IJSEM Papers in Press. Published June 18, 2010 as doi:10.1099/ijs.0.024661-0 1 Marinomonas brasilensis sp. nov. isolated from the coral Mussismilia 2 hispida and reclassification of Marinomonas basaltis as a later synonym 3 of Marinomonas communis 4 5 Luciane A. Chimetto1,2,3, Ilse Cleenwerck3, Marcelo Brocchi1, Anne Willems4, 6 Paul De Vos3,4, and Fabiano L. Thompson2* 7 8 1Departament of Genetics, Evolution and Bioagents, Institute of Biology, State University of 9 Campinas (UNICAMP), Brazil 10 11 2Department of Genetics, Institute of Biology, Federal University of Rio de Janeiro (UFRJ), 12 Brazil 13 14 3BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, 15 Belgium 16 17 4Laboratory of Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B- 18 9000 Ghent, Belgium 19 20 *Corresponding author: Fabiano L. Thompson. Phone/FAX: +55-21-25626567. 21 E-mail: [email protected] 22 23 Running Title: 24 Marinomonas brasilensis sp. nov. 25 Subject Category: New taxa, Proteobacteria 26 27 Footnote: The GenBank/EMBL accession number for the 16S rRNA gene 28 sequence of strain R-40503T is GU929940. 29 30 31 32 33 34 35 36 37 38 39 40 1 41 Abstract 42 A Gram-negative, aerobic bacterium, designated R-40503T was isolated from 43 mucus of the reef builder coral, Mussismilia hispida, located in the São 44 Sebastião Channel, São Paulo, Brazil. Phylogenetic analyses revealed that 45 strain R-40503T belongs to the genus Marinomonas. The 16S rRNA gene 46 sequence similarity of R-40503T was above 97 % with the type strains of 47 Marinomonas vaga, M. basaltis, M. communis and M. pontica, and below 97 % 48 with all other the Marinomonas type strains. Strain R-40503T showed less than 49 35 % DNA-DNA hybridization (DDH) similarity with the type strains of the 50 phylogenetically closest Marinomonas species, demonstrating it should be 51 classified into a novel species. Amplified Fragment Length Polymorphism 52 (AFLP), chemotaxonomic and phenotypic analyses provided further evidence 53 for the classification of the new species. Concurrently, a close genomic 54 relationship between M. basaltis and M. communis was observed. The type 55 strains of these two species showed 78 % DDH similarity and 63 % AFLP 56 pattern similarity. Their phenotypic features were very similar, and their DNA 57 G+C content was identical (46.3 mol%). Collectively, these data demonstrates 58 unambiguously the synonymy of Marinomonas basaltis and Marinomonas 59 communis. Several phenotypic features can be used to discriminate 60 Marinomonas species. The novel strain R-40503T is clearly distinguishable 61 species from its neighbours. For instance, it shows oxidase and urease activity, 62 utilizes L-asparagine, has the fatty acid C12:1 3-OH (but lacks C10:0 and 63 C12:0). The name Marinomonas brasilensis sp. nov. is proposed (type strain is 64 R-40503T = LMG 25434T = CAIM 1459T). The DNA G+C content of the type 65 strain R-40503T is 46.5 mol%. 2 66 67 Mussismilia hispida is one of the major reef-builders corals along the 68 northeastern Brazilian coast, and it also has the widest geographic distribution 69 among its genus (from Maranhão to Santa Catarina states, ca. 5000 km) (Leão 70 & Kikuchi, 2005). The ability of Mussismilia to survive in different regions 71 indicates its adaptation to wide environmental gradients, such as temperature, 72 water turbidity and pollution. However, recent studies have revealed that M. 73 hispida and M. braziliensis are threatened by extinction (Castro et al., 2010; 74 Francini-Filho et al., 2008). Microorganisms appear to play a key role in coral 75 health. Microorganisms and the coral compose the holobiont (Rosenberg et al., 76 2007). The holobiont microbiota appears to protect its host by providing 77 nourishment and antibiotics (Raina et al., 2009; Shnit-Orland & Kushmaro, 78 2009). It is also recognized that the holobiont harbours a vast microbial 79 diversity. In the last 10 years a growing number of studies have focused on the 80 characterization of the coral microbiota diversity and ecology (Alves et al., 2009; 81 Dinsdale et al. 2008; Rohwer et al. 2001). 82 83 The genus Marinomonas was created in 1983 to accommodate Alteromonas 84 communis and Alteromonas vaga (Baumann et al., 1972), which were distinct 85 from the other species of Alteromonas (van Landschoot & De Ley, 1983). The 86 genus Marinomonas comprises 15 species, mainly originating from sea-water of 87 different geographical locations. M. communis and M. vaga (Baumann et al., 88 1972; van Landschoot & De Ley, 1983), were isolated from the Pacific Ocean, 89 M. pontica (Ivanova et al., 2005) from the Black Sea, M. dokdonensis (Yoon et 90 al., 2005) from the East Sea of Korea, and M. mediterranea (Solano & 3 91 Sanchez-Amat, 1999) and M. aquimarina (Macián et al., 2005) from the 92 Mediterranean Sea. M. polaris (Gupta et al., 2006) and M. ushuaiensis 93 (Prabagaran et al., 2005) were isolated from the subantarctic region, while M. 94 primoryensis (Romanenko et al., 2003) and M. arctica (Zhang et al., 2008) were 95 isolated from sea-ice. M. ostreistagni (Lau et al., 2006) and some M. aquimarina 96 strains (Macián et al., 2005) were isolated from oysters. M. basaltis (Chang et 97 al., 2008) and M. arenicola (Romanenko et al., 2009) were isolated from marine 98 sediment, while M. balearica and M. pollencensis (Espinosa et al., 2009) were 99 isolated from seagrass Posidonia oceanica. 100 101 In the present study, one isolate (R-40503T), obtained from mucus of apparently 102 healthy coral Mussismilia hispida, located in the rocky shore of Grande beach 103 (coordinate 23°50´25´´S; 045°24´59´´W) in São Sebastião Channel, São Paulo, 104 Brazil, in the summer of 2005, during a survey of the heterotrophic bacterial 105 diversity associated with cnidarians in São Paulo (Brazil) (Chimetto et al., 2008, 106 2009), was investigated using a polyphasic taxonomic approach. The strain was 107 isolated using the nitrogen-free (NFb) selective medium supplemented with 3 % 108 NaCl after 4 days of incubation at 28 °C. 109 110 Five strains (R-236, R-237, R-249, R-256, and R-278) isolated at the time of 111 collection as described in Chimetto et al. (2008) clustered together in this new 112 taxa by 16S rRNA gene sequences, but only one strain (R-278 = R-40503T) 113 maintained viability. The 16S rRNA gene sequence of R-40503T (1425 nt), 114 accession number GU929940, was obtained as described previously (Chimetto 115 et al., 2008, 2009). The raw sequence data were transferred to the ChromasPro 116 ver. 1.34 software (Technelysium Pty. Ltd, Tewantin, Australia) where 4 117 consensus sequences were determined. The sequence was aligned with 118 sequences from EMBL using the ClustalW software (Chenna et al., 2003). 119 Pairwise similarities were calculated with the BioNumerics 4.61 software 120 (Applied Maths, Sint-Martiens-Latem Belgium), using an open gap penalty of 121 100 % and a unit gap penalty of 0 %. Similarity matrices and phylogenetic trees 122 were constructed using the MEGA ver. 4.0 (Tamura et al., 2007) and the 123 BioNumerics 4.61 software (Applied Maths, Belgium). Trees were drawn using 124 the Neighbour-Joining (Saitou & Nei, 1987) and Maximum Parsimony methods 125 (Eck & Dayhoff, 1966). The robustness of the topologies of the trees were 126 checked by bootstrap replications (Felsenstein, 1985). The gene sequence data 127 obtained in this study are also available through our website TAXVIBRIO 128 (http://www.taxvibrio.lncc.br/). 129 130 The novel strain R-40503T was closely related to M. vaga, with 97.9% 16S 131 rRNA gene sequence similarity. R-40503T had 97.2% 16S rRNA gene 132 sequence similarity towards M. basaltis, M. communis, M. aquimarina (Fig. 1 133 and Supplementary Figure S2). DNA-DNA hybridizations were performed 134 between the novel strain R-40503T and the type strains of the closest 135 phylogenetic neighbours, i.e. M. vaga, M. basaltis, M. communis and M. 136 aquimarina (Table 1), using the microplate method described by Ezaki et al. 137 (1989) with minor modifications (Willems et al., 2001). Hybridizations were 138 performed at 40.7 °C in the presence of 50 % formamide. Reciprocal reactions 139 were performed for every DNA pair and their variation was within the limits of 140 this method (Goris et al., 1998). The DDH relatedness between R-40503T and 141 the tested type strains was below 70 % (Table 1). The DDH demonstrated that it 5 142 represents a novel species in the genus Marinomonas (Wayne et al., 1987; 143 Stackebrandt & Ebers, 2006). The DDH relatedness between Marinomonas 144 basaltis LMG 25279T and Marinomonas communis LMG 2864T was above 70 % 145 (i.e. 78 %), which suggests that these species are synonymous. Chang et al. 146 (2008) obtained 56.2 % DDH similarity between the same pair of type strains, 147 but the additional data of the present study (see below) support the value of 78 148 %. The authenticity of M. basaltis LMG 25279T (GU929941) and M. communis 149 LMG 2864T used in this study were verified by means of their 16S rRNA 150 sequences. The sequences of both type strains (1501 nt for LMG 25279T and 151 1499 nt for LMG 2864T showed 100 % similarity with those deposited in the 152 GenBank M. basaltis J63T (EU143359) and M. communis LMG 2864T 153 (DQ011528) respectively, indicating the authenticity of the LMG strains (Figure 154 1). The 16S rRNA gene sequence similarity between M. basaltis LMG 25279T 155 and M. communis LMG 2864T was 98.7 %. Giving further support for the 156 synonymy between, Marinomonas basaltis LMG 25279T and Marinomonas 157 communis LMG 2864T had identical GC contents and related AFLP patterns.