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1 NASA/NSSTC, VP-62, Astrobiology Laboratory, 320 Sparkman Dr., Huntsville, AL 35805, USA
2 Department of Biology, University of Alabama at Birmingham, AL 35294, USA
3 Laboratory for Structural Biology, The University of Alabama in Huntsville, MSB, 203C, Huntsville, AL 35899, USA
4 Department of Microbiology, University of Georgia, Athens, GA 30602-2605, USA
5 American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110, USA
6 Mitretek Systems, 3150 Fairview Park Drive South, Falls Church, VA 22042, USA
Correspondence
Elena V. Pikuta
elenapikuta{at}hotmail.com
Richard B. Hoover
Richard.Hoover{at}NASA.GOV
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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AF394926 (16S rRNA gene sequence of strain PmagG1T), DQ485314 and DQ485315 (16S RNA gene upstream regions of strain PmagG1T and T. collinsii ATCC BAA-296T, respectively) and DQ485316 and DQ485317 (partial sequence of the family A DNA polymerase genes of strain PmagG1T and T. collinsii ATCC BAA-296T, respectively).
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and, after reclassification of some species of the genera Lactosphaera and Ruminococcus, an emended description of the genus was published (Liu et al., 2002). Currently, the genus Trichococcus includes four species: Trichococcus flocculiformis (the type species), T. palustris, T. pasteurii and T. collinsii. All of these species are psychrotolerant mesophiles and are able to grow at low temperatures. All species in this genus have the same morphology, with typical coccoid-shaped cells (sometime elongated ellipses) occurring singularly, in pairs, in short chains or as irregular conglomerates. This pleomorphic nature is a common characteristic within the genus. All species of the genus Trichococcus are facultative anaerobes and, like species of the genus Carnobacterium, are capable of reducing resazurin in aerobic media during growth. Another interesting characteristic has been noted at the genotypic level: all species of this genus have a highly similar (99.9–100 %) 16S rRNA gene sequence (Liu et al., 2002). In bacteriology, there are cases where phylogenetic analysis demonstrates 100 % 16S rRNA gene sequence similarity, but, at the same time, DNA–DNA hybridization shows relatedness values of <70 % (enough for separation of strains into novel species). This situation is rare, but it raises methodological questions in bacterial taxonomy (Keswani & Whitman, 2001; Stackebrandt et al., 2002; Coenye et al., 2005).
In the present work, we describe a novel species of the genus Trichococcus capable of growth at sub-zero temperatures. Phylogenetic analysis of this novel isolate showed an identical 16S rRNA gene sequence (100 % similarity) to that of T. collinsii ATCC BAA-296T, but the phenotypes of these two strains had significant physiological differences. We have previously described the psychrotolerant characteristics of this novel isolate, PmagG1T (Hoover et al., 2002; Pikuta et al., 2003a; Pikuta & Hoover, 2003, 2004). DNA–DNA hybridization data supported the basis for the separation of this isolate into a novel species.
Strain PmagG1T was isolated from a sample of guano of the Magellanic penguin (Spheniscus magellanicus), an endemic species that inhabits the southern Patagonia region of the South American continent. The guano was taken from the bottom of a shallow marine tidal pool at the Seno Otway Magellanic penguin colony in southern Chile. Guano with surrounding seawater was collected aseptically in a Corning 50 ml centrifuge tube and immediately transferred to a cooler and maintained at +4 °C during transport to the Astrobiology Laboratory at the NASA Marshall Space Flight Center.
Homogenized sample material (0.5 ml) was injected into Hungate tubes containing the following medium (g l–1): NaCl, 30.0; KCl, 0.3; KH2PO4, 0.3; MgSO4.7H2O, 0.1; NH4Cl, 1.0; CaSO4.7H2O, 0.0125; NaHCO3, 0.4; Na2S.9H2O, 0.4; resazurin, 0.0001; yeast extract, 0.1; D-glucose, 5.0, with 2 ml vitamin solution (Wolin et al., 1963) l–1 and 1 ml trace mineral solution (Whitman et al., 1982) l–1. The final pH was 7.8. High-purity nitrogen was used as the gas phase. A pure culture was obtained by serial dilution and incubation at 4 °C. The ninth dilution of the morphologically monotypic culture was chosen for growth of colonies on an agar medium. Isolation of colonies was performed on the above-described medium with 3 % (w/v) agar by the roll-tube method. After incubation for 14 days at 4 °C, colonies of strain PmagG1T were circular (on the surface) or convex lens-shaped (convex–convex lens-shaped in deep agar), white in colour and had a diameter of 0.5–4.0 mm. The surface of the large, circular colonies had an inner raised greyish circle. The margins were entire, smooth and thinner than the centre. The consistency of the colonies was mucoid and slimy. Young colonies were dry, but the older ones were shiny. The morphology of colonies grown at –5 °C was different (Fig. 1d, e). Cells on the surface of the colonies grown at –5 °C were covered by an excreted mucoid substance that probably maintains the encapsulated cells in an unfrozen state.
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The morphology of the novel isolate was observed under a phase-contrast microscope (Fisher Micromaster) and a FEI Quanta 600FEG field emission scanning electron microscope was used to examine the ultramicrostructure of the cell surfaces (Fig. 1
). Cells of strain PmagG1T were coccoid or ovoid (olive)-shaped and at sub-zero temperatures were surrounded by mucoid matter (Fig. 1d, e). Cells occur singly, in pairs, in short chains or gathered in irregular conglomerates. The diameter of cells was 1.3–2.0 µm. Cells were motile and slow moving, with a peritrichous type of locomotion. Gram staining showed a variable reaction. Spores were never observed.
Growth of a pure culture was determined by direct cell counting under a phase-contrast microscope or by turbidimetry by measuring optical density changes at 595 nm (Genesis 5; Spectronic Instruments). Catalase activity was determined by the reaction with hydrogen peroxide (Gerhardt et al., 1994). Growth substrates at concentrations of 3 g l–1 were added separately to the medium, which contained 0.1 g yeast extract l–1. Metabolic end products of glucose fermentation in the liquid phase were determined by HPLC (Pikuta et al., 2005). Separation was done on an Aminex HPX-87H (Bio-Rad) column with 5 mM H2SO4 as the mobile phase. Gases were measured using a gas chromatograph 3700 (Varian) equipped with a Porapak Q column and thermal conductivity detector. Nitrogen was used as the gas carrier.
The novel isolate was a facultative anaerobe and grew well under both aerobic and anaerobic conditions. During growth under aerobic conditions, resazurin was reduced (i.e. became colourless). The same effect is observed during cultivation of Carnobacterium species (Pikuta et al., 2005; Franzmann et al., 1991). Strain PmagG1T had a negative catalase reaction. It could grow without NaCl in the medium if sodium salts were replaced with potassium salts, with a prolonged lag phase, indicating that strain PmagG1T was not dependent on the Na+ ion. The NaCl range for growth was 0–6.5 % (w/v) and optimum growth was observed at 0.5 % (w/v). No growth was observed at 10 % (w/v) NaCl. Strain PmagG1T grew in the pH range 6.0–10.0, with optimum growth at pH 8.5 (Fig. 2b), indicating that it is a slightly alkaliphilic bacterium. The temperature range for growth of strain PmagG1T was from –5 to 35 °C with optimal growth at 28–30 °C. Growth was never observed at –7 °C or 40 °C (Fig. 2a).
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Metabolic end products of the culture grown on D-glucose were lactate (3.3 mM), formate (1.5 mM), acetate (0.5 mM) and ethanol (1.3 mM) in the liquid phase and CO2 in the gas phase.
The sensitivity of strain PmagG1T to ampicillin, kanamycin, gentamicin, tetracycline and rifampicin (all at 250 µg ml–1) and chloramphenicol (125 µg ml–1) was tested. Strain PmagG1T was sensitive to all of these antibiotics.
For extraction of fatty acid methyl esters (FAMEs), the culture was incubated for 4 days at 22 °C on the liquid medium described above. The extraction and analysis procedures have been described previously (Pikuta et al., 2003b). The major fatty acids for strain PmagG1T were C14 : 0, C16 : 1cis7, C16 : 0, C18 : 1cis9 and C18 : 0. The percentages of each are shown in Table 1.
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with slight modifications. Each biomass (0.1 g) was resuspended in 1.5 ml TE buffer (pH 8.0) and washed twice following centrifugation at 14 000 g for 5 min. Each pellet was then resuspended in 1 ml TE buffer (pH 8.0) and sonicated for 4 min. Before preparation of the mixture for electrophoresis, each suspension was homogenized for 1 min. The total volume of each mixture was 12 µl, including 1 µl blue/orange 6x loading dye (Promega) and an aliquot of distilled water. Dilutions were as follows: 3 µl protein standard; first dilutions (threefold) contained 4 µl cell extract; second dilutions (sixfold) contained 2 µl of each suspension. SDS-PAGE was performed using a 12 % Tris/HCl gel (Bio-Rad), which was subsequently stained with Coomassie blue (Fig. 3). Differences were observed between strain PmagG1T and T. collinsii ATCC BAA-296T in the intensities of protein bands b and d; strain PmagG1T (Fig. 3, lanes 2 and 5) contained band b but did not have band d, which was found in T. collinsii ATCC BAA-296T (Fig. 3, lanes 3 and 6). In addition, strain PmagG1T contained an additional protein band between bands g and h, which was absent in T. collinsii ATCC BAA-296T. Band k was missing in the reference strain but was present in strain PmagG1T (Fig. 3, lanes 2 and 3).
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). The 16S rRNA gene of strain PmagG1T was amplified and sequenced as described previously (Hoover et al., 2003).
A sequence covering 1483 nt of the 16s rRNA gene was obtained, corresponding to positions 27–1492 of the Escherichia coli 16S rRNA gene sequence. The G+C content of this sequence was 53.4 mol%. The sequence was compared with all GenBank sequences and appeared to be highly similar to the sequences of Trichococcus species. The sequence was aligned with closely related sequences using CLUSTAL W (Thompson et al., 1994). Pairwise distances were computed from 1371 common nucleotide sites with MEGA version 3.1 (Kumar et al., 2004) using the Jukes–Cantor model (Jukes & Cantor, 1969). Unrooted phylogenetic trees showing the position of strain PmagG1T among all currently known species of the genus Trichococcus and some species of the genera Carnobacterium and Isobaculum were constructed with the same program using neighbour-joining (Fig. 4), minimum evolution and maximum-parsimony methods. All trees shared the same topology. According to the pairwise distance table (not shown), strain PmagG1T appeared closest to T. collinsii ATCC BAA-296T, T. pasteurii ATCC 35945T, T. palustris DSM 9172T and T. flocculiformis DSM 2094T, with similarities of 100.0, 99.9, 99.4 and 99.0 %, respectively.
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Melting temperatures (Tm) of total genomic DNA from strain PmagG1T, T. pasteurii ATCC 35945T and T. collinsii ATCC BAA-296T were determined as described previously (De Ley et al., 1970; Gillis et al., 1970). DNA was prepared and purity determined as described by Ausubel et al. (1987). The Tm of the genomic DNA was 78±3 °C (mean±SD, n=4) for strain PmagG1T, 86±2 °C (n=4) for T. collinsii ATCC BAA-296T and 85±1 °C for T. pasteurii ATCC 49837 (n=4).
DNA–DNA hybridization analysis of genomic DNA of strain PmagG1T and T. collinsii ATCC BAA-296T and of strain PmagG1T and T. pasteurii ATCC 49837T was performed at 5 °C above the Tm of the DNA from each species, followed by calculation of the initial reassociation kinetics using linear regression analysis (De Ley et al., 1970; Johnson, 1985). The results showed DNA–DNA relatedness values of 47±1.5 % (mean±SD, n=3) between the genomes of strain PmagG1T and T. pasteurii ATCC 49837T and 45±1 % (n=3) between the genomes of strain PmagG1T and T. collinsii ATCC BAA-296T.
The G+C content of the genomic DNA of strain PmagG1T was determined by HPLC as described previously (Pikuta et al., 2003b; Mesbah et al., 1989) and was found to be 45.8±0.1 mol% (mean±SD, n=4).
To evaluate further the degree of separation between isolate PmagG1T and T. collinsii ATCC BAA-296T, additional sequence information was acquired and analysed. A sequence upstream of the 16S rRNA gene was obtained using multiplex restriction site PCR (Weber et al., 1998) with nested primers 5'-GTTACTCACCCGTCCGCCAC-3' and 5'-CTCAGTGGAAGCAAGCTTCC-3'. For strain PmagG1T, the sequence was 435 nt, including 31 nt of the 16S rRNA gene. For T. collinsii ATCC BAA-296T, the sequence was 524 nt long, including 55 nt of the 16S rRNA gene. A pairwise comparison of these sequences revealed 20 differences in the upstream region of the 16S rRNA gene.
A partial sequence of a family A DNA polymerase gene was amplified by PCR from PmagG1T and T. collinsii ATCC BAA-296T using the degenerate primers 5'-AARACNAARACNGGNTAYWSNAC-3' and 5'-ATRTCNGCNGCNSWNCCYTGDATNGG-3', which were designed based on available family A DNA polymerase sequences from 11 members of the order Lactobacillales. PCR products were cloned into a sequencing vector (StrataClone PCR cloning kit; Stratagene) and sequenced in both directions using vector-specific sequencing primers. Primer sequences were removed from the consensus sequences. For both strains, a sequence of 722 nt was obtained, encoding a 240 aa sequence. Pairwise comparison revealed 48 differences at the nucleotide level and five differences at the amino acid level.
Our novel isolate demonstrated 100 % similarity to the reference strain T. collinsii ATCC BAA-296T by 16S rRNA gene sequence analysis, but had a DNA–DNA relatedness value of <70 % by hybridization analysis. This situation is not unique and has been discussed previously (Liu et al., 2002). Of course, this situation suggests the need for additional differentiating experiments. Here, we added data from gel electrophoresis of total cell proteins and a comparison of the sequence upstream of the 16S rRNA gene, which were also different. In addition, the partial sequence of a family A DNA polymerase gene from the two compared strains showed differences at the nucleotide and amino acid levels.
According to Cohan (2001), strain PmagG1T and other species of the genus Trichococcus may have experienced the same environmental stress during evolution and perhaps developed a strong conservative mechanism in their genomes that prevented the development of separate ‘ecotypes' within this group.
From all checked strains of psychrophilic and psychrotolerant bacteria in our laboratory collection, only strain PmagG1T grew at sub-zero temperatures. This is the first bacterial representative known to be able to grow at –5 °C in pure culture (Hoover et al., 2002). Strain PmagG1T was obtained from guano of the Magellanic penguin, an endemic bird species that has evolved over a long period of time in the same geographical area. Table 2 shows a comparison of the distinguishing features of all known species of the genus Trichococcus. The pH and NaCl maxima were found to be highest for strain PmagG1T. In addition, the optimum pH for growth of strain PmagG1T was higher than that of T. collinsii and T. flocculiformis. Notwithstanding the identical 16S rRNA gene sequences of the novel isolate and T. collinsii ATCC 296T, their metabolic features showed significant differences: strain PmagG1T was incapable of growth on D-trehalose, whereas T. collinsii ATCC 296T and T. pasteurii ATCC 35945T both fermented this sugar and, in contrast to T. collinsii ATCC 296T, strain PmagG1T could grow on D-ribose and maltose. The G+C content of the genomic DNA of strain PmagG1T and T. collinsii ATCC 296T differed by 1.2 mol% and the Tm differed by 8 °C. In comparison with T. pasteurii ATCC 35945T, strain PmagG1T differed in its Tm (by 7 °C), its ability to ferment D-ribose and its ability to grow on D-trehalose.
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). DNA–DNA hybridization of the genomic DNA of strain PmagG1T and T. collinsii ATCC 296T and of strain PmagG1T and T. pasteurii ATCC 35945T exhibited DNA–DNA relatedness values of <50 %. Initially, we thought that the low minimum temperature for growth was exclusive to strain PmagG1T, but it was determined that reference strain T. collinsii ATCC 296T was also capable of growth at –5 °C, requiring the species description to be emended.
On the basis of phenotypic and genotypic characteristics (cell morphology, facultatively anaerobic and fermentative metabolism, psychrotolerant and slightly alkalitolerant physiology, independence from NaCl, 16S rRNA gene sequence, DNA–DNA hybridization, a sequence upstream of the 16S rRNA gene and a region of the family A DNA polymerase gene), strain PmaG1T is proposed as the type strain of a novel species of the genus Trichococcus, for which the name Trichococcus patagoniensis sp. nov. is proposed.
Emended description of Trichococcus collinsii Liu et al. 2002
The minimum temperature for growth is –5 °C (under both aerobic and anaerobic conditions).
Description of Trichococcus patagoniensis sp. nov.
Trichococcus patagoniensis (pa.ta.go.ni.en'sis. N.L. masc. adj. patagoniensis pertaining to Patagonia, the region of South America where the sample for the type strain was collected).
Cells are motile cocci with a diameter of 1.3–2.0 µm. Cells are spherical, ovoid or olive-shaped and occur as single cells, in pairs, in chains or as irregular conglomerates. Gram-variable. Growth occurs between –5 and 35 °C (optimum 28–30 °C). Slightly alkalophilic: pH range for growth is 6.0–10.0, with optimum growth at pH 8.5. Range of NaCl for growth is 0–6.5 % (w/v); optimum growth occurs at 0.5 % NaCl. Facultative anaerobe and catalase-negative. Heterotrophic growth occurs with D-glucose, D-fructose, maltose, D-mannitol, D-mannose, D-ribose, D-arabinose, sucrose, starch, pyruvate and citrate. The metabolic end products are lactate, formate, acetate, ethanol and CO2 (in the gas phase). Sensitive to ampicillin, kanamycin, gentamicin, tetracycline, rifampicin and chloramphenicol. The G+C content of the genomic DNA of the type strain is 45.82 mol%.
The type strain, strain PmagG1T (=ATCC BAA-756T=JCM 12176T=CIP 108035T), was isolated from a guano sample of Magellanic penguins (Spheniscus magellanicus) inhabiting the southern region of Chilean Patagonia.
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ACKNOWLEDGEMENTS |
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