Complete genome sequence and description of Salinispira pacifica gen. nov., sp. nov., a novel spirochaete isolated form a hypersaline microbial mat

Strains and cultivation conditions

Strain L21-RPul-D2^T was isolated from an anaerobic enrichment culture inoculated with slurries of a cyanobacterial
mat retrieved from the hypersaline Lake 21 on the Kiritimati atoll (Northern Line
Islands, Republic of Kiribati). The location of the sampling site and details of the
isolation method were described elsewhere 7].

For the preparation of media and incubation under anoxic conditions the anaerobe cultivation
technique of Hungate 10] with the modifications introduced by Bryant 11] was used. The basal medium for the characterization of strain L21-RPul-D2^T included per liter: sea salts (50.0 g), yeast extract (2.0 g), Biotrypticase (2.0 g),
L-cysteine-HCl × H₂O (0.5 g), Balch trace element solution (10.0 ml) 12] and 1.0 ml of a 0.1% (w/v) solution of resazurin. The pH was adjusted to 7.2 with
10 M KOH solution and the medium was boiled under a stream of O₂ free N₂ gas and cooled to room temperature. Aliquots of 5 ml were dispensed into Hungate-type
tubes, degassed under 80% N₂ and 20% CO₂ gas mixture, and subsequently sterilized by autoclaving at 120°C for 20 min. Before
inoculation aliquots of the following sterile anoxic stock solutions were injected
into the tubes containing 5 ml of medium: 0.1 ml of 10% (w/v) NaHCO₃, 0.1 ml of 2% (w/v) Na₂S × 9H₂O, and 0.05 ml of 30% (w/v) MgCl₂ × 6 H₂O. For routine cultivation 20 mM D-glucose was added to the medium from a 1 M sterile
anoxic stock solution.

For comparison the following type strains of related alkaliphilic Spirochaeta species were obtained from the DSMZ culture collection (Braunschweig, Germany): S. asiatica DSM 8901^T, S. africana DSM 8902^T, and S. dissipatitropha DSM 23605^T. All of these strains were cultured according to the recommendations given in the
DSMZ catalogue of strains 13] except that the DSMZ medium 1263 was used instead of DSMZ medium 700 for growing
the strains DSM 8901^T and DSM 8902^T.

Phylogeny

The determination of the almost complete 16S rRNA gene sequence of strain L21-RPul-D2^T was already described in 7] and deposited in the GenBank/EMBL/DDBJ databases under the accession number KC665949.
Phylogenetic trees based on almost complete 16S rRNA gene sequences with a minimum
length of 1300 nucleotides were reconstructed using distance matrix (neighbor-joining),
parsimony and maximum-likelihood programs included in the ARB package 14]. The dataset of aligned and almost complete 16S rRNA gene sequences was based on
the ARB SILVA database release 111 (July 2012) 15].

Based on the comparative sequence analysis of 16S rRNA genes strain L21-Rpul-D2^T was most closely related to Spirochaeta species. The genus Spirochaeta comprises currently more than 20 species with validly published names 16]. Members of this genus are Gram-negative free-living bacteria that are widely spread
in aquatic habitats, especially mud or sediments. The hallmark of spirochaetes is
their slender helical cell shape combined with polar inserted periplasmic flagella
conferring a spiral motility with bending and undulating movements. Despite a common
morphotype free-living spirochaetes are metabolic versatile and include highly diverse
phenotypes like obligately anaerobic, aerotolerant, halophilic, alkaliphilic, thermophilic
and psychrophilic representatives. The observed phenotypic diversity is reflected
in a large phylogenetic and genotypic divergence. The genomic DNA G?+?C content varies
within the Spirochaeta from 39 – 65 mol% 17], which exceeds the variation typically found among representatives of one genus by
far 18]. Accordingly, the 16S rRNA gene sequence identity values among type strains of the
genus Spirochaeta are in the range of 99.6 to 82%, which is typically found among strains related at
family or order level, but not within a genus. Several recent taxonomic studies have
identified a 16S rRNA gene identity value of 95% or above for strains belonging to
one genus 8,9,19]. This indicates that the genus Spirochaeta is currently only loosely defined on the molecular level and comprises several misclassified
species. In reconstructed phylogenetic trees based on 16S rRNA gene sequences strain
L21-Rpul-D2^T was affiliated with high bootstrap support to a clade of Spirochaeta species isolated from alkaline or saline environments (Figure 1). A BLAST search against the nr/nt database including 16S rRNA gene sequences of
non-cultured bacteria resulted in several hits with identity values above 97%. The
best matches were found with two clone sequences retrieved from a hypersaline microbial
mat (JN449894 and JN440289) indicating a preference of strains related to L21-RPul-D2^T for microbial mats. Another more distantly related cloned 16S rRNA gene sequence
(HQ916637) with an identity value of 97% was retrieved from a terrestrial mud volcano
in Taiwan. Based on 16S rRNA sequence identity values the closest related type strains
were S. africana DSM 8902^T (90%), S. asiatica Z-7591^T (89%) and S. dissipatitropha ASpC2^T (89%), which form together with strain L21-RPul-D2^T a common lineage supported by high bootstrap values above 80%. Five further stable
clades could be identified by bootstrap analyses, of which two were so far only represented
by one species with a validly published name (Figure 1).

Figure 1. Phylogenetic tree highlighting the position of strain L21-RPul-D2^T within the family Spirochaetaceae. The dendrogram is based on almost complete 16S rRNA gene sequences and was reconstructed
with a neighbor-joining distance matrix program as implemented in the ARB package
using phylogenetic distances calculated with the algorithm of Jukes and Cantor. No
filter or weighting masks were used to constrain the used positions of the alignment.
In addition, trees were reconstructed using the PHYLIP maximum parsimony program of
ARB and the RAxML maximum likelihood program. Support of a distinct branching by bootstrap
analyses is indicated by symbols. Closed circles at a distinct node indicate that
bootstrap values of 80% or above (percentages of 1000 resamplings) were obtained with
three different reconstruction methods, while open circles indicate that values of
80% or above were obtained with only two reconstruction methods. Stable clades of
Spirochaeta species formed by two or more type strains are marked with brackets. The sequence
of Leptospira interrogans (acc. no. Z12817) was used as outgroup (not shown). Type strains with genome sequencing
projects registered in GOLD 20] are labeled with one asterisk, those also listed as ‘Complete and Published’ with
two asterisks (see 21-23], CP002541 for S. globosa, CP003155 for S. pleomorpha, CP001841 for Treponema azotonutricium, and CP002696 for T. brennaborense). The bar represents an estimated sequence divergence of 5%.

Morphology and physiology

Shape and pigmentation

The Gram reaction of cells of strain L21-RPul-D2^T was determined with air-dried smears of liquid cultures that were fixed with methanol
and stained with DIFCO kit reagents. For electron microscopy, exponentially grown
cells were negatively stained with 1% sodium phosphotungstic acid (pH 7.2). Whole
cells and cross-sections were observed with a Zeiss EM 912 electron microscope at
an accelerating voltage of 75 kV. The cross-sections were obtained by fixation of
cells with 10% glutaraldehyde and 0.1 M cacodylate buffer, a post fixation staining
with 2% osmium tetroxide and subsequent inclusion in epoxy resin (EMbed 812). The
presence of spores was analyzed by phase contrast microscopic observations of young
and old cultures and pasteurization tests, performed at 80, 90 and 100°C for 10 and
20 min. Cells of strain L21-RPul-D2^T stained Gram-negative, had a slender helical shape and exhibited a rotating, undulatory
motility typical of spirochaetes. In exponential growth phase the size of cells was
0.2 – 0.25 ?m in width and 8–9 ?m in length with an approximate wavelength of 1 ?m
(Figure 2A). Sometimes cells up to 25 ?m in length were observed. Coccoid bodies resembling
spheroplasts appeared in stationary phase cultures and had a diameter ranging from
1.5 to 2.8 ?m (Figure 2B). The ultrastructure of cells is characterized by two periplasmic flagella that
overlap in the middle of the cell (Figure 2C).

Figure 2. Morphological characteristics of cells. Phase contrast micrographs of strain L21-RPul-D2^T showing the typical helical shape of exponentially grown cells (A) and spherical bodies during late stationary phase (B). Electron micrograph of a transversal cross-section through the middle of a cell
showing a bulging outer sheath encasing two putative periplasmic flagella (C).

Pigments were extracted for spectroscopic analyses from strain L21-RPul-D2^T and related type strains of Spirochaeta with a mixture of acetone/methanol (7:2 v/v) as described previously 24]. Spectra were recorded with a Thermo BioMate 6 UV–VIS split beam spectrophotometer.
Pigments were formed under anaerobic and semiaerobic incubation conditions and gave
cells a yellow to light orange color. Absorption spectra of the pigments extracted
with acetone/methanol were characteristic for carotenoids with maxima at 439 and 468 nm
and a shoulder at 487 nm. Carotenoid-like pigments could be also extracted from cell
pellets of the type strains of S. africana and S. dissipatitropha, but not of S. asiatica.

Growth conditions

The pH, temperature and NaCl concentration ranges for growth were determined using
basal medium supplemented with 20 mM D-glucose. Different pH values (5 to 9) of the
medium were adjusted in increments of around 0.5 by injecting aliquots of anoxic stock
solutions of 100 mM HCl (acidic pH values), 10% (w/v) NaHCO₃ or Na₂CO₃ (basic pH values) in Hungate-type tubes containing 5 ml of medium. Water baths were
used for incubating bacterial cultures from 15 to 55°C. NaCl requirement was determined
by directly weighing NaCl in Hungate-type tubes before dispensing modified basal medium
containing per liter: Na₂SO₄ (5.70 g), KCl (1.00 g), KBr (0.04 g), yeast extract (2.00 g), Biotrypticase (2.00 g),
L-cysteine-HCl (0.50 g), NaHCO₃ (0.30 g), resazurin (1.00 mg) and 10.00 ml of Balch trace element solution. Strain
L21-RPul-D2^T was moderately halophilic and grew optimally at a salinity of 5% (w/v). It required
at least 2% (w/v) NaCl for growth and tolerated salinities up to 15% (w/v), which
is the highest known salt tolerance of any known member of the free-living spirochaetes
and grew at temperatures ranging from 20 to 45°C, with an optimum at 35°C. The pH
range for growth was 6.5–8.4, with an optimum at pH 6.9-7.0.

Substrate utilization

Substrates (D-glucose, D-ribose, sucrose, D-fructose, D-xylose, D-arabinose, lactose,
D-maltose, D-mannose, D-melibiose, D-cellobiose, D-trehalose, glycerol, ethanol, methanol,
formate, acetate, pyruvate, fumarate, DL-lactate, succinate, DL-malate, citrate, butyrate,
propionate) were tested at a final concentration of 20 mM in glucose-free basal medium.
Casamino acids, peptone, starch and pullulan were tested at a final concentration
of 2 g l^-1 and 80% H₂ and 20% CO₂ gas mixture with and without acetate (2 mM) was tested under 2 bars of overpressure.
To test for electron acceptors, sodium thiosulfate (20 mM), sodium sulfate (20 mM),
sodium sulfite (2 mM), elemental sulfur (10 g l^-1), sodium nitrate (20 mM), or sodium nitrite (2 mM) were added to the medium. Cultures
were subcultured at least twice under the same experimental conditions before determination
of growth rates. H₂S production was determined photometrically as colloidal CuS according to Cord-Ruwisch
25]. End-products of metabolism were measured by high pressure liquid chromatography
(HPLC) after 2 days of incubation at 35°C 26]. Strain L21-RPul-D2^T had a strictly fermentative-type of metabolism and required yeast extract or Trypticase
peptone for growth, but no vitamins. It was saccharolytic and used pullulan, starch,
N-acetylglucosamine, D-fructose, D-glucose, D-maltose, D-mannose, and D-trehalose
for growth, but not peptides or amino acids. In addition the carboxylic acids fumarate
and pyruvate were utilized as electron donors. No positive growth response was obtained
with chitin, arabinose, cellobiose, D-galactose, lactose, melibiose, D-ribose, sucrose,
D-xylose, acetate, butyrate, casamino acids, citrate, formate, lactate, malate, propionate,
succinate, ethanol, glycerol, D-mannitol, methanol, and H₂/CO₂ (8:2 v/v). The end-products resulting from D-glucose fermentation were acetate, lactate,
ethanol, CO₂ and H₂. Sulfate, sulfite, elemental sulfur, nitrate and nitrite were not used as terminal
electron acceptors. Thiosulfate and nitrate were not reduced during anaerobic growth.
Despite a negative reaction of cells in tests for oxidase and catalase activity, growth
did not depend on prereduced media. Oxygen concentrations of up to 10% were tolerated
in the headspace gas atmosphere of statically incubated cultures. During semiaerobic
cultivation (5 – 10% O₂ in the headspace gas atmosphere) the growth yield did not increase compared to anaerobic
incubation and the pattern of fermentation products did not change significantly,
hence oxygen was not used as electron acceptor for respiratory metabolism.

Resistance to antibiotics

Susceptibility to antibiotics was tested by adding sterile anoxic stock solutions
of antibiotics to the complete medium prior to inoculation. Rifampicin and chloramphenicol
were dissolved in methanol, while stock solutions of all other antibiotic compounds
were prepared in distilled water. Final concentrations of the respective antibiotics
in cultivation media were 10, 100 and 1000 mg l^-1 for ampicillin, carbenicillin, D-cycloserin, gentamicin, kanamycin A and penicillin
G; 10, 100 and 500 mg l^-1 for tetracycline; 10 and 100 mg l^-1 for rifampicin; 20 and 200 mg l^-1 for chloramphenicol. Strain L21-RPul-D2^T was resistant to the antibiotics rifampicin (10–100 mg l^-1) and kanamycin A (10–1000 mg l^-1), but susceptible to ampicillin (1000 mg l^-1), carbenicillin (1000 mg l^-1), penicillin G (1000 mg l^-1), D-cycloserin (1000 mg l^-1), chloramphenicol (20 mg l^-1), gentamicin (1000 mg l^-1), and tetracycline (500 mg l^-1). A summary of the classification and general features of strain L21-RPul-D2^T is presented in Table 1 and a list of diagnostic traits allowing differentiation from related type strains
is given in Additional file 1.

Additional file 1. Differential phenotypic characteristics of Salinispira pacifica strain L21-RPul-D2^T and type strains of the phylogenetically closest related Spirochaeta species, as well as S. halophila and S. smaragdinae. +, positive; -, negative; (+), weakly positive; *, results obtained in this study;
ND, no data available. Strains and sources of data: Salinispira pacifica L21-RPul-D2^T (this study); S. africana Z-7692^T59]; S. asiatica Z-7591^T59]; S. dissipatitropha ASpC2^T60]; S. halophila RS-1^T61]; S. smaragdinae SEBR 4228^T62].

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Chemotaxonomy

The cellular fatty acid pattern of strain L21-RPul-D2^T was determined from cells grown to early stationary phase in TYG medium 7]. For comparison additional cellular fatty acid patterns were determined from related
type strains cultured in DSMZ medium 1263 under the conditions given in the DSMZ catalogue
of strains 13]. The preparation and extraction of fatty acid methyl esters from biomass and their
subsequent separation and identification by gas chromatography was done as described
elsewhere 35]. Polar lipid analyses were carried out by the DSMZ Identification Service according
to the published protocols 36]. Diagnostic diamino acids of the cell wall peptidoglycan were detected in hydrolysates
(4 N HCl, 100°C, 16 h) of whole cells by using GC/MS as described by Schumann 37]. The cellular fatty acid composition of strain L21-RPul-D2^T in comparison with the profiles of phylogenetically related Spirochaeta strains is shown in Additional file 2. The major cellulary fatty acids (5% of the total abundance) of the novel isolate
were C_14:0, C_16:0, iso-C_15:0, and C_18:0. Unique characteristics of strain L21-RPul-D2^T compared to all related Spirochaeta strains were a predominance of the fatty acids C_18:0 (9.7%) and iso-C_15:0 (11.7%) combined with the presence of the branched fatty acid ante-C_15:0. The determined polar lipid pattern revealed major amounts of phosphatidylglycerol,
an unidentified aminolipid, an unidentified phospholipid and two distinct glycolipids
(Additional file 3). The cell wall peptidoglycan contained L-ornithine as diagnostic diamino acid (type
A1? according to the classification of Schleifer and Kandler 38]), which is a characteristic of the family Spirochaetaceae30].

Additional file 2. Cellular fatty acid patterns of Salinispira pacifica strain L21-RPul-D2^T and phylogenetically related type strains of Spirochaeta. Values are percentages of total fatty acids. Major fatty acids (5% of total amount)
are given in bold. Fatty acids that were detected only in trace amounts (0.5% of
the total amount) are not shown. ALDE, aldehyde; DMA, dimethyl acetal; c, cis isomer; iso and ante indicate iso- and anteiso-branched fatty acids, respectively.
* Summed features are groups of fatty acids that could not be separated under the
conditions used: summed feature 9, iso-C_16:0 3OH and/or unknown fatty acid DMA with ECL 17.157.

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Additional file 3. Polar lipid pattern of strain L21-RPul-D2^T revealed after two dimensional thin layer chromatography. Staining of the chromatogram was done with molybdatophosphoric acid. Abbreviations:
PG, phosphatidylglycerol; AL, unidentified aminolipid; PL, unidentified phospholipid;
GL1 and GL2, unidentified glycolipids.

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