Reclassification of the members of the genus
Tetrathiobacter Ghosh et al. 2005 to the genus
Advenella Coenye et al. 2005
A. Gibello,1A. I. Vela,1,2M. Martı ´n,3A. Barra-Caracciolo,4P. Grenni4
and J. F. Ferna ´ndez-Garayza ´bal1,2
J. F. Ferna ´ndez-Garayza ´bal
1Dpto Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
2Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Facultad de Veterinaria, Universidad
Complutense, 28040 Madrid, Spain
3Dpto Bioquı ´mica y Biologı ´a Molecular IV, Facultad de Veterinaria, Universidad Complutense,
28040 Madrid, Spain
4Water Research Institute – National Research Council, Via Reno 1, 00198 Rome, Italy
The taxonomic position of the genera Advenella and Tetrathiobacter was examined. 16S rRNA
gene sequence analysis revealed that the two genera are closely related, representing a
monophyletic cluster with high sequence similarity (98.1–99.7%) within the family
Alcaligenaceae. The phenotypic characteristics of the type strains of Advenella incenata,
Tetrathiobacter kashmirensis and Tetrathiobacter mimigardefordensis were re-examined using
the API 20NE, API ZYM and API 50CH systems. Phylogenetic data together with similarities in
phenotypic characteristics, G+C content and cellular acid composition suggest that they should
be classified in the same genus. On the basis of the data presented, the two species of the genus
Tetrathiobacter should be transferred to the genus Advenella, since this genus has nomenclatural
priority. Therefore, Tetrathiobacter kashmirensis and Tetrathiobacter mimigardefordensis should
be transferred to the genus Advenella as Advenella kashmirensis comb. nov. (type strain WT001T
5LMG 22695T5MTCC7002T) and Advenella mimigardefordensis comb. nov. (type strain
DPN7T5DSM 17166T5LMG 22922T). Emended descriptions of Advenella incenata and the
genus Advenella are also presented.
In the course of a study of environmental bacteria able to
use n-triazines, we isolated several strains from ground-
water contaminated by terbutylazine. Preliminary phylo-
genetic analysis of the 16S rRNA gene sequences of these
strains (about 700 nt) revealed that their closest relatives
were members of the genera Advenella and Tetrathiobacter.
The genus Advenella was proposed by Coenye et al. (2005)
to accommodate Gram-negative, rod-shaped to coccoid,
oxidase-positive bacteria isolated from various human and
veterinary clinical samples. Advenella incenata is the type
and single species of this genus. The genus Tetrathiobacter,
with the type species Tetrathiobacter kashmirensis, was
created by Ghosh et al. (2005) to describe Gram-negative,
non-flagellated, oval to coccoid-shaped bacteria occurring
singly or in pairs, chains, branched chains or clusters
isolated from bulk soils of a temperate orchard in Srinagar,
Jammu and Kashmir, India. Another species, Tetrathio-
bacter mimigardefordensis, was described soon after by
Wu ¨bbeler et al. (2006). Both genera are members of the
family Alcaligenaceae (De Ley et al., 1986). In this study, we
present the results of the phenotypic and phylogenetic
characterization of the environmental bacterial isolates and
a critical taxonomic evaluation of the members of the two
genera, and propose the combination of the genus
Tetrathiobacter with the genus Advenella, since the latter
has nomenclatural priority.
The environmental isolates 4GA-2008, 6GA-2008 and
7GA-2008 were isolated from groundwater contaminated
by terbutylazine, located in Assisi, in central Italy. Primary
isolation was achieved on minimal medium (MM) [l21;
1.6 g K2HPO4, 0.4 g KH2PO4, 0.1 g CaSO4.2H2O, 1 g
MgSO4.7H2O, 0.02 g FeSO4.7H2O, 2 g (NH4)2SO4, 15 g
agar], supplemented with 2 p.p.m. terbutylazine and
0.03% Casamino acids, after incubation at 30 uC for 48 h
under aerobic conditions. After primary isolation and
further subculture on Luria–Bertani (LB) agar plates at
30 uC for 48 h, isolates were stored at 220 uC as glycerol
suspensions (20% v/v). A. incenata CCUG 45225T, T.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequences of A. incenata strains CCUG 45225Tand 4GA-2008 are
AM944734 and AM944735.
International Journal of Systematic and Evolutionary Microbiology (2009), 59, 1914–1918
1914007443G2009 IUMS Printed in Great Britain
kashmirensis LMG 22695T
mimigardefordensis LMG 22922Twere obtained from the
respective culture collections, grown aerobically at 30 uC
on LB agar plates for 48 h and stored at 220 uC as glycerol
suspensions (20% v/v).
and LMG 22696 and T.
Phylogenetic analysis of the environmental isolates was
performed by comparative 16S rRNA gene sequence
analysis as described previously (Vela et al., 2006). The
sequence of a large fragment of the 16S rRNA gene of the
three isolates (approx. 1440 bases) as well as that of A.
incenata CCUG 45225T(1341 bp; the sequence available
previously in GenBank had only 734 nucleotides) was
obtained bidirectionally. 16S rRNA gene sequence analysis
revealed 100% similarity among the three environmental
strains. Sequence searches of GenBank using the program
FASTA (Pearson, 1994) confirmed the preliminary sequen-
cing results and confirmed that the environmental isolates
were phylogenetically most closely related to T. kashmir-
ensis WT001T(99.7% sequence similarity), A. incenata
CCUG 45225T(99.4% to the new, longer sequence) and T.
mimigardefordensis DPN7T(99.0%). These sequences and
those of other representative members of the family
Alcaligenaceae were retrieved from GenBank and aligned
with the newly determined sequence by using the program
DNATools (Rasmussen, 1995). Phylogenetic trees were
constructed according to three different methods, a
neighbour-joining algorithm (Saitou & Nei, 1987), per-
formed with the programs DNATools and TreeView (Page,
1996), maximum-likelihood analysis done using the PHYML
software (Guindon & Gascuel, 2003) and maximum-
parsimony method carried out using the MEGA software
package version 3.1 (Kumar et al., 2004). Genetic distances
for the neighbour-joining and maximum-likelihood algo-
rithms were calculated by Kimura’s two-parameter model
(Kimura, 1980) and close-neighbour interchange (search
level52, random additions5100) was applied in maximum-
parsimony analysis. The stability of the groupings was
estimated by bootstrap analysis (1000 replications). The
members of the genera Advenella and Tetrathiobacter formed
a monophyletic cluster with 100% bootstrap support and
were readily differentiated from other genera of the family
Alcaligenaceae (Fig. 1). Within this cluster, T. mimigarde-
fordensis DPN7Tformed a distinct subline from that formed
by A. incenata CCUG 45225Tand T. kashmirensis WT001T
with 99% bootstrap support. This tree topology was
confirmed by the three phylogenetic algorithms. Pairwise
16S rRNA gene sequence similarity values within this cluster
ranged between 98.1 and 99.7%; these values are typical of
members of the same genus. The G+C contents of
members of the genera Advenella (53.5–58.0 mol%) and
compositions, with 16:0 and 18:1v7c as the predominant
fatty acids, are very similar (Coenye et al., 2005; Ghosh et al.,
2005; Wu ¨bbeler et al., 2006).
Fig. 1. Phylogenetic tree inferred from 16S
rRNA gene sequence comparison using the
neighbour-joining method, showing the rela-
tionships of the members of the genus
Advenella with other
Alcaligenaceae. The sequence of Zoogloea
ramigera ATCC 19324 (GenBank accession
no. D14257) was used as an outgroup (not
shown). Bootstrap values (expressed as per-
centages of 1000 replications) higher than
50% are given at branching points. Filled
circles indicate that the corresponding nodes
(groupings) were also obtained in maximum-
likelihood trees. Open circles indicate that the
corresponding nodes (groupings) were also
obtained in maximum-likelihood and parsimony
trees. Bar, 1% sequence divergence.
taxa of the family
Reclassification of the members of the genus Tetrathiobacter
The phenotypic characteristics described for A. incenata, T.
kashmirensis and T. mimigardefordensis are not directly
comparable, because the same characteristics were not
determined for the three species (Coenye et al., 2005;
Ghosh et al., 2005; Wu ¨bbeler et al., 2006). In this study, the
environmental isolates were characterized phenotypically
and the phenotypic characteristics of A. incenata CCUG
45225T, T. kashmirensis LMG 22695Tand T. mimigarde-
fordensis LMG 22922Twere re-examined using commercial
kits. Biochemical and enzyme characteristics were deter-
mined using the API 20NE and API ZYM systems
(bioMe ´rieux) according to the manufacturer’s instructions.
Carbohydrate assimilation was essayed by using API 50CH
strips (bioMe ´rieux) which were inoculated with a 0.5
McFarland suspension of bacterial cells in AUX medium
(bioMe ´rieux). The API 50CH strips were read for up to
4 days of incubation at 30 uC. Assimilation of DL-lactate
was determined in MM broth containing 1% (w/v) DL-
lactate (Sigma). The environmental isolates exhibited
almost identical phenotypic characteristics, which matched
those exhibited by A. incenata CCUG 45225Texcept that
they did not hydrolyse urea (A. incenata CCUG 45225Twas
positive). A. incenata CCUG 45225T, T. kashmirensis LMG
22695Tand T. mimigardefordensis LMG 22922Talso
exhibited many common characteristics, although several
tests can be used for their differentiation. The results are
given in the species descriptions and in Table 1.
The distinct phylogenetic position of T. mimigardefordensis
and its separate species status with respect to T. kashmirensis
were supported by DNA–DNA hybridization experiments
and by differences in biochemical and chemotaxonomic
characteristics (Wu ¨bbeler et al., 2006). Therefore, genomic
relatedness was examined to determine the species status of
the environmental strains with respect to A. incenata and T.
kashmirensis and between A. incenata and both species of
Tetrathiobacter. DNA–DNA hybridization experiments were
carried out between isolate 4GA-2008 and isolates 6GA-2008
and 7GA-2008, between isolate 4GA-2008 and its nearest
phylogenetic neighbours A. incenata CCUG 45225Tand T.
kashmirensis LMG 22695T, between the two latter strains and
between A. incenata CCUG 45225Tand T. kashmirensis LMG
22696 and T. mimigardefordensis LMG 22922T. DNA was
purified by chromatography on hydroxyapatite as described
by Cashion et al. (1977). DNA–DNA hybridization was
carried out as described by De Ley et al. (1970) under
consideration of the modifications described by Huß et al.
(1983) using a Cary 100 Bio UV/Vis spectrophotometer
equipped with a Peltier-thermostatted 666 multicell changer
and a temperature controller with in situ temperature probe
(Varian). Preparation of high-molecular-mass DNA and
DNA–DNA hybridization experiments were performed by
the DSMZ Identification Service (Braunschweig, Germany).
DNA–DNA hybridization between isolate 4GA-2008 and
isolates 6GA-2008 and 7GA-2008 showed DNA relatedness
values of 95.1 and 84.9%, respectively. The DNA–DNA
reassociation values between isolate 4GA-2008 and A.
incenata CCUG 45225T, T. kashmirensis LMG 22695Tand
T. kashmirensis LMG 22696 were 93.9, 44.8 and 31.4%,
respectively, demonstrating that the environmental isolates
are members of the species A. incenata (Wayne et al., 1987).
DNA–DNA reassociation values between A. incenata CCUG
45225Tand T. kashmirensis LMG 22695T, T. kashmirensis
LMG 22696 and T. mimigardefordensis LMG 22922Twere
48.3, 44.0 and 27.7%, respectively. These values are below the
recommended threshold value of 70%, confirming that they
merit their separate species status (Wayne et al., 1987).
Considering the phenotypic similarities, phylogenetic
position and genetic and chemotaxonomic data, A.
incenata, T. kashmirensis and T. mimigardefordensis should
be members of the same genus. The names Advenella and
Tetrathiobacter were published in the same year but,
according to the Bacteriological Code (Rule 24b), the
genus Advenella has priority and, consequently, the two
species of the genus Tetrathiobacter should be reclassified as
members of the genus Advenella.
Emended description of the genus Advenella
Coenye et al. 2005
Advenella (Ad.ven.el9la. L. n. advena a stranger, a foreigner;
L. dim. ending -ella; N.L. fem. n. Advenella the little
stranger, referring to the fact that the source of the first
strains was unknown).
The description is as given by Coenye et al. (2005) with the
following modifications. Some members of the genus do
not assimilate DL-lactate, D-mannose or maltose. The type
species is Advenella incenata.
Emended description of Advenella incenata
Coenye et al. 2005
Advenella incenata (in.ce.na9ta. L. fem. adj. incenata that
has not dined, fasting, referring to the fact that this
organism shows little biochemical activity).
Table 1. Phenotypic characteristics that differentiate type
strains of the genus Advenella
Strains: 1, A. incenata CCUG 45225T; 2, A. kashmirensis comb. nov.
LMG 22695T; 3, A. mimigardefordensis comb. nov. LMG 22922T. Data
were obtained in this study.
Esterase (C4) activity
A. Gibello and others
1916 International Journal of Systematic and Evolutionary Microbiology 59
The description remains that given by Coenye et al. (2005)
with the following additions. Assimilates adipate, glycerol,
D- and L-xylose, D-arabinose, galactose, ribose, rhamnose,
D- and L-fucose, gluconate and 2-ketogluconate, but does
not assimilate erythritol, D-adonitol, turanose, tagatose, D-
lyxose, xylitol, gentiobiose, glycogen, sucrose, melibiose,
D- or L-arabitol, methyl b-D-glucopyranoside, methyl a-D-
mannopyranoside, sorbitol, starch, raffinose, inulin, dulci-
L-sorbose, methyl b-D-xylopyranoside,
fructose, trehalose or melezitose.
The type strain is CCUG 45225T5LMG 22250T, isolated
from human sputum in Sweden.
Description of Advenella kashmirensis
Advenella kashmirensis (kash.mir.en9sis. N.L. masc. adj.
kashmirensis of Kashmir, after the name of the province
from where the original strains of the species were
Basonym: Tetrathiobacter kashmirensis Ghosh et al. 2005.
The description is as given for Tetrathiobacter kashmirensis
by Ghosh et al. (2005) with the following additions or
modifications. Assimilates adipate, D- and L-xylose, D-
arabinose, galactose, ribose, rhamnose, D- and L-fucose,
gluconate and fructose, but does not assimilate erythritol,
D-adonitol, turanose, tagatose, D-lyxose, xylitol, gentio-
biose, glycogen, sucrose, melibiose, D- or L-arabitol, methyl
b-D-glucopyranoside, methyl a-D-mannopyranoside, sor-
bitol, starch, raffinose, inulin, dulcitol, inositol, L-sorbose,
methyl b-D-xylopyranoside, glycerol, trehalose, 2-ketoglu-
conate or melezitose. Esterase (C4), acid phosphatase
(weak reaction) and alkaline phosphatase activities are
detected. No activity is detected for esterase lipase (C8) and
5MTCC7002T), isolated from bulk soil of a temperate
orchard in Srinagar, Jammu and Kashmir, India.
typestrain is WT001T
Description of Advenella mimigardefordensis
M.L. masc. adj. mimigardefordensis of Mimegardefordum,
a medieval name of Mu ¨nster, where the type strain was
Basonym: Tetrathiobacter mimigardefordensis Wu ¨bbeler
et al. 2006.
The description remains that given for Tetrathiobacter
mimigardefordensis by Wu ¨bbeler et al. (2006) with the
following additions. Assimilates glycerol, DL-lactate, adi-
pate, D- and L-xylose, D-arabinose, D-galactose, ribose, D-
and L-fucose, gluconate and melezitose, but does not
assimilate erythritol, D-adonitol, turanose, tagatose, D-
lyxose, xylitol, gentiobiose, glycogen, sucrose, melibiose,
methyl a-D-mannopyranoside, sorbitol, raffinose, inulin,
dulcitol, inositol, L-sorbose, methyl b-D-xylopyranoside, D-
Alkaline phosphatase (weak reaction), acid phosphatase
and esterase lipase (C8) are detected. No activity is detected
for esterase (C4) or cystine arylamidase.
L-arabitol, methyl b-D-glucopyranoside,
The type strain is DPN7T(5DSM 17166T5LMG 22922T),
isolated from a sample of matured compost from a
compost plant in Mu ¨nster (Germany).
The authors thank A. Casamayor for technical assistance and Juncal
Ferna ´ndez-Garayza ´bal for her assistance with the English reviewing of
the manuscript. This work was funded by project CTM-2007-66306-
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