Thauera butanivorans sp. nov., a C₂–C₉ alkane-oxidizing bacterium previously referred to as ‘Pseudomonas butanovora’

Abstract

The placement of ‘Pseudomonas butanovora’ in the genus Thauera was proposed previously, based on 16S rRNA gene sequence analysis, upon further studies of taxonomical characteristics. In this study, physiological characteristics and DNA–DNA reassociation data are presented and the transfer of ‘P. butanovora’ to the genus Thauera is proposed. The original description of the strain (strain Bu-B1211) indicated that it was capable of denitrification but not anaerobic growth. ‘P. butanovora’ is capable of anaerobic respiration and growth, utilizing nitrate as a terminal electron acceptor during the oxidation of organic acids and alcohols, but not aromatic hydrocarbons or open-chain terpenoids. The total fatty acid composition supported the assignment of strain Bu-B1211 to the Betaproteobacteria and resembled that of members of the genus Thauera. The combination of 16S rRNA gene phylogenetic evidence, physiological and taxonomical characteristics and DNA–DNA reassociation data supported the placement of ‘Pseudomonas butanovora’ Bu-B1211 in the genus Thauera as representing a novel species, for which the name Thauera butanivorans sp. nov. is proposed. The type strain is Bu-B1211^T (=IAM 12574^T=ATCC 43655^T=DSM 2080^T).

The genus Thauera was described originally by Macy et al. (1993) and comprises eight species with validly published names: Thauera selenatis (Macy et al., 1993), Thauera aromatica (Anders et al., 1995), Thauera chlorobenzoica (Song et al., 2001), Thauera linaloolentis (Foss & Harder, 1998), Thauera mechernichensis (Scholten et al., 1999), Thauera terpenica (Foss & Harder, 1998), Thauera aminoaromatica (Mechichi et al., 2002) and Thauera phenylacetica (Mechichi et al., 2002). ‘Pseudomonas butanovora’ strain Bu-B1211 (=IAM 12574=ATCC 43655=DSM 2080) was isolated originally from activated sludge sampled from an oil-refining plant in Japan by enrichment with n-butane as the sole source of carbon and energy (Takahashi et al., 1980). The initial characterization of the isolate placed the bacterium in the genus Pseudomonas, based solely on morphology and phenotypic characteristics (Takahashi et al., 1980). A comprehensive study of the phylogenetic affiliation of the pseudomonads using 16S rRNA gene analysis showed that, phylogenetically, ‘P. butanovora’ was a member of the Rhodocyclus group, with the closest relatives belonging to the genus Thauera (Anzai et al., 2000). The level of 16S rRNA gene sequence similarity between ‘P. butanovora’ and recognized Thauera species ranged between 96.2 and 98.7 %, prompting the authors to propose the transfer of ‘P. butanovora’ to the genus Thauera based upon additional taxonomic studies.

Growth of ‘P. butanovora’ (ATCC 43655), T. aromatica S100 (DSM 14793) and T. linaloolentis 47Lol^T (DSM 12138^T) was carried out using the method and conditions described in Sluis et al. (2002), with the exception that, for aerobic growth, and when alkane was supplied as the sole source of carbon energy, the medium was amended with 100 μM Fe³⁺-EDTA. Anaerobic conditions were established in 50 ml growth medium in serum vials (160 ml) by sparging with O₂-free nitrogen, and crimp sealing with butyl-rubber septa followed by sparging of the headspace. Organic acid stock solutions (1 M) were neutralized (pH 7.0) and made anaerobic as described above.

The fatty acid profile of ‘P. butanovora’ (grown in LB medium) was determined in this study by Microbial ID (Newark, DE, USA). The profile was composed of summed feature 3 (C_16 : 1ω6c/C_16 : 1ω7c; 43.6 %), C_16 : 0 (27.6 %), C_18 : 1 (14.8 %), C_10 : 0 3-OH (4.7 %) and C_12 : 0 (4.6 %). The main components of the profile were in the range of those of recognized Thauera species (Song et al., 2001) and agreed with the 16S rRNA gene phylogenetic placement in the class Betaproteobacteria. The DNA G+C content (67.3 mol%) determined by Takahashi et al. (1980) was also similar to those of members of the genus Thauera (Song et al., 2001).

In the original paper describing the isolation of ‘P. butanovora’, the bacterium was reported to be capable of reduction and denitrification, but not anaerobic growth (Takahashi et al., 1980). Growth medium for assessing terminal electron acceptors for anaerobic respiration was supplemented with 10 mM NH₄Cl as a supplementary nitrogen source. ‘P. butanovora’ was capable of utilizing KNO₃ (10 mM), NaNO₂ (10 mM) and N₂O as terminal electron acceptors for growth when butyrate was supplied as the electron donor. Utilization of N₂O as the terminal electron acceptor required complete reduction of the growth medium (5 mM ascorbate, which is not utilized as a source of carbon under anaerobic conditions). Selenate did not serve as an electron acceptor for anaerobic growth of ‘P. butanovora’.

The utilization of organic substrates as carbon sources for growth of ‘P. butanovora’ was assessed under aerobic and anaerobic conditions. The results are summarized in Table 1. Takahashi et al. (1980) reported previously that aerobic growth occurred with n-alkanes (C₂–C₉), primary alcohols and carboxylic acids (C₂–C₄). The pathway for butane assimilation was determined to occur via the terminal alcohol (1-butanol), with subsequent oxidation to butyraldehyde and butyrate. Presumably, butyrate was metabolized further to acetyl-CoA via the β-oxidation pathway (Arp, 1999). One major characteristic of recognized Thauera species is the utilization of aromatic or terpenoid hydrocarbons under denitrifying conditions (Anders et al., 1995; Foss & Harder, 1998; Mechichi et al., 2002; Scholten et al., 1999; Song et al., 2001). ‘P. butanovora’ was not capable of growth on aromatic or terpenoid hydrocarbons when was supplied as the terminal electron acceptor. Interestingly, aerobic growth with the branched hydrocarbons isobutane and isopentane was possible due to a soluble butane monooxygenase (Dubbels et al., 2007; Sluis et al., 2002). Cell-free extracts, prepared from aerobic, acetate-grown ‘P. butanovora’ and assayed according to the protocol of Alber et al. (2006), showed isocitrate lyase activity indicating a functional glyoxylate cycle and the ability to synthesize precursors of amino acids and sugars from the carboxylic acid end products of alkane oxidation.

Table 1.

Characteristics that differentiate strain Bu-B1211^T and two species of the genus Thauera

Taxa: 1, strain Bu-B1211^T (T. butanivorans sp. nov.); 2, T. aromatica; 3, T. linaloolentis 47Lol^T. All taxa grew under aerobic conditions with butyrate. Strain Bu-B1211^T and T. aromatica grew under aerobic conditions with acetate, propionate and succinate/fumarate; no data are available for T. linaloolentis 47Lol^T. All taxa grew under denitrifying () conditions with acetate, propionate, butyrate, succinate/fumarate and ethanol (1–5 mM; Takahashi et al., 1980 and this study). nd, No data available; +, positive; −, negative; d, different reactions from strains of the same species.

Characteristic	1	2	3
Aerobic growth
Ethanol	+	d*	nd
Benzoate	–	+*	nd
Butane	+†	–	–
Isobutane	+	–	–
Isopentane	+	–	–
Leucine	–	+*	nd
Valine	–‡	–*	nd
Denitrifying growth
Benzoate	–	+*	–*
Phenol	–	d*	nd
Toluene	–	+*	–*
Geraniol	–	nd	+*
Linalool	–	nd	+*

*Data from Heider & Fuchs (2005).

†Data from Takahashi et al. (1980).

‡Takahashi et al. (1980) reported growth on valine. Growth was not observed under the conditions employed in this study.

DNA–DNA reassociation was performed as described by Simbahan et al. (2004) and Urbance et al. (2001) with DNA from two species of Thauera (T. aromatica S100 and T. linaloolentis 47Lol^T) that exhibit high 16S rRNA gene sequence similarity to ‘P. butanovora’ (97 and 98.7 %, respectively). The results showed DNA–DNA reassociation values of about 20 % between ‘P. butanovora’ and either Thauera species examined. Based on the data above and the established classification that two strains within the same genus represent distinct species when the level of DNA–DNA reassociation is below 70 % (Stackebrandt & Liesack, 1993; Stackebrandt & Goebel, 1994; Wayne et al., 1987), we propose a novel species with the name Thauera butanivorans sp. nov.

Description of Thauera butanivorans sp. nov.

Thauera butanivorans (bu.tan.i.vo′rans. N.L. n. butanum butane; L. part. adj. vorans devouring; N.L. part. adj. butanivorans butane-devouring).

Morphology and general characteristics are as described for the genus. Cells are Gram-negative, rod-shaped (0.6–0.8 μm wide and 1.1–2.4 μm long), and motile by means of a polar flagellum. Fatty acid profile comprises summed feature 3 (C_16 : 1ω6c/C_16 : 1ω7c), C_16 : 0, C_18 : 1, C_10 : 0 3-OH and C_12 : 0. Shows facultatively anaerobic chemo-organotrophic metabolism. O₂, , and N₂O (requires reduction of medium) are utilized as terminal electron acceptors. Capable of utilizing C₂–C₉ alkanes for carbon and energy, aerobically. Negative for anaerobic catabolism of aromatic hydrocarbons (benzoate, toluene or phenol) or open-chain terpenoids (linalool or geraniol). The DNA G+C content of the type strain is 67.3 mol%.

The type strain, Bu-B1211^T (=IAM 12574^T=ATCC 43655^T=DSM 2080^T), was isolated from activated sludge sampled from an oil-refining plant in Japan. Originally isolated and identified as ‘Pseudomonas butanovora’ by Takahashi et al. (1980).