- Email: [email protected]
Herminiimonas fonticola gen. nov., sp. nov., a Betaproteobacterium isolated from a source of bottled mineral water
ARTICLE IN PRESS
Systematic and Applied Microbiology 28 (2005) 596–603 www.elsevier.de/syapm
Herminiimonas fonticola gen. nov., sp. nov., a Betaproteobacterium isolated from a source of bottled mineral water$ Chantal Fernandesa, Fred A. Raineyb, M. Fernanda Nobrec, Isabel Pinhala, Fa´tima Folhasa, Milton S. da Costaa, a
Departamento de Bioquı´mica, Centro de Neurocieˆncias e Biologia Celular, Departamento de Zoologia, Universidade de Coimbra, 3001-401 Coimbra, Portugal b Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA c Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal Received 24 March 2004
Abstract Several yellowish-pigmented bacteria with an optimum growth temperature of about 30 1C, were recovered from the source (borehole) of bottled mineral water in the Serra da Estrela in Eastern Portugal. Phylogenetic analyses of the 16S rRNA gene sequence of strains S-94T, S-97, S-99 and S-92 indicated that these organisms represent a new species of the Betaproteobacteria that is not closely related to any other known species. The major fatty acids of the strains are 16:1 o7c and 16:0. Ubiquinone 8 is the major respiratory quinone. The new isolates are strictly organotrophic and aerobic. The new strains only assimilated organic acids, glycine and alanine. Casamino acids and a mixture of all natural amino acids are not used as sole carbon and nitrogen sources; these are used as nitrogen source in the presence of organic acids. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we are of the opinion that strains S-94T, S-97, S-99 and S-92 represent a new species of a novel genus for which we propose the name Herminiimonas fonticola gen. nov., sp. nov. r 2005 Elsevier GmbH. All rights reserved. Keywords: Herminiimonas fonticola; Betaproteobacteria; Mineral water; Borehole
Introduction Mineral or spring water bottled in Europe always contains a culturable aerobic population of bacteria, because these waters cannot be disinfected to eliminate $ Nucleotide sequence accession numbers: Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers: AY676462 for strain S-94T , DQ011676 for strain S-92, DQ011677 for strain S-97 and DQ011678 for strain S-99. Corresponding author. Tel.: +351 239824024; fax: +351 239826798. E-mail address: [email protected] (M.S. da Costa).
0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.03.013
microorganisms [1]. This population is small at source (borehole) and immediately after bottling, but increases rapidly during storage of bottles at room temperature to about 104–105 colony forming units (CFU) per ml within 3–7 days [11,20,25]. Many aerobic bacteria have been isolated from bottled mineral water or from their borehole sources. Most of these organisms belong to species that also colonize other aquatic environments [20], but a few bacteria represent new species that have, as yet, only been encountered in bottled mineral water or in their source water. Several new recently described species all belong to the genus Pseudomonas namely
ARTICLE IN PRESS C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
P. veroni, P. jessenii, P. mandelli, P. brennerii and P. grimontii [2,3,9,35]. The description of several new species of the genus Pseudomonas, could be misconstrued as indicating that the major aerobic bacterial populations of bottled mineral water or their aquifers are members of the Gammaproteobacteria or even the genus Pseudomonas. However, recent studies indicate that the aerobic heterotrophic bacterial populations of bottled mineral water are composed largely of Betaproteobacteria (W. Beisker et al., unpublished results). We recovered several isolates from a bottled mineral water in Portugal that formed transparent yellowish pinpoint colonies. These organisms were found to belong to a species of the Betaproteobacteria whose closest relatives are two undescribed isolates, designated ND5 from urban soil in Japan [16], and Um1 from an undisclosed source. On the basis of the phylogenetic analysis and physiological, and biochemical characteristics we are of the opinion that these isolates belong to an undescribed species of a novel genus for which we propose the name Herminiimonas. fonticola gen. nov., sp. nov. for strain S-94T (LMG 22527T ¼ CIP 108398T).
Materials and methods Isolation and bacterial strains Strains S-94T, S-97, S-99 and S-92 were isolated, on one occasion on two different plates, from a borehole used as source of water for a mineral water bottling plant located in the Serra da Estrela in Eastern Portugal. Water samples were transported without temperature control and filtered the same day through membrane filters (Gelman type GN-6, pore size 0.2 mm, diameter 47 mm). The filters were placed on R2A agar (Difco) and incubated at 22 1C for 15 days. The organisms were purified on R3A agar [30]. The isolates were stored at 70 1C in Nutrient Broth (Difco) with 15% (w/v) glycerol.
Growth, morphological, biochemical and tolerance characteristics Growth of the organisms was examined in several media that included R2A agar (Difco), Nutrient Agar (Difco), Tryptic Soy Agar (Difco), Plate Count Agar (PCA, Difco), PCA diluted 10 times, Thermus medium [37], Thermus medium diluted 10 times. Thiosulfate, 0.5 g l1 or L-cysteine, 0.2 g l1 were added to these media in an attempt to improve growth, but did not. Heterotrophic medium H3P ( ¼ DSMZ medium 428; www.dsmz.de/media/med428.htm) was also used; growth was consistently better in medium 428, which was adopted to grow the organisms, unless otherwise
597
stated. Cells dimensions and mobility were determined by phase-contrast microscopy. The number and the position of the flagella were visualized by light microscopy after staining the cells with the Ryu stain [13]. The growth temperature range of strains S-94T was determined by measuring the turbidity (610 nm) of cultures incubated in 300 ml metal-capped Erlenmeyer flasks, containing 100 ml of 428 medium in a refrigerated orbital shaker. The pH range for growth was determined at 30 1C in the same medium using MES 20 mM for pH values between 4.5 and 6.5, Tris 20 mM for pH values between 7.0 and 8.5, and CAPSO 20 mM for pH values between 9.0 and 10.5. The pH of each buffer was adjusted with NaOH or HCl. Control media, containing each buffer adjusted to pH 7.0, were used to assess possible inhibitory effects of the buffering agents. Unless otherwise stated, all biochemical and physiological tests were performed as described previously [28,31] in liquid medium 428 at pH 8,0 and at 30 1C for up 5 d. Single-carbon-source assimilation tests were performed in a minimal medium composed of 428 medium without solutions D, which contains yeast extract, acetate, succinate and malate and without solution E, which contains lactate, pyruvate, mannitol, and glucose. Yeast extract (0.1 g l1), the carbon source (2.0 g l1) and ammonium chloride (0.1 g l1) or potassium nitrate (0.5 g l1) were added to the basal salts medium. Growth was determined daily by measuring the turbidity (610 nm) of cultures incubated at 30 1C for up to 5 d in 20 ml screw-capped tubes containing 10 ml of the medium. Positive and negative control cultures were grown in medium 428 and in the minimal medium without carbon source. The utilization of casamino acids (Difco) at a final concentration of 2.0 g l1 and the amino acid mixture of Hudson [15] containing all natural amino acids was also examined. The assimilation of citrate was also performed in Simmons citrate (Difco). Nitrate reduction under anaerobic conditions was examined in NB and in 428 medium to which 2.5 g l1 of agar and 1.0 g l1 of NaNO3 was added. Nitrate reduction was determined as previously described [32]. Hydrolysis of casein, starch, elastin and gelatin was performed in 428 solid medium without solutions D and E, as described previously [32]. Acid production from glucose was assessed in NB with 0.03 g l1 of bromothymol blue, 2.0 g l1 of agar and 5.0 g l1 of glucose for up to 8 d. Enzymatic activities were tested using the API ZYM and the API 20 NE (BioMerieux, Marcy L’Etoile, France) at 30 1C, following manufacturer’s instruction.
Polar lipid, lipoquinone and fatty acid composition The cultures used for polar lipid analysis were grown in medium 428 at 30 1C until the exponential phase of
ARTICLE IN PRESS 598
C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
growth. Harvesting of the cultures and extraction of lipids was performed as described previously [8]. The individual polar lipids were separated by two-dimensional TLC on silica gel G plates (Merck; 0.25 mm thickness) with two solvent systems consisting of chloroform/methanol/water (65:25:4, v/v) followed by chloroform/methanol/acetic acid/water (80:12:15:4, v/v). Lipoquinones were extracted from freeze-dried cells grown on 428 medium and purified by TLC as described by Tindall [34]. They were separated with a Gilson HPLC apparatus by using a reverse-phase column (RP18, Spherisorb, S5 ODS2) with methanol/heptane (10:2, v/ v) as the mobile phase and were detected at 269 nm. Cultures for fatty acid analyses were grown on medium 428 agar plates incubated in sealed plastic bags submerged in a water-bath at 30 1C for 48 h. Fatty acid methyl esters (FAMEs) were extracted as described previously [26]. Identification and quantification of the FAMEs, as well as the numerical analysis of the fatty acid profiles, were performed by using the standard MIS Library Generation Software (Microbial ID).
Determination of G+C content of DNA, random amplified polymorphic DNA (RAPD), 16 rRNA gene sequence and phylogenetic analysis DNA was isolated as described by Nielsen et al. [27]. The G+C content of DNA was determinated by HPLC as described by Mesbah et al. [24]. Crude cell lysates were used as DNA template for RAPD as described by Wiedmann-al-Ahmad et al. [36]. Amplification reactions were performed with a total volume of 50 ml containing as follows: 0.3 U of Taq polymerase, 1.5 mM MgCl2 (Pharmacia Biotech), 0.2 mM each dNTP, 0.6 mM primer OPA3 (50 AGTCAGCCAC30 ) and 2.0 ml of crude cell lysates. Samples were subjected to 45 cycles of amplification (Perkin-Elmer, model 240) as follow: 1 min at 94 1C, 1 min at 34 1C, 2 min at 72 1C followed by a final extension step of 7 min at 94 1C. The fragments were analyzed by electrophoresis in 2% agarose gel in Tris–acetate–EDTA buffer. Extraction of genomic DNA for 16 rRNA gene sequence determination, PCR amplification of the 16 rRNA gene and sequencing of the purified PCR products was carried out as described previously [29]. Purified sequencing-reaction products were electrophoresed using a model 3100 DNA sequencer (Applied Biosystems). The 16S rRNA gene sequence obtained in this study was aligned against previously determined bProteobacteria sequences available from the public databases using the ae2 editor [23]. The method of Jukes and Cantor [18] was used to calculate evolutionary distances. Phylogenetic dendrograms were generated using various treeing algorithms contained in the PHYLIP package [10].
Results Morphological and biochemical characteristics Strains S-94T, S-92, S-97 and S-99 formed thin short rod-shaped cells measuring 1–4 mm in length and about 0.7 mm in width. Gram stain was negative and the cells possessed one polar flagellum. Colonies were yellowishpigmented and translucent. The growth temperature range of strain S-94T and S-92 in 428 medium was between 10 and 35 1C with an optimum growth temperature of about 30 1C. The pH range for growth was between 6.0 and 9.5, with an optimum around 7.5–8.0. Strains S-94T, S-92, S-97 and S-99 were strictly aerobic and did not reduce nitrate or nitrite under anaerobic conditions. These strains were catalase and oxidase positive. These organisms did not hydrolyze elastin, casein, gelatin or starch. The organisms possessed alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase, valine arylaminase, trypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase in the API ZYM test strips (Table 1). The strains of the new species assimilated acetate, citrate, pyruvate, fumarate, malate, lactate, succinate and oxaloacetate, but did not assimilate any single carbohydrate or polyol tested (Table 1). No growth of the organisms occurred on any of the amino acids examined except on alanine and weakly on glycine with or without the addition of ammonium chloride. Moreover, the organism did not grow on casamino acids or the amino acid mixture of Hudson et al. [15]. However, casamino acids and the amino acid mixture served as nitrogen source in minimal medium containing citrate or succinate. Several single amino acids, such as proline, glutamate and aspartate promoted the growth of the organism on succinate or citrate, but did not by themselves, result in growth (Table 2).
Chemotaxonomic characteristics The polar lipid pattern on TLC of strains S-94T, S-92, S-97 and S-99 consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, among other minor phospholipids. The major respiratory quinone was ubiquinone 8. The fatty acid composition of all the isolates was fairly simple and was dominated by 16:1 o7c (45%) and by 16:0 (26%) at the optimum growth temperature (Table 3).
16S rRNA gene sequence comparison, RAPD analysis and guanine plus cytosine (G+C) content of DNA The mole G+C ratio of the DNA of strains S-92, S94T and S-97 was 52.3, 52.2 and 51.9, respectively.
ARTICLE IN PRESS C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
Table 1. Phenotypic characteristics of strains S-94T, S-92, S97, S-99 Characteristic
S94
S92
S97
S99
Presence of Alkaline phosphatase Esterase (C 4) Esterase lipase (C 8) Lipase (C 14) Leucine arylamidase Valine arylaminase Cystine arylamidase Trypsin a-Chymotrypsin Acid phosphatase Naphthol phosphohydrolase a-Galactosidase b-Galactosidase b-Glucuronosidase a-Glucosidase b-Glucosidase N-Acetyl-b-glucosaminidase a-Mannosidase a-Fucosidase Arginine dihydrolase Urease
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
Utilization of Glucose Fructose Xylose Galactose Mannose L-Rhamnose Sucrose Cellobiose Maltose Trehalose Ethanol Glycerol i-Erythritol Arabitol Ribitol Xylitol Mannitol Sorbitol m-Inositol Acetate Citrate Pyruvate
+ + +
+ + +
+ + +
+ + +
Formation of indole Nitrate reduction Fermentation Hydrolysis of Gelatin Elastin Casein Starch
Table 1. (continued ) Characteristic
Strains T
599
Fumarate Malate Lactate Succinate Oxaloacetate a-Ketoglutarate Alanine Glycine Cysteine Serine Asparagine Aspartate Threonine Glutamate Glutamine Leucine Methionine Proline Valine Arginine Histidine Isoleucine Lysine Phenylalanine Tryptophan Ornitine Tyrosine
Strains S94T
S92
S97
S99
+ + + + + + w
+ + + + + + w
+ + + + + + w
+ + + + + + w
+, positive or presence; , negative or absence; w, weak positive reaction.
RAPD analysis indicated that strains S-94T, S-92, S-97 and S-99 belonged to the same clone (Results not shown). Almost complete 16S rRNA gene sequences of 1427–1442 nucleotides were obtained from these four strains, which had identical sequences. Comparison of the 16S rRNA gene sequences of the four new isolates with those available in the public databases showed these organisms to fall within the radiation of the Betaproteobacteria and the proposed family ‘‘Oxalobacteraceae’’ (www.bergeysoutline.com). This family level cluster contains species of the genera Duganella, Herbaspirillum, Janthinobacterium, Oxalobacter and Telluria and a number of novel isolates and environmental clone sequences (Fig. 1). Strains S-94T, S-92, S97 and S-99 cluster with strains ND5 (AB008506) and Um1 (AY367012) with 97.8% pairwise sequence similarity. The next closest relatives based on 16S rRNA gene sequence comparison are two environmental clone sequences with 96% sequence similarity (Fig. 1). Sequence similarities of new isolates to the type species of the genera Duganella, Herbaspirillum, Janthinobacterium
ARTICLE IN PRESS 600
C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
Table 2. The effect of casamino acids, the amino acid mixture of Hudson et al. [15], NH4Cl and KNO3 on the growth of strain S-94T in the presence or absence of organic acids Substrates/mediuma
Turbidityb
— Casamino acids Casamino acids Casamino acids Casamino acids Casamino acids Casamino acids — — — — Amino acid mixture [15]
— — — Citrate Succinate Citrate Succinate Citrate Succinate Citrate Succinate
NH4Cl — NH4Cl — — NH4Cl NH4Cl NH4Cl NH4Cl KNO3 KNO3
Medium 428
0.07 0.08 0.09 0.34 0.50 0.24 0.66 0.37 0.36 0.29 0.20 0.08 0.77
a
Yeast extract (0.1 g l1) was added to all media, except medium 428 which contained 1.0 g l1. b The turbidity of the cultures was estimated at 610 nm.
Table 3. Fatty acid composition (%) of strain S-94T, S-97, S99 and S-92 grown at 30 1C Fatty acids
10:0 10:0 14:0 16:1 16:1 16:0 17:0 18:1 18:0 19:0
3OH o7c o5c Cyclo o7c Cyclo o8c
Strains S-94T
S-97
S-99
S-92
0.5 6.0 4.8 45.6 0.8 26.1 7.9 7.3 0.3 0.4
— 6.0 4.8 45.4 0.8 25.4 9.2 7.4 0.2 0.4
0.5 5.6 4.5 44.1 0.9 26.2 8.1 8.9 0.3 0.6
0.5 7.1 4.8 44.0 0.8 23.1 11.3 7.2 0.2 0.6
are in the range 95.6–96.5%. A much lower 16S rRNA gene sequence similarity is found between strains S-94T, S-97, S-99 and S-92 and the rapidly evolving Telluria species (93.0%).
Discussion Strains S-94T, S-97, S-99 and S-92 are closely related to undescribed organisms, designated ultramicrobacterium strains ND5 and Um1 (16S rRNA gene sequences AB008506 and AY367012, respectively). Strain ND5 was isolated from urban soil in Japan, while the source of Um1 is unknown. These results indicate that organisms closely related to strain S-94T have been
isolated from environments other than ground water. The 16S rRNA gene sequence-based phylogenetic analyzes clearly show that the strains of the new species do not fall within any of the previously described genera of the Betaproteobacteria (Fig. 1). The inability of the new strains to assimilate carbohydrates is not surprising since other betaproteobacteria are also unable to use them. We recently described two species of the genus Tepidimonas that were not able to use any of the carbohydrates tested; these organisms only used organic acids and amino acids as single sources of carbon and energy [12,26]. Other bacteria, namely Thermonema lapsum and T. rossianum are examples of bacteria that grow on complex mixtures of amino acids, but which seem unable to grow on any single amino acid [15,33]. However, the ability to use only two amino acids, namely alanine and glycine (weak growth), as a source of carbon and energy is a much rarer phenomenon. To our surprise strain S-94T did not even grow on casamino acids or a mixture of all natural amino acids as a source of carbon, energy and nitrogen. Moreover, casamino acids and the amino acid mixture of Hudson et al. [15] promoted the growth of the organism on citrate and succinate in the minimal medium without ammonium salts, appearing therefore, to serve as a source of nitrogen. We initially thought that casamino acids could contain ammonium salts from the spontaneous breakdown of amino acids or nitrogenous organic compounds that served as nitrogen source for growth of the organism in the presence of organic acids. However, the filter-sterilized amino acid mixture of Hudson et al. [15] also promoted the growth of the organism in the minimal medium containing succinate or citrate and presumably did not contain large amounts of breakdown products. It turned out that the casamino acids and the amino acid mixture [15] contained only very low concentrations of alanine, and even lower levels of glycine, that could not support visible growth. It is, however, difficult to explain the promotion of growth on succinate and citrate by proline, glutamate and glutamine, none of which serve as sole source of carbon, energy and nitrogen. We speculate that amino acids are taken up very slowly by transport systems, while organic acids are rapidly taken up. The low rates of amino acid uptake could provide enough ammonia for biosynthesis and growth of the organism on organic acids, but would not provide enough carbon and energy to promote growth within the time used to assess carbon source assimilation. The species of the other genera within the family ‘‘Oxalobacteriaceae’’ utilize carbohydrates for growth and have higher G+C mole ratios of the DNA. In common with the proposed new genus are the major fatty acids, the presence of 10:0 3OH fatty acid and quinone composition and the inability to grow under anaerobic conditions (Table 4). On the basis of the
ARTICLE IN PRESS C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
601
Fig. 1. Phylogenetic dendrogram based on 16S rRNA gene sequence comparisons. The dendrogram was reconstructed from evolutionary distances by using the neighbour-joining methods. The scale bar represents 5 nucleotide substitutions per 100 positions.
Table 4.
Differential characteristics of the new genus and other genera within the family ‘‘Oxalobacteriaceae’’
Characteristics
New genus
Janthinobacteriuma Herbaspirillumb
Telluriac
Duganellad
Morphology Flagella
Rods Single polar
Rods Single polar
30
Vibroid or helical 1–5 flagella, on 1 or 2 poles 25–37
Rods Single polar
Optimum growth temperature Optimum growth pH Aerobic growth Anaerobic growth Fermentation Oxidase Catalase Major respiratory quinine Major fatty acids
Rods Single polar or 1–4 subpolar 25
30–35
28
8.0 + + + Q-8
7.0–8.0 + + + Q-8
+ + + Q-8
+ ND + + Q-8
7.0 + ND V V Q-8
16:1 o7c;16:0
16:1 o7c; 16:0;
16:1 o7c; 16:0;
ND
16:1 o7c; 16:0
OH-fatty acids
10:0 3OH
10:0 3OH; 12:0 2OH
ND
10:0 3OH; 12:0 2OH; 12:0 3OH
Nitrate reduction Assimilation of carbohydrates Nitrogen fixation G+C content of DNA (mol%)
V +
10:0 3OH; 12:0 2OH; 12:0 3OH; 14:0 2OH V +
+
+
ND 51–52
ND 61–67
V 57–65
ND 67–72
ND 63–64
5.0–8.0
7.0
ND, Not determined; +, positive; , negative; V, variable results among the species. a Data from [6,22]. b Data from [4,7,17,19,22]. c Data from [5]. d Data from [14,21].
physiological and biochemical parameters, and 16S rRNA gene sequence analyzes, strain S-94T represents a new genus and species for which we suggest the name Herminiimonas. fonticola gen. nov., sp. nov.
Description of Herminiimonas gen. nov. Herminiimonas (Her. mi. ni. i. mo’ nas, L. masc. n. Mons Herminius, a mountain range of Lusitania; L. fem.
ARTICLE IN PRESS 602
C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
n. monas, a unit, monad; N. L. fem. n. Herminiimonas). Herminiimonas forms rod-shaped cells that stain Gramnegative. Endospores are not formed. Mesophilic. Strictly aerobic, oxidase and catalase positive. Fatty acids are straight-chained; major phospholipids are phosphatydilethanolamine, and phosphatydilglycerol; ubiquinone 8 is the major respiratory quinone. Chemoorganotrophic. Organic acids and a few amino acids are used as carbon and energy sources. The species of this genus belong to the Betaproteobacteria. The type species is H. fonticola.
Description of Herminiimonas fonticola sp. nov. H. fonticola (fon. ti’ co. la, L. masc. n. fontis, a spring, fountain; L. suf. -cola; N. L. n. fonticola, an inhabitant of a fountain). H. fonticola forms short rod-shaped cells 1.0–4.0 mm in length and 0.7 mm in diameter. Gram stain is negative. The cells are motile by one polar flagellum. Colonies are yellow-pigmented, translucent and less than 1 mm in diameter after 72 h of growth. Growth occurs between 10 and 35 1C; the optimum growth temperature is about 30 1C. The optimum pH is between 7.5 and 8.0; growth does not occur at pH below 6.0 or above 9.5. Strictly aerobic; cytochrome oxidase and catalase are positive. The major fatty acids are 16:1 o7c and 16:0. Yeast extract, or growth factors, are required for growth. Strain S-94T does not reduce nitrate. The strains of this species are chemoorganotrophic. Starch, casein, elastin, and fibrin are not degraded. Several organic acids are utilized for growth; carbohydrates are not used for growth. Alanine and glycine are the only amino acids used as carbon, energy source and nitrogen; amino acids are used as nitrogen source in the presence of a metabolizable carbon source. The DNA of strain S-94T has a G+C content of 52.2 mol%. This bacterium was isolated from a borehole in the Serra da Estrela in Portugal. The type strain, S94T, has been deposited in the Collection of the Institute Pasteur, Paris, France, as strain CIP 108398T and in the BCCM/LMG Bacteria Collection, Ghent, Belgium as strain LMG 22527T.
Acknowledgements This work was supported, in part by the PRAXIS XXI Program (PRAXIS/PCNA/BIO/46/96) and POCTI 35029/99, Portugal. FAR was in part supported by NSF award DEB-971427. The authors would like to thank Dr. Jean Euzeby for the etymology of the epithets of the new species.
References [1] Anonymous, Directive du Conseil du 15 Juillet 1980 relative au rapprochement des le´gislations des Etats membres concernant l’exploitation et la mise dans le commerce des eaux mine´rales naturelles (80/777/CEE), Journal Officiel des Communaute´s Europe´ennes L 229 (1980) 1–10. [2] N. Baida, A. Yazourh, E. Singer, D. Izard, Pseudomonas brenneri sp. nov., a new species isolated from natural mineral waters, Res. Microbiol. 152 (2000) 493–502. [3] N. Baida, A. Yazourh, E. Singer, D. Izard, Pseudomonas grimontii sp. nov., Int. J. Syst. Evol. Microbiol. 52 (2002) 1497–1503. [4] J.I. Baldani, B. Pot, G. Kirchhof, E. Falsen, V.L.D. Baldani, F.L. Olivares, B. Hoste, K. Kersters, A. Hartmann, M. Gillis, J. Dobereiner, Emended description of Herbaspirillum; inclusion of [Pseudomonas] rubrisubalbicans, a mild plant pathogen, as Herbaspirillum rubrisubalbicans comb. nov.; and classification of a group of clinical isolates (EF group 1) as Herbaspirillum species 3, Int. J. Syst. Bacteriol. 46 (1996) 802–810. [5] J.P. Bowman, L.I. Sly, A.C. Hayward, Y. Spiegel, E. Stackebrandt, Telluria mixta (Pseudomonas mixta Bowman, Sly, and Hayward 1988) gen. nov., comb. nov., and Telluria chitinolytica sp. nov., soil-dwelling organisms which actively degrade polysaccharides, Int. J. Syst. Bacteriol. 43 (1993) 120–124. [6] J. De Ley, P. Segers, M. Gillis, Intra- and intergeneric similarities of Chromobacterium and Janthinobacterium ribosomal ribonucleic acid cistrons, Int. J. Syst. Bacteriol. 28 (1978) 154–168. [7] L. Ding, A. Yokota, Proposals of Curvibacter gracilis gen. nov., sp. nov. and Herbaspirillum putei sp. nov. for bacterial strains isolated from well water and reclassification of [Pseudomonas] huttiensis, [Pseudomonas] lanceolata, [Aquaspirillum] delicatum and [Aquaspirillum] autotrophicum as Herbaspirillum huttiense comb. nov., Curvibacter lanceolatus comb. nov., Curvibacter delicatus comb. nov. and Herbaspirillum autotrophicum comb. nov., Int. J. Syst. Evol. Microbiol. 54 (2004) 2223–2230. [8] M.M. Donato, E.A. Seleiro, M.S. da Costa, Polar lipid and fatty acid composition of strains of the genus Thermus, Syst. Appl. Microbiol. 13 (1990) 234–239. [9] M. Elomari, L. Coroler, B. Hoste, M. Gillis, D. Izard, H. Leclerc, DNA relatedness among Pseudomonas strains isolated from natural mineral waters and proposal of Pseudomonas veronii sp. nov., Int. J. Syst. Bacteriol. 46 (1996) 1138–1144. [10] J. Felsenstein, PHYLIP (Phylogenetic Inference Package) Version 3.5.1, Department of Genetics, University of Washington, Seattle, 1993. [11] A.C. Ferreira, P. Vasconcellos Morais, M.S. da Costa, Alteration in total bacteria, iodonitrophenyltetrazolium (INT)-positive bacteria, and heterotrophic plate counts of bottled mineral water, Can. J. Microbiol. 40 (1994) 72–77. [12] M. Freitas, F.A. Rainey, M.F. Nobre, A.J.D. Silvestre, M.S. da Costa, Tepidimonas aquatica sp. nov., a new slightly thermophilic b-Proteobacterium isolated from a hot water tank, Syst. Appl. Microbiol. 26 (2003) 376–381.
ARTICLE IN PRESS C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
[13] M.E. Heimbrook, W.L.L. Wang, G. Campbell, Staining bacterial flagella easily, J. Clin. Microbiol. 27 (1989) 2612–2615. [14] A. Hiraishi, Y.K. Shin, J. Sugiyama, Proposal to reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov., Int. J. Syst. Bacteriol. 47 (1997) 1249–1252. [15] J.A. Hudson, K.M. Schofield, H.W. Morgan, R.M. Daniel, Thermonema lapsum gen. nov., sp. nov., a thermophilic gliding bacterium, Int. J. Syst. Bacteriol. 39 (1989) 485–487. [16] T. Iizuka, S. Yamanaka, T. Nishiyama, A. Hiraishi, Isolation and phylogenetic analysis of aerobic copiotrophic ultramicrobacteria from urban soil, J. Gen. Appl. Microbiol. 44 (1998) 75–84. [17] W.T. Im, H.S. Bae, A. Yokota, S.T. Lee, Herbaspirillum chlorophenolicum sp. nov., a 4-chlorophenol-degrading bacterium, Int. J. Syst. Evol. Microbiol. 54 (2004) 851–855. [18] T.H. Jukes, C.R. Cantor, Evolution of protein molecules, In: H.N. Munro (Ed.), Mammalian Protein Metabolism, Academic Press, New York, 1969, pp. 21–132. [19] G. Kirchhof, B. Eckert, M. Stoffels, J.I. Baldani, V.M. Reis, A. Hartmann, Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4fibre plants, Int. J. Syst. Evol. Microbiol. 51 (2001) 157–168. [20] H. Leclerc, M.S. da Costa, The microbiology of natural water, In: D.A.G. Senior, P.R. Ashurst (Eds.), Technology of Bottled Water, first ed., Sheffield Academic Press, 1998, pp. 223–263. [21] W.J. Li, Y.Q. Zhang, D.J. Park, C.T. Li, L.H. Xu, C.J. Kim, C.L. Jiang, Duganella violaceinigra sp. nov., a novel mesophilic bacterium isolated from forest soil, Int. J. Syst. Evol. Microbiol. 54 (2004) 1811–1814. [22] S.P. Lincoln, T.R. Fermor, B.J. Tindall, Janthinobacterium agaricidamnosum sp. nov., a soft rot pathogen of Agaricus bisporus, Int. J. Syst. Bacteriol. 49 (1999) 1577–1589. [23] B.L. Maidak, J.R. Cole, T.G. Lilburn, C.T.JR. Parker, P.R. Saxman, R.J. Farris, J.M. Garrity, G.J. Olsen, T.M. Schmidt, J.M. Tiedje, The RDP-II (Ribosomal Database Project), Nucleic Acids Res. 29 (2001) 173–174. [24] M. Mesbash, U. Premachandran, W.B. Whitman, Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography, Int. J. Syst. Bacteriol. 39 (1989) 159–167. [25] P.V. Morais, M.S. da Costa, Alteration in the major heterotrophic bacterial populations isolated from a still bottled mineral water, J. Appl. Bacteriol. 69 (1990) 750–757.
603
[26] C. Moreira, F.A. Rainey, M.F. Nobre, M.T. da Silva, M.S. da Costa, Tepidimonas ignava gen. nov., sp. nov., a new chemolithoheterotrophic and slightly thermophilic member of the b-Proteobacteria, Int. J. Syst. Evol. Microbiol. 50 (2000) 735–742. [27] P. Nielsen, D. Fritze, F.G. Priest, Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species, Microbiology 141 (1995) 1745–1761. [28] O.C. Nunes, M.M. Donato, M.S. da Costa, Isolation and characterization of Rhodothermus strains from S. Miguel, Azores, Syst. Appl. Microbiol. 15 (1992) 92–97. [29] F.A. Rainey, N. Ward-Rainey, R.M. Kroppenstedt, E. Stackebrandt, The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov., Int. J. Syst. Bacteriol. 46 (1996) 1088–1092. [30] D.J. Reasoner, E.E. Geldreich, A new medium for the enumeration and subculture of bacteria from potable water, Appl. Environ. Microbiol. 49 (1985) 1–7. [31] M.A. Santos, R.A.D. Williams, M.S. da Costa, Numerical taxonomy of Thermus isolates from hot springs in Portugal, Syst. Appl. Microbiol. 12 (1989) 310–315. [32] R.M. Smibert, N.R. Krieg, General characterization, In: P. Gerhardt, R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg, G.B. Phillips (Eds.), Manual Methods for General Bacteriology, Washington DC, 1981, pp., 409–443. [33] S. Tenreiro, M.F. Nobre, F.A. Rainey, C. Miguel, S.M. da Costa, Thermonema rossianum sp. nov., a new thermophilic and slightly halophilic species from hot springs in Naples, Italy, Int. J. Syst. Bacteriol. 47 (1997) 122–126. [34] B.J. Tindall, Fully saturated menaquinones in the archaebacterium Pyrobaculum islandicum, FEMS Microbiol. Lett. 60 (1989) 251–254. [35] S. Verhille, N. Baida, F. Dabboussi, D. Izard, H. Leclerc, Taxonomic study of bacteria isolated from natural mineral waters: proposal of Pseudomonas jessenii sp. nov. and Pseudomonas mandelii sp. nov., Syst. Appl. Microbiol. 22 (1999) 45–58. [36] M. Wiedmann-al-Ahmad, H.V. Tichy, G. Schon, Characterization of Acinetobacter type strains and isolates obtained from wastewater treatment plants by PCR fingerprinting, Appl. Environ. Microbiol. 60 (1994) 4066–4071. [37] R.A.D. Williams, M.S. da Costa, The genus Thermus and related microorganisms, In: A. Balows, H.G. Truper, M. Dworkin, W. Harder, K.H. Schleifer (Eds.), The Prokaryotes: a Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, Second ed., Springer, New York, 1992, pp. 478–490.
Systematic and Applied Microbiology 28 (2005) 596–603 www.elsevier.de/syapm
Herminiimonas fonticola gen. nov., sp. nov., a Betaproteobacterium isolated from a source of bottled mineral water$ Chantal Fernandesa, Fred A. Raineyb, M. Fernanda Nobrec, Isabel Pinhala, Fa´tima Folhasa, Milton S. da Costaa, a
Departamento de Bioquı´mica, Centro de Neurocieˆncias e Biologia Celular, Departamento de Zoologia, Universidade de Coimbra, 3001-401 Coimbra, Portugal b Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA c Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal Received 24 March 2004
Abstract Several yellowish-pigmented bacteria with an optimum growth temperature of about 30 1C, were recovered from the source (borehole) of bottled mineral water in the Serra da Estrela in Eastern Portugal. Phylogenetic analyses of the 16S rRNA gene sequence of strains S-94T, S-97, S-99 and S-92 indicated that these organisms represent a new species of the Betaproteobacteria that is not closely related to any other known species. The major fatty acids of the strains are 16:1 o7c and 16:0. Ubiquinone 8 is the major respiratory quinone. The new isolates are strictly organotrophic and aerobic. The new strains only assimilated organic acids, glycine and alanine. Casamino acids and a mixture of all natural amino acids are not used as sole carbon and nitrogen sources; these are used as nitrogen source in the presence of organic acids. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we are of the opinion that strains S-94T, S-97, S-99 and S-92 represent a new species of a novel genus for which we propose the name Herminiimonas fonticola gen. nov., sp. nov. r 2005 Elsevier GmbH. All rights reserved. Keywords: Herminiimonas fonticola; Betaproteobacteria; Mineral water; Borehole
Introduction Mineral or spring water bottled in Europe always contains a culturable aerobic population of bacteria, because these waters cannot be disinfected to eliminate $ Nucleotide sequence accession numbers: Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers: AY676462 for strain S-94T , DQ011676 for strain S-92, DQ011677 for strain S-97 and DQ011678 for strain S-99. Corresponding author. Tel.: +351 239824024; fax: +351 239826798. E-mail address: [email protected] (M.S. da Costa).
0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.03.013
microorganisms [1]. This population is small at source (borehole) and immediately after bottling, but increases rapidly during storage of bottles at room temperature to about 104–105 colony forming units (CFU) per ml within 3–7 days [11,20,25]. Many aerobic bacteria have been isolated from bottled mineral water or from their borehole sources. Most of these organisms belong to species that also colonize other aquatic environments [20], but a few bacteria represent new species that have, as yet, only been encountered in bottled mineral water or in their source water. Several new recently described species all belong to the genus Pseudomonas namely
ARTICLE IN PRESS C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
P. veroni, P. jessenii, P. mandelli, P. brennerii and P. grimontii [2,3,9,35]. The description of several new species of the genus Pseudomonas, could be misconstrued as indicating that the major aerobic bacterial populations of bottled mineral water or their aquifers are members of the Gammaproteobacteria or even the genus Pseudomonas. However, recent studies indicate that the aerobic heterotrophic bacterial populations of bottled mineral water are composed largely of Betaproteobacteria (W. Beisker et al., unpublished results). We recovered several isolates from a bottled mineral water in Portugal that formed transparent yellowish pinpoint colonies. These organisms were found to belong to a species of the Betaproteobacteria whose closest relatives are two undescribed isolates, designated ND5 from urban soil in Japan [16], and Um1 from an undisclosed source. On the basis of the phylogenetic analysis and physiological, and biochemical characteristics we are of the opinion that these isolates belong to an undescribed species of a novel genus for which we propose the name Herminiimonas. fonticola gen. nov., sp. nov. for strain S-94T (LMG 22527T ¼ CIP 108398T).
Materials and methods Isolation and bacterial strains Strains S-94T, S-97, S-99 and S-92 were isolated, on one occasion on two different plates, from a borehole used as source of water for a mineral water bottling plant located in the Serra da Estrela in Eastern Portugal. Water samples were transported without temperature control and filtered the same day through membrane filters (Gelman type GN-6, pore size 0.2 mm, diameter 47 mm). The filters were placed on R2A agar (Difco) and incubated at 22 1C for 15 days. The organisms were purified on R3A agar [30]. The isolates were stored at 70 1C in Nutrient Broth (Difco) with 15% (w/v) glycerol.
Growth, morphological, biochemical and tolerance characteristics Growth of the organisms was examined in several media that included R2A agar (Difco), Nutrient Agar (Difco), Tryptic Soy Agar (Difco), Plate Count Agar (PCA, Difco), PCA diluted 10 times, Thermus medium [37], Thermus medium diluted 10 times. Thiosulfate, 0.5 g l1 or L-cysteine, 0.2 g l1 were added to these media in an attempt to improve growth, but did not. Heterotrophic medium H3P ( ¼ DSMZ medium 428; www.dsmz.de/media/med428.htm) was also used; growth was consistently better in medium 428, which was adopted to grow the organisms, unless otherwise
597
stated. Cells dimensions and mobility were determined by phase-contrast microscopy. The number and the position of the flagella were visualized by light microscopy after staining the cells with the Ryu stain [13]. The growth temperature range of strains S-94T was determined by measuring the turbidity (610 nm) of cultures incubated in 300 ml metal-capped Erlenmeyer flasks, containing 100 ml of 428 medium in a refrigerated orbital shaker. The pH range for growth was determined at 30 1C in the same medium using MES 20 mM for pH values between 4.5 and 6.5, Tris 20 mM for pH values between 7.0 and 8.5, and CAPSO 20 mM for pH values between 9.0 and 10.5. The pH of each buffer was adjusted with NaOH or HCl. Control media, containing each buffer adjusted to pH 7.0, were used to assess possible inhibitory effects of the buffering agents. Unless otherwise stated, all biochemical and physiological tests were performed as described previously [28,31] in liquid medium 428 at pH 8,0 and at 30 1C for up 5 d. Single-carbon-source assimilation tests were performed in a minimal medium composed of 428 medium without solutions D, which contains yeast extract, acetate, succinate and malate and without solution E, which contains lactate, pyruvate, mannitol, and glucose. Yeast extract (0.1 g l1), the carbon source (2.0 g l1) and ammonium chloride (0.1 g l1) or potassium nitrate (0.5 g l1) were added to the basal salts medium. Growth was determined daily by measuring the turbidity (610 nm) of cultures incubated at 30 1C for up to 5 d in 20 ml screw-capped tubes containing 10 ml of the medium. Positive and negative control cultures were grown in medium 428 and in the minimal medium without carbon source. The utilization of casamino acids (Difco) at a final concentration of 2.0 g l1 and the amino acid mixture of Hudson [15] containing all natural amino acids was also examined. The assimilation of citrate was also performed in Simmons citrate (Difco). Nitrate reduction under anaerobic conditions was examined in NB and in 428 medium to which 2.5 g l1 of agar and 1.0 g l1 of NaNO3 was added. Nitrate reduction was determined as previously described [32]. Hydrolysis of casein, starch, elastin and gelatin was performed in 428 solid medium without solutions D and E, as described previously [32]. Acid production from glucose was assessed in NB with 0.03 g l1 of bromothymol blue, 2.0 g l1 of agar and 5.0 g l1 of glucose for up to 8 d. Enzymatic activities were tested using the API ZYM and the API 20 NE (BioMerieux, Marcy L’Etoile, France) at 30 1C, following manufacturer’s instruction.
Polar lipid, lipoquinone and fatty acid composition The cultures used for polar lipid analysis were grown in medium 428 at 30 1C until the exponential phase of
ARTICLE IN PRESS 598
C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
growth. Harvesting of the cultures and extraction of lipids was performed as described previously [8]. The individual polar lipids were separated by two-dimensional TLC on silica gel G plates (Merck; 0.25 mm thickness) with two solvent systems consisting of chloroform/methanol/water (65:25:4, v/v) followed by chloroform/methanol/acetic acid/water (80:12:15:4, v/v). Lipoquinones were extracted from freeze-dried cells grown on 428 medium and purified by TLC as described by Tindall [34]. They were separated with a Gilson HPLC apparatus by using a reverse-phase column (RP18, Spherisorb, S5 ODS2) with methanol/heptane (10:2, v/ v) as the mobile phase and were detected at 269 nm. Cultures for fatty acid analyses were grown on medium 428 agar plates incubated in sealed plastic bags submerged in a water-bath at 30 1C for 48 h. Fatty acid methyl esters (FAMEs) were extracted as described previously [26]. Identification and quantification of the FAMEs, as well as the numerical analysis of the fatty acid profiles, were performed by using the standard MIS Library Generation Software (Microbial ID).
Determination of G+C content of DNA, random amplified polymorphic DNA (RAPD), 16 rRNA gene sequence and phylogenetic analysis DNA was isolated as described by Nielsen et al. [27]. The G+C content of DNA was determinated by HPLC as described by Mesbah et al. [24]. Crude cell lysates were used as DNA template for RAPD as described by Wiedmann-al-Ahmad et al. [36]. Amplification reactions were performed with a total volume of 50 ml containing as follows: 0.3 U of Taq polymerase, 1.5 mM MgCl2 (Pharmacia Biotech), 0.2 mM each dNTP, 0.6 mM primer OPA3 (50 AGTCAGCCAC30 ) and 2.0 ml of crude cell lysates. Samples were subjected to 45 cycles of amplification (Perkin-Elmer, model 240) as follow: 1 min at 94 1C, 1 min at 34 1C, 2 min at 72 1C followed by a final extension step of 7 min at 94 1C. The fragments were analyzed by electrophoresis in 2% agarose gel in Tris–acetate–EDTA buffer. Extraction of genomic DNA for 16 rRNA gene sequence determination, PCR amplification of the 16 rRNA gene and sequencing of the purified PCR products was carried out as described previously [29]. Purified sequencing-reaction products were electrophoresed using a model 3100 DNA sequencer (Applied Biosystems). The 16S rRNA gene sequence obtained in this study was aligned against previously determined bProteobacteria sequences available from the public databases using the ae2 editor [23]. The method of Jukes and Cantor [18] was used to calculate evolutionary distances. Phylogenetic dendrograms were generated using various treeing algorithms contained in the PHYLIP package [10].
Results Morphological and biochemical characteristics Strains S-94T, S-92, S-97 and S-99 formed thin short rod-shaped cells measuring 1–4 mm in length and about 0.7 mm in width. Gram stain was negative and the cells possessed one polar flagellum. Colonies were yellowishpigmented and translucent. The growth temperature range of strain S-94T and S-92 in 428 medium was between 10 and 35 1C with an optimum growth temperature of about 30 1C. The pH range for growth was between 6.0 and 9.5, with an optimum around 7.5–8.0. Strains S-94T, S-92, S-97 and S-99 were strictly aerobic and did not reduce nitrate or nitrite under anaerobic conditions. These strains were catalase and oxidase positive. These organisms did not hydrolyze elastin, casein, gelatin or starch. The organisms possessed alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase, valine arylaminase, trypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase in the API ZYM test strips (Table 1). The strains of the new species assimilated acetate, citrate, pyruvate, fumarate, malate, lactate, succinate and oxaloacetate, but did not assimilate any single carbohydrate or polyol tested (Table 1). No growth of the organisms occurred on any of the amino acids examined except on alanine and weakly on glycine with or without the addition of ammonium chloride. Moreover, the organism did not grow on casamino acids or the amino acid mixture of Hudson et al. [15]. However, casamino acids and the amino acid mixture served as nitrogen source in minimal medium containing citrate or succinate. Several single amino acids, such as proline, glutamate and aspartate promoted the growth of the organism on succinate or citrate, but did not by themselves, result in growth (Table 2).
Chemotaxonomic characteristics The polar lipid pattern on TLC of strains S-94T, S-92, S-97 and S-99 consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, among other minor phospholipids. The major respiratory quinone was ubiquinone 8. The fatty acid composition of all the isolates was fairly simple and was dominated by 16:1 o7c (45%) and by 16:0 (26%) at the optimum growth temperature (Table 3).
16S rRNA gene sequence comparison, RAPD analysis and guanine plus cytosine (G+C) content of DNA The mole G+C ratio of the DNA of strains S-92, S94T and S-97 was 52.3, 52.2 and 51.9, respectively.
ARTICLE IN PRESS C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
Table 1. Phenotypic characteristics of strains S-94T, S-92, S97, S-99 Characteristic
S94
S92
S97
S99
Presence of Alkaline phosphatase Esterase (C 4) Esterase lipase (C 8) Lipase (C 14) Leucine arylamidase Valine arylaminase Cystine arylamidase Trypsin a-Chymotrypsin Acid phosphatase Naphthol phosphohydrolase a-Galactosidase b-Galactosidase b-Glucuronosidase a-Glucosidase b-Glucosidase N-Acetyl-b-glucosaminidase a-Mannosidase a-Fucosidase Arginine dihydrolase Urease
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
Utilization of Glucose Fructose Xylose Galactose Mannose L-Rhamnose Sucrose Cellobiose Maltose Trehalose Ethanol Glycerol i-Erythritol Arabitol Ribitol Xylitol Mannitol Sorbitol m-Inositol Acetate Citrate Pyruvate
+ + +
+ + +
+ + +
+ + +
Formation of indole Nitrate reduction Fermentation Hydrolysis of Gelatin Elastin Casein Starch
Table 1. (continued ) Characteristic
Strains T
599
Fumarate Malate Lactate Succinate Oxaloacetate a-Ketoglutarate Alanine Glycine Cysteine Serine Asparagine Aspartate Threonine Glutamate Glutamine Leucine Methionine Proline Valine Arginine Histidine Isoleucine Lysine Phenylalanine Tryptophan Ornitine Tyrosine
Strains S94T
S92
S97
S99
+ + + + + + w
+ + + + + + w
+ + + + + + w
+ + + + + + w
+, positive or presence; , negative or absence; w, weak positive reaction.
RAPD analysis indicated that strains S-94T, S-92, S-97 and S-99 belonged to the same clone (Results not shown). Almost complete 16S rRNA gene sequences of 1427–1442 nucleotides were obtained from these four strains, which had identical sequences. Comparison of the 16S rRNA gene sequences of the four new isolates with those available in the public databases showed these organisms to fall within the radiation of the Betaproteobacteria and the proposed family ‘‘Oxalobacteraceae’’ (www.bergeysoutline.com). This family level cluster contains species of the genera Duganella, Herbaspirillum, Janthinobacterium, Oxalobacter and Telluria and a number of novel isolates and environmental clone sequences (Fig. 1). Strains S-94T, S-92, S97 and S-99 cluster with strains ND5 (AB008506) and Um1 (AY367012) with 97.8% pairwise sequence similarity. The next closest relatives based on 16S rRNA gene sequence comparison are two environmental clone sequences with 96% sequence similarity (Fig. 1). Sequence similarities of new isolates to the type species of the genera Duganella, Herbaspirillum, Janthinobacterium
ARTICLE IN PRESS 600
C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
Table 2. The effect of casamino acids, the amino acid mixture of Hudson et al. [15], NH4Cl and KNO3 on the growth of strain S-94T in the presence or absence of organic acids Substrates/mediuma
Turbidityb
— Casamino acids Casamino acids Casamino acids Casamino acids Casamino acids Casamino acids — — — — Amino acid mixture [15]
— — — Citrate Succinate Citrate Succinate Citrate Succinate Citrate Succinate
NH4Cl — NH4Cl — — NH4Cl NH4Cl NH4Cl NH4Cl KNO3 KNO3
Medium 428
0.07 0.08 0.09 0.34 0.50 0.24 0.66 0.37 0.36 0.29 0.20 0.08 0.77
a
Yeast extract (0.1 g l1) was added to all media, except medium 428 which contained 1.0 g l1. b The turbidity of the cultures was estimated at 610 nm.
Table 3. Fatty acid composition (%) of strain S-94T, S-97, S99 and S-92 grown at 30 1C Fatty acids
10:0 10:0 14:0 16:1 16:1 16:0 17:0 18:1 18:0 19:0
3OH o7c o5c Cyclo o7c Cyclo o8c
Strains S-94T
S-97
S-99
S-92
0.5 6.0 4.8 45.6 0.8 26.1 7.9 7.3 0.3 0.4
— 6.0 4.8 45.4 0.8 25.4 9.2 7.4 0.2 0.4
0.5 5.6 4.5 44.1 0.9 26.2 8.1 8.9 0.3 0.6
0.5 7.1 4.8 44.0 0.8 23.1 11.3 7.2 0.2 0.6
are in the range 95.6–96.5%. A much lower 16S rRNA gene sequence similarity is found between strains S-94T, S-97, S-99 and S-92 and the rapidly evolving Telluria species (93.0%).
Discussion Strains S-94T, S-97, S-99 and S-92 are closely related to undescribed organisms, designated ultramicrobacterium strains ND5 and Um1 (16S rRNA gene sequences AB008506 and AY367012, respectively). Strain ND5 was isolated from urban soil in Japan, while the source of Um1 is unknown. These results indicate that organisms closely related to strain S-94T have been
isolated from environments other than ground water. The 16S rRNA gene sequence-based phylogenetic analyzes clearly show that the strains of the new species do not fall within any of the previously described genera of the Betaproteobacteria (Fig. 1). The inability of the new strains to assimilate carbohydrates is not surprising since other betaproteobacteria are also unable to use them. We recently described two species of the genus Tepidimonas that were not able to use any of the carbohydrates tested; these organisms only used organic acids and amino acids as single sources of carbon and energy [12,26]. Other bacteria, namely Thermonema lapsum and T. rossianum are examples of bacteria that grow on complex mixtures of amino acids, but which seem unable to grow on any single amino acid [15,33]. However, the ability to use only two amino acids, namely alanine and glycine (weak growth), as a source of carbon and energy is a much rarer phenomenon. To our surprise strain S-94T did not even grow on casamino acids or a mixture of all natural amino acids as a source of carbon, energy and nitrogen. Moreover, casamino acids and the amino acid mixture of Hudson et al. [15] promoted the growth of the organism on citrate and succinate in the minimal medium without ammonium salts, appearing therefore, to serve as a source of nitrogen. We initially thought that casamino acids could contain ammonium salts from the spontaneous breakdown of amino acids or nitrogenous organic compounds that served as nitrogen source for growth of the organism in the presence of organic acids. However, the filter-sterilized amino acid mixture of Hudson et al. [15] also promoted the growth of the organism in the minimal medium containing succinate or citrate and presumably did not contain large amounts of breakdown products. It turned out that the casamino acids and the amino acid mixture [15] contained only very low concentrations of alanine, and even lower levels of glycine, that could not support visible growth. It is, however, difficult to explain the promotion of growth on succinate and citrate by proline, glutamate and glutamine, none of which serve as sole source of carbon, energy and nitrogen. We speculate that amino acids are taken up very slowly by transport systems, while organic acids are rapidly taken up. The low rates of amino acid uptake could provide enough ammonia for biosynthesis and growth of the organism on organic acids, but would not provide enough carbon and energy to promote growth within the time used to assess carbon source assimilation. The species of the other genera within the family ‘‘Oxalobacteriaceae’’ utilize carbohydrates for growth and have higher G+C mole ratios of the DNA. In common with the proposed new genus are the major fatty acids, the presence of 10:0 3OH fatty acid and quinone composition and the inability to grow under anaerobic conditions (Table 4). On the basis of the
ARTICLE IN PRESS C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
601
Fig. 1. Phylogenetic dendrogram based on 16S rRNA gene sequence comparisons. The dendrogram was reconstructed from evolutionary distances by using the neighbour-joining methods. The scale bar represents 5 nucleotide substitutions per 100 positions.
Table 4.
Differential characteristics of the new genus and other genera within the family ‘‘Oxalobacteriaceae’’
Characteristics
New genus
Janthinobacteriuma Herbaspirillumb
Telluriac
Duganellad
Morphology Flagella
Rods Single polar
Rods Single polar
30
Vibroid or helical 1–5 flagella, on 1 or 2 poles 25–37
Rods Single polar
Optimum growth temperature Optimum growth pH Aerobic growth Anaerobic growth Fermentation Oxidase Catalase Major respiratory quinine Major fatty acids
Rods Single polar or 1–4 subpolar 25
30–35
28
8.0 + + + Q-8
7.0–8.0 + + + Q-8
+ + + Q-8
+ ND + + Q-8
7.0 + ND V V Q-8
16:1 o7c;16:0
16:1 o7c; 16:0;
16:1 o7c; 16:0;
ND
16:1 o7c; 16:0
OH-fatty acids
10:0 3OH
10:0 3OH; 12:0 2OH
ND
10:0 3OH; 12:0 2OH; 12:0 3OH
Nitrate reduction Assimilation of carbohydrates Nitrogen fixation G+C content of DNA (mol%)
V +
10:0 3OH; 12:0 2OH; 12:0 3OH; 14:0 2OH V +
+
+
ND 51–52
ND 61–67
V 57–65
ND 67–72
ND 63–64
5.0–8.0
7.0
ND, Not determined; +, positive; , negative; V, variable results among the species. a Data from [6,22]. b Data from [4,7,17,19,22]. c Data from [5]. d Data from [14,21].
physiological and biochemical parameters, and 16S rRNA gene sequence analyzes, strain S-94T represents a new genus and species for which we suggest the name Herminiimonas. fonticola gen. nov., sp. nov.
Description of Herminiimonas gen. nov. Herminiimonas (Her. mi. ni. i. mo’ nas, L. masc. n. Mons Herminius, a mountain range of Lusitania; L. fem.
ARTICLE IN PRESS 602
C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
n. monas, a unit, monad; N. L. fem. n. Herminiimonas). Herminiimonas forms rod-shaped cells that stain Gramnegative. Endospores are not formed. Mesophilic. Strictly aerobic, oxidase and catalase positive. Fatty acids are straight-chained; major phospholipids are phosphatydilethanolamine, and phosphatydilglycerol; ubiquinone 8 is the major respiratory quinone. Chemoorganotrophic. Organic acids and a few amino acids are used as carbon and energy sources. The species of this genus belong to the Betaproteobacteria. The type species is H. fonticola.
Description of Herminiimonas fonticola sp. nov. H. fonticola (fon. ti’ co. la, L. masc. n. fontis, a spring, fountain; L. suf. -cola; N. L. n. fonticola, an inhabitant of a fountain). H. fonticola forms short rod-shaped cells 1.0–4.0 mm in length and 0.7 mm in diameter. Gram stain is negative. The cells are motile by one polar flagellum. Colonies are yellow-pigmented, translucent and less than 1 mm in diameter after 72 h of growth. Growth occurs between 10 and 35 1C; the optimum growth temperature is about 30 1C. The optimum pH is between 7.5 and 8.0; growth does not occur at pH below 6.0 or above 9.5. Strictly aerobic; cytochrome oxidase and catalase are positive. The major fatty acids are 16:1 o7c and 16:0. Yeast extract, or growth factors, are required for growth. Strain S-94T does not reduce nitrate. The strains of this species are chemoorganotrophic. Starch, casein, elastin, and fibrin are not degraded. Several organic acids are utilized for growth; carbohydrates are not used for growth. Alanine and glycine are the only amino acids used as carbon, energy source and nitrogen; amino acids are used as nitrogen source in the presence of a metabolizable carbon source. The DNA of strain S-94T has a G+C content of 52.2 mol%. This bacterium was isolated from a borehole in the Serra da Estrela in Portugal. The type strain, S94T, has been deposited in the Collection of the Institute Pasteur, Paris, France, as strain CIP 108398T and in the BCCM/LMG Bacteria Collection, Ghent, Belgium as strain LMG 22527T.
Acknowledgements This work was supported, in part by the PRAXIS XXI Program (PRAXIS/PCNA/BIO/46/96) and POCTI 35029/99, Portugal. FAR was in part supported by NSF award DEB-971427. The authors would like to thank Dr. Jean Euzeby for the etymology of the epithets of the new species.
References [1] Anonymous, Directive du Conseil du 15 Juillet 1980 relative au rapprochement des le´gislations des Etats membres concernant l’exploitation et la mise dans le commerce des eaux mine´rales naturelles (80/777/CEE), Journal Officiel des Communaute´s Europe´ennes L 229 (1980) 1–10. [2] N. Baida, A. Yazourh, E. Singer, D. Izard, Pseudomonas brenneri sp. nov., a new species isolated from natural mineral waters, Res. Microbiol. 152 (2000) 493–502. [3] N. Baida, A. Yazourh, E. Singer, D. Izard, Pseudomonas grimontii sp. nov., Int. J. Syst. Evol. Microbiol. 52 (2002) 1497–1503. [4] J.I. Baldani, B. Pot, G. Kirchhof, E. Falsen, V.L.D. Baldani, F.L. Olivares, B. Hoste, K. Kersters, A. Hartmann, M. Gillis, J. Dobereiner, Emended description of Herbaspirillum; inclusion of [Pseudomonas] rubrisubalbicans, a mild plant pathogen, as Herbaspirillum rubrisubalbicans comb. nov.; and classification of a group of clinical isolates (EF group 1) as Herbaspirillum species 3, Int. J. Syst. Bacteriol. 46 (1996) 802–810. [5] J.P. Bowman, L.I. Sly, A.C. Hayward, Y. Spiegel, E. Stackebrandt, Telluria mixta (Pseudomonas mixta Bowman, Sly, and Hayward 1988) gen. nov., comb. nov., and Telluria chitinolytica sp. nov., soil-dwelling organisms which actively degrade polysaccharides, Int. J. Syst. Bacteriol. 43 (1993) 120–124. [6] J. De Ley, P. Segers, M. Gillis, Intra- and intergeneric similarities of Chromobacterium and Janthinobacterium ribosomal ribonucleic acid cistrons, Int. J. Syst. Bacteriol. 28 (1978) 154–168. [7] L. Ding, A. Yokota, Proposals of Curvibacter gracilis gen. nov., sp. nov. and Herbaspirillum putei sp. nov. for bacterial strains isolated from well water and reclassification of [Pseudomonas] huttiensis, [Pseudomonas] lanceolata, [Aquaspirillum] delicatum and [Aquaspirillum] autotrophicum as Herbaspirillum huttiense comb. nov., Curvibacter lanceolatus comb. nov., Curvibacter delicatus comb. nov. and Herbaspirillum autotrophicum comb. nov., Int. J. Syst. Evol. Microbiol. 54 (2004) 2223–2230. [8] M.M. Donato, E.A. Seleiro, M.S. da Costa, Polar lipid and fatty acid composition of strains of the genus Thermus, Syst. Appl. Microbiol. 13 (1990) 234–239. [9] M. Elomari, L. Coroler, B. Hoste, M. Gillis, D. Izard, H. Leclerc, DNA relatedness among Pseudomonas strains isolated from natural mineral waters and proposal of Pseudomonas veronii sp. nov., Int. J. Syst. Bacteriol. 46 (1996) 1138–1144. [10] J. Felsenstein, PHYLIP (Phylogenetic Inference Package) Version 3.5.1, Department of Genetics, University of Washington, Seattle, 1993. [11] A.C. Ferreira, P. Vasconcellos Morais, M.S. da Costa, Alteration in total bacteria, iodonitrophenyltetrazolium (INT)-positive bacteria, and heterotrophic plate counts of bottled mineral water, Can. J. Microbiol. 40 (1994) 72–77. [12] M. Freitas, F.A. Rainey, M.F. Nobre, A.J.D. Silvestre, M.S. da Costa, Tepidimonas aquatica sp. nov., a new slightly thermophilic b-Proteobacterium isolated from a hot water tank, Syst. Appl. Microbiol. 26 (2003) 376–381.
ARTICLE IN PRESS C. Fernandes et al. / Systematic and Applied Microbiology 28 (2005) 596–603
[13] M.E. Heimbrook, W.L.L. Wang, G. Campbell, Staining bacterial flagella easily, J. Clin. Microbiol. 27 (1989) 2612–2615. [14] A. Hiraishi, Y.K. Shin, J. Sugiyama, Proposal to reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov., Int. J. Syst. Bacteriol. 47 (1997) 1249–1252. [15] J.A. Hudson, K.M. Schofield, H.W. Morgan, R.M. Daniel, Thermonema lapsum gen. nov., sp. nov., a thermophilic gliding bacterium, Int. J. Syst. Bacteriol. 39 (1989) 485–487. [16] T. Iizuka, S. Yamanaka, T. Nishiyama, A. Hiraishi, Isolation and phylogenetic analysis of aerobic copiotrophic ultramicrobacteria from urban soil, J. Gen. Appl. Microbiol. 44 (1998) 75–84. [17] W.T. Im, H.S. Bae, A. Yokota, S.T. Lee, Herbaspirillum chlorophenolicum sp. nov., a 4-chlorophenol-degrading bacterium, Int. J. Syst. Evol. Microbiol. 54 (2004) 851–855. [18] T.H. Jukes, C.R. Cantor, Evolution of protein molecules, In: H.N. Munro (Ed.), Mammalian Protein Metabolism, Academic Press, New York, 1969, pp. 21–132. [19] G. Kirchhof, B. Eckert, M. Stoffels, J.I. Baldani, V.M. Reis, A. Hartmann, Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4fibre plants, Int. J. Syst. Evol. Microbiol. 51 (2001) 157–168. [20] H. Leclerc, M.S. da Costa, The microbiology of natural water, In: D.A.G. Senior, P.R. Ashurst (Eds.), Technology of Bottled Water, first ed., Sheffield Academic Press, 1998, pp. 223–263. [21] W.J. Li, Y.Q. Zhang, D.J. Park, C.T. Li, L.H. Xu, C.J. Kim, C.L. Jiang, Duganella violaceinigra sp. nov., a novel mesophilic bacterium isolated from forest soil, Int. J. Syst. Evol. Microbiol. 54 (2004) 1811–1814. [22] S.P. Lincoln, T.R. Fermor, B.J. Tindall, Janthinobacterium agaricidamnosum sp. nov., a soft rot pathogen of Agaricus bisporus, Int. J. Syst. Bacteriol. 49 (1999) 1577–1589. [23] B.L. Maidak, J.R. Cole, T.G. Lilburn, C.T.JR. Parker, P.R. Saxman, R.J. Farris, J.M. Garrity, G.J. Olsen, T.M. Schmidt, J.M. Tiedje, The RDP-II (Ribosomal Database Project), Nucleic Acids Res. 29 (2001) 173–174. [24] M. Mesbash, U. Premachandran, W.B. Whitman, Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography, Int. J. Syst. Bacteriol. 39 (1989) 159–167. [25] P.V. Morais, M.S. da Costa, Alteration in the major heterotrophic bacterial populations isolated from a still bottled mineral water, J. Appl. Bacteriol. 69 (1990) 750–757.
603
[26] C. Moreira, F.A. Rainey, M.F. Nobre, M.T. da Silva, M.S. da Costa, Tepidimonas ignava gen. nov., sp. nov., a new chemolithoheterotrophic and slightly thermophilic member of the b-Proteobacteria, Int. J. Syst. Evol. Microbiol. 50 (2000) 735–742. [27] P. Nielsen, D. Fritze, F.G. Priest, Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species, Microbiology 141 (1995) 1745–1761. [28] O.C. Nunes, M.M. Donato, M.S. da Costa, Isolation and characterization of Rhodothermus strains from S. Miguel, Azores, Syst. Appl. Microbiol. 15 (1992) 92–97. [29] F.A. Rainey, N. Ward-Rainey, R.M. Kroppenstedt, E. Stackebrandt, The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov., Int. J. Syst. Bacteriol. 46 (1996) 1088–1092. [30] D.J. Reasoner, E.E. Geldreich, A new medium for the enumeration and subculture of bacteria from potable water, Appl. Environ. Microbiol. 49 (1985) 1–7. [31] M.A. Santos, R.A.D. Williams, M.S. da Costa, Numerical taxonomy of Thermus isolates from hot springs in Portugal, Syst. Appl. Microbiol. 12 (1989) 310–315. [32] R.M. Smibert, N.R. Krieg, General characterization, In: P. Gerhardt, R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg, G.B. Phillips (Eds.), Manual Methods for General Bacteriology, Washington DC, 1981, pp., 409–443. [33] S. Tenreiro, M.F. Nobre, F.A. Rainey, C. Miguel, S.M. da Costa, Thermonema rossianum sp. nov., a new thermophilic and slightly halophilic species from hot springs in Naples, Italy, Int. J. Syst. Bacteriol. 47 (1997) 122–126. [34] B.J. Tindall, Fully saturated menaquinones in the archaebacterium Pyrobaculum islandicum, FEMS Microbiol. Lett. 60 (1989) 251–254. [35] S. Verhille, N. Baida, F. Dabboussi, D. Izard, H. Leclerc, Taxonomic study of bacteria isolated from natural mineral waters: proposal of Pseudomonas jessenii sp. nov. and Pseudomonas mandelii sp. nov., Syst. Appl. Microbiol. 22 (1999) 45–58. [36] M. Wiedmann-al-Ahmad, H.V. Tichy, G. Schon, Characterization of Acinetobacter type strains and isolates obtained from wastewater treatment plants by PCR fingerprinting, Appl. Environ. Microbiol. 60 (1994) 4066–4071. [37] R.A.D. Williams, M.S. da Costa, The genus Thermus and related microorganisms, In: A. Balows, H.G. Truper, M. Dworkin, W. Harder, K.H. Schleifer (Eds.), The Prokaryotes: a Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, Second ed., Springer, New York, 1992, pp. 478–490.