The genus Chelatococcus was first described by Auling et al. (1993) and later by Egli and Auling (2005) as consisting of gram negative bacteria with the distinctive characteristics of utilizing the metal-chelating aminopolycarboxylic acid nitrilotriacetic acid (NTA). Following the first description of species C. asaccharovorans (Auling et al. 1993; Egli and Auling 2005), two distinct species were recently affiliated to the genus, C. daeguensis (Yoon et al. 2008) and C. sambhunathii (Panday and Das 2010). Ecologically, strains of the genus were isolated from different aquatic and waste treatment environments (Wilberg et al. 1993; Egli and Auling 2005; Ibrahim et al. 2010; Xu et al. 2014).
New physiological characteristics were reported for the new isolates, MW9T, MW10, MW11, MW12, MW13 and MW14, such as high optimum growth temperature, star-shaped cell aggregates, and PHA accumulation. The 16S rRNA gene sequences of these isolates are very similar and place them in the genus Chelatococcus (Ibrahim et al. 2010).
These new unusual characteristics made it important to conduct a more detailed study for a precise affiliation of these isolates within the genus Chelatococcus, possibly as a new species. Moreover, a clear differentiation between the new isolates is needed.
SSC aggregates have previously been found in several taxa within the Alphaproteobacteria (Biebl et al. 2007). However, this aggregation was not previously reported for any of the Chelatococcus species, possibly because growth was usually studied in nutrient-rich media. In the present study and during cultivation in mineral salts medium, the type strains C. daeguensis LMG 25471T and C. sambhunathii LMG 26063T showed SSC aggregation during growth.
Little information about PHA accumulation was reported for strains belonging to related Chelatococcus species: PHB granules were observed in the type strain of C.
asaccharovorans (Egli and Auling 2005) (Table 1) and very recently, a new thermophilic denitrifying bacterial strain of C. daeguensis, TAD1, was reported to accumulate poly(3-hydroxybutyrate) at 45 and 50 °C from glucose (Xu et al. 2014). PHA accumulation can be regarded as a characteristic attribute of the genus Chelatococcus, especially after the confirmation of high PHB content in cells of strains C. daeguensis LMG 25471T and C. sambhunathii LMG 26063T in the present study, and the high content reported for our new thermophilic isolates (Ibrahim et al. 2010).
The identity of rep-PCR profiles and partial dnaK gene sequences between the new isolates together with the high similarity of their 16S rRNA gene sequences would suggest that they may not be classified as separate organisms, but rather be different colonies with slightly differences in carbohydrates preference (Ibrahim et al. 2010). We therefore chose the glucose utilizing isolate MW9T as representative of the new thermophilic isolates and describe it as a new species given the differences in 16S rRNA, rep-PCR, and partial dnaK gene sequences with other Chelatococcus species. This isolate was used for further investigations.
The dnaK gene sequence of the type strain of the type species C. asaccharovorans showed less similarity to all new Chelatococcus strains (Fig. 4), even though it did belong to the Chelatococcus cluster in 16S rRNA gene phylogeny (Ibrahim et al. 2010), indicating its dnaK sequence may be affected by lateral gene transfer. Comparison of the complete dnaK gene might clarify this possibility.
Utilization of NTA as the sole carbon and nitrogen source was investigated for the new strain MW9T. However, this strain was not able to grow on NTA alone. Similar results were also reported for strain C. daeguensis (Yoon et al. 2008). The emended genus description by Yoon et al. (2008) also stated that this characteristic could be positive or negative variable within different species of Chelatococcus.
DNA G+C content, together with the physiological properties summarized in Table 1, are in accordance with the genus amendment of Chelatococcus reported by Yoon et al. (2008). Considering the 70.0 mol% DNA–DNA relatedness cut-off point recommended for bacterial species delineation (Wayne et al. 1987), strain MW9T should be regarded as representing a novel species of the genus Chelatococcus.
The phylogenetic distinctiveness and low DNA–DNA relatedness with other Chelatococcus type strains reported here as well as phenotypic differences (Table 1), justify the creation of a new Chelatococcus species for strain MW9T, for which the name Chelatococcus
thermostellatus sp. nov. is proposed. The species was isolated from aerobic organic waste compost in Germany, and DNA sequence data indicate it may be present in other compost systems. Indeed, a FASTA search with the 16S rRNA gene sequence of MW9T in the EMBL database Environmental subsection revealed highly similar Chelatococcus sequences from asparagus straw compost from China (99.9 %, accession number JQ740271, clone ASC373), from a pilot scale municipal drum compost from Finland (99.8 %, FN667527, clone PS3657), from dairy manure and rice chaff composting samples in China [99.6 %, JQ337129, clone NT-3-59 (Tian et al. 2013)], and from fermented composting material in China [99.3 %, FJ930065, clone 6 (Wang et al. 2011)].
As a conclusion, the present study, while presenting a new species in the genus Chelatococcus, also confirmed PHA accumulation as a new genus-wide feature and showed that the formation of SSC aggregates can be used to describe many strains of Chelatococcus species. Strain MW9T is capable of growing and producing high content of PHB at higher temperature compared to other related strains. This makes this new Chelatococcus strain a potent candidate for bioplastic production at thermophilic condition, where production costs can be minimized. Further physiological and proteomic investigations should facilitate a precise differentiation between isolates, MW9T, MW10, MW11, MW12, MW13 and MW14.
Description of Chelatococcus thermostellatus sp. nov
Chelatococcus thermostellatus [ther.mo.ste’llatus. Gr. adj. thermos, hot; N.L. v. stellatus, to form star-shape cell aggregates; N.L. part. adj. thermostellatus, referring to the formation of star-shaped cell aggregates at high temperatures].
Cells are Gram-negative, oxidase positive motile rods (0.2–0.4 × 1.0–2.5 μm), aerobic, and non-spore forming. Good growth occurs on LB broth, nutrients broth, standard I nutrient broth, and MSM-gluconate/glucose/glycerol after 1–3 days at 50 °C. No growth was detected below 37 °C or above 55 °C. Optimum pH 7.0–7.5. NTA was not utilized as a sole source for carbon and nitrogen. During optimum growth on MSM, star-shaped cell aggregates, and PHB intracellular granules were observed as well. Colonies on MSM are circular with bright white to beige color and 0.5–1.0 mm in diameter. The major fatty acids comprises (>10 %) 18:1 ω7c, 19:0 cyclo ω8c and summed feature 2 (12:0 aldehyde, 16:1 iso I and/or 14:0 3OH). The DNA G+C content of the type strain is 67.1 mol%.
The type strain MW9T (=LMG 27009T = DSM 28244T) was isolated from aerobic organic waste compost in Münster, Germany.