Download A new radiation-resistant bacterium from Azores

FEMS Microbiology Letters 247 (2005) 161–169
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Truepera radiovictrix gen. nov., sp. nov., a new radiation
resistant species and the proposal of Trueperaceae fam. nov.
Luciana Albuquerque a, Catarina Simões a, M. Fernanda Nobre b, Nicole M. Pino c,
John R. Battista c, Manuel T. Silva d, Fred A. Rainey c, Milton S. da Costa a,*
a
Departamento de Bioquı́mica and Centro de Neurociências, Universidade de Coimbra, 3001-401 Coimbra, Portugal
Departamento de Zoologia and Centro de Neurociências, Universidade de Coimbra, 3004-517 Coimbra, Portugal
c
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
d
Instituto de Biologia Molecular e Celular, Universidade do Porto, R. do Campo Alegre, 4150 Porto, Portugal
b
Received 10 April 2005; received in revised form 2 May 2005; accepted 2 May 2005
First published online 17 May 2005
Edited by A. Oren
Abstract
Two isolates, belonging to a new species of a novel genus of the Phylum ‘‘Deinococcus/Thermus ’’, were recovered from hot spring
runoffs on the Island of São Miguel in the Azores. Strains RQ-24T and TU-8 are the first cultured representatives of a distinct phylogenetic lineage within this phylum. These strains form orange/red colonies, spherical-shaped cells, have an optimum growth temperature of about 50 C, an optimum pH for growth between about 7.5 and 9.5, and do not grow at pH below 6.5 or above pH 11.2.
These organisms grow in complex media without added NaCl, but have a maximum growth rate in media with 1.0% NaCl and grow
in media containing up to 6.0% NaCl. The organisms are extremely ionizing radiation resistant; 60% of the cells survive 5.0 kGy.
These strains are chemoorganotrophic and aerobic; do not grow in Thermus medium under anaerobic conditions with or without
nitrate as electron acceptor and glucose as a source of carbon and energy, but ferment glucose to D-lactate without formation of gas.
The organisms assimilate a large variety of sugars, organic acids and amino acids. Fatty acids are predominantly iso- and anteisobranched; long chain 1,2 diols were also found in low relative proportions; menaquinone 8 (MK-8) is the primary respiratory quinone. Peptidoglycan was not detected. Based on 16S rRNA gene sequence analysis, physiological, biochemical and chemical analysis
we describe a new species of one novel genus represented by strain RQ-24T (CIP 108686T = LMG 22925T = DSM 17093T) for which
we propose the name Truepera radiovictrix. We also propose the family Trueperaceae fam. nov. to accommodate this new genus.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Truepera radiovictrix; Deinococcus; Thermaceae; Radiation resistance; ‘‘Deinococcus/Thermus’’; Trueperaceae
1. Introduction
The phylum ‘‘Deinococcus/Thermus’’ represents an
ancient line of descent within the domain Bacteria that
includes the extremely radiation resistant bacteria of
the family Deinococcaceae [1] represented by the species
*
Corresponding author. Tel.: +351 239824024; fax: +351 239826798.
E-mail address: [email protected] (M.S. da Costa).
of the genus Deinococcus [2,3] and the slightly thermophilic or thermophilic members of the family Thermaceae [4] represented by the species of the genera
Thermus [5,6]; Meiothermus [7,8], Marinithermus [9],
Vulcanithermus [10] and Oceanithermus [11,12]. This
phylogenetic lineage also includes a number of environmental 16S rRNA gene sequences, several of which are
not closely related to any cultured strains and form distinct branches of uncultured strains (e.g. AY905381,
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.05.002
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L. Albuquerque et al. / FEMS Microbiology Letters 247 (2005) 161–169
AY250871, AF513964 and AJ853517). The species of
the genus Deinococcus and the genera of the family
Thermaceae share few easily discernible characteristics;
the major respiratory quinone is menaquinone 8 (MK8) and ornithine is the diamino acid of the peptidoglycan
of all species examined. The species of the genus Deinococcus have optimum growth temperatures that range
between about 9 and 50 C, form spherical or rodshaped cells that generally stain Gram-positive because
of a thick multilayered cell wall. These organisms are
also the archetypal extremely gamma (c) radiation-resistant bacteria [2]. The species of the family Thermaceae
have optimum temperatures between about 50 and
70 C, form rod-shaped cells and filaments that stain
Gram-negative, and possess a thin layer of peptidoglycan surrounded by a corrugated outer membrane-like
structure [5,7]. The type strain of the species Thermus
aquaticus has only recently been shown to be slightly c
radiation resistant, but the degree of resistance of the
other species remains unknown [13]. The species of
Meiothermus and Thermus are generally isolated from
fresh water thermal environments, although strains of
T. thermophilus are isolated from terrestrial or marine
thermal environments venting saline water [5]. The species Marinithermus hydrothermalis, Vulcanithermus
mediatlanticus, Oceanithermus profundus and O. desulfurans have been exclusively recovered from abyssal marine hot springs [9–12]. The species of the genus
Deinococcus, on the other hand, have been isolated from
a variety of sources subjected to c irradiation, hydrothermal areas and extremely dry environments where
their diversity appears to be large [2,14–16].
We recently isolated a large number of strains from
two thermal sites on the Island of São Miguel in the
Azores at 50 C. Some of these organisms were red-pigmented and formed spherical cells and had an optimum
growth temperature of about 50 C. 16S rRNA gene sequence analysis showed that these organisms represent a
distinct lineage of organisms within the Phylum ‘‘Deinococcus/Thermus’’. Based on phylogenetic analysis,
physiological and biochemical parameters, and radiation-resistance, we are of the opinion that strains RQ24T and TU-8 represent a novel genus and species for
which we propose the name Truepera radiovictrix, the
type strain being RQ-24T (CIP 108686T = LMG
22925T = DSM 17093T). We are also of the opinion that
these organisms represent a new family for which we
propose the name Trueperaceae fam. nov.
2. Materials and methods
2.1. Isolation and bacterial strains
Strains RQ-24T (T = type strain) and TU-8 were isolated from a hot spring runoff at Ribeira Quente and
from a hot spring at Furnas on the Island of São Miguel
in the Azores. Water samples were transported and
maintained without temperature control for 6 days,
and then filtered through membrane filters (Gelman type
GN-6; pore size 0.45 lm; diameter 47 mm). The filters
were placed on the surface of agar-solidified Thermus
medium [5], wrapped in plastic bags and incubated at
50 C. The isolates were stored at 70 C in Thermus
medium with 15% (w/v) glycerol. The type strains of
Deinococcus murrayi ALT-1bT (=DSM 11303T), D.
radiodurans RT1 (ATCC 13939T), ‘‘Meiothermus timidus’’
SPS-243T (=LMG 22897T) and Thermus aquaticus YT1T (=ATCC 25104T) were used for comparison.
2.2. Morphology, growth, biochemical and physiological
characteristics
Electron microscopy was performed on exponential
phase cells of strain RQ-24T in medium containing
1.0% NaCl [17,18]. Cell morphology and motility were
examined by phase contrast microscopy during the
exponential growth phase. The salt range of the organisms was determined in Thermus liquid medium and in
Degryse medium 162 [19], with NaCl ranging between
0.0% and 8.0%. The growth temperature range of the
strains was performed in liquid Thermus medium containing 1.0% NaCl [14]. The pH range for growth was
examined at 50 C in the same medium by using
100 mM MES for pH values between 6.0 and 6.5,
100 mM HEPES for pH values between 7.0 and 7.5,
100 mM TAPS for pH values between 8.0 and 8.5,
100 mM CAPSO for pH values between 9.0 and 10.0
and 100 mM CAPS for pH values between 10.5 and
11.5; the pH of each buffer was adjusted with HCl or
NaOH.
Unless otherwise stated, all biochemical and tolerance
tests were performed, as described previously [8], in Thermus liquid medium or Thermus agar containing 1% NaCl
at 50 C for up 5 days. Anaerobic growth was assessed in
cultures in Thermus medium containing NaCl (10.0 g l 1),
glucose (2.0 g l 1), KNO3 (1.0 g l 1) and TAPS (50 mM;
pH 8.2), or in the same medium without glucose, incubated in anaerobic chambers (GENbox anaer, bioMérieux). Single-carbon source assimilation tests were
performed in a minimal medium composed of Thermus
basal salts containing 1.0% NaCl to which filter-sterilized
ammonium sulfate (0.5 g l 1) and the carbon source
(2.0 g l 1) were added. Growth of the strains on single carbon sources was examined by measuring the turbidity of
cultures incubated at 50 C in 20 ml screw capped tubes
containing 10 ml of medium for 5 days. The production
of acid from carbohydrates was determined with the
API 50 CHL system (bioMérieux) according to the manufacturerÕs instructions, using medium API 50 CHB/E
containing 1.0% NaCl. Results were recorded after 24 h,
48 h and 5 days of incubation at 50 C. Fermentation
L. Albuquerque et al. / FEMS Microbiology Letters 247 (2005) 161–169
products were determined by growing the cultures in
Thermus liquid medium containing glucose (11.1 mmol)
and TAPS (50 mM, pH 8.2) overnight. The cultures were
washed by centrifugation and incubated at 50 C in
50 mM phosphate buffer, pH 8.2 containing glucose
(11.1 mmol) at a turbidity of 7–8 (OD 610 nm) for 24 h
with periodic samplings and centrifuged. Glucose utilization was determined using the glucose oxidase kit (Sigma/
Aldrich), the production of D- and L-lactate, and ethanol
was determined using test kits (R-Biopharm, Darmstadt,
Germany). The samples were also analyzed using standard proton-nuclear magnetic resonance (1H NMR) on
a Bruker (AMX300, Germany) spectrometer.
2.3. Ionizing radiation resistance
Stationary phase cultures (1 · 108–3 · 108 cfu ml 1)
of strains RQ-24T, TU-8, Thermus aquaticus YT-1T,
and Deinococcus radiodurans RT1 were exposed to
5000 Gy of c radiation. Irradiation was conducted at
25 C using a Model 484R 60Co irradiator (J.L. Shepherd & Associates, San Fernando, CA) at a rate of
30 Gy min 1. Survival was determined by plating serial
dilutions of irradiated cultures in triplicate on TGY
(5.0 g l 1 tryptone, 1.0 g l 1 glucose, 3.0 g l 1 yeast extract) plates, incubating at 50 C and the colonies were
counted three and five days after plating.
2.4. Peptidoglycan analysis and polar lipid, lipoquinone
and fatty acid compositions
Purified cell wall preparations were obtained and the
identification of the peptidoglycan type was attempted
using previously described methods [20,21]. The cultures
used for polar lipid analysis were grown in 1 l Erlenmeyer flasks with 250 ml of 1% NaCl Thermus medium
at 50 C. Harvesting of the cultures, extraction of the
lipids and single dimensional thin-layer chromatography
were performed as described previously [22]. Lipoquinones were extracted from freeze-dried cells, purified
by thin-layer chromatography, and separated by high
performance liquid chromatography [14]. Cultures for
fatty acid analysis were grown on solidified Thermus
medium, in sealed plastic bags submerged in a water
bath at 50 C for 48 h. Fatty acid methyl esters
(FAMEs) were obtained and separated, identified and
quantified with the standard MIS Library Generation
Software (Microbial ID Inc) as described previously [6].
2.5. Determination of G + C content of DNA and 16S
rRNA gene sequence determination and phylogenetic
analyses
The DNA for the determination of the G + C content
of the DNA was isolated as described previously [23].
The G + C content of DNA was determined by high-
163
performance liquid chromatography as described by
Mesbah et al. [24].
The extraction of genomic DNA for 16S rRNA gene
sequence determination, PCR amplification of the 16S
rRNA gene and sequencing of the purified PCR products were carried out as described previously [25]. Purified reactions were electrophoresed using a model 310
Genetic Analyzer (Applied Biosystems). The 16S rRNA
gene sequences were aligned against representative reference sequences of members of the Phylum ‘‘Deinococcus/
Thermus’’ using the ae2 editor [26]. The method of Jukes
and Cantor [27] was used to calculate evolutionary distances. Phylogenetic dendrograms and bootstrap analyses were generated using various algorithms contained in
the PHYLIP package [28].
2.6. Nucleotide sequence accession numbers
The 16S rRNA gene sequences determined in this
study were deposited in GenBank under the accession
numbers: RQ-24T (DQ022076) and TU-8 (DQ022077).
3. Results
3.1. Isolation of strains RQ-24T and TU-8
Strain RQ-24T was isolated from a hot spring within
a geothermal area located along an almost vertical wall
and dry bed of a stream known as Ribeira Quente located about 500 m south-east of the geothermal area
on the eastern edge of the town of Furnas where strain
TU-8 was isolated. The hot springs in both areas discharge neutral to slightly alkaline water that may reach
the boiling point. There is also a small solfataric area in
the Furnas geothermal area with heated acid soil and
mud. Strain RQ-24T was isolated from a runoff with a
temperature of 52 C and a pH of 7.0; one vent nearby
had a Na+ content of 474.2 mg l 1 (unpublished results).
Strain TU-8 was isolated from a site in the Furnas area
with a temperature of 70 C and pH 7.5. Thermal
springs in this area have Na+ contents of 44.5 to
63.0 mg l 1 [29]. Several other organisms were recovered
from the plates incubated at 50 C, some of which belonged to a new species of the genus Meiothermus [8].
3.2. Morphology, growth, biochemical and physiological
characteristics
The red-pigmented organisms formed non-motile
spherical cells about 1.25–2.0 lm in diameter, most of
which formed pairs or tetrads. The organisms stained
light brown with the Gram stain. The cytoplasm of
strain RQ-24T had ribosomes, a fibrillar nucleoid and
tubular structures of unknown nature (Fig. 1(a)). The
cytoplasmic membrane had a symmetric triple-layered
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L. Albuquerque et al. / FEMS Microbiology Letters 247 (2005) 161–169
profile. The cell wall had three distinct layers (Fig. 1(b));
the innermost layer (L1) appeared as a thin (6–9 nm)
electron-dense layer. The outermost layer (L3) was a
thick (20–90 nm) electron-dense layer and was homogeneous except for the irregular surface. Between these
two electro-dense layers there was an electron-transparent layer with fibrillar material (L2). A periplasmic space
was visible between the cytoplasmic membrane and the
L1 layer of the cell wall (arrowhead).
Strains RQ-24T and TU-8 had an optimum growth
temperature in the neighbourhood of 50 C and did
not grow at 20 or 60 C (results not shown). Growth
took place in Thermus medium which contains 8 mg l 1
NaCl or Degryse medium 162 which does not contain
added NaCl, but the growth rate increased in media
containing 1.0% NaCl and weak growth still occurred
in medium with 6.0% NaCl (Supplementary Fig. 1a).
The optimum pH of strain RQ-24T and TU-8 was between 7.5 and 9.5; the organisms grew at pH 11.2, but
no growth was detected at pH 6.0 or 11.5 (Supplementary Fig. 1b). These organisms were oxidase and catalase
positive and used carbohydrates, organic acids and
amino acids as single carbon and energy sources; yeast
extract or co-factor were not necessary for growth in a
minimal medium (Table 1). Nitrate was not reduced
and anaerobic growth in the presence of glucose with
or without the addition of nitrate was not observed.
The organisms produced acid from several sugars. Glucose (5.8 mmol) was fermented to D-lactate (12 mmol);
gas and ethanol were not produced, however, minor levels of succinate and acetate (4.6% and 4.7% of lactate,
respectively) were also detected by 1H NMR.
Strains RQ-24T and TU-8 behaved identically exhibiting 60% survival following exposure to 5.0 kGy of c
irradiation. Cultures of D. radiodurans showed no loss
of viability at this dose, whereas the culture of T. aquaticus was eradicated by this treatment (results not
shown).
3.3. Peptidoglycan, polar lipids, respiratory quinones and
fatty acids
Two attempts were made on each of the two strains
to detect and identify the peptidoglycan type, but they
failed. The polar lipid pattern of strains RQ-24T and
TU-8 consisted of a complex mixture of glycolipids
and phospholipids. The major respiratory quinone of
all strains was menaquinone 8 (MK-8). The fatty acids
were predominantly iso- and anteiso-branched fatty
acids of which 15:0 anteiso, 17:0 iso and 17:0 anteiso
predominated (Table 2). One acyl compound had and
equivalent chain length (ECL) consistent with isobranched 1,2-diol with 18 carbons (iso C18:0 diol) and
another compound with ECL 16.090 probably represents iso C15:0 diol.
3.4. 16S rRNA gene sequence comparison
Fig. 1. Ultrathin section of strain RQ-24T. (a) A pair of cells during
division with symmetric triple-layered cytoplasmic membrane and the
complex multi-layered cell wall. Bar = 0.25 lm. (b) Higher magnification of the envelopes of strain RQ-24T, showing a symmetric triplelayered cytoplasmic membrane (CM), and the three layers of the cell
wall (L1, L2 and L3). The arrowhead indicates the periplasmic space.
Bar = 0.12 lm.
Almost complete 16S rRNA gene sequences comprising 1461 nucleotide positions were determined for
strains RQ-24T and TU-8. These two 16S rRNA gene sequences were found to be identical. Phylogenetic analyses showed these strains to fall within the radiation of
the ‘‘Deinococcus/Thermus’’ lineage. The strains represent the first cultured members of a phylogenetic lineage
comprised of environmental 16S rRNA gene sequences
(Fig. 2). Within this lineage the new isolates represent
a distinct branch within a cluster of 3 environmental
16S rRNA gene sequences recovered from diverse habitats. The 16S rRNA gene sequence to which the strains
RQ-24T and TU-8 are most closely related (95% sequence similarity) was recovered from a lichen dominated cryptoendolithic community in the McMurdo
Dry Valleys, Antarctica [30]. The reconstructed phylogeny shown in Fig. 2 and the corresponding pairwise similarity values show this novel lineage to be equidistant to
L. Albuquerque et al. / FEMS Microbiology Letters 247 (2005) 161–169
165
Table 1
Characteristics of strains RQ-24T and TU-8a
Morphology
Size (lm)
Pigmentation
Gram reaction
NaCl range (%)
NaCl optimum (%)
Growth temperature
range (C)
Optimum growth
temperature (C)
pH range
Optimal pH
Reduction of NO3 to
NO2
Anaerobic growth
Degradation of:
Esculin
Arbutin
Gelatin
Casein
Elastin
Starch
Xylan
Tween 20
Tween 40
Tween 60
Tween 80
a
Spherical
1.25–2.0
Orange–red
Indeterminate
0–7
1
25–55
Assimilation of:
D-Glucose
+
D-Mannose
+
D-Galactose
+
D-Fructose
+
L-Sorbose
D-Arabinose
+
D-Mannitol
+
myo-Inositol
Glycerol
Succinate
a-Ketoglutarate
+
+
50
L-Arabinose
+
DL-Lactate
6.5–11.2
7.5–9.5
D-Ribose
+
+
+
+
+
+
+
+
D-Xylose
Sucrose
+
+
+
+
+
+
+
+
Maltose
Lactose
D-Trehalose
D-Cellobiose
D-Melibiose
L-Rhamnose
D-Raffinose
L-Fucose
Ribitol
Xylitol
Sorbitol
L-Erythritol
D-Arabitol
+
+
+
Acid production from:
Glycerol
Erythritol
D-Arabinose
L-Arabinose
D-Ribose
D-Xylose
+
+
+
+
D-Lactose
+
D-Galactose
+
Inulin
Malate
Pyruvate
Citrate
+
w
D-Glucose
+
+
+
D-Raffinose
Starch
Glycogen
+
+
+
Acetate
Aspartate
L-Glutamate
L-Alanine
L-Asparagine
Glycine
L-Histidine
L-Lysine
L-Glutamine
L-Arginine
L-Serine
L-Valine
+
+
+
Xylitol
Gentiobiose
D-Turanose
D-Lyxose
D-Tagatose
D-Fucose
L-Fucose
D-Arabitol
L-Arabitol
Potassium Gluconate
Potassium 2-Ketogluconate
Potassium 5-Ketogluconate
+
+
+
+
+
w
+
+
L-Arabitol
L-Sorbose
L-Rhamnose
+
w
+
w
w
Dulcitol
Inositol
D-Mannitol
D-Sorbitol
Methyl-a-D-Glucopyranoside
N-Acetylglucosamine
Amygdalin
Arbutin
Esculin
Salicin
D-Maltose
+
+
w
+
+
+
+
D-Melibiose
D-Sucrose
D-Trehalose
+
+
+
+
+
+
+
, Negative result; +, positive result; w, weakly positive result.
Fatty acidsa
15:0 iso
15:0 anteiso
16:0 iso
16:1 x7c
16:0
ECL 16.090 (15:0 iso diol)
17:1 x9c iso
17:1 x9c anteiso
17:0 iso
17:0 anteiso
17:0
18:0 iso
18:1 x7c
18:0
19:0 iso
19:0 anteiso
18:0 iso diol
a
D-Mannose
D-Cellobiose
+
Table 2
Mean fatty acid composition of strain RQ-24T and strain TU-8 grown
at 50 C
b
D-Fructose
+
% Of the total in:
RQ-24T
TU-8
1.5
38.6
0.9
0.9
6.9
1.4
1.6
0.6
16.6
17.2
4.2
1.9
1.2
4.3
0.8
1.7
–
1.8
36.3
0.7
–b
5.2
1.5
1.6
0.6
22.5
17.0
4.3
1.4
0.7
3.0
1.1
1.4
0.7
Values for acids present at levels of less than 0.5% are not shown.
Not detected.
the lineages of the families Deinococcaceae and Thermaceae. The 16S rRNA gene sequence similarity values between strain RQ-24T and the species that fall into the
families Deinococcaceae and Thermaceae are in the
ranges 87.0–89.4% and 86.4–88.2%, respectively.
4. Discussion
The phylogenetic analysis of the 16S rRNA gene sequences clearly indicates that strains RQ-24T and TU8 are cultured representatives of a distinct phylogenetic
lineage within the Phylum ‘‘Deinococcus/Thermus’’ comprised until now of environmental 16S rRNA gene sequences that, based on 16S rRNA gene sequence
comparisons, is equidistantly related to the lineages
comprising the families Deinococcaceae and Thermaceae
at the base of the branch leading to the order Deinococcales [3,31]. The closest phylogenetic relationship at the
95% similarity level was found to be a 16S rRNA gene
sequence recovered from a cryptoendolithic community
from an environment considered to be extreme and
shown to have a limited microbial diversity [30]. This level of 16S rRNA gene sequence similarity indicates that
the environmental 16S rRNA gene sequence recovered
from this environment could represent an additional as
yet uncultured species of this novel genus.
The new organisms have an optimum growth temperature of about 50 C, which is similar to that of M. chliarophilus, D. murrayi and D. geothermalis [7,14]. All
other members of the family Thermaceae have higher
growth temperature ranges while the other species of
the genus Deinococcus have lower growth temperature
ranges (Supplementary Table 1). It came as a surprise
that strains RQ-24T and TU-8 were alkaliphilic and
slightly halophilic having been isolated from hot springs
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L. Albuquerque et al. / FEMS Microbiology Letters 247 (2005) 161–169
Fig. 2. Phylogenetic tree based on a comparison of the 16S rRNA gene sequences of strains RQ-24T and representatives of the main lineages within
the domain Bacteria. Ionizing radiation resistant species have been included in the tree. Additional species of the families Deinoccaceae and
Thermaceae were included to establish the relationship of this novel lineage to these previously described families. The tree was reconstructed from
distance values using the neighbor-joining method. Scale bar, 10 inferred nucleotide substitutions per 100 nucleotides.
and runoffs that had neutral pH and low levels of sodium. These new Azorean organisms are, in fact, the
most alkaliphilic members of the Phylum ‘‘Deinococcus/Thermus’’. Strains RQ-24T and TU-8 grow at pH
11.2, and may be considered extremely alkaliphilic. We
also did not expect strains RQ-24T and TU-8 to be facultatively halophilic and capable of growing in medium
containing up to about 6.0% NaCl. This salinity range
for growth is rivaled only by strains of T. thermophilus,
Marinithermus, Vulcanithermus and Oceanithermus, but
not among the species of Deinococcus.
The peptidoglycan of all organisms of the Phylum
‘‘Deinococcus/Thermus’’ examined belongs to the A3b
type [20]. However, peptidoglycan was not found in
either of the two new strains (Peter Schumann, personal
communication), indicating that the cell wall contains
other polymers or that the peptidoglycan layer is a
minor component of the cell wall that cannot be detected by the methods used. The brownish Gram-indeterminate stain indicates that some of the polymerized
crystal violet-iodine is retained by the cell wall, unlike
D. grandis which stains Gram-negative.
Long chain 1,2 diols, primarily iso- and anteiso-18:0
diols and an unknown acyl compound with ECL of
16.090, which may represent iso-15:0 diol, are found in
several species of the genus Thermus and Meiothermus
[32], but diols have never been detected in any of the
strains of Deinococcus. Strains RQ-24T and TU-8 also
possess iso-18 diol and, likely, iso-15 diol, indicating
that these acyl chains have an ancient origin within the
Phylum ‘‘Deinococcus/Thermus’’.
The organisms were isolated without c irradiation of
the water samples and the phylogenetic position of this
lineage did not necessarily argue for radiation-resistance, however, strains RQ-24T and TU-8 are nearly
as radiation resistant as those of the genus Deinococcus
[2]. The vast majority of the species of the family Thermaceae have not been examined for radiation resistance,
but the type strain of T. aquaticus has been recently
found to be more resistant to c radiation than E. coli,
but much more sensitive than D. radiodurans [13]. The
only other extremely radiation resistant organisms
known, in addition to the species of the genus Deinococcus, and strains RQ-24T and TU-8, are those of the
actinobacterial genus Rubrobacter [33].
After so many surprising and unexpected characteristics, it was bewildering to find that these organisms fermented glucose to lactate without the formation of gas.
The homolactic fermentation which is emblematic of
some lactic acid bacteria has now been found to occur
in the most unlikely bacteria of a phylum that does
not contain known fermentative organisms. However,
growth was not observed under anaerobic conditions
with glucose.
L. Albuquerque et al. / FEMS Microbiology Letters 247 (2005) 161–169
The species represented by strains RQ-24T and TU-8
share many characteristics with the species of Deinococcus, namely the cell wall structure, the coccoid cell shape
which is found in several of the species of this genus and
the extreme radiation resistance (Supplementary Table
1). However, the absence of detectable peptidoglycan,
the alkaliphilic and the slightly halophilic nature of the
organisms, the homolactic fermentation, the presence
of long chain 1,2 diols which are unknown in the species
of Deinococcus and the distinct phylogenetic position
strongly argues for the proposal of a novel genus and
species which we name Truepera radiovictrix gen. nov.,
sp. nov. Moreover, the equidistant phylogenetic position
with respect to the families Deinococcaceae and Thermaceae argues not only for a novel genus but, in fact, for a
novel family for which we propose the name Trueperaceae fam. nov.
5. Description of Trueperaceae fam. nov. (Rainey, da
Costa and Albuquerque)
Trueperaceae (true.pe.raÕce.ae, N. L. fem. n. Truepera, the type genus of the family; L. suff. – aceae,
ending to denote a family; N. L. fem. pl. n. Trueperaceae, the Truepera family). Cells are Gram indeterminate and spherical-shaped. Endospores not formed.
Slightly thermophilic. Fatty acids are iso and anteiso-branched chained: 1,2 diols are present; major
respiratory quinone is menaquinone 8 (MK-8). Chemoorganotrophic. Aerobic and respiratory; homolactic
fermentation takes place. Carbohydrates, polyols, organic acids and amino acids are used as carbon and
energy sources. Extremely ionizing radiation resistant.
The mole G + C of the DNA of the type genus is 67–
68%. The family Trueperaceae belongs to the Phylum
‘‘Deinococcus/Thermus’’. The type genus of this family
is Truepera.
6. Description of Truepera gen. nov. (da Costa, Rainey
and Albuquerque)
Truepera (trueÕpe.ra, N. L. fem. n. Truepera, named
in honor of the German microbiologist Hans G. Trüper). Truepera forms spherical-shaped cells that are
Gram indeterminate. Colonies are orange/red pigmented. Endospores are not formed. Alkaliphilic,
slightly thermophilic and, slightly halophilic. Fatty acids
are iso and anteiso-branched: long chain 1,2 diols are
present; major respiratory quinone is menaquinone 8
(MK-8). Oxidase and catalase positive. Chemoorganotrophic, aerobic and respiratory; homolactic fermentation takes place. Sugars, organic acids and amino
acids are used as carbon and energy sources. Extremely
ionizing radiation resistant. The mole G + C of the
167
DNA of the type species is 67–68%. The type species
is Truepera radiovictrix.
7. Description of Truepera radiovictrix sp. nov.
(Albuquerque, da Costa and Rainey)
Truepera radiovictrix (ra.di.o.vicÕ trix. L. n. radius,
beam, N. L. prefix radio- pertaining to radiation, L.
fem. n. victrix, female winner, N. L. fem. n. radiovictrix, the vanquisher of radiation). Truepera radiovictrix forms spherical shaped cells 1.25–2.0 lm in
diameter. Pairs and tetrads are very common. The
cells stain Gram-indeterminate and are not motile.
Colonies are orange/red pigmented. The optimum
growth temperature is about 50 C, growth does not
occur at 20 and 60 C; the optimum pH is between
7.5 and 9.5, does not grow below pH 6.5, maximum
pH growth is about 11.2. Extremely ionizing radiation
resistant. The predominant fatty acids are 15:0 anteiso, 17:0 iso and 17:0 anteiso, long chain 1,2 isobranched diols are present. Cytochrome oxidase and
catalase positive. The organism is facultatively halophilic. Chemoorganotrophic, aerobic with respiratory
metabolism; growth does not occur under anaerobic
conditions in Thermus medium with glucose as carbon
and energy source and nitrate as electron acceptor
even though glucose is fermented to lactic acid; gas
is not produced. Growth factors are not necessary
for growth. A large number of sugars, polyols, amino
acids and organic acids serve as single carbon sources.
The mole G + C ratio of the DNA is 67.6–67.8%.
This bacterium was isolated from the geothermal area
at Ribeira Quente on the Island of São Miguel. The type
strain, RQ-24T, has been deposited in the Deutsche
Sammlung von Mikroorganismen und Zellkulturen,
Braunschweig, Germany as strain DSMZ 17093, in the
BCCM/LMG Bacteria Collection, Ghent, Belgium as
strain LMG 22925 and in the Collection of the Institute
Pasteur, Paris, France as strain CIP 108686. Strain TU-8
(=DSMZ 17094 = LMG 22926 = CIP 108687) is an
additional strain of this species.
Acknowledgements
This work was supported, in part by POCTI 35029/
99, Portugal and COOP-CT-2003-508644, European
Commission. FAR was in part supported by NSF award
MCB 9977882. We thank Peter Schumann (Braunschweig, Germany) for the peptidoglycan analysis. We
also thank Helen Santos, João Cavalheiro and Claudia
Sanchez (ITQB, Oeiras, Portugal) for the determination
of fermentation products by NMR.
168
L. Albuquerque et al. / FEMS Microbiology Letters 247 (2005) 161–169
Appendix A. Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/
j.femsle.2005.05.002.
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