Rheinheimera texasensis sp. nov., a halointolerant freshwater oligotroph

The genus Rheinheimera was first proposed by Brettar et al. (2002) and, at the time of writing, contains three species, Rheinheimera baltica (Brettar et al., 2002), Rheinheimera pacifica (Romanenko et al., 2003) and Rheinheimera perlucida (Brettar et al., 2006), all of which are of marine origin. On the basis of 16S rRNA gene sequence comparisons, members of the genus Rheinheimera are related to a clinical isolate, Alishewanella fetalis (Fonnesbech Vogel et al., 2000), and an alkaliphilic psychrophile, ‘Arsukibacterium ikkense’ (Schmidt et al., 2007). A new isolate, strain A62-14B^T, is described in this paper and is proposed as a novel member of the genus Rheinheimera on the basis of the recommendations for the description of a novel species given by Kämpfer et al. (2003).

Strain A62-14B^T was isolated from Spring Lake in San Marcos, Texas, USA (2 ° 53′ 26″ N 9 ° 56′ 1″ W), during a study of freshwater bacteria capable of colonizing dialysis tubing in response to acylated homoserine lactones (McLean et al., 2005). The strain was isolated on R2A agar (Difco) after incubation for 48 h at 30 °C and produced white, semi-transparent colonies. Subculturing was performed on R2A agar incubated for 48 h at 25 °C and 7 days at 20 °C. On R2A agar, strain A62-14B^T was able to grow at temperatures in the range 20–37 °C. No growth was seen at 4, 42 or 55 °C. Limited growth was seen on dilute (10 %, w/v) tryptone soy agar after 48 h at 30 °C. No other media tested [brain-heart infusion agar, full-strength tryptone soy agar, Luria–Bertani agar, nutrient agar (all from Difco) and Biolog universal growth media (Biolog)] (Amy et al., 1992) could support growth of strain A62-14B^T. The organism grew between pH 6.5 and 9.6, with optimal growth occurring at pH 7.5–8.0. No growth was observed at pH 5.0. The novel strain could not grow in the presence of NaCl at concentrations greater than 1 % (w/v). Gram staining was performed using a standard protocol (Doetsch, 1981).

Cell morphology was observed using a Zeiss microscope at a magnification of ×1000, using cells grown overnight on R2A agar at 30 °C. Motility was observed by examining a wet-mount slide preparation of strain A62-14B^T with a Zeiss microscope at ×800, using both phase-contrast and dark-field optics. Dilute suspensions of strain A62-14B^T and related organisms, namely R. pacifica KMM 1406^T [obtained from the Culture Collection of the University of Göteborg, Sweden, and cultured overnight at 30 °C on brain-heart infusion agar (Difco)] and Alishewanella fetalis ATCC BAA-284^T (obtained from the American Type Culture Collection, USA, and cultured overnight at 37 °C on brain-heart infusion agar), were examined for the presence of flagella and pili by using negative staining and transmission electron microscopy (Tolson et al., 1995). The transmission electron microscopy examination revealed that cells of strain A62-14B^T have either single polar flagella (Fig. 1a⇓) or multiple polar and lateral flagella; occasionally, filaments were seen between cells lying in close proximity (Fig. 1b⇓). Cells of R. pacifica KMM 1406^T had single polar flagella, but no flagella were seen on cells of Alishewanella fetalis ATCC BAA-284^T (data not shown).

For 16S rRNA gene sequencing, cultures were grown for 16 h at 30 °C in R2A broth. DNA was extracted using the DNeasy tissue kit (Qiagen). The 16S rRNA gene was amplified using primers EUB7F and 1492R, as described elsewhere (Lane, 1991). The PCR products were purified using a minElute gel extraction kit (Qiagen). Sequencing was performed using a DNA sequencer (model 377; Applied Biosystems) and a BigDye cycle sequencing kit (version 3.1; Perkin-Elmer Biosystems); the 1447 bp sequence was aligned with related sequences obtained from GenBank and EMBL by using blast and fasta searches (Altschul et al., 1997; Pearson & Lipman, 1988) using Sequencher 4.2.2 (Gene Codes Corporation) and clustal_x (Thompson et al., 1997). Using the MrModeltest program, version 2.2 (Nylander, 2004), the GTR+I+G model of sequence evolution was found to best fit this dataset. Phylogenetic analyses (Fig. 2⇓) were performed using paup 4.0b10 with distance-based neighbour-joining, maximum-parsimony and maximum-likelihood methods (Swofford, 2002). Confidence in the topology obtained from these analyses was gauged using bootstrap resampling methods in paup and included 10 000 replications (1000 replications for the maximum-likelihood tree) and a full heuristic search. Only those bootstrap percentages of at least 70 % were retained, as they demonstrate good support (Hillis & Bull, 1993). Bayesian analysis was completed using mrbayes (version 3.0) and a 90 % majority rule consensus tree was generated from this treefile in paup (Huelsenbeck & Ronquist, 2001). Fig. 2⇓ shows a representative analysis of closely related cultured species (see Supplementary Fig. S1, available at IJSEM Online, for a full analysis with cultured and uncultured bacterial sequences).

Strain A62-14B^T most closely resembled an unclassified, filament-producing, gammaproteobacterium designated strain F8 (GenBank accession number AY077611) that had also been isolated from a freshwater environment (Böckelmann et al., 2006). Here, the three recognized species of the genus Rheinheimera and strain A62-14B^T partitioned into four distinct clades. Only the Bayesian analyses partially resolved the relationships between the recognized species of the genus Rheinheimera and their close relatives. As noted by Brettar et al. (2006), the phylogenetic position of strain A62-14B^T was unstable with respect to the genus Alishewanella, as demonstrated by a lack of bootstrap support in the neighbour-joining, maximum-parsimony and maximum-likelihood analyses.

Metabolic profiles for preferred carbon, nitrogen, phosphorous and sulfur sources were obtained by the use of the phenotype microarray technique (Bochner et al., 2001). An isolate of strain A62-14B^T was cultured on R2A agar and shipped to Biolog (USA) for phenotype microarray testing of approximately 1000 characteristics. Duplicate phenotype microarray readings were recorded for 24 h and kinetic data were analysed with proprietary software. The cellular fatty acids were determined at MIDI Laboratories (USA) by using fatty acid methyl ester analysis (Norton & LeChevallier, 2000). Strain A62-14B^T grew on R2A agar, but, unlike R. baltica, R. pacifica, R. perlucida and Alishewanella fetalis, did not grow on marine agar or Luria–Bertani agar. As a result, comparison of the fatty acid profiles of these species is limited by differences in growth conditions. The metabolic profiles are summarized in Table 1⇓ and in the species description. As strain A62-14B^T is generally salt-intolerant, we provide an emended description for the genus Rheinheimera based on the current study and that of Brettar et al. (2002). On the basis of the data from this study, strain A62-14B^T represents a novel species of the genus Rheinheimera, for which the name Rheinheimera texasensis sp. nov. is proposed.