Systematic and Applied Microbiology 38 (2015) 161–168
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Aeromonas aquatica sp. nov., Aeromonas finlandiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish waters associated with cyanobacterial blooms夽 R. Beaz-Hidalgo a , F. Latif-Eugenín a , M.J. Hossain b , K. Berg c , R.M. Niemi d , J. Rapala e , C. Lyra c , M.R. Liles b , M.J. Figueras a,∗ a Unitat de Microbiologia, Departament de Ciènces Médiques Bàsiques, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain b Department of Biological Sciences, Auburn University, Auburn, AL, United States c Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland d Finnish Environment Institute, Ecosystem Change, Helsinki, Finland e Ministry of Social Affairs and Health, Department of Promotion of Welfare and Health, Helsinki, Finland
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Article history: Received 31 March 2014 Received in revised form 18 February 2015 Accepted 27 February 2015 Keywords: Aeromonas New taxa Polyphasic ANI Water
a b s t r a c t Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093 bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751 bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all 94%) independent clusters from the rest of the species of the genus. The new candidate species Aeromonas lusitana which has not yet been formally described was included in the analysis in order to demonstrate that the three new species do not belong to this species. The nearest phylogenetic neighbors were different than those obtained with the 16S rRNA phylogeny which is not strange considering the low bootstrap values obtained with the latter gene (Figs. 1 and 2). The most related species in the MLSA for A. finlandiensis sp. nov. were A. allosaccharophila, A. australiensis and A. veronii; for A. aquatica sp. nov., A. encheleia and A. eucrenophila and for A. lacus sp. nov., A. jandaei (Figs. 2 and 3). As can be seen in the present study and in many others [3,4,7,9,15,27,30] the resolution of the MLSA is better than the one provided by the 16S rRNA gene and the overall phylogenetic relatedness is more robust (bootstrap values of 100% for all the species clusters). With the 7 concatenated genes, the inter-species ranges of substitution of the new species with their closest relatives were: 2.67–3.41% for A. finlandiensis with A. allosaccharophila (3.04% between the type strains), 2.97–3.61% for A. finlandiensis with A. veronii (3.30% between the type strains), 4.10–4.61% for A. aquatica and A. encheleia (4.61% between type strains), 4.15–4.47% for A. aquatica and A. eucrenophila (4.17% between the type strains) and 2.67–3.31% for A. lacus and A. jandaei (2.87% between the type strains). Other species within the genus share similar or lower inter-species ranges like A. allosaccharophila and A. veronii (2.87–3.53%, 3.21% between type strains) or A. bestiarum and A. piscicola (2.36–3.34%, 2.63% between type strains) as has previously been demonstrated [29]. To confirm these results a more complete analysis was performed using the concatenated sequence of 15 genes of the type strains of the new species with those of their closest relatives using A. piscicola and A. bestiarum as reference of the lowest inter-species separation within the genus [29]. The additional genes cpn60, dnaK, gltA, mdh, radA, rpoB, tsf and zipA were retrieved from the genome sequences performing a BLAST search using the Basic Local Alignment Search Tool (from the NCBI web interface). The new tree confirmed that the 3 proposed species formed new independent phylogenetic lines within the genus (Fig. 3) because the length of the branches that separate them from the closest relatives were equivalent to those shown by other accepted species of the genus (i.e. A. piscicola and A. bestiarum). The inter-species nucleotide substitution rates for these 15 genes between the type strains of the new species and their close relatives were similar to those obtained with 7 genes, i.e. A. finlandiensis 4287DT -A. allosaccharophila CECT 4199T (3.32%), A. finlandiensis 4287DT -A. veronii CECT 4257T (3.60%), A. aquatica AE235T -A. encheleia CECT 4342T (4.9%) and A. lacus AE122T -A. jandaei CECT 4228T (2.85%). However, it was slightly lower (by 0.44%) for A. aquatica AE235T -A. eucrenophila CECT 4224T (3.73%). As in the 7 gene analysis all inter-species values between the type strains and those of their close relatives were lower than those obtained for A. bestiarum CECT 4227T -A. piscicola CECT 7443T (2.56%), and all except one (A. lacus-A. jandaei) were also lower than A. allosaccharophila CECT 4199T – A. veronii CECT 4257T (2.97%). These highly similar inter-species nucleotide substitution values could be an indication that the rate of evolution of the different genes between those species is constant. The genome sequences of strains A. aquatica AE235T and A. lacus AE122T have recently been published [20] and using the same methodology we also obtained the genome of A.
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Fig. 1. Unrooted neighbour joining phylogenetic tree derived from the 16S rRNA sequences (1405 bp) showing the relationships of the type strains of the 3 new species with other type strains of all Aeromonas species. Numbers at the node indicate bootstrap values (>50%). Bar, 0.002 estimated substitutions per site.
finlandiensis 4287DT (accession number JRGK00000000). Genomes of the type strains of the most closely related species for each new species on the basis of the 16S rRNA gene phylogeny and the MLSA, were retrieved from the GenBank and ANI calculations were performed using three platforms: the ANI calculator web-interface (http://enve-omics.ce.gatech.edu/ani/index),
the EzGenome (http://www.ezbiocloud.net/ezgenome/ani) and the JSpecies software (http://www.imedea.uib.es/jspecies). The ANI results are shown in Table 3 and all were 50%). Bar, 0.01 estimated substitutions per site.
faecal contamination, and is used as a water source to produce drinking water. Description of Aeromonas finlandiensis sp. nov. Aeromonas finlandiensis (fin.lan.di.en’sis. N.L. fem. Adj. finlandiensis of belonging to Finland) Cells are motile Gram-negative rods, non-encapsulated and non spore forming, 1.7–1.9 m long and 0.5–0.7 m wide with a polar flagella of 9.6–16.1 nm wide. The 7 strains show reactions that are typical of the genus, being positive for cytochrome oxidase,
reducing nitrates to nitrites, positive for glucose oxidation and fermentation, resistant to the vibriostatic agent O/129 (150 G) and growing at 0% NaCl but not at 6% NaCl. No brown diffusible pigment is observed on TSA and all strains are able to grow on TCBS and on McConkey agar without fermenting lactose. Haemolysis is observed on sheep blood agar in 6 of the 7 strains. The strains show -galactosidase activity and are positive for ADH and LDC (except strain HE110 that is LDC negative), the production of gas from glucose, indole from trytptohan and MR. However, they are negative for ODC, VP, urocanic acid, do not oxidize gluconate and do not produce sulphydric acid. All strains are able to grow in KCN medium, use citrate (except strain AE119 that is unable to use citrate) and hydrolyze gelatin, starch, Tween 80, casein, DNA (except strain HE110 that was negative for DNA) and egg yolk (except strain HE40) but not aesculine, elastase, arbutin or SDS (except strains HE40 and HE110 that are positive for SDS). The 7 strains grow at 15 and 30 ◦ C but not at 4 ◦ C or 45 ◦ C in TSA. All of them also grow in nutrient agar containing 3% NaCl but not at 4.5% NaCl. Growth is observed at pH 5–9 but not at pH 3 and 4 in TSB. Optimal growth conditions are at 30 ◦ C in TSA and at pH 8.5–9 in TSB. Acid is produced in nutrient broth from glycerol, cellobiose, d-mannose, maltose, trehalose, dextrin, dgalactose and glucose. Acid production is observed for the following carbohydrates with the API 50CH kit: d-ribose, d-fructose, starch, glycogen and N-acetylglucosamine. Variable response on the production of acid is observed for d-mannitol (only strain HE110 is negative), methyl--d-glucopyranoside (only strains AE 93 and HE40 are negative), potassium gluconate (strains 4287DT , 4301 C and HE40 are negative), l-fucose (strains 4287DT , 4301C, 4318B, AE119 and HE403 are negative), inositol (only strains 4287DT and AE93 are positive and can also use it as a sole carbon source), d-melibiose and gentiobiose (only strain HE 110 is positive for both tests). Acid is not produced in nutrient broth from raffinose, dulcitol, d-saccharose, d-sorbitol, lactose, l-proline, salicine, dllactate, rhamnose, l-arabinose and glycine. Acid production is not observed for the following carbohydrates with the API 50CH kit: erythritol, d-arabinose, d-xylose, l-xylose, d-adonitol, methyl-d-xylopiranoside, l-sorbose, methyl-␣d-mannopyranoside, amygdalin, d-melibiose, inulin, d-melezitose, xylitol, d-turanose, d-lyxose, d-tagatose, d-fucose, d-arabitol, l-arabitol and 2- and 5-ketogluconate. Habitat description The 7 strains belonging to A. finlandiensis sp. nov. were isolated from 6 different Finnish lakes and from a river in 7 different
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municipalities (Table S1). The type strain of the species is 4287DT (=CECT 8028T = LMG 26709T ) and was isolated from the Lake Pyhälampi (municipality Hauho), which showed elevated nitrogen content.
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Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.syapm.2015.02.005.
Description of Aeromonas lacus sp. nov. References Aeromonas lacus (la’cus. L. gen. n. lacus, of a lake) Cells are motile Gram-negative rods, non-encapsulated and non spore forming, 1.3–2.3 m long and 0.6–0.8 m wide with a polar flagella of 17.9–20.3 nm wide. Both strains (AE122T , AE204) are cytochrome oxidase positive, reduce nitrates to nitrites, are positive for glucose oxidation and fermentation, are resistant to the vibriostatic agent O/129 (150 G) and grow at 0% NaCl but not at 6% NaCl. No brown diffusible pigment is observed on TSA and both strains are able to grow on TCBS and on McConkey agar without fermenting lactose. Haemolysis on sheep blood agar, LDC and production of indole from tryptohan are variable and the type strain is positive for haemolysis and LDC but negative for indole. Both strains are positive for ADH and VP. However, they are negative for ODC, MR, urocanic acid, do not produce sulphydric acid and do not grow in KCN medium. The two strains produce gas from glucose, are able to use citrate, oxidize gluconate and both have -galactosidase activity. The strains hydrolyse gelatin, starch, Tween 80, DNA, egg yolk and casein but not esculin, elastase, arbutin or SDS. The 2 strains show growth at 15 and 30 ◦ C but not at 4 ◦ C or 45 ◦ C in TSA. Both strains also grow in nutrient agar containing 3% NaCl but not at 4.5% NaCl. Growth is observed at pH 5–9 but not at pH 3, 4 in TSB. Optimal growth conditions are at 30 ◦ C and pH 7–9. Acid is produced in nutrient broth from glycerol, cellobiose, d-mannose, d-mannitol, maltose, trehalose, dextrin, d-galactose and glucose. Acid production is observed for the following carbohydrates with the API 50CH kit: d-ribose, d-fructose, N-acetylglucosamine, starch and glycogen. Variable response for the production of acid is observed in methyl--d-glucopyranoside and d-melobiose, to which the type strain AE122T is positive and negative, respectively, as opposed to strain AE 204 after 48 h incubation. Acid is not produced in nutrient broth from raffinose, dulcitol, d-saccharose, d-sorbitol, lactose, l-proline, salicine, dl-lactate, inositol (they also do not use it as a sole carbon source), l-arabinose, rhamnose and glycine. Acid production is not observed for the following carbohydrates with the API 50CH kit: erythritol, d-arabinose, d-xylose, l-xylose, d-adonitol, methyl--d-xylopiranoside, lsorbose, methyl-␣d-mannopyranoside, amygdalin, inulin, dmelezitose, xylitol, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, gentiobiose and 2- and 5-ketogluconate. Habitat description The strains belonging to A. lacus sp nov. were recovered from two lakes (Table S1) and the type strain of the species AE122T (=CECT 8024T = LMG 26710T ) was isolated from the small humic lake Huutjärvi (municipality Pyhtää), which has elevated nitrogen levels. Acknowledgements ˜ for her technical assistance. The authors thank Catalina Núnez This work was supported in part by the projects AGL2011-30461C02-02 of the Ministerio de Ciencia e Innovación (Spain) and JPIW2013-095-CO3 from the Ministerio de Economía y Competitividad (WATER JPI).
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Aeromonas aquatica sp. nov., Aeromonas finlandiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish waters associated with cyanobacterial blooms夽 R. Beaz-Hidalgo a , F. Latif-Eugenín a , M.J. Hossain b , K. Berg c , R.M. Niemi d , J. Rapala e , C. Lyra c , M.R. Liles b , M.J. Figueras a,∗ a Unitat de Microbiologia, Departament de Ciènces Médiques Bàsiques, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain b Department of Biological Sciences, Auburn University, Auburn, AL, United States c Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland d Finnish Environment Institute, Ecosystem Change, Helsinki, Finland e Ministry of Social Affairs and Health, Department of Promotion of Welfare and Health, Helsinki, Finland
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Article history: Received 31 March 2014 Received in revised form 18 February 2015 Accepted 27 February 2015 Keywords: Aeromonas New taxa Polyphasic ANI Water
a b s t r a c t Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093 bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751 bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all 94%) independent clusters from the rest of the species of the genus. The new candidate species Aeromonas lusitana which has not yet been formally described was included in the analysis in order to demonstrate that the three new species do not belong to this species. The nearest phylogenetic neighbors were different than those obtained with the 16S rRNA phylogeny which is not strange considering the low bootstrap values obtained with the latter gene (Figs. 1 and 2). The most related species in the MLSA for A. finlandiensis sp. nov. were A. allosaccharophila, A. australiensis and A. veronii; for A. aquatica sp. nov., A. encheleia and A. eucrenophila and for A. lacus sp. nov., A. jandaei (Figs. 2 and 3). As can be seen in the present study and in many others [3,4,7,9,15,27,30] the resolution of the MLSA is better than the one provided by the 16S rRNA gene and the overall phylogenetic relatedness is more robust (bootstrap values of 100% for all the species clusters). With the 7 concatenated genes, the inter-species ranges of substitution of the new species with their closest relatives were: 2.67–3.41% for A. finlandiensis with A. allosaccharophila (3.04% between the type strains), 2.97–3.61% for A. finlandiensis with A. veronii (3.30% between the type strains), 4.10–4.61% for A. aquatica and A. encheleia (4.61% between type strains), 4.15–4.47% for A. aquatica and A. eucrenophila (4.17% between the type strains) and 2.67–3.31% for A. lacus and A. jandaei (2.87% between the type strains). Other species within the genus share similar or lower inter-species ranges like A. allosaccharophila and A. veronii (2.87–3.53%, 3.21% between type strains) or A. bestiarum and A. piscicola (2.36–3.34%, 2.63% between type strains) as has previously been demonstrated [29]. To confirm these results a more complete analysis was performed using the concatenated sequence of 15 genes of the type strains of the new species with those of their closest relatives using A. piscicola and A. bestiarum as reference of the lowest inter-species separation within the genus [29]. The additional genes cpn60, dnaK, gltA, mdh, radA, rpoB, tsf and zipA were retrieved from the genome sequences performing a BLAST search using the Basic Local Alignment Search Tool (from the NCBI web interface). The new tree confirmed that the 3 proposed species formed new independent phylogenetic lines within the genus (Fig. 3) because the length of the branches that separate them from the closest relatives were equivalent to those shown by other accepted species of the genus (i.e. A. piscicola and A. bestiarum). The inter-species nucleotide substitution rates for these 15 genes between the type strains of the new species and their close relatives were similar to those obtained with 7 genes, i.e. A. finlandiensis 4287DT -A. allosaccharophila CECT 4199T (3.32%), A. finlandiensis 4287DT -A. veronii CECT 4257T (3.60%), A. aquatica AE235T -A. encheleia CECT 4342T (4.9%) and A. lacus AE122T -A. jandaei CECT 4228T (2.85%). However, it was slightly lower (by 0.44%) for A. aquatica AE235T -A. eucrenophila CECT 4224T (3.73%). As in the 7 gene analysis all inter-species values between the type strains and those of their close relatives were lower than those obtained for A. bestiarum CECT 4227T -A. piscicola CECT 7443T (2.56%), and all except one (A. lacus-A. jandaei) were also lower than A. allosaccharophila CECT 4199T – A. veronii CECT 4257T (2.97%). These highly similar inter-species nucleotide substitution values could be an indication that the rate of evolution of the different genes between those species is constant. The genome sequences of strains A. aquatica AE235T and A. lacus AE122T have recently been published [20] and using the same methodology we also obtained the genome of A.
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Fig. 1. Unrooted neighbour joining phylogenetic tree derived from the 16S rRNA sequences (1405 bp) showing the relationships of the type strains of the 3 new species with other type strains of all Aeromonas species. Numbers at the node indicate bootstrap values (>50%). Bar, 0.002 estimated substitutions per site.
finlandiensis 4287DT (accession number JRGK00000000). Genomes of the type strains of the most closely related species for each new species on the basis of the 16S rRNA gene phylogeny and the MLSA, were retrieved from the GenBank and ANI calculations were performed using three platforms: the ANI calculator web-interface (http://enve-omics.ce.gatech.edu/ani/index),
the EzGenome (http://www.ezbiocloud.net/ezgenome/ani) and the JSpecies software (http://www.imedea.uib.es/jspecies). The ANI results are shown in Table 3 and all were 50%). Bar, 0.01 estimated substitutions per site.
faecal contamination, and is used as a water source to produce drinking water. Description of Aeromonas finlandiensis sp. nov. Aeromonas finlandiensis (fin.lan.di.en’sis. N.L. fem. Adj. finlandiensis of belonging to Finland) Cells are motile Gram-negative rods, non-encapsulated and non spore forming, 1.7–1.9 m long and 0.5–0.7 m wide with a polar flagella of 9.6–16.1 nm wide. The 7 strains show reactions that are typical of the genus, being positive for cytochrome oxidase,
reducing nitrates to nitrites, positive for glucose oxidation and fermentation, resistant to the vibriostatic agent O/129 (150 G) and growing at 0% NaCl but not at 6% NaCl. No brown diffusible pigment is observed on TSA and all strains are able to grow on TCBS and on McConkey agar without fermenting lactose. Haemolysis is observed on sheep blood agar in 6 of the 7 strains. The strains show -galactosidase activity and are positive for ADH and LDC (except strain HE110 that is LDC negative), the production of gas from glucose, indole from trytptohan and MR. However, they are negative for ODC, VP, urocanic acid, do not oxidize gluconate and do not produce sulphydric acid. All strains are able to grow in KCN medium, use citrate (except strain AE119 that is unable to use citrate) and hydrolyze gelatin, starch, Tween 80, casein, DNA (except strain HE110 that was negative for DNA) and egg yolk (except strain HE40) but not aesculine, elastase, arbutin or SDS (except strains HE40 and HE110 that are positive for SDS). The 7 strains grow at 15 and 30 ◦ C but not at 4 ◦ C or 45 ◦ C in TSA. All of them also grow in nutrient agar containing 3% NaCl but not at 4.5% NaCl. Growth is observed at pH 5–9 but not at pH 3 and 4 in TSB. Optimal growth conditions are at 30 ◦ C in TSA and at pH 8.5–9 in TSB. Acid is produced in nutrient broth from glycerol, cellobiose, d-mannose, maltose, trehalose, dextrin, dgalactose and glucose. Acid production is observed for the following carbohydrates with the API 50CH kit: d-ribose, d-fructose, starch, glycogen and N-acetylglucosamine. Variable response on the production of acid is observed for d-mannitol (only strain HE110 is negative), methyl--d-glucopyranoside (only strains AE 93 and HE40 are negative), potassium gluconate (strains 4287DT , 4301 C and HE40 are negative), l-fucose (strains 4287DT , 4301C, 4318B, AE119 and HE403 are negative), inositol (only strains 4287DT and AE93 are positive and can also use it as a sole carbon source), d-melibiose and gentiobiose (only strain HE 110 is positive for both tests). Acid is not produced in nutrient broth from raffinose, dulcitol, d-saccharose, d-sorbitol, lactose, l-proline, salicine, dllactate, rhamnose, l-arabinose and glycine. Acid production is not observed for the following carbohydrates with the API 50CH kit: erythritol, d-arabinose, d-xylose, l-xylose, d-adonitol, methyl-d-xylopiranoside, l-sorbose, methyl-␣d-mannopyranoside, amygdalin, d-melibiose, inulin, d-melezitose, xylitol, d-turanose, d-lyxose, d-tagatose, d-fucose, d-arabitol, l-arabitol and 2- and 5-ketogluconate. Habitat description The 7 strains belonging to A. finlandiensis sp. nov. were isolated from 6 different Finnish lakes and from a river in 7 different
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municipalities (Table S1). The type strain of the species is 4287DT (=CECT 8028T = LMG 26709T ) and was isolated from the Lake Pyhälampi (municipality Hauho), which showed elevated nitrogen content.
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Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.syapm.2015.02.005.
Description of Aeromonas lacus sp. nov. References Aeromonas lacus (la’cus. L. gen. n. lacus, of a lake) Cells are motile Gram-negative rods, non-encapsulated and non spore forming, 1.3–2.3 m long and 0.6–0.8 m wide with a polar flagella of 17.9–20.3 nm wide. Both strains (AE122T , AE204) are cytochrome oxidase positive, reduce nitrates to nitrites, are positive for glucose oxidation and fermentation, are resistant to the vibriostatic agent O/129 (150 G) and grow at 0% NaCl but not at 6% NaCl. No brown diffusible pigment is observed on TSA and both strains are able to grow on TCBS and on McConkey agar without fermenting lactose. Haemolysis on sheep blood agar, LDC and production of indole from tryptohan are variable and the type strain is positive for haemolysis and LDC but negative for indole. Both strains are positive for ADH and VP. However, they are negative for ODC, MR, urocanic acid, do not produce sulphydric acid and do not grow in KCN medium. The two strains produce gas from glucose, are able to use citrate, oxidize gluconate and both have -galactosidase activity. The strains hydrolyse gelatin, starch, Tween 80, DNA, egg yolk and casein but not esculin, elastase, arbutin or SDS. The 2 strains show growth at 15 and 30 ◦ C but not at 4 ◦ C or 45 ◦ C in TSA. Both strains also grow in nutrient agar containing 3% NaCl but not at 4.5% NaCl. Growth is observed at pH 5–9 but not at pH 3, 4 in TSB. Optimal growth conditions are at 30 ◦ C and pH 7–9. Acid is produced in nutrient broth from glycerol, cellobiose, d-mannose, d-mannitol, maltose, trehalose, dextrin, d-galactose and glucose. Acid production is observed for the following carbohydrates with the API 50CH kit: d-ribose, d-fructose, N-acetylglucosamine, starch and glycogen. Variable response for the production of acid is observed in methyl--d-glucopyranoside and d-melobiose, to which the type strain AE122T is positive and negative, respectively, as opposed to strain AE 204 after 48 h incubation. Acid is not produced in nutrient broth from raffinose, dulcitol, d-saccharose, d-sorbitol, lactose, l-proline, salicine, dl-lactate, inositol (they also do not use it as a sole carbon source), l-arabinose, rhamnose and glycine. Acid production is not observed for the following carbohydrates with the API 50CH kit: erythritol, d-arabinose, d-xylose, l-xylose, d-adonitol, methyl--d-xylopiranoside, lsorbose, methyl-␣d-mannopyranoside, amygdalin, inulin, dmelezitose, xylitol, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, gentiobiose and 2- and 5-ketogluconate. Habitat description The strains belonging to A. lacus sp nov. were recovered from two lakes (Table S1) and the type strain of the species AE122T (=CECT 8024T = LMG 26710T ) was isolated from the small humic lake Huutjärvi (municipality Pyhtää), which has elevated nitrogen levels. Acknowledgements ˜ for her technical assistance. The authors thank Catalina Núnez This work was supported in part by the projects AGL2011-30461C02-02 of the Ministerio de Ciencia e Innovación (Spain) and JPIW2013-095-CO3 from the Ministerio de Economía y Competitividad (WATER JPI).
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