Characterization of Tetragenococcus strains from sugar thick juice reveals a novel species, Tetragenococcus osmophilus sp. nov., and divides Tetragenococcus halophilus into two subspecies, T. halophilus subsp. halophilus subsp. nov. and T. halophilus subsp. flandriensis subsp. nov. A. Juste ´, 1,2,3 S. Van Trappen, 4 C. Verreth, 1,2,3 I. Cleenwerck, 4 P. De Vos, 4 B. Lievens 1,2,33 and K. A. Willems 1,2,33 Correspondence B. Lievens bli@scientiaterrae.org K. A. Willems kwi@scientiaterrae.org 1 Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Consortium for Industrial Microbiology and Biotechnology (CIMB), Department of Microbial and Molecular Systems (M 2 S), KULeuven Association, Lessius University College, De Nayer Campus, B-2860 Sint-Katelijne-Waver, Belgium 2 Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium 3 Leuven Food Science and Nutrition Research Centre (LFoRCe); B-3001 Leuven, Belgium 4 BCCM/LMG Bacteria Collection, Laboratory for Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium Most bacteria recovered so far from sugar thick juice during storage represent strains of the species Tetragenococcus halophilus. Recently, several Gram-positive, non-motile, non-spore- forming cocci with other physiological and genetic traits were isolated from sugar thick juice samples from different origins. In this study, representative isolates were investigated using a polyphasic taxonomic approach. The 16S rRNA gene sequence similarity between these isolates and their closest relative, Tetragenococcus muriaticus, was 97.4 %. The level of DNA–DNA relatedness between isolate T1 T , representing the newly found Tetragenococcus isolates, and T. muriaticus was 57 %. Isolate T1 T had a DNA G+C content of 36.7 mol%. Phylogenetic data and genomic and phenotypic features demonstrated that the isolates represent a novel species, for which the name Tetragenococcus osmophilus sp. nov. is proposed with T1 T as the type strain (5LMG 26041 T 5DSM 23765 T ). Additionally, T. halophilus isolates from high-salt and high- sugar environments showed clear differences in several physiological and genetic characteristics like RAPD fingerprints and 16S rRNA gene sequences. DNA–DNA hybridizations, however, showed 79 to 80 % relatedness between osmophilic and halophilic T. halophilus isolates, demonstrating that the different strains belong to the same species. Based on the phenotypic and genotypic differences observed, as well as the different origins of the strains and the industrial relevance of thick juice degradation, two subspecies of T. halophilus are described in this manuscript: T. halophilus subsp. halophilus subsp. nov. for the strains isolated from salt media and T. halophilus subsp. flandriensis subsp. nov. for the strains isolated from sugar-rich environments, which were first isolated in Flanders, Belgium. The type strains for the subspecies are IAM 1676 T (5LMG 11490 T 5DSM 20339 T ) and T5 T (5LMG 26042 T 5DSM 23766 T ), respectively. The genus Tetragenococcus was described by Collins et al. (1990) through reclassification of the halophilic lactic acid bacterium Pediococcus halophilus as Tetragenococcus halo- philus. Strains of this species were isolated from habitats rich in salt and proteins such as salted anchovies (Villar et al., 1985), soy sauce mash (Ro ¨ling & van Verseveld, 3These authors contributed equally to this work. Abbreviations: RAPD, random amplified polymorphic DNA; TV, tetra- zolium violet. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences of strains T170, T179 and T181 are HQ123368– HQ123370, respectively. International Journal of Systematic and Evolutionary Microbiology (2012), 62, 129–137 DOI 10.1099/ijs.0.029157-0 029157 G 2012 IUMS Printed in Great Britain 129