Limnohabitans australis sp. nov., isolated from a freshwater pond, and emended description of the genus Limnohabitans

A chemo-organotrophic, aerobic, non-motile strain, MWH-BRAZ-DAM2DT, isolated from a freshwater pond in Brazil, was characterized phenotypically, phylogenetically and chemotaxonomically. Phylogenetic analysis of 16S rRNA gene sequences indicated affiliation of the strain with the genus Limnohabitans (Comamonadaceae, Betaproteobacteria). 16S rRNA gene sequence similarities between the isolate and Limnohabitans curvus MWH-C5T, representing the type species of the genus, and the type strains of Limnohabitans parvus and Limnohabitans planktonicus were 98.2, 96.5 and 97.0 %, respectively. DNA–DNA reassociation analyses with DNA of the type strains of all three previously described Limnohabitans species revealed similarity values in the range 26.2–44.6 %. The predominant fatty acids of the isolate were C16 : 1ω7c/ω6c, C16 : 0, C12 : 0 and C8 : 0 3-OH, the major quinone was ubiquinone Q-8 and the DNA G+C content was 55.8 mol%. The isolate could be discriminated from the type strains of the three Limnohabitans species by several phenotypic traits including differences in the utilization of several carbon sources. Based on the phylogeny of the isolate and its differences from the three most closely related species, the isolate represents a novel species for which the name Limnohabitans australis sp. nov. is proposed. The type strain is MWH-BRAZ-DAM2DT (=DSM 21646T=CCUG 56719T).

The genus Limnohabitans (Comamonadaceae, Betaproteobacteria) was established by Hahn et al. (2010) and currently contains three described species . The genus represents a phylogenetic cluster formed mainly by environmental sequences obtained by cultivationindependent methods from numerous freshwater systems (e.g. Zwart et al., 2002;Crump & Hobbie, 2005;Percent et al., 2008;Shaw et al., 2008), which was previously designated the 'Rhodoferax sp. BAL47 cluster' (Zwart et al., 2002). Investigations using fluorescence in situ hybridization probes specific for the R-BT065 subcluster of the 'Rhodoferax sp. BAL47 cluster ' (Šimek et al., 2001), which is represented by two of the Limnohabitans species , revealed the following relevant characteristics: a free-living, planktonic lifestyle (Šimek et al., 2001); a significant contribution to total cell numbers of freshwater bacterioplankton (up to 30 %; Šimek et al., 2010); a broad habitat range including, for instance, acidic and alkaline systems (Šimek et al., 2010); and the potential for rapid growth under in situ conditions (Šimek et al., 2006).
The type strains of all three Limnohabitans species described so far were all isolated from the water columns of stagnant freshwater systems located in Central Europe. Here, a novel strain, isolated from a pond located in subtropical South America, which is closely related to previously described Limnohabitans species, as well as to numerous uncultured bacteria represented by environmental sequences, is characterized and it is proposed that this strain represents a novel species, Limnohabitans australis sp. nov.
Carbon source utilization tests and other phenotypic characterizations were performed as described previously (Hahn et al., 2009). Briefly, growth enabled by utilization of specific substrates was determined by comparison of optical density established in liquid one-tenth-strength NSY medium (0.3 g l 21 ) with and without the respective test substrate (0.5 g l 21 ). Optical density differences of ,10 %, 10-50 % and .50 % of the optical density established on medium without test substrate were scored after 10 days of growth as no utilization (2), weak utilization (W) and good utilization (+), respectively. Analysis of the phylogenetic position of the novel isolate was performed by 16S rRNA gene sequence analysis as Table 1. Phenotypic traits of strain MWH-BRAZ-DAM2D T (Limnohabitans australis sp. nov.) and other members of the genus Limnohabitans Strains: 1, Limnohabitans australis sp. nov. strain MWH-BRAZ-DAM2D T ; 2, L. curvus strain MWH-C5 T ; 3, L. parvus strain II-B4 T ; 4, L. planktonicus strain II-D5 T . Substrate utilization tests were performed for all four strains under the same conditions. All four strains grew under anoxic conditions, were oxidase-positive, non-motile and unpigmented, and possessed ubiquinone Q-8 as major quinone. Furthermore, all strains were positive for utilization of D-glycerate, butyrate and a-ketoglutarate and none of the four strains utilized oxalate, DL-lactate, L-arginine, L-sorbose, N-acetylglucosamine, L-carnitine, betaine or spermidine. 2, Negative; +, positive; W, weakly positive.
Cell morphology Curved rods Curved rods Short rods Rods Cell length (mm) 55.8 55.5 59.9 59.9 described previously (Hahn et al., 2009). Determination of the DNA G+C content, analysis of major respiratory lipoquinones and DNA-DNA reassociation experiments, to ascertain whether the novel isolate belongs to a previously described Limnohabitans species (Wayne et al., 1987), were all carried out by the Identification Service and B. J. Tindall, DSMZ, Braunschweig, Germany. Fatty acid profiles were characterized by using the MIS Sherlock automatic identification system (MIDI) and the Sherlock Aerobic Bacterial Database (TSBA60) as described by Greenblatt et al. (1999). Biomass of duplicate cultures obtained by growing the strain in NSY medium (3 g l 21 ) for 2 days at 21 u C was analysed. Results of the phenotypic and chemotaxonomic investigations are presented in Tables 1 and 2. BLAST searches (Altschul et al., 1990) (Shaw et al., 2008;Crump & Hobbie, 2005). A phylogenetic analysis of the relationship of cultivated Limnohabitans strain and environmental sequences representing the so-called 'Rhodoferax sp. BAL47 cluster' was presented previously .
Phylogenetic analyses with sequence sets representing the most closely related recognized species by using the neighbour-joining (NJ) and the maximum-likelihood (ML) methods consistently revealed the affiliation of strain MWH-BRAZ-DAM2D T with the genus Limnohabitans (Fig. 1). The 16S rRNA gene sequence of the isolate possessed sequence similarities of 98.2, 97.0 and 96.5 % to those of the type strains of Limnohabitans curvus, Limnohabitans parvus and Limnohabitans planktonicus, respectively. DNA-DNA reassociation analyses with DNA of the type strains of the three previously described Limnohabitans species resulted in similarity values (mean values of duplicate measurements) of 26.2 % (L. parvus), 30.0 % (L. planktonicus) and 44.6 % (L. curvus). The duplicate measurements performed for each of the three pairings differed by 2.6 % (L. curvus) to 5.0 % (L. planktonicus). The predominant fatty acids of the isolate were C 16 : 1 v7c/v6c (73.5 %), C 8 : 0 3-OH (8.2 %), C 16 : 0 (7.7 %) and C 12 : 0 (7.4 %). The major quinone was ubiquinone Q-8 and the DNA G+C content was 55.8 mol% (Tables 1 and 2).
Strain MWH-BRAZ-DAM2D T can be distinguished from the type strain of L. curvus  by its ability to utilize malonate and its inability to utilize ethanol, propionate, malate, citrate, D-ribose, D-galactose, D-mannose and sucrose, as well as by a lower maximum NaCl concentration that supports growth and differences in the minimum and maximum growth temperatures (Table 1). In addition, the two strains differ in the presence of minor fatty acid compounds (Table 2). Differential traits that separate strain MWH-BRAZ-DAM2D T from all three previously described Limnohabitans species Kasalický et al., 2010) are utilization of malonate and no utilization of malate or citrate, as well as the absence of the minor fatty acids C 16 : 1 v5c, C 18 : 0 , 11-Me-C 18 : 1 v7c and C 18 : 1 v9c, a catalase-negative reaction, a lower maximum NaCl concentration that supports growth and higher minimum and maximum growth temperatures (Tables 1  and 2). Differences in thermal adaptation may reflect differences in adaptation to local climate conditions at the sites of origin of the four strains (subtropical versus temperate climate) and may not represent a trait shared by all members of the proposed species L. australis sp. nov. (Hahn & Pöckl, 2005).
The phylogenetic analysis, as well as several phenotypic and chemotaxonomic similarities, suggest that strain MWH-BRAZ-DAM2D T belongs to the genus Limnohabitans. The results of the DNA-DNA reassociation analyses demonstrate that the strain does not belong to one of the previously described Limnohabitans species when the recommendation of a threshold value of 70 % DNA-DNA similarity for delineation of prokaryotic species (Wayne et al., 1987) is Table 2. Whole-cell fatty acid composition of Limnohabitans australis sp. nov. and other members of the genus Limnohabitans Strains: 1, Limnohabitans australis sp. nov. strain MWH-BRAZ-DAM2D T ; 2, L. curvus strain MWH-C5 T ; 3, L. parvus strain II-B4 T ; 4, L. planktonicus strain II-D5 T . All strains were cultivated under identical conditions [NSY medium (3 g l 21 ) at 21 u C for 2 days]. The presented data are percentages of the summed fatty acids and represent mean values from analysed duplicate cultures. ND, Not detected. The description of the genus Limnohabitans is as given previously Kasalický et al., 2010), but with the following amendment. Members of the genus can be catalase-positive or catalase-negative.
Description of Limnohabitans australis sp. nov.
Limnohabitans australis (aus.tra9lis. L. masc. adj. australis southern, relating to the region in which the organism was isolated).
The type strain is MWH-BRAZ-DAM2D T (5DSM 21646 T 5CCUG 56719 T ), isolated from Monjolinho Pond, São Carlos, Brazil. The DNA G+C value of the type strain is 55.8 mol%.