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1 Département de Biochimie-Microbiologie, Unité de Formation et de Recherches en Sciences de la Vie et de la Terre, Université de Ouagadougou, 03 BP 7021, Ouagadougou 03, Burkina Faso
2 Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Avenida Michoacán y la Purisima s/n, Col. Vicentina, 09340 México DF, Mexico
3 Institut de Recherche pour le Développement (IRD), Cicerón 609, Col. Los Morales, 11530 México DF, Mexico
4 Laboratoire de Microbiologie IRD, IFR-BAIM, Universités de Provence et de la Méditerranée, ESIL case 925, 163, avenue de Luminy, 13288 Marseille cedex 9, France
Correspondence
Aboubakar S. Ouattara
ouattabs{at}univ-ouaga.bf
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ABSTRACT |
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-Proteobacteria, most closely related to Bosea thiooxidans DSM 9653T (similarity of 98·88 %). Methylobacterium organophilum JCM 2833T, Methylobacterium mesophilicum JCM 2829T, Afipia clevelandensis DSM 7315T, Afipia felis DSM 7326T, Afipia broomeae DSM 7327T, Blastobacter denitrificans LMG 8443T and Bradyrhizobium japonicum DSM 30131T showed significant 16S rRNA gene sequence similarities to strain AMX 51T. The DNA G+C composition of strain AMX 51T was 68·5 mol%. DNA–DNA hybridization analysis revealed 44·2 and 15·1 % relatedness between strain AMX 51T and the respective type strains of Bosea thiooxidans and A. felis. Overall results suggest that strain AMX 51T (=DSM 13099T=ATCC 700918T=CIP 106457T) represents a novel species of the genus Bosea; the name Bosea minatitlanensis sp. nov. is proposed.
The GenBank accession number for the 16S rRNA gene sequence of strain AMX 51T is AF273081.
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MAIN TEXT |
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-Proteobacteria (Das et al., 1996
; Stubner et al., 1998). It was initially described with one species, Bosea thiooxidans, isolated from agricultural soils as a free-living micro-organism that was capable of oxidizing reduced inorganic sulfur compounds (Das et al., 1996). Later, a strain designated 5Z2111, which was closely related to B. thiooxidans (97·3 % 16S rRNA sequence similarity), was isolated from rice field soil (Stubner et al., 1998). Isolate 5Z2111 differs from the type strain of B. thiooxidans in its ability to grow autotrophically with CO2 as a carbon source. Comparing all the characteristics of the type strain of B. thiooxidans and strain 5Z2111, the authors assumed that the latter probably represented a separate species and perhaps genus. To our knowledge, the taxonomic status of strain 5Z2111 as a novel species of Bosea has not been established, and this strain is not available in any public collection.
During a study of the role of strictly aerobic bacteria in anaerobic digesters, enumeration and identification of these micro-organisms were performed using a laboratory-scale upflow anaerobic sludge blanket (UASB) reactor fed with the wastewater of a petrochemical company producing purified terephthalic acid. Eighty-five strains were isolated and subjected to identification by classical biochemical methods, analysis of the cellular fatty acids (CFA) and/or partial 16S rRNA gene sequence analysis. The taxonomic methods used could identify 87 % of the isolates accurately. Comparison of the CFA profiles of the unknown strains by unweighted arithmetic average clustering allowed separation of the strains into two groups. All of the strains in the first group were later shown to belong to one species, Stenotrophomonas acidaminiphila (Assih et al., 2002). The second group was represented by a single isolate, strain AMX 51T (=DSM 13099T=ATCC 700918T=CIP 106457T). This strain was then subjected to more-detailed taxonomic study. Its characterization is reported as the type strain of a novel species of the genus Bosea, Bosea minatitlanensis sp. nov.
Strain AMX 51T was isolated from the anaerobic sludge of a laboratory-scale UASB reactor using R2A medium (Oxoid). Purification and culture procedures were described elsewhere (Assih et al., 2002). B. thiooxidans DSM 9653T and Afipia felis DSM 7326T were obtained from the DSMZ. Procedures for analytical techniques, determination of general phenotypic characteristics, antibiotic susceptibility, DNA G+C content and CFA composition, 16S rRNA sequencing and DNA–DNA hybridization are described elsewhere (Assih et al., 2002). Thiosulfate oxidation was tested in basal medium (Assih et al., 2002) with maltose or Casamino acids as substrate. Additional biochemical analysis was performed by inoculating Biotype 100 strips (bioMérieux) according to the manufacturer's instructions. Biotype medium 1 was used for inoculum preparation. For phylogeny, a non-redundant BLASTN search (Altschul et al., 1997) of the full sequence through GenBank and EMBL (Benson et al., 1999) identified its closest relatives. Sequence data were imported into the sequence editor BioEdit version 5.0.9 (Hall, 1999); the bases were examined and a contiguous consensus sequence was generated for each isolate. The full sequence was aligned using the RDP Sequence Aligner program (Maidak et al., 2001). The consensus sequence was then adjusted manually to conform to the 16S rRNA secondary structure model (Winker & Woese, 1991). Sequences used in the phylogenetic analysis were obtained from the RDP (Maidak et al., 2001) and GenBank (Benson et al., 1999). Positions of sequence and alignment ambiguity were omitted and pairwise evolutionary distances based on 1350 unambiguous nucleotides (16S rRNA) were calculated using the method of Jukes & Cantor (1969). Dendrograms were constructed using the neighbour-joining method (Saitou & Nei, 1987). Confidence in the tree topology was determined using 100 bootstrapped trees (Felsenstein, 1985).
After 2–10 days at 30 °C, strain AMX 51T formed non-pigmented, circular colonies on trypticase soy or R2A agar. Growth was not accompanied by odour. Further attempts to obtain other colonies or strains with phenotypic characteristics similar to those of strain AMX 51T were unsuccessful. Such results suggest that this bacterium was probably only a transient micro-organism in the reactor, which was fed with non-sterilized wastewater, from which it was isolated. Cells of isolate AMX 51T were straight to curved rods, slowly motile and stained Gram-negative. Electron microscopy observations showed that the cell wall structure of the isolate was typical of Gram-negative bacteria and indicated a monotrichous polar flagellation type (data not shown). Spore formation was not observed. Cells occurred singly or in pairs and were 0·5x1·5–2·0 µm in size. Strain AMX 51T was strictly aerobic, as shown by the absence of growth after 1 month incubation at 35 °C in an anaerobic jar on R2A medium. Growth was observed between 15 and 42 °C, with optimum growth at 37 °C. No growth occurred at 
4 or 
45 °C. The pH range for growth was 5·0–8·5, with optimum growth at pH 6·0. The maximum growth rate determined in rich medium (Assih et al., 2002) was approximately 0·237 h-1 at 37 °C, pH 7·0, and 0·201 h-1 at 35 °C, pH 6·0. Strain AMX 51T was positive for oxidase, catalase, urease and amylase and weakly proteolytic. Tests for nitrite reductase, nitrate reductase, indole production, aesculin, Simmons' citrate, ONPG, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, DNase and Tween 80 esterase were negative. The substrate utilization tests performed on API 20NE and Biotype 100 strips or in defined basal liquid medium (Assih et al., 2002) showed that strain AMX 51T was able to use several amino acids, organic acids and methanol (for detailed list, see below). Discrepancies in the utilization of substrates were observed between the different tests. Whereas utilization of L-histidine, L-serine, L-tyrosine and succinate was positive in Biotype 100, these tests were negative in the defined medium, and vice versa for L-aspartate, fumarate, glycerol and propionate. Assimilation of all other substrates present on Biotype 100 (i.e. 74 out of 99) was negative. Negative results were also obtained for assimilation of choline, L-isoleucine, DL-leucine, L-methionine, D-ornithine, DL-threonine, DL-valine, oxalate and starch tested in basal liquid medium and adipate tested with API 20NE. With the API 50CH strips, none of the substrates was acidified after 9 days incubation. Thiosulfate oxidation was observed in the presence of succinate and Casamino acids. The striking feature of these results is that, under all culture conditions tested (API 20NE, API 50CH, Biotype 100, defined basal medium), none of the sugars tested could be used as a substrate by strain AMX 51T.
Phenotypic similarities shared by B. thiooxidans DSM 9653T and strain AMX 51T included cell shape, the presence of catalase and oxidase, thiosulfate oxidation, utilization of L-glutamate, D-gluconate, L-malate, D-malate, L-proline, DL-alanine, L-arginine, L-cysteine, L-glutamine, L-asparagine, acetate and pyruvate and the absence of indole production and arginine dihydrolase. Up to 21 phenotypic differences could be observed between B. thiooxidans DSM 9653T and strain AMX 51T, including nitrate reductase activity, Simmons' citrate test (citrate assimilation), starch and gelatin hydrolysis, utilization of lactate, adipate, phenylacetate and sugars and growth above 41 °C (Table 1). Susceptibility of strain AMX 51T to the following antibiotics was observed: imipenem (10 µg), cephalothin (30 µg), cefotaxime (30 µg), ticarcillin (75 µg), tobramycin (10 µg), amikacin (30 µg), gentamicin (10 IU), netilmicin (30 µg), colistin (300 IU), piperacillin/tazobactam (75/10 µg) and trimethoprim/sulfamethoxazole (1·25/23·75 µg). Isolate AMX 51T was resistant to ceftazidime (30 µg), ofloxacin (5 µg) and ciprofloxacin (5 µg). Intermediate responses were observed with piperacillin (75 µg), amoxicillin (25 µg) and amoxicillin/clavulanic acid (25/10 µg).
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7c (56·56 %), C19 : 0 cyclo8c (13·27 %), C16 : 0 (9·64 %), C16 : 0 3-OH (4·64 %) and C18 : 17c 11-methyl (4·55 %). This CFA pattern is similar to that of B. thiooxidans DSM 9653T (Table 2). Notable differences in the their CFA profiles are in levels of C18 : 17c 11-methyl, C17 : 18c and C17 : 0 (Table 2). The Euclidian distance of 12 between the CFA profiles of isolate AMX 51T and B. thiooxidans DSM 9653T suggests that they are linked at the genus level, but not at the species level. The CFA profile of the novel isolate was significantly different from that of A. felis, which can be considered as a representative of the genus Afipia (data not shown). The Euclidian distance found during CFA analysis between strain AMX 51T, B. thiooxidans and A. felis was about 50–52. Such results confirm that these species cannot belong to the same genus, which is consistent with other phenotypic data.
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) and a closely related bacterium, strain 5Z2111 (66·45±0·05 %) (Stubner et al., 1998). A total of 1482 positions of its 16S rRNA gene was sequenced. Phylogenetic analysis revealed that strain AMX 51T is member of the -2 subgroup of Proteobacteria. Among species with validly published names, its closest relatives were B. thiooxidans DSM 9653T, Methylobacterium organophilum JCM 2833T, Methylobacterium mesophilicum JCM 2829T, Afipia clevelandensis DSM 7315T and A. felis DSM 7326T, with respective similarity levels of 98·88, 92·70, 92·61, 91·67 and 91·59 % (Fig. 1). As isolate AMX 51T shares 91–93 % 16S rRNA sequence similarity with M. organophilum JCM 2833T, M. mesophilicum JCM 2829T, A. clevelandensis DSM 7315T and A. felis DSM 7326T, it cannot be member of any of these species (Stackebrandt & Goebel, 1994). The phenotypic differences observed between the isolate and the above-mentioned species are sufficient to exclude its affiliation to the genera Methylobacterium and Afipia; this result correlates with the phylogeny results. The high 16S rRNA sequence similarity (98·88 %) found between strain AMX 51T and B. thiooxidans DSM 9653T correlates with the similarities observed in phenotypic traits and CFA profiles. In accordance with this, it is proposed that strain AMX 51T should be affiliated to the genus Bosea. Since the relatively high 16S rRNA sequence similarity (98·88 %) did not allow discrimination of strain AMX 51T from B. thiooxidans DSM 9653T at the species level (Stackebrandt & Goebel, 1994), DNA–DNA hybridization analysis was used to resolve the genomic relationships. DNA–DNA hybridization revealed 44·2 % similarity with B. thiooxidans DSM 9653T. This value is high enough to support affiliation of strain AMX 51T to the genus Bosea. However, since the DNA–DNA hybridization value observed is significantly below 70 %, it is concluded that strain AMX 51T represents a separate species (Stackebrandt et al., 2002; Wayne et al., 1987). In contrast, the DNA–DNA hybridization values found between AMX 51T and A. felis (15·1 %) and between B. thiooxidans DSM 9653T and A. felis (19·6 %) are very low. Such results, combined with general phenotypic characteristics, CFA profiles and 16S rRNA sequence similarity values, support the conclusion that the novel isolate and B. thiooxidans are not members of the genus Afipia.
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Cells are straight to curved rods, 0·5x1·5–2·0 µm. Gram-negative, non-sporulating, motile, strictly aerobic bacterium. Monotrichous polar flagellation. Circular, non-pigmented colonies on trypticase soy or R2A agar. Growth is not accompanied by odour. Thiosulfate oxidation is observed in presence of succinate or Casamino acids. Positive for oxidase, catalase and urease; weakly positive for amylase and proteolysis; negative for nitrite reductase, nitrate reductase, indole production, Simmons's citrate, ONPG, aesculin, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, DNase and Tween 80 esterase. All API 50CH tests are negative. Amino acids (Casamino acids, L-alanine, L-arginine, L-asparagine, L-cysteine, L-glutamate, L-glutamine, L-phenylalanine and L-proline) and organic acids (acetate, 4-aminobutyrate, crotonate, formate, D-galacturonate, D-gluconate, D-glucuronate, glutarate, DL-glycerate, 3-hydroxybutyrate, 2-ketogluconate, 2-oxoglutarate, DL-lactate, D-malate, L-malate and pyruvate) are utilized as substrates, as well as methanol, but sugars are not. Casamino acids are required as nitrogen source. Susceptible to imipenem, cephalothin, cefotaxime, ticarcillin, tobramycin, amikacin, gentamicin, netilmicin, piperacillin/tazobactam, trimethoprim/sulfamethoxazole and colistin. Resistant to ceftazidime, ofloxacin and ciprofloxacin. Intermediate responses are observed for piperacillin and amoxicillin/clavulanic acid. Growth is observed between 15 and 42 °C, with an optimum at 37 °C. No growth at 4 or 45 °C. The pH range for growth is 5·0–8·5; optimum pH 6·0. Predominant CFA are C18 : 17c, C19 : 0 cyclo8c, C16 : 0, C16 : 0 3-OH and C18 : 17c 11-methyl. The G+C content of the type strain is 68·5±0·4 mol%.
The type strain, AMX 51T (=DSM 13099T=ATCC 700918T=CIP 106457T), is an environmental bacterium, probably originating from soil or water, that was isolated as a transient micro-organism from anaerobic sludge of a lab-scale UASB reactor treating the petrochemical wastewater of a purified terephthalic acid plant in Minatitlán (Mexico).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Assih, E. A., Ouattara, A. S., Thierry, S., Cayol, J.-L., Labat, M. & Macarie, H. (2002). Stenotrophomonas acidaminiphila sp. nov., a strictly aerobic bacterium isolated from an upflow anaerobic sludge blanket (UASB) reactor. Int J Syst Evol Microbiol 52, 559–568.[Abstract]
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Das, S. K., Mishra, A. K., Tindall, B. J., Rainey, F. A. & Stackebrandt, E. (1996). Oxidation of thiosulfate by a new bacterium, Bosea thiooxidans (strain BI-42) gen. nov., sp. nov.: analysis of phylogeny based on chemotaxonomy and 16S ribosomal DNA sequencing. Int J Syst Bacteriol 46, 981–987.
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