IJSEM Papers in Press. Published May 8, 2014 as doi:10.1099/ijs.0.064576-0
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Roseiarcus fermentans gen. nov. sp. nov., a Bacteriochlorophyll a - Containing
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Fermentative Bacterium Phylogenetically Related to Alphaproteobacterial
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Methanotrophs and Description of the Family Roseiarcaceae fam. nov.
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Irina S. Kulichevskaya1, Olga V. Danilova1, Vera M. Tereshina1, Vadim V.
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Kevbrin1, and Svetlana N. Dedysh1
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Russia
S.N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow 117312,
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Author for correspondence: Svetlana N. Dedysh
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Tel: 7 (499) 135 0591. Fax: 7 (499) 135 6530. Email: [email protected]
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Journal: International Journal of Systematic and Evolutionary Microbiology (IJSEM)
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Contents Category: New Taxa - Proteobacteria
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Running title: Roseiarcus fermentans gen. nov., sp. nov.
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the partial
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sequence of the nifH gene of Roseiarcus fermentans Pf56T are KJ406703 and KJ406704,
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respectively.
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ABSTRACT
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A light-pink-pigmented, microaerophilic bacterium was obtained from a methanotrophic
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consortium enriched from acidic Sphagnum peat and designated strain Pf56T. Cells of this
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bacterium are Gram-negative, non-motile, thick curved rods, which contain a vesicular
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intracytoplasmic membrane system characteristic for some purple non-sulfur
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Alphaproteobacteria. Absorption spectrum of the acetone: methanol extracts of cells grown
7
in the light shows maxima at 363, 475, 505, 601 and 770 nm; the peaks 363 and 770 nm are
8
characteristic for bacteriochlorophyll a. However, in contrast to purple non-sulfur
9
bacteria, strain Pf56T is unable to grow phototrophically under anoxic conditions in the
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light. Best growth occurs on some sugars and organic acids under micro-oxic conditions by
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means of fermentation. The fermentation products are propionate, acetate, and hydrogen.
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Slow chemo-organotrophic growth is also observed under fully oxic conditions. Light
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stimulates growth. C1 substrates are not utilized. Strain Pf56T grows in the pH range 4.0-
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7.0 (optimum 5.5-6.5) and at 15- 30 C (optimum 22–28 °C). The major cellular fatty acids
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are 19:0 CYCLO ω8c and 18:1ω7c; quinones are represented by ubiquinone Q10. The
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G+C content of the DNA is 70.0 mol%. Strain Pf56 displays 93.6-94.7 and 92.7-93.7% 16S
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rRNA gene sequence similarity to members of the families Methylocystaceae and
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Beijerinckiaceae, respectively, and belongs to a large cluster of environmental sequences
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retrieved in cultivation-independent studies from various wetlands and forest soils.
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Phenotypic, genotypic and chemotaxonomic characteristics of strain Pf56 T suggest that it
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represents a novel genus and species of bacteriochlorophyll a-containing fermentative
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bacteria, for which the name Roseiarcus fermentans is proposed. Strain Pf56T (=DSM
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24875T = VKM B-2876T) is the type strain, which is also the first characterized member of
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a novel family within the class Alphaproteobacteria, Roseiarcaceae fam. nov.
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Keywords: Roseiarcus fermentans gen. nov., sp. nov., Roseiarcaceae fam. nov.,
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bacteriochlorophyll a-containing bacteria, fermentative growth, acidic wetlands.
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The class Alphaproteobacteria accommodates a wide variety of microorganisms with the
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phototrophic lifestyle, including the purple non-sulfur bacteria and the aerobic
6
bacteriochlorophyll a-containing bacteria (Imhoff, 2006; Swingley et al., 2009). Purple non-
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sulfur bacteria are metabolically versatile anoxygenic phototrophs that grow phototrophically
8
under anoxic conditions in the light or chemotrophically under microoxic to oxic conditions in
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the dark (Imhoff, 1995, 2001). Many purple non-sulfur bacteria are also capable of fermentative
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growth under anoxic conditions in the dark (Uffen & Wolfe, 1970; Madigan et al., 1980; Schultz
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& Weaver, 1982). By contrast, aerobic bacteriochlorophyll a-containing bacteria are incapable of
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anaerobic photosynthesis and light stimulates a transient enhancement of aerobic growth after a
13
shift from the dark to illumination (Yurkov & Beatty, 1998; Yurkov, 2001). In our study, we
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describe an unusual, microaerophilic, bacteriochlorophyll a-possessing isolate, strain Pf56T,
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which prefers to grow by means of fermentation and is phenotypically different from both the
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purple non-sulfur bacteria and the earlier described aerobic bacteriochlorophyll a-containing
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bacteria.
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Strain Pf56T was isolated from a methanotrophic enrichment culture that was established
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from an acidic peat soil (pH 3.8) sampled at a depth of 10 cm of Sphagnum peat bog Staroselsky
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moss (56o34’ N, 32o46’ E), Tver region, Russia, in August 2008. A methanotrophic enrichment
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culture was obtained from this peat sample using liquid mineral medium M2 of pH 5.0 (Dedysh
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et al., 2000) and incubation with 30% (vol/vol) methane. A cell suspension of this enrichment
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culture was serially diluted and used to inoculate deep-agar cultures prepared with semi-liquid
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agar (0.1%, w/v) medium M2 in 25-ml test tubes. The inoculated tubes were sealed with
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Parafilm M and incubated in jars with 30% (vol/vol) methane under light. After 1.5 months of
3
incubation, intensive development of a cream-colored methanotrophic biofilm was observed in
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the surface (0-3 mm depth) agar layer of these tubes. A methanotrophic bacterium, strain S284,
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isolated this biofilm was further described as a member of a novel species of the genus
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Methylocystis, Methylocystis bryophila (Belova et al., 2013). At a depth of 1-3 cm below this
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biofilm, however, we noticed the development of brownish-red-colored colonies of 2-3 mm in
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diameter. Microscopic examination of a cell material taken from these colonies revealed the
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presence of thick curved rods, which occurred singly or in pairs (Fig. 1A). These cells showed
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bright probe-conferred signal when hybridized to the probe M-450 with reported group
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specificity for Methylosinus/Methylocystis-like methanotrophs (Eller et al., 2001) and, therefore,
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were preliminary identified as belonging to the family Methylocystaceae. Given these
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identification results, the routine procedure for methanotroph isolation was employed. The
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respective cell material was spread plated onto the medium M2 solidified with Phytagel and the
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plates were incubated at 20°C in desiccators under a methane/air (50 : 50) gas mixture. The
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unpigmented colonies that developed on these plates after 6 weeks of incubation were again
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examined by means of phase-contrast microscopy and whole-cell hybridization with the probe
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M-450. The selected colonies were picked and purified by successive re-streaking until the target
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organism, designated strain Pf56T, was obtained in a pure culture. Partial sequencing of the 16S
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rRNA gene from this isolate showed that it is affiliated with the Alphaproteobacteria and is
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equally divergent (92-94% sequence similarity) from members of the two methanotroph-
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accommodating families, Methylocystaceae and Beijerinckiaceae.
4
1
Unexpectedly, our further attempts to maintain this isolate as a methanotrophic
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bacterium, in screw-cap 500 ml serum bottles containing 100 ml liquid medium M2 and 10-20%
3
(v/v) CH4 in the headspace, were unsuccessful. No growth was observed also when strain Pf56T
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was re-streaked on plates with medium M2 solidified with agar and incubated in a desiccator
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under a methane/air gas mixture. Apparently, development of this bacterium on the isolation
6
medium could only be explained by Phytagel utilization. This was further confirmed by
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cultivating strain Pf56T in 160 ml screw-cap serum bottles filled with 20 ml liquid medium M2
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and supplemented with 0.05% (wt/vol) Phytagel as a carbon source. These growth conditions,
9
however, were not fully optimal for strain Pf56T since only a relatively poor growth was
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observed. Further search for the optimal growth conditions revealed that strain Pf56T can be
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maintained in flasks filled by 4/5th - 9/10th with a liquid medium M3 containing (g per liter of
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distilled water): KH2PO4, 0.1; NH4Cl, 0.2; MgSO4 × 7H2O, 0.1; CaCl2× 2H2O, 0.02; yeast
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extract, 0.1; glucose (or malate) 0.5; and 1 ml of trace element solution ‘SLA’ (Imhoff, 2006),
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water 1L; pH 5.8-6.0. Growth in this medium was observed after 15-20 days of incubation in
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static conditions. Incubation in the light caused apparent (approximately by 20-30%) stimulation
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of growth.
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Liquid cultures of strain Pf56T grown in the light displayed homogenous turbidity and
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were slightly pinkish in color. This color intensity was not comparable to that in known purple
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bacteria, which form intensively colored, purplish red cell suspensions when incubated in the
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light under anoxic conditions. Nonetheless, cell pellets of strain Pf56T collected from the cultures
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incubated in the light were red and, therefore, were examined for the presence of pigments. The
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absorption spectra of living cells were recorded with a LOMO SPh-56 spectrophotometer. For
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these measurements, the cells were suspended in 50% glycerol. In addition, pigments were
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extracted with acetone/methanol (7:1, v/v) and the absorption spectra of these extracts were
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recorded. Carotenoids were analyzed by HPLC as described previously (Kulichevskaya et al.,
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2006). The absorption spectrum of the acetone: methanol extracts of cells grown in the light
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showed maxima at 363, 475, 505, 601 and 770 nm (Fig. 2). The peaks 363 and 770 nm are
5
characteristic for bacteriochlorophyll a. Absorption maxima of living cells were at 455, 489, 528,
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593, 806 and 865 nm. Carotenoid analysis by HPLC identified spirilloxanthin (35.3% of total
7
carotenoids), rhodopin (34.8%), and 3,4- didehydrorhodopin (15.5%) as the major carotenoids of
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strain Pf56T. Lycopene (8.1%) and anhydrorhodovibrin (3.3%) were also detected. Cell pellets of
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strain Pf56T collected from the cultures incubated in the dark were light-cream;
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bacteriochlorophyll a was not detected in these cells.
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Morphological observations and cell-size measurements were made with a Zeiss Axioplan 2
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microscope and Axiovision 4.2 software (Zeiss). Cells of strain Pf56T were Gram-negative, non-
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motile, non-spore-forming, thick curved rods with an outer diameter of 2.7-4.0 µm and a width
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of 0.6-1.2 µm, that occurred singly or in pairs (Fig. 1A). For preparation of ultrathin sections,
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cells of the exponentially growing cultures were collected by centrifugation and pre-fixed with
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1.5% (w/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 6.5) for 1 h at 4oC and then fixed in
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1% (w/v) OsO4 in the same buffer for 4 h at 20oC. After dehydration in an ethanol series, the
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samples were embedded in a Spurr epoxy resin. Thin sections were cut on an LKB-4800
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microtome, stained with 3% (w/v) uranyl acetate in 70% (v/v) ethanol. The specimen samples
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were examined with a JEM-100C transmission electron microscope. Electron microscopy of
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ultrathin sections revealed the presence of a vesicular intracytoplasmic membrane system (Fig.
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1B), which is characteristic for some purple non-sulfur alphaproteobacteria (Imhoff, 2006).
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Granules of polyhydroxybutyrate and polyphosphate were also observed in cells of strain Pf56T.
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Tests for photo-organotrophic growth in the light under anoxic conditions were performed in
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screw-cap 120 ml serum bottles containing 100 ml of medium M3 with 0.05% (w/v) acetate,
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malate, succinate, glucose, fructose or pyruvate. Before autoclaving, these flasks were flushed
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with N2 for 10 min. No growth of strain Pf56T was observed under these conditions. Negative
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results were also obtained in tests for photo-lithotrophic growth, which were performed in screw-
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cap 120 ml serum bottles containing 30 ml medium M3 without a carbon source and a mixture of
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H2 and CO2 (8 : 2, v/v) in a gas phase. Apparently, strain Pf56T was incapable of phototrophic
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growth under anoxic conditions.
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Tests for aerobic growth in the dark were performed in screw-cap 120 ml serum bottles
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containing 10 ml of medium M3 with 0.05% (w/v) concentrations of the following carbon
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sources: glucose, fructose, xylose, sucrose, cellobiose, trehalose, acetate, malate, citrate,
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succinate, pyruvate, α-oxoglutarate, propionate, galacturonic and glucuronic acids, ethanol,
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methanol, formate, and Phytagel. The bottles were incubated on a shaker (120 r.p.m.) and the
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growth was monitored by measuring OD600 and comparing to a negative control. Under these
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conditions, only a slow growth (generation time of 40-60 h) of strain Pf56T was observed on
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glucose, fructose, xylose, malate, pyruvate, galacturonic acid and Phytagel. The cultures grown
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under fully aerobic conditions contained forked and misshapen cells indicating that these
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conditions are not optimal for this bacterium (Suppl. Fig. S1).
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The same range of growth substrates was examined during incubations in the dark in micro-
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oxic conditions (bottles filled with a liquid medium by 9/10th of total volume with ambient air in
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the headspace). Best growth (doubling time 25-30 h) occurred with glucose, fructose, xylose, and
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galacturonic acid, while strain Pf56T grew also with trehalose, malate, succinate, pyruvate, and
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Phytagel. Under these conditions, the growth occurred by means of fermentation. Fermentation
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products (organic acids, alcohols) and substrates (sugars) were assayed using a Stayer HPLC
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chromatograph (Aquilon, Russia) equipped with a refractometric detector (Knauer, Germany)
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and an Aminex HPX-87H column (Bio-Rad, USA), operated isocratically with 5 mM H2SO4 as
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eluent at 0.6 ml/min. The products of fructose fermentation by strain Pf56T were acetate,
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propionate, and hydrogen (Suppl. Fig. S2). The same fermentation products were detected in
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micro-oxic cultures of strain Pf56T incubated in the light. Notably, the growth yield in the light
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was somewhat (approximately 20-30%) higher than that in the dark.
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Nitrogen sources were tested by replacing NH4Cl in a medium M3 with 0.01% (w/v) KNO3, NaNO2, urea, alanine, asparagine, histidine, lysine, glutamate or yeast extract. Strain Pf56T
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utilized ammonium salts, histidine, glutamate and yeast extract as nitrogen sources. It was also
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capable of growth in a liquid nitrogen-free medium. Partial fragment of the nifH gene (encoding
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dinitrogenase reductase) was amplified using primers and reaction conditions described by
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Dedysh et al. (2004b). The sequences of nifH gene fragments from this bacterium displayed
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highest similarity (90-92% nucleotide sequence similarity and 95-96% derived amino acid
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sequence identity) to the corresponding gene fragments from various strains of
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Rhodopseudomonas palustris.
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Despite its isolation from a methanotrophic enrichment culture, strain Pf56T was unable to
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grow on methane. The presence of a key enzyme of aerobic methanotrophs, the particulate
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methane monooxygenase (pMMO), in this bacterium could not be confirmed by any of the
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commonly used approaches. Cells of this isolate did not contain intracytoplasmic membranes
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characteristic of all pMMO-possessing methanotrophic proteobacteria. Our attempts to amplify a
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pmoA gene fragment from DNA of strain Pf56T using any of the two primer sets for this gene,
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i.e. A189/A682 (Holmes et al., 1995) and A189/Mb661r (Costello & Lidstrom, 1999), were
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unsuccessful. The mmoX gene, coding for the soluble methane monooxygenase (sMMO), also
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could not be amplified from DNA of our novel isolate with any of the previously described
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mmoX-targeted primers (McDonald et al., 1995; Miguez et al., 1997; Shigematsu et al., 1999;
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Auman et al., 2000; Vorobev et al., 2011). Methanol and formate also did not support the growth
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of strain Pf56T. In summary, we did not find any evidence for the presence of C1 metabolic
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capabilities in this bacterium.
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Physiological tests were performed in the liquid medium M3 with glucose under micro-
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oxic conditions in the dark. Growth of strain Pf56T was monitored for 10-14 days under a variety
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of conditions, including temperatures of 10-37oC, pH 3.5-7.5 and NaCl concentrations of 0.01-
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3.0 % (w/v). Variations in the acidity level were achieved by mixing 0.1M solutions of HCl and
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KOH. Strain Pf56T grew in the pH range 4.0-7.0 with a pH optimum of 5.5-6.5 (Fig. 3). The
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temperature range for growth was 15- 30 C, with an optimum at 22–28 °C. Growth inhibition
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was observed in the presence of NaCl in the medium above 0.5% (w/v).
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The presence of catalase in strain Pf56T was tested by using the Method 1 described by
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Gerhardt et al. (1981). Oxidase was tested using the commercial kit (bioMérieux). Strain Pf56T
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was cytochrome oxidase-negative and catalase-possitive.
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Cell biomass for cellular fatty acid, polar lipid, isoprenoid quinone analyses and for DNA
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extraction was obtained from batch cultures grown in liquid medium M3 with glucose under
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micro-oxic conditions in the light at 20ºC for 2 weeks. This corresponds to the late exponential
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growth phase. The fatty acid profiles were analyzed at the Identification Service of the Deutsche
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Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) as
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described by Kämpfer & Kroppenstedt (1996), using an Agilent 6890N GC, Sherlock MIS
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version 6.0 and the library TSBA40 4.10. The cellular fatty acid composition of strain Pf56T is
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shown in Table 1. Similar to many alphaproteobacteria, strain Pf56T contained significant
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amounts (26.3%) of 11-cys-octadecenoic acid (18:1ω7c), which is also typical for members of
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the families Methylocystaceae and Beijerinckiaceae. However, the major component of the fatty
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acid profile (39% of total fatty acids) in our novel isolate was 19:0 CYCLO ω8c, which is
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absent from methanotrophs of the Methylocystaceae (Bodelier et al., 2009) and was detected in
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significant amounts (up to 13%) only in a very few members of the Beijerinckiaceae, such as
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Methylocella tundrae (Dedysh et al. 2004a).
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For polar lipid analysis, the cells were washed with distilled water, re-suspended in isopropanol and incubated for 30 min at 70°C. The biomass residue was removed by filtration
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and extracted twice with isopropanol–chloroform (1 : 1) (Nichols, 1963). The combined extract
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was dried on a rotor evaporator; the residue was dissolved in 3 ml of chloroform–methanol (1:
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1), and the water-soluble compounds were removed by addition of 2.5% sodium chloride (4 ml).
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The chloroform layer was separated, dehydrated by passing through anhydrous sodium sulfate,
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dried in a rotor evaporator, and vacuum-dried to a constant weight. The obtained residue was
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dissolved in chloroform–methanol (1 : 1) and stored at –21°C. The separation of phospho- and
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sphingolipids was performed by two-dimensional TLC on glass plates (Merck, Germany) using
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chloroform–methanol–water (65 : 25 : 4) in the first direction and chloroform–acetone–
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methanol–acetic acid–water (50 : 20 : 10 : 10 : 5) in the second direction (Benning et al., 1995).
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Several plates with increased amount of the analyzed material were used in order to ensure the
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detection of lipids present in minor amounts. The chromatograms were developed by spraying
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with 5% sulfuric acid in ethanol with subsequent heating at 180°C. The lipids were identified
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using the individual markers and qualitative reactions with ninhydrin (for the presence of amino
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groups), the Dragendorff reagent (for choline), and α-naphthol (for glycolipids). The
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sphingolipid nature of glycolipids was determined by the saponification method (Kates, 1972).
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Lipid analyses were carried out using the following standards: phosphatidylcholine (Sigma,
3
USA) for phospholipids and glycoceramide mixture (Larodan, Sweden) for sphingolipids. The
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polar lipids of strain Pf56T consisted of phosphatidylcholine, phosphatidylethanolamine,
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phosphatidylglycerol, cardiolipin and sphingolipids (Suppl. Fig. S3). This polar lipid
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composition is characteristic for phototrophic bacteria of the family Rhodospirillaceae (Imhoff et
7
al., 1982).
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Isoprenoid quinones were extracted according to Collins (1985) and analyzed using a
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tandem-type MS LCQ ADVANTAGE MAX and ionization MS Finnigan Mat 8430 with
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atmospheric pressure chemical ionization. The mass spectra were first recorded in MS mode and
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then analyzed using MS/MS mode. Similarly to many phototrophic representatives of the
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Alphaproteobacteria, cells of strain Pf56T contained ubiquinone Q10.
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The DNA base composition of strain Pf56T was determined by thermal denaturation using a
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Cary 100Bio spectrophotometer (Varian, USA) at a heating rate of 0.5°C min-1. The mol % G+C
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value was calculated according to Owen et al. (1969). The G+C content of the DNA of strain
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Pf56T was 70.0 mol%, which is higher than in all currently characterized members of the
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Methylocystaceae and Beijerinckiaceae (GC content in the range of 55-65 mol%). At the same
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time, it is within the range of high GC content characteristic for DNA from purple nonsulfur
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bacteria (GC content in the range of 59-72 mol%) (Imhoff, 1995).
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PCR-mediated amplification of the 16S rRNA gene was performed using primers 9f and
21
1492r and reaction conditions described by Weisburg et al. (1991). Phylogenetic analysis was
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carried out using the ARB program package (Ludwig et al., 2004). The trees were constructed
23
using distance-based (neighbor-joining), maximum-likelihood (DNAml), and maximum-
11
1
parsimony methods. The significance levels of interior branch points obtained in neighbor-
2
joining analysis were determined by bootstrap analysis (1000 data re-samplings) using PHYLIP
3
(Felsenstein, 1989). A phylogenetic tree constructed on the basis of 16S rRNA gene sequences
4
(Fig. 4) indicated that strain Pf56T belongs to the class Alphaproteobacteria and forms a lineage,
5
which is most closely related to the family Methylocystaceae (93.6-94.7% sequence similarity).
6
Interestingly, the probe M-450 with reported group specificity for Methylosinus/Methylocystis-
7
like methanotrophs (Eller et al., 2001) matched the corresponding target region of 16S rRNA
8
gene of strain Pf56T, and the cells of this bacterium showed bright probe-conferred signal when
9
hybridized to this probe. This may cause an overestimation of methanotroph abundance in
10
environments colonized with strain Pf56T-like bacteria. The 16S rRNA gene sequence similarity
11
of strain Pf56T with members of the family Beijerinckiaceae was between 92.7 and 93.7%. The
12
most closely related phototrophs which, nonetheless, belong to a separate lineage, are acidophilic
13
purple bacteria Rhodoblastus acidophilus and Rhodoblastus sphagnicola (93.4-93.7% 16S rRNA
14
gene similarity). At the same time, strain Pf56T displayed high (96.3-98.6%) 16S rRNA gene
15
similarity to a large number of environmental sequences that were retrieved in cultivation-
16
independent studies from various boreal and tropical peatlands (GenBank accession numbers
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FR720624, FR720611, AM162440, AM162436, JX504960, AY163571, GQ402716, GQ402766,
18
GQ402819), forest soil (FJ624894) and some other environments such as volcanic deposits
19
(AY425766). The phylogenetic cluster formed by these environmental 16S rRNA gene
20
sequences and the corresponding gene sequence from strain Pf56T was stable independently of
21
the algorithm used for the tree construction and was supported by a bootstrap value of 100%
22
(Fig. 4).
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1
In summary, we showed that strain Pf56T is phenotypically distinct from all phylogenetically
2
related alphaproteobacteria. It does not possess methanotrophic capabilities and, therefore,
3
cannot be placed in the family Methylocystaceae, which is phenotypically uniform and
4
accommodates aerobic, pMMO-containing methanotrophs (Bowman, 2000). The family
5
Beijerinckiaceae is more heterogeneous and contains both organotrophs and methano-
6
/methylotrophs (Tamas et al., 2014) but all these organisms are strict aerobes. Finally, strain
7
Pf56T is different from anaerobic phototrophs of the genus Rhodoblastus in possessing
8
fermentative metabolism and inability to grow phototrophically under anoxic conditions. We
9
therefore suggest that strain Pf56T (=DSM 24875T=VKM B-2876T) should be classified as a
10
novel genus and species of bacteriochlorophyll a-possessing, fermentative bacteria, for which the
11
name Roseiarcus fermentans is proposed. We also propose a novel family to accommodate this
12
novel genus, Roseiarcaceae fam. nov.
13 14
Description of Roseiarcus gen. nov.
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Roseiarcus (Ro.se.i.ar’cus. L. adj. roseus, rose, pink; L. masc. n. arcus, arch, bow; N. L. masc. n.
16
Roseiarcus, pink bow).
17
Gram-negative, non-motile, non-spore-forming, pink-pigmented, thick curved rods that occur
18
singly or in pairs. Reproduce by normal cell division. Cells contain bacteriochlorophyll a and a
19
vesicular intracytoplasmic membrane system when grown in the light. Grow best in micro-oxic
20
conditions by means of fermentation. Growth substrates are some sugars and organic acids.
21
Unable to grow phototrophically under anoxic conditions. Capable of slow aerobic
22
chemoorganotrophic growth. Incubation in the light stimulates growth. C1 compounds are not
23
utilized. Capable of dinitrogen fixation. Moderately acidophilic and mesophilic. The major
13
1
cellular fatty acids are 19:0 CYCLO ω8c and 18:1ω7c. The polar lipids consist of
2
phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and
3
sphingolipids. Quinones are represented by ubiquinone Q10. Belong to the family
4
Roseiarcaceae, class Alphaproteobacteria. Found in peatlands and soils. The type species is
5
Roseiarcus fermentans.
6 7
Description of Roseiarcus fermentans sp. nov.
8
Roseiarcus fermentans (fer.men’tans. L. part. adj. fermentans fermenting).
9
The description is as for the genus but with the following additional traits. Cells are 2.7-4.0
10
µm in lenght and 0.6-1.2 µm in wight. Liquid cultures are slightly pinkish or colorless when
11
grown in the light and in the dark, respectively. Absorption maxima of living cells grown in the
12
light are at 455, 489, 528, 593, 806 and 865 nm. Bacteriochlorophyll a is produced only in the
13
light. Spirilloxanthin, rhodopin and 3,4- didehydrorhodopin are the major carotenoids. Preferred
14
mode of growth is fermentation of some sugars and organic acids under micro-oxic conditions.
15
Carbon sources utilized include glucose, fructose, xylose, trehalose, malate, succinate, pyruvate,
16
galacturonic acid, and Phytagel. Methane, methanol and formate are not utilized. Nitrogen
17
sources utilized include ammonium salts, histidine, glutamate, yeast extract and N2. Oxidase-
18
negative and catalase-positive. Grows in the pH range 4.0-7.0 (optimum 5.5-6.5) and at 15- 30 C
19
(optimum 22–28 °C). NaCl inhibits growth at concentrations above 0.5% (w/v). The DNA G+C
20
content is 70.0 mol%. The type strain, Pf56T (=DSM 24875T = VKM B-2876T), was isolated
21
from the acidic Sphagnum peat bog Staroselsky moss, Tver region, Russia.
o
22 23
Description of Roseiarcaceae fam. nov.
14
1
Roseiarcaceae (Ro.se.i.ar.ca.ce’ae. N.L. masc. n. Roseiarcus type genus of the family; -aceae
2
ending to denote a family; N.L. fem. pl. n. Roseiarcaceae the Roseiarcus family).
3
Gram-negative, non-spore-forming bacteria. Aerobes capable of fermentation under micro-oxic
4
conditions. Mesophilic and mildly acidophilic. The family Roseiarcaceae belongs to the class
5
Alphaproteobacteria, order Rhizobiales. The type genus is Roseiarcus.
6 7 8
ACKNOWLEDGMENTS
9
This research was supported by the Program “Molecular and Cell Biology” and the Russian Fund
10
of Basic Research (project No 12-04-00768). The authors thank Vladimir M. Gorlenko for
11
helpful discussions and valuable advices, Natalia E. Suzina for electron microscopy analysis and
12
E.N. Detkova for DNA G+C content analysis.
15
1
REFERENCES
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Eller, G., Stubner, S. & Frenzel, P. (2001). Group-specific 16S rRNA targeted probes for the
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photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
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bacteria of the Rhodospirillaceae and Chromatiaceae families. J Bacteriol 150, 1192-1201.
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Kates, M. (1972). Techniques of Lipidology. New York: Elsevier.
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Kulichevskaya, I.S., Guzev, V.S., Gorlenko, V.M., Liesack, W. & Dedysh, S.N. (2006).
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Rhodoblastus sphagnicola sp. nov., a novel acidophilic purple non-sulfur bacterium from
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Sphagnum peat bog. Int J Syst Evol Microbiol 56, 1397-1402.
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Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A.,
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Lai, T., Steppi, S., Jobb, G. & other authors (2004). ARB: a software environment for
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sequence data. Nucleic Acids Res 32, 1363-1371.
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Madigan, M.T., Cox, J.C., & Gest, H. (1980). Physiology of dark fermentative growth of
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Rhodopseudomonas capsulata. J Bacteriol 142, 908-915.
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McDonald, I.R., Kenna, E.M. & Murrell, J.C. (1995). Detection of methanotrophic bacteria in
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environmental samples with the PCR. Appl Environ Microbiol 61, 116-121.
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Miguez, C.B., Bourque, D., Sealy, J.A., Greer, C.W. & Groleau, D. (1997). Detection and
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isolation of methanotrophic bacteria possessing soluble methane monooxygenase (sMMO) genes
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using the polymerase chain reaction (PCR). Microb Ecol 33, 21-31.
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Nichols, B.W. (1963). Separation of the lipids of photosynthetic tissues; improvement in
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analysis by thin-layer chromatography, Biochim. Biophys. Acta 4145, 417–422.
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Owen, R.J., Lapage, S.P. & Hill, L.R. (1969). Determination of base composition from melting
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Schultz, J.E. & Weaver, P.F. (1982). Fermentation and anaerobic respiration by
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Rhodospirillum rubrum and Rhodopseudomonas capsulata. J Bacteriol 149, 181-190.
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Shigematsu, T., Hanada, S., Eguchi, M., Kamagata, Y., Kanagawa, T. & Kurane, R. (1999).
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Soluble methane monooxygenase gene clusters fron trichloroethylene-degrading Methylomonas
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sp. strains and detection of methanotrophs during in situ bioremediation. Appl Environ Microbiol
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Swingley, W.D., Blankenship, R.E. & Raymond, J. (2009). Evolutionary relationships among
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purple photosynthetic bacteria and the origin of proteobacterial photosynthetic systems. Pp. 1718
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Tamas, I., Smirnova, A.V., He, Z. & Dunfield, P.F. (2014). The (d)evolution of
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methanotrophy in the Beijerinckiaceae – a comparative genomic analysis. The ISME Journal 8,
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369-382.
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Uffen, R.L., & Wolfe, R.S. (1970). Anaerobic growth of purple nonsulfur bacteria under dark
7
conditions. J Bacteriol 104, 462-472.
8
Vorobev, A.V., Baani, M., Doronina, N.V., Brady, A.L., Liesack, W., Dunfield, P.F. &
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Dedysh, S.N. (2011). Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately
10
methanotrophic bacterium possessing only a soluble methane monooxygenase. Int J Syst Evol
11
Microbiol 61, 2456-2463.
12
Weisburg, W.G., Barns, S.M., Pelletier, D.A. & Lane, D.J. (1991). 16S ribosomal DNA
13
amplification for phylogenetic study. J Bacteriol 173, 697-703.
14
Yurkov, V.V. & Beatty, J.T. (1998). Aerobic anoxygenic phototrophic bacteria. Microbiol
15
Molec Biol Rev 62, 695-724.
16
Yurkov, V.V. (2001). Aerobic phototrophic proteobacteria. In The Prokaryotes: an Evolving
17
Electronic Resource for the Microbiological Community, 3rd edn, release 3.6, 22 June 2001.
18
Edited by M. Dworkin & others. New York: Springer. http://141.150.157.117:8080/
19
prokPUB/index.htm.
19
1
Table 1. Cellular fatty acid composition of strain Pf56T. Values are percentages of total fatty acids.
2
Major fatty acids are shown in bold.
Fatty acid
Strain Pf56T
12:0
0.3
13:0
0.4
14:0
0.5
15:0
0.7
16:1ω7c
0.3
16:0
9.3
17:1ω8c
1.4
17:0
6.5
18:1ω9c
0.8
18:1ω7c
26.3
18:1ω5c
0.6
11 methyl 18:1ω7c
0.9
17:0 3OH
0.6
19:0 CYCLO ω8c
38.9
18:0 3OH
2.5
20:2ω6,9c
0.5
20
1
FIGURE CAPTIONS
2
Figure 1. (A) Phase-contrast micrograph of cells of stain Pf56T, bar, 10 µm. (B) Electron
3
micrograph of an ultrathin section showing vesicular intracytoplasmic membranes, granules of
4
polyhydroxybutyrate and polyphosphate, bar, 1 µm.
5
Figure 2. Absorption spectrum of the acetone/ methanol extract of cells of phototrophically
6
grown strain Pf56T showing the characteristic peaks of BChl a at 363 and 770 nm.
7
Figure 3. Influence of medium pH on the growth of strain Pf56T.
8
Figure 4. 16S rRNA gene-based neighbor-joining tree showing the phylogenetic position of
9
strain Pf56T in relation to taxonomically characterized members of the families
10
Methylocystaceae and Beijerinckiaceae, as well as to some environmental clone sequences
11
retrieved in cultivation-independent studies from various wetlands and soils. Bootstrap values
12
(percentages of 1000 data resamplings) >50% are shown. Black circles indicate that the
13
corresponding nodes were also recovered in the maximum-likelihood and maximum-parsimony
14
trees. Four members of the Gammaproteobacteria, Methylomicrobium album (X72777),
15
Methylobacter luteus (AF304195), Methylomonas methanica S1 (AF304196) and Methylococcus
16
capsulatus Texas (AJ563935) were used as an outgroup. Bar, 0.05 substitutions per nucleotide
17
position.
18
Suppl. Figure S1. Phase-contrast micrographs of cells of stain Pf56T grown under fully oxic (a)
19
and micro-oxic conditions (b); bar, 10 µm.
20
Suppl. Figure S2. (A) Growth of strain Pf56 in anoxic conditions in the dark with (1) and
21
without (2) fructose. (B) Dynamics of fructose (1), propionate (2) and acetate (3) during
22
anaerobic fermentation of fructose by strain Pf56 T. 21
1
Suppl. Figure S3. Polar lipids of strain Pf56 T after separation by two dimensional TLC.
2
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol;
3
CL, cardiolipin ; SL1 and SL2, sphingolipids; PL, unidentified phospholipid.
4
5 6
7
22
1 2 3
23
1
24
1
Roseiarcus fermentans gen. nov. sp. nov., a Bacteriochlorophyll a - Containing
2
Fermentative Bacterium Phylogenetically Related to Alphaproteobacterial
3
Methanotrophs and Description of the Family Roseiarcaceae fam. nov.
4 5
Irina S. Kulichevskaya1, Olga V. Danilova1, Vera M. Tereshina1, Vadim V.
6
Kevbrin1, and Svetlana N. Dedysh1
7 8
1
9
Russia
S.N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow 117312,
10 11
Author for correspondence: Svetlana N. Dedysh
12
Tel: 7 (499) 135 0591. Fax: 7 (499) 135 6530. Email: [email protected]
13 14 15
Journal: International Journal of Systematic and Evolutionary Microbiology (IJSEM)
16
Contents Category: New Taxa - Proteobacteria
17 18
Running title: Roseiarcus fermentans gen. nov., sp. nov.
19
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the partial
20
sequence of the nifH gene of Roseiarcus fermentans Pf56T are KJ406703 and KJ406704,
21
respectively.
1
1
ABSTRACT
2
A light-pink-pigmented, microaerophilic bacterium was obtained from a methanotrophic
3
consortium enriched from acidic Sphagnum peat and designated strain Pf56T. Cells of this
4
bacterium are Gram-negative, non-motile, thick curved rods, which contain a vesicular
5
intracytoplasmic membrane system characteristic for some purple non-sulfur
6
Alphaproteobacteria. Absorption spectrum of the acetone: methanol extracts of cells grown
7
in the light shows maxima at 363, 475, 505, 601 and 770 nm; the peaks 363 and 770 nm are
8
characteristic for bacteriochlorophyll a. However, in contrast to purple non-sulfur
9
bacteria, strain Pf56T is unable to grow phototrophically under anoxic conditions in the
10
light. Best growth occurs on some sugars and organic acids under micro-oxic conditions by
11
means of fermentation. The fermentation products are propionate, acetate, and hydrogen.
12
Slow chemo-organotrophic growth is also observed under fully oxic conditions. Light
13
stimulates growth. C1 substrates are not utilized. Strain Pf56T grows in the pH range 4.0-
14
7.0 (optimum 5.5-6.5) and at 15- 30 C (optimum 22–28 °C). The major cellular fatty acids
15
are 19:0 CYCLO ω8c and 18:1ω7c; quinones are represented by ubiquinone Q10. The
16
G+C content of the DNA is 70.0 mol%. Strain Pf56 displays 93.6-94.7 and 92.7-93.7% 16S
17
rRNA gene sequence similarity to members of the families Methylocystaceae and
18
Beijerinckiaceae, respectively, and belongs to a large cluster of environmental sequences
19
retrieved in cultivation-independent studies from various wetlands and forest soils.
20
Phenotypic, genotypic and chemotaxonomic characteristics of strain Pf56 T suggest that it
21
represents a novel genus and species of bacteriochlorophyll a-containing fermentative
22
bacteria, for which the name Roseiarcus fermentans is proposed. Strain Pf56T (=DSM
23
24875T = VKM B-2876T) is the type strain, which is also the first characterized member of
24
a novel family within the class Alphaproteobacteria, Roseiarcaceae fam. nov.
o
2
1
Keywords: Roseiarcus fermentans gen. nov., sp. nov., Roseiarcaceae fam. nov.,
2
bacteriochlorophyll a-containing bacteria, fermentative growth, acidic wetlands.
3 4
The class Alphaproteobacteria accommodates a wide variety of microorganisms with the
5
phototrophic lifestyle, including the purple non-sulfur bacteria and the aerobic
6
bacteriochlorophyll a-containing bacteria (Imhoff, 2006; Swingley et al., 2009). Purple non-
7
sulfur bacteria are metabolically versatile anoxygenic phototrophs that grow phototrophically
8
under anoxic conditions in the light or chemotrophically under microoxic to oxic conditions in
9
the dark (Imhoff, 1995, 2001). Many purple non-sulfur bacteria are also capable of fermentative
10
growth under anoxic conditions in the dark (Uffen & Wolfe, 1970; Madigan et al., 1980; Schultz
11
& Weaver, 1982). By contrast, aerobic bacteriochlorophyll a-containing bacteria are incapable of
12
anaerobic photosynthesis and light stimulates a transient enhancement of aerobic growth after a
13
shift from the dark to illumination (Yurkov & Beatty, 1998; Yurkov, 2001). In our study, we
14
describe an unusual, microaerophilic, bacteriochlorophyll a-possessing isolate, strain Pf56T,
15
which prefers to grow by means of fermentation and is phenotypically different from both the
16
purple non-sulfur bacteria and the earlier described aerobic bacteriochlorophyll a-containing
17
bacteria.
18
Strain Pf56T was isolated from a methanotrophic enrichment culture that was established
19
from an acidic peat soil (pH 3.8) sampled at a depth of 10 cm of Sphagnum peat bog Staroselsky
20
moss (56o34’ N, 32o46’ E), Tver region, Russia, in August 2008. A methanotrophic enrichment
21
culture was obtained from this peat sample using liquid mineral medium M2 of pH 5.0 (Dedysh
22
et al., 2000) and incubation with 30% (vol/vol) methane. A cell suspension of this enrichment
23
culture was serially diluted and used to inoculate deep-agar cultures prepared with semi-liquid
3
1
agar (0.1%, w/v) medium M2 in 25-ml test tubes. The inoculated tubes were sealed with
2
Parafilm M and incubated in jars with 30% (vol/vol) methane under light. After 1.5 months of
3
incubation, intensive development of a cream-colored methanotrophic biofilm was observed in
4
the surface (0-3 mm depth) agar layer of these tubes. A methanotrophic bacterium, strain S284,
5
isolated this biofilm was further described as a member of a novel species of the genus
6
Methylocystis, Methylocystis bryophila (Belova et al., 2013). At a depth of 1-3 cm below this
7
biofilm, however, we noticed the development of brownish-red-colored colonies of 2-3 mm in
8
diameter. Microscopic examination of a cell material taken from these colonies revealed the
9
presence of thick curved rods, which occurred singly or in pairs (Fig. 1A). These cells showed
10
bright probe-conferred signal when hybridized to the probe M-450 with reported group
11
specificity for Methylosinus/Methylocystis-like methanotrophs (Eller et al., 2001) and, therefore,
12
were preliminary identified as belonging to the family Methylocystaceae. Given these
13
identification results, the routine procedure for methanotroph isolation was employed. The
14
respective cell material was spread plated onto the medium M2 solidified with Phytagel and the
15
plates were incubated at 20°C in desiccators under a methane/air (50 : 50) gas mixture. The
16
unpigmented colonies that developed on these plates after 6 weeks of incubation were again
17
examined by means of phase-contrast microscopy and whole-cell hybridization with the probe
18
M-450. The selected colonies were picked and purified by successive re-streaking until the target
19
organism, designated strain Pf56T, was obtained in a pure culture. Partial sequencing of the 16S
20
rRNA gene from this isolate showed that it is affiliated with the Alphaproteobacteria and is
21
equally divergent (92-94% sequence similarity) from members of the two methanotroph-
22
accommodating families, Methylocystaceae and Beijerinckiaceae.
4
1
Unexpectedly, our further attempts to maintain this isolate as a methanotrophic
2
bacterium, in screw-cap 500 ml serum bottles containing 100 ml liquid medium M2 and 10-20%
3
(v/v) CH4 in the headspace, were unsuccessful. No growth was observed also when strain Pf56T
4
was re-streaked on plates with medium M2 solidified with agar and incubated in a desiccator
5
under a methane/air gas mixture. Apparently, development of this bacterium on the isolation
6
medium could only be explained by Phytagel utilization. This was further confirmed by
7
cultivating strain Pf56T in 160 ml screw-cap serum bottles filled with 20 ml liquid medium M2
8
and supplemented with 0.05% (wt/vol) Phytagel as a carbon source. These growth conditions,
9
however, were not fully optimal for strain Pf56T since only a relatively poor growth was
10
observed. Further search for the optimal growth conditions revealed that strain Pf56T can be
11
maintained in flasks filled by 4/5th - 9/10th with a liquid medium M3 containing (g per liter of
12
distilled water): KH2PO4, 0.1; NH4Cl, 0.2; MgSO4 × 7H2O, 0.1; CaCl2× 2H2O, 0.02; yeast
13
extract, 0.1; glucose (or malate) 0.5; and 1 ml of trace element solution ‘SLA’ (Imhoff, 2006),
14
water 1L; pH 5.8-6.0. Growth in this medium was observed after 15-20 days of incubation in
15
static conditions. Incubation in the light caused apparent (approximately by 20-30%) stimulation
16
of growth.
17
Liquid cultures of strain Pf56T grown in the light displayed homogenous turbidity and
18
were slightly pinkish in color. This color intensity was not comparable to that in known purple
19
bacteria, which form intensively colored, purplish red cell suspensions when incubated in the
20
light under anoxic conditions. Nonetheless, cell pellets of strain Pf56T collected from the cultures
21
incubated in the light were red and, therefore, were examined for the presence of pigments. The
22
absorption spectra of living cells were recorded with a LOMO SPh-56 spectrophotometer. For
23
these measurements, the cells were suspended in 50% glycerol. In addition, pigments were
5
1
extracted with acetone/methanol (7:1, v/v) and the absorption spectra of these extracts were
2
recorded. Carotenoids were analyzed by HPLC as described previously (Kulichevskaya et al.,
3
2006). The absorption spectrum of the acetone: methanol extracts of cells grown in the light
4
showed maxima at 363, 475, 505, 601 and 770 nm (Fig. 2). The peaks 363 and 770 nm are
5
characteristic for bacteriochlorophyll a. Absorption maxima of living cells were at 455, 489, 528,
6
593, 806 and 865 nm. Carotenoid analysis by HPLC identified spirilloxanthin (35.3% of total
7
carotenoids), rhodopin (34.8%), and 3,4- didehydrorhodopin (15.5%) as the major carotenoids of
8
strain Pf56T. Lycopene (8.1%) and anhydrorhodovibrin (3.3%) were also detected. Cell pellets of
9
strain Pf56T collected from the cultures incubated in the dark were light-cream;
10
bacteriochlorophyll a was not detected in these cells.
11
Morphological observations and cell-size measurements were made with a Zeiss Axioplan 2
12
microscope and Axiovision 4.2 software (Zeiss). Cells of strain Pf56T were Gram-negative, non-
13
motile, non-spore-forming, thick curved rods with an outer diameter of 2.7-4.0 µm and a width
14
of 0.6-1.2 µm, that occurred singly or in pairs (Fig. 1A). For preparation of ultrathin sections,
15
cells of the exponentially growing cultures were collected by centrifugation and pre-fixed with
16
1.5% (w/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 6.5) for 1 h at 4oC and then fixed in
17
1% (w/v) OsO4 in the same buffer for 4 h at 20oC. After dehydration in an ethanol series, the
18
samples were embedded in a Spurr epoxy resin. Thin sections were cut on an LKB-4800
19
microtome, stained with 3% (w/v) uranyl acetate in 70% (v/v) ethanol. The specimen samples
20
were examined with a JEM-100C transmission electron microscope. Electron microscopy of
21
ultrathin sections revealed the presence of a vesicular intracytoplasmic membrane system (Fig.
22
1B), which is characteristic for some purple non-sulfur alphaproteobacteria (Imhoff, 2006).
23
Granules of polyhydroxybutyrate and polyphosphate were also observed in cells of strain Pf56T.
6
1
Tests for photo-organotrophic growth in the light under anoxic conditions were performed in
2
screw-cap 120 ml serum bottles containing 100 ml of medium M3 with 0.05% (w/v) acetate,
3
malate, succinate, glucose, fructose or pyruvate. Before autoclaving, these flasks were flushed
4
with N2 for 10 min. No growth of strain Pf56T was observed under these conditions. Negative
5
results were also obtained in tests for photo-lithotrophic growth, which were performed in screw-
6
cap 120 ml serum bottles containing 30 ml medium M3 without a carbon source and a mixture of
7
H2 and CO2 (8 : 2, v/v) in a gas phase. Apparently, strain Pf56T was incapable of phototrophic
8
growth under anoxic conditions.
9
Tests for aerobic growth in the dark were performed in screw-cap 120 ml serum bottles
10
containing 10 ml of medium M3 with 0.05% (w/v) concentrations of the following carbon
11
sources: glucose, fructose, xylose, sucrose, cellobiose, trehalose, acetate, malate, citrate,
12
succinate, pyruvate, α-oxoglutarate, propionate, galacturonic and glucuronic acids, ethanol,
13
methanol, formate, and Phytagel. The bottles were incubated on a shaker (120 r.p.m.) and the
14
growth was monitored by measuring OD600 and comparing to a negative control. Under these
15
conditions, only a slow growth (generation time of 40-60 h) of strain Pf56T was observed on
16
glucose, fructose, xylose, malate, pyruvate, galacturonic acid and Phytagel. The cultures grown
17
under fully aerobic conditions contained forked and misshapen cells indicating that these
18
conditions are not optimal for this bacterium (Suppl. Fig. S1).
19
The same range of growth substrates was examined during incubations in the dark in micro-
20
oxic conditions (bottles filled with a liquid medium by 9/10th of total volume with ambient air in
21
the headspace). Best growth (doubling time 25-30 h) occurred with glucose, fructose, xylose, and
22
galacturonic acid, while strain Pf56T grew also with trehalose, malate, succinate, pyruvate, and
23
Phytagel. Under these conditions, the growth occurred by means of fermentation. Fermentation
7
1
products (organic acids, alcohols) and substrates (sugars) were assayed using a Stayer HPLC
2
chromatograph (Aquilon, Russia) equipped with a refractometric detector (Knauer, Germany)
3
and an Aminex HPX-87H column (Bio-Rad, USA), operated isocratically with 5 mM H2SO4 as
4
eluent at 0.6 ml/min. The products of fructose fermentation by strain Pf56T were acetate,
5
propionate, and hydrogen (Suppl. Fig. S2). The same fermentation products were detected in
6
micro-oxic cultures of strain Pf56T incubated in the light. Notably, the growth yield in the light
7
was somewhat (approximately 20-30%) higher than that in the dark.
8 9
Nitrogen sources were tested by replacing NH4Cl in a medium M3 with 0.01% (w/v) KNO3, NaNO2, urea, alanine, asparagine, histidine, lysine, glutamate or yeast extract. Strain Pf56T
10
utilized ammonium salts, histidine, glutamate and yeast extract as nitrogen sources. It was also
11
capable of growth in a liquid nitrogen-free medium. Partial fragment of the nifH gene (encoding
12
dinitrogenase reductase) was amplified using primers and reaction conditions described by
13
Dedysh et al. (2004b). The sequences of nifH gene fragments from this bacterium displayed
14
highest similarity (90-92% nucleotide sequence similarity and 95-96% derived amino acid
15
sequence identity) to the corresponding gene fragments from various strains of
16
Rhodopseudomonas palustris.
17
Despite its isolation from a methanotrophic enrichment culture, strain Pf56T was unable to
18
grow on methane. The presence of a key enzyme of aerobic methanotrophs, the particulate
19
methane monooxygenase (pMMO), in this bacterium could not be confirmed by any of the
20
commonly used approaches. Cells of this isolate did not contain intracytoplasmic membranes
21
characteristic of all pMMO-possessing methanotrophic proteobacteria. Our attempts to amplify a
22
pmoA gene fragment from DNA of strain Pf56T using any of the two primer sets for this gene,
23
i.e. A189/A682 (Holmes et al., 1995) and A189/Mb661r (Costello & Lidstrom, 1999), were
8
1
unsuccessful. The mmoX gene, coding for the soluble methane monooxygenase (sMMO), also
2
could not be amplified from DNA of our novel isolate with any of the previously described
3
mmoX-targeted primers (McDonald et al., 1995; Miguez et al., 1997; Shigematsu et al., 1999;
4
Auman et al., 2000; Vorobev et al., 2011). Methanol and formate also did not support the growth
5
of strain Pf56T. In summary, we did not find any evidence for the presence of C1 metabolic
6
capabilities in this bacterium.
7
Physiological tests were performed in the liquid medium M3 with glucose under micro-
8
oxic conditions in the dark. Growth of strain Pf56T was monitored for 10-14 days under a variety
9
of conditions, including temperatures of 10-37oC, pH 3.5-7.5 and NaCl concentrations of 0.01-
10
3.0 % (w/v). Variations in the acidity level were achieved by mixing 0.1M solutions of HCl and
11
KOH. Strain Pf56T grew in the pH range 4.0-7.0 with a pH optimum of 5.5-6.5 (Fig. 3). The
12
temperature range for growth was 15- 30 C, with an optimum at 22–28 °C. Growth inhibition
13
was observed in the presence of NaCl in the medium above 0.5% (w/v).
o
14
The presence of catalase in strain Pf56T was tested by using the Method 1 described by
15
Gerhardt et al. (1981). Oxidase was tested using the commercial kit (bioMérieux). Strain Pf56T
16
was cytochrome oxidase-negative and catalase-possitive.
17
Cell biomass for cellular fatty acid, polar lipid, isoprenoid quinone analyses and for DNA
18
extraction was obtained from batch cultures grown in liquid medium M3 with glucose under
19
micro-oxic conditions in the light at 20ºC for 2 weeks. This corresponds to the late exponential
20
growth phase. The fatty acid profiles were analyzed at the Identification Service of the Deutsche
21
Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) as
22
described by Kämpfer & Kroppenstedt (1996), using an Agilent 6890N GC, Sherlock MIS
23
version 6.0 and the library TSBA40 4.10. The cellular fatty acid composition of strain Pf56T is
9
1
shown in Table 1. Similar to many alphaproteobacteria, strain Pf56T contained significant
2
amounts (26.3%) of 11-cys-octadecenoic acid (18:1ω7c), which is also typical for members of
3
the families Methylocystaceae and Beijerinckiaceae. However, the major component of the fatty
4
acid profile (39% of total fatty acids) in our novel isolate was 19:0 CYCLO ω8c, which is
5
absent from methanotrophs of the Methylocystaceae (Bodelier et al., 2009) and was detected in
6
significant amounts (up to 13%) only in a very few members of the Beijerinckiaceae, such as
7
Methylocella tundrae (Dedysh et al. 2004a).
8 9
For polar lipid analysis, the cells were washed with distilled water, re-suspended in isopropanol and incubated for 30 min at 70°C. The biomass residue was removed by filtration
10
and extracted twice with isopropanol–chloroform (1 : 1) (Nichols, 1963). The combined extract
11
was dried on a rotor evaporator; the residue was dissolved in 3 ml of chloroform–methanol (1:
12
1), and the water-soluble compounds were removed by addition of 2.5% sodium chloride (4 ml).
13
The chloroform layer was separated, dehydrated by passing through anhydrous sodium sulfate,
14
dried in a rotor evaporator, and vacuum-dried to a constant weight. The obtained residue was
15
dissolved in chloroform–methanol (1 : 1) and stored at –21°C. The separation of phospho- and
16
sphingolipids was performed by two-dimensional TLC on glass plates (Merck, Germany) using
17
chloroform–methanol–water (65 : 25 : 4) in the first direction and chloroform–acetone–
18
methanol–acetic acid–water (50 : 20 : 10 : 10 : 5) in the second direction (Benning et al., 1995).
19
Several plates with increased amount of the analyzed material were used in order to ensure the
20
detection of lipids present in minor amounts. The chromatograms were developed by spraying
21
with 5% sulfuric acid in ethanol with subsequent heating at 180°C. The lipids were identified
22
using the individual markers and qualitative reactions with ninhydrin (for the presence of amino
23
groups), the Dragendorff reagent (for choline), and α-naphthol (for glycolipids). The
10
1
sphingolipid nature of glycolipids was determined by the saponification method (Kates, 1972).
2
Lipid analyses were carried out using the following standards: phosphatidylcholine (Sigma,
3
USA) for phospholipids and glycoceramide mixture (Larodan, Sweden) for sphingolipids. The
4
polar lipids of strain Pf56T consisted of phosphatidylcholine, phosphatidylethanolamine,
5
phosphatidylglycerol, cardiolipin and sphingolipids (Suppl. Fig. S3). This polar lipid
6
composition is characteristic for phototrophic bacteria of the family Rhodospirillaceae (Imhoff et
7
al., 1982).
8
Isoprenoid quinones were extracted according to Collins (1985) and analyzed using a
9
tandem-type MS LCQ ADVANTAGE MAX and ionization MS Finnigan Mat 8430 with
10
atmospheric pressure chemical ionization. The mass spectra were first recorded in MS mode and
11
then analyzed using MS/MS mode. Similarly to many phototrophic representatives of the
12
Alphaproteobacteria, cells of strain Pf56T contained ubiquinone Q10.
13
The DNA base composition of strain Pf56T was determined by thermal denaturation using a
14
Cary 100Bio spectrophotometer (Varian, USA) at a heating rate of 0.5°C min-1. The mol % G+C
15
value was calculated according to Owen et al. (1969). The G+C content of the DNA of strain
16
Pf56T was 70.0 mol%, which is higher than in all currently characterized members of the
17
Methylocystaceae and Beijerinckiaceae (GC content in the range of 55-65 mol%). At the same
18
time, it is within the range of high GC content characteristic for DNA from purple nonsulfur
19
bacteria (GC content in the range of 59-72 mol%) (Imhoff, 1995).
20
PCR-mediated amplification of the 16S rRNA gene was performed using primers 9f and
21
1492r and reaction conditions described by Weisburg et al. (1991). Phylogenetic analysis was
22
carried out using the ARB program package (Ludwig et al., 2004). The trees were constructed
23
using distance-based (neighbor-joining), maximum-likelihood (DNAml), and maximum-
11
1
parsimony methods. The significance levels of interior branch points obtained in neighbor-
2
joining analysis were determined by bootstrap analysis (1000 data re-samplings) using PHYLIP
3
(Felsenstein, 1989). A phylogenetic tree constructed on the basis of 16S rRNA gene sequences
4
(Fig. 4) indicated that strain Pf56T belongs to the class Alphaproteobacteria and forms a lineage,
5
which is most closely related to the family Methylocystaceae (93.6-94.7% sequence similarity).
6
Interestingly, the probe M-450 with reported group specificity for Methylosinus/Methylocystis-
7
like methanotrophs (Eller et al., 2001) matched the corresponding target region of 16S rRNA
8
gene of strain Pf56T, and the cells of this bacterium showed bright probe-conferred signal when
9
hybridized to this probe. This may cause an overestimation of methanotroph abundance in
10
environments colonized with strain Pf56T-like bacteria. The 16S rRNA gene sequence similarity
11
of strain Pf56T with members of the family Beijerinckiaceae was between 92.7 and 93.7%. The
12
most closely related phototrophs which, nonetheless, belong to a separate lineage, are acidophilic
13
purple bacteria Rhodoblastus acidophilus and Rhodoblastus sphagnicola (93.4-93.7% 16S rRNA
14
gene similarity). At the same time, strain Pf56T displayed high (96.3-98.6%) 16S rRNA gene
15
similarity to a large number of environmental sequences that were retrieved in cultivation-
16
independent studies from various boreal and tropical peatlands (GenBank accession numbers
17
FR720624, FR720611, AM162440, AM162436, JX504960, AY163571, GQ402716, GQ402766,
18
GQ402819), forest soil (FJ624894) and some other environments such as volcanic deposits
19
(AY425766). The phylogenetic cluster formed by these environmental 16S rRNA gene
20
sequences and the corresponding gene sequence from strain Pf56T was stable independently of
21
the algorithm used for the tree construction and was supported by a bootstrap value of 100%
22
(Fig. 4).
12
1
In summary, we showed that strain Pf56T is phenotypically distinct from all phylogenetically
2
related alphaproteobacteria. It does not possess methanotrophic capabilities and, therefore,
3
cannot be placed in the family Methylocystaceae, which is phenotypically uniform and
4
accommodates aerobic, pMMO-containing methanotrophs (Bowman, 2000). The family
5
Beijerinckiaceae is more heterogeneous and contains both organotrophs and methano-
6
/methylotrophs (Tamas et al., 2014) but all these organisms are strict aerobes. Finally, strain
7
Pf56T is different from anaerobic phototrophs of the genus Rhodoblastus in possessing
8
fermentative metabolism and inability to grow phototrophically under anoxic conditions. We
9
therefore suggest that strain Pf56T (=DSM 24875T=VKM B-2876T) should be classified as a
10
novel genus and species of bacteriochlorophyll a-possessing, fermentative bacteria, for which the
11
name Roseiarcus fermentans is proposed. We also propose a novel family to accommodate this
12
novel genus, Roseiarcaceae fam. nov.
13 14
Description of Roseiarcus gen. nov.
15
Roseiarcus (Ro.se.i.ar’cus. L. adj. roseus, rose, pink; L. masc. n. arcus, arch, bow; N. L. masc. n.
16
Roseiarcus, pink bow).
17
Gram-negative, non-motile, non-spore-forming, pink-pigmented, thick curved rods that occur
18
singly or in pairs. Reproduce by normal cell division. Cells contain bacteriochlorophyll a and a
19
vesicular intracytoplasmic membrane system when grown in the light. Grow best in micro-oxic
20
conditions by means of fermentation. Growth substrates are some sugars and organic acids.
21
Unable to grow phototrophically under anoxic conditions. Capable of slow aerobic
22
chemoorganotrophic growth. Incubation in the light stimulates growth. C1 compounds are not
23
utilized. Capable of dinitrogen fixation. Moderately acidophilic and mesophilic. The major
13
1
cellular fatty acids are 19:0 CYCLO ω8c and 18:1ω7c. The polar lipids consist of
2
phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and
3
sphingolipids. Quinones are represented by ubiquinone Q10. Belong to the family
4
Roseiarcaceae, class Alphaproteobacteria. Found in peatlands and soils. The type species is
5
Roseiarcus fermentans.
6 7
Description of Roseiarcus fermentans sp. nov.
8
Roseiarcus fermentans (fer.men’tans. L. part. adj. fermentans fermenting).
9
The description is as for the genus but with the following additional traits. Cells are 2.7-4.0
10
µm in lenght and 0.6-1.2 µm in wight. Liquid cultures are slightly pinkish or colorless when
11
grown in the light and in the dark, respectively. Absorption maxima of living cells grown in the
12
light are at 455, 489, 528, 593, 806 and 865 nm. Bacteriochlorophyll a is produced only in the
13
light. Spirilloxanthin, rhodopin and 3,4- didehydrorhodopin are the major carotenoids. Preferred
14
mode of growth is fermentation of some sugars and organic acids under micro-oxic conditions.
15
Carbon sources utilized include glucose, fructose, xylose, trehalose, malate, succinate, pyruvate,
16
galacturonic acid, and Phytagel. Methane, methanol and formate are not utilized. Nitrogen
17
sources utilized include ammonium salts, histidine, glutamate, yeast extract and N2. Oxidase-
18
negative and catalase-positive. Grows in the pH range 4.0-7.0 (optimum 5.5-6.5) and at 15- 30 C
19
(optimum 22–28 °C). NaCl inhibits growth at concentrations above 0.5% (w/v). The DNA G+C
20
content is 70.0 mol%. The type strain, Pf56T (=DSM 24875T = VKM B-2876T), was isolated
21
from the acidic Sphagnum peat bog Staroselsky moss, Tver region, Russia.
o
22 23
Description of Roseiarcaceae fam. nov.
14
1
Roseiarcaceae (Ro.se.i.ar.ca.ce’ae. N.L. masc. n. Roseiarcus type genus of the family; -aceae
2
ending to denote a family; N.L. fem. pl. n. Roseiarcaceae the Roseiarcus family).
3
Gram-negative, non-spore-forming bacteria. Aerobes capable of fermentation under micro-oxic
4
conditions. Mesophilic and mildly acidophilic. The family Roseiarcaceae belongs to the class
5
Alphaproteobacteria, order Rhizobiales. The type genus is Roseiarcus.
6 7 8
ACKNOWLEDGMENTS
9
This research was supported by the Program “Molecular and Cell Biology” and the Russian Fund
10
of Basic Research (project No 12-04-00768). The authors thank Vladimir M. Gorlenko for
11
helpful discussions and valuable advices, Natalia E. Suzina for electron microscopy analysis and
12
E.N. Detkova for DNA G+C content analysis.
15
1
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Sphagnum peat bog. Int J Syst Evol Microbiol 56, 1397-1402.
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19
prokPUB/index.htm.
19
1
Table 1. Cellular fatty acid composition of strain Pf56T. Values are percentages of total fatty acids.
2
Major fatty acids are shown in bold.
Fatty acid
Strain Pf56T
12:0
0.3
13:0
0.4
14:0
0.5
15:0
0.7
16:1ω7c
0.3
16:0
9.3
17:1ω8c
1.4
17:0
6.5
18:1ω9c
0.8
18:1ω7c
26.3
18:1ω5c
0.6
11 methyl 18:1ω7c
0.9
17:0 3OH
0.6
19:0 CYCLO ω8c
38.9
18:0 3OH
2.5
20:2ω6,9c
0.5
20
1
FIGURE CAPTIONS
2
Figure 1. (A) Phase-contrast micrograph of cells of stain Pf56T, bar, 10 µm. (B) Electron
3
micrograph of an ultrathin section showing vesicular intracytoplasmic membranes, granules of
4
polyhydroxybutyrate and polyphosphate, bar, 1 µm.
5
Figure 2. Absorption spectrum of the acetone/ methanol extract of cells of phototrophically
6
grown strain Pf56T showing the characteristic peaks of BChl a at 363 and 770 nm.
7
Figure 3. Influence of medium pH on the growth of strain Pf56T.
8
Figure 4. 16S rRNA gene-based neighbor-joining tree showing the phylogenetic position of
9
strain Pf56T in relation to taxonomically characterized members of the families
10
Methylocystaceae and Beijerinckiaceae, as well as to some environmental clone sequences
11
retrieved in cultivation-independent studies from various wetlands and soils. Bootstrap values
12
(percentages of 1000 data resamplings) >50% are shown. Black circles indicate that the
13
corresponding nodes were also recovered in the maximum-likelihood and maximum-parsimony
14
trees. Four members of the Gammaproteobacteria, Methylomicrobium album (X72777),
15
Methylobacter luteus (AF304195), Methylomonas methanica S1 (AF304196) and Methylococcus
16
capsulatus Texas (AJ563935) were used as an outgroup. Bar, 0.05 substitutions per nucleotide
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position.
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Suppl. Figure S1. Phase-contrast micrographs of cells of stain Pf56T grown under fully oxic (a)
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and micro-oxic conditions (b); bar, 10 µm.
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Suppl. Figure S2. (A) Growth of strain Pf56 in anoxic conditions in the dark with (1) and
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without (2) fructose. (B) Dynamics of fructose (1), propionate (2) and acetate (3) during
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anaerobic fermentation of fructose by strain Pf56 T. 21
1
Suppl. Figure S3. Polar lipids of strain Pf56 T after separation by two dimensional TLC.
2
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol;
3
CL, cardiolipin ; SL1 and SL2, sphingolipids; PL, unidentified phospholipid.
4
5 6
7
22
1 2 3
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1
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