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1 Escola Superior de Biotecnologia, Universidade Católica Portuguesa, 4200-072 Porto, Portugal
2 Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal
3 LEPAE – Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, 4200-465 Porto, Portugal
Correspondence
Célia M. Manaia
cmmanaia{at}esb.ucp.pt
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain E4FC31T is AM295491.
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; http://www.bacterio.cict.fr/classifgenerafamilies.html#Neisseriaceae). Members of these genera have been isolated from human and animal clinical specimens, and from soil and water (Patureau et al., 1998; Yuen et al., 2001; Tønjum, 2005; Vela et al., 2005; Lau et al., 2006). Major distinguishing features among genera of the family Neisseriaceae are nutritional pattern, biochemical characteristics, such as the production of indole or arginine dihydrolase, and G+C content of the genomic DNA, supported by low 16S rRNA gene sequence similarities (Grimes et al., 1997; Yuen et al., 2001; Chern et al., 2004; Tønjum, 2005; Lau et al., 2006). Members of some of these genera, such as Microvirgula and Laribacter (Patureau et al., 1998; Yuen et al., 2001), do not have the ability to oxidize, ferment or assimilate sugars and have been referred to as asaccharolytic (Yuen et al., 2001).
This paper describes a member of the family Neisseriaceae, strain E4FC31T, that was isolated from treated wastewater on m-faecal coliform (m-FC) agar medium, during a survey of antibiotic resistance patterns of members of the Enterobacteriaceae (Ferreira da Silva et al., 2007). The isolate was purified by subculturing, and was maintained on plate count agar (PCA). Long-term preservation was in modified Luria–Bertani (MLB) medium (Tiago et al., 2004) supplemented with 15 % (v/v) glycerol, at –80 °C. Preliminary characterization, based on analysis of colony and cell morphology, Gram-staining, presence of endospores, poly-
-hydroxybutyrate granules, presence of a capsule, motility, number and position of flagella, cytochrome c oxidase and catalase, was based on the methodologies of Murray et al. (1994) and Smibert & Krieg (1994) as described by Manaia et al. (2003). Unless stated otherwise, all biochemical and physiological tests were performed in MLB medium, incubated at 30 °C. The pH range for growth was examined in culture medium containing 12 mM MES (Sigma) to adjust the pH to 5.5 and 12 mM CAPS (Sigma) to adjust the pH to 9.0 or 10.0. NaCl tolerance and temperature range for growth were assayed, respectively, in culture medium supplemented with 1 and 3 % NaCl (w/v) or incubated at 15, 22, 30, 40, 45 and 50 °C. The production of extracellular amylases, gelatinases and tweenases (Tween 80) was tested as described by Tiago et al. (2004).
The ability to grow anaerobically was tested in culture medium supplemented with 0.1 % KNO3 (w/v), under a N2-saturated atmosphere. Growth in minimal medium was tested in mineral medium B (Barreiros et al., 2003) supplemented with 4 mM (NH4)2SO4 and 5 mM malic acid, in 100-ml screw-capped Erlenmeyer flasks containing 20 ml medium, with shaking at 120 r.p.m. Other biochemical and nutritional tests were performed by using the API 20E, API 20NE and API 50CH galleries (bioMérieux) following the manufacturer's instructions. The API 50CH gallery was inoculated with three different culture media: the medium recommended to test acid production (50 CHB/E; bioMérieux), mineral medium B with (NH4)2SO4 and bioMérieux AUX medium recommended for the inoculation of API 20NE galleries. The ability to use other amino acids and organic acids was assayed in mineral medium B with (NH4)2SO4 supplemented with 20 mM (amino acids) or 5 mM (organic acids) of the test substrate. Growth and colony morphology on selective/differential media were tested on m-FC, eosin methyl blue (EMB) agar, MacConkey agar, glucose broth and Columbia agar with 5 % sheep blood. Antibiotic susceptibility was assayed as described by Ferreira da Silva et al. (2006), following the interpretation criteria proposed by the Comité de l'Antibiogramme de la Société Française de Microbiologie (1998).
For the determination of the DNA G+C content, genomic DNA was extracted based on the method of Cashion et al. (1977) and G+C ratios were estimated by using the HPLC method as described by Mesbah et al. (1989). For analysis of respiratory quinones, freeze-dried cells were extracted according to Tindall (1989) and extracts were analysed by UV HPLC (Knauer) at 260 nm with a Lichrosphere 5-µm RP-18 column (Merck). A mixture of methanol/hexane (75 : 25, v/v) was used as the mobile phase at a flow rate of 1 ml min–1. Retention times were compared with known standards. Fatty acid methyl esters (FAMEs) were analysed in 24-h cultures on trypticase casein soy agar. Cell harvesting and preparation of FAMEs were performed as described by Kuykendall et al. (1988). Separation, identification and quantification of individual FAMEs were achieved with the Sherlock version 4.6 microbial identification system (MIS-MIDI). FAMEs were extracted and analysed twice.
The nucleic acid sequence of the 16S rRNA gene was determined after PCR amplification of total DNA extracts as described by Rainey et al. (1996). The 16S rRNA gene sequence was compared with others available in the GenBank/EMBL/DDBJ database via BLASTN from NCBI and aligned with reference sequences included in those databases. The phylogenetic analysis was conducted by using MEGA software, version 3.1 (Kumar et al., 2004). Sequence similarity was estimated based on the model of Jukes & Cantor (1969) and a phylogenetic tree was constructed with the neighbour-joining method. Other methods, namely maximum-parsimony, minimum-evolution and unweighted pair group analysis with arithmetic means, were used to assess tree stability. A total of 1335 nucleotide positions in each 16S rRNA gene were included in the analysis. Non-homologous and ambiguous nucleotide positions were excluded from the calculations.
Strain E4FC31T was isolated on m-FC agar medium. Whereas faecal coliform bacteria produce typical blue colonies on this selective and differential medium, strain E4FC31T produced atypical, greenish colonies. Subsequent DNA typing of the m-FC isolates revealed that strain E4FC31T presented a unique genotype, distinct from those obtained for the other isolates, subsequently identified as members of the family Enterobacteriaceae (Ferreira da Silva et al., 2007). 16S rRNA gene sequence analysis showed that strain E4FC31T represented a member of the class Betaproteobacteria, branching within the family Neisseriaceae. The low levels of 16S rRNA gene sequence similarity (<94 %) with other members of the Neisseriaceae and the distinctive phenotypic characteristics of strain E4FC31T suggested its placement within a new genus (Table 1, Fig. 1). The closest phylogenetic neighbours of strain E4FC31T were Aquitalea magnusonii TRO-001DR8T (93.9 % 16S rRNA gene sequence similarity) and Chromobacterium violaceum ATCC 12472T (93.5 % similarity).
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Strain E4FC31T was able to grow, after subculturing, in mineral medium with malic acid as the only source of carbon and energy, indicating that this organism has no specific growth requirements, e.g. vitamins, amino acids or nucleosides. Strain E4FC31T did not assimilate any of the API 50CH carbon sources when tested in mineral medium or in bioMérieux AUX medium. Similarly, strain E4FC31T did not produce acid from any of the carbon sources when tested in API 50CHB/E medium. Acid was not produced in this same culture medium, even when sodium malate was added as the single carbon source. Strain E4FC31T did not ferment or oxidize any of the API 20E carbon sources. In the carbon source assimilation tests with the API 20NE system, strain E4FC31T was able to use malate and caprate. Other carbon sources assimilated were organic acids and amino acids, namely fumarate, lactate, succinate, glutamic acid, L-leucine and L-proline. The nutritional pattern observed for isolate E4FC31T was consistent with the asaccharolytic profile described for Laribacter hongkongensis and Microvirgula denitrificans, but contrasts with that of its close phylogenetic neighbour C. violaceum (Patureau et al., 1998; Yuen et al., 2001; Gillis & Logan, 2005). Although A. magnusonii can also be considered to be asaccharolytic, its ability to use glucose distinguishes it from strain E4FC31T (Lau et al., 2006). With regard to the key diagnostic tests of the API 20E gallery, strain E4FC31T displayed positive reactions for arginine dihydrolase, indole production and the Voges–Proskauer reaction. It showed low 16S rRNA gene sequence similarity (<94 %) with its closest neighbours A. magnusonii LMG 23054T and C. violaceum LMG 1267T, while the latter two type strains shared a sequence similarity of about 95 %. The chemotaxonomic characterization of strain E4FC31T, namely FAME profile (Table 2) and the presence of ubiquinone 8 as the major respiratory quinone, confirmed its affiliation to the family Neisseriaceae (Wilkinson, 1988; Chern et al., 2004; Yang et al., 2005; Lau et al., 2006). However, the low levels of 16S rRNA gene sequence similarity with recognized taxa in this family and the identification of distinctive phenotype characteristics support the proposal of a new genus to accommodate this organism. Characteristics that differentiate strain E4FC31T from A. magnusonii LMG 23054T include the inability to assimilate glucose, serine, histidine, asparagine, aspartic acid, propionic acid and citric acid, the ability to grow at 45 °C and a higher G+C DNA content (Table 1
).
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Description of Gulbenkiania gen. nov.
Gulbenkiania [Gul.ben.ki.a'ni.a. N.L. fem. n. Gulbenkiania in honour of Calouste Gulbenkian (1869–1955), a protector of the arts and sciences in Portugal, and founder of the Fundação Calouste Gulbenkian].
Cells are non-spore-forming, Gram-negative, motile, curved rods. Catalase-, oxidase- and arginine dihydrolase-positive. Mesophilic. Chemoorganoheterotrophic with aerobic respiratory metabolism. No specific organic growth factors are required. Nitrate is reduced to nitrite. Amino acids and organic acids, but not sugars, are used as single carbon sources. The major respiratory quinone is ubiquinone 8; ubiquinone 9 is a minor component. The DNA G+C content of the type strain of the type species is 63 mol%. C16 : 1/iso-C15 : 0 2-OH and C16 : 0 are the predominant fatty acids. Phylogenetically, the genus belongs to the family Neisseriaceae. The type species is Gulbenkiania mobilis.
Description of Gulbenkiania mobilis sp. nov.
Gulbenkiania mobilis (mo'bi.lis. L. fem. adj. mobilis movable, motile).
Cells are short rods (0.95±0.17 µm in length and 0.38±0.11 µm in width), motile by means of a polar flagellum. No capsule is seen. Produces indole and acetoin. Growth occurs between 15 and 45 °C and between pH 5.5 and 9. Growth occurs in the presence of 1 % NaCl but not with 3 %. Anaerobic growth occurs in the presence of nitrate. None of the API 50CH carbon sources supports growth or acid production. Malate, caprate, fumarate, lactic acid, succinate, glutamic acid, L-leucine and L-proline support growth as single carbon sources. Susceptible to amoxicillin (25 µg), gentamicin (10 µg), ciprofloxacin (5 µg), tetracycline (30 µg), SXT (sulfamethoxazole/trimethoprim; 23.75/1.25 µg), sulfamethoxazole (25 µg), cephalothin (30 µg), streptomycin (10 µg), ticarcillin (75 µg), ceftazidime (30 µg), meropenem (10 µg) and colistin sulphate (50 µg).
The type strain, E4FC31T (=DSM 18507T=LMG 23770T), was isolated from treated municipal wastewater.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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-D-mannopyranoside, methyl 
5c, C15 : 0, iso-C15 : 0, C15 : 1, C16 : 1, iso-C17 : 1, anteiso-C17 : 1, iso-C17 : 0, C17 : 0 and iso-C20 : 0. –, Not detected.