Algoriphagus hitonicola sp. nov., isolated from an athalassohaline lagoon

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY on 01 February 2008, available at: https://doi.org/10.1099/ijs.0.65433-0 .” 1 Algoriphagus hiticola sp. nov., isolated from an athalassohaline lagoon 1 2 José L. Copa-Patiño1, María Arenas1, Juan Soliveri1, Cristina Sánchez-Porro2 and 3 Antonio Ventosa2 4 5 1Departamento de Microbiología y Parasitología, Facultad de Farmacia, 6 Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain 7 2Departamento de Microbiología y Parasitología, Facultad de Farmacia, 8 Universidad de Sevilla, 41012 Sevilla, Spain 9 10 Running title: Algoriphagus hiticola sp. nov. 11 12 Subject category: New Taxa. Bacteroidetes 13 14 Author for correspondence: José L. Copa-Patiño. Tel: +34 91 885 46 34. 15 e-mail: [email protected] 16 17 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence 18 of strain 7-UAHT is EF488486. 19 2 20 21 A Gram-negative, heterotrophic, aerobic, reddish-orange pigmented, non-sporing-22 forming and non-motile bacterial strain, designated 7-UAHT, was isolated from 23 salty water from the lagoon at “El Hito”, located in a central area of Spain. The 24 strain grew optimally at 37 ºC, pH 7·0 and in the presence of 2·5 % NaCl. We 25 carried out a polyphasic taxonomic study in order to characterize it in detail. 26 Phylogenetic analyses based on the 16S rRNA gene sequence comparison 27 indicated that strain 7-UAHT clustered within the branch constituted by species of 28 Hongiella, that recently have been transferred to the genus Algoriphagus. Polar 29 lipids analysis and DNA G+C contend further support placement of strain 7-UAHT 30 in the genus Algoriphagus. On the basis of the phenotypic, chemotaxonomic, and 31 phylogenetic distinctiveness, a novel species is proposed to accommodate the 32 strain, for which the name Algoriphagus hiticola sp. nov. is proposed. The type 33 strain is 7-UAHT (= CECT 7267T = CCM 7449T). 34 35 36 The genus Algoriphagus was proposed by Bowman et al. (2003) for heterotrophic, 37 Gram-negative, non-motile, strictly aerobic, saccharolytic, pink-pigmented rod-like 38 cells. They are cold-adapted strains isolated from sea ice and from saline lake 39 cyanobacterial mats collected in Antarctica. Phylogenetically this genus is 40 distantly related to the genus Cyclobacterium within the phylum Bacteroidetes and 41 was represented by a single species, Algoriphagus ratkowskyi. Currently this 42 genus comprises ten species (Bowman et al., 2003; Nedashkovskaya et al., 2004; 43 Van Trappen et al., 2004; Yoon et al., 2005a, 2005b; Yoon et al., 2006; 44 3 Nedashkovskaya et al., papers in press). The genus Hongiella was described by 45 Yi & Chun (2004), it is closely related to the genera Algoriphagus and 46 Chimaereciella (Tiago et al., 2006) and comprises three species of pink–orange-47 pigmented, aerobic, non-motile, heterotrophic and mesophilic bacteria from 48 marine sediments and sea water from Korea. The close relation between these 49 genera has recently led to the inclusion of the species of the genera Hongiella and 50 Chimaereciella in the genus Algoriphagus (Nedashkovskaya et al., papers in 51 press). 52 53 The lagoon of “El Hito” is located in Castilla-La Mancha, a central region of Spain 54 (39°51'58''N 02°41'32''W). With a surface of 573 ha it is a temporary, endorreic, 55 saline steppe lagoon and a Ramsar site (no. 1259). As it is only rain-fed, it 56 regularly dries out and may occasionally remain dry for large periods of time, 57 especially in summer. During the characterization of microbial diversity in water 58 samples from “El Hito” lagoon, strain 7-UAHT was isolated at 37 oC by the dilution 59 plating technique on RV medium (Rodriguez-Valera et al., 1981), containing 1·5% 60 salts mixture and 0·1% yeast extract, adjusted to pH 7·2. Subcultivation was done 61 on marine agar 2216 (MA; Difco). 62 63 Cellular morphology was examined by phase-contrast microscopy (model Axiolab; 64 Zeiss) and scanning electron microscopy (SEM) (model DSM950; Zeiss) after 65 growth on MA medium at 37 oC. Gram reaction was performed as described by 66 Gerhardt et al. (1994). Gliding motility was observed by direct microscopic 67 examination of the edge of colonies and motility was observed by the hanging-68 drop technique for cells in exponential phase in marine broth 2216 (MB, Difco). 69 4 Growth under anaerobic conditions was determined after incubation in an 70 anaerobic chamber with anaerobically prepared MA medium. Growth at various 71 NaCl concentrations (0–12 % at 0·5 % intervals) was investigated in CSY-3 72 medium (Sawabe et al., 1998). Growth at various temperatures was tested over 73 the range 5–50 oC at intervals of 5 oC using MA medium. Growth at different pH 74 values (4.0–11.0) was examined on MA medium with the appropriate biological 75 buffers. In vivo pigment absorption-spectrum analysis was performed 76 spectrophotometrically (Hitachi model U-2001), as described by Yoon et al. 77 (2004). The presence of flexirubin-like pigments was tested according to 78 Reichenbach (1989). Catalase activity was determined by adding a 1% (w/v) H2O2 79 solution to colonies on MA medium. Oxidase activity was detected using a 1% 80 solution of tetramethyl-p-phenylendiamine (Difco) (Kovacs, 1956). Hydrolysis of 81 starch, aesculin, gelatin and Tween 80 were determined as described by Cowan 82 & Steel (1974). Xylan and pullulan hydrolysis were carried out as described by 83 Ensor et al. (1999). Urease activity was studied in Christensen’s medium 84 (Christensen, 1946). Citrate utilization was determined on Simmons citrate 85 medium. Indole production was determined with Kovacs’ reagent on 1% Tryptone 86 broth. Nitrate and nitrite reduction was tested on nitrate broth containing 0.2% 87 KNO3 (Skerman, 1967). H2S production was determined on Kligler Iron Agar 88 (Difco). All these tests were carried out on media supplemented with 2,5 % salts. 89 For the determination of acid production from different carbohydrates a medium 90 containing 0.5% peptone, 2.5% NaCl and 0.001% phenol red was used 91 supplemented with 0.7% of each carbohydrate (Cowan & Steel, 1974). Additional 92 biochemical assays were performed using the API 50CH and API ZYM kits 93 (bioMérieux). Strips were inoculated with a heavy bacterial suspension in a 2·5 % 94 5 salts solution. Antibiotic susceptibility was determined according to the 95 conventional Kirby-Bauer method (Bauer et al., 1966). 96 Morphological, physiological and biochemical characteristics are shown in Table 1 97 or are given in the species description (see bellow). Cells were rod-shaped with 98 rounded ends, no spores or flagella were observed (Figure 1). Absorption 99 maximum of crude extracts of strain 7-UAHT was approximately 481 nm. 100 Bathychromatic shift, a characteristic of flexirubins, was not observed. 101 102 For the determination of the DNA G+C content of strain 7-UAHT, genomic DNA 103 extraction and purification were performed according to Marmur (1961). The G+C 104 content was determined using the thermal denaturation method (Marmur & Doty, 105 1962; Owen & Hill, 1979). The phylogenetic position of strain 7-UAHT was 106 determined by 16S rRNA gene sequence analysis. The 16S rRNA gene was 107 amplified using oligonucleotide primers complementary to highly conserved 108 regions of bacterial 16S rRNA genes. The forward primer and the reverse primer 109 were 27F and 1492R (Reysenbach et al., 2000). PCR products were purified 110 using an UltraClean PCR Clean-up kit (Mo Bio), according to the manufacturer’s 111 instructions. Purified PCR products were sequenced using the ABI Prism Big Dye 112 Terminator cycle sequencing ready reaction kit and an Applied Biosystems 3130 113 Genetic Analyzer, using the protocols of the manufacturer (Applied Biosystems). 114 Sequence assembly was performed using the CAP3 program (Huang & Madan, 115 1999). Phylogenetic analysis was performed using the MEGA software package 116 version 3·1 (Kumar et al., 2004) after multiple alignment of data sequences by 117 CLUSTALX (Thompson et al., 1997). Distances (distance options according to the 118 Kimura two-parameter model) and clustering with the neighbour-joining methods 119 6 were determined. The bootstrap values based on 1000 replications were 120 calculated. 121 The 16S rRNA sequence of strain 7-UAHT was a continuous stretch of 1394 bp 122 and was used for an initial BLAST search against GenBank, EMBL, DDBJ and 123 PDB databases. The 16S rRNA sequence of strain 7-UAHT and sequences of 124 closely related strains were aligned and the phylogenetic tree showed that strain 125 7-UAHT cluster together with Hongiella, Algoriphagus and Chimaereicella species 126 (Figure 2). The sequence identity matrix indicate that the closest relatives of strain 127 7-UAHT were H. ornithinivorans (93·8 % sequence similarity), H. mannitolivorans 128 (93·2 %), A. vanfongensis (93·0 %), A. halophilus (91·9 %), A. ratkowskyi (90·9 %) 129 and H. marincola (90·0 %). Sequence similarities to all other species included in 130 the phylogenetic analysis were bellow 90 %. The G+C content of strain 7-UAHT 131 was 43·4 mol%, similar to the values of 43, 42 and 38 mol% described for H. 132 marincola, H. mannitolivorans and H. ornithinivorans, respectively (Yi & Chun, 133 2004; Yoon et al., 2004). 134 135 Fatty acid were analysed by using GC at the Belgian Coordinated Collections of 136 Microorganisms, Laboratory of Microbiology of Gent (BCCM/LMG), Gent, Belgium 137 (Kämpfer & Kroppenstedt, 1996; Miller, 1982). Cells were cultured on MA medium 138 for 24 h and 37ºC. Strain 7-UAHT had a fatty acid profile similar to those described 139 for the type strains of the closely related Algoriphagus species (Table 2). The 140 predominant fatty acids are iso-C15:0, summed feature 3 and iso-C17:1ω9c. 141 142 Based on these combined phenotypic and phylogenetic analyses, strain 7-UAHT 143 is not closely affiliated with any previously described species. This organism is 144 7 closely related to the species of the genus Hongiella; however, since the species 145 of this genus have been transferred to the genus Algoriphagus, we propose to 146 include it as a new species of Algoriphagus and name it as Algoriphagus hiticola 147 sp. nov. 148 149 Description of Algoriphagus hiticola sp. nov. 150 Algoriphagus hiticola (hi.ti’co.la L. gen. n. hito of the “El Hito” lagoon; L. suff. -cola 151 inhabitant, dweller; N.L. n. hiticola inhabitant of the “El Hito”). 152 153 Cells are rods, 0·2-0·4 x 0·8-1·2 μm on MA medium. Non-motile. Gram-negative. 154 Does not form endospores. Strictly aerobic. Colonies are circular, smooth, 155 convex, glistening and 0·5-1·5 mm in diameter after 3 days of incubation at 37 ºC 156 on MA medium. Produces a non-diffusible reddish-orange pigment. Flexirubin 157 pigments are not produced. Growth occurs at 20-40 ºC (optimum 35-37 ºC), 158 between 1·5 and 5 % NaCl (optimum 2·5 %). Growth is observed between pH 6·0 159 and 10·0. Optimal pH for growth is pH 7·0. Acid is produced from D-glucose, D-160 fructose, D-mannose, N-acetylglucosamine, D-cellobiose, lactose, sucrose, 161 maltose, D-trehalose, D-raffinose, D-melibiose, amygdalin, arbutin, salicin, 162 melibose, gentiobiose or turanose, but not from D-sorbitol, myo-inositol, D-ribose, 163 D-adonitol, D- and L-xylose, D- and L-arabinose, glycerol, D- and L-fucose or L-164 rhamnose. Starch, DNA, pullulan, and aesculin are hydrolyzed. Does not 165 hydrolyze xylan, Tween 80, mannan or carboxymethylcellulose. Produces β-166 galactosidase, alkaline phosphatase, arylamidase, trypsin, α-chemotrypsin and N-167 acetyl-β-glucosaminidase. Nitrate is reduced to nitrite. H2S and indole production 168 are negative. Simmons citrate is negative. Sensitive to (μg per disc): erythromycin 169 8 (15), chloranphenicol (30) and vancomycin (30). Intermediate resistance to (μg 170 per disc): trimetoprim/sulfamethoxazole (1.25/23.75) and polimyxim B (300). 171 Resistant to (μg per disc): nalidixic acid (30), streptomycin (10), bacitracin (10 172 units), penicillin (10 units), kanamycin (30) and neomycin (10). The predominant 173 fatty acids are iso-C15:0, summed feature 3 and iso-C17:1ω9c. The type strain is 174 strain 7-UAHT (= CECT 7267T = CCM 7449T); the DNA G + C content of the type 175 strain is 43·4 mol%. Isolated from a salty lagoon water sample at “El Hito”, Spain. 176 177 Acknowledgements 178 179 We are grateful to ADINMAC (“Asociación para el Desarrollo integral de los 180 Municipios de la Mancha Alta Conquense”) for helping us to take samples from El 181 Hito lagoon. This research was supported by a grant of University of Alcala-182 Ibercaja (XZ032). We thank Dr Pilar Rubio (Molecular Biology Unit University of 183 Alcala) for helping with the sequencing work. 184 185 References 186 187 Bauer, A. W., Kirby, W. M. M., Sherris, J. C. & Turck, M. (1966). Antibiotic 188 susceptibility testing by a standarized single disk method. Am J Clin Pathol 45, 189 493-496. 190 191 Bowman, J. P., Nichols, C. M. & Gibson, J. A. E. (2003). Algoriphagus 192 ratkowskyi gen. nov., sp. nov., Brumimicrobium glaciale gen. nov., sp. nov., 193 Cryomorpha ignava gen. nov., sp. nov. and Crocinitomix catalasitica gen. nov., 194 15 Algoriphagus winogradskyi KMM 3956 AJ575263) T ( Algoriphagus locisalis MSS-170 (AY835922) T Algoriphagus yeomjeoni MSS-160 (AY699794) T Algoriphagus chordae KMM 3957 (AJ575265) T Algoriphagus antarcticus LMG 21980 (AJ577141) T Algoriphagus aquamarinus KMM 3958 (AJ575264 ) T Algoriphagus ratkowskyi LMG 21435 (AJ608641) T Algoriphagus halophilus IMSNU 14013 (AY264839) T Algoriphagus terrigena DS-44 (DQ178979) T Chimaereicella boritolerans T 22 (AB197852) T Chimaereicella alkaliphila AC74 (AJ717393) T Algoriphagus hiticola 7-UAH (EF488486) T Hongiella marincola SW-2 (AY533663) T Hongiella mannitolivorans IMSNU 14012 (AY264838) T Hongiella ornithinivorans IMSNU 104014 (AY264840) T Algoriphagus vanfongensis KMM 6241 (EF392675) T Cyclobacterium marinum DSM 745 (AY533665) T Belliella baltica BA134 (AJ564643) T Bacteroides fragilis ATCC 25285 (X83935) T 69 98 71 51 69 53 98 96 99 95 96 65 100 77 83 66 002 · Fig. 2. Neighbour-joining tree based on 16S rRNA gene sequences, showing the 310 phylogenetic positions of strain 7-UAHT and representatives of the related taxa 311 (GenBank/EMBL/DDBJ accession numbers are shown in parentheses). Numbers 312 at branch points indicate the level of bootstrap support, based on 1000 313 resamplings. Bacteroides fragilis was used as outgroup. Bar, 0·02 expected 314 nucleotide substitutions per position. 315 16 Table 1. Phenotypic characteristics of Algoriphagus hiticola strain 7-UAHT and the 316 closely related species: Hongiella marincola, H. mannitolivorans, H. 317 ornithinivorans and A. ratkowskyi. 318 Species: 1, Algoriphagus hiticola; 2, H. marincola (Yoon et al., 2004); 3, H. 319 mannitolivorans (Yi & Chun, 2004); 4, H. ornithinivorans (Yi & Chun, 2004); 5, A. 320 ratkowskyi (Bowman et al., 2003). The strain was positive for catalase, alkaline 321 phosphatase, trypsin, α-chemotrypsin and N-acetyl-β-glucosaminidase. The strain 322 was negative for: gliding motility; flexirubin pigments; negative for oxidase; H2S 323 and indole production; hydrolysis of cellulose (carboximethylcellulose) and urea; 324 utilization of citrate. +, Positive; -, negative; W, weakly positive; ND, not 325 determined; v, variable between strains. 326 Characteristic 1 2 3 4 5 Pigment absorption max. (nm) 481 486 480 480 ND Growth at 0 % NaCl - - + + - Growth at 45 ºC - + - + ND Nitrate reduction to nitrite + - + - - Hydrolisis: DNA + + + + - Pullulan + + + + ND Xylan - - - - ND Starch + + + + v Tween 80 - + - + - Utilization of: D-Fructose + + - + + D-Mannitol - - + - + Enzyme activity (with API ZYM system): Esterase (C4) w + - - ND Esterase lipase (C8) w + - - ND Cystine arylamidase + + - - ND α-Galactosidase - - + + + β-Galactosidase - + + w + α-Glucosidase w + - + + β-Glucosidase w w - + + DNA G+C content (mol%) 43·4 43 42 38 36 17 Table 2. Cellular fatty acid composition (%) of Algoriphagus hiticola, Hongiella 327 marincola, H. mannitolivorans and H. ornithinivorans. 328 329 Species: 1, A. hiticola 7-UAHT; 2, H. marincola DSM 16067T; 3, H. 330 mannitolivorans DSM 15301T; 4, H. ornithinivorans DSM 15282T. 331 332 333 *Summed features are groups of fatty acids which were not resolved 334 chromatographically; summed feature 3 consists of C16:1 ω7c and/or iso-C15:0 2-335 OH; summed feature 4 consists of iso-C17:1 I and/or anteiso-C17:1 B. 336 Fatty acid composition 1 2 3 4 anteiso-C11:0 1·8 - 1·3 - Unknown 13.565 1·2 0·8 1·0 1·1 iso-C15:1 G 2·6 ND ND ND iso-C15:0 35·2 31·9 41·7 37·1 anteiso-C15:0 4·4 4·8 4·9 4·5 C15:1 ω6c 2·0 2·5 2·0 1·5 Unknown 14.959 0·4 2·0 1·3 0·7 iso-C16:1 H 4·1 3·8 1·7 3·2 iso-C16:0 3·0 8·8 5·6 7·5 Summend feature 3* 12·4 12·7 11·7 12·3 C16:1 ω5c 1·8 2·4 1·2 2·0 iso-C15:0 3-OH 2·8 2·2 2·9 1·6 iso-C17:1 ω9c 10·0 4·1 3·1 10·1 Summend feature 4* 4·0 2·7 1·8 4 C17:1 ω6c 1·4 4·4 1·9 2·5 iso-C16:0 3-OH 2·8 4·7 4·5 2·0 iso-C17:0 3-OH 5·2 5·8 5·9 5·0