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Aquirufa antheringensis gen. nov., sp. nov. and Aquirufa
nivalisilvae sp. nov., representing a new genus of widespread
freshwater bacteria
Alexandra Pitt,*Johanna Schmidt, Ulrike Koll and Martin W. Hahn
Abstract
Three bacterial strains, 30S-ANTBAC, 103A-SOEBACH and 59G- WUEMPEL, were isolated from two small freshwater creeks
and an intermittent pond near Salzburg, Austria. Phylogenetic reconstructions with 16S rRNA gene sequences and, genome
based, with amino acid sequences obtained from 119 single copy genes showed that the three strains represent a new
genus of the family Cytophagaceae within a clade formed by the genera Pseudarcicella,Arcicella and Flectobacillus.BLAST
searches suggested that the new genus comprises widespread freshwater bacteria. Phenotypic, chemotaxonomic and
genomic traits were investigated. Cells were rod shaped and were able to glide on soft agar. All strains grew
chemoorganotrophically and aerobically, were able to assimilate pectin and showed an intense red pigmentation putatively
due to various carotenoids. Two strains possessed genes putatively encoding proteorhodopsin and retinal biosynthesis.
Genome sequencing revealed genome sizes between 2.5 and 3.1 Mbp and G+C contents between 38.0 and 42.7 mol%. For the
new genus we propose the name Aquirufa gen. nov. Pairwise-determined whole-genome average nucleotide identity values
suggested that the three strains represent two new species within the new genus for which we propose the names Aquirufa
antheringensis sp. nov. for strain 30S-ANTBAC
T
(=JCM 32977
T
=LMG 31079
T
=DSM 108553
T
) as type species of the genus, to
which also belongs strain 103A-SOEBACH (=DSM 108555=LMG 31082) and Aquirufa nivalisilvae sp. nov. for strain 59G-
WUEMPEL
T
(=LMG 31081
T
=DSM 108554
T
).
The phylum Bacteroidetes contains Gram-negative bacteria,
which occur in a broad variety of habitats all over the world
and show different lifestyles and physiologies [1]. The Cyto-
phaga–Flavobacteria cluster belongs to this phylum and is
characterized by rod-shaped, non-spore-forming, frequently
pigmented and gliding, chemoorganotrophic bacteria [2].
They are often capable of degrading biopolymers such as
agar, cellulose, chitin, pectin and keratin [3]. Bacteria
belonging to the Cytophaga–Flavobacteria cluster occur in
high abundances in many freshwater and marine systems
and play an important role in these ecosystems in respect to
the uptake and degradation of dissolved organic material
[2]. A large family within this cluster are the Cytophagaceae,
which comprise 30 genera and about 150 species. Phyloge-
netic analyses based on 16S rRNA gene sequences suggested
that this family encompass divergent clades which cannot
be well distinguished from members of the families Cyclo-
bacteriaceae and Flammeovirgaceae [4]. Genome-scale
phylogenies inferred from whole proteomes revealed that
the Cytophagaceae as a family and some of their genera are
not monophyletic [1].
Within a cooperation project between schools and science
(Sparkling Science program), which pursues the aim to iso-
late and taxonomically describe new bacterial species, 125
teenagers from high schools took samples from various
freshwater habitats around the city of Salzburg (Austria),
measured basic water chemistry parameters and inoculated
agar plates with the collected water samples. By screening
these plates, we found several strains belonging to an obvi-
ously undescribed genus affiliated with the family Cytopha-
gaceae. From nine pure cultures, three were selected for
further analysis. Some students joined the lab and helped
with the phenotypic characterizations of the strains. Fur-
thermore, some students created, with the help of a nomen-
clature advisor, the proposed genus and species names.
Author affiliation: Research Department for Limnology, University of Innsbruck, Mondseestrasse 9, A-5310 Mondsee, Austria.
*Correspondence: Alexandra Pitt, Alexandra.Pitt@uibk.ac.at
Keywords: Aquirufa;Cytophagaceae; freshwater; genome; whole genome average nucleotide identity (gANI).
Abbreviations: DTS, tryptone–soyotone; gANI, whole-genome average nucleotide identity; IMG/ER, Integrated Microbial Genomes/Expert Review;
NSY, nutrient broth–soyotone–yeast extract; R2A, Reasoner’s 2A.
The Whole Genome Shotgun project and 16S rRNA gene sequences have been deposited at DDBJ/ENA/GenBank. The accession numbers of strain
30S-ANTBAC
T
are SEWZ00000000 (genome) and MK449343 (16S rRNA gene), strain 103A-SOEBACH SEWY00000000 (genome) and MK449347 (16S
rRNA gene), strain 59G-WUEMPEL
T
SEWX00000000 (genome) and MK449345 (16S rRNA gene).
Two supplementary figures and one supplementary table are available with the online version of this article.
TAXONOMIC DESCRIPTION
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
DOI 10.1099/ijsem.0.003554
003554 ã2019 The Authors
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited. 2739
So, here we describe a new genus belonging to the Cytopha-
gaceae which seems to be common in various aquatic fresh-
water habitats. As type species for this new genus we
propose Aquirufa antheringensis gen. nov., sp. nov. with its
type strain 30S-ANTBAC
T
. Furthermore, we propose to
establish an additional species within this genus, Aquirufa
nivalisilvae for strain 59G-WUEMPEL
T
.
HOME HABITATS AND ISOLATION
Strain 30S-ANTBAC
T
was isolated from Antheringer Bach,
a small freshwater creek running through the town Anther-
ing, Austria, the approximate geographic coordinates are
47.877 N 13.006 E. Water sampled in November 2017 had a
pH of 6.9 and a conductivity of 435 µS cm
1
. Strain 103A-
SOEBACH was isolated from Soellheimer Bach, a small
freshwater creek located in Salzburg, Austria, the approxi-
mate geographic coordinates are 47.826 N 13.049 E. Water
sampled in May 2018 had a pH of 6.9 and a conductivity of
515 µS cm
1
. Strain 59G-WUEMPEL
T
was isolated from a
small intermittent freshwater pond located in a forest in
Schneegattern (Lengau), Austria, the approximate geo-
graphic coordinates are 48.029 N 13.299 E. Water sampled
in April 2018 had a pH of 7.5 and a conductivity of 163.7 µS
cm
1
. All three strains were isolated by filtrating the sam-
ples and subsequent plating on agar plates. For strains 30S-
ANTBAC
T
and 103A-SOEBACH filters with 0.45 µm pore
size and for strain 59G-WUEMPEL
T
filters with 0.65 µm
pore size were used. Nutrient broth–soyotone–yeast extract
(NSY) agar plates [5] were utilized for 103A-SOEBACH,
while Reasoner’s 2A (R2A) agar plates [6] were used for
strain 30S-ANTBAC
T
and tryptone–soyotone (DTS) plates
[7] with very low nutrient concentrations were used for
strain 59G-WUEMPEL
T
for the first cultivation steps,
respectively. Nevertheless, all strains grew on NSY plates
and in liquid NSY medium at pH 7.2 and were purified
using these media.
PHENOTYPIC AND CHEMOTAXONOMIC
CHARACTERIZATION
The temperature range for growth was tested on NSY agar
plates exposed to increasing temperatures in 1 C steps start-
ing at 5 C until no growth was observed. NaCl tolerance
was tested using agar plates with various NaCl concentra-
tions in 0.1 % (w/v) steps. For testing anaerobic growth, an
anaerobic chamber and standard NSY agar plates as well as
NSY plates supplemented with 2 g l
-1
NaNO
3
were used.
For determination of cell morphology and cell dimensions,
well-growing liquid cultures were fixed with 2 % parafor-
maldehyde, stained with DAPI (4¢,6-diamidino-2-phenylin-
dole) and investigated by using an epifluorescence
microscope (UV filter). To test the ability of the strains to
glide, soft agar plates (1 g l
1
yeast extract, 0.1 g l
1
K
2
HPO
4
, 2.0 g l
1
agar) were used [8]. One drop of a well-growing
culture was placed in the centre of these test plates, as well
as on standard NSY plates, respectively, incubated at room
temperature and observed for several days. Assimilation of
various substrates was tested using GEN III MicroPlates
(Biolog), which detect utilization of substrates as electron
donors by the subsequent reduction of a tetrazolium redox
dye. Cells from well-growing liquid cultures were centri-
fuged and added to the inoculum medium so that the OD of
the culture correspond to 0.07 at a wavelength of 590 nm.
The absorption was measured with a Multiskan FC
(Thermo Scientific) at a wavelength of 595 nm after 48 h
incubation at 20 C. After subtracting the value of the nega-
tive control (without substrate), obtained values from 0.016
to 0.05 were regarded as weak utilization and for >0.05 as
positive. The chemotaxonomic characterization of the
strains included analyses of the composition of whole-cell
fatty acids, polar lipids and respiratory quinones. They were
carried out by the Identification Service, Leibniz-Institut
DSMZ-Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH, Braunschweig, Germany. For all che-
motaxonomic analyses cells were inoculated into liquid
NSY medium and harvested after 3 days of growth by cen-
trifugation. For the whole-cell fatty acid composition, an
Agilent Technologies 6890 N instrument, the Microbial
Identification System (MIDI) Sherlock version 6.1 and the
TSBA 40 database were used as described by Sasser [9].
Polar lipids and respiratory quinones were extracted and
analysed as described by Tindall [10, 11] based on the
method by Bligh and Dyer [12]. To investigate if the pig-
mentation of the cultures was caused by flexirubin, tests
with 20 % KOH and 12 M HCl were performed [13].
Cells of all investigated strains were slim rods. They formed
bright red, in older stages dark-red coloured, circular, and
convex colonies with smooth surface on agar plates and
showed a strong orange-red colouring in liquid media.
Strain 59G-WUEMPEL
T
grew up to a temperature of 35 C,
the other two strains stopped growing at lower temperatures
(Table 1). All strains only tolerated low salt concentrations,
showed no anaerobic growth and were able to glide and
showed spreading over the whole soft agar plates within
periods of 7 days (Table 1). Despite various efforts accord-
ing to Bernardet et al. [13] the flexirubin test was negative
for all investigated strains, maybe caused by other overlay-
ing pigments. All three investigated strains assimilated pec-
tin and Tween 40 and showed weak assimilation of
acetoacetic acid, glucuronamide, and D-fructose-6-PO
4
(Table 1). Strain 59G-WUEMPEL
T
showed weak assimila-
tion of additional substrates (Table 1). All three investigated
strains contained nearly the same fatty acids but in various
amounts, C
15 : 0
was only found in strain 30S-ANTBAC
T
(Table 2). The amounts of the major fatty acids differed
remarkably between strain 30S-ANTBAC
T
and strain 103A-
SOEBACH, which should be classified as the same species
(see below). For example, iso-C
15 : 0
constituted 20.3 % and
39.5 % of fatty acids in strains 30S-ANTBAC
T
and 103A-
SOEBACH, respectively (Table 2). These finding suggested,
that the relative fatty acid amounts can vary a lot among
strains belonging to the same species grown under compa-
rable conditions. A study describing a new Polynucleobacter
species (Betaproteobacteria) within the cryptic species com-
plex PnecC based on six investigated isolates came to the
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
2740
same conclusion [14]. Regarding the polar lipids, all three
strains presented here contained phosphatidylethanolamine
and various unidentified polar lipids like aminolipids, phos-
pholipids and aminophospolipids (Table 2, Fig. S1, available
in the online version of this article). While for strain 30S-
ANTBAC
T
seven polar lipids could be separated, strain
59G-WUEMPEL
T
showed a more diverse pattern with three
additional unidentified polar lipids. The main respiratory
quinone for all three strains presented here was MK7. Addi-
tionally, strain 30S-ANTBAC
T
and strain 103A-SOEBACH
contained traces of MK6 (Table 2).
GENOMIC CHARACTERIZATION
DNA extraction and genome sequencing were performed as
described previously [15]. A shotgun library was paired-end
sequenced on an Illumina MiSeq instrument (2300 bp).
De novo assemblies were performed by using the software
SPAdes version 3.13.0 [16], details are given in Table 3. The
obtained genome sequences were annotated by the NCBI
Prokaryotic Genome Annotation Pipeline and for further
comparative analyses by the Integrated Microbial Genomes/
Expert Review (IMG/ER) annotation pipeline and incorpo-
rated in the IMG database [17]. The IMG Genome ID and
GenBank accession numbers of all three investigated strains
and strain HME7025 (Kim et al., ‘Pseudarcicella sp.’
HME7025 genome sequencing and assembly, unpublished
data), which is closely related to the three investigated
strains, are listed in Table 3.
The size of the genomes of strain 30S-ANTBAC
T
and strain
103A-SOEBACH was 2.5 Mbp, while strain 59G-WUEM-
PEL
T
had a size of 3.1 Mbp (Table 3). All strains had rela-
tively low G+C contents of less than 45 mol% (Table 3).
Interestingly, members of the nearest relatives of the new
genus had genome sizes twice as big (Table 4). Additionally,
among 60 genomes of members of the family Cytophagaceae
and related taxa, strain 30S-ANTBAC
T
and strain 103A-
SOEBACH not only possessed the smallest genome size but
also the highest coding density. While coding densities of
the other 58 genomes ranged from 85.6 % to 93.5 % coding
bases (average 89.5 %, median 89.4 %), these two genomes
were characterized by a value of 94.8 %. On the other hand,
the G+C content of their genomes of 43 mol% was close to
the average of the other Cytophagaceae genomes (range
35–56 mol%, average 45 mol%), suggesting that the genomes
of these two strains underwent evolutionary genome
streamlining, which was decoupled from changes in
G+C content.
The gene content of the three investigated strains and strain
HME7025, which is closely related to strain 59G-WUEM-
PEL
T
(see above), showed interesting pattern, some genes
had all strains in common and some differed between them
(Table 5). All four strains had genes putatively encoding
proteins associated with gliding motility, which confirmed
the observed gliding on soft agar plates. Only strain 30S-
ANTBAC
T
and strain 103A-SOEBACH possessed genes
putatively encoding a proteorhodopsin and a b-carotene
Table 1. Traits characterizing the three strains
All strains have the following substrate usage characteristics in common: assimilation of pectin and Tween 40; weak assimilation of acetoacetic
acid, glucuronamide and D-fructose-6-PO
4
; no assimilation of L-histidine, propionic acid, D-lactic acid methyl ester, L-alanine, L-glutamic acid, L-
aspartic acid, D-glucose-6-PO
4
, dextrin, D-glucuronic acid, D-fructose, D-arabitol, a-D-glucose, a-hydroxy-butyric-acid, D-galactose, methyl b-D-gluco-
side, D-galacturonic acid, 3-methyl glucose, L-rhamnose, L-galactonic acid lactone, D-mannitol, formic acid, L-malic acid, D-gluconic acid, N-acetyl-D-
glucosamine, N-acetyl-b-D-mannosamine, mucic acid, myo-inositol, trehalose, inosine, maltose, gentiobiose, b-hydroxyl-D,L-butyric acid, cellobiose,
glycyl-L-proline, L-pyroglutamic acid, N-acetyl-D-galacosamine, glycerol, L-fucose, a-keto-glutaric acid, melibiose, lactose, D-fucose, L-lactic acid, tura-
nose, N-acetyl neuraminic acid, quinic acid, D-sorbitol, D-malic acid, p-hydroxy-phenylacetic acid, raffinose, g-amino-butryric acid, L-arginine, sta-
chyose, gelatin, D-serine, D-saccharic acid, methyl pyruvate, a-keto-butyric acid, bromo-succinic acid, L-serine, citric acid and acetic acid. +, Positive;
W, weak; -, negative
Characteristic Strain 30S-ANTBAC
T
Strain 103A-SOEBACH Strain 59G-WUEMPEL
T
Cell morphology Rods Rods Rods
Mean cell length (µm) 1.7 1.8 1.6
Mean cell width (µm) 0.6 0.4 0.5
Temperature range for growth (C) 5–32 (W) 5–31 (W) 5–35 (W)
NaCl tolerance (%, w/v) 0–0.3 (W) 0–0.2 (W) 0–0.4
Anaerobic growth on NSY plates
Anaerobic growth on NSY+nitrate
Gliding ability + + +
Pigmentation Red Red Red
Flexirubin test
Assimilation of:
Sucrose W
D-Mannose W
D-Salicin W
D-Aspartic acid W
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
2741
Table 2. Chemotaxonomy: polar lipids, respiratory quinones and fatty acid composition (%) of the three investigated strains
Fatty acids with values less than 0.5 percent for all strains are not listed. +, Detected; , not detected, TR, trace.
Chemotaxonomic property Strain 30S-ANTBAC
T
Strain 103A-SOEBACH Strain 59G-WUEMPEL
T
Polar lipids:
Phosphatidylethanolamine + + +
Unidentified aminolipids 1 1
Unidentified amino-phospho-lipids 2 2 3
Unidentified polar lipids 4 4 5
Respiratory quinones:
MK7 + + +
MK6 TR TR
Fatty acids:
C
14 : 0
2.9 1.0 0.4
C
15 : 0
2.2
C
15 : 1
!6c1.5 2.0 1.1
C
16 : 1
!5c11.9 6.8 3.0
C
17 : 1
!6c1.1 1.3 1.4
iso-C
11 : 0
3.6 2.6 1.2
iso-C
13 : 0
0.8 0.8 1.4
iso-C
15 : 0
20.3 39.5 34.4
anteiso-C
11 : 0
0.5 0.3 0.2
anteiso-C
13 : 0
0.5 0.4 0.7
anteiso-C
15 : 0
5.6 8.3 10.7
C
16 : 0
3-OH 1.0 0.5 0.3
Iso-C
15 : 0
3-OH 13.4 11.7 10.5
Iso-C
17 : 0
3-OH 1.1 2.5 3.3
Iso-C
17 : 1
!9c0.4 0.8 2.0
Summed feature 1* 0.3 1.0 1.0
Summed feature 2* 1.3 0.5 0.2
Summed feature 3* 24.7 9.2 16.8
Summed feature 4* 1.1 2.9 3.0
Unknown 14.959†2.4 4.5 5.0
*Summed features represent groups of fatty acids which could not separated by GLC and the MIDI system. Summed feature 1 contains iso-C
15 : 1
h,
C
13 : 0
3-OH, iso-C
15 : 1
I I/H and iso-C
15 : 1
I; summed feature 2 contains C
12 : 0
ALDE, unknown 10.928, iso-C
16 : 1
I, C
14 : 0
3-OH and iso-C
16 : 1
I; summed
feature 3 contains iso-C
15 : 0
2-OH and C
16 : 1
!7c; summed feature 4 contains iso-C
17 : 1
I and anteiso-C
17 : 1
B/I I.
†Unknown 14.959 is an unknown compound with an ECL of 14.959.
Table 3. Genome characteristics of the three investigated strains and strain HME7025
Characteristic Strain 30S-ANTBAC
T
Strain 103A-SOEBACH Strain 59G-WUEMPEL
T
Strain HME7025
Number of scaffolds 14 17 40 –
K-mer coverage 260 235 107 –
N50 value (Mbp) 0.64 0.58 0.78 –
Genome size (Mbp) 2.5 2.5 3.1 3.1
G+C content (mol%) 42.6 42.7 38.0 37.9
ANI with 30S-ANTBAC
T
100.0 96.5 70.8 71.0
ANI with 103A-SOEBACH 96.5 100.0 71.0 71.1
ANI with 59G-WUEMPEL
T
70.8 71.0 100.0 97.4
ANI with HME7025 71.0 71.1 97.4 100.0
Accession number GenBank SEWZ00000000 SEWY00000000 SEWX00000000 CP029346
IMG/ER Genome ID 2816332120 2816332126 2816332125 2811994884
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
2742
Table 4. Comparison of the proposed genus Aquirufa and the three most closely related genera
Features for all genera in common: identified polar lipid, phosphatidylethanolamine; major respiratory quinone, MK-7. ++, Genes putatively associated
with gliding, ND, not determined.
Feature Aquirufa gen. nov. Arcicella Pseudarcicella Flectobacillus
Number of species 2 4 1 6
Cell morphology Rods Various* Rods Rods
Range of cell length (µm) 1.6–1.8 2.5–6.3 1.0–2.0 1.5–10.0
Range of cell width (µm) 0.4–0.6 0.5–0.75 0.9–1.1 0.3–1.0
Pigmentation Red Pink or orange Pink Pink or orange
Flexirubins No No ND No
Carotinoids All strains†One species ND Some species
Motility Gliding /++†No/++†No/++†No/++†
Temperature range of growth (C) 5–34 4–40 10–36 4–40
NaCl tolerance (%NaCl, w/v) 0–0.4 0–3.0 ND 0–4.0
Predominant fatty acids Iso-C
15 : 0
3-OH, iso-C
15 : 0
C
16 : 1
!5c, iso-C
15 : 0
C
16 : 1
!5c, C
18 : 1
!7cC
16 : 1
!5c, iso-C
15 : 0
Summed feature C
16 : 1
!7c, C
16 : 1
!7c, iso-C
15 : 0
2-OH C
16 : 1
!7c, C
16 : 1
!6cC
16 : 1
!7c, iso-C
15 : 0
2-OH
G+C content (mol%) 38–43 34–44 38†38–40
Genome size (Mbp) 2.5–3.1†5.9†6.2†6.2†
Genome available (Table 3, S1) All strains Arcicella aurantiaca Pseudarcicella hirudinis Flectobacillus major
References This study [40] [35] [41–44]
*Rods, vibrioid, curved or spiral-shaped.
†Based on genome data.
Table 5. Comparison of the presence and absence of selected genes of the three investigated strains and strain HME7025
+, Present; , absent.
Genes putatively encoding Strain 30S-
ANTBAC
T
Strain 103A-
SOEBACH
Strain 59G-
WUEMPEL
T
Strain
HME7025
Motility:
Proteins associated with gliding + + + +
Utilization of light:
Proteorhodopsin + +
Biosynthesis of 7,8-dihydro-b-carotene + + + +
b-Carotene 1, 15¢-monooxygenase + +
Pigments:
Biosynthesis of b,gand z-carotene + + + +
Biosynthesis of b-cryptoxanthin, canthaxanthin, phoenicoxanthin and
astaxanthin
+ + + +
Transport systems:
ABC-type: phospholipid, lipoprotein + + + +
myo-Inositol, lipopolysaccaride + +
MFS-transporter: nitrate/nitrite + + +
Inorganic nutrients:
Nitrate reductase (assimilatory) + + +
Nitrite reductase (assimilatory) + + +
Anaerobiosis:
Nitrous oxide reductase + + +
Oxidative stress:
Catalase-peroxidase (EC:1.11.1.21) + +
Cytochrome c peroxidase (EC:1.11.1.5) + +
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
2743
15,15¢-monooxygenase. But all four strains had genes puta-
tively encoding enzymes for the complete biosynthesis of
7,8-dihydro-b-carotene. The latter one serves as substrate
for the monooxygenase, which produces retinal, the cofactor
for the light driven proton pump proteorhodopsin [18].
Furthermore, only strain 30S-ANTBAC
T
and strain 103A-
SOEBACH possessed genes putatively encoding a catalase-
peroxidase and a cytochrome c peroxidase, which could
protect bacteria from oxidative stress.
All three investigated strains and strain HME7025 encoded
genes annotated for the biosynthesis of various carotenoids
(Table 5). Concerning the ABC-type transport systems the
four strains showed different patterns (Table 5). Regarding
nitrogen metabolism the four strains showed some shared
genes with an interesting distribution pattern among them.
All strains except 103A-SOEBACH possessed a gene cluster,
which encompassed genes putatively necessary for the
uptake and assimilation of nitrate and nitrite (Table 5). All
strains except 30S-ANTBAC
T
possessed a gene cluster with
genes putatively encoding enzymes responsible for the
reduction of nitrous oxide to nitrogen, which is the last step
of the dissimilatory denitrification (see below).
PHYLOGENY
Phylogenetic and phylogenomic reconstructions were per-
formed by using almost full length sequences of the 16S
rRNA gene and an aligned concatenated set of 119 single
copy marker proteins [19], respectively.
For the phylogenetic tree in Fig. 1, 16S rRNA gene sequen-
ces from the type species of all genera currently classified as
Cytophagaceae [1, 20], of further species closely related to
the investigated strains, of Prevotella melaninogenica serving
as outgroup, and of the investigated strains were used. The
sequence set was aligned by using the software MEGA X [21].
The trimmed alignment consisted of 1336 alignment posi-
tions. With the same software the data set was tested for the
best fitting model and thus a maximum-likelihood phyloge-
netic tree was calculated using Kimura’s two-parameter
model [22] as follows: discrete gamma distribution (G=5),
500 replications, gaps were partial deleted, which resulted in
1306 used positions. In addition, neighbour-joining and
maximum-parsimony trees were calculated by using the
corresponding algorithms.
Although the 16S rRNA gene tree showed that the major
branching close to the root of the family Cytophagceae were
only supported by low bootstrap values, the clade compris-
ing species of the genera Flectobacillus,Arcicella,Pseudarci-
cella and the new taxon represented by the investigated
strains was very well supported in all calculated trees.
Within this clade, the phylogenetic tree showed a clear
structure supported by high bootstrap values. The new
taxon appeared to be closest related to Pseudarcicella hirudi-
nis. The 16S rRNA gene sequence of the type strain of this
species had a similarity of 93 % to that of strain 30S-ANT-
BAC
T
. Nevertheless, it was not clear if the 16S rRNA gene
tree could fully resolve the positions of the strains belonging
to the new taxon. The fact, that 16S rRNA gene sequences
of different species within a genus are quite similar was
reported from other taxonomic groups [23].
To improve the phylogenetic placement of the members of
the new genus, genome data sets were used to calculate a
RAxML tree [24]. Of the 120 protein-encoding genes
recommended by Parks et al. [19], one gene (protein family
TIGR00095) was not present in any of the available
genomes of members of the Cytophagaceae. Sequences of
the remaining 119 protein families were concatenated and
aligned by using MAFFT [25]. This resulted in a sequence
alignment of 50 907 amino acid positions. This alignment
was filtered by GBLOCKS (version 0.91b) [26] for highly vari-
able positions. The chosen settings resulted in reduction of
the alignment to 37 796 positions (74 % of positions) in 556
selected blocks. A maximum-likelihood tree (Fig. 2) was
reconstructed using RAxML 8.2.10 [24], as implemented on
the CIPRES Science Gateway version 3.3 [27], under the Pro-
tein CAT model with auto selection of a protein substitution
matrix (model settings PROTCATAUTO), and 100 bootstrap-
ping iterations.
The phylogenetic reconstruction revised the placements of
the members of the Cytophagaceae and confirmed the clade
comprising members of the genus Flectobacillus,Arcicella,
Pseudarcicella and the new taxon. The latter was now clearly
separated from the other three genera. (Fig. 2).
ECOLOGY
The three strains presented in this study were isolated from
either standing or running freshwater systems. All home
habitats had in common that they were small ones. Thus, all
habitats were in close contact to terrestrial systems. This
corresponded with the fact that all strains are obviously able
to degrade the polymer pectin, which is a component of the
cell wall of terrestrial plants. The ability of all investigated
strains to glide could also be a hint to their lifestyle in small
and not permanent waters, which potentially allow them to
move on moist surfaces. Strains 30S-ANTBAC
T
and 103A-
SOEBACH could have, via proteorhodopsin, the opportu-
nity to use sunlight as a supplementary energy source,
which indicates that the strains live in at least temporarily
light exposed habitats. All investigated members of the new
genus-like taxon possessed a gene cluster putatively encod-
ing for enzymes responsible for the reduction of nitrous
oxide to nitrogen. This last step of the dissimilatory nitrate
reduction is used by some bacteria to transfer protons across
the membrane for energy conservation and was considered
as the least oxygen tolerant step of nitrogen respiration [28].
However, a recent study [29] from a marine system revealed
that N
2
O-consuming bacteria were also present and active
in oxygenated surface water, this might be attributed to
anoxic micro-environments created by particles. Even
though the members of the new taxon grew aerobically, the
potential ability to use N
2
O could also be an advantage at
the water sediment interface of small or ephemeral water
bodies, where anaerobic conditions may occur.
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
2744
BLAST searches with the 16S rRNA gene sequences of
30S-ANTBAC
T
and 59G-WUEMPEL
T
revealed that the
new genus-like taxon represents a common and wide-
spread group of freshwater bacteria. Even when taking
only the 100 % and 99 % hits into account, more than
100 sequences from uncultured and cultured bacteria
originating from all over the world were reported (Fig.
S2). This included detections from various inland water
systems like rivers, lakes, ponds, groundwater and lake
sediments in the USA, Canada, the Arctic, China, Japan,
Africa and Europe. For instance, sequences from envi-
ronmental samples putatively belonging to the new
genus-like taxon were found in high-altitude lakes of the
eastern Tibetan plateau [30], the temperate Ipswich and
Parker River in Massachusetts, USA [31], the humic
lake Grosse Fuchskuhle, Germany [32], and in sediments
of the freshwater Lake Kasumigaura, Japan [33]. Never-
theless, only a very few isolates were found in databases
corresponding to the 16S rRNA genes from strains 59G-
WUEMPEL
T
and 30S-ANTBAC
T
with identities of more
Flectobacillus lacus KCCM 42271T (DQ112352)
79/97/86
99/100/99
94/97/97
98/96/99
100/100/100
83/-/82
88/85/88
98/100/100
99/100/99
100/100/100
97/97/96
100/100/100
90/100/100
95/98/92
85/-/-
87/-/68
99/100/98
89/-/73
61/68/55
94/78/98
62/-/-
100/100/100 70/-/-
99/100/99
82/82/86
80/57/92
64/-/65
82/87/34
0.1
Flectobacillus pallidus BCRC 80975T(LT223123)
Flectobacillus fontis KCTC 33763T (LN890295)
Flectobacillus major ATCC 29496T (M62787)
Flectobacillus rhizosphaerae JC289T (LN810638)
Flectobacillus roseus BCRC 17834T (EU420062)
Arcicella aquatica DSM 17092T (AJ535729)
Arcicella rigui KCTC 23307T (HM357635)
Arcicella rosea CCM 7523T (AM948969)
Arcicella aurantiaca DSM 22214T (FJ593908)
Pseudarcicella hirudinis CCM 7988T (HE585218)
Aquirufa antheringensis 103A-SOEBACH (MK449347)
Aquirufa antheringensis 30S-ANTBACT (MK449343)
Aquirufa nivalisilvae 59G-WUEMPELT (MK449345)
Strain HME7025 (genome locus tag 00303)
Siphonobacter aquaeclarae DSM 21668T (FJ177421)
Runella slithyformis ATCC 29530T (M62786)
Dyadobacter fermentans DSM 18053T (NR_074368)
Telluribacter humicola JCM 31133T (KT630891)
Rhabdobacter roseus JCM 30685T (KP324792)
Persicitalea jodogahamensis NBRC 103568T (AB272165)
Ravibacter arvi JCM 31920T (NR_159219)
Larkinella insperata LMG 22510T (AM000022)
Fibrella aestuarina DSM 22563T (NR_102471)
Rudanella lutea DSM 19387T (EF635010)
Fibrisoma limi DSM 22564T (GQ355622)
Spirosoma linguale DSM 74T (NR_074369)
Huanghella arctica CCTCC 2010418T (JQ303016)
Nibrella saemangeumensis JCM 17927T (JN607159)
Arsenicibacter rosenii KCTC 52624T (KT989310)
Emticicia oligotrophica DSM 17448T (AY904352)
Articibacterium luteifluviistationis KCTC 42716T (KU529276)
Taeseokella kangwonensis KACC 16933T (JX426065)
Jiulongibacter sediminis MCCC 1A00733T (KT345953)
Fluviimonas pallidilutea KCTC 32035T (HE793031)
Lacihabitans soyangensis KCTC 23259T (HM590831)
Leadbetterella byssophila NBRC 106382T (AB682398)
Rhodocytophaga aerolata DSM 22190T (EU004198)
Sporocytophaga myxococcoides DSM 11118T (AJ310654)
Cytophaga hutchinsonii ATCC 33406T (NR_102866)
Flexibacter flexilis DSM 6793T (M62794)
Fig. 1. Reconstruction of the phylogenetic position of the investigated strains based on almost full length 16S rRNA gene sequences
(1336 alignment positions). Sequences from all genus type species of the family Cytophagaceae, and further species closely related to
the investigated strains were used. Shown is the maximum-likelihood tree. Bootstrap values are shown from left to right for maxi-
mum-likelihood, neighbour-joining, and maximum- parsimony trees calculated with the same sequence set. Bar, 0.01 substitutions per
nucleotide position. The tree was rooted with Prevotella melaninogenica DSM 7089
T
(not shown, AY323525).
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
2745
than 98.7 %, which is according to Chun et al. [34] a
similarity value potentially separating different species.
In the case of 59G-WUEMPEL
T
six strains sharing 16S
rRNA gene similarities of >99.7 % were isolated from a
mesotrophic lake in the Republic of Korea (strains
HME7025, IMCC25912 and HMD1017), from the sedi-
ment of a freshwater lake in Japan [33], from an
unknown source in China (strain D11), and from fresh-
water in Taiwan (strain CAR-16). In case of strain 30S-
ANTBAC
T
, only two isolates sharing 16S rRNA gene
similarities of >99.3 %, one from an artificial mesotro-
phic lake in the Republic of Korea (strain HME7208)
and one from Lake Maarsseveen, The Netherlands
(strain MI3-53), could be found.
PROPOSAL OF THE NEW GENUS AQUIRUFA
GEN NOV. AND THE TWO SPECIES AQUIRUFA
ANTHERINGENSIS SP. NOV. AND AQUIRUFA
NIVALISILVAE SP. NOV.
In the 16S rRNA gene tree, strains 30S-ANTBAC
T
,
103A-SOEBACH and 59G-WUEMPEL
T
formed their
own clade, well separated from the closest related gen-
era. With their nearest relative Pseudarcicella hirudinis
they showed low 16S rRNA gene sequence similarities
of less than 94.5 %. The separation of the clade formed
by the new strains was confirmed and more pronounced
by the phylogenomic tree (Fig. 2). Pairwise whole
genome average nucleotide identity (gANI) values
FIectobaciIIus major DSM 103T
*
*
*
*
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*
**
*
*
*
97
0.1
79
99
76
65 88
*
*
*
*
*
Arcicella aurantiaca DSM 22214T
Pseudarcicella hirudinis DSM 25647T
Siphonobacter aquaeclarae DSM 21668T
Aquirufa antheringensis 103A-SOEBACH
Aquirufa antheringensis 30S-ANTBACT
Aquirufa nivalisilvae 59G-WUEMPELT
Strain HME7025
Runella limosa DSM 17973T
Runella slithyformis DSM 19594T
Runella zeae DSM 19591T
Dyadobacter alkalitolerans DSM 23607T
Dyadobacter ginsengisoli DSM 21015T
Dyadobacter jiangsuensis DSM 29057T
Dyadobacter fermentans DSM 18053T
Dyadobacter beijingensis DSM 21582T
Dyadobacter crusticola DSM 16708T
Dyadobacter tibetensis JCM 18589T
Larkinella arboricola DSM 21851T
Arsenicibacter rosenii KCTC 52624T
Fibrella aestuarina DSM 22563T
Rudanella lutea DSM 19387T
Fibrisoma limi DSM 22564T
Spirosoma oryzae DSM 28354T
Spirosoma panaciterrae DSM 21099T
Spirosoma endophyticum DSM 26130T
Spirosoma radiotolerans JCM 19447T
Spirosoma fluviale DSM 29961T
Spirosoma linguale DSM 74T
Spirosoma luteum DSM 19990T
Spirosoma spitsbergense DSM 19989T
Emticicia oligotrophica DSM 17448T
Arcticibacterium luteifluviistationis KCTC 42716T
Jiulongibacter sediminis KCTC 42153T
Leadbetterella byssophila DSM 17132T
Sporocytophaga myxococcoides DSM 11118T
Cytophaga hutchinsonii ATCC 33406T
Cytophaga aurantiaca DSM 3654T
Flexibacter flexilis DSM 6793T
Fig. 2. Phylogenomic RAxML tree calculated with amino acid sequences obtained from 119 single copy genes from genomes of spe-
cies belonging to the family Cytophagaceae. Asterisk, bootstrap value 100 %. Bar, 0.2 substitutions per nucleotide position. Accession
numbers of the genome sequences are listed in Tables S1 and 3. The tree was rooted with Prevotella melaninogenica DSM 7089
T
(not
shown).
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
2746
calculated with the type strains of Pseudarcicella hirudi-
nis and Arcicella aurantiaca were only slightly higher
(71.8 %) than pairwise values calculated (i) for any of
the three new strains and the P. hirudinis type strain
(in all three cases about 69 %), and (ii) for any of the
three new strains and the A. aurantiaca type strain (in
all three cases 69–70 %). A comparison of the new
taxon with the closest related genera Pseudarcicella,Arci-
cella and Flectobacillus is given in Table 4. The new
taxon differed in pigmentation, in predominant fatty
acid composition and in the combination of some other
features from all other genera (Table 4). The patterns of
the polar lipids of the three investigated strains (S1) and
Pseudarcicella hirudinis [35] differed strongly. The most
striking point is the genome size of the new taxon,
which is approximately half of that of these nearest
related genera. These points and the phylogenetic recon-
structions (Fig. 1, Fig. 2), as well as 16S rRNA gene
similarities and gANI values suggested that these three
strains represent a new genus, for which we propose the
name Aquirufa gen. nov.
To test which of the three investigated strains belong to the
same species, pairwise gANI values were calculated
(Table 3). While strains 30S-ANTBAC
T
and 103A-SOE-
BACH had a gANI value of 96.5 % which is slightly higher
than the proposed cut-off of 95–96 % separating two species
[34, 36–39], indicated the remaining gANI values of
rounded 71 % (Table 3) that the three investigated strains
represented two different species [34, 36–39]. These find-
ings were confirmed by the phylogenomic reconstructions
with multiple amino acid sequences (Fig. 2), which sepa-
rated the proposed novel species on different branches with
branch length, i.e. evolutionary distances, similar to those
between other type strains belonging to the same genus
within the family Cytophagaceae (Fig. 2). The pairwise
gANI value between strains 59G-WUEMPEL
T
and
HME7025 of 97.4 % suggested, that these strains need to be
considered as members of the same species. At first glance,
the results of the gene content concerning the nitrogen
metabolism disagree with the classification of the investi-
gated strains into two species, since strains 30S-ANTBAC
T
and 103A-SOEBACH classified as one species differed in
possessing gene clusters putatively encoding for nitrate/
nitrite assimilation and nitrous oxide reduction, respectively
(see above). According to the pangenome concept, these
two gene clusters could be regarded as auxiliary genes repre-
senting parts of the flexible genome of the strains. Such
genes could be exchanged by intra- or even interspecific
horizontal gene transfer [15], which might explain, why the
clusters are present or absent in strains belonging to the
same species. In the case of the predicted assimilatory
nitrate reductase, the gene sequence similarity between
strains HME7025 and 59G-WUEMPEL
T
, probably belong-
ing to a single species, was relatively high (97 %), while the
latter and strain 30S-ANTBAC
T
showed 70 %. In contrast,
for the predicted nitrous oxide reductase and flanking genes,
the sequence similarity between strains HME7025 and 59G-
WUEMPEL
T
was the same (97 %), while the latter and
strain 103A-SOEBACH showed 88 % similarity of the genes.
This value is much higher than the whole genome sequence
similarity of 71 % between these two strains and could imply
that for this gene cluster horizontal gene transfer across spe-
cies boundaries might be possible.
Some features distinguished the three strains from each
other. Only strain 59G-WUEMPEL
T
weakly assimilated
sucrose, D-mannose, D-salicin and D-aspartic acid (Table 1).
Furthermore, while strain 30S-ANTBAC
T
had only four
unidentified polar lipids, strain 59G-WUEMPEL
T
had five.
The two strains 30S-ANTBAC
T
and 103A-SOEBACH con-
sidered as belonging to the same species, shared highly simi-
lar G+C values but differed in this characteristic from the
third strain.
DESCRIPTION OF AQUIRUFA GEN. NOV.
Aquirufa gen. nov. [A.qui.ru¢fa. L. n. aqua, water; L. adj.
rufus, red; N.L. fem. n. Aquirufa, a red (bacterium) isolated
from water].
Cells form rods and grow chemoorganotrophically and aer-
obically. Colonies grown on NSY or R2A agar are bright
red, in older stages dark red pigmented, circular and convex
with smooth surface. Liquid cultures in NSY or R2A
medium have an intense red-orange colouring. Cells are
able to glide on soft agar. Major respiratory quinone is MK-
7, predominant fatty acid is iso-C
15 : 0
, identified polar lipid
is phosphatidylethanolamine. Based on phylogenetic recon-
structions with 16S rRNA gene sequences and amino acid
sequences obtained from 119 single copy genes, respectively,
the genus belongs to the family Cytophagaceae. G+C content
is in the range of 38–43 mol%. The type species of the new
genus is Aquirufa antheringensis.
DESCRIPTION OF AQUIRUFA
ANTHERINGENSIS SP. NOV.
Aquirufa antheringensis (an.the.rin.gen¢sis. N.L. fem. adj.
antheringensis, isolated from Antheringer Creek).
Cells form rods, about 1.7 µm long and 0.6 µm wide. Colo-
nies grown on NSY or R2A agar are bright red, in older
stages dark red, pigmented, circular and convex with
smooth surface. Liquid cultures in NSY or R2A medium
have an intense red-orange colouring. Cells are able to glide
on soft agar. Growth occurs at 5–32 C and in 0–0.3 % (w)
NaCl. Cells assimilate pectin and Tween 40, weakly assimi-
late acetoacetic acid, glucuronamide and D-fructose-6-PO
4
,
and do not assimilate L-histidine, propionic acid, D-lactic
acid methyl ester, L-alanine, L-glutamic acid, L-aspartic acid,
D-glucose-6-PO
4
, dextrin, D-glucuronic acid, D-fructose, D-
arabitol, a-D-glucose, a-hydroxy-butyric-acid, D-galactose,
methyl b-D-glucoside, D-galacturonic acid, 3-methyl glu-
cose, L-rhamnose, L-galactonic acid lactone, D-mannitol,
formic acid, L-malic acid, D-gluconic acid, N-acetyl-D-glu-
cosamine, N-acetyl-b-D-mannosamine, mucic acid, myo-
inositol, trehalose, inosine, maltose, gentiobiose, b-
Pitt et al., Int J Syst Evol Microbiol 2019;69:2739–2749
2747
hydroxyl-D,L-butyric acid, cellobiose, glycyl-L-proline, L-
pyroglutamic acid, N-acetyl-D-galacosamine, glycerol, L-
fucose, a-keto-glutaric acid, melibiose, lactose, D-fucose, L-
lactic acid, turanose, N-acetyl neuraminic acid, quinic acid,
D-sorbitol, D-malic acid, p-hydroxy-phenylacetic acid, raffi-
nose, g-amino-butryric acid, L-arginine, stachyose, gelatin,
D-serine, D-saccharic acid, methyl pyruvate, a-keto-butyric
acid, bromo-succinic acid, L-serine, citric acid, sucrose, D-
mannose, D-salicin, D-aspartic acid and acetic acid. Major
fatty acids are C
16 : 1
!5c, iso-C
15 : 0
, iso-C
15 : 0
3-OH and
summed feature 3 (iso-C
15 : 0
2-OH and C
16 : 1
!7c). Polar
lipids are phosphatidylethanolamine, unidentified aminoli-
pids, unidentified amino-phospho-lipids and unidentified
polar lipids. Respiratory quinones are MK7 and traces of
MK6.
The type strain is 30S-ANTBAC
T
(=JCM 32977
T
=LMG
31079
T
=DSM 108553
T
), which was isolated from a small
creek with medium conductivity and nearly neutral pH
located in Anthering, Austria. The genome of the type strain
is characterized by a size of 2.5 Mbp and a G+C content of
42.6 mol%.
DESCRIPTION OF AQUIRUFA NIVALISILVAE
SP. NOV.
Aquirufa nivalisilvae [ni.va.li.sil¢vae. L. adj. nivalis, snow
covered; L. n. silva, forest; N.L. gen. n. nivalisilvae, from the
forest of Schnee (snow)gattern].
Cells form rods, about 1.6 µm long and 0.5 µm wide. Colo-
nies grown on NSY or R2A agar are bright red, in older
stages dark red, pigmented, circular and convex with
smooth surface. Liquid cultures with NSY or R2A medium
have an intense red-orange colouring. Cells are able to glide
on soft agar. Growth occurs at 5–35 C and in 0–0.4 % (w)
NaCl. Cells assimilate pectin and Tween 40, weakly assimi-
late acetoacetic acid, glucuronamide, D-fructose-6-PO
4
,
sucrose, D-mannose, D-salicin, and D-aspartic acid, but do
not assimilate L-histidine, propionic acid, D-lactic acid
methyl ester, L-alanine, L-glutamic acid, L-aspartic acid, D-
glucose-6-PO
4
, dextrin, D-glucuronic acid, D-fructose, D-
arabitol, a-D-glucose, a-hydroxy-butyric-acid, D-galactose,
methyl b-D-glucoside, D-galacturonic acid, 3-methyl glu-
cose, L-rhamnose, L-galactonic acid lactone, D-mannitol,
formic acid, L-malic acid, D-gluconic acid, N-acetyl-D-glu-
cosamine, N-acetyl-b-D-mannosamine, mucic acid, myo-
inositol, trehalose, inosine, maltose, gentiobiose, b-hydrox-
yl-D,L-butyric acid, cellobiose, glycyl-L-proline, L-pyrogluta-
mic acid, N-acetyl-D-galacosamine, glycerol, L-fucose, a-
keto-glutaric acid, melibiose, lactose, D-fucose, L-lactic acid,
turanose, N-acetyl neuraminic acid, quinic acid, D-sorbitol,
D-malic acid, p-hydroxy-phenylacetic acid, raffinose, g-
amino-butryric acid, L-arginine, stachyose, gelatin, D-serine,
D-saccharic acid, methyl pyruvate, a-keto-butyric acid,
bromo-succinic acid, L-serine, citric acid and acetic acid.
Major fatty acids are iso-C
15 : 0
, anteiso-C
15 : 0
, iso-C
15 : 0
3-
OH and summed feature 3 (iso-C
15 : 0
2-OH and C
16 : 1
!7c).
Polar lipids are phosphatidylethanolamine, one unidentified
aminolipid, three unidentified amino-phospho-lipids and
five unidentified polar lipids. The respiratory quinone is
MK7.
The type strain is 59G-WUEMPEL
T
(=LMG 31081
T
=DSM
108554
T
), which was isolated from a small intermittent
freshwater pond with low conductivity and nearly neutral
pH located near Lengau, Austria. The genome of the type
strain is characterized by a size of 3.1 Mbp and a
G+C content of 38.0 mol%.
Funding information
This study was supported by ‘Sparkling Science’project SPA 06/065
funded by the Austrian Federal Ministry of Education, Science and
Research (BMBWF) and the program ‘Talente’of the Austrian
Research Promotion Agency (FFG).
Acknowledgements
We thank Rabia Eskil, Florian Groemer and Simon Hallinger for taking
and handling water samples. We also thank Anita Hatheuer, Lea
Emberger, Dilara Yildirim, Isabella Robl, Amely Sikula, Leonie Kittl, Vik-
toria Fuchs and Oliver Millgrammer for their help in the lab. We thank
Eva Schnitzlbaumer, Carina Schiel, Gerhild Bach, Valentina Gruber,
Lena Roider, Lena Beitscheck and Sarah Jelov
can for creating the
genus and species names, Johanna Poettler for her organizational
support and Bernhard Schink for advice concerning the nomenclature.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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