Methylobacterium hispanicum sp. nov. and Methylobacterium aquaticum sp. nov., isolated from drinking water

The genus Methylobacterium includes a group of pink-pigmented facultatively methylotrophic bacteria with the ability to grow on one-carbon compounds such as formate, formaldehyde and methanol as sole carbon and energy sources, as well as on a wide range of multi-carbon growth substrates (Green, 1992). This genus comprises 15 species with the type species Methylobacterium organophilum, which is not able to grow on methane. A recently described species isolated from poplar tissues, Methylobacterium populi (Van Aken et al., 2004), is able to utilize methane as the sole source of carbon and energy. Methylotrophic bacteria that do not utilize methane are classified into two groups that use either the ribulose monophosphate or serine pathways for formaldehyde assimilation (Anthony, 1982). Members of the genus Methylobacterium that utilize methanol have the serine pathway for formaldehyde assimilation. 16S rRNA gene sequence analyses have shown that the genus Methylobacterium belongs to the α-subclass of the Proteobacteria, whereas ribulose monophosphate methylotrophs belong to the β-subclass (Bratina et al., 1992; Brusseau et al., 1994; Tsuji et al., 1990).

The 15 species that belong to the genus Methylobacterium are Methylobacterium aminovorans (Urakami et al., 1993), Methylobacterium chloromethanicum (McDonald et al., 2001), Methylobacterium dichloromethanicum (Doronina et al., 2000), Methylobacterium extorquens (Bousfield & Green, 1985), Methylobacterium fujisawaense (Green et al., 1988), Methylobacterium lusitanum (Doronina et al., 2002), Methylobacterium mesophilicum (Green & Bousfield, 1983), M. organophilum (Patt et al., 1976), M. populi (Van Aken et al., 2004), Methylobacterium radiotolerans (Green & Bousfield, 1983), Methylobacterium rhodesianum (Green et al., 1988), Methylobacterium rhodinum (Green & Bousfield, 1983), Methylobacterium suomiense (Doronina et al., 2002), Methylobacterium thiocyanatum (Wood et al., 1998) and Methylobacterium zatmanii (Green et al., 1988). In addition, the novel species Methylobacterium nodulans has recently been described (Jourand et al., 2004). Members of the genus Methylobacterium are distributed among a wide variety of natural habitats, including soil, dust, air, freshwater and aquatic sediments. These bacteria also occur in man-made environments, including potable water supplies, air-conditioning systems and masonry bathrooms and washstands, where they sometimes produce pink ropy masses of growth (Hiraishi et al., 1995; Trotsenko et al., 2001; Ultee et al., 2004). Some species have been described as opportunistic human pathogens (Truant et al., 1998; Hornei et al., 1999). On the other hand, methylotrophic bacteria are frequently associated with terrestrial and aquatic plants, colonizing roots and leaf surfaces (Austin et al., 1978; Yoshimura, 1982; Corpe & Rheem, 1989; Trotsenko et al., 2001; Lidstrom & Chistoserdova, 2002). It is important to note that most of the Methylobacterium strains isolated from aquatic environments are highly resistant to chlorine (Hiraishi et al., 1995). The chlorine tolerance of methylotrophic bacteria may explain why these organisms frequently occur in human-related environments.

Recent studies focusing on determination of the bacterial population in the drinking water of a municipal water supply in Seville (Spain) during 2003 permitted us to isolate a large number of bacteria. In the present paper, we have described the features of five novel isolates and shown that they constitute two novel species of the genus Methylobacterium, for which we propose the names Methylobacterium hispanicum sp. nov. and Methylobacterium aquaticum sp. nov.

The five strains, designated GR16^T and GR18 (isolated from culture medium R2A; Difco), and GP22, GP32 and GP34^T [isolated from plate count agar (PCA); Difco], were studied in detail. They were Gram-negative rods, strictly aerobic and motile (Table 1⇓), occurring singly, in pairs or in rosettes (Fig. 1⇓). Isolates were routinely maintained on PCA.

These bacteria, isolated from chlorinated water, were relatively slow growing and required between 5 and 7 days at 28 °C to form detectable colonies. The same growth characteristics were found in the chlorine-resistant strains isolated by Hiraishi et al. (1995). The methods used for phenotypic characterization have been described previously in detail (Doronina et al., 1998). Because these bacteria are slow growing, identification by physiological testing is laborious. Moreover, the different species of this genus share a great number of phenotypic characteristics (Doronina et al., 2002; McDonald et al., 2001; Urakami et al., 1993; Wood et al., 1998; Van Aken et al., 2004) and high chemotaxonomic homogeneity is also observed in the genus.

As with all other Methylobacterium species, the five isolates were strict aerobes, catalase positive and able to produce urease. Indole and H₂S were not produced, and methyl red and Voges–Proskauer tests were negative. The five isolates possessed a number of phenotypic similarities. Colonies were pink to red and convex. All were capable of hydrolysing starch but not gelatin, aesculin, casein or DNA. Only strains GR16^T, GP22 and GP32 hydrolysed Tween 80. Nitrate was reduced to nitrite, Simmons' citrate test was positive and acid was oxidatively produced from d-arabinose and not from d-glucose, d-galactose, d-mannose or maltose. No growth occurred in the presence of 1·0 % NaCl.

The nutritional features of the isolates were determined using Biolog MicroPlates. Strains were grown on isolate medium (Biolog) at 28 °C for 72 h and suspended in sterile saline medium, within the density range specified by the manufacturer with a Biolog photometer model 21101. Immediately after suspending the cells in the saline solution, the suspensions were transferred into sterile multichannel pipetter reservoirs (Biolog) and the Biolog GN MicroPlates were inoculated with 125 μl of the cell suspension per well by means of an eight-channel repeating pipetter (Biolog). The inoculated plates were incubated at 28 °C for 7 days and the results were read with a MicroPlate Reader using Microlog 3.59 computer software to perform automated reading. The results of the nutritional tests are shown in the species descriptions and indicated a wide nutritional versatility of the five isolates.

Chromosomal DNA of the five strains was isolated and purified according to the methods described by Wilson (1987) and Marmur (1961) and partially modified by Hood et al. (1987). The 16S rRNA gene of the five isolates was amplified by PCR using two universal primers as described previously (Mellado et al., 1995) and almost-complete nucleotide sequences (approx. 1400 bp) were determined. The arb software package (Ludwig & Strunk, 1996) was used for 16S rRNA gene sequence analysis. Base-frequency filters were applied in the sequence comparison analysis and the effects on the results were evaluated.

16S rRNA gene phylogenetic analysis performed based on the neighbour-joining method (Saitou & Nei, 1987) clearly showed the position of this group of strains within the genus Methylobacterium. Maximum-parsimony- and maximum-likelihood-based trees using the full dataset or a selection of sequences were also obtained showing the same phylogenetic position of the group of isolates in the genus Methylobacterium, forming two clusters separated from the other species of this genus (Fig. 2⇓).

Strains GP34^T and GR18 grouped together and their closest relatives were M. organophilum (95·6 % sequence similarity), M. mesophilicum (95·7 %), M. fujisawaense (96·4 %) and M. radiotolerans (96·6 %). Strains GR16^T, GP32 and GP22 also clustered together. Strain GR16^T was most closely related to M. extorquens (96·0 %), M. rhodesianum (95·8 %), M. zatmanii (95·5 %) and M. thiocyanatum (95·2 %). In addition, the 16S rRNA gene sequence similarity of strain GR16^T with respect to strains GP34^T and GR18 was 94·2 and 94·1 %, respectively. The 16S rRNA gene sequence similarity of strain GP34^T with respect to strains GR16^T, GP22 and GP32 was not greater than 94·5 %. According to these phylogenetic data, the isolates belonged to the genus Methylobacterium, but did not show similarity values higher than 96 % to the type strains of species of Methylobacterium, indicating that they constituted novel species of this genus.

The G+C content of genomic DNA was determined from the mid-point value (T_m) of the thermal denaturation profile (Marmur & Doty, 1962) using the equation of Owen & Hill (1979). The five isolates were shown to be very similar in their G+C content, ranging between 66·5 and 67·8 mol%. The G+C content of strains GP34^T and GR18 was 67·7 and 66·5 mol%, respectively, and of strains GR16^T, GP22 and GP32 was 67·5, 67·7 and 67·8 mol%, respectively.

Sequence similarity values obtained for these two groups of strains isolated from drinking water and all Methylobacterium species with validly published names were low enough to propose their placement in two novel species within this genus. To confirm these results, DNA–DNA hybridization studies were performed following the competition procedure of Johnson (1994), described in detail in Mormile et al. (1999). The hybridization temperature was 60 °C, which was within the limit of validity for the filter method (De Ley & Tijtgat, 1970), and the percentage of hybridization was calculated according to Johnson (1994). DNA–DNA hybridization values between these five strains and the type strains of the Methylobacterium species that were more closely related phylogenetically are shown in Table 2⇓. Our strains were found to have low levels of hybridization, showing relatedness values not higher than 45 % with the other Methylobacterium species studied. In contrast, a DNA–DNA hybridization value of 85 % was found between strain GP34^T and isolate GR18. In addition, strain GR16^T exhibited levels of DNA–DNA hybridization equal to or greater than 80 % with strains GP22 and GP32. These data indicated that the five isolates were genotypically distinct from the phylogenetically related type strains of Methylobacterium species. Furthermore, we have provided clear evidence that these novel isolates form two phylogenetic and genotypic groups, showing DNA–DNA hybridization values not higher than 45 % and 16S rRNA gene sequence similarities below 96 % with respect to previously described species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). On the basis of these results, two novel species within the genus Methylobacterium are proposed, with the names Methylobacterium hispanicum sp. nov. (with type strain GP34^T) and Methylobacterium aquaticum sp. nov. (with type strain GR16^T). The phenotypic characteristics that differentiate the two novel species from their phylogenetically closest relatives are summarized in Table 1⇑.