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

The genus Limnohabitans (Comamonadaceae, Betaproteobacteria) was established by Hahn et al. (2010) and currently contains three described species (Kasalický et al., 2010). The genus represents a phylogenetic cluster formed mainly by environmental sequences obtained by cultivation-independent 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 (Kasalický et al., 2010), 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.

Strain MWH-BRAZ-DAM2D^T was isolated from the subtropical Monjolinho Pond (2 ° 59′ 10.14″ S 4 ° 52′ 50.28″ W) located on the university campus in São Carlos, São Paulo state, Brazil. The strain was isolated by using the dilution-acclimatization method and NSY medium (Hahn et al., 2004, 2005). Strain MWH-BRAZ-DAM2D^T grew on a variety of solidified complex media including Luria–Bertani agar (Difco BD), Casitone agar (Difco BD), R2A agar (Remel) and NSY agar (Hahn et al., 2004), forming unpigmented, smooth, convex colonies.

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⁻¹) with and without the respective test substrate (0.5 g l⁻¹). 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 (−), 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 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⁻¹) for 2 days at 21 °C was analysed. Results of the phenotypic and chemotaxonomic investigations are presented in Tables 1⇓ and 2⇓.

blast searches (Altschul et al., 1990) against the database with the 16S rRNA gene sequence of the novel isolate in December 2009 resulted in five and 81 hits with >99 % and >97 % sequence similarities, respectively. Thirteen out of the 81 hits represented cultivated strains, whereas the majority represented uncultivated bacteria. No cultivated strains were among the hits with >99 % sequence similarity. This group was represented by environmental sequences obtained from two estuary systems (Delaware and Chesapeake Bays, USA), as well as from Ipswich River, MA, USA (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 (Hahn et al., 2010).

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 : 1ω7c/ω6c (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 (Hahn et al., 2010) 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 (Hahn et al., 2010; 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 : 1ω5c, C_18 : 0, 11-Me-C_18 : 1ω7c and C_18 : 1ω9c, 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 considered. Therefore, it is proposed that the novel species Limnohabitans australis sp. nov. be established to accommodate strain MWH-BRAZ-DAM2D^T.