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
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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-tospiral-shaped, oxidase- and catalase- positive and facultatively aerobic bacterium, designated HS6T, was isolated from marine sediment of Yellow Sea, China. It can reduce nitrate to nitrite and grow well in marine broth 2216 (MB, Hope Biol-Technology Co., Ltd) with an optimal temperature for growth of 30–33 °C (range 12–45 °C) and in the presence of 2–3 % (w/v) NaCl (range 0.5–7 %, w/v). The pH range for growth was pH 6.2–9.0, with an optimum at 6.5–7.0. Phylogenetic analysis based on 16S rRNA gene sequences
The GenBank accession number for the 16S rRNA gene sequence of Motiliproteus sediminis HS6T is KF953945.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0232-2) contains supplementary material, which is available to authorized users. Z.-J. Wang C. Wang Z.-J. Du (&) G.-J. Chen College of Marine Science, Shandong University at Weihai, Weihai 264209, China e-mail: [email protected] G.-J. Chen e-mail: [email protected] Z.-H. Xie Key Laboratory of Coastal Biology and Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China Z.-J. Du G.-J. Chen State key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
demonstrated that the novel isolate was 93.3 % similar to the type strain of Neptunomonas antarctica, 93.2 % to Neptunomonas japonicum and 93.1 % to Marinobacterium rhizophilum, the closest cultivated relatives. The polar lipid profile of the novel strain consisted of phosphatidylethanolamine, phosphatidylglycerol and some other unknown lipids. Major cellular fatty acids were summed feature 3 (C16:1 x7c/iso-C15:0 2-OH), C18:1 x7c and C16:0 and the main respiratory quinone was Q-8. The DNA G?C content of strain HS6T was 61.2 mol %. Based on the phylogenetic, physiological and biochemical characteristics, strain HS6T represents a novel genus and species and the name Motiliproteus sediminis gen. nov., sp. nov., is proposed. The type strain is HS6T (=ATCC BAA-2613T=CICC 10858T). Keywords Motiliproteus sediminis gen. nov., sp. nov. Polyphasic taxonomy 16S rRNA gene Marine bacteria Coastal sediment
Introduction The family Oceanospirillaceae within the phylum Proteobacteria was described by Garrity et al. (2005) in Bergey’s Manual of Systematic Bacteriology. It contains 18 genera (LPSN, http://www.bacterio.net/classifphyla.html) at the time of writing and most of them are strictly respiratory (except for Neptunomonas
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which gives weak fermentation reactions) aquatic organisms of primarily marine origin. The type genus of the family is Oceanospirillum (Hylemon et al. 1973) with O. linum as the type species. The family Oceanospirillaceae has a close relationship with the genus Marinobacterium (Gonza´lez et al. 1997), a member of family Alteromonadaceae. In the present study, the novel isolate was characterized by phenotypic and phylogenetic analyses and is proposed to be a member of novel species in a new genus of family Oceanospirillaceae. The novel genus forms a cluster with Marinobacterium which supports the view of Kim et al. (2007) that the genus Marinobacterium should be reclassified into the order Oceanospirillales.
Materials and methods Organism, maintenance and cultural conditions During the study of diversity of the multi-drug resistant bacteria in coastal marine environments, a novel heterotrophic, facultatively aerobic, olive to brown, motile, Gram-negative bacterial strain HS6T was isolated on marine agar 2216 (MA, Hope BiolTechnology Co., Ltd) at 28 °C. The sample was collected from the coastal of Yellow Sea (121°70 5700 E, 37°370 5300 N) around Yantai, China. For isolation of bacterial strains, 1 g wet sediment was blended to 99 mL sterilized seawater with glass beads and shaken vigorously. The suspension was serially diluted to 10-6 with sterilized seawater and 0.1 mL aliquots of each dilution were spread onto MA. The plates were incubated at 28 °C for 5–7 days. Strain HS6T was isolated and then stored at -80 °C in sterile 1 % (w/v) saline supplemented with 15 % (v/v) glycerol. The strain was routinely cultivated at 33 °C on MA or MB. Morphological, physiological and biochemical analysis The morphological and physiological features of HS6T were examined after incubation at 33 °C for 2–3 days on MA. The transmission electron microscope was used to observe the cell size, morphology and flagella. Meanwhile, the light microscopy (E600; Nikon) was used as supplement. Motility was assessed with the method of hanging-drop. Gram reaction was examined following the standard Gram procedure
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(Murray et al. 1994). The effects of different NaCl concentrations were assessed by modified MA (seawater replaced with distilled water) containing different concentrations of NaCl (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 %, w/v). Growth at different temperature was tested on MA at 4, 8, 12, 17, 24, 28, 30, 33, 37, 42 and 45 °C. To test the effect of pH on growth, MB was adjusted to different pH with MES (pH, 5.5, 6.0, 6.2), PIPES (pH, 6.5, 7.0), HEPES (pH, 7.5, 8.0), Tricine (pH, 8.5) and CAPSO (pH, 9.0, 9.5) buffers and each at a concentration of 20 mM and then measured the value of OD600. Anaerobic growth was determined at 33 °C in an anaerobic chamber on MA with or without 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 commercial oxidase reagent (bioMe´rieux). Hydrolysis of starch, lipid and algin was tested on MA supplemented with 0.2 % (w/v) soluble starch, 1 % Tween 80 (v/v) and 2 % sodium alginate respectively (Cowan and Steel 1965). Tests for other physiological or biochemical characteristics were performed using API 20E, API 20NE, API ZYM kits, API 50CH and Biolog according to the manufacturer’s instructions except that the salinity was adjusted to 3 %. All the API and Biolog tests were performed at least twice. Susceptibility to antibiotics was tested using filter-paper discs containing various antibiotics on cultures incubated at 33 °C on MA (since strain HS6T showed poor growth on Mueller–Hinton agar) for 2–3 days. Determination of G?C content of DNA, 16S rRNA gene sequencing and phylogenetic analysis Genomic DNA of strain HS6T was obtained from 60 h old cultures on MA using a genomic DNA extraction kit (Sangon, China) and the DNA G?C content was determined by HPLC (Mesbah et al. 1989). The 16S rRNA gene of strain HS6T was amplified from the genomic DNA by PCR with the universal primers 27F and 1492R (Lane 1991). PCR products were purified using a PCR product purification kit (Tiangen Biotech Co., Ltd, Beijing, China) and then ligated to the vector of pGM-T (Tiangen Biotech Co., Ltd, Beijing, China). Sequencing was performed by Shanghai Sunny Biotechnology Co., Ltd, China. To identify the taxonomic status of HS6T, a near complete sequence (1,466 bp) was obtained and submitted to GenBank/EMBL/ DDBJ datebase to search for similar sequences using
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BLAST algorithm. EzTaxon server (http://eztaxone. ezbiocloud.net/; Kim et al. 2012) was used to achieve the similarity values of sequences. Phylogenetic analysis was performed using MEGA version 5.2 (Tamura et al. 2011) after multiple alignments of data by CLUSTAL W (Larkin et al. 2007) and manual edition (to remove gaps at 30 and 50 ends and ambiguous bases) using BioEdit version 7.0 (Hall 1999). The phylogenetic trees were constructed using the neighbor-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) algorithms. Bootstrap values were calculated based on 1,000 replicates. Chemotaxonomic analysis The cells cultured on MA at 33 °C for 3 days (end of the logarithmic phase) were used to determine fatty acids. The method was described by Sasser (1990) previously. And then the extract was separated using Sherlock Microbial Identification System (MIS) (MIDI, Microbial ID, Newark, DE 19711 U.S.A.). Peaks were automatically integrated and fatty acid names and percentages calculated by the MIS Standard Software (Microbial ID). Respiratory lipoquinones were extracted from freeze dried cells and the methods were described by Tindall (1990a, b). They were separated into different classes by thin layer chromatography on silica gel and removed from the plate and further analyzed by HPLC. Polar lipids were separated by two dimensional silica gel thin layer chromatography. Total lipid material was detected using molybdatophosphoric acid and specific functional groups detected using spray reagents specific for defined functional groups, Full details are given in Tindall (2007). All procedures of analysis were carried out by the Identification Service, DSMZ, Braunschweig, Germany.
Results Phenotypic characteristics Strain HS6T can form circular, viscid and olive to brown (the color would change over time) colonies whose diameter were about 1.5 mm after 60 h incubation on MA at 33 °C. The cells of the strain were found to be Gram-negative, rod-to-spiral-shaped, motile and 0.5–0.8 9 1.5–6.0 lm in size, in contrast
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to the closest phylogenetic neighbors as shown in Table 1. It characteristically showed bipolar flagella which is different from most of the members of family Oceanospirillaceae and there were 1–2 flagella attached to the polar or subpolar of the HS6T (Fig. 1). The characteristics of morphology of HS6T are significant different from the relative genera shown in Table 1 and this can distinguish it from other genera partially. Growth was found to occur in 0.5–7 % (w/v) NaCl (optimum 2–3 %), at 15–45 °C (optimum 30–33 °C) and at pH 6.2–9.0 (optimum pH 6.5–7.0). There was no visible colonies formed under anaerobic conditions on MA supplemented with 0.1 % (w/v) NaNO3. But it can reduce nitrate to nitrite with or without oxygen which can distinguish HS6T from most of the members in the family Oceanospirillaceae. The strain was positive for oxidase and catalase activities and negative for hydrolysis of starch, Tween 80 and algin. The positive of gelatinase also can distinguish the new isolate from the genera Marinobacterium, Neptunomonas, Neptuniibacter and Nitrincola. Other cultural, physiological and biochemical characteristics of the novel strain will be given in the species description. And the different characteristics that can distinguish HS6T from its closest phylogenetic relatives were displayed in Table 1. Molecular phylogenetic analysis The nearly full-length (1,466 bp) 16S rRNA gene sequence of strain HS6T was obtained and the comparison revealed that strain HS6T had highest similarity to the type strain of Neptunomonas antarctica (93.3 %), followed by Neptunomonas japonica (93.2 %) (Miyazaki et al. 2008) and Marinobacterium rhizophilum (93.1 %) (Kim et al. 2008). Addition to these, the similarities with other relative neigbourhoods were all below 93 %. That suggested the strain HS6T may represent a novel genus in family Oceanospirillaceae. The further studies of phylogenetic trees (Fig. 2) proved that. In the NJ tree, the strain HS6T formed a separate branch with the genera Marinobacterium, Neptunomonas, Neptuniibacter and Nitrincola. Although the genus Marinobacterium belongs to the family Alteromonadaceae according to NCBI (http://www.ncbi. nlm.nih.gov/) and LPSN, we still insisted the strain HS6T should be classified to the family
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Table 1 Phenotypic comparison of HS6T and related genera HS6T
Neptunomonas
Cell shape
rods to spiral
rods
rods
rods to bent rods
rods
Size (lm)
0.5–0.8 9 1.5–6.0
0.6–1.0 9 1.2–3.0
0.3–0.8 9 0.6–3.0
0.2–0.8 9 1.5–2.3
0.5 9 1.3–1.6
Flagella Growth at
Bipolar
Single Polar
Single Polar
Single Polarb
Single Polar
–
?a
-a
–
–
a
?a
–
ND
Ana
An
F
Characteristic
4 °C 40 °C
Marinobacterium
Neptuniibacter
Nitrincola
?
-
F
F
–
-a
-a
–
–
Tween 80
–
-
a
?a
–
ND
Gelatin
?
-a
-a
–
–
Nitrate Reduction
?
?a
-a
–
–
G?C Content (%)
61.2
43.6–48.2
54.9–62.5
46.6–54.2
47.4
Quinone(s)
Q8/Q7
Q8/Q9b
Q8b
Q8
ND
O2 metabolism Hydrolysis of Starch
HS6T (this study); Neptunomonas (Hedlund et al. 1999; Miyazaki et al. 2008; Zhang et al. 2010; Lee et al. 2012; Liu et al. 2013); Marinobacterium (Baumann et al. 1983; Gonza´lez et al. 1997; Chang et al. 2007; Kim et al. 2007; Kim et al. 2008; Huo et al. 2009; Kim et al. 2009a; Kim et al. 2009b; Kim et al. 2010; Chimetto et al. 2011); Neptuniibacter (Arahal et al. 2007; Chen et al. 2012); Nitrincola (Dimitriu et al. 2005) ? positive, - negative, F facultatively aerobic, An strictly anaerobic a b
[50 % negative/positive The information is incomplete because some papers missed the relevant information
Oceanospirillaceae. Besides the higher similarity of 16S rRNA gene sequence and phenotypic characteristics, the EzTaxon also classified the genus Marinobacterium to the family of Oceanospirillaceae. And we also added the 16S rRNA gene sequences of families Alterononadaceae and Shewanellaceae to our phylogenetic tree, they formed a separate cluster which is far from the clusters including the Marinobacterium. The result supported the view of Kim et al. (2007) that the genus Marinobacterium should be reclassified into the order Oceanospirillales. The ML tree and the MP tree were similar and all of them supported our conclusion—the strain HS6T may represent a novel genus of the family Oceanospirillaceae. The DNA G?C content of the novel isolate was 61.2 mol % which is similar to Marinobacterium but much higher than other relatives (Table 1). Chemotaxonomic characteristics
T
Fig. 1 Transmission electron micrograph of HS6 . Cells grown on MA at 33 °C for 12 h. Bar = 1.0 lm
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The predominant respiratory quinone detected in strain HS6T was Q-8 (85 %) which is compatible with the most members of family Oceanospirillaceae
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619 Marinobacterium coralli CAIM 1449T (GU183820)
73
Marinobacterium sediminicola JCM 15524T (EU573966)
79 97
Marinobacterium maritimum KCTC 22254T (EU399548) Marinobacterium stanieri ATCC 27130T (AB021367)
96
Marinobacterium georgiense ATCC 700074T (U58339)
78
Marinobacterium halophilum DSM 17586T (AY563030)
100
Marinobacterium jannaschii ATCC 27135T (AB006765) Marinobacterium nitratireducens JCM 15523T (EU573965) Marinobacterium rhizophilum DSM 18822T (EF192391) Nitrincola lacisaponensis ATCC BAA-920T (AY567473)
81
Marinobacterium marisflavi KCTC 12757T (EF468717) Marinobacterium litorale KCTC 12756T (DQ917760)
52
Marinobacterium lutimaris DSM 22012T (FJ230839)
99
Neptuniibacter caesariensis CCUG 52065T (AY136116) Neptuniibacter halophilus BCRC 80079T (GQ131677)
98
Neptunomonas naphthovorans ATCC 700637T (AF053734) 76
Neptunomonas concharum JCM 17730T (JF748732)
86
Neptunomonas antarctica KACC 14056T (FJ713802)
67
Neptunomonas japonica DSM 18939T (AB288092) 70 97
Neptunomonas qingdaonensis KCTC 23686T (JF747202) Motiliproteus sediminis HS6T (KF953945)
63
Oceanospirillum linum ATCC 11336T (M22365) Oleispira antarctica DSM 14852T (AJ426420)
100
Bermanella marisrubri CCUG 52064T (AY136131)
95
Oceaniserpentilla haliotis DSM 19503T (AM747817)
97
Thalassolituus oleivorans DSM 14913T (AJ431699) Oceanobacter kriegii ATCC 27133T (AB006767)
99
Oleibacter marinus NBRC 105760T (FJ167390)
99
Shewanella putrefaciens ATCC 8071T (X81623) Alishewanella fetalis ATCC BAA-284T (AF144407) Catenovulum agarivorans DSM 23111T (GQ262000) 53
Agarivorans albus NBRC 102603T (AB076561)
100
Aliagarivorans marinus BCRC 17888T (FJ167390)
65
Bowmanella denitrificans BCRC 17491T (DQ343294) Aestuariibacter salexigens DSM 15300T (AY207502)
74 99
Glaciecola punicea ATCC 700756T (U85853) Alteromonas macleodii DSM 6062T (Y18228)
55 88
Salinimonas chungwhensis DSM 16280T (AY553295)
0.01
Fig. 2 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of HS6T and its closely relatives. Type strains of families Alteromonadaceae and Shewanellaceae were
used to root the tree. Bootstrap values ([50 %) based on 1,000 replications were shown at branching points. Bar, 0.01 substitutions per nucleotide position
(Garrity et al. 2005; Zhang et al. 2010; Miyazaki et al. 2008 and Arahal et al. 2007) and the genus Marinobacterium (Kim et al. 2008). A minor amount of Q-7 (11 %) was also detected, which seems to be less abundant or absent in close relatives. The remaining 4 % was difficult to identify. The existence of Q-7 can distinguish HS6T from the genera shown in Table 1. The major fatty acids (relative amount [ 1 %) found
in strain HS6T were summed feature 3 (C16:1 x7c/isoC15:0 2-OH,34.7 %), C18:1 x7c (24.1 %), C16:0 (21.3 %), C10:0 3-OH (5.0 %), C18:1 x6c (4.1 %), C14:0 3-OH/iso-C16:1 (3.8 %), C12:0 (2.4 %) and C14:0 (1.0 %). The components of fatty acids of HS6T were similar to its close relatives, however relatives proportions of individual and components differ considerably. The polar lipids found in strain HS6T
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were very complex. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and an unknown phospholipid (PL2) were major polar lipids. Two unknown phospholipids (PL1, PL4) and an unknown aminophospholipid (PN) were moderate and some other unknown lipids (AP, L1-3, PL3, PL5) were minor components. The detailed information are displayed in supplementary (Fig. S1). Polyphasic taxonomic conclusion The results of the phenotypic analysis, molecular phylogenetic analysis and chemotaxonomic analysis all support the view that strain HS6T represents a novel genus and species of the family Oceanospirillaceae, for which the name Motiliproteus sediminis is proposed. Description of Motiliproteus gen. nov Motiliproteus (Mo.ti.li.pro’teus. L. adj. motilis, motile; L. masc. n. Proteus, a mythical figure able to assume different forms; N.L. masc. n. Motiliproteus, a motile bacteria of various forms) Cells are Gram-negative, rod-to-spiral-shaped, facultatively aerobic and motile with bipolar flagella. Both catalase and oxidase are positive. No growth occurs in the absence of salt. Nitrate can be reduced to nitrite with or without oxygen. The major cellular fatty acids are C16:1 x7c/iso-C15:0 2-OH, C18:1 x7c and C16:0 and the main ubiquinone system is Q-8. Description of Motiliproteus sediminis sp. nov Motiliproteus sediminis (se.di.mi’nis. L. n. sedimeninis, sediment; L. gen. n. sediminis, of sediment) Besides the characteristics described above, the following characteristics are also observed for the type species. Cells are 0.5–0.8 lm in diameters and 1.5–6.0 lm in length. Forms circular, viscid and olive to brown (the color would change over time) colonies on MA after 60 h incubation at 33 °C. Growth occurs in 0.5–7 % (w/v) NaCl (optimum 2–3 %), at 15–45 °C (optimum 30–33 °C) and at pH 6.2–9.0 (optimum 6.5–7.0). Does not hydrolyse starch, Tween-80, alginate and cellulose. According to the API ZYM kits, cells are positive for acid and alkaline phosphatase,
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esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase and naphthol-AS-BI-phosphohydrolase but negative for lipase (C14), cystine arylamidase, trypsin, a-chymotrypsin, a-and b-galactosidase, a-and b-glucosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase. Arginine dihydrolase, citrate utilization and gelatin hydrolase are positive according to the API 20E tests. Lysine decarboxylase, ornithine decarboxylase, urease and tryptophan deaminase are negative. Indole, acetoin (Voges–Proskauer reaction) and H2S are not produced. Acid can be produced from D-ribose, L-xylose, L-sorbose, esculin, D-lyxose, D-tagatose, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate and can be weakly produced from D/L-arabinose and D-lactose (bovine origin) according to API 50CH test results. Myo-inositol, gelatin, L-arginine, L-aspartic acid, L-histidine, glucuronamide, a-keto-glutaric acid are utilized and sucrose, D-glucose-6-phosphate, D-fructose-6-phosphate, D-glucuronic acid, D/L-malic acid, b-hydroxyD,L-butyric acid, a-keto-butyric acid are weakly utilized as sole sources of carbon and energy according to Biolog GNIII MicroPlates assays. Cells are susceptible to streptomycin, ampicillin, penicillin G and chloramphenicol, moderately susceptible to vancomycin, gentamycin and resistant to clindamycin. The major cellular fatty acids are summed feature 3 (C16:1 x7c/ iso-C15:0 2-OH), C18:1 x7c and C16:0. Major respiratory quinone is Q-8. The major polar lipids present include phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and an unknown phospholipid (PL2). The DNA G?C content of the type strain is 61.2 mol %. The type strain, HS6T (=ATCC BAA-2613T=CICC 10858T) was isolated from marine sediment of Yellow Sea, China. Acknowledgments This work was supported by the National Natural Science Foundation of China (31370057, 31290231, 31370108) and the China Ocean Mineral Resources R & D Association (COMRA) Special Foundation (DY125-15-T-05). Z. J. Wang acknowledges grant of Graduate Innovation Foundation of Shandong University at WeiHai, GIFSDUWH yjs12037.
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621 Kim JM, Lee SH, Jung JY, Jeon CO (2010) Marinobacterium lutimaris sp. nov., isolated from a tidal flat. Int J Syst Evol Microbiol 60:1828–1831 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948 Lee HW, Shin NR, Lee J, Roh SW, Whon TW, Bae JW (2012) Neptunomonas concharum sp. nov., isolated from a dead ark clam, and emended description of the genus Neptunomonas. Int J Syst Evol Microbiol 62:2657–2661 Liu A, Zhang XY, Chen CX, Xie BB, Qin QL, Liu C, Li GW, Li H, Xu Z, Chen XL, Zhou BC, Zhang YZ (2013) Neptunomonas qingdaonensis sp. nov., isolated from intertidal sand. Int J Syst Evol Microbiol 63:1673–1677 Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G?C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 38:159–167 Miyazaki M, Nogi Y, Fujiwara Y, Kawato M, Kubokawa K, Horikoshi K (2008) Neptunomonas japonica sp. nov., an Osedax japonicus symbiont-like bacterium isolated from sediment adjacent to sperm whale carcasses off Kagoshima, Japan. Int J Syst Evol Microbiol 58:866–871 Murray RGE, Doetsch RN, Robinow F (1994) Determinative and cytological light microscopy. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 21–41 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. MIDI, Inc, Newark Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Tindall BJ (1990a) A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13:128–130 Tindall BJ (1990b) Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Letts 66:199–202 Tindall BJ, Sikorski J, Smibert RM, Kreig NR (2007) Phenotypic characterization and the principles of comparative systematics. In: Reddy CA, Beveridge TJ, Breznak JA, Marzluf G, Schmidt TM, Snyder LR (eds) Methods for general and molecular microbiology, 3rd edn. ASM press, Washington DC, pp 330–393 Zhang XY, Zhang YJ, Yu Y, Li HJ, Gao ZM, Chen XL, Chen B, Zhang YZ (2010) Neptunomonas antarctica sp. nov., isolated from marine sediment. Int J Syst Evol Microbiol 60:1958–1961
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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-tospiral-shaped, oxidase- and catalase- positive and facultatively aerobic bacterium, designated HS6T, was isolated from marine sediment of Yellow Sea, China. It can reduce nitrate to nitrite and grow well in marine broth 2216 (MB, Hope Biol-Technology Co., Ltd) with an optimal temperature for growth of 30–33 °C (range 12–45 °C) and in the presence of 2–3 % (w/v) NaCl (range 0.5–7 %, w/v). The pH range for growth was pH 6.2–9.0, with an optimum at 6.5–7.0. Phylogenetic analysis based on 16S rRNA gene sequences
The GenBank accession number for the 16S rRNA gene sequence of Motiliproteus sediminis HS6T is KF953945.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0232-2) contains supplementary material, which is available to authorized users. Z.-J. Wang C. Wang Z.-J. Du (&) G.-J. Chen College of Marine Science, Shandong University at Weihai, Weihai 264209, China e-mail: [email protected] G.-J. Chen e-mail: [email protected] Z.-H. Xie Key Laboratory of Coastal Biology and Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China Z.-J. Du G.-J. Chen State key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
demonstrated that the novel isolate was 93.3 % similar to the type strain of Neptunomonas antarctica, 93.2 % to Neptunomonas japonicum and 93.1 % to Marinobacterium rhizophilum, the closest cultivated relatives. The polar lipid profile of the novel strain consisted of phosphatidylethanolamine, phosphatidylglycerol and some other unknown lipids. Major cellular fatty acids were summed feature 3 (C16:1 x7c/iso-C15:0 2-OH), C18:1 x7c and C16:0 and the main respiratory quinone was Q-8. The DNA G?C content of strain HS6T was 61.2 mol %. Based on the phylogenetic, physiological and biochemical characteristics, strain HS6T represents a novel genus and species and the name Motiliproteus sediminis gen. nov., sp. nov., is proposed. The type strain is HS6T (=ATCC BAA-2613T=CICC 10858T). Keywords Motiliproteus sediminis gen. nov., sp. nov. Polyphasic taxonomy 16S rRNA gene Marine bacteria Coastal sediment
Introduction The family Oceanospirillaceae within the phylum Proteobacteria was described by Garrity et al. (2005) in Bergey’s Manual of Systematic Bacteriology. It contains 18 genera (LPSN, http://www.bacterio.net/classifphyla.html) at the time of writing and most of them are strictly respiratory (except for Neptunomonas
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which gives weak fermentation reactions) aquatic organisms of primarily marine origin. The type genus of the family is Oceanospirillum (Hylemon et al. 1973) with O. linum as the type species. The family Oceanospirillaceae has a close relationship with the genus Marinobacterium (Gonza´lez et al. 1997), a member of family Alteromonadaceae. In the present study, the novel isolate was characterized by phenotypic and phylogenetic analyses and is proposed to be a member of novel species in a new genus of family Oceanospirillaceae. The novel genus forms a cluster with Marinobacterium which supports the view of Kim et al. (2007) that the genus Marinobacterium should be reclassified into the order Oceanospirillales.
Materials and methods Organism, maintenance and cultural conditions During the study of diversity of the multi-drug resistant bacteria in coastal marine environments, a novel heterotrophic, facultatively aerobic, olive to brown, motile, Gram-negative bacterial strain HS6T was isolated on marine agar 2216 (MA, Hope BiolTechnology Co., Ltd) at 28 °C. The sample was collected from the coastal of Yellow Sea (121°70 5700 E, 37°370 5300 N) around Yantai, China. For isolation of bacterial strains, 1 g wet sediment was blended to 99 mL sterilized seawater with glass beads and shaken vigorously. The suspension was serially diluted to 10-6 with sterilized seawater and 0.1 mL aliquots of each dilution were spread onto MA. The plates were incubated at 28 °C for 5–7 days. Strain HS6T was isolated and then stored at -80 °C in sterile 1 % (w/v) saline supplemented with 15 % (v/v) glycerol. The strain was routinely cultivated at 33 °C on MA or MB. Morphological, physiological and biochemical analysis The morphological and physiological features of HS6T were examined after incubation at 33 °C for 2–3 days on MA. The transmission electron microscope was used to observe the cell size, morphology and flagella. Meanwhile, the light microscopy (E600; Nikon) was used as supplement. Motility was assessed with the method of hanging-drop. Gram reaction was examined following the standard Gram procedure
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(Murray et al. 1994). The effects of different NaCl concentrations were assessed by modified MA (seawater replaced with distilled water) containing different concentrations of NaCl (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 %, w/v). Growth at different temperature was tested on MA at 4, 8, 12, 17, 24, 28, 30, 33, 37, 42 and 45 °C. To test the effect of pH on growth, MB was adjusted to different pH with MES (pH, 5.5, 6.0, 6.2), PIPES (pH, 6.5, 7.0), HEPES (pH, 7.5, 8.0), Tricine (pH, 8.5) and CAPSO (pH, 9.0, 9.5) buffers and each at a concentration of 20 mM and then measured the value of OD600. Anaerobic growth was determined at 33 °C in an anaerobic chamber on MA with or without 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 commercial oxidase reagent (bioMe´rieux). Hydrolysis of starch, lipid and algin was tested on MA supplemented with 0.2 % (w/v) soluble starch, 1 % Tween 80 (v/v) and 2 % sodium alginate respectively (Cowan and Steel 1965). Tests for other physiological or biochemical characteristics were performed using API 20E, API 20NE, API ZYM kits, API 50CH and Biolog according to the manufacturer’s instructions except that the salinity was adjusted to 3 %. All the API and Biolog tests were performed at least twice. Susceptibility to antibiotics was tested using filter-paper discs containing various antibiotics on cultures incubated at 33 °C on MA (since strain HS6T showed poor growth on Mueller–Hinton agar) for 2–3 days. Determination of G?C content of DNA, 16S rRNA gene sequencing and phylogenetic analysis Genomic DNA of strain HS6T was obtained from 60 h old cultures on MA using a genomic DNA extraction kit (Sangon, China) and the DNA G?C content was determined by HPLC (Mesbah et al. 1989). The 16S rRNA gene of strain HS6T was amplified from the genomic DNA by PCR with the universal primers 27F and 1492R (Lane 1991). PCR products were purified using a PCR product purification kit (Tiangen Biotech Co., Ltd, Beijing, China) and then ligated to the vector of pGM-T (Tiangen Biotech Co., Ltd, Beijing, China). Sequencing was performed by Shanghai Sunny Biotechnology Co., Ltd, China. To identify the taxonomic status of HS6T, a near complete sequence (1,466 bp) was obtained and submitted to GenBank/EMBL/ DDBJ datebase to search for similar sequences using
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BLAST algorithm. EzTaxon server (http://eztaxone. ezbiocloud.net/; Kim et al. 2012) was used to achieve the similarity values of sequences. Phylogenetic analysis was performed using MEGA version 5.2 (Tamura et al. 2011) after multiple alignments of data by CLUSTAL W (Larkin et al. 2007) and manual edition (to remove gaps at 30 and 50 ends and ambiguous bases) using BioEdit version 7.0 (Hall 1999). The phylogenetic trees were constructed using the neighbor-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) algorithms. Bootstrap values were calculated based on 1,000 replicates. Chemotaxonomic analysis The cells cultured on MA at 33 °C for 3 days (end of the logarithmic phase) were used to determine fatty acids. The method was described by Sasser (1990) previously. And then the extract was separated using Sherlock Microbial Identification System (MIS) (MIDI, Microbial ID, Newark, DE 19711 U.S.A.). Peaks were automatically integrated and fatty acid names and percentages calculated by the MIS Standard Software (Microbial ID). Respiratory lipoquinones were extracted from freeze dried cells and the methods were described by Tindall (1990a, b). They were separated into different classes by thin layer chromatography on silica gel and removed from the plate and further analyzed by HPLC. Polar lipids were separated by two dimensional silica gel thin layer chromatography. Total lipid material was detected using molybdatophosphoric acid and specific functional groups detected using spray reagents specific for defined functional groups, Full details are given in Tindall (2007). All procedures of analysis were carried out by the Identification Service, DSMZ, Braunschweig, Germany.
Results Phenotypic characteristics Strain HS6T can form circular, viscid and olive to brown (the color would change over time) colonies whose diameter were about 1.5 mm after 60 h incubation on MA at 33 °C. The cells of the strain were found to be Gram-negative, rod-to-spiral-shaped, motile and 0.5–0.8 9 1.5–6.0 lm in size, in contrast
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to the closest phylogenetic neighbors as shown in Table 1. It characteristically showed bipolar flagella which is different from most of the members of family Oceanospirillaceae and there were 1–2 flagella attached to the polar or subpolar of the HS6T (Fig. 1). The characteristics of morphology of HS6T are significant different from the relative genera shown in Table 1 and this can distinguish it from other genera partially. Growth was found to occur in 0.5–7 % (w/v) NaCl (optimum 2–3 %), at 15–45 °C (optimum 30–33 °C) and at pH 6.2–9.0 (optimum pH 6.5–7.0). There was no visible colonies formed under anaerobic conditions on MA supplemented with 0.1 % (w/v) NaNO3. But it can reduce nitrate to nitrite with or without oxygen which can distinguish HS6T from most of the members in the family Oceanospirillaceae. The strain was positive for oxidase and catalase activities and negative for hydrolysis of starch, Tween 80 and algin. The positive of gelatinase also can distinguish the new isolate from the genera Marinobacterium, Neptunomonas, Neptuniibacter and Nitrincola. Other cultural, physiological and biochemical characteristics of the novel strain will be given in the species description. And the different characteristics that can distinguish HS6T from its closest phylogenetic relatives were displayed in Table 1. Molecular phylogenetic analysis The nearly full-length (1,466 bp) 16S rRNA gene sequence of strain HS6T was obtained and the comparison revealed that strain HS6T had highest similarity to the type strain of Neptunomonas antarctica (93.3 %), followed by Neptunomonas japonica (93.2 %) (Miyazaki et al. 2008) and Marinobacterium rhizophilum (93.1 %) (Kim et al. 2008). Addition to these, the similarities with other relative neigbourhoods were all below 93 %. That suggested the strain HS6T may represent a novel genus in family Oceanospirillaceae. The further studies of phylogenetic trees (Fig. 2) proved that. In the NJ tree, the strain HS6T formed a separate branch with the genera Marinobacterium, Neptunomonas, Neptuniibacter and Nitrincola. Although the genus Marinobacterium belongs to the family Alteromonadaceae according to NCBI (http://www.ncbi. nlm.nih.gov/) and LPSN, we still insisted the strain HS6T should be classified to the family
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Table 1 Phenotypic comparison of HS6T and related genera HS6T
Neptunomonas
Cell shape
rods to spiral
rods
rods
rods to bent rods
rods
Size (lm)
0.5–0.8 9 1.5–6.0
0.6–1.0 9 1.2–3.0
0.3–0.8 9 0.6–3.0
0.2–0.8 9 1.5–2.3
0.5 9 1.3–1.6
Flagella Growth at
Bipolar
Single Polar
Single Polar
Single Polarb
Single Polar
–
?a
-a
–
–
a
?a
–
ND
Ana
An
F
Characteristic
4 °C 40 °C
Marinobacterium
Neptuniibacter
Nitrincola
?
-
F
F
–
-a
-a
–
–
Tween 80
–
-
a
?a
–
ND
Gelatin
?
-a
-a
–
–
Nitrate Reduction
?
?a
-a
–
–
G?C Content (%)
61.2
43.6–48.2
54.9–62.5
46.6–54.2
47.4
Quinone(s)
Q8/Q7
Q8/Q9b
Q8b
Q8
ND
O2 metabolism Hydrolysis of Starch
HS6T (this study); Neptunomonas (Hedlund et al. 1999; Miyazaki et al. 2008; Zhang et al. 2010; Lee et al. 2012; Liu et al. 2013); Marinobacterium (Baumann et al. 1983; Gonza´lez et al. 1997; Chang et al. 2007; Kim et al. 2007; Kim et al. 2008; Huo et al. 2009; Kim et al. 2009a; Kim et al. 2009b; Kim et al. 2010; Chimetto et al. 2011); Neptuniibacter (Arahal et al. 2007; Chen et al. 2012); Nitrincola (Dimitriu et al. 2005) ? positive, - negative, F facultatively aerobic, An strictly anaerobic a b
[50 % negative/positive The information is incomplete because some papers missed the relevant information
Oceanospirillaceae. Besides the higher similarity of 16S rRNA gene sequence and phenotypic characteristics, the EzTaxon also classified the genus Marinobacterium to the family of Oceanospirillaceae. And we also added the 16S rRNA gene sequences of families Alterononadaceae and Shewanellaceae to our phylogenetic tree, they formed a separate cluster which is far from the clusters including the Marinobacterium. The result supported the view of Kim et al. (2007) that the genus Marinobacterium should be reclassified into the order Oceanospirillales. The ML tree and the MP tree were similar and all of them supported our conclusion—the strain HS6T may represent a novel genus of the family Oceanospirillaceae. The DNA G?C content of the novel isolate was 61.2 mol % which is similar to Marinobacterium but much higher than other relatives (Table 1). Chemotaxonomic characteristics
T
Fig. 1 Transmission electron micrograph of HS6 . Cells grown on MA at 33 °C for 12 h. Bar = 1.0 lm
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The predominant respiratory quinone detected in strain HS6T was Q-8 (85 %) which is compatible with the most members of family Oceanospirillaceae
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619 Marinobacterium coralli CAIM 1449T (GU183820)
73
Marinobacterium sediminicola JCM 15524T (EU573966)
79 97
Marinobacterium maritimum KCTC 22254T (EU399548) Marinobacterium stanieri ATCC 27130T (AB021367)
96
Marinobacterium georgiense ATCC 700074T (U58339)
78
Marinobacterium halophilum DSM 17586T (AY563030)
100
Marinobacterium jannaschii ATCC 27135T (AB006765) Marinobacterium nitratireducens JCM 15523T (EU573965) Marinobacterium rhizophilum DSM 18822T (EF192391) Nitrincola lacisaponensis ATCC BAA-920T (AY567473)
81
Marinobacterium marisflavi KCTC 12757T (EF468717) Marinobacterium litorale KCTC 12756T (DQ917760)
52
Marinobacterium lutimaris DSM 22012T (FJ230839)
99
Neptuniibacter caesariensis CCUG 52065T (AY136116) Neptuniibacter halophilus BCRC 80079T (GQ131677)
98
Neptunomonas naphthovorans ATCC 700637T (AF053734) 76
Neptunomonas concharum JCM 17730T (JF748732)
86
Neptunomonas antarctica KACC 14056T (FJ713802)
67
Neptunomonas japonica DSM 18939T (AB288092) 70 97
Neptunomonas qingdaonensis KCTC 23686T (JF747202) Motiliproteus sediminis HS6T (KF953945)
63
Oceanospirillum linum ATCC 11336T (M22365) Oleispira antarctica DSM 14852T (AJ426420)
100
Bermanella marisrubri CCUG 52064T (AY136131)
95
Oceaniserpentilla haliotis DSM 19503T (AM747817)
97
Thalassolituus oleivorans DSM 14913T (AJ431699) Oceanobacter kriegii ATCC 27133T (AB006767)
99
Oleibacter marinus NBRC 105760T (FJ167390)
99
Shewanella putrefaciens ATCC 8071T (X81623) Alishewanella fetalis ATCC BAA-284T (AF144407) Catenovulum agarivorans DSM 23111T (GQ262000) 53
Agarivorans albus NBRC 102603T (AB076561)
100
Aliagarivorans marinus BCRC 17888T (FJ167390)
65
Bowmanella denitrificans BCRC 17491T (DQ343294) Aestuariibacter salexigens DSM 15300T (AY207502)
74 99
Glaciecola punicea ATCC 700756T (U85853) Alteromonas macleodii DSM 6062T (Y18228)
55 88
Salinimonas chungwhensis DSM 16280T (AY553295)
0.01
Fig. 2 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of HS6T and its closely relatives. Type strains of families Alteromonadaceae and Shewanellaceae were
used to root the tree. Bootstrap values ([50 %) based on 1,000 replications were shown at branching points. Bar, 0.01 substitutions per nucleotide position
(Garrity et al. 2005; Zhang et al. 2010; Miyazaki et al. 2008 and Arahal et al. 2007) and the genus Marinobacterium (Kim et al. 2008). A minor amount of Q-7 (11 %) was also detected, which seems to be less abundant or absent in close relatives. The remaining 4 % was difficult to identify. The existence of Q-7 can distinguish HS6T from the genera shown in Table 1. The major fatty acids (relative amount [ 1 %) found
in strain HS6T were summed feature 3 (C16:1 x7c/isoC15:0 2-OH,34.7 %), C18:1 x7c (24.1 %), C16:0 (21.3 %), C10:0 3-OH (5.0 %), C18:1 x6c (4.1 %), C14:0 3-OH/iso-C16:1 (3.8 %), C12:0 (2.4 %) and C14:0 (1.0 %). The components of fatty acids of HS6T were similar to its close relatives, however relatives proportions of individual and components differ considerably. The polar lipids found in strain HS6T
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were very complex. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and an unknown phospholipid (PL2) were major polar lipids. Two unknown phospholipids (PL1, PL4) and an unknown aminophospholipid (PN) were moderate and some other unknown lipids (AP, L1-3, PL3, PL5) were minor components. The detailed information are displayed in supplementary (Fig. S1). Polyphasic taxonomic conclusion The results of the phenotypic analysis, molecular phylogenetic analysis and chemotaxonomic analysis all support the view that strain HS6T represents a novel genus and species of the family Oceanospirillaceae, for which the name Motiliproteus sediminis is proposed. Description of Motiliproteus gen. nov Motiliproteus (Mo.ti.li.pro’teus. L. adj. motilis, motile; L. masc. n. Proteus, a mythical figure able to assume different forms; N.L. masc. n. Motiliproteus, a motile bacteria of various forms) Cells are Gram-negative, rod-to-spiral-shaped, facultatively aerobic and motile with bipolar flagella. Both catalase and oxidase are positive. No growth occurs in the absence of salt. Nitrate can be reduced to nitrite with or without oxygen. The major cellular fatty acids are C16:1 x7c/iso-C15:0 2-OH, C18:1 x7c and C16:0 and the main ubiquinone system is Q-8. Description of Motiliproteus sediminis sp. nov Motiliproteus sediminis (se.di.mi’nis. L. n. sedimeninis, sediment; L. gen. n. sediminis, of sediment) Besides the characteristics described above, the following characteristics are also observed for the type species. Cells are 0.5–0.8 lm in diameters and 1.5–6.0 lm in length. Forms circular, viscid and olive to brown (the color would change over time) colonies on MA after 60 h incubation at 33 °C. Growth occurs in 0.5–7 % (w/v) NaCl (optimum 2–3 %), at 15–45 °C (optimum 30–33 °C) and at pH 6.2–9.0 (optimum 6.5–7.0). Does not hydrolyse starch, Tween-80, alginate and cellulose. According to the API ZYM kits, cells are positive for acid and alkaline phosphatase,
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esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase and naphthol-AS-BI-phosphohydrolase but negative for lipase (C14), cystine arylamidase, trypsin, a-chymotrypsin, a-and b-galactosidase, a-and b-glucosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase. Arginine dihydrolase, citrate utilization and gelatin hydrolase are positive according to the API 20E tests. Lysine decarboxylase, ornithine decarboxylase, urease and tryptophan deaminase are negative. Indole, acetoin (Voges–Proskauer reaction) and H2S are not produced. Acid can be produced from D-ribose, L-xylose, L-sorbose, esculin, D-lyxose, D-tagatose, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate and can be weakly produced from D/L-arabinose and D-lactose (bovine origin) according to API 50CH test results. Myo-inositol, gelatin, L-arginine, L-aspartic acid, L-histidine, glucuronamide, a-keto-glutaric acid are utilized and sucrose, D-glucose-6-phosphate, D-fructose-6-phosphate, D-glucuronic acid, D/L-malic acid, b-hydroxyD,L-butyric acid, a-keto-butyric acid are weakly utilized as sole sources of carbon and energy according to Biolog GNIII MicroPlates assays. Cells are susceptible to streptomycin, ampicillin, penicillin G and chloramphenicol, moderately susceptible to vancomycin, gentamycin and resistant to clindamycin. The major cellular fatty acids are summed feature 3 (C16:1 x7c/ iso-C15:0 2-OH), C18:1 x7c and C16:0. Major respiratory quinone is Q-8. The major polar lipids present include phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and an unknown phospholipid (PL2). The DNA G?C content of the type strain is 61.2 mol %. The type strain, HS6T (=ATCC BAA-2613T=CICC 10858T) was isolated from marine sediment of Yellow Sea, China. Acknowledgments This work was supported by the National Natural Science Foundation of China (31370057, 31290231, 31370108) and the China Ocean Mineral Resources R & D Association (COMRA) Special Foundation (DY125-15-T-05). Z. J. Wang acknowledges grant of Graduate Innovation Foundation of Shandong University at WeiHai, GIFSDUWH yjs12037.
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