Microcella alkaliphila sp. nov., a novel member of the family Microbacteriaceae isolated from a non-saline alkaline groundwater, and emended description of the genus Microcella

The ophiolite-like geological context of the aquifer at Cabeço de Vide in southern Portugal, together with its chemical characteristics, strongly suggests serpentinization activity. The groundwater vents at a temperature of 20.5 °C and has high alkalinity (pH 11.4) associated with an extremely low ionic concentration, with Ca²⁺ and OH⁻ as major chemical constituents (Tiago et al., 2004). The majority of the bacterial isolates recovered from Cabeço de Vide groundwater belonged to the recently described species Microcella putealis (Tiago et al., 2005). In this study, we report the taxonomic characterization of another Cabeço de Vide isolate that represents a novel species of the genus Microcella.

Strain AC4r^T was isolated from borehole water at Cabeço de Vide on alkaline buffered medium 2 (ABM2), adjusted to pH 9.5, at 30 °C, as described previously (Tiago et al., 2004). The isolate was routinely cultured under the same conditions and maintained at −70 °C in the same medium supplemented with 15 % glycerol. Unless otherwise stated, all morphological, biochemical and tolerance tests were performed on that medium at 35 °C, with incubation for up to 6 days as described previously (Tiago et al., 2005).

The growth-temperature range of strain AC4r^T was examined in ABM2 liquid medium buffered at pH 9.5; the pH range for growth was determined at 35 °C in the same medium, and growth in the presence of NaCl was examined in liquid medium at pH 9.5 and 35 °C as described previously (Tiago et al., 2005).

Assimilation of single carbon sources was determined using API 50 CH test strips (bioMérieux), with 0.1 M carbonate/bicarbonate buffer (pH 9.5) supplemented with 0.3 % (w/v) agar (Difco), 0.05 % NH₄Cl (Merck) and macronutrient and micronutrient solutions as described previously (Tiago et al., 2005). Anaerobic growth was assessed at 35 °C in anaerobic chambers with a H₂/CO₂ atmosphere (bioMérieux).

Strain AC4r^T formed yellow-pigmented colonies and Gram-positive, small, rod-shaped, non-motile cells (0.3 μm in width and 3–4 μm in length). The isolate had an optimum growth temperature of 35 °C and did not grow at 20 or 45 °C. The optimum pH for growth was 9.5; no growth was observed at pH 7.5 or pH 10.5. Optimal growth was observed in the absence of NaCl, but growth occurred in ABM2 containing up to 8.0 % NaCl. Despite having the same optimal temperature as the type strain of M. putealis, strain AC4r^T had a narrower temperature range for growth, a higher optimal pH for growth and did not grow below pH 8.0 (Tiago et al., 2005).

Strain AC4r^T and the type strain of M. putealis utilized several sugars and proteinaceous substrates, but several differences between the two strains were detected that can be used to distinguish the strains (Table 1⇓).

Purified cell-wall preparations were obtained by using the method of Schleifer & Kandler (1972). The amino acids and peptides in cell-wall hydrolysates were analysed as described previously (Schleifer & Kandler, 1972; Schleifer, 1985). Lipoquinones were extracted (from 300 mg freeze-dried cells) and detected as described previously (Tindall, 1989). Cultures used for fatty acid analyses were grown on ABM2 medium, adjusted to pH 9.5, in sealed plastic bags submerged in a water bath at 35 °C for 24 h. Fatty acid methyl esters were obtained from fresh wet biomass by saponification, methylation and extraction as described previously and were separated, identified and quantified with standard MIS Library Generation Software as described by the manufacturer (Microbial ID).

The peptidoglycan of strain AC4r^T was of the B2β type (according to the nomenclature of Schleifer & Kandler, 1972), with l-homoserine in position 3 of the tetrapeptide and an interpeptide bridge containing glycine and d-ornithine. Although the peptidoglycan structure determined for the novel strain and for M. putealis CV2^T is of type B, the diagnostic diamino acid of M. putealis strain CV2^T is Lys, although the complete peptidoglycan structure of M. putealis remains unknown (Tiago et al., 2005). The respiratory quinones found in strain AC4r^T were menaquinones: MK-13 (47 %), MK-14 (35 %) and MK-12 (18 %). The menaquinone composition and the relative amounts of the menaquinones constitute additional distinctive features of the novel isolate in comparison with the species M. putealis (Table 1⇑). The fatty acid composition of strain AC4r^T was characterized by the predominance of branched fatty acids, namely anteiso-15 : 0, iso-16 : 0, iso-14 : 0 and iso-15 : 0, which constituted 32.6, 30.8, 12.5 and 9.8 %, respectively, of the total fatty acids. The fatty acid profiles of AC4r^T and of the type strain of M. putealis are similar (see Supplementary Table S1 available in IJSEM Online).

DNA for determination of the G+C content was isolated as described by Nielsen et al. (1995). The G+C content of the DNA was determined by HPLC as described by Mesbah et al. (1989). The DNA relatedness between the type strain of M. putealis and novel strain AC4r^T was determined by DNA–DNA hybridization. For this purpose, DNA was isolated and purified as described by Cashion et al. (1977) and reassociation reactions were carried out as described by De Ley et al. (1970) with the modifications described by Huß et al. (1983).

The 16S rRNA gene was amplified and sequenced as described by Tiago et al. (2004) and phylogenetic analysis was performed using the software package mega3 (Kumar et al., 2004) after multiple alignments of the sequence data had been obtained using clustal_x (Thompson et al., 1997). Evolutionary distances were calculated by using the method of Jukes & Cantor (1969), phylogenetic dendrograms were constructed using the neighbour-joining method (Saitou & Nei, 1987) and tree topologies were evaluated by performing a bootstrap analysis (Felsenstein, 1985) of 1000 datasets.

The DNA G+C content determined for strain AC4r^T was 67.1 mol%. Comparative analyses of 1505 nucleotide positions of the 16S rRNA gene sequence of strain AC4r^T with respect to representatives of the main lines of descent within the domain Bacteria indicated that this strain is a member of the family Microbacteriaceae (see Supplementary Fig. S1 available in IJSEM Online). The most closely related organism was M. putealis CV2^T (99.2 % similarity). Despite the close phylogenetic relationship, the two strains exhibited a DNA–DNA reassociation value of only 38.3 %. This fact, together with the distinctive phenotypic characteristics of strain AC4r^T, clearly indicates that it represents a novel species of the genus Microcella, for which we propose the name Microcella alkaliphila sp. nov.