Abstract
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain ULPAs1T is AY728038.
Footnotes
,†,The arsenic oxanion arsenite (As[III]) can be used by prokaryotes as an electron donor. Cells couple the oxidation of arsenite to the reduction of either oxygen or nitrate and use the energy derived either to fix CO2 into organic cellular material and achieve growth for chemolithoautotrophic prokaryotes or to help in the resistance to arsenic for heterotrophic organisms (Oremland & Stolz, 2003). The microbiological oxidation of As[III] to arsenate (As[V]) impacts the mobility and the speciation of arsenic in the environment. More than 30 strains representing at least nine genera of the Bacteria and Archaea, including members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, DeinococcusThermus and Crenarchaeota, have been reported to be involved in arsenite oxidation. Physiologically diverse, these micro-organisms include both heterotrophic arsenite oxidizers and chemolithoautotrophic arsenite oxidizers (Santini et al., 2000; Oremland & Stolz, 2003; Silver & Phung, 2005). Heterotrophic oxidation of As[III] is viewed primarily as a detoxification reaction that converts As[III] encountered on the outer membrane of the cell into the less toxic and less mobile form As[V], perhaps making it less likely to enter the cell (Anderson et al., 1992).
A Gram-negative, aerobic bacterial strain, designated ULPAs1T, was isolated from an industrial wastewater treatment plant contaminated with arsenic (0.47 mmol kg1) and other metals (Weeger et al., 1999). This strain was able to tolerate 5 mM As[III] and was able to oxidize it to As[V] either in free suspension (Weeger et al., 1999; Muller et al., 2003) or immobilized in a calcium alginate gel (Simeonova et al., 2005). Moreover, strain ULPAs1T was able to reduce As[V] to As[III] and to synthesize at least two arsenate reductases (Carapito et al., 2006). Finally, it was also found to be resistant to numerous heavy metals such as Se[IV], Mn[II], Cr[III], Cd[II], Sb[III] and Ni[II] (Muller et al., 2003). Based on partial 16S rRNA gene sequence analysis, strain ULPAs1T was provisionally identified to be closely related to the species Duganella zoogloeoides (former Zoogloea ramigera IAM 12670) (Weeger et al., 1999) and was tentatively given the name Caenibacter arsenoxydans (Carapito et al., 2006). The present study completes the phenotypic and genotypic characterization of this strain. Our results show that this bacterium represents a novel species of the recently described genus Herminiimonas (Fernandes et al., 2005).
Strain ULPAs1T was isolated after aerobic enrichment on a chemically defined medium (CDM) supplemented with 1.33 mM As[III] (Weeger et al., 1999). Briefly, CDM was prepared as follows: 100 ml solution A [81.2 mM MgSO4.7H2O (Sigma), 187 mM NH4Cl (99.8 % purity; Merck), 70 mM Na2SO4 (99 %; Prolabo), 0.574 mM K2HPO4 (97 %; Prolabo), 4.57 mM CaCl2.2H2O (99.5 %; Merck), 446 mM sodium lactate (98 %; Sigma)], 2.5 ml solution B [4.8 mM Fe2SO4.7H2O (99 %; Prolabo)] and 10 ml solution C [950 mM NaHCO3 (99.5 %; Prolabo)] were mixed and made up to 1 litre with water. The final pH of the medium was about 7.2. Cells of strain ULPAs1T were Gram-negative, and pale yellow to straw-coloured convex colonies with entire margins were observed when the strain was grown on CDM agar. Transmission electron microscopic observations showed that cells were slightly curved or straight rods with rounded ends, approximately 12.5 µm long and 0.50.7 µm wide, harbouring a single polar flagellum (Fig. 1). Cell motility was observed on low-concentration agar plates, with a swarming rate of 1520 mm in 48 h.
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Different carbon substrates were tested and the results are listed in Table 1. Growth was not affected when the medium was supplemented with As[III] (1.3 mM). Chemolithoautotrophic growth was tested on CDM in the absence of lactate or other organic carbon sources; no growth was observed, indicating that strain ULPAs1T possesses a chemoorganotrophic metabolism. Moreover, under anaerobic growth conditions (BBL GasPak pouch; Becton Dickinson) on CDM containing a carbon source (lactate or acetate) and one metal (2.5 mM selenate, 2.5 mM arsenate), no growth was observed after 2 weeks, indicating that these metals are not used as electron acceptors. Finally, strain ULPAs1T showed mesophilic growth between 4 and 30 °C with optimum growth at 25 °C. Above 30 °C, growth was inhibited. Optimum pH for growth was between 7 and 8.5. Antibiotic resistance was tested by the disc diffusion method (Courvalin et al., 1985). Growth of the isolate on CDM agar was not inhibited by tetracycline or ampicillin, but was inhibited by kanamycin, chloramphenicol, streptomycin and trimethoprim-sulfamethoxazol.
Table 1. Physiological characteristics that differentiate strain ULPAs1T and related strains Strains: 1, strain ULPAs1T; 2, H. fonticola S-94T (data from Fernandes et al., 2005); 3, H. aquatilis CCUG 44693T (Kämpfer et al., 2006); 4, strain ND5 (data from this study unless indicated). With the exception of microscopic observations and metal resistance tests, all characteristics were tested by using the API 20NE and API 50CH systems for strains ULPAs1T and ND5. Arsenite oxidase activity was tested by AgNO3 staining (Lett et al., 2001; Simeonova et al., 2004). All four strains are positive for oxidase activity and assimilation of lactate. Strains ULPAs1T and ND5 showed negative results for glucose fermentation, aesculin hydrolysis, gelatin hydrolysis, β-galactosidase activity and assimilation of mannose, mannitol, N-acetylglucosamine, caprate, citrate, glycerol, erythritol, arabinose, D-ribose, xylose, methyl β-D-xylopyranoside, galactose, fructose, sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, amygdalin, arbutin, aesculin, salicin, D-cellobiose, D-maltose, D-lactose, D-trehalose, D-melezitose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, fucose, arabitol, gluconate, 2-ketoglutarate and 5-ketoglutarate. +, Positive; , negative; ND, no data.
16S rRNA gene fragments were amplified by PCR from DNA extracts by using the eubacterial universal primers specific for this gene (P8: 5'-AGAGATTTGATCCTGGCTCAG-3'; Pc1544: 5'-AAGGAGGTGATCCAGCCGCA-3'). Phylogenetic analyses based on the 16S rRNA gene sequence (1455 nt) further supported the conclusion that the strain belongs to the Betaproteobacteria and that its nearest phylogenetic relatives are members of the family Oxalobacteraceae in the order Burkholderiales (Garrity et al., 2001): Herbaspirillum huttiense ATCC 14670T (96 % sequence similarity), Herbaspirillum rubrisubalbicans ATCC 19308T (96 %), uncultured Duganella clone CTHB-18 (96 %), Paucimonas lemoignei LMG 2207T (95 %), Duganella zoogloeoides IAM 12670T (94 %) and Telluria mixta ACM 1762T (93 %).
Strain ULPAs1T exhibited 16S rRNA gene sequence similarity of 98.56 % to the recently described species Herminiimonas fonticola S-94T (Fernandes et al., 2005), 98 % to Herminiimonas aquatilis CCUG 44693T (Kämpfer et al., 2006) and 98.6 % to the partially characterized strain ND5, isolated from a soil in Tokyo (Iizuka et al., 1998). A phylogenetic tree based on 16S rRNA gene sequences detailing the relationship between strain ULPAs1T and its closest relatives is shown in Fig. 2. In order to determine further the position of strain ULPAs1T, DNADNA hybridization experiments were performed as described by Riegel et al. (1994). Strain ULPAs1T showed levels of DNADNA hybridization of 3 % with H. fonticola S-94T, 5 % with H. aquatilis CCUG 36956T and 11 % with strain ND5. These values are clearly lower than the recommended 70 % cut-off value used to delineate genomic species (Sneath, 1984; Stackebrandt & Goebel, 1994).
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Phenotypic data were obtained by classical methods of carbohydrate utilization determined either on nutrient broth or with the API 20NE and API 50CH systems. As with the two recently described Herminiimonas species, strain ULPAs1T utilized few compounds as unique carbon sources, whereas strain ND5 was able to assimilate a greater variety of carbon sources (Table 1). H. aquatilis CCUG 36956T and strain ND5 have been shown to grow on nutrient-rich media (Iizuka et al., 1998; Kämpfer et al., 2006), but these media did not support the growth of strain ULPAs1T. The minimal inhibitory concentration (MIC) of different metals was determined by following the procedure described by Lim & Cooksey (1993). Briefly, bacterial suspensions were transferred in triplicate from microtitre plates to solid CDM plates supplemented with increasing concentrations of metals. The MIC, defined as the metal concentration that inhibited confluent growth on plates after 3 days at 30 °C, was then determined (Table 1). Growth of H. fonticola, H. aquatilis and strain ND5 was inhibited by 2 mM As[III], whereas strain ULPAs1T exhibited growth at up to 5 mM As[III]. Differences in MIC for different metals allowed the differentiation of ULPAs1T from H. fonticola and H. aquatilis (Table 1). Taken together, these data demonstrate that, despite a significant level of conservation in their 16S rRNA gene sequences, some significant phenotypic differences allowed the differentiation of strain ULPAs1T from H. fonticola, H. aquatilis and the partially characterized strain ND5 (Table 1).
Standard procedures were used to determine the G+C content of the genomic DNA of strain ULPAs1T. The DNA G+C content determined was 54.3 mol%, similar to the value for H. fonticola (52 %; Fernandes et al., 2005). Fatty acid analysis was performed by the Belgian Co-ordinated Collections of Microorganisms (BCCMTM/LMG, University of Gent, Belgium) and the results are presented in Table 2. The cellular fatty acid compositions of strain ULPAs1T, H. fonticola and H. aquatilis differ notably from those of other members of the Oxalobacteraceae by the absence of dodecanoic fatty acids. As with H. fonticola and H. aquatilis, strain ULPAs1T contained large amounts of C16 : 0 and C16 : 1ω7c fatty acids. Strain ULPAs1T differed from H. aquatilis by producing C17 : 0 cyclo and C14 : 0. Differences in cellular fatty acid composition allowing the differentiation of strain ULPAs1T from H. fonticola and H. aquatilis are shown in Table 2.
Table 2. Fatty acid composition of Herminiimonas species Strains: 1, strain ULPAs1T; 2, H. fonticola S-94T (data from Fernandes et al., 2005); 3, H. aquatilis CCUG 44693T (Kämpfer etal., 2006). Data are percentages of total fatty acids.
Taken together, our results suggest that strain ULPAs1T is a member of the Betaproteobacteria, order Burkholderiales, and is the type strain of a novel species of the genus Herminiimonas, for which the name Herminiimonas arsenicoxydans sp. nov. is proposed.
Description of Herminiimonas arsenicoxydans sp. nov.
Herminiimonas arsenicoxydans (ar.se.nic.ox'y.dans. N.L. n. arsenicum arsenic; N.L. v. oxydare to oxidize; N.L. part. adj. arsenicoxydans arsenic-oxidizing).
Cells are slightly curved or straight rods with rounded ends, approximately 12 µm long and 0.50.7 µm wide. Cells stain Gram-negative and harbour a single polar flagellum. Cells do not form spores. Colonies on CDM agar are convex with entire margins and are pale yellow to straw coloured. Positive for oxidase and catalase activity. Most alcohols (e.g. ethanol, methanol), sugars (e.g. fructose, glucose) and sugar acids (e.g. gluconate) and most rich media (e.g. gelatin, trypticase soy, MRS and LuriaBertani broths) do not support growth. Phototrophic or chemolithotrophic growth is not observed. Exhibits aerobic chemo-organotrophic metabolism using oxygen as a terminal electron acceptor. Optimal growth occurs at between pH 7 and 8.5. Growth occurs at 430 °C, optimal growth being at approximately 25 °C. Major fatty acids include C16 : 0, C14 : 0 and cyclo C17 : 0. The hydroxylated fatty acid C10 : 0 3-OH is also present but dodecanoic acid is not. Cells are resistant to tetracycline and ampicillin and to heavy metals: As[III] (5 mM), As[V] (>50 mM), Se[IV] (>10 mM) and Mn[II] (>10 mM). Able to oxidize arsenite to arsenate as well to reduce arsenate to arsenite. The DNA G+C content is 54.3 mol%.
The type strain, ULPAs1T (=CCM 7303T=DSM 17148T=LMG 22961T), was isolated from an enrichment culture inoculated with a liquid sample from an industrial wastewater treatment plant contaminated with arsenic in Germany.
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