Jiella Aquimaris Gen. Nov., Sp. Nov., Isolated from Offshore Surface Seawater

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1127–1132 DOI 10.1099/ijs.0.000067

Jiella aquimaris gen. nov., sp. nov., isolated from offshore surface seawater

Jing Liang,1 Ji Liu1 and Xiao-Hua Zhang1,2

Correspondence 1College of Marine Life Sciences, Ocean University of China, Qingdao 266003, PR China Xiao-Hua Zhang 2Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, PR China [email protected]

A Gram-stain-negative, strictly aerobic and rod-shaped motile bacterium with peritrichous flagella, designated strain LZB041T, was isolated from offshore surface seawater of the East China Sea. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain LZB041T formed a lineage within the family ‘Aurantimonadaceae’ that was distinct from the most closely related genera Aurantimonas (96.0–96.4 % 16S rRNA gene sequence similarity) and Aureimonas (94.5–96.0 %). Optimal growth occurred in the presence of 1–7 % (w/v) NaCl, at pH 7.0–8.0 and at 28–37 6C. Ubiquinone-10 was the predominant respiratory quinone. The major fatty acids

(.10 % of total fatty acids) were C18 : 1v7c and/or C18 : 1v6c (summed feature 8) and cyclo-

C19 : 0v8c. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, one unknown aminolipid, one unknown phospholipid and one unknown polar lipid. The DNA G+C content of strain LZB041T was 71.3 mol%. On the basis of polyphasic analysis, strain LZB041T is considered to represent a novel species of a new genus in the class Alphaproteobacteria, for which the name Jiella aquimaris gen. nov., sp. nov. is proposed. The type strain of the type species is LZB041T (5JCM 30119T5MCCC 1K00255T).

Over recent decades, many novel taxa within the class Fulvimarina was first proposed for a novel bacterium Alphaproteobacteria have been described, and at the time of isolated from the Atlantic Ocean (Cho & Giovannoni, writing, this class comprises 13 orders. The order Rhizo- 2003), and subsequently emended by Rathsack et al. (2011). biales is a phenotypically heterogeneous assemblage within The genus Martelella was proposed by Rivas et al. (2005) the class Alphaproteobacteria, and is divided into 13 families with a single species, Martelella mediterranea, and at the time based on 16S rRNA gene sequence analyses. The family of writing comprises four species. ‘Aurantimonadaceae’ is affiliated to the order Rhizobiales Members of the family ‘Aurantimonadaceae’ were isolated (Kuykendall, 2005) and, at the time of writing, comprises from various sources including diverse marine environ- the genera Aurantimonas, Aureimonas, Fulvimarina and ments, water, soil, air, tidal flats, plant tissues and rusty iron Martelella. The genus Aurantimonas was originally described plates. In the course of identifying dimethylsulfoniopropio- by Denner et al. (2003) with Aurantimonas coralicida as the nate (DMSP)-utilizing bacteria in the marine environ- type species. Subsequently, five species have been character- ment, a peritrichously flagellated bacterial strain, designated ized. However, based on the evident heterogeneity to other LZB041T, was isolated from the offshore surface seawater species in the genus Aurantimonas, Rathsack et al. (2011) of the East China Sea at station ME3 (28u 43.9319 N reclassified Aurantimonas altamirensis, Aurantimonas frigi- 122u 34.9049 E) during the expedition of the R/V ‘Dong daquae and Aurantimonas ureilytica into a novel genus Fang Hong 2’ in July 2013. The aim of the present study was named Aureimonas as Aureimonas altamirensis, Aureimonas to determine the exact taxonomic position of strain LZB041T frigidaquae and Aureimonas ureilytica, respectively, with by using a polyphasic characterization that included the Aureimonas altamirensis as the type species. The genus determination of chemotaxonomic and phenotypic prop- erties, and detailed phylogenetic investigation based on 16S Abbreviations: DMSP, dimethylsulfoniopropionate; DPG, diphospha- rRNA gene sequences. tidylglycerol; ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbour-joining; PC, phosphatidylcholine; PE, phosphatidylethanolamine; Strain LZB041T was isolated by the standard dilution PG, phosphatidylglycerol; PME, phosphatidylmonomethylethanolamine. spreading method on marine agar 2216 (MA; Becton The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene Dickinson) incubated at 28 uC for up to 1 week. A yellow sequence of strain LZB041T is KJ620984. colony, designated strain LZB041T, was isolated and sub- Three supplementary figures are available with the online Supple- sequently purified three times on MA at 28 uC. Working mentary Material. cultures were routinely maintained on MA at 28 uC and

000067 G 2015 IUMS Printed in Great Britain 1127 J. Liang, J. Liu and X.-H. Zhang stocks were preserved at 280 uC as a suspension in sterile synthetic marine ZoBell broth (5 g peptone, 1 g yeast extract 0.85 % (w/v) saline supplemented with 15 % (w/v) glycerol. and 0.1 g FePO4 in 1 l water). NaCl concentrations were Aurantimonas coralicida DSM 14790T and Aureimonas adjusted to 0–20.0 % (w/v, at intervals of 1.0 %). Growth altamirensis DSM 21988T were chosen as reference strains was evaluated at 0, 4, 10, 16, 25, 28, 32, 37, 42 and 46 uCon for phenotypic characterization and fatty acid analysis. MA and at pH 2.0–11.0 in marine broth 2216 (MB; Becton Dickinson) using the following buffer systems: Na HPO / Genomic DNA extraction, and PCR amplification, cloning 2 4 citric acid (for pH 2.0–7.0), Tris/HCl (pH 8.0–9.0) and and sequencing of the 16S rRNA gene were performed Na2CO3/NaHCO3 (pH 10.0–11.0). To test for anaerobic according to Yu et al. (2013). The almost complete 16S T growth, strain LZB041 was cultured at 28 uC for 1 month rRNA gene sequence (1445 nt) was manually checked and on MA with resazurin (0.02 %, w/v) added as an indicator of submitted to the GenBank database. Pairwise similarity T anaerobic condition. Inoculated plates were incubated in an values between strain LZB041 and closely related type anaerobic jar filled with nitrogen and an AneroPack bag strains were calculated using the EzTaxon-e server (http:// (Mitsubishi Gas Chemical Co.). Phenotypic characteristics eztaxon-e.ezbiocloud.net/; Kim et al., 2012). 16S rRNA possessed by strain LZB041T and two reference strains were gene sequences of related strains were downloaded from tested according to standard approaches (Tindall et al., the NCBI database (http://www.ncbi.nlm.nih.gov). Phylo- 2007) with sterile seawater substituted for distilled water; genetic analysis was performed by the software package tested characteristics included: activities of catalase, oxidase MEGA version 5.0 (Tamura et al., 2011) after multiple align- (method 2) and hydrolysis of starch, casein, gelatin and ment of the sequence data with CLUSTAL X (Thompson et al., Tweens 20, 40 and 80 (method 2). DNase agar (Qingdao 96 1997). Phylogenetic trees based on the neighbour-joining Hope Bio-technology Co.) prepared with sterile seawater (NJ), maximum-likelihood (ML) and maximum-parsimony was used to detect the DNase activity. Chitin (1 %, w/v) and (MP) algorithms were reconstructed and the genetic dis- sodium alginate (2 %, w/v) were added to MA to determine tances were calculated by using Kimura’s two-parameter their degradation by the formation of clear zones around model (Kimura, 1980). The topology of the phylogenetic colonies directly or after flooding with appropriate solutions trees was evaluated by the bootstrap resampling method of (Teather & Wood, 1982). Activities of constitutive enzymes, Felsenstein (1981) with 1000 replicates. the fermentation/oxidation profile, acid production, and Strain LZB041T showed the highest 16S rRNA gene sequence substrate utilization as sole carbon and energy source were similarity with members of the genera Aurantimonas (96.0– performed using API 20E, API 20NE, API 50CH, API ZYM 96.4 %), Aureimonas (94.5–96.0 %), Fulvimarina (93.9–95.1 %) strips (bioMe´rieux) and the GN2 MicroPlate kits (Biolog) and Martelella (92.6–93.0 %) in the family ‘Aurantimo- according to the manufacturers’ instructions except that nadaceae’, with lower similarities observed to other alp- sterile seawater was used to prepare the inocula. The morphological, physiological and biochemical characteris- haproteobacterial species including those of the genera T Rhizobium (92.6–94.5 %), Pseudochrobactrum (92.6–92.9 %), tics of strain LZB041 are given in Table 1, Fig. 2 and the Cucumibacter (92.6 %) and Devosia (91.5 %). The NJ tree, species description. T showing phylogenetic relationships among strain LZB041 For cellular fatty acid analysis, strain LZB041T and the two and selected type strains of species in the closest families, is reference strains were grown on MA at 28 uC for 48 h until T presentedinFig.1.StrainLZB041 formed a distinct lineage the bacterial communities reached the late exponential in the family ‘Aurantimonadaceae’. The branch pattern of the stage of growth according to the four quadrants streak clades was identical to that in phylogenetic trees obtained method (Sasser, 1990). Fatty acid methyl esters were by the ML algorithm (Fig. S1, available in the online prepared and analysed according to the standard protocol Supplementary Material) and the MP algorithm (Fig. S2); of MIDI (Sherlock Microbial Identification System, version major branches revealed by the different tree-making 6.10), and identified by the TSBA6.0 database of the methods are indicated by filled circles. Considering the Microbial Identification System (Sasser, 1990). For analysis branching patterns from phylogenetic trees along with low of respiratory quinones and polar lipids, cells were harvested sequence similarity values to other type species, strain from MB after incubation at 28 uC for 48 h and freeze-dried. LZB041T should be assigned to a novel genus in the family Polar lipids were extracted according to Minnikin et al. ‘Aurantimonadaceae’. (1984), separated by two-dimensional TLC on silica gel 60 The cellular morphology of strain LZB041T was determined F254 plates (Merck), and identified by spraying with the by transmission electron microscopy (JEOL; JEM-1200EX) appropriate detection reagents (Komagata & Suzuki, 1987). The respiratory quinones of strain LZB041T were extracted after cells in exponential phase had been negatively stained with chloroform/methanol (2 : 1, v/v) and identified by with 1 % (w/v) phosphotungstic acid. Gram-staining and HPLC as described by Xie & Yokota (2003). The genomic flagellum staining were performed according to Beveridge DNA of strain LZB041T was extracted according to Moore et al. (2007). Salinity and pH ranges supporting growth were et al. (1999) and the G+C content was determined by investigated in 96-well microplates by measuring the optical reversed-phase HPLC according to Mesbah et al. (1989). densities at 590 nm. In the salinity experiment, artificial + seawater (Lyman & Fleming, 1940) with Na replaced by Like other species belonging to the family ‘Aurantimo- + appropriate K and distilled water were used to prepare nadaceae’, the predominant cellular fatty acid (.10 % of

1128 International Journal of Systematic and Evolutionary Microbiology 65 Jiella aquimaris gen. nov., sp. nov.

Jiella aquimaris LZB041T (KJ620984) 60 Aurantimonas coralicida WP1T (AJ786361) 100 Aurantimonas manganoxydans SI85-9A1T (AAPJ01000003) 0.01 99 ‘Aurantimonas litoralis’ HTCC2156 (AY178863) Aureimonas frigidaquae CW5T (EF373540) 98 S21BT (DQ372921) 51 Aureimonas altamirensis CC-CFT023T (JQ864240) 97 Aureimonas ferruginea 93 Aureimonas rubiginis CC-CFT034T (JQ864241) 84 Aureimonas ureilytica 5715S-12T (DQ883810) 72 Aureimonas phyllosphaerae L9-753T (JQ346806) 52 52 Aureimonas jatrophae L7-484T (JQ346805) Fulvimarina pelagi HTCC2506T (AATP01000004) 97 Fulvimarina manganoxydans 8047T (HQ622550) Pseudochrobactrum kiredjianiae CCUG 49584T (AM263420) 96 Rhizobium pseudoryzae J3-A127T (DQ454123) 87 58 ‘Rhizobium qilianshanense’ CCNWQLS01 (JX117881) 60 Martelella mangrovi BM9-1T (KF560340) 100 Martelella mediterranea MACL11T (AY649762) 86 Martelella endophytica YC6887T (HM800924) 78 Martelella radicis BM5-7T (KF560339) Cucumibacter marinus DSM 18995T (EF211830) 98 Devosia pacifica NH131T (KF111722) Ancylobacter oerskovii NS05T (AM778407) 76 Amorphus suaedae YC6899T (KC006961) Escherichia coli ATCC 11775T (X80725)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain LZB041T, members of the family Aurantimonadaceae and other families of the class Alphaproteobacteria. Bootstrap percentages (.50 %) based on 1000 replicates are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in trees generated with the maximum-likelihood and maximum-parsimony algorithms. Escherichia coli ATCC 11775T (GenBank accession no. X80725) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.

Table 1. Differential characteristics between strain LZB041T and the type strains of phylogenetically related species of genera in the family Aurantimonadaceae

Strains: 1, LZB041T (data from this study); 2, Aurantimonas coralicida DSM 14790T (this study); 3, Aureimonas altamirensis DSM 21988T (this study); 4, Fulvimarina pelagi HTCC2506T (Cho & Giovannoni, 2003); 5, Martelella mediterranea MACL11T (Rivas et al., 2005). All strains are strictly aerobic and positive for catalase, oxidase and urease activities. +, Positive; 2, negative; w, weakly positive; ND, no data available.

Characteristic 1 2 3 4 5

Flagellation Peritrichous .1, polar 22 2 Growth temperature (uC) 4–42 4–37 10–40 4–40 4–37 NaCl range for growth(%, w/v) 0–15 0–7 0–5 0–10 0–5 Glucose fermentation (API 20E) ++2 ND ND Assimilation of (API 20E): Citrate + 22ND 2 N-Acetylglucosamine + 2 + ND 2 Enzyme activity (API ZYM) Valine arylamidase + 22ND + a-Chymotrypsin + 22ND 2 a-Glucosidase + 22ND +

Major fatty acids C18 : 1v7c and/or C18 : 1v7c, cyclo-C19 : 0v8c, C18 : 1v7c, C18 : 1v7c C18 : 1v7c, cyclo- C18 : 1v6c, cyclo- C16 : 0 C16 : 0 C19 : 0v8c,C16 : 0, C19 : 0v8c 11-methyl C18 : 1v7c DNA G+C content (mol%) 71.3 66.3*a 71.8b 57.6 57.4 Major respiratory quinone Q-10 Q-10, Q-9a Q-10b Q-10 Q-10

*Data from: a, Denner et al. (2003); b, Jurado et al. (2006). http://ijs.sgmjournals.org 1129 J. Liang, J. Liu and X.-H. Zhang

(a) (b) Table 2. Cellular fatty acid contents (%) of strain LZB041T and the reference strains

Strains: 1, LZB041T;2,Aurantimonas coralicida DSM 14790T;3, Aureimonas altamirensis DSM 21988T. All data are from this study. Only fatty acids .1 % of the total fatty acids of at least one of the strains are shown. Major fatty acids (.10 %) are indicated in bold type. TR, Trace amount (,1 % of total); 2, not detected.

Fatty acid 1 2 3

Straight chain

C16 : 0 5.91 13.71 11.41 C18 : 0 1.54 5.31 5.1 Hydroxy

C18 : 1 2-OH 2 2.71 2 Unsaturated Fig. 2. Transmission electron micrographs of negatively stained C20 : 1v7c 2 TR 1.08 T 6 cells of strain LZB041 cultured on MA at 28 C for 24 h. Bar, cyclo-C19 : 0v8c 21.44 3.48 10.44 0.5 mm. 11-methyl C18 : 1v7c Tr 22 Summed feature* 2 1.13 2 1.09 total fatty acids) of strain LZB041T was summed feature 8 5 TR 1.18 1.14 T 8 64.07 68.4 60.73 (C18 : 1v7c and/or C18 : 1v6c). However, in strain LZB041 , the amount of the second predominant cellular fatty acid, *As indicated by Montero-Calasanz et al. (2013), summed features are cyclo-C19 : 0v8c, was significantly higher than that observed in members of the genera Aurantimonas and Aureimonas. groups of two or three fatty acids that are treated together for the Differences in fatty acid composition and the proportion of purpose of evaluation in the MIDI system, and include both peaks some components were identified between strain LZB041T with discrete equivalent chain-lengths (ECLs) as well as those where and the reference strains (Table 2). The predominant isopren- the ECLs are not reported separately. Summed feature 5 was listed as oid quinone detected in strain LZB041T was ubiquinone-10 C18 : 2v6,9c and/or anteiso-C18 : 0; summed feature 8 was listed as v v (Q-10), which is consistent with its placement in the family C18 : 1 7c and/or C18 : 1 6c. Summed feature 2 was listed as C12 : 0 aldehyde, unknown 10.928, C 3-OH and iso-C I. ‘Aurantimonadaceae’. The major polar lipids detected in strain 14 : 0 16 : 1 LZB041T were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), phosphatidy- representing a novel species of a new genus in the family lethanolamine (PE), phosphatidylmonomethylethanolamine ‘Aurantimonadaceae’, for which the name Jiella aquimaris (PME), one unknown aminolipid, one unknown phospho- gen. nov., sp. nov. is proposed. lipid and one unknown polar lipid (Fig. S3). This polar lipid profile was mostly similar to the reference strains (Rathsack Description of Jiella gen. nov. et al., 2011) but there were some differences in strain LZB041T including the absence of sulfoquinovosyldiacylgly- Jiella (Ji.el9la. N.L. dim. fem. n. Jiella of Ji, in honour of the cerol (SQDG) and the unidentified lipids. The DNA G+C marine microbiologist Weishang Ji from Ocean University content of strain LZB041T was 71.3 mol%, which falls of China). within the range for members of the genus Aureimonas Cells with peritrichous flagella are Gram-stain-negative, (63.9–71.8 mol%; Lin et al., 2013; Madhaiyan et al., 2013). strictly aerobic rods. Oxidase-, catalase- and urease-positive. The conclusion drawn from phylogenetic analysis that strain The major respiratory quinone is Q-10. Predominant T LZB041 represents a novel genus of the family ‘Auranti- cellular fatty acids are summed feature 8 (C18 : 1v7c and/or monadaceae’ was supported by morphological and physio- C18 : 1v6c) and cyclo-C19 : 0v8c. The major polar lipids are logical characteristics that distinguished the novel strain PG, DPG, PC, PE, PME, one unknown aminolipid, one from other bacteria in the family ‘Aurantimonadaceae’ unknown phospholipid and one unknown polar lipid. including: (i) the presence of peritrichous flagella; (ii) the Phylogenetically, the genus belongs to the family ‘Auran- high tolerance of NaCl for growth; (iii) the proportion of timonadaceae’ in the class Alphaproteobacteria. The type cyclo-C19 : 0v8c was significantly high among total fatty species is Jiella aquimaris. acids; (iv) the evident different results in citrate assimilation, valine arylamidase, a-chymotrypsin and a-glucosidase tests. Description of Jiella aquimaris sp. nov. Other characteristics that differentiate strain LZB041T from members of related genera are shown in Table 1. It can Jiella aquimaris (a.qui.ma9ris. L. n. aqua water; L. gen. n. be concluded after combining phenotypic, phylogenetic maris of the sea; N.L. gen. n. aquimaris of the water of the and genetic data, strain LZB041T should be classified as sea).

1130 International Journal of Systematic and Evolutionary Microbiology 65 Jiella aquimaris gen. nov., sp. nov.

Displays the following characteristics in addition to those Acknowledgements m given in the genus description. Cells are 0.6–0.7 min This work was supported by the National Natural Science Foundation width and 2.3–2.8 mm in length. Growth occurs on MA of China through grants 41476112 and 41221004. and R2A medium. Colonies on MA are yellow, regular, opaque and smooth with an entire margin of 0.5–1 mm in diameter after incubation for 3 days at 28 uC. Growth References occurs at 4 to 42 uC (optimum 28–37 uC) and at pH 7.0– Beveridge, T. J., Lawrence, J. G. & Murray, R. G. E. (2007). 10.0 (optimum pH 7.0–8.0). The NaCl range for growth is Sampling and staining for light microscopy. In Methods for General and Molecular 0–15 % (optimum 1–7 %). Starch, alginate and Tweens 20, Microbiology, pp. 19–33. Edited by C. A. Reddy, T. J. Beveridge, 40 and 80 are hydrolysed, but casein, gelatin, chitin and J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. DNA are not. 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