International Journal of Systematic and Evolutionary (2013), 63, 1464–1470 DOI 10.1099/ijs.0.039982-0

Iodobacter limnosediminis sp. nov., isolated from Arctic lake sediment

Wei Su, Zhichao Zhou, Fan Jiang, XuLu Chang, Ying Liu, Shaohua Wang, Wenjing Kan, Mengchen Xiao, Ming Shao, Fang Peng and Chengxiang Fang

Correspondence China Center for Type Culture Collection (CCTCC), College of Life Sciences, Wuhan University, Fang Peng Wuhan 430072, PR China [email protected]

A Gram-reaction-negative, motile, non-violet-pigmented, rod-shaped bacterial strain, designated E1T, was isolated from Arctic lake sediment. Growth occurred at 4 6C–28 6C (optimum, 18 6C), at pH 4–11(optimum, 9–10) and in the presence of 0–1 % (w/v) NaCl. The taxonomic position of E1T was analysed using a polyphasic approach. Strain E1T exhibited 16S rRNA gene sequence similarity value of 98.1 % with respect to the type strain of Iodobacter fluviatilis, but no more than 93 % with the type strains of other recognized species. A further DNA–DNA hybridization experiment was conducted, which demonstrated unambiguously that strain E1T was distinct from I. fluviatilis ATCC 33051T (51.3 % relatedness). The DNA G+C content of strain E1T was 52.3 mol%. Chemotaxonomic data [Q-8 as the monospecific respiratory quinone and summed

feature 3 (C16 : 1v7c and/or C16 : 1v6c, 56.1 %) and C16 : 0 (18.8 %) as the major cellular fatty acids] supported the affiliation of strain E1T to the genus Iodobacter. However, the results of physiological and biochemical tests allowed phenotypic differentiation of strain E1T from I. fluviatilis ATCC 33051T. On the basis of phenotypic and genotypic properties, strain E1T represents a novel species of genus Iodobacter, for which the name Iodobacter limnosediminis sp. nov. is proposed. The type strain is E1T (5CCTCC AB 2010224T5NRRL B-59456T).

Chromobacterium fluviatile was initially proposed by Moss determine the exact taxonomic rank of an Arctic bacterial et al. (1978). However, as its great discrepancy in the strain using prospective polyphasic characterization. content of guanine-plus-cytosine with Chromobacterium A sediment sample collected from an Arctic lake on species and low levels of DNA–rRNA hybridization with Blomstrand Island (78u57995.0’’ N12u04’ 22.3’’ E), was Chromobacterium (Gillis & Logan, 2005; Sneath, 1984a; diluted serially in sterile 0.85 % (w/v) NaCl solution for Moss & Ryall, 1981; Sneath, 1974) and Janthinobacterium isolation. The resulting supernatant was spread on (Sneath, 1984b) from data of Moss & Bryant (1982), the R2A agar (BD) plates and incubated at 18 uC for 7 days. genus Iodobacter, residing in the family , Strain E1T was subsequently obtained from the plates. was established by Logan (1989) to accommodate Chromobacterium fluviatile (Moss et al., 1978). Logan Genomic DNA of strain E1T was extracted using a Bacteria (1989, 2005) described the violacein-producing isolates Genomic DNA Isolation kit (Shanghai Chaoshi Bio Iodobacter fluviatile from running fresh water in England Technologies). PCR was performed with the forward and Scotland (even reported from Antarctic lakes) (Wynn- primer 16F27 (59-AGAGTTTGATCCTGGCTCAG-3’) and Williams, 1983). This organism is Gram-negative, straight, the reverse primer 16R1492 (5’-TACCTTGTTACGACTT- round-ended rod-shaped, growing with spreading colonies, 3’), with sets of primers encompassing a whole 16S rRNA motile with a single polar and one or more lateral gene. PCR conditions were: pre-denaturation 94uC for 10 flagella, which has 50–52 mol% G+C content. The min; 30 cycles each of denaturation at 94uC for 60 s, original spelling of the type strain belonging to genus annealing at 55uC for 60 s and elongation at 72uC for 60 s; Iodobacter has been corrected to Iodobacter fluviatilis by and a final stage of 72uC for 10 min. Sequencing was Euze´by since 1997. The purpose of the present work was to conducted with an established method (Lane, 1991). PCR-amplified DNA was sequenced by Invitrogen Biotechnology. The 16S rRNA gene sequence of strain The GenBank/EMBL/DDBJ accession number for 16S rRNA gene E1T was compared with those of related taxa retrieved from sequence of strain E1T is HM031078. GenBank databases. Gene sequence identity with related A supplementary figure is available with the online version of this paper. species was calculated by pairwise alignment obtained from

1464 039982 G 2013 IUMS Printed in Great Britain Iodobacter limnosediminis sp. nov. the EzTaxon database (Chun et al., 2007). Multiple Tween-20, pH 7.8). The volume of 100 ml chromogen alignments were performed with the CLUSTAL_X software solution (TMB, ZOMANBIO) was added to the wells; the (Thompson et al., 1997). Phylogenetic trees were con- microtitre plate was then incubated at room temperature structed by neighbour-joining (Saitou & Nei, 1987) and for 4 min, the enzyme reaction was blocked by adding 21 maximum-parsimony (Fitch, 1971) tree-making algo- 50 mlH2SO4 solution (2 M l ). The intensity of the yellow rithms using the MEGA package version 4.0 (Tamura et al., colour was measured at 450 nm with a microplate reader 2007). In each case, bootstrap values were calculated based (Benchmark model; BioTek). The percentage DNA relat- on 1000 resamplings (Felsenstein, 1985). The evolutionary edness was calculated according to the description of distance matrix for the neighbour-joining method was Christensen et al. (2000). Each experiment was performed generated according to the Kimura two-parameter method with eight replicates. In the DNA hybridization experi- (Kimura, 1980). Close-neighbour-interchange (search ments, the criteria for acceptance of value used for level52, random additions5100) was applied in the calculation of DNA similarity were within 15 % standard maximum-parsimony analysis. deviation, calculated as a percentage of the mean for eight T Comparative 16S rRNA gene sequence analysis exhibited replicate micro-wells. The DNA of strain E1 exhibited a that strain E1T shared the highest sequence identity with I. relatedness value of 51.3 % with the DNA from the type T strain of I. fluviatilis. The DNA relatedness difference of fluviatilis ATCC 33051 (98.1 %). Additionally, a similarity T T search indicated that the strain E1T had sequence similarity interchange of E1 and I. fluviatilis ATCC 33051 used for of 92.8 % to Chitinibacter alvei TNR-14T (Yang et al., 2010) covalent binding and hybridization was 2.9 %. T and 91.4 % to Silvimonas amylolytica ir6-4 (Muramatsu Analyses of respiratory quinones were performed for the et al., 2010). Based on the complete 16S rRNA gene isolate E1T and the type strain of I. fluviatilis with HPLC T sequence, phylogenetic analysis revealed that strain E1 according to standard procedures described by Xie & T formed a robust clade with I. fluviatilis ATCC 33051 in Yokota (2003). Q-8 was determined to be the monospecific the neighbour-joining tree (Fig. 1) and approached to quinone, which is in accord with previous reports of the T related species Chitiniphilus shinanonensis SAY3 (Sato type species of the genus Iodobacter and other members of T et al., 2009) and koreensis R2A43-10 (Kim the family Neisseriaceae (Table 1). et al., 2006) (Fig. 1) from the genealogical relationship. The overall topology of the neighbour-joining tree was Cell biomass for analysis of cellular fatty acid was obtained supported by the tree built using maximum-parsimony from cells grown to late-exponential phase on R2A agar (not shown). In conclusion, 16S rRNA sequence clearly (BD) at 18 uC. For this purpose, extraction of cellular fatty suggested that strain E1T represented a possible novel acids was using the protocol of the Sherlock Microbial species belonging to the genus Iodobacter. Identification System (MIDI) version 6.0. Cellular fatty acids were analysed with a Hewlett Packard 6890N gas DNA–DNA hybridizations was applied as a more sensitive T chromatograph with MIDI Sherlock TSBA6 (Sasser, 1990). tool for genetic recognition of strain E1 and type strain of The results documented that the major fatty acid patterns I. fluviatilis. The level of DNA relatedness between them of strain E1T and I. fluviatilis ATCC 33051T were similar, were determined with a method modified from that of differing from those of other genera belonging to family Ezaki et al. (1989), using peroxidase as the enzyme system Neisseriaceae (Table 2). Compared with the profile of fatty instead of the b-galactosidase system. Therefore, the acids of I. fluviatilis ATCC 33051T, strain E1T contained genomic DNA of E1T and I. fluviatilisATCC 33051T were larger amount of summed in feature 3 (C16 : 1v7c and /or extracted with the procedure of Yoon et al. (1996). The C16 : 1v6c) (56.1 %) and C16 : 0 (18.8 %) as major fatty acids. covalent binding to the NucleoLink surface was achieved T However, strain E1 lacked iso-C13 : 0 3-OH, iso-C15 : 0, iso- with 1 mg DNA each well. Hybridization was carried out [ T C17 : 0 I. fluviatilis ATCC 33051 contained iso-C13 : 0 3-OH for 16 h with 100 ng photo-activatable biotin-labelled ] (1.2 %), iso-C15 : 0 (1.1 %) and iso-C17 : 0 (2.2 %) and other DNA per well at 40 uC due to the low G+C content [ T T ] fatty acids in trace amounts (data not shown). In addition E1 (52.3 %), I. fluviatilis ATCC 33051 (49.6 %) . to evidence based on 16S rRNA-gene-sequence-based The hybridization temperature was calculated from the phlyogenetic analysis and DNA hybridization described formulae described by Schildkraut & Lifson (1965) above, the data on cellular fatty acids allowed strain E1T to m and Wetmur & Davidson (1968). An aliquot of 100 l be distinguished from I. fluviatilis ATCC 33051T defined by streptavidin-conjugated horseradish peroxidase (sigma, Logan (1989) at the species level since differentiations of 1 mg ml21) diluted 1 : 8000 in buffer solution the cellular fatty acid components was perceived as the (13 mM NaCl, 0.3 mM KCl, 3.8 mM Na HPO . 12H O, 2 4 2 criterion to investigate . 0.2 mM NaH2PO4 .2H2O, 0.3 mM MgCl2, 0.5 mM BSA, 0.2 mM ethylmercurithiosalicylic acid, 0.2 mM The DNA base composition of isolate E1T was determined gentamicin sulfate, pH 7.6) was added to every well; the by the method of Mesbah et al. (1989) with the microtitre plates were then incubated at 37 uC for 10 min. modification that DNA was hydrolysed using nuclease P1 After incubation, the plates were washed with washing (Sigma) and the resultant nucleotides were analysed by buffer (38 mM NaCl, 0.3 mM KCl, 3.8 mM reversed-phase HPLC (UltiMate 3000, Dionex). In order to Na2HPO4 . 12H2O, 0.2 mM NaH2PO4 .2H2O, 0.8 mM accomplish this goal, Genomic DNA was isolated and http://ijs.sgmjournals.org 1465 W. Su and others

T 91 Chitinibacter alvei TNR-14 (FJ593907) Chitinibacter tainanensis BCRC 17254T (AY264287) c14T (DQ314581) 100 Chitinilyticum aquatile Chitinilyticum litopenaei c1T (EU682455) Deefgea chitinilytica Nsw-4T (FJ347759) 100 T Deefgea rivuli WB 3.4-79 (AM397080) 99 Andreprevotia chitinilytica JS11-7T(DQ836355) 76 GFC-1T (EU287926) Andreprevotia lacus T Jeongeupia naejangsanensis BIO -TAS4 - 2 (FJ669617) 73 T 100 Silvimonas terrae KM -45 (AB194302) Silvimonas amylolytica ir6-4T (AB326111) T 92 70 Silvimonas iriomotensis ir6 -1 (AB326110) Chitiniphilus shinanonensis SAY3T (AB453176) DSM 6150T (Y17602) 78 Formivibrio citricus 0.02 Iodobacter limnosediminis E1T (HM031078 ) 100 ATCC 33051T (M22511) Iodobacter fluviatilis T Chitinimonas taiwanensis cf (AY323827) 100 T R2A43-10 (DQ256728) 100 TRO-001DR8T (DQ018117) Aquitalea magnusonii T Aquitalea denitrificans 5YN1-3 (EU594330) 100 Chromobacterium piscinae CCM 3329T (AJ871127) T 99 Chromobacterium aquaticum CC-SEYA-1 (EU109734) 95 T Chromobacterium haemolyticum MDA0585 (DQ785104) 86 Leeia oryzae HW7T (DQ280369) 78 Microvirgula aerodenitrificans SGLY2T (U89333) 95 Laribacter hongkongensis HKU1T (AF389085) Limnobacter thiooxidans CS-K2T (AJ289885) T Azonexus fungiphilus BS-58 (AF011350) T 89 100 caeni EMB43 (DQ413148) A-7T (AB201043) Zoogloea oryzae 83 Zoogloea ramigera IAM 12136T (D14254) T 100 Glaciimonas immobilis Cr9-30 (GU441679) T Collimonas arenae NCCB 100031 (AY281146) MY14T (AB008503) Oxalicibacterium solurbis T Escherichia albertii LMG 20976 (AJ508 775)

Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the positions of strain E1T and some related taxa. Bootstrap percentage values (1000 replications) greater than or equal to 70 % are shown at nodes. Escherichia albertii LMG 20976T (AJ508775) was used as an outgroup. Bar, 0.02 substitutions per nucleotide position. purified according to the method of Yoon et al. (1996), GN2 system. The Gram-reaction experiment was performed applying RNase A to minimize contamination with RNA and using the classical Gram method described by Doetsch proteinase K to remove peptides. The DNA G+C content of (1981). Gliding motility was observed as described by strain E1T was 52.3 mol%, in contrast to that of the type Bowman (2000). Growth under anaerobic conditions was strain of I. fluviatilis (49.6 mol%), which is slightly different tested on R2A agar (BD) at 18 uC for two weeks. The from the values (50–52 mol%) reported for I. fluviatilis with morphology and flagellation were observed using phase- melting temperature according to Moss et al. (1978). contrast microscopy (Olympus B) and transmission electron microscopy (Hitachi H-8100) respectively, with cells from To investigate the basic physiological and biochemical exponentially growing cultures (growth for two days). The characteristics, standard experiments were performed as temperature range (4, 10, 18, 28, 30, 37 and 42 uC), pH follows: Oxidase activity was estimated by the oxidation of range (2–11, using increments of 1 pH unit) and salt 1 % (w/v) tetramethyl-p-phenylenediamine, activ- tolerance [0, 1, 2, 3, 4 and 5 %, (w/v)] were determined using ity was tested by measurement of bubble production after R2A medium (BD) for up to one week. To test pigment- the addition of 3 % (v/v) H2O2 solution, hydrolysis of production, the colour changes following the addition of casein and starch were conducted on R2A agar (BD) after either 10 % (v/v) H2SO4 or 10 % (w/v) NaOH to a drop of incubation for 1 week according to Smibert & Krieg an ethanolic solution of pigment in a test tube were recorded (1994). In addition, commercial tests (API 20NE, API ZYM (Moss et al., 1978). Susceptibility to antibiotics was and API 50CH test kits) were also used according to the performed on R2A agar (BD) plates for 7 days at 18 uC recommendation of the manufacturer (bioMe´rieux). using filter-paper discs (diameter, 6.35 mm) contained the Carbon-source oxidation was carried out by the Biolog following antibiotics (concentration per disc): ceftazidime

1466 International Journal of Systematic and Evolutionary Microbiology 63 Iodobacter limnosediminis sp. nov.

Table 1. Differential characteristics of strain E1T and members of related species

Species: 1, E1T (all data were obtained in this study); 2, I. fluviatilis ATCC 33051T (data were obtained in this study); 3, Janthinobacterium agaricidamnosum DSM 9628T (Lincoln et al., 1999); 4, Chromobacterium aquaticum CCUG 55175T (Young et al., 2008); 5, Chitinimonas koreensis KACC 11467T (Kim et al., 2006); 6, Chitiniphilus shinanonensis NBRC 104970T (Sato et al., 2009). Cells of all taxa were rod-shaped, Gram-reaction- negative and motile. +, Positive; W, weakly positive; 2, negative; ND, no data. Data for rows ‘Nitrate reduction’ down to ‘Potassium gluconate’ were obtained from API 20NE tests.

Character 1 2 3 4 5 6

Flagella ++ND ++ + Violet pigmentation 2 + 222ND Spreading growth ++ND + ND ND Colony colour Yellowish Violet Buff Tan Milky Pale yellow Colony surface Rough Rough Smooth Smooth ND Smooth Irregular colony edge ++2 ND + ND Temperature range for growth (uC) 4–28 4–30 2–30 25–40 10–40 15–45 Optimum temperature for growth (uC) 18 28 ND 32 ND 30–37 NaCl tolerance (w/v) 0–1 % 0–1 % 0–2.9 % 0–3.5 % 0–1 % 0–3 % Catalase W ++ 2 ++ Oxidase ++++++ Nitrate reduction ++2 ++ + Indole production 222222 Glucose fermentation 222+ 22 Arginine dihydrolase 222+ 2 ND Urease 222222 b-Glucosidase 222+ 22 Gelatin hydrolysis 2 + 2 ++ 2 b-Galactosidase 222222 Assimilation of : Glucose 2 ++ ++ + Arabinose + 22 22 2 Mannose +++2 ++ Mannitol 22+ 22 2 N-acetylglucosamine + 22 + 2 + Maltose ++22+ 2 Potassium gluconate ++2 + 2 ND Quinone(s) Q-8 Q-8 Q-8 ND Q-8 Q-8 DNA G+C content (mol%) 52.3 49.6 64.2 62.3 65 67.6

(30 mg), chloromycetin (30 mg), tobramycin (10 mg), clafor- The cells of strain E1T were motile rod-shaped cells with anvial (30 mg), trobicin (100 mg), amoxicillin (10 mg), dimensions of 0.7 mm61.4–1.6 mm, observed singly and in cefazolin (30 mg), tetracycline (30 mg), penicillin G pairs; occasionally elongated forms may be observed. One or (10 mg), neomycin (30 mg), carbenicillin (100 mg), cefotax- more polar flagella and lateral flagella were observed under ime (30 mg), polymyxin B (30 mg), ampicillin (10 mg), transmission electron microscopy (Fig. S1 available in IJSEM norfloxacin (10 mg), lincomycin (2 mg), rifampicin (5 mg), Online). In terms of several physiological and biochemical clindamycin (2 mg), oxacillin (1 mg), rocephin (30 mg), features, strain E1T was distinct from I. fluviatilis ATCC aztreonam (30 mg), vancomycin (30 mg), amikacin 33051T, the closest phylogenetic neighbour with the highest (30 mg), imipenem (10 mg), cefepime (30 mg), cefoperazone 16S rRNA gene sequence similarity (strain E1T was unable to (75 mg), roxithromycin (15 mg), azithromycin (15 mg), grow anaerobically, to hydrolyse gelatin, to grow at 30 uC, to bacitracin (0.04 mg), gentamicin (10 mg), erythrocin assimilate glucose and to resist claforanvial, amoxicillin, (15 mg), cefuroxime (30 mg), phosphonomycin (100 mg), cefazolin, carbenicillin, cefotaxime, rocephin, cefoperazone streptomycin (10 mg), cefoxitin (30 mg), piperacillin or piperacillin, but was able to assimilate arabinose and resist (100 mg), nalidixic acid (30 mg), ofloxacin (5 mg) and ceftazidime). For the purple pigment, violacein was synthe- medemycin (30 mg). After 7 days, Signs of growth inhibition sized by Janthinobacterium lividum, Chromobacterium were interpreted as sensitivity to those antibiotics. Resistance violaceum, some members of the genus Pseudoalteromonas to an antimicrobial drug was indicated if no inhibition zone as well as the type strain I. fluviatilis ATCC 33051T.However, was observed. The egg yolk reaction was examined by the it was possible to isolate and culture Janthinobacterium method of Sneath (1984a). lividum and Chromobacterium violaceum strains in a http://ijs.sgmjournals.org 1467 W. Su and others

Table 2. Cellular fatty acid composition (%) of strain E1T and Description of Iodobacter limnosediminis sp. nov. closely related strains Iodobacter limnosediminis (lim.no.se.di9mi.nis. Gr. n. limneˆ Strains: 1, E1T (all data were obtained in this study); 2, I. fluviatilis lake; L.n. sedimen -inis sediment; N.L. gen. n. limnosedi- ATCC 33051T (data were obtained in this study); 3, minis of/from lake sediment). T Janthinobacterium agaricidamnosum CCUG 43140 (Ka¨mpfer et al., Cells are aerobic, Gram-reaction-negative, non-violacein- 2007); 4, Chromobacterium aquaticum CCUG 55175T [data from ] producing, rod-shaped (0.7 mm61.4–1.7 mm), motile with Young et al. (2008) unless otherwise indicated ;5,Chitinimonas one or more polar flagella and lateral flagella, occurring koreensis KACC 11467T (Kim et al., 2006); 6, Chitiniphilus T T singly, in pairs and, occasionally, as long filaments. shinanonensis NBRC 104970 (Sato et al., 2009). Strains E1 and I. Colonies are yellowish, very thin, slightly roughened, with fluviatilis ATCC 33051T were cultivated on R2A agar (BD) plates at irregular edges, spreading to 1–6 mm in diameter. Some 18 uC and cells at late-exponential phase were harvested in this study. colonies may be gelatinous. Colonies are butyrous in Values are percentages of the total fatty acids. Summed feature 3 consistency and easily emulsifiable in water. Optimal contains C v7c and/or C v6c; Summed feature 8 contains 16 : 1 16 : 1 growth occurs at 18 uC; the range of growth temperature C v7c. –, Not detected; ND, no data; 18 : 1 is from 4 to 28 uC, but does not grow at 30 uC. Growth occurs at pH 4 and 11, not at pH 3; optimal growth is at Fatty acid 1 2 3 4 5 6 pH 9–10. Growth occurs in the presence of 0–1 % (w/v) T C10 : 0 3-OH 1.6 2.0 7.8 4.6 ND ND NaCl, not in 2 % (w/v) NaCl. Strain E1 is oxidase- C12 : 0 0.4 0.5 3.0 8.8 – 3.0 positive, but is weakly positive for catalase activity and C12 : 0 2-OH – – 2.2 0.2 3.3 ND hydrolysis of casein, negative for hydrolysis of starch. On C12 : 0 3-OH 2.4 3.1 ND 4.4 ND 5.3 API 20NE strips, nitrate reduction; assimilation of C14 : 0 6.7 9.0 0.6 2.6 1.7 1.4 mannose, potassium gluconate and maltose are present; C15 : 0 ––ND 0.6 ND 3.1 tryptophan, arginine dihydrolase, urease, b-glucosidase, anteiso-C15 : 0 0.3 0.2 ND ND ND ND protease, b-galactosidase, assimilation of mannitol, capric C15 : 1v6c 0.2 0.3 ND ND ND 2.4 acid, adipic acid, malate, trisodium citrate and phenylacetic C16 : 0 18.8 15.8 34.8 25.8 33.8 31.2 acid are absent. In assays with the API ZYM system, there C 2-OH – – ND ND 3.0 ND 16 : 0 are positive reactions for alkaline phosphatase, esterase Summed feature 3 56.1 50.9 9.7 33.4 39.5 32.5 (C4), esterase lipase, leucine arylamidase, naphthol-AS-BI- C v5c 0.3 0.2 ND 0.3* – 0.3 16 : 1 phosphohydrolase and a-glucosidase and negative reac- C cyclo – – 34.2 –* ND 6.9 17 : 0 tions for lipase, valine arylamidase, cystine arylamidase, C17 : 1v8c 0.3 0.4 ND ND ND ND trypsin, a-chymotrypsin, acid phosphatase, a-galactosidase, C17 : 0 0.6 0.4 ND ND ND 1.8 b b b a C18 : 1v9c 1.8 1.2 ND ND ND ND -galactosidase, -glucuronidase, -glucosidase, -manno- Summed feature 8 4.6 3.3 4.5 18.8 4.6 9.9 sidase and a-fucosidase. In the API 50CH, acid production

C18 : 0 4.2 2.7 ND 0.5* 1.9 1.2 is observed from D-ribose, D-glucose, D-fructose, D- mannose, N-acetylglucosamine maltose, sucrose, trehalose *Data from Ka¨mpfer et al. (2009). and potassium gluconate but not from glycerol, erythritol, D-arabinose, D-xylose, L-xylose, D-adonitol, methyl-b-D- xylopyranoside, D-galactose, L-sorbose, L-rhamnose, dulci- non-pigmented state (Logan & Moss, 1992; Shivaji et al., tol, D-mannitol, D-sorbitol, methyl-a-D-mannopyranoside, 1991; Sivendra et al., 1975). Non-violet-pigmented species methyl-a-D-glucopyranoside, amygdalin, arbutin, aesculin, Chromobacterium aquaticum CCUG 55175T (Young et al., salicin, cellobiose, lactose, melibiose, inulin, melezitose, 2008) and Janthinobacterium agaricidamnosum DSM 9628T raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D- (Lincoln et al., 1999) were also isolated. In the test for lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, pot- pigmentation of strain E1T, no colour changes were observed assium 2-ketogluconate, potassium 5-ketogluconate. In the tests for Biolog GN2 Microplate substrates: dextrin, N- after adding either H2SO4 or NaOH solution, which illustrated unambiguously that the strain is non-violacein- acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-fruct- a D b D producing. These differential phenotypic characters were ose, - -glucose, maltose, -methyl- -glucoside, sucrose, able to distinguish strain E1T from the type strain of I. trehalose, pyruvic acid methyl ester, D-gluconic acid, L- glutamic acid and D-glucose-6-phosphate are utilized; but fluviatilis phenotypically at the species level. The other T a-cyclodextrin, glycogen, Tween 40, Tween 80, adonitol, D- phenotypic properties of strain E1 are listed in Table 1 and arabitol, cellobiose, i-erythritol, L-fucose, D-galactose, in the species description. gentiobiose, myo-inositol, a-lactose, lactulose, D-mannitol, In summary, the phenotypic and genotypic characteristics melibiose, D-psicose, raffinose, L-rhamnose, D-sorbitol, of strain E1T are sufficient to distinguish it from recognized turanose, xylitol, succinic acid methyl ester, acetic acid, typical Iodobacter species and other phylogenetic neigh- cis-aconitic acid, citric acid, formic acid, D-galactonic acid bours, supporting its classification as a novel species lactone, D-galacturonic acid, D-glucosaminic acid, D- belonging to genus Iodobacter, for which the name glucuronic acid, a-hydroxybutyric acid, b-hydroxybutyric Iodobacter limnosediminis sp. nov. is proposed. acid, c-hydroxybutyric acid, p-hydroxyphenylacetic acid,

1468 International Journal of Systematic and Evolutionary Microbiology 63 Iodobacter limnosediminis sp. nov. itaconic acid, a-ketobutyric acid, a-ketoglutaric acid, a- which radioisotopes are used to determine genetic relatedness among ketovaleric acid, DL-lactic acid, malonic acid, quinic acid, bacterial strains. Int J Syst Bacteriol 39, 224–229. D-saccharic acid, sebacic acid, succinic acid, bromosuccinic Felsenstein, J. (1985). Confidence limits on phylogenies: an approach acid, succinamic acid, glucuronamide, L-alaninamide, D- using the bootstrap. Evolution 39, 783–791. alanine, L-alanine, L-alanyl-glycine, L-asparagine, L-aspartic Fitch, W. M. (1971). Towards defining the course of evolution: acid, glycyl-L-aspartic acid, glycyl-L-glutamic acid, L- minimum change for a specific tree topology. Syst Zool 20, 406–416. histidine, hydroxy-L-proline, L-leucine, L-ornithine, L- Gillis, M. & Logan, N. A. (2005). Genus IV. 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