Antonie van Leeuwenhoek (2014) 106:615–621 DOI 10.1007/s10482-014-0232-2 ORIGINAL PAPER Motiliproteus sediminis gen. nov., sp. nov., isolated from coastal sediment Zong-Jie Wang • Zhi-Hong Xie • Chao Wang • Zong-Jun Du • Guan-Jun Chen Received: 3 April 2014 / Accepted: 4 July 2014 / Published online: 20 July 2014 Ó Springer International Publishing Switzerland 2014 Abstract A novel Gram-stain-negative, rod-to- demonstrated that the novel isolate was 93.3 % similar spiral-shaped, oxidase- and catalase- positive and to the type strain of Neptunomonas antarctica, 93.2 % facultatively aerobic bacterium, designated HS6T, was to Neptunomonas japonicum and 93.1 % to Marino- isolated from marine sediment of Yellow Sea, China. bacterium rhizophilum, the closest cultivated rela- It can reduce nitrate to nitrite and grow well in marine tives. The polar lipid profile of the novel strain broth 2216 (MB, Hope Biol-Technology Co., Ltd) consisted of phosphatidylethanolamine, phosphatidyl- with an optimal temperature for growth of 30–33 °C glycerol and some other unknown lipids. Major (range 12–45 °C) and in the presence of 2–3 % (w/v) cellular fatty acids were summed feature 3 (C16:1 NaCl (range 0.5–7 %, w/v). The pH range for growth x7c/iso-C15:0 2-OH), C18:1 x7c and C16:0 and the main was pH 6.2–9.0, with an optimum at 6.5–7.0. Phylo- respiratory quinone was Q-8. The DNA G?C content genetic analysis based on 16S rRNA gene sequences of strain HS6T was 61.2 mol %. Based on the phylogenetic, physiological and biochemical charac- teristics, strain HS6T represents a novel genus and The GenBank accession number for the 16S rRNA gene T species and the name Motiliproteus sediminis gen. sequence of Motiliproteus sediminis HS6 is KF953945. nov., sp. nov., is proposed. The type strain is HS6T T T Electronic supplementary material The online version of (=ATCC BAA-2613 =CICC 10858 ). this article (doi:10.1007/s10482-014-0232-2) contains supple- mentary material, which is available to authorized users. Keywords Motiliproteus sediminis gen. nov., sp. nov. Á Polyphasic taxonomy Á 16S rRNA gene Á Marine Z.-J. Wang Á C. Wang Á Z.-J. Du (&) Á G.-J. Chen College of Marine Science, Shandong University at bacteria Á Coastal sediment Weihai, Weihai 264209, China e-mail: [email protected] G.-J. Chen e-mail: [email protected] Introduction Z.-H. Xie Key Laboratory of Coastal Biology and Utilization, The family Oceanospirillaceae within the phylum Yantai Institute of Coastal Zone Research, Chinese Proteobacteria was described by Garrity et al. (2005) Academy of Sciences, Yantai 264003, China in Bergey’s Manual of Systematic Bacteriology.It contains 18 genera (LPSN, http://www.bacterio.net/- Z.-J. Du Á G.-J. Chen State key Laboratory of Microbial Technology, Shandong classifphyla.html) at the time of writing and most of University, Jinan 250100, China them are strictly respiratory (except for Neptunomonas 123 616 Antonie van Leeuwenhoek (2014) 106:615–621 which gives weak fermentation reactions) aquatic (Murray et al. 1994). The effects of different NaCl organisms of primarily marine origin. The type genus concentrations were assessed by modified MA (sea- of the family is Oceanospirillum (Hylemon et al. water replaced with distilled water) containing differ- 1973) with O. linum as the type species. The family ent concentrations of NaCl (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, Oceanospirillaceae has a close relationship with the 9, and 10 %, w/v). Growth at different temperature genus Marinobacterium (Gonza´lez et al. 1997), a was tested on MA at 4, 8, 12, 17, 24, 28, 30, 33, 37, 42 member of family Alteromonadaceae. In the present and 45 °C. To test the effect of pH on growth, MB was study, the novel isolate was characterized by pheno- adjusted to different pH with MES (pH, 5.5, 6.0, 6.2), typic and phylogenetic analyses and is proposed to be PIPES (pH, 6.5, 7.0), HEPES (pH, 7.5, 8.0), Tricine a member of novel species in a new genus of family (pH, 8.5) and CAPSO (pH, 9.0, 9.5) buffers and each at Oceanospirillaceae. The novel genus forms a cluster a concentration of 20 mM and then measured the with Marinobacterium which supports the view of value of OD600. Anaerobic growth was determined at Kim et al. (2007) that the genus Marinobacterium 33 °C in an anaerobic chamber on MA with or without should be reclassified into the order Oceanospirillales. 0.1 % NaNO3 for 5–7 days. Catalase activity was detected by bubble production in 3 % (v/v) H2O2 solution and oxidase activity was evaluated by using Materials and methods commercial oxidase reagent (bioMe´rieux). Hydrolysis of starch, lipid and algin was tested on MA supple- Organism, maintenance and cultural conditions mented with 0.2 % (w/v) soluble starch, 1 % Tween 80 (v/v) and 2 % sodium alginate respectively (Cowan During the study of diversity of the multi-drug and Steel 1965). Tests for other physiological or resistant bacteria in coastal marine environments, a biochemical characteristics were performed using API novel heterotrophic, facultatively aerobic, olive to 20E, API 20NE, API ZYM kits, API 50CH and Biolog brown, motile, Gram-negative bacterial strain HS6T according to the manufacturer’s instructions except was isolated on marine agar 2216 (MA, Hope Biol- that the salinity was adjusted to 3 %. All the API and Technology Co., Ltd) at 28 °C. The sample was Biolog tests were performed at least twice. Suscepti- collected from the coastal of Yellow Sea (121°705700E, bility to antibiotics was tested using filter-paper discs 37°3705300N) around Yantai, China. For isolation of containing various antibiotics on cultures incubated at bacterial strains, 1 g wet sediment was blended to 33 °C on MA (since strain HS6T showed poor growth 99 mL sterilized seawater with glass beads and shaken on Mueller–Hinton agar) for 2–3 days. vigorously. The suspension was serially diluted to 10-6 with sterilized seawater and 0.1 mL aliquots of Determination of G?C content of DNA, 16S each dilution were spread onto MA. The plates were rRNA gene sequencing and phylogenetic analysis incubated at 28 °C for 5–7 days. Strain HS6T was isolated and then stored at -80 °C in sterile 1 % (w/v) Genomic DNA of strain HS6T was obtained from 60 h saline supplemented with 15 % (v/v) glycerol. The old cultures on MA using a genomic DNA extraction strain was routinely cultivated at 33 °C on MA or MB. kit (Sangon, China) and the DNA G?C content was determined by HPLC (Mesbah et al. 1989). The 16S Morphological, physiological and biochemical rRNA gene of strain HS6T was amplified from the analysis genomic DNA by PCR with the universal primers 27F and 1492R (Lane 1991). PCR products were purified The morphological and physiological features of using a PCR product purification kit (Tiangen Biotech HS6T were examined after incubation at 33 °C for Co., Ltd, Beijing, China) and then ligated to the vector 2–3 days on MA. The transmission electron micro- of pGM-T (Tiangen Biotech Co., Ltd, Beijing, China). scope was used to observe the cell size, morphology Sequencing was performed by Shanghai Sunny Bio- and flagella. Meanwhile, the light microscopy (E600; technology Co., Ltd, China. To identify the taxonomic Nikon) was used as supplement. Motility was assessed status of HS6T, a near complete sequence (1,466 bp) with the method of hanging-drop. Gram reaction was was obtained and submitted to GenBank/EMBL/ examined following the standard Gram procedure DDBJ datebase to search for similar sequences using 123 Antonie van Leeuwenhoek (2014) 106:615–621 617 BLAST algorithm. EzTaxon server (http://eztaxone. to the closest phylogenetic neighbors as shown in ezbiocloud.net/; Kim et al. 2012) was used to achieve Table 1. It characteristically showed bipolar flagella the similarity values of sequences. Phylogenetic ana- which is different from most of the members of family lysis was performed using MEGA version 5.2 (Tamura Oceanospirillaceae and there were 1–2 flagella et al. 2011) after multiple alignments of data by attached to the polar or subpolar of the HS6T CLUSTAL W (Larkin et al. 2007) and manual edition (Fig. 1). The characteristics of morphology of HS6T (to remove gaps at 30 and 50 ends and ambiguous are significant different from the relative genera bases) using BioEdit version 7.0 (Hall 1999). The shown in Table 1 and this can distinguish it from phylogenetic trees were constructed using the neigh- other genera partially. bor-joining (NJ), maximum-likelihood (ML) and Growth was found to occur in 0.5–7 % (w/v) NaCl maximum-parsimony (MP) algorithms. Bootstrap (optimum 2–3 %), at 15–45 °C (optimum 30–33 °C) values were calculated based on 1,000 replicates. and at pH 6.2–9.0 (optimum pH 6.5–7.0). There was no visible colonies formed under anaerobic conditions Chemotaxonomic analysis on MA supplemented with 0.1 % (w/v) NaNO3. But it can reduce nitrate to nitrite with or without oxygen The cells cultured on MA at 33 °C for 3 days (end of which can distinguish HS6T from most of the members the logarithmic phase) were used to determine fatty in the family Oceanospirillaceae. The strain was acids. The method was described by Sasser (1990) positive for oxidase and catalase activities and nega- previously. And then the extract was separated using tive for hydrolysis of starch, Tween 80 and algin. The Sherlock Microbial Identification System (MIS) positive of gelatinase also can distinguish the new (MIDI, Microbial ID, Newark, DE 19711 U.S.A.).