| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan
2 Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan
Correspondence
Yuji Sekiguchi
y.sekiguchi{at}aist.go.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains IMO-1T, YMTK-2T and KIBI-1T are AB109437–AB109439.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
; Hugenholtz et al., 1998; Rappé & Giovannoni, 2003). The bacterial phylum Chloroflexi has also been recognized as a typical bacterial lineage that contains a number of environmental 16S rRNA gene sequences with a few cultured representatives. The phylum Chloroflexi is currently known to be divided phylogenetically into at least five major classes (subphyla), including the classes ‘Chloroflexi’ and Thermomicrobia. The class ‘Chloroflexi’ was proposed by Garrity & Holt (2001) as the best characterized class in the phylum, in which various photosynthetic bacteria (previously known as green non-sulphur bacteria) as well as non-photosynthetic microbes are classified. The class Thermomicrobia, with which two cultured species, Thermomicrobium roseum and Sphaerobacter thermophilus, are affiliated (Hugenholtz & Stackebrandt, 2004), was recently transferred to the phylum Chloroflexi. In addition to these formally proposed taxa, Hugenholtz & Stackebrandt (2004) informally proposed the remaining three subphylum-level taxonomic groups of the phylum Chloroflexi, the classes ‘Anaerolineae’ and ‘Dehalococcoidetes’ and a clone cluster (at subphylum level) called ‘subphylum IV’ (SAR202 cluster) (Morris et al., 2004).
Among these classes, the ‘Anaerolineae’ contained a vast number of environmental 16S rRNA gene sequences with only a few cultured strains. Since ‘Anaerolineae’-type clones have been found frequently within various ecosystems, the organisms within the group have been thought to be ubiquitous and to play important roles in these ecosystems (Hugenholtz et al., 1998). Two thermophilic, multicellular filamentous organisms, Anaerolinea thermophila and Calidilinea aerophila, were recently isolated from an anaerobic granular sludge and a hot spring, respectively, and were characterized in detail (Sekiguchi et al., 2003); these two strains are the only cultured representatives of the subphylum. In addition to the two thermophiles, we recently reported one thermophilic and two mesophilic filamentous strains belonging to the ‘Anaerolineae’ newly isolated from mesophilic and thermophilic upflow anaerobic sludge blanket (UASB) sludge granules (Yamada et al., 2005).
In this paper, we surveyed the characteristics of these three filamentous strains, IMO-1T, KIBI-1T and YMTK-2T, in more detail to outline their taxonomic placements. Based on the morphological, physiological, chemotaxonomic and phylogenetic analysis, we propose novel species to include each strain, two of which are placed in new genera. In addition, we formally propose two classes, Anaerolineae classis nov. and Caldilineae classis nov., and also propose several subordinate taxa from order- to family-level lineages.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). Strains KIBI-1T and YMTK-2T were isolated from sludge granules of mesophilic UASB reactors treating an actual wastewater discharged from a sugar-processing plant and an artificial wastewater mainly composed of sucrose and volatile fatty acids (Yamada et al., 2005). Detailed isolation procedures for all the strains were described in our previous paper (Yamada et al., 2005). Methanospirillum hungatei DSM 864T and Methanothermobacter thermautotrophicus DSM 1053T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Braunschweig, Germany).
Cultivation conditions.
Cultivation of all filamentous microbes was performed with the basal medium used in previous studies (Sekiguchi et al., 2000; Yamada et al., 2005). Cultivation of strains KIBI-1T and YMTK-2T was carried out at 37 °C, whereas strain IMO-1T was cultivated at 55 °C. Neutralized substrates were added to the vials from stock solutions prior to inoculation. Methanospirillum hungatei DSM 864T and Methanothermobacter thermautotrophicus DSM 1053T were incubated in the medium mentioned above, with an exception that hydrogen (approx. 1 Pa) was included in the gas phase (80 % N2, 20 % CO2, v/v) in the vials as an energy source. These cultures were incubated at 37 or 55 °C. Solid medium was prepared by adding purified agar (Agar noble; Difco) to the medium described above at a final concentration of 20 g l–1. PE medium (Hanada et al., 1995) was used for phototrophic and aerobic growth tests. All the tests were performed under optimal pH and temperature conditions.
Effect of pH and temperature.
To determine the optimal pH for growth, the pH of the basal medium was adjusted at room temperature to 5.0–9.0 by adding HCl or NaOH under a 100 % N2 atmosphere. pH-adjusted media for strains IMO-1T and KIBI-1T were supplemented with sucrose (20 mM) and yeast extract (0.1 %), whereas media for strain YMTK-2T were supplemented with yeast extract (0.1 %) as carbon and energy source. All cultivations were performed in triplicate (2 % inoculum per medium volume) and growth was evaluated by both the increase in optical density (OD400) and production of hydrogen and acetate in the vials.
To judge the optimal temperature and temperature range for growth, we prepared the medium described above (i.e. gas phase of 80 % N2 and 20 % CO2, v/v; pH25 °C 7.0). Temperature tests for the two mesophilic isolates were carried out at 15, 25, 30, 35, 37, 40, 45, 50, 55 and 60 °C, whereas those for the thermophilic strain were performed at 25, 37, 40, 42, 45, 50, 52, 55, 60, 65 and 70 °C. All tests were done in triplicate (2 % inoculum per medium volume). Growth was evaluated by measuring both OD400 and the production of hydrogen and acetate in the vials.
Substrate utilization and syntrophic growth.
To examine the substrate utilization of the isolates, they were cultivated in medium (containing 0.01 % yeast extract) supplemented individually with 36 autoclaved or filter-sterilized compounds such as proteins, sugars, alcohols, aromatic compounds and volatile fatty acids (see below for details). All substrates and electron acceptors, including ferric nitrilotriacetate [Fe(III)-NTA] (Roden & Lovley, 1993), were prepared as described previously (Sekiguchi et al., 2000). In all tests, cultivation was performed for 1 month under optimal growth conditions for each strain (2 % inoculum).
Syntrophic growth was tested for strains IMO-1T and KIBI-1T using medium supplemented with sucrose and yeast extract, whereas strain YMTK-2T was evaluated using medium with yeast extract. For the two mesophilic strains (strains KIBI-1T and YMTK-2T), pre-grown cells of Methanospirillum hungatei DSM 864T (2 % inoculum) were added as the hydrogen-/formate-consuming organism in the vials prior to cultivation. For the thermophilic strain (IMO-1T), cells of Methanothermobacter thermautotrophicus DSM 1053T (2 % inoculum) were added. Substrate utilization and syntrophic growth were examined with production of methane and acetate, as well as increase in OD400 in the vials.
Analytical methods.
Short-chain fatty acids, methane, hydrogen and carbon dioxide were determined by GC as described previously (Imachi et al., 2000, 2002). Alcohols and other compounds were determined by HPLC as described previously (Imachi et al., 2000, 2002). Carbohydrates such as glucose and sucrose were measured by HPLC using an SCR-101-H column (Sekiguchi et al., 2001).
Microscopy.
Cell morphology of the strains was observed with a phase-contrast microscope (Olympus AX80T). For microscopy, agar-coated slides were prepared and microbial cells were immobilized on the slides (Pfennig & Wagener, 1986). Gram-staining was done by Hucker's method (Doetsch, 1981). Observation of the strains by transmission electron microscopy (TEM) was performed according to Sekiguchi et al. (2003).
Determination of DNA base content.
DNA was extracted and purified as described previously (Kamagata & Mikami, 1991). The G+C content of all isolates was determined by HPLC (Shimadzu LC-6A) with a UV detector (Shintani et al., 2000).
Determination of quinones and fatty acid methyl ester (FAME) analysis.
Quinones were determined as described previously (Zhang et al., 2003). For FAME analysis, cells of strains KIBI-1T and IMO-1T were harvested from cultures grown on medium containing sucrose (20 mM) plus yeast extract (0.1 %), whereas FAMEs of strain YMTK-2T were prepared from cultures grown on medium with yeast extract (0.1 %). All of the isolates were cultivated under optimal growth conditions. Fatty acids were converted to methyl esters by using HCl/methanol and analysed by GC with a mass spectrometer (Hitachi M7200A FC/3DQMS system) (Hanada et al., 2002).
Sequencing and phylogenetic analyses.
16S rRNA gene sequences of all three strains were reported previously (Yamada et al., 2005). The sequence data were aligned in an ARB dataset using the ARB program package, and the aligned data were corrected manually by using the editing tool in the package (Ludwig et al., 2004). Phylogenetic trees based on the 16S rRNA gene dataset and various outgroups were constructed by three different algorithms, the neighbour-joining method (Saitou & Nei, 1987) within the ARB program package (Ludwig et al., 2004), the maximum-parsimony method within the PAUP* 4.0 package (Swofford, 2002) and maximum-likelihood method within the TREEFINDER program package (Jobb, 2005). Bootstrap resampling analyses (Felsenstein, 1985) for 1000 replicates were performed for the neighbour-joining, maximum-likelihood and maximum-parsimony analyses to estimate the confidence of tree topologies. In addition, posterior probabilities of branching points were estimated by Bayesian inference using MrBayes version 3.1 (Ronquist & Huelsenbeck, 2003) as mentioned previously (Zhang et al., 2003).
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). In all cultures, small, colourless, lens-shaped colonies, 0.1–0.2 mm in diameter, were formed on anaerobic solid medium supplemented with sucrose (20 mM) and yeast extract (0.1 %) after 2–4 weeks of incubation (Yamada et al., 2005).
All three strains formed flexible filaments of indefinite length (generally longer than 100 µm). The cell widths for strains IMO-1T and KIBI-1T were respectively 0.3–0.4 µm and 0.4–0.5 µm. Cells of strain YMTK-2T were found to be much thinner (0.15–0.2 µm) than those of the other strains (Figs 1 and 2). The strains stained Gram-negative. TEM also indicated a Gram-negative-type cell-wall structure, showing multicellular forms in filaments (Fig. 2). The electron micrographs also suggested that the strains did not possess a clearly visible sheath-like structure (Fig. 2). Individual cells of all three strains were found to be longer than 2 µm (Fig. 2). Based on observations of cell movement under a microscope and of colony morphology of all isolates formed on solid media, the strains were found to have no gliding motility. Spore formation was not observed under any culture conditions.
|
|
Utilization of various substrates (added at final concentrations of 20 mM unless specified) was tested in the presence of yeast extract (0.01 %). All three strains utilized glucose, fructose, ribose, xylose, sucrose, raffinose and tryptone (0.1 %). Strain IMO-1T also utilized mannose, galactose, arabinose and pyruvate, strain KIBI-1T utilized pyruvate and strain YMTK-2T utilized mannose, xylan, pectin and betaine. Casamino acids (0.1 %) supported weak growth and acid formation for all three strains, as did the following compounds: starch (5 g l–1), xylan, pectin and peptone (0.1 %) for strain IMO-1T, xylan, pectin, peptone (0.1 %), betaine, mannose and galactose for strain KIBI-1T and starch (5 g l–1), peptone (0.1 %), galactose, arabinose and pyruvate for strain YMTK-2T. None of the strains utilized crotonate, H2/CO2 (1 atm) plus acetate (10 mM), lactate, glycerol, fumarate, acetate, propionate, butyrate, malate, succinate, ethanol, methanol, 1-propanol, ethylene glycol, hydroquinone (1 mM), phenol (1 mM), benzoate (5 mM) or formate plus acetate (10 mM). Strain IMO-1T did not utilize betaine.
Major fermentative products in medium containing glucose (20 mM) and yeast extract (0.01 %) were acetate, lactate and hydrogen for strain IMO-1T, acetate, formate and hydrogen with a small amount of lactate for strain KIBI-1T and acetate, lactate, pyruvate and hydrogen with small amounts of succinate and formate for strain YMTK-2T. The three strains could not utilize any of the following compounds as electron acceptors within 4 weeks of incubation (glucose and yeast extract medium): 20 mM sulphate, 1 mM sulphite, 20 mM thiosulphate, 20 mM elemental sulphur, 20 mM nitrate, 20 mM fumarate and 5 mM Fe(III)-NTA.
The optimal temperatures for growth were 50 °C (range 42–55 °C) for strain IMO-1T and around 37 °C (range 25–50 °C) for strains KIBI-1T and YMTK-2T. The optimal pH for growth was around pH 7.0 for all three strains; the strains grew at pH 6.0–7.5 (strain IMO-1T) or pH 6.0–7.2 (strains KIBI-1T and YMTK-2T).
Growth of strain IMO-1T was found to stagnate after an amount of hydrogen (approx. 10 kPa) accumulated in medium supplemented with glucose and yeast extract. No significant improvement of growth of strain KIBI-1T or YMTK-2T was observed in co-cultivation with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T (Yamada et al., 2005). NaCl was slightly inhibitory for growth of all three strains at 2.5 g l–1; growth of strains IMO-1T and YMTK-2T was inhibited completely by 15 g NaCl l–1, whereas growth of strain KIBI-1T was inhibited only slightly by 30 g NaCl l–1.
Chemotaxonomic analyses
The DNA G+C content of strain IMO-1T was 53.3 mol%, and those of strains KIBI-1T and YMTK-2T were respectively 59.5 and 48.2 mol%. FAME analysis showed that cells of strain IMO-1T contained anteiso-C17 : 0 (21 %), iso-C15 : 0 (19 %), C16 : 0 (16 %) and anteiso-C15 : 0 (12 %) as the major fatty acids, with C18 : 0 (6 %), iso-C17 : 0 (5 %), iso-C13 : 0 (5 %), C14 : 0 (5 %), anteiso-C13 : 0 (3 %), C17 : 0 (3 %), iso-C16 : 0 (2 %), C15 : 0 (2 %) and C12 : 0 (1 %) as minor components. Cells of strain KIBI-1T contained branched C14 : 0 (30 %), iso-C15 : 0 (22 %), C16 : 0 (14 %) and branched C17 : 0 (13 %) as the major fatty acids, with C16 : 1 (
9) cis (8 %), C12 : 0 (4 %), branched C13 : 0 (4 %), branched C14 : 0 (3 %), C18 : 0 (1 %) and branched C15 : 0 (1 %) as minor fatty acids. Cells of strain YMTK-2T contained branched C17 : 0 (41 %), C16 : 0 (25 %), C14 : 0 (11 %) and C17 : 0 (9 %) as the major fatty acids, with branched C15 : 0 (5 %), C12 : 0 (2 %), branched C13 : 0 (2 %), C16 : 1 (9) cis (2 %), anteiso-C15 : 0 (1 %), branched C16 : 0 (1 %) and branched-C19 : 0 (1 %) as minor components. No quinones were detected from cells of any of the strains cultivated under anaerobic conditions.
Phylogenetic analysis
In our previous study, the most closely related, previously characterized micro-organism to all the strains was A. thermophila (16S rRNA gene sequence similarity of strains IMO-1T, KIBI-1T and YMTK-2T with A. thermophila UNI-1T of respectively 93, 88 and 88 %; Yamada et al., 2005). In addition, the 16S rRNA gene sequence similarity among the three strains was found previously to be lower than 89 % (Yamada et al., 2005), indicating at least species-level novelty of each strain.
To examine the monophyly of the tentatively named, subphylum-level group ‘Anaerolineae’ (Hugenholtz & Stackebrandt, 2004), we performed more detailed phylogenetic analyses including 16S rRNA gene sequences of members of the class Thermomicrobia (containing the genera Thermomicrobium, Sphaerobacter and Thermobaculum), with sequences of a variety of bacteria representing multiple phyla as outgroups (Fig. 3). In addition to posterior probabilities of branching points based on Bayesian inference, bootstrap values on the basis of maximum-parsimony and maximum-likelihood indicated that the node support of the group ‘Anaerolineae’ was lower than 90 %, although the node support estimated with neighbour-joining analysis was 94 % (Fig. 3). These facts suggested that the group ‘Anaerolineae’ might not be a monophyletic taxon as suggested by Hugenholtz & Stackebrandt (2004). In particular, the Caldilinea cluster often did not form a clade with other members of the group ‘Anaerolineae’ within four phylogenetic algorithms. In addition, the group ‘Anaerolineae’ clearly exhibited monophyletic taxon status in the same estimations when members affiliated with the Caldilinea cluster were excluded (data not shown). Thus, it was concluded that the group ‘Anaerolineae’ should be subdivided phylogenetically into two class-level taxa (Fig. 3).
|
, 2003). The phenotypic features of strain IMO-1T were very similar to those of A. thermophila. However, comparative analysis of the 16S rRNA gene sequence of strain IMO-1T with that of A. thermophila suggested that strain IMO-1T should be differentiated from A. thermophila at the species level (Stackebrandt & Goebel, 1994). In addition, strain IMO-1T can be differentiated from A. thermophila in several phenotypic respects (Table 1): (i) the presence of branched fatty acids as major cellular fatty acids; (ii) slightly thicker filaments; (iii) clearly faster growth. These genotypic and phenotypic differences justify the creation of a novel species of the genus Anaerolinea to accommodate strain IMO-1T, for which we propose the name Anaerolinea thermolimosa sp. nov.
|
). In addition, the mesophilic strains KIBI-1T and YMTK-2T also exhibited several distinct phenotypic features: (i) cells of strain KIBI-1T were clearly thicker than those of strain YMTK-2T; (ii) strain KIBI-1T had a higher G+C content than strain YMTK-2T; (iii) substrate utilization for the two strains was slightly different. Phylogenetic analyses with 16S rRNA gene sequences of strains KIBI-1T and YMTK-2T indicated that the two organisms formed a cluster independent from A. thermophila and strain IMO-1T, and the sequence similarity of the strains with strains of the genus Anaerolinea was lower than 90 %. Furthermore, the 16S rRNA gene sequence similarity between strain KIBI-1T and YMTK-2T (91 %) was too low to classify both strains into a single genus. These findings justify the creation of the new genera and species Levilinea saccharolytica gen. nov., sp. nov. and Leptolinea tardivitalis gen. nov., sp. nov. for strains KIBI-1T and YMTK-2T, respectively. Both genera should be affiliated with the same family as the genus Anaerolinea.
Subdivision of the group ‘Anaerolineae’
The putative class- (subphylum)-level group ‘Anaerolineae’ now contains four cultured genera, Anaerolinea, Caldilinea, Levilinea and Leptolinea (Sekiguchi et al., 2003). Based on the phylogenetic placements of these organisms, we propose to subdivide ‘Anaerolineae’ into two new classes, Anaerolineae classis nov. and Caldilineae classis nov. (Fig. 3). The notable phenotypic property that clearly distinguishes members of Anaerolineae (the genera Anaerolinea, Levilinea and Leptolinea) from Caldilineae (the genus Caldilinea) is oxygen respiration: members of the class Anaerolineae are obligately anaerobic, while members of the class Caldilineae are facultatively aerobic. More strains belonging to the two classes (as well as comparative genomics of the two classes) might lend further support to the distinctiveness of these classes.
We also propose subordinate taxa from order (Anaerolineales ord. nov. and Caldilineales ord. nov.) to family (Anaerolineaceae fam. nov. and Caldilineaceae fam. nov.) for the classes Anaerolineae and Caldilineae.
Description of Anaerolineae classis nov.
Anaerolineae (An.ae.ro.li.ne'ae. N.L. fem. pl. n. Anaerolineales type order of the class; -ae ending to denote a class; N.L. fem. pl. n. Anaerolineae the Anaerolineales class).
The class Anaerolineae is defined on the basis of a phylogenetic tree by comparative 16S rRNA gene sequence analysis from four representatives and a wide variety of environmental clones. Gram-negative. Cells are not coloured. Negative for gliding motility. Multicellular filamentous organisms. Spores are not formed. No photosynthetic growth is observed. Grow under strictly anaerobic conditions. Chemo-organotrophs. No quinone is detected. The type order is Anaerolineales.
Description of Anaerolineales ord. nov.
Anaerolineales (An.ae.ro.li.ne.al'es. N.L. fem. n. Anaerolinea type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Anaerolineales order of the genus Anaerolinea).
The description is the same as for the class Anaerolineae. The type genus is Anaerolinea.
Description of Anaerolineaceae fam. nov.
Anaerolineaceae (An.ae.ro.li.ne.a'ce.ae. N.L. fem. n. Anaerolinea type genus of the family; -aceae ending to denote a family; N.L. fem. adj. Anaerolineaceae family of the genus Anaerolinea).
The description is the same for the class Anaerolineae. The type genus is Anaerolinea.
Emended description of the genus Anaerolinea Sekiguchi et al. 2003
Anaerolinea (An.ae.ro.li.ne'a. Gr. pref. an not; Gr. masc. n. aer air; L. fem. n. linea line; N.L. fem. n. Anaerolinea line-shaped organisms not living in air).
Gram-negative. Cells are non-motile and filamentous. Spores are not formed. Thermophilic. Grow under strictly anaerobic conditions. Neither photosynthetic nor aerobic growth is observed. The cellular fatty acids consist mainly of straight-chain saturated fatty acids (e.g. C16 : 0 and C18 : 0) and branched-chain fatty acids (e.g. anteiso-C17 : 0). The G+C content of genomic DNA is 53.3–54.5 mol%. Phylogenetic position is in the family Anaerolineaceae, order Anaerolineales, class Anaerolineae of the phylum Chloroflexi. The type species is Anaerolinea thermophila.
Description of Anaerolinea thermolimosa sp. nov.
Anaerolinea thermolimosa (ther.mo.li.mo'sa. Gr. adj. thermos hot; L. fem. adj. limosa muddy, pertaining to sludge; N.L. fem. adj. thermolimosa living in thermophilic sludge).
Has the following properties in addition to those described for the genus. The main fatty acids are anteiso-C17 : 0, iso-C15 : 0, C16 : 0 and anteiso-C15 : 0. The G+C content of genomic DNA is 53.3 mol%. Multicellular filaments are longer than 100 µm in length and 0.3–0.4 µm in width. Growth occurs between 42 and 55 °C with optimum growth at 50 °C. Growth is observed at pH 6.0–7.5 with optimum growth at pH 7.0. The doubling time for growth is 48 h under optimal growth conditions. Growth is stimulated significantly when the organism is co-cultivated with hydrogenotrophic methanogens. Yeast extract is required for growth. In the presence of 0.01 % yeast extract, growth and substrate utilization are observed with glucose, mannose, galactose, fructose, ribose, xylose, arabinose, sucrose, raffinose, tryptone and pyruvate. Weak growth occurs with the following substrates with medium supplemented with 0.01 % yeast extract: Casamino acids, starch, xylan, pectin and peptone. The following substrates are not utilized in the presence of 0.01 % yeast extract: crotonate, H2/CO2 plus acetate, betaine, lactate, glycerol, fumarate, acetate, propionate, butyrate, malate, succinate, ethanol, methanol, 1-propanol, ethylene glycol, hydroquinone, phenol, benzoate and formate plus acetate. Sulphate, sulphite, thiosulphate, elemental sulphur, nitrate, fumarate and Fe(III)-NTA are not utilized as electron acceptors.
The type strain is strain IMO-1T (=JCM 12577T=DSM 16554T), isolated from sludge from a thermophilic UASB reactor in which wastewater from manufacture of a Japanese distilled alcohol (shochu) is treated.
Description of Levilinea gen. nov.
Levilinea (Le.vi.li'ne.a. L. adj. levis smooth; L. fem. n. linea line; N.L. fem. n. Levilinea smooth, line-shaped organism).
Gram-negative. Cells are non-motile. Multicellular and filamentous morphology with indefinite length. Mesophiles. Spores are not formed. Grow under strictly anaerobic conditions. No photosynthetic growth is observed. The main fatty acids are branched C14 : 0, iso-C15 : 0, C16 : 0 and branched C17 : 0. The G+C content of genomic DNA is 59.5 mol%. The phylogenetic position is in the family Anaerolineaceae. The type species is Levilinea saccharolytica.
Description of Levilinea saccharolytica sp. nov.
Levilinea saccharolytica (sac.cha.ro.ly'ti.ca. Gr. n. saccharon sugar; N.L. fem. adj. lytica from Gr. adj. lutikos able to loosen; N.L. fem. adj. saccharolytica saccharolytic, using various sugars).
Multicellular filaments are longer than 100 µm and 0.4–0.5 µm wide. Growth occurs between 25 and 50 °C with optimum growth at 37 °C. Growth is observed at pH 6.0–7.2 with optimum growth at pH 7.0. Doubling time for growth is 56 h under optimal growth conditions. Yeast extract is required for growth. In the presence of 0.01 % yeast extract, growth is observed with glucose, fructose, xylose, ribose, sucrose, raffinose, tryptone, pyruvate and pectin. Weak growth occurs with the following substrates with medium supplemented with 0.01 % yeast extract: Casamino acids, peptone, betaine, mannose, galactose and xylan. The following substrates are not utilized in the presence of yeast extract: crotonate, H2/CO2 plus acetate, lactate, glycerol, fumarate, acetate, propionate, butyrate, malate, succinate, ethanol, methanol, 1-propanol, arabinose, starch, ethylene glycol, hydroquinone, phenol, benzoate and formate plus acetate. Sulphate, sulphite, thiosulphate, elemental sulphur, nitrate, fumarate and Fe(III)-NTA are not utilized as electron acceptors.
The type strain is KIBI-1T (=JCM 12578T=DSM 16555T), isolated from sludge in a mesophilic UASB reactor in which sugar-manufacturing wastewater is treated.
Description of Leptolinea gen. nov.
Leptolinea (Lep.to.li'ne.a. Gr. adj. leptus fine; L. fem. n. linea line; N.L. fem. n. Leptolinea fine, line-shaped organism).
Gram-negative. Cells are non-motile, forming very fine multicellular filaments (0.2 µm wide or less). Mesophiles. Spores are not formed. Grow under strictly anaerobic conditions. No photosynthetic growth is observed. The main fatty acids are branched C17 : 0, C16 : 0, C14 : 0 and C17 : 0. The G+C content of genomic DNA is 48.2 mol%. The phylogenetic position is in the family Anaerolineaceae. The type species is Leptolinea tardivitalis.
Description of Leptolinea tardivitalis sp. nov.
Leptolinea tardivitalis (tar.di.vi.ta'lis. L. adj. tardus slow; L. fem. adj. vitalis vital, alive; N.L. fem. adj. tardivitalis having a slow lifestyle).
Multicellular filaments are longer than 100 µm and thinner than 0.2 µm. Growth occurs between 25 and 50 °C with optimum growth at 37 °C. Growth is observed at pH 6.0–7.2 with optimum growth at pH 7.0. Doubling time for growth is 50 h under optimal growth conditions. In the presence of 0.01 % yeast extract, growth is observed with glucose, mannose, fructose, xylose, ribose, sucrose, raffinose, xylan, pectin, tryptone and betaine. Weak growth occurs with the following substrates with medium supplemented with 0.01 % yeast extract: Casamino acids, peptone, galactose, arabinose, starch and pyruvate. The following substrate are not utilized in the presence of 0.01 % yeast extract: crotonate, H2/CO2 plus acetate, lactate, glycerol, fumarate, acetate, propionate, butyrate, malate, succinate, ethanol, methanol, 1-propanol, ethylene glycol, hydroquinone, phenol, benzoate and formate plus acetate. Sulphate, sulphite, thiosulphate, elemental sulphur, nitrate, fumarate and Fe(III)-NTA are not utilized as electron acceptors.
The type strain is strain YMTK-2T (=JCM 12579T=DSM 16556T), isolated from sludge in a mesophilic UASB reactor in which an artificial wastewater composed mainly of peptone, sucrose, propionate and acetate is treated.
Description of Caldilineae classis nov.
Caldilineae (Cal.di.li'ne.ae. N.L. fem. pl. n. Caldilineales type order of the class; -ae ending to denote a class; N.L. fem. pl. n. Caldilineae the Caldilineales class).
The description is the same as that for the genus Caldilinea given by Sekiguchi et al. (2003). The type order is Caldilineales.
Description of Caldilineales ord. nov.
Caldilineae (Cal.di.li.ne.al'es. N.L. fem. n. Caldilinea type genus of the order; -ales ending to denote a order; N.L. fem. pl. n. Caldilineales order of the genus Caldilinea).
The description is the same as that for the genus Caldilinea given by Sekiguchi et al. (2003). The type genus is Caldilinea.
Description of Caldilineaceae fam. nov.
Caldilineaceae (Cal.di.li.ne.a'ce.ae. N.L. fem. n. Caldilinea type genus of the family; -aceae ending to denote a family; N.L. fem. n. Caldilineaceae family of the genus Caldilinea).
The description is the same as that for the genus Caldilinea given by Sekiguchi et al. (2003). The type genus is Caldilinea.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Garrity, G. M. & Holt, J. G. (2001). Phylum BVI. Chloroflexi phy. nov. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, p. 427. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springer.
Hanada, S., Hiraishi, A., Shimada, K. & Matsuura, K. (1995). Isolation of Chloroflexus aurantiacus and related thermophilic phototrophic bacteria from Japanese hot springs using an improved isolation procedure. J Gen Appl Microbiol 41, 119–130.
Hanada, S., Takaichi, S., Matsuura, K. & Nakamura, K. (2002). Roseiflexus castenholzii gen. nov., sp. nov., a thermophilic, filamentous, photosynthetic bacterium that lacks chlorosomes. Int J Syst Evol Microbiol 52, 187–193.[Abstract]
Hugenholtz, P. (2002). Exploring prokaryotic diversity in the genomic era. Genome Biol 3, 1–8.[Medline]
Hugenholtz, P. & Stackebrandt, E. (2004). Reclassification of Sphaerobacter thermophilus from the subclass Sphaerobacteridae in the phylum Actinobacteria to the class Thermomicrobia (emended description) in the phylum Chloroflexi (emended description). Int J Syst Evol Microbiol 54, 2049–2051.
Hugenholtz, P., Goebel, B. M. & Pace, N. R. (1998). Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180, 4765–4774.
Imachi, H., Sekiguchi, Y., Kamagata, Y., Ohashi, A. & Harada, H. (2000). Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. Appl Environ Microbiol 66, 3608–3615.
Imachi, H., Sekiguchi, Y., Kamagata, Y., Hanada, S., Ohashi, A. & Harada, H. (2002). Pelotomaculum thermopropionicum gen. nov., sp. nov., an anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium. Int J Syst Evol Microbiol 52, 1729–1735.[Abstract]
Jobb, G. (2005). TREEFINDER version of June 2005. Distributed by the author. http://www.treefinder.de
Kamagata, Y. & Mikami, E. (1991). Isolation and characterization of a novel thermophilic Methanosaeta strain. Int J Syst Bacteriol 41, 191–196.
Ludwig, W., Strunk, O., Westram, R. & 29 other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.
Morris, R. M., Rappé, M. S., Urbach, E., Connon, S. A. & Giovannoni, S. J. (2004). Prevalence of the Chloroflexi-related SAR202 bacterioplankton cluster throughout the mesopelagic zone and deep ocean. Appl Environ Microbiol 70, 2836–2842.
Pfennig, N. & Wagener, S. (1986). An improved method of preparing wet mounts for photomicrographs of microorganisms. J Microbiol Methods 4, 303–306.[CrossRef]
Rappé, M. S. & Giovannoni, S. J. (2003). The uncultured microbial majority. Annu Rev Microbiol 57, 369–394.[CrossRef][Medline]
Roden, E. E. & Lovley, D. R. (1993). Dissimilatory Fe(III) reduction by the marine microorganism Desulfuromonas acetoxidans. Appl Environ Microbiol 59, 734–742.
Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sekiguchi, Y., Kamagata, Y., Nakamura, K., Ohashi, A. & Harada, H. (2000). Syntrophothermus lipocalidus gen. nov., sp. nov., a novel thermophilic, syntrophic, fatty-acid-oxidizing anaerobe which utilizes isobutyrate. Int J Syst Evol Microbiol 50, 771–779.[Abstract]
Sekiguchi, Y., Takahashi, H., Kamagata, Y., Ohashi, A. & Harada, H. (2001). In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I. Appl Environ Microbiol 67, 5740–5749.
Sekiguchi, Y., Yamada, T., Hanada, S., Ohashi, A., Harada, H. & Kamagata, Y. (2003). Anaerolinea thermophila gen. nov., sp. nov. and Caldilinea aerophila gen. nov., sp. nov., novel filamentous thermophiles that represent a previously uncultured lineage of the domain Bacteria at the subphylum level. Int J Syst Evol Microbiol 53, 1843–1851.
Shintani, T., Liu, W.-T., Hanada, S., Kamagata, Y., Miyaoka, S., Suzuki, T. & Nakamura, K. (2000). Micropruina glycogenica gen. nov., sp. nov., a new Gram-positive glycogen-accumulating bacterium isolated from activated sludge. Int J Syst Evol Microbiol 50, 201–207.[Abstract]
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Swofford, D. L. (2002). PAUP* – Phylogenetic Analysis Using Parsimony* and other methods, version 4. Sunderland, MA: Sinauer Associates.
Yamada, T., Sekiguchi, Y., Imachi, H., Kamagata, Y., Ohashi, A. & Harada, H. (2005). Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules. Appl Environ Microbiol 71, 7493–7503.
Zhang, H., Sekiguchi, Y., Hanada, S., Hugenholtz, P., Kim, H., Kamagata, Y. & Nakamura, K. (2003). Gemmatimonas aurantiaca gen. nov., sp. nov., a Gram-negative, aerobic, polyphosphate-accumulating micro-organism, the first cultured representative of the new bacterial phylum Gemmatimonadetes phyl. nov. Int J Syst Evol Microbiol 53, 1155–1163.
This article has been cited by other articles:
|
|
 |
|
  E. Cardenas, W.-M. Wu, M. B. Leigh, J. Carley, S. Carroll, T. Gentry, J. Luo, D. Watson, B. Gu, M. Ginder-Vogel, et al. Microbial Communities in Contaminated Sediments, Associated with Bioremediation of Uranium to Submicromolar Levels Appl. Envir. Microbiol., June 15, 2008; 74(12): 3718 - 3729. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yamada, H. Imachi, A. Ohashi, H. Harada, S. Hanada, Y. Kamagata, and Y. Sekiguchi Bellilinea caldifistulae gen. nov., sp. nov. and Longilinea arvoryzae gen. nov., sp. nov., strictly anaerobic, filamentous bacteria of the phylum Chloroflexi isolated from methanogenic propionate-degrading consortia Int J Syst Evol Microbiol, October 1, 2007; 57(10): 2299 - 2306. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
