Fluviispira multicolorata gen. Nov., sp. nov. and Silvanigrella paludirubra sp. nov., isolated from freshwater habitats

Abstract

Strain 33A1-SZDPT was isolated from a small creek located in Puch, Austria. Strain SP-Ram-0.45-NSY-1T was obtained from a small pond located in Schönramer Moor, Germany. 16S rRNA gene sequence similarities between the type strain of Silvanigrella aquatica, currently the only member of the family Silvanigrellaceae, and strains 33A1-SZDPT and SP-Ram-0.45-NSY-1T of 94.1 and 99.1 %, respectively, suggested affiliation of the two strains with this family. Phylogenetic reconstructions with 16S rRNA gene sequences and phylogenomic analyses with amino acid sequences obtained from 103 single-copy genes suggested that the strains represent a new genus and a new species in the case of strain 33A1-SZDPT (=JCM 32978T=DSM 107810T), and a new species within the genus Silvanigrella in the case of strain SP-Ram-0.45-NSY-1T (=JCM 32975T=DSM 107809T). Cells of strain 33A1-SZDPT were motile, pleomorphic, purple-pigmented on agar plates, putatively due to violacein, and showed variable pigmentation in liquid media. They grew chemoorganotrophically and aerobically and tolerated salt concentrations up to 1.2 % NaCl (v/w). The genome size of strain 33A1-SZDPT was 3.4 Mbp and the G+C content was 32.2 mol%. For this new genus and new species, we propose the name Fluviispira multicolorata gen. nov., sp. nov. Cells of strain SP-Ram-0.45-NSY-1T were motile, pleomorphic, red-pigmented and grew chemoorganotrophically and aerobically. They tolerated salt concentrations up to 1.1 % NaCl (v/w). The genome size of strain SP-Ram-0.45-NSY-1T was 3.9 Mbp and the G+C content 29.3 mol%. For the new species within the genus Silvanigrella we propose the name Silvanigrella paludirubra sp. nov.

Full text

1630
Fluviispira multicolorata gen. nov., sp. nov. and Silvanigrella
paludirubra sp. nov., isolated from freshwaterhabitats
AlexandraPitt*, UlrikeKoll, JohannaSchmidt and Martin W.Hahn
TAXONOMIC DESCRIPTION
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
DOI 10.1099/ijsem.0.003947
Author aliations: 1Research Department for Limnology, University of Innsbruck, Mondseestrasse 9, A-5310 Mondsee, Austria.
*Correspondence: Alexandra Pitt, Alexandra. Pitt@ uibk. ac. at
Keywords: Silvanigrellaceae; Silvanigrella; Fluviispira; freshwater.
Abbreviations: gANI, whole genome average nucleotide identity; NSY, nutrient broth–soytone–yeast extract; R2A, Reasoner's 2A.
The Whole Genome Shotgun projects and 16S rRNA gene sequences have been deposited at DDBJ/ENA/GenBank. The accession numbers for
strain 33A1- SZDPT are WFLN00000000 and MN574303, respectively. The accession numbers for strain SP- Ram-0.45- NSY-1T WFLM00000000 and
MN565732, respectively.
Two supplementary figures and two supplementary tables are available with the online version of this article.
003947 © 2020 The Authors
This is an open- access article distributed under the terms of the Creative Commons Attribution NonCommercial License.
Abstract
Strain 33A1- SZDPT was isolated from a small creek located in Puch, Austria. Strain SP- Ram-0.45- NSY-1T was obtained from
a small pond located in Schönramer Moor, Germany. 16S rRNA gene sequence similarities between the type strain of Silvani-
grella aquatica, currently the only member of the family Silvanigrellaceae, and strains 33A1- SZDPT and SP- Ram-0.45- NSY-1T
of 94.1 and 99.1 %, respectively, suggested aliation of the two strains with this family. Phylogenetic reconstructions with 16S
rRNA gene sequences and phylogenomic analyses with amino acid sequences obtained from 103 single- copy genes suggested
that the strains represent a new genus and a new species in the case of strain 33A1- SZDPT (=JCM 32978T=DSM 107810T), and
a new species within the genus Silvanigrella in the case of strain SP- Ram-0.45- NSY-1T (=JCM 32975T=DSM 107809T). Cells of
strain 33A1- SZDPT were motile, pleomorphic, purple- pigmented on agar plates, putatively due to violacein, and showed variable
pigmentation in liquid media. They grew chemoorganotrophically and aerobically and tolerated salt concentrations up to 1.2 %
NaCl (v/w). The genome size of strain 33A1- SZDPT was 3.4 Mbp and the G+C content was 32.2 mol%. For this new genus and
new species, we propose the name Fluviispira multicolorata gen. nov., sp. nov. Cells of strain SP- Ram-0.45- NSY-1T were motile,
pleomorphic, red- pigmented and grew chemoorganotrophically and aerobically. They tolerated salt concentrations up to 1.1 %
NaCl (v/w). The genome size of strain SP- Ram-0.45- NSY-1T was 3.9 Mbp and the G+C content 29.3 mol%. For the new species
within the genus Silvanigrella we propose the name Silvanigrella paludirubra sp. nov.
e family Silvanigrellaceae was described by Hahn et al. [1]
as a novel family belonging to the novel order Silvanigrel-
lales, assigned to the class Oligoexia, phylum Proteobacteria
[2]. Until now, the only validly described species within this
family is Silvanigrella aquatica, with the type strain MWH-
Nonnen- W8redT, which was isolated from a small humic
lake located in the Black Forest Mountains, Germany [1]. It
is characterized by aerobic, chemoorganotrophic growing,
pleomorphic, motile and red- pigmented cells, which tolerate
salt concentrations up to 1.0 % NaCl (w/v). Data are available
for only one close relative of S. aquatica. is ‘Candidatus
Spirobacillus cienkowskii', a pathogen of water eas of the
genus Daphnia, shares 96 % of its 16S rRNA gene sequence
with the type strain of S. aquatica. is taxon was morpho-
logically and ecologically characterized almost 130 years ago
[3], phylogenetically characterized in 2008 [4] and genome
sequenced recently [5].
Within a Citizen Science project (Sparkling Science program),
a cooperation between scientists and students from high
schools, which pursues the aim to isolate and describe new
bacterial taxa [6], water samples from various freshwater
habitats were ltered and subsequently spread on agar plates.
We found two isolates among the puried strains, which were
aliated with the family Silvanigrellaceae. Strain SP- Ram-
0.45- NSY-1T, which was red- pigmented, originated from
a humic pond and obviously belonged to the genus Silva-
nigrella. Strain 33A1- SZDPT, which originated from a small
creek, showed a striking purple pigmentation and evidently
represented a new genus of the family Silvanigrellaceae.
HOME HAbITAT AND ISOlATION
e home habitat of strain 33A1- SZDPT is a small creek,
which ows through Puch as a tributary and discharges in
the river Salzach, Austria. Water sampled in November 2017

1631
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
at the approximate coordinates 47.7092 N and 13.0894 E, was
characterized by a pH value of 6.6 and conductivity of 323
µS cm−1. A second measurement at the same site in July 2018
revealed pH 8.2 and 332 µS cm−1. e sample taken in 2017
was ltered through a 0.2 µm pore size lter and subsequently
plated on Reasoner's 2A (R2A) agar plates [7]. e strain was
puried by using nutrient broth–soytone–yeast extract (NSY)
medium [8].
e home habitat of strain SP- Ram-0.45- NSY-1T is a pond
located at 47.8980 N and 12.8508 E in the Schönramer Moor
in Bavaria, Germany, a disturbed peat bog. Water sampled in
July 2017 was characterized by pH 6.7, conductivity of 28.6
µS cm−1, temperature of 22.5 °C and oxygen saturation of
93.6 % (7.7 mg l
−1
). However, at other samplings of this habitat
(n=16) pH values in the range pH 5.1–6.6 were obtained. e
sampled water was ltered through a 0.45 µm pore size lter
and subsequently spread on NSY agar plates [8] and puried
by using this medium.
PHENOTyPIC AND CHEMOTAXONOMIC
CHARACTERIzATION
All phenotypic and chemotaxonomic characterizations were
performed as described previously [6] using NSY medium
(pH 7.2) and an incubation temperature of 22 °C. In brief, the
temperature range of growth was tested on NSY agar plates
exposed at dierent temperatures in 1 °C steps, temperatures
under 6 °C were not investigated. NaCl tolerance was tested
by using NSY agar plates supplemented with various NaCl
concentrations 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 NaNO3 were
used. Cell morphologies and cell dimensions were investigated
by using DAPI (4′,6- diamidino-2- phenylindole) staining and
an epiuorescence microscope (UV lter). Assimilation of
various substrates was tested using GEN III MicroPlates
(Biolog). e absorption was measured with a Multiskan FC
apparatus (ermo Scientic) at a wavelength of 595 nm aer
48 h incubation at 22 °C. Aer subtracting the value of the
negative control (without substrate), obtained values below
0.015 were regarded as negative, values from 0.015 to 0.03
as weak utilization and >0.03 as positive. e chemotaxo-
nomic characterizations of the strains included analyses of
the composition of whole- cell fatty acids, polar lipids, and
respiratory quinones. ese investigations were carried out by
the Identication Service, Leibniz- Institut DSMZ- Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany. For all chemotaxonomic analyses,
cells were inoculated into liquid NSY medium (pH 7.2), incu-
bated at 22 °C and harvested aer 3 days of growth by centri-
fuge. For the whole- cell fatty acid investigations, an Agilent
Technologies 6890 N instrument, the Microbial Identication
System () Sherlock version 6.1, and the  40 database
were used as described by Sasser [9]. Polar lipids and respira-
tory quinones were extracted and analysed as described by
Tindall [10, 11] based on the method of Bligh and Dyer [12].
For comparison, some of the investigations were performed
with the type strain of S. aquatica, MWH- Nonnen- W8redT.
For temperature and salinity growth tests, as well as for the
chemotaxonomic analysis, data were taken from previous
investigations published elsewhere [1]. ese investigations
were performed in the same lab under the same conditions.
One deviation was an incubation temperature of 28 °C for
generation of biomass for the fatty acid investigations.
Phenotypic traits characterizing the three strains are given
in Table1. Strains 33A1- SZDPT and SP- Ram-0.45- NSY-1T
showed variable morphologies (Fig.1) similar to the morphol-
ogies of the type strain of S. aquatica (Fig. S1, available in the
online version of this article). All strains occurred as rods
of variable length and width, as well as laments, straight,
curved or spiral forms. Examples of the dierent morpholo-
gies are given in Fig.1. Spirals and long laments occurred
in greater numbers when the strain was cultured on so agar.
Eorts to assign the various morphologies to dierent growth
phases were unsuccessful, partly because the dierent forms
co- occurred in samples taken at dierent times. Nevertheless,
it seemed that cultures newly activated from glycerol stocks
initially grew predominantly as short rods, while longer la-
ments and spirals appeared in greater numbers when the
cultures were repeatedly sub- cultured. Strain SP- Ram-0.45-
NSY-1
T
was grey- red pigmented in liquid cultures and formed
red colonies on agar plates (Table1). Strain 33A1- SZPDT
showed various colours in liquid NSY media and purple colo-
nies on agar plates (Table1, Fig.2), putatively due to synthesis
of violacein (see below). For S. aquatica it was reported that
experiments concerning the phenotypic characterization of
the strain were also not reliable [1]. In respect to anaerobic
growth, we also found that experiments gave variable results
and were not replicable (Table1). Concerning the assimilation
patterns of substrates, the S. aquatica type strain used a broad
variety of substrates, while strains SP- Ram-0.45- NSY-1T and
33A1- SZDPT assimilated a smaller number (Table 1). e
results of the fatty acid analysis are given in Table2. Strains
33A1- SZDPT and SP- Ram-0.45- NSY-1T contained menaqui-
none 8 and, as identied polar lipids, phosphatidyglycerol and
phosphatidylethanolamine (Fig. S2). In strain SP- Ram-0.45-
NSY-1T, two unidentied lipids, two unidentied glycolipids,
one unidentied aminolipid and one unidentied phospho-
lipid were also detected, while strain 33A1- SZDPT only had
one additional unidentied lipid (Fig. S2) .
GENOMIC CHARACTERIzATION
DNA extraction and genome sequencing were performed as
described previously [13]. A shotgun library was paired- end
sequenced on an Illumina MiSeq instrument (2×300 bp).
De novo assemblies were performed by using the soware
SPAdes version 3.13.0 [14]. For strain SP- Ram-0.45- NSY-1T,
the k- mer coverage value was 43×, the N50 value was 0.6 Mbp
and the L50 value was 3. For strain 33A1- SZDPT the k- mer
coverage value was 95×, the N50 value was 0.5 Mbp and the
L50 value was also 3. e obtained genome sequences were
annotated by using the  Prokaryotic Genome Annotation
Pipeline and deposited at GenBank. For further comparative

1632
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
Table 1. Traits characterizing strains 33A1- SZDPT, SP- Ram-0.45- NSY-1T and MWH- Nonnen- W8redT
Strains: 1, 33A1- SZDPT; 2, SP- Ram-0.45- NSY-1T; 3, Silvanigrella aquatica MWH- Nonnen- W8redT. As regards the assimilation patterns, only results
which were at least for one strain positive are shown, for the rest of the substrates of the GEN III MicroPlates (Biolog) all three strains were negative
(see species description). +, Positive; −, negative; , weak.
Characteristic 1 2 3
Cell morphology Pleomorphic Pleomorphic Pleomorphic
Pigmentation, liquid media Purple, grey, bright orange Grey- red Red
Pigmentation, agar plates Purple Red Red
Motility on so agar + + +
Temperature range of growth (°C) 6–34 () 6–36 () 10–32 ()
NaCl tolerance (%, v/w) 1.0–1.2 () 1.0–1.1 () 0–1.0 ()
Anaerobic growth:
NSY medium −//+* −/−* −/−*
NSY medium with nitrate −/−/+* −/* −/*
Assimilation of:
Glucuronamide − + +
Pectin − − +
- Histidine + + +
Dextrin − − +
α- - Glucose −  +
Tween 40 + + +
Mucic acid − − +
Propionic acid − − 
Formic acid − − 
- Glucose-6- PO4 + 
Acetic acid − − 
- Glucuronic acid − − 
Gelatin − − 
α-Keto- glutaric acid − + 
- Galacturonic acid − − 
Acetoacetic acid −  
Quinic acid − − 
- Lactic acid methyl ester + − 
α-Keto- butyric acid − − 
- Galactose −  
- Mannose − − 
- Glutamic acid − + 
- Alanine − − 
Lactose − − 
- Gluconic acid  − −
Continued

1633
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
analyses, the genomes were also annotated by the Integrated
Microbial Genomes and Microbiomes Expert Review (IMG/
MER) annotation pipeline and incorporated in the IMG data-
base [15]. An overview of the newly sequenced genomes and
of related genomes [strain MWH- Nonnen- W8red
T
[1], strain
RF1110005 (Shintani M and Kimbara K, unpublished) and
'Candidatus Spirobacillus cienkowskii' binning01 [5]] is given
in Table S1. Analyses with the IMG/MER system [15] revealed
some interesting features. All ve genomes contained genes
putatively encoding for agella compounds and assembly,
as well as pilus assembly (Table S1), which conrmed the
observed motility of our strains on so agar plates (Table1). In
respect to the pigmentation, the ve genomes included genes
likely required for the biosynthesis of lycopene and ζ-carotene,
especially crtl phytoene desaturases, putatively causal for the
red colouring. Only strain 33A1- SZDPT contained a gene
putatively encoding for the biosynthesis of violacein, VioE,
which matched the purple pigmentation of the strain. Only
strains 33A1- SZDPT and RF1110005 possessed genes that
probably encoding for a formate dehydrogenase.
PHylOGENy
Phylogenetic trees were calculated by using the almost full-
length sequences of the 16S rRNA genes (Fig.3) and by using
the amino acid sequences of 103 single copy marker genes out
of the 120 genes recommended by Parks et al. [16] (Fig.4).
e sequences of these marker genes were obtained from
the genome sequences of the strains. e 17 genes, which
are lacking in at least one of the analysed genomes are listed
in Table S2. For the phylogenetic trees based on 16S rRNA
genes (Fig.3), the sequences were aligned, analysed for the
best tting substitution model and used for reconstruction
of phylogenetic trees with the three methods maximum-
likelihood, neighbour- joining and maximum- parsimony.
e shown maximum- likelihood tree was calculated by
Characteristic 1 2 3
ß-Hydroxy-,- butyric acid  − −
- Malic acid + − −
- Arginine  − −
Glycerol  − −
- Fructose −  −
Melibiose  − −
- Fucose − + −
*Results from dierent experiments.
Table 1. Continued
Fig. 1. Examples of the dierent morphologies of strains 33A1- SZPDT (a, first row) and SP- Ram-0.45- NSY- 1T (b, second row). Cells from
various cultures were stained with DAPI and observed under an epifluorescence microscope (UV filter). Bar, 1µm.

1634
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
using the Kimura two- parameter substitution model [17],
gamma distribution (ve categories), invariant sites, and
1000 bootstrap replications, by using the soware X
[18]. All positions of the 1338 aligned positions with less
than 95 % site coverage were eliminated, which resulted in
1226 used positions. For the phylogenomic tree the amino
acid sequences of the used 103 protein- encoding single- copy
genes were concatenated and aligned by using  [19].
e soware  (version 0.91b) [20] was used to lter
out highly variable positions, which resulted in a reduction
of the alignment length from 47 886 to 27 328 positions in
452 selected blocks, which corresponded to 57 % of the initial
alignment positions. A RAxML tree [21] (Fig.4) with 100
bootstrap replications was calculated using the  Science
Gateway version 3.3 [22].
Both phylogenetic trees showed the same structure, never-
theless, the two earliest nodes in the 16S rRNA gene tree
(Fig.3) were only present in the tree established by using
the maximum- likelihood algorithm. All other bootstrap
values were very high; in the genome- based phylogenetic
reconstruction (Fig.4) all internal nodes were supported by
bootstrap values of 100 %. e phylogenetic reconstructions
assigned both new strains to the family Silvanigrellaceae,
placed strain SP- Ram-0.45- NSY-1T on a branch beside
S. aquatica and separated strain 33A1- SZDPT from the
Silvanigrella/Spirobacillus group.
ECOlOGy
As reported previously [1], the currently known members
of the family Silvanigrellaceae seem to be rather rare species.
For strain 33A1- SZPDT,  searches with the 16S rRNA
gene sequence, considering the cut o for new species of
98.7 % [23], revealed neither sequences from environmental
samples nor from cultures that could be assigned to the
proposed novel species represented by the strain. Regarding
the novel proposed genus, only one hit was found. Strain
RF1110005, which seems to belong to the same genus as strain
33A1- SZDPT (Figs3 and 4), was isolated from brackish Lake
Sanaru in Hamamatsu, Japan (Shintani M and Kimbara K,
unpublished). Interestingly, this eutrophic brackish lake [24]
shares with the home habitat of 33A1- SZPDT in comparison
to the habitats of the Silvanigrella strains a much higher
conductivity (>10 fold) and an alkaline pH. In the case of
strain SP- Ram-0.45- NSY-1T, ve bacterial strains isolated
from amphibian skin [25] shared 99.8 % 16S rRNA gene
sequence identity and likely belonged to the same species.
Taking the mean cut- o of 94.6 % separating two genera [26]
into account, three uncultured clones which seemed to belong
to the genus Silvanigrella were found: from Yellostone Lake,
USA (accession number HM856518) [27], from a peat bog
habitat in Tierra del Fuego, Argentina (JF907335) (Kip N et
al., unpublished) and from a subsurface karst water pool in
Switzerland (HE998901) [28]. e physical–chemical condi-
tions of the home habitats of strains SP- Ram-0.45- NSY-1T and
MWH- Nonnen- W8red
T
had the occurrence of humic matter,
low conductivity and pH slightly below neutral in common.
One could speculate that the genus Silvanigrella occurs more
frequently under humic and low conductivity conditions and
members of the genus Fluviispira more frequently in systems
with higher conductivity and pH above neutral; however,
more data are needed to support these postulated trends.
An interesting feature is the violacein synthesis predicted for
strain 33A1- SZDPT. is pigment is known for antibacterial
activity and potential antitumour eects, and was shown
to act against some diseases caused by eukaryotic parasites
[29]. A study suggested that presence of violacein- producing
bacteria on frog skin prevented morbidity and mortality
caused by a lethal skin fungus [30]. e ecological role of
violacein in strain 33A1- SZDPT is currently unknown, and it
is also not known how widespread genes for synthesis of this
pigment are among Silvanigrellales bacteria.
Another interesting point related to the ecology of the
strains investigated so far is the morphological vari-
ability. They share this trait with the parasitic ‘Candidatus
Fig. 2. Pigmentation of strain 33A1- SZDPT grown in liquid NSY medium (left) and on an NSY agar plate (right).

1635
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
Spirobacillus cienkoskii’. However, it remains unclear
if the observed variations in morphologies of the non-
parasitic strains are an artificial effect of the cultivation
conditions or if they occur as well under natural condi-
tions. If so, the ecological significance could be that the
diverse forms allow these bacteria species to adapt to
variable environmental conditions. Such morphological
stages could represent resting stages, migratory stages, or
in the case of larger filaments or large spirilla, stages less
sensitive to protistan predation (morphological defence
strategies). However, more investigations are needed to
prove the links between growth conditions, for example
substrate availability, and morphological stages of the
Silvanigrellaceae strains.
PROPOSAl Of Silvanigrella paludirubra
SP. NOv.
Both phylogenetic reconstructions with 16S rRNA gene
sequences and with 103 amino acid sequences of single-
copy genes, placed strain SP- Ram-0.45- NSY-1T in the family
Silvanigrellaceae on a branch beside S. aquatica. To test if the
new strain represents a new species pairwise whole genome
average nucleotide identity (gANI) values were calculated
with the IMG/MER system [15] (Table S1). Although the
16S rRNA gene sequence similarity between the two taxa is
relatively high, i.e. 99.1 % (Table S1), the pairwise calculated
gANI value of 75.6 % indicated clearly that the new strain
does not belong to the species S. aquatica [31–34]. Further-
more, several phenotypic and chemotaxonomic features
distinguished the two taxa. While strain SP- Ram-0.45-
NSY-1T was grey- red pigmented in liquid cultures, utilized
only a small number of substrates (Table1) and grew up to
36 °C, strain MWH- Nonnen- W8redT (S. aquatica) showed
an intense red colouring, utilized a much broader variety
of substrates and grew only up to a temperature of 32 °C.
Even though the biomass for the fatty acids was grown at
dierent temperatures, the fatty acid pattern of the two taxa
were nearly the same but diered slightly in the amount (see
Table2). While MK8 was identied as the menaquinone
detected in strain SP- Ram-0.45- NSY-1
T
no known quinone
could be identied for strain MWH- Nonnen- W8redT. So,
we assume that strain SP- Ram-0.45- NSY-1T represents a
new species of the genus Silvanigrella, for which we propose
the name Silvanigrella paludirubra sp. n o v.
PROPOSAl Of FluviiSpira multicolorata
GEN. NOv., SP. NOv.
Both phylogenetic reconstructions with 16S rRNA gene
sequences and with 103 amino acid sequences of single- copy
genes placed strain 33A1- SZDPT in the family Silvanigrel-
laceae on a separated branch but close to S. aquatica. e 16S
rRNA sequence similarity between the latter and 33A1- SZDPT
was 94.3 % and therefore slightly lower than the threshold
sequence identity of 94.5 % proposed by Yarza et al. [26] for
separation of two genera. Some phenotypic and chemotaxo-
nomic features distinguished strain 33A1- SZDP
T
from the
two strains belonging to the genus Silvanigrella. While the
latter were basically red pigmented, showed the new strain
various pigmentations in liquid medium and a purple colour
on agar plates (Table 3). Furthermore, the assimilation
patterns of substrates diered between the two taxa (Table3).
So, we assume that strain 33A1- SZDPT represents a new genus
and a new species, for which we propose the name Fluviispira
multicolorata gen. nov, sp. nov.
DESCRIPTION Of Silvanigrella
paludirubra SP. NOv.
Silvanigrella paludirubra sp. nov. ( pa. lu. di. ru′bra L. fem. n.
palus, bog; L. fem. adj. rubra, red; N.L. fem. n. paludirubra,
the red from the bog).
Table 2. Composition of the fatty acids of strains SP- Ram-0.45- NSY-1T,
33A1- SZDPT and MWH- Nonnen- W8redT
Strains: 1, 33A1- SZDPT; 2, SP- Ram-0.45- NSY-1T; 3, Silvanigrella aquatica
MWH- Nonnen- W8redT. Only fatty acids with results of higher than 0.5 %
in at least one of the strains are listed. Data for strain 3 were published
elsewhere [1].
Fatty acid 1 2 3
iso- C15 : 0 20.8 33.1 33.1
C17 : 1ω8c12.0 8.1 9.1
iso- C14 : 0 11.1 5.9 1.3
iso- C16 : 0 9.8 1.2 1.1
anteiso- C15 : 0 9.3 5.7 11.2
C15 : 0 6.6 5.8 3.6
C17 : 0 5.8 2.9 6.5
Summed feature 3* 4.5 5.0 5.7
C15 : 1ω6c3.1 3.8 2.7
iso- C13 : 0−3OH 2.8 0.1 –
iso- C14 : 0−3OH 2.3 10.6 5.0
C16 : 0 2.3 1.8 4.3
C17 : 0−3OH 1.9 0.6 0.6
iso- C17 : 0 1.1 1.6 5.6
C14 : 0 0.9 0.8 1.0
Unknown 13.565* 0.5 5.4 1.4
iso- C17 : 0−3OH 0.5 0.5 1.3
anteiso- C17 : 0 0.5 0.3 0.8
Summed feature 2* 0.4 2.6 0.4
iso- C15 : 0−3OH – – 1.2
iso- C11 : 0−3OH – 0.2 0.7
*Unknown 13.565, unknown fatty acid with equivalent chain length
of 13.565; summed feature 2, iso- C16 : 0 I, C 14 : 0−3OH; summed
feature 3, C16 : 1ω6c, C16 : 1ω7c, iso- C15 : 1−2OH, iso- C15 : 0−2OH.

1636
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
Cells are motile and pleomorphic, including rods and laments
of various length and formation. ey grow chemoorgano-
trophically and aerobically. Cells grown on NSY agar form
red- pigmented, small and circular colonies. In liquid NSY
medium they appear grey- red- pigmented. Growth occurs up
to 36 °C and 1.1 % NaCl (v/w). Cells assimilate glucurona-
mide, - histidine, α- - glucose, Tween 40, - glucose-6- PO4,
α-keto- glutaric acid, acetoacetic acid, - galactose, - glutamic
acid, - fructose and - fucose; do not assimilate N- acetyl- -
glucosamine, 3- methyl glucose, lactose, glycerol, - fucose,
melibiose, maltose, acetic acid, ranose, - aspartic acid,
glycyl- - proline, - sorbitol, dextrin, cellobiose, - lactic acid
methyl ester, - glucuronic acid, turanose, inosine, gentio-
biose, N- acetyl- - galactosamine, - mannose, myo- inositol,
- serine, trehalose, - alanine, α-keto- butyric acid, γ-amino-
butryric acid, - arabitol, gelatin, - rhamnose, - gluconic
acid, stachyose, - fructose-6 -PO4, - galactonic acid lactone,
methyl β- - glucoside, propionic acid, - galacturonic acid,
- arginine, sucrose, - lactic acid, - aspartic acid, β-hydroxy-
,- butyric acid, quinic acid, - malic acid, - mannitol, pectin,
N- acetyl-β- - mannosamine, - salicin, - serine, mucic acid,
N- acetyl neuraminic acid, - pyroglutamic acid, - saccharic
Fig. 3. Reconstruction of the phylogenetic position of the investigated strains within the class Oligoflexia based on almost full- length
16S rRNA gene sequences. Shown is a maximum- likelihood tree. Bootstrap values are shown from left to right for maximum- likelihood,
neighbour- joining and maximum- parsimony trees calculated with the same sequence set. Bar, 0.05 substitutions per nucleotide position.
Fig. 4. Reconstruction of the phylogenetic position of the investigated strains within the class Oligoflexia based on amino acid sequences
obtained from 103 single- copy genes as a RAxML tree. Bar, 0.2 substitutions per nucleotide position.

1637
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
acid, α-hydroxy- butyric acid, citric acid, formic acid, bromo-
succinic acid, methyl pyruvate, p- hydroxy- phenylacetic acid
and - malic acid. Major fatty acids (more than 5%) are iso-
C
15 : 0
, C
17 : 1
ω8c, iso- C
14 : 0
, anteiso- C
15 : 0
, C
15 : 0
, iso- C
14 : 0
−3OH and
an unknown component with an equivalent chain length of
13.565. e respiratory quinone is MK-8. e polar lipids
are phosphatidylglycerol, phosphatidylethanolamine, one
unidentied phospholipid, two unidentied glycolipids, two
unidentied lipids and one aminolipid.
e type strain is SP- Ram-0.45- NSY-1T (=JCM 32975T=DSM
107809
T
), which was isolated from a small pond located in
Schönramer Moor, Bavaria, Germany. e G+C content of
the genomic DNA of the type strain is 29.3 % and the genome
size is 3.9 Mbp.
DESCRIPTION Of FluviiSpira GEN. NOv.
Fluviispira gen. nov. ( Flu. vi. i. spi′ra. L. masc. n. uvius, river; L.
fem. n. spira, coil; N.L. fem. n. Fluviispira, a coil from a river).
Cells are motile and pleomorphic, including rods and la-
ments of various length and formation. ey grow chem-
oorganotrophically and aerobically and occur in various
pigmentations. e respiratory quinone is MK-8. e polar
lipids are phosphatidylglycerol and phosphatidylethan-
olamine. e major fatty acid is iso- C15 : 0. Based on phylo-
genetic reconstructions with 16S rRNA gene sequences
and amino acid sequences obtained from 103 single-
copy genes, respectively, the genus belongs to the family
Silvanigrellaceae. e G+C content of the genomic DNA is
approximately 33 mol% and the genome size 3.5 Mbp. e
type species of the genus is Fluviispira multicolorata.
DESCRIPTION Of FluviiSpira
multicolorata SP. NOv.
F. multicolorata sp. nov. ( mul. ti. co. lo. ra′ta. L. masc. adj.
multus, many; L. past part. coloratus, coloured; N.L. fem.
adj. multicolorata, multi- coloured).
Cells are motile and pleomorphic, including rods and la-
ments of various length and formation. ey grow chemoor-
ganotrophically and aerobically. Cells grown on NSY agar
form purple- pigmented, circular colonies. Liquid cultures
appear either purple, grey or orange. Growth occurs up to
34 °C and 1.2 % (v/w) NaCl. Cells assimilate - histidine,
Tween 40, - glucose-6- PO4, - lactic acid methyl ester,
- gluconic acid, β-hydroxy-,- butyric acid, - malic
acid, - arginine, glycerol and melibiose; do not assimi-
late - fructose, - fucose, glucuronamide, α- - glucose,
N- acetyl- - glucosamine, α-keto- glutaric acid, acetoacetic
acid, 3- methyl glucose, lactose, - galactose, - glutamic
acid, - fucose, maltose, acetic acid, ranose, - aspartic
acid, glycyl- - proline, - sorbitol, dextrin, cellobiose,
- glucuronic acid, turanose, inosine, gentiobiose, N- acetyl-
- galactosamine, - mannose, myo- inositol, - serine,
trehalose, - alanine, α-keto- butyric acid, γ-amino- butryric
acid, - arabitol, gelatin, - rhamnose, stachyose, - fructose-
6- PO4, - galactonic acid lactone, methyl β- - glucoside,
propionic acid, - galacturonic acid, sucrose, - lactic acid,
- aspartic acid, quinic acid, - mannitol, pectin, N- acetyl-
β- - mannosamine, - salicin, - serine, mucic acid, N- acetyl
neuraminic acid, - pyroglutamic acid, - saccharic acid,
α-hydroxy- butyric acid, citric acid, formic acid, bromo-
succinic acid, methyl pyruvate, p- hydroxy- phenylacetic
acid and - malic acid. Major fatty acids (more than 5 %)
are iso- C15 : 0, C17 : 1ω8c, iso- C14 : 0, iso- C16 : 0, anteiso- C15 : 0, C15 : 0
and C17 : 0. e respiratory quinone is MK-8. e polar lipids
are phosphatidylglycerol, phosphatidylethanolamine and
one unidentied lipid.
e type strain is 33A1- SZDPT (=JCM 32978T=DSM
107810T), which was isolated from a small creek owing
through Puch as an inow to the river Salzach, Austria.
e G+C content of the genomic DNA of the type strain is
32.2 mol% and the genome size is 3.4 Mbp.
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 are grateful to Tamina Busse for taking and handling the water
sample from which strain 33A1- SZDPT was isolated and Eva Schni-
zlbaumer for creating the taxonomic names for this strain. We are
also grateful to Sabrina Pitt for taking the water sample, isolation and
naming of strain SP- Ram-0.45- NSY-1T. We thank Anita Hatheuer, Lea
Table 3. Features dierentiating the genus Fluviispira from the genus
Silvanigrella
, Weak; +, positive; −, negative.
Characteristic Fluviispira Silvanigrella
Pigmentation, liquid media Purple, grey, bright
orange
Grey- red
Pigmentation, agar plates Purple Red
Assimilation of:
Glucuronamide − +
α- - Glucose − +
α-Keto- glutaric acid − +
Acetoacetic acid − 
- Galactose − 
- Glutamic acid − +
- Gluconic acid  −
ß-Hydroxy-,- butyric acid  −
- Malic acid + −
- Arginine  −
Glycerol  −
Melibiose  −

1638
Pitt etal., Int. J. Syst. Evol. Microbiol. 2020;70:1630–1638
Emberger, Dilara Yildirim, Isabella Robl, Amely Sikula, Leonie Kittl,
Viktoria Fuchs and Oliver Millgrammer for their contributions to the
characterizations of the strains. We thank Jannik Pitt for editing the
pictures. Special thanks to Bernhard Schink for advice concerning the
genus and species names and the nomenclature.
Conflicts of interest
The authors declare that there are no conflicts of interest.
References
1. Hahn MW, Schmidt J, Koll U, Rohde M, Verbarg S et al. Silvani-
grella aquatica gen. nov., sp. nov., isolated from a freshwater lake,
description of Silvanigrellaceae fam. nov. and Silvanigrellales ord.
nov., reclassification of the order Bdellovibrionales in the class
Oligoflexia, reclassification of the families Bacteriovoracaceae and
Halobacteriovoraceae in the new order Bacteriovoracales ord. nov.,
and reclassification of the family Pseudobacteriovoracaceae in the
order Oligoflexales. Int J Syst Evol Microbiol 2017;67:2555–2568.
2. Nakai R, Nishijima M, Tazato N, Handa Y, Karray F etal. Oligoflexus
tunisiensis gen. nov., sp. nov., a gram- negative, aerobic, filamen-
tous bacterium of a novel proteobacterial lineage, and description
of Oligoflexaceae fam. nov., Oligoflexales ord. nov. and Oligoflexia
classis nov. Int J Syst Evol Microbiol 2014;64:3353–3359.
3. Metchniko E. Contributions l'étude Du pleomorphisme des bacte-
riens. Annales De l'Institut Pasteur 1889;3:61–68.
4. Rodrigues JLM, Duy MA, Tessier AJ, Ebert D,
Mouton L et al. Phylogenetic characterization and prevalence of
"Spirobacillus cienkowskii," a red- pigmented, spiral- shaped bacte-
rial pathogen of freshwater Daphnia species. Appl Environ Microbiol
2008;74:1575–1582.
5. Bresciani L, Lemos LN, Wale N, Lin JY, Strauss AT et al. Draft
genome sequence of "Candidatus Spirobacillus cienkowskii," a
pathogen of freshwater Daphnia species, reconstructed from
hemolymph metagenomic reads. Microbiol Resour Ann 2018;7:2.
6. Pitt A, Schmidt J, Koll U, Hahn MW. Aquirufa antheringensis gen.
nov., sp. nov. and Aquirufa nivalisilvae sp. nov., representing a new
genus of widespread freshwater bacteria. Int J Syst Evol Microbiol
2019;69:2739–2749.
7. Reasoner DJ, Geldreich EE. A new medium for the enumeration
and subculture of bacteria from potable water. Appl Environ Micro-
biol 1985;49:1–7.
8. Hahn MW, Stadler P, Wu QL, Pöckl M. The filtration- acclimatization
method for isolation of an important fraction of the not readily
cultivable bacteria. J Microbiol Methods 2004;57:379–390.
9. Sasser M. Identification of bacteria by gas chromatography of
cellular fatty acids. USFCC Newsl 1990;20:16.
10. Tindall BJ. A comparative study of the lipid composition of Halo-
bacterium saccharovorum from various sources. Syst Appl Microbiol
1990;13:128–130.
11. Tindall BJ. Lipid composition of Halobacterium lacusprofundi. FEMS
Microbiol Lett 1990;66:199–202.
12. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and puri-
fication. Can J Biochem Physiol 1959;37:911–917.
13. Hoetzinger M, Schmidt J, Jezberová J, Koll U, Hahn MW. Microdi-
versification of a pelagic Polynucleobacter species is mainly driven
by acquisition of genomic islands from a partially interspecific
gene pool. Appl Environ Microbiol 2017;83:19.
14. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al.
SPAdes: a new genome assembly algorithm and its applica-
tions to single- cell sequencing. Journal of Computational Biology
2012;19:455–477.
15. Chen I- MA, Chu K, Palaniappan K, Pillay M, Ratner A etal. IMG/M
v.5.0: an integrated data management and comparative analysis
system for microbial genomes and microbiomes. Nucleic Acids Res
2019;47:D666–D677.
16. Parks DH, Chuvochina M, Waite DW, Rinke C, Skarsh-
ewski A etal. A standardized bacterial taxonomy based on genome
phylogeny substantially revises the tree of life. Nat Biotechnol
2018;36:996–1004.
17. Kimura M. A simple method for estimating evolutionary rates
of base substitutions through comparative studies of nucleotide
sequences. J Mol Evol 1980;16:111–120.
18. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular
evolutionary genetics analysis across computing platforms. Mol
Biol Evol 2018;35:1547–1549.
19. Katoh K, Kuma K- ichi, Toh H, Miyata T. MAFFT version 5: improve-
ment in accuracy of multiple sequence alignment. Nucleic Acids
Res 2005;33:511–518.
20. Castresana J. Selection of conserved blocks from multiple
alignments for their use in phylogenetic analysis. Mol Biol Evol
2000;17:540–552.
21. Stamatakis A. RAxML version 8: a tool for phylogenetic anal-
ysis and post- analysis of large phylogenies. Bioinformatics
2014;30:1312–1313.
22. Miller MA, Pfeier W, Schwartz T. Creating the CIPRES science
gateway for inference of large phylogenetic trees. Proceedings of
the Gateway Computing Environments Workshop. New Orleans, LA:
IEEE; 2010. pp. 1–8.
23. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR et al.
Proposed minimal standards for the use of genome data
for the taxonomy of prokaryotes. Int J Syst Evol Microbiol
2018;68:461–466.
24. Qian Y, Hiroyuki F, Satoshi M. Quantitative analysis of water quality
for controlling lake pollution — Case study of two lakes in Japan
and China —. JJAP Conference Proceedings 2016:011406:4.
25. Walke JB, Becker MH, Hughey MC, Swartwout MC, Jensen RV
et al. Most of the dominant members of amphibian skin bacte-
rial communities can be readily cultured. Appl Environ Microbiol
2015;81:6589–6600.
26. Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W et al.
Uniting the classification of cultured and uncultured bacteria
and archaea using 16S rRNA gene sequences. Nat Rev Microbiol
2014;12:635–645.
27. Clingenpeel S, Macur RE, Kan J, Inskeep WP, Lovalvo D et al.
Yellowstone lake: high- energy geochemistry and rich bacterial
diversity. Environ Microbiol 2011;13:2172–2185.
28. Shabarova T, Widmer F, Pernthaler J. Mass eects meet
species sorting: transformations of microbial assemblages
in epiphreatic subsurface karst water pools. Environ Microbiol
2013;15:2476–2488.
29. Numan M, Bashir S, Mumtaz R, Tayyab S, Rehman NU etal. Ther-
apeutic applications of bacterial pigments: a review of current
status and future opportunities. 3 Biotech 2018;8:207.
30. Harris RN, Brucker RM, Walke JB, Becker MH, Schwantes CR etal.
Skin microbes on frogs prevent morbidity and mortality caused by
a lethal skin fungus. Isme J 2009;3:818–824.
31. Kim M, Oh H- S, Park S- C, Chun J. Towards a taxonomic coherence
between average nucleotide identity and 16S rRNA gene sequence
similarity for species demarcation of prokaryotes. Int J Syst Evol
Microbiol 2014;64:346–351.
32. Konstantinidis KT, Tiedje JM. Genomic insights that advance
the species definition for prokaryotes. Proc Natl Acad Sci U S A
2005;102:2567–2572.
33. Konstantinidis KT, Ramette A, Tiedje JM. The bacterial species
definition in the genomic era. Philos Trans R Soc Lond B Biol Sci
2006;361:1929–1940.
34. Rosselló-Móra R, Amann R. Past and future species definitions for
bacteria and archaea. Syst Appl Microbiol 2015;38:209–216.