobacter species.

The nitrogenase enzyme complex, responsible for nitrogen-fixation, is composed of nifHDK and other gene fragments. A 336 bp region encoding dinitrogenase reductase, nifH, was amplified from strain RG1^T using degenerate primers 19F and 407R (Franke et al., 1998) and sequenced. Presence of nifH gene further confirmed the nitrogen-fixing ability of strain RG1^T.

To determine genomic relatedness of the novel isolate, dot-blot hybridization experiments were carried out with DIG-labelled DNA as described previously (Labrenz et al., 2000) using the detection kit from Roche Applied Sciences and following the manufacturer's instructions. Colorimetric quantification of dot intensities was done using the Molecular Analyst software (Bio-Rad) by determining mean pixel densities in equal sized circles. A genomic DNA probe was prepared from the novel strain RG1^T, digested with EcoRI and run on 0.7 % agarose gel. Total DNA digests were transferred from gels to nylon membrane by Southern blotting. Hybridization was performed at a temperature of 75 °C for 16 h and the membrane was washed under high stringency conditions (twice with 2× SSC/0.1 % SDS at room temperature for 10 min; once with 0.1× SSC/0.1 % SDS at 75 °C for 15 min). A low level of genomic DNA relatedness (DNA–DNA hybridization values less than 50 %) was observed between the phylogenetically closest species and genera. Strain RG1^T showed low DNA–DNA relatedness with the type strains of A. oeni (24 %), A. aceti (22 %), A. cerevisiae (19 %), A. cibinongensis (18.91 %), A. orientalis (18.9 %), A. estunensis (11 %), A. orleansis (10.37 %), A. malorum (7.31 %), A. indonesiensis (3.71 %) and A. tropicalis (2 %).

Although the limitations of 16S rRNA gene sequencing to differentiate closely related species have been documented (Fox et al., 1992), it is suggested that strains sharing greater than 97 % similarity might belong to the same species but a lower similarity value could form the basis of delineation of bacterial species (Stackebrandt & Goebel, 1994). Also a DNA–DNA relatedness level below 70 % indicates a distinct species (Stackebrandt & Goebel, 1994). In the present study, these considerations were consistent with both the 16S rRNA gene sequence similarity levels and the low levels of DNA–DNA relatedness exhibited within the Acetobacter species. The genetic distance of different closely related acetobacters involving strain RG1^T was found to be greater than 0.5 in all comparisons and such a distance indicates a novel species; hence strain RG1^T represent a novel species of the genus Acetobacter.

In view of low physiological, biochemical, phylogenetic and genetic similarities among different closely related members of the genus Acetobacter, we recommend that nitrogen-fixing strain RG1^T described here should be assigned to a novel species of the genus Acetobacter, for which the name Acetobacter nitrogenifigens sp. nov. is proposed.