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Department of Biology, Texas State University – San Marcos, 601 University Drive, San Marcos, TX 78666, USA
Correspondence
Robert J. C. McLean
McLean{at}txstate.edu
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7c, 38.56 %; C16 : 0, 19.04 %; C12 : 0 3-OH, 12.83 %; C18 : 17c, 7.70 %) and the motility of strain A62-14BT support its affiliation to the genus Rheinheimera. The salt intolerance of strain A62-14BT, together with the results of other physiological and biochemical tests, allowed the differentiation of this strain from the three species of the genus Rheinheimera with validly published names. Therefore strain A62-14BT represents a novel species of the genus Rheinheimera, for which the name Rheinheimera texasensis sp. nov. is proposed. The type strain is A62-14BT (=ATCC BAA-1235T=DSM 17496T). The description of the genus Rheinheimera is emended to reflect the halointolerance and freshwater origin of strain A62-14BT.
Present address: Qiagen Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355, USA. 
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A62-14BT is AY701891.
An expanded phylogenetic tree for strain A62-14BT and related taxa is available with the online version of this paper.
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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 A62-14BT, 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 A62-14BT 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 A62-14BT 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 A62-14BT. 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 x1000, using cells grown overnight on R2A agar at 30 °C. Motility was observed by examining a wet-mount slide preparation of strain A62-14BT with a Zeiss microscope at x800, using both phase-contrast and dark-field optics. Dilute suspensions of strain A62-14BT and related organisms, namely R. pacifica KMM 1406T [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-284T (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 A62-14BT 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 1406T had single polar flagella, but no flagella were seen on cells of Alishewanella fetalis ATCC BAA-284T (data not shown).
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). 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).
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). Here, the three recognized species of the genus Rheinheimera and strain A62-14BT 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 A62-14BT 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 A62-14BT 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 A62-14BT 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 A62-14BT 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 A62-14BT represents a novel species of the genus Rheinheimera, for which the name Rheinheimera texasensis sp. nov. is proposed.
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The description of the genus Rheinheimera is as given by Brettar et al. (2002) but with the following changes. Of freshwater, estuarine or marine origin. NaCl supports growth, except in the case of one species, but strains are not tolerant of high salinities (>6 %).
Description of Rheinheimera texasensis sp. nov.
Rheinheimera texasensis (tex.as.en'sis. N.L. fem. adj. texasensis pertaining to Texas, the location from which the organism was first isolated).
Cells are Gram-negative, rod-shaped, 1.2–2.5 µm long and 0.7–0.8 µm wide, with single polar flagella or several polar and lateral flagella. Facultatively anaerobic. Network of filaments noted among cells lying in close proximity. After 24 h growth on R2A agar at 37 °C, colonies are 1–2 mm in diameter, smooth, non-pigmented, transparent, raised and circular with entire margins. Optimal growth occurs between 30 and 37 °C, with slow growth occurring at 20–25 °C and no growth above 40 °C. Withstands pH levels from 6.5 to 9.6, with optimal growth occurring at pH 7.5–8.0. No growth occurs at NaCl concentrations above 1 % (w/v). Carbon sources utilized include N-acetylglucosamine, Tweens 20, 40 and 80, D-glucose, D-lactose, D-galactose, sucrose, trehalose, methyl pyruvate, 
-cyclodextrin, laminarin, glycogen and palatinose; citrate is not utilized. The type strain is positive in tests for starch and gelatin hydrolysis, nitrate reduction and oxidase and catalase activities. Indole production is not detected. The DNA G+C content of the type strain is 48.2 mol%. The predominant fatty acids are C16 : 17c (38.6 %), C16 : 0 (19.0 %), C12 : 0 3-OH (12.8 %), C18 : 17c (7.7 %), C17 : 18c (3.8 %), C12 : 0 (2.0 %) and C18 : 0 (2.0 %).
The type strain, A62-14BT (=ATCC BAA-1235T=DSM 17496T), was isolated from Spring Lake, a freshwater lake in San Marcos, Texas, USA.
Note added in proof
Since this paper was accepted for publication, two further novel species of the genus Rheinheimera have been described, Rheinheimera aquimaris (Yoon et al., 2007) and Rheinheimera chironomi (Halpern et al., 2007).
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ACKNOWLEDGEMENTS |
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