Downloaded from www.microbiologyresearch.org by IP: 54.162.133.179 On: Mon, 29 Feb 2016 17:29:16 Defluvicoccus vanus gen. nov., sp. nov., a novel Gram-negative coccus/coccobacillus in the ‘Alphaproteobacteria’ from activated sludge A. M. Maszenan, 1 3 R. J. Seviour, 1 B. K. C. Patel, 2 P. H. Janssen 3 and J. Wanner 4 Correspondence R. J. Seviour r.seviour@latrobe.edu.au 1 Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3550, Australia 2 School of Biological and Biomedical Sciences, Faculty of Science and Technology, Griffith University, Nathan, Queensland 4111, Australia 3 Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia 4 Department of Water Technology and Environmental Engineering, Prague Institute of Chemical Technology, Praha 6, Czech Republic A novel Gram-negative coccus/coccobacillus, strain Ben 114 T , growing in tetrads, clusters or aggregates, was isolated from activated sludge by micromanipulation. 16S rRNA gene sequence analysis revealed that it belonged to the ‘Alphaproteobacteria’, with no close relatives among cultured bacterial isolates. On the basis of phylogenetic data, this organism is considered to belong to a new genus, Defluvicoccus, represented by the species Defluvicoccus vanus sp. nov., a name chosen because of the distinctive staining properties of this organism; only the cell wall stained strongly with a wide range of stains, giving the cell a hollow and empty appearance. No intracellular polyphosphate granules could be detected after staining, but poly-b-hydroxyalkanoate inclusions were detected using Nile blue A staining. Because of its taxonomic distance from its closest relatives among the ‘Alphaproteobacteria’, namely members of the genera Azospirillum, Phaeospirillum, Rhodospirillum, Rhodocista, Magnetospirillum and Rhodospira, D. vanus is considered to represent a new phylogenetic lineage within subgroup 1 of the ‘Alphaproteobacteria’, the D. vanus subgroup. The type strain is Ben 114 T (=NCIMB 13612 T =CIP 107350 T ). The application of molecular techniques to the study of activated sludge systems has revealed the presence of many previously uncultured bacteria (Seviour & Blackall, 1999; Loy et al., 2002; Wagner & Loy, 2002). However, the techniques have not always provided a clearer explanation for how these systems might operate, a comment particu- larly relevant to processes of enhanced biological phos- phorus removal (EBPR) (Loy et al., 2002; Seviour et al., 2003). Molecular studies on community structure of EBPR systems suggest that members of the Actinobacteria and ‘Betaproteobacteria’ closely related to members of the genus Rhodocyclus are probably responsible for phosphate removal in some sludge plants (Hesselmann et al., 1999; Crocetti et al., 2000; Zilles et al., 2002; Seviour et al., 2003). There is some evidence that other bacteria under certain conditions can out-compete the polyphosphate-accumulating organ- isms (PAO) during anaerobic substrate uptake, eventually leading to accumulation of glycogen-like carbohydrates instead of polyphosphate (polyP). Bacteria referred to as the ‘glycogen-accumulating organisms’ (GAO) are one such group (Seviour et al., 2000; Crocetti et al., 2002). They were originally called ‘G-bacteria’ because they dominated plants fed glucose (Cech & Hartman, 1993), and exist often as distinctive coccal cells in tetrads or clusters (Seviour et al., 2000). Pure cultures of these tetrad-forming organisms (TFO) from different countries were described as separate species of a novel genus Amaricoccus, in subgroup 3 of the ‘Alphaproteobacteria’ (Maszenan et al., 1997). Several other phylogenetically different TFO have also been isolated from activated sludge (Nakamura et al., 1995; Yoshimi et al., 1996; Maszenan et al., 1999a, b, 2000). Some, like the actinobacterial Tetrasphaera species (Maszenan et al., 2000) and the betaproteobacterium Quadricoccus australiensis (Maszenan et al., 2002), appear to accumulate polyP in 3Present address: Environmental Engineering Research Centre, School of Civil and Environmental Engineering, Nanyang Technological Univer- sity, Singapore 639798. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Ben 114 T is AF179678. Abbreviations: EBPR, enhanced biological phosphorus removal; GAO, glycogen-accumulating organisms; PAO, polyphosphate-accumulating organisms; PHA, poly-b-hydroxyalkanoate; polyP, polyphosphate; TFO, tetrad-forming organisms. 02332 G 2005 IUMS Printed in Great Britain 2105 International Journal of Systematic and Evolutionary Microbiology (2005), 55, 2105–2111 DOI 10.1099/ijs.0.02332-0