International Journal of Systematic and Evolutionary Microbiology (2016), 66, 1976–1985 DOI 10.1099/ijsem.0.

000974

Longimicrobium terrae gen. nov., sp. nov., an

oligotrophic bacterium of the under-represented
phylum Gemmatimonadetes isolated through a
system of miniaturized diffusion chambers
Javier Pascual,3 Marina Garcı́a-López, Gerald F. Bills4 and Olga Genilloud
Correspondence Fundación MEDINA, Avenida del Conocimiento 3, Health Sciences Technology Park,
Olga Genilloud 18016 Granada, Spain
olga.genilloud@
medinaandalucia.es
A novel chemo-organoheterotroph bacterium, strain CB-286315T, was isolated from a
Mediterranean forest soil sampled at the Sierra de Tejeda, Almijara and Alhama Natural Park,
Spain, by using the diffusion sandwich system, a device with 384 miniature diffusion chambers.
16S rRNA gene sequence analyses identified the isolate as a member of the under-represented
phylum Gemmatimonadetes, where ‘Gemmatirosa kalamazoonensis’ KBS708, Gemmatimonas
aurantiaca T-27T and Gemmatimonas phototrophica AP64T were the closest relatives, with
respective similarities of 84.4, 83.6 and 83.3 %. Strain CB-286315T was characterized as a
Gram-negative, non-motile, short to long rod-shaped bacterium. Occasionally, some cells
attained an unusual length, up to 35–40 mm. The strain showed positive responses for catalase
and cytochrome-c oxidase and division by binary fission, and exhibited an aerobic metabolism,
showing optimal growth under normal atmospheric conditions. Strain CB-286315T was also
able to grow under micro-oxic atmospheres, but not under anoxic conditions. The strain is a
slowly growing bacterium able to grow under low nutrient concentrations. Major fatty acids
included iso-C17 : 1v9c, summed feature 3 (C16 : 1v7c and/or iso-C15 : 0 2-OH), C16 : 0 and
iso-C17 : 0. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine,
phosphatidylcholine, two unidentified glycolipids and three phospholipids. The major isoprenoid
quinone was MK-8 and the diagnostic diamino acid was meso-diaminopimelic acid. The DNA
G+C content was 67.0 mol%. Based on a polyphasic taxonomic characterization, strain
CB-286315T represents a novel genus and species, Longimicrobium terrae gen. nov., sp. nov.,
within the phylum Gemmatimonadetes. The type strain of Longimicrobium terrae is strain
CB-286315T ( 5 DSM 29007T 5 CECT 8660T). In order to classify the novel taxon within the
existing taxonomic framework, the family Longimicrobiaceae fam. nov., order Longimicrobiales
ord. nov. and class Longimicrobia classis nov. are also proposed.

The phylum Gemmatimonadetes is a cosmopolitan bacterial niches (DeBruyn et al., 2011; Hanada & Sekiguchi, 2014;
taxon, members of which inhabit a wide variety of ecological Zhang et al., 2003) including terrestrial and marine environ-
ments (Kamagata, 2010). Although they are found world-
wide, members of the phylum are usually found at low
3Present address: Department of Microbial Ecology and Diversity frequency in soil microbial communities, with relative abun-
Research, Leibniz Institute DSMZ – German Collection of Microorganisms
dances ranging from 0.2 to 6.5 % (DeBruyn et al., 2011).
and Cell Cultures, Inhoffenstr. 7B, 38124 Braunschweig, Germany.
Presently, .13 000 environmental 16S rRNA gene
4Present address: Texas Therapeutics Institute, The Brown Foundation sequences available in the SILVA SSU Ref 123 database are
Institute of Molecular Medicine, The University of Texas Health Science likely to be associated with the phylum Gemmatimonadetes
Center, 1881 East Road, 3SCR6.4676, Houston TX 77054, USA.
(Quast et al., 2013). According to the environmental 16S
Abbreviations: DSS, diffusion sandwich system; FAME, fatty acid methyl rRNA gene sequences, the phylum Gemmatimonadetes can
ester. be divided into five major lineages at the class level
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene (groups 1–5) (Hanada & Sekiguchi, 2014). Group 1 (class
sequence of strain CB-286315T is LN833202. Gemmatimonadetes) is the numerically dominant lineage,
Three supplementary tables and two supplementary figures are available and the majority of sequences are associated with soils,
with the online Supplementary Material. while the remaining sequences have been retrieved from
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activated sludge in wastewater treatment systems (Hanada &
Sekiguchi, 2014). The class Gemmatimonadetes is the only
one represented by cultured strains to date. Within this
class, two genera, Gemmatimonas and ‘Gemmatirosa’, have
been proposed. The first genus harbours two species,
Gemmatimonas aurantiaca (Zhang et al., 2003) and Gemmati-
monas phototrophica (Zeng et al., 2015), both only represented
by the type strains. Gemmatimonas aurantiaca T-27T was iso-
lated from a laboratory-scale anaerobic–aerobic sequential
batch reactor operated under excess biological phosphorus
removal (Zhang et al., 2003), whereas Gemmatimonas
phototrophica AP64T, a microaerophilic, bacteriochloro-
phyll a-containing bacterium, was isolated from a fresh-
water lake (Zeng et al., 2015). The genus ‘Gemmatirosa’
comprises a single species, ‘Gemmatirosa kalamazoonensis’,
of which the proposed type strain KBS708 was isolated
from an organically managed agricultural soil (DeBruyn
et al., 2013). However, the names of the genus and species
have not yet been validly published (http://www.bacterio.
net/index.html). Almost all of the environmental sequences
from groups 2, 3 and 5 have a terrestrial origin, whereas
group 4 includes sequences recovered from marine sedi-
ments and sponges (Hanada & Sekiguchi, 2014).
The taxonomic and, probably, the metabolic diversity of the
members of this phylum may be as extensive as that of other
well-studied phyla such as Proteobacteria and Actinobacteria
(Hanada & Sekiguchi, 2014). Despite the progress made so
far to understand the ecology of the Gemmatimonadetes
through high-throughput sequencing technologies, full
exploration of their metabolic and physiological capacities
still requires in vitro culture. Because of the difficulty in cul-
turing members of the Gemmatimonadetes, improved and/
or alternative culture strategies are needed (Alain & Querel-
lou, 2009; Overmann, 2013). In this context, our group
developed the diffusion sandwich system (DSS), an array
of 384 miniature diffusion chambers, to isolate recalcitrant
bacteria. DSS is based on the concept of the Ichip (Ling
et al., 2015; Nichols et al., 2010), but it is engineered in a
standard Society for Biomolecular Sciences (SBS) format
to facilitate high-throughput microbial isolation and sub-
sequent transfers. The device consists of a sandwich of
three perforated stainless-steel layers and two polycarbonate
semipermeable membranes that retain bacterial cells
embedded in a gel matrix in each miniature chamber, pre-
venting microbial cells from migrating in and out, but
allowing diffusion of nutrients, signal molecules and waste
products between the chambers and the environment.
After incubation buried in a simulated soil environment,
the DSS is disassembled and individual gel plugs containing
grown bacterial colonies are removed from the miniaturized
chambers by pushing them out using a stainless steel repli-
cator and dispersing them in 384-well culture microplates
filled with R2A broth medium.
In the course of the DSS proof-of-concept validation, strain
CB-286315T was isolated from a soil sample collected from
the Sierra de Tejeda, Almijara and Alhama Natural Park, a
typical Mediterranean forest ecosystem located in Granada,
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Longimicrobium terrae gen. nov., sp. nov.
Spain (368 549 34.40 N 38 519 52.90 W, 972 m above sea-
level), in February 2013. At the time of collection, the tem-
perature was 10 8C and the soil temperature (,10 cm
depth) was 7 8C. The soil pH was 8.5 in deionized water,
and its water content was 11.5 %. For bacterial isolation,
cells were embedded in 2.5 % (w/v) gellan gum (Gelzan
CM Gelrite; Sigma-Aldrich) at an average of one cell per
diffusion chamber. The DSS was buried 10 cm deep in a
simulated soil environment that consisted of a 20-l plastic
box filled with soil from the same location, and incubated
under an alternating 12-h cycle of 10 and 18 8C for
2 months. Strain CB-286315T was isolated from one
gellan-gum plug by transferring the material from the dif-
fusion chamber to synthetic R2A broth medium (DSMZ
medium 830). The 384-well culture microplate was initially
incubated in the dark for 12 weeks at 18 8C and 60 % rela-
tive humidity with 150 r.p.m. orbital shaking and then
transferred to fresh medium and incubated for another
4 weeks under the same conditions. Finally, strain CB-
286315T was streaked onto R2A medium solidified with
1.5 % (w/v) noble agar (Sigma-Aldrich) and its purity
was checked throughout the study by Gram staining and
colonial micromorphology. The diffusion of low-molecu-
lar-mass compounds, e.g. nutrients available in the natural
environment or signal molecules produced by other micro-
organisms, that is favoured by the DSS suggests that
the isolation of CB-286315T might not be the result of
a dilution-to-extinction factor favoured by the DSS,
where numerically dominant taxa are isolated at higher
frequency, but instead is a true enrichment facilitated by
biotic and abiotic conditions reproduced from its natural
environment.
The reference strains used in this taxonomic study were
Gemmatimonas aurantiaca DSM 14586T and ‘Gemmatirosa
kalamazoonensis’ ATCC BAA-2150. Unless otherwise
noted, CB-286315T and the reference strains were grown
under normal atmospheric conditions at 25 8C using R2A
medium solidified with 1.5 % (w/v) noble agar or in R2A
broth for 15 days at 28 8C in the dark for inoculum or
sample preparation. Since Gemmatimonas phototrophica
AP64T only grew well on agar plates (Zeng et al., 2015),
its inclusion in parallel analyses based on broth cultures
was not possible. However, Gemmatimonas phototrophica
AP64T was characterized by Zeng et al. (2015) under com-
parable growth conditions.
Strain CB-286315T was found to be a Gram-negative bac-
terium according to a 3 % KOH assay (Powers, 1995)
and Gram staining and oxidase-positive and catalase-posi-
tive based on tests with 1 % (w/v) N,N,N9,N9-tetramethyl
p-phenylenediamine dihydrochloride and 3 % (v/v) hydro-
gen peroxide solutions, respectively.
Cell morphology was examined using phase-contrast,
transmission electron and scanning electron microscopy
(see supplementary methods available in the online Sup-
plementary Material). Motility was checked by observing
the bacteria in a wet mount with phase-contrast
1977
J. Pascual and others
microscopy and using semi-solid agar stabs (2 g agar l21)
(Tittsler & Sandholzer, 1936). Cells were found to be
non-motile, unlike Gemmatimonas aurantiaca DSM
14586T and Gemmatimonas phototrophica AP64T (Table 1).
Cells were irregular-rod-shaped (1.3–15 mm long, 0.4–0.8 mm
wide) (Table 1). Occasionally, some cells reached an unu-
sual length, up to 35–40 mm, during the exponential
phase (Fig. 1). Septa could not be observed in elongated
cells with transmission electron microscopy, suggesting
these cells to be single cell units (Fig. 1). Further molecular
studies based on targeting membrane proteins present in
cell division septa will be carried out in order to confirm
the unusual cell size. Cells divided by binary fission and,
unlike members of the genera Gemmatimonas and ‘Gem-
matirosa’, membrane vesicles with lipid bilayers budding
off the cells were not evident with scanning or transmission
electron microscopy (Fig. 1). In addition, strain CB-
286315T synthesized an unknown granular material intra-
cellularly that was excreted externally. This material
adhered to the cell surface as an extracellular matrix and,
together with pilus-like structures, led to the formation
of cellular aggregates (Fig. 1). The composition of the gran-
ular material, how it is excreted, the ultrastructure of the
pilus-like structures and the biological role of this coating
remain to be investigated. Capsules and endospores were
not observed after staining the cells with Indian ink and
malachite green, respectively. Intracellular polyphosphate
accumulation was suggested by DAPI (4,6-diamidino-2-
phenylindole) staining (DeBruyn et al., 2013; Zhang
et al., 2003) and by the presence of electron-dense inclusion
bodies in transmission electron micrographs (Fig. 1). These
polyphosphate granules may represent a reservoir of inor-
ganic phosphorus that can be used by bacteria under low-
phosphate conditions. Gemmatimonas aurantiaca DSM
14586T and ‘Gemmatirosa kalamazoonensis’ ATCC BAA-
2150 are also characterized by their capacity to accumulate
polyphosphate.
After 15 days on solid R2A noble agar, growth appeared as
small, circular, convex, entire, translucent, pale salmon-
pigmented colonies, 0.9–1.3 mm in diameter. In broth
medium, cells did not flocculate and showed a whitish-
pink colour. ‘Gemmatirosa kalamazoonensis’ ATCC BAA-
2150 flocculated in broth medium, whereas Gemmatimonas
phototrophica AP64T was reported to be unable to grow in
liquid media (Zeng et al., 2015).
Strain CB-286315T showed an aerobic, chemo-organo-
heterotrophic metabolism, and was unable to reduce
nitrate or to ferment glucose. Although it grew optimally
under aerobic conditions, growth was also observed
under microaerophilic conditions. Growth was not
observed under anaerobic conditions. A candle jar was
used to test microaerophilic bacterial growth on solid
medium in an environment with decreased O2 and elevated
CO2 (Salim et al., 2014), and growth was characterized as
microaerobic. For anaerobic conditions, an anaerobic
atmosphere generation bag (Sigma-Aldrich 68061) was
used in an anaerobe jar. To test bacterial growth under
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microaerobic and anaerobic atmospheres, bacterial cultures
were incubated on solid R2A noble agar at 25 8C for
30 days. Gemmatimonas aurantiaca and ‘Gemmatirosa
kalamazoonensis’ are also chemo-organoheterotrophic bac-
teria, but the former grew optimally under aerobic con-
ditions (Zhang et al., 2003), like strain CB-286315T,
whereas the latter preferred micro-oxic conditions (DeB-
ruyn et al., 2013). Gemmatimonas phototrophica is a micro-
aerophilic, bacteriochlorophyll a-containing bacterium
with a facultatively photoheterotrophic metabolism. Gem-
matimonas phototrophica AP64T is the only known repre-
sentative of the phylum Gemmatimonadetes able to
undergo photosynthesis. The organization and phylogeny
of its photosynthesis genes suggest an ancient acquisition
of the photosynthesis gene cluster via horizontal transfer
from purple phototrophic bacteria (Zeng et al., 2014).
The inability to grow under anoxic conditions and the
lack of a fermentative metabolism are traits shared by all
known members of the phylum.
Growth ranges and optima of temperature, salinity and pH
were determined under normal atmospheric conditions in
R2A broth medium at 25 8C. For each pH range, the
appropriate buffer was used: MES for pH 5, 6 and 6.5,
HEPES for pH 7, 7.5 and 8, HEPPS for pH 8.5 and
CHES for pH 9 and 10 (from Sigma-Aldrich; 10 mM
each). To test the salinity range, R2A broth medium was
supplemented with increasing concentrations of NaCl, ran-
ging from 0 to 1.5 % in increments of 0.1 % (w/v). Growth
was determined by measuring the OD660. Optimal growth
was defined as $75 % of the highest growth rate achieved.
Strain CB-286315T was able to grow between 10 8C (weak
growth) and 33 8C with a temperature optimum of 25–
28 8C. Comparable range and optimum values were
reported for other species of the Gemmatimonadetes
(Table 1). Growth of strain CB-286315T was observed
between pH 6.0 and 9.0 and was optimal at pH 7.0–7.5,
these results also being similar to those reported for
other species of the Gemmatimonadetes (Table 1). Strain
CB-286315T was able to grow when R2A medium was sup-
plemented with up to 0.4 % (w/v) NaCl (final concen-
tration). Optimum growth was reached with 0.1 % (w/v)
NaCl. None of the other strains tolerated NaCl concen-
trations higher than 1 % (w/v), with Gemmatimonas aur-
antiaca DSM 14586T being the most halotolerant [up to
0.7 % (w/v) NaCl]. All strains showed optimal growth at
the lowest NaCl concentrations tested (Fig. 1). Doubling
time of growth under optimal conditions was 13.5 h, and
strain CB-286315T could therefore be considered a slowly
growing bacterium, like Gemmatimonas aurantiaca T-27T
(12 h; Zhang et al., 2003).
For physiological tests, commercial miniaturized API
20NE, API 20E and API ZYM galleries (bioMérieux) were
used in triplicate, following the instructions of the manu-
facturer. API 20NE and API 20E tests were examined
after 48 h, except for assimilation/oxidation of substrates,
which was examined after 2 weeks, and exoenzymic activi-
ties of the API ZYM gallery were revealed after 5 h of
International Journal of Systematic and Evolutionary Microbiology 66
 Table 1. Differential characteristics of strain CB-286315T and type strains of other species of the phylum Gemmatimonadetes

Strains: 1, CB-286315T; 2, Gemmatimonas aurantiaca T-27T; 3, Gemmatimonas phototrophica AP64T (data from Zeng et al., 2015); 4, ‘Gemmatirosa kalamazoonensis’ KBS708. Data for growth,
oxygen requirement, phenotypic properties and enzyme activities in columns 2 and 4 were obtained in this study from Gemmatimonas aurantiaca DSM 14586T and ‘Gemmatirosa kalamazoonensis’
ATCC BAA-2150 under comparable growth conditions; other data were taken from the original species descriptions (Zhang et al., 2003; DeBruyn et al., 2013). +, Positive; 2, negative; W , weakly
positive; V , variable response; NA , no data available; DAP, diaminopimelic acid. All strains form circular colonies.

Characteristic 1 2 3 4

Isolation
Source Forest soil Wastewater reactor Freshwater lake Agricultural soil

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Method DSS NM-1 agar plate Half-strength standard R2A agar plate VL55 noble agar plate
Incubation time (days) 60 14 21–42 36
Colonies on solid media Convex, entire, translucent, pale salmon, Smooth, faintly orange to pink, Tiny (,0.3 mm), smooth, red Smooth and convex, opaque, pink,
0.9–1.3 mm diameter 1–2 mm diameter ,2 mm diameter
Growth in broth media Does not flocculate, whitish pink NA Does not grow well Flocculates, light to dark pink
Cell shape Short to long rods Short rods Short to long rods Short to long rods
Cell size (mm) 0.4–0.661.3–15* 0.762.5–3.5 0.3–0.561–12 0.5–0.761–16
Budding morphology 2 + + +
Motility 2 + + 2
Catalase production + + + 2
Temperature range (optimum) (8C) 10–33 (25–28) 22–33 (30)D 16–30 (25–30) 18–37 (33)d
pH range (optimum) 6.0–9.0 (7.0–7.5) 6.5–9.5 (7.0)§ 6.0–9.0 (7.0–7.5) 5.0–7.0 (6.0)
NaCl concentration range (%, w/v) 0–0.4 0–0.7|| 0–0.2 0–0.4
Metabolism Chemoheterotroph Chemoheterotroph Facultative photoheterotroph Chemoheterotroph
Doubling time (h) 14 12 NA NA

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Oxygen requirement
Preferred mode Aerobic Aerobic Microaerophilic Microaerophilic
Other tolerated mode Microaerophilic Microaerophilic None Aerobic

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Hydrolysis of gelatin + V NA +
Enzyme activities
Esterase (C4) + + NA 2

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Esterase lipase (C8) + + NA 2
Valine arylamidase 2 + NA W
a-Chymotrypsin 2 + NA 2
Acid phosphatase + W NA W
N-Acetyl-b-glucosaminidase 2 + NA W
Major quinone MK-8 MK-9 MK-8 MK-9
Diagnostic diamino acid meso-DAP No DAP isomers detected NA NA
DNA G+C content (mol%) 67.0 64.3" 64.4" 72.0"

*Cells can occasionally elongate up to 35–40 mm.

DReported as 22–33 8C (optimum 30 8C) by Zhang et al. (2003).
dReported as 20–40 8C (optimum 37 8C) by DeBruyn et al. (2013).
§Reported as pH 6.0–10.0 (optimum pH 7.0–7.5) by Zeng et al. (2015).
||Reported as 0–0.8 % (w/v) NaCl by Zeng et al. (2015).
"Based on published genome sequences.

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Longimicrobium terrae gen. nov., sp. nov.
J. Pascual and others

(a) (b) (c)

UP

PI

(d) (e) (f)

PLI
UP

PI

(g) (h)

PLI

Fig. 1. Cell morphology of strain CB-286315T grown on R2A agar or in R2A broth at 28 8C for 15 days. (a, b) Phase-con-
trast photomicrographs of cells immobilized on agarose-covered slides (1 %, w/v). The images were taken with a Zeiss Axio-
skop microscope equipped with an Olympus DP-12 camera (61000 magnification). (c, d) Transmission electron
micrographs. (e–h) Scanning electron micrographs. PI, Polyphosphate inclusions, which appear as electron-dense inclusion
bodies; PLI, pilus-like structures; UP, unidentified polymer. The unidentified polymer with a granular structure is synthetized
intracellularly and excreted outside and adheres to the cell surface. The polymer, along with pilus-like structures, forms an
extracellular matrix that leads to the formation of cellular aggregates. Bars, 10 mm (a, b), 1 mm (c, h), 2 mm (d, f), 500 nm
(e) and 5 mm (g).

incubation. Strain CB-286315T showed a negative response the assimilation of D -mannitol and maltose and, in API
for reduction of nitrates, indole production, fermentation 20E, it showed a variable response for oxidation of L -arabi-
of glucose, arginine dihydrolase, lysine decarboxylase, nose. The small number of carbon sources assimilated or
ornithine decarboxylase, citrate utilization, H2S pro- oxidized in API 20NE or API 20E, respectively, might be
duction, tryptophan deaminase, acetoin production and caused by either the high concentration of nutrients pro-
urease. However, it showed a positive result for gelatinase, vided in these commercial kits or its inability to use the
like ‘Gemmatirosa kalamazoonensis’ ATCC BAA-2150 and provided substrates. Among the exoenzymic activities,
Gemmatimonas aurantiaca DSM 14586T (Table 1). In API strain CB-286315T showed a preference towards phosphate
20NE, strain CB-286315T showed variable responses for esters (alkaline and acid phosphatases and naphthol-AS-BI-
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phosphohydrolase), esterases [esterase (C4) and esterase
lipase (C8)] and leucyl arylamidase, showing variable
activity for a-glucosidase. Gemmatimonas aurantiaca
DSM 14586T and ‘Gemmatirosa kalamazoonensis’ ATCC
BAA-2150 showed a positive response for alkaline phos-
phatase, naphthol-AS-BI-phosphohydrolase and leucyl
arylamidase and a weak response for acid phosphatase
(Table 1). In addition, Gemmatimonas aurantiaca DSM
14586T showed positive responses for esterase (C4), ester-
ase lipase (C8), a-chymotrypsin, valine arylamidase and
N-acetyl-b-glucosaminidase and weak activity for cystine
arylamidase and trypsin. ‘Gemmatirosa kalamazoonensis’
ATCC BAA-2150 showed weak responses for valine aryl-
amidase and N-acetyl-b-glucosaminidase. Other exoenzy-
mic activities were reported as negative.
Individual carbon substrates used for growth were ana-
lysed in triplicate using the basal medium VL70 (DSMZ
medium 1266 without added glucose and buffered at
pH 7.0 with 10 mM HEPES). The final concentration of
each substrate is detailed in Table S1, available in the
online Supplementary Material. Substrates were scored
as supporting growth when the ratio between the final
OD660 (mean of parallel incubations) and the initial
OD660 control value (without the addition of substrate)
was higher than 1.5 after 30 days of incubation. Strain
CB-286315T grew on 56 of 73 carbon sources tested,
showing weak growth on 11 of them. The strain was
able to utilize a wide range of substrates, mainly complex
protein substrates, simple sugars, sugar alcohols, organic
acids and complex carbohydrate polymers such as cellu-
lose, laminarin, starch and xylan. The full list of sole sub-
strates used by strain CB-286315T is detailed in the species
description. Gemmatimonas aurantiaca DSM 14586T and
‘Gemmatirosa kalamazoonensis’ ATCC BAA-2150 showed
narrower ranges of preferred sole carbon sources (Table
S2). Gemmatimonas phototrophica AP64T was character-
ized as using just a few substrates for growth, mainly pro-
tein-containing complex substrates such as yeast extract
and peptone (Zeng et al., 2015).
The ability of strain CB-286315T to grow on serially diluted
complex culture media was tested in order to quantify its
oligotrophic metabolism. It was able to grow in the follow-
ing media: 16R2A broth, 1/5 R2A broth, 1/10 R2A broth,
1/50 R2A broth, 16nutrient broth (NB; BD Difco), 1/10
NB, 1/10 trypticase soy broth (TSB; BD Difco) and
1/10 brain heart infusion broth (BD Difco), showing a
weak response on 1/100 R2A broth. No growth was
observed in 16TSB or 16brain heart infusion broth.
These results confirm the oligotrophic metabolism of
strain CB-286315T, which grows only at low nutrient con-
centrations and not in rich media.
The molar G+C content of genomic DNA was analysed by
HPLC by the Identification Service of the DSMZ (https://
www.dsmz.de/) according to Mesbah et al. (1989) and
Tamaoka & Komagata (1984). The genomic DNA G+C
content of strain CB-286315T was 67.0 mol%, within the
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Longimicrobium terrae gen. nov., sp. nov.
range reported for other members of the Gemmatimona-
detes, i.e. Gemmatimonas aurantiaca T-27T (64.3 mol%),
Gemmatimonas phototrophica AP64T (64.4 mol%) and
‘Gemmatirosa kalamazoonensis’ KBS708 (72.6 mol%)
(DeBruyn et al., 2013; Zeng et al., 2015; Zhang et al.,
2003) (Table 1).
For fatty acid methyl ester (FAME) analysis, fatty acids
were extracted, saponified and methylated according to
standard protocols (MIDI Microbial Identification
System) (Sasser, 1990), and individual FAMEs were ident-
ified using the Microbial Identification System using the
TSBA50 library (MIDI). The major fatty acids of strain
CB-286315T were iso-C17 : 1v9c (47.5 %), summed feature 3
(C16 : 1v7c and/or iso-C15 : 0 2-OH; 13.6 %), C16 : 0
(7.7 %), iso-C17 : 0 (7.4 %) and iso-C15 : 0 (6.1 %) (Table S3).
The FAME profile of Gemmatimonas aurantiaca DSM
14586T reported in this study was comparable to that orig-
inally reported by Zhang et al. (2003), and was character-
ized by major amounts of iso-C15 : 0, summed feature 3
(C16 : 1v7c and/or iso-C15 : 0 2-OH) and iso-C13 : 0. How-
ever, Zeng et al. (2015) reported a FAME profile of Gem-
matimonas aurantiaca T-27T different from that reported
by Zhang et al. (2003). Zeng et al. (2015) speculated that
the reason for these differences could be the use of different
growth conditions (e.g. bacteria grown on solid medium vs
in liquid medium). However, since our results are in agree-
ment with those reported by Zhang et al. (2003), we
hypothesize that the differences in this specific case were
more related to how the fatty acids were processed and
further analysed than to the growth conditions. Gemmati-
monas phototrophica AP64T was characterized by major
amounts of C16 : 1, C14 : 1, C18 : 1v9c and C16 : 0. In the
case of ‘Gemmatirosa kalamazoonensis’ KBS708, the domi-
nant FAMEs were iso-C17 : 1v9c, iso-C15 : 0 and C16 : 1.
Based on these results, we confirmed that members of the
phylum Gemmatimonadetes are characterized by large
amounts of C16 : 1v7c. The methyl-branched unsaturated
fatty acid iso-C17 : 1v9c was a major FAME in strain CB-
286315T and ‘Gemmatirosa kalamazoonensis’ KBS708, and
the methyl-branched saturated fatty acid iso-C15 : 0 was
also a major FAME in Gemmatimonas aurantiaca DSM
14586T and ‘Gemmatirosa kalamazoonensis’ KBS708.
Polar lipid composition, isoprenoid quinones and the
diagnostic diamino acid of the peptidoglycan were ana-
lysed by the Identification Service of the DSMZ. The
polar lipid composition of strain CB-286315T was analysed
by two-dimensional TLC (Bligh & Dyer, 1959; Tindall
et al., 2007). The major polar lipids of the strain were
phosphatidylglycerol, phosphatidylethanolamine, phos-
phatidylcholine, two unidentified glycolipids and three
phospholipids. In addition, four unidentified lipids and
one atypical glycolipid were reported at lower concen-
trations (Fig. S1). The large number of unidentified
polar lipids synthesized by strain CB-286315T, including
an atypical lipid, confirmed its taxonomic novelty. Further
characterization of these unidentified polar lipids will be
carried out in a future study. The polar lipid profiles of
1981
J. Pascual and others
other members of the Gemmatimonadetes have not yet
been analysed, and the profile of strain CB-286315T there-
fore represents the only data available for the phylum. Iso-
prenoid quinones were extracted from dried biomass with
chloroform/methanol (2 : 1, v/v) (Collins & Jones, 1981)
and analysed by HPLC (Tindall, 1990). The predominant
respiratory quinone of CB-286315T was MK-8 (100 %),
which is in agreement with Gemmatimonas phototrophica
AP64T. In contrast, the quinone of Gemmatimonas auran-
tiaca T-27T and ‘Gemmatirosa kalamazoonensis’ KBS708
differed by one isoprenoid unit (MK-9) (Table 1). The
diagnostic diamino acid of the peptidoglycan was deter-
mined according to Schumann (2011). The peptidoglycan
synthesized by strain CB-286315T was characterized
by meso-diaminopimelic acid. This is a key difference
compared with Gemmatimonas aurantiaca T-27T, the
Gram-negative wall of which lacks diaminopimelic acid
isomers (Zhang et al., 2003). Other representatives of the
phylum have not yet been analysed for this character,
even though it could be a relevant chemotaxonomic
marker.
The nearly full-length 16S rRNA gene sequence of strain
CB-286315T was amplified and sequenced following the
methods detailed in the supplementary methods. The
resulting sequence was 1413 nt long. Additional 16S
rRNA gene sequences used in this study were acquired
from public databases (Munoz et al., 2011; Quast et al.,
2013) and their accession numbers are provided with
the phylogenetic trees calculated with the neighbour-join-
ing, maximum-parsimony and maximum-likelihood
algorithms (Figs 2 and S2). According to the EzTaxon-e
database (Kim et al., 2012), the closest type strains to
strain CB-286315T were ‘Gemmatirosa kalamazoonensis’
KBS708 (GenBank accession no. HM154525), Gemmati-
monas aurantiaca T-27T (AB072735) and Gemmatimonas
phototrophica AP64T (KF481682), sharing 84.4, 83.6 and
83.3 % 16S rRNA gene sequence similarity, respectively.
This result confirmed the phylogenetic placement of
strain CB-286315T as representing a novel genomospecies
within the phylum Gemmatimonadetes (Kim et al., 2014;
Stackebrandt & Goebel, 1994). Independently of the algor-
ithm used, strain CB-286315T was placed into group 3
defined by Hanada & Sekiguchi (2014). The arrangement
of the major groups was supported by high bootstrap
values. In the trees, the nearest neighbour of strain CB-
286315T was clone FCPS453 (GenBank accession no.
EF516348), which was recovered from a grassland soil,
sharing 16S rRNA gene sequence identity of 92.7 %.
Group 3 appeared as an external lineage to groups 2, 4
and 5. Whereas groups 2, 3 and 5 were considered
terrestrial groups, group 4 is a marine benthic group
(Hanada & Sekiguchi, 2014). The class Gemmatimonadetes
(group 1), including the genera Gemmatimonas and ‘Gem-
matirosa’, appeared as an external clade. To the best of
our knowledge, strain CB-286315T is the first cultured
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representative of group 3, and therefore represents an
important discovery taking into account the great phylo-
genetic distance from its closest cultured relatives (group 1).
This discovery demonstrates the usefulness of the DSS as
a tool for the isolation and further in vitro ‘domestication’
of recalcitrant bacterial taxa.
According to the thresholds delineated by Yarza et al.
(2014) and Rosselló-Móra & Amann (2015) to delimit
higher taxonomic levels based on 16S rRNA gene sequence
similarities, strain CB-286315T belongs to an independent
class within the phylum Gemmatimonadetes (,86 %
sequence identity). In addition to this phylogenetic con-
clusion, this phylogenetic distance was correlated with a
large number of significant chemotaxonomic and phenoty-
pic differences from Gemmatimonas aurantiaca T-27T and
Gemmatimonas phototrophica AP64T. Among the more
important differences were the size of cells, reproduction
by budding, motility, type of metabolism, oxygen require-
ment, major quinone and presence of a diaminopimelic
acid isomer in the cellular walls.
Comparison of the morphological, physiological, chemo-
taxonomic and phylogenetic characteristics of strain CB-
286315T with those of the species of the genera Gemmati-
monas and ‘Gemmatirosa’ supported the conclusion
that the isolate represents a novel species of a new
genus, for which we propose the name of Longimicrobium
terrae gen. nov., sp. nov. In addition, we propose the
formal description of the novel family Longimicrobiaceae
fam. nov., order Longimicrobiales ord. nov. and class
Longimicrobia classis nov. to accommodate the genus
Longimicrobium.
Description of Longimicrobium gen. nov.
Longimicrobium (Lon.gi.mi.cro9bi.um. L. adj. longus long;
N.L. neut. n. microbium microbe; N.L. neut. n. Longimicro-
bium long microbe, referring to the capacity of the type
species to form very long rods).
Gram-negative, non-spore-forming, non-motile, short to
long rod-shaped bacteria. Cells divide by binary fission.
Aerobic chemo-organoheterotrophs with optimal growth
under normal atmospheric conditions. Also able to grow
under micro-oxic atmospheres, but not under anoxic con-
ditions. Store inorganic phosphorus as intracellular poly-
phosphate granules. Mesophilic, neutrophilic or slightly
alkaliphilic, and non-halophilic bacteria. Positive for cata-
lase and cytochrome-c oxidase. Slow-growing bacteria
able to grow at low nutrient concentrations. Major fatty
acids include iso-C17 : 1v9c, summed feature 3 (C16 : 1v7c
and/or iso-C15 : 0 2-OH), C16 : 0 and iso-C17 : 0. Major
polar lipids are phosphatidylglycerol, phosphatidylethano-
lamine, phosphatidylcholine, two unidentified glycolipids
and three phospholipids. The major isoprenoid quinone
is MK-8. The diagnostic diamino acid is meso-diaminopi-
melic acid. The type species is Longimicrobium terrae.
International Journal of Systematic and Evolutionary Microbiology 66
 Longimicrobium terrae gen. nov., sp. nov.

100 Longimicrobium terrae CB-286315T (LN833202)

0.05 Grassland soil clone FCPS453 (EF516348)
99 Class Longimicrobia
Gas hydrate clone AT425_EubC11 (AY053483) (=Group 3)
78 Farm soil clone AKYG1759 (AY921941)
66 Forest soil clone DUNssu028 (AY913247)

100 Tallgrass prairie soil clone FFCH12021 (EU134893)

95
97 Rhizosphere clone 2h-20 (FJ444650)
Group 5, terrestrial group
100 Farm soil clone AKYG1662 (AY921853)
Forest soil clone S0134 (AF507712)

68 Deep-sea sediment cloner BD7-2 (AB015578)

98 Sponge symbiont TK19 (AJ347028)
96 Group 2, terrestrial group
100 Deep-sea sediment BD2-11 (AB015540)
Forest soil clone DUNssu145 (AY913352)
76 Lake clone SINI1011 (HM126679)
Sponge symbiont PAUC51f (AF186416) Group 4, marine benthic group
100
94 Sponge symbiont PAUC43f (AF186415)
59 Saltmarsh clone LCP -68 (AF286033)

53 Soil clone KCM-C-27 (AJ582060)

97 Soil clone 0319-7E19 (AF234139)
100 Grassland soil clone FCPN548 (EF516935)
Heavy metal-contaminated soil clone a13154 (AY102340)
‘Gemmatirosa kalamazoonensis’ KBS708 (HM154525)
99
Forest soil clone NOS7.157WL (AF432607)

100 Soil isolate strain Ellin7146 (AY673312)

91
98 Soil isolate strain Ellin5220 (AY234571)
Soil isolate strain Ellin5290 (AY234641)
Class Gemmatimonadetes
Arid soil clone 0319-6A19 (AF234125)
(=Group 1)
100 Soil isolate strain Ellin5301 (AY234652)

60 Tallgrass prairie soil clone FFCH3881 (EU134882)

Forested wetland clone FW125 (AF524020)
Forest soil clone C114 (AF507703)
Farm soil clone AKYH1347 (AY921725)
Gemmatimonas phototrophica AP64T (KF481682)

100 Sludge clone SBRH63 (AF268993)

99 Sludge clone Ebpr21 (AF255632)
100 Gemmatimonas aurantiaca T-27T (AB072735)

Fibrobacter succinogenes ATCC 19169T (AJ496032)

Fig. 2. Neighbour-joining tree illustrating the phylogenetic position of strain CB-286315T and related members of the phylum
Gemmatimonadetes, including cultured representatives and environmental clones, based on almost full-length 16S rRNA
gene sequences. Bar, 0.05 fixed nucleotide substitutions per site. Fibrobacter succinogenes ATCC 19169T was used as an
outgroup. Bootstrap values above 50 % (of 1000 resamplings) are indicated at branching points. Accession numbers are
provided in parentheses.

Description of Longimicrobium terrae sp. nov. (1.3–15 mm long and 0.4–0.6 mm wide) that occur as cellu-
lar aggregates. Occasionally, cells can elongate up to 35–
Longimicrobium terrae (ter9rae. L. gen. n. terrae of the soil,
40 mm. On solid medium, colonies are small, circular,
the source of the type strain).
convex, entire, translucent and pale salmon-pigmented
Has the following characteristics in addition to the charac- (0.9–1.3 mm in diameter). In liquid medium, cells do not
teristics that define the genus. Cells are short to long rods flocculate and are a whitish pink. Growth is observed at
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J. Pascual and others
10–33 uC (optimum 25–28 uC), at pH 6.0–9.0 (optimum
pH 7.0–7.5) and in media containing up to 0.4 % (w/v)
NaCl [optimum 0.1 % (w/v) NaCl]. Doubling time under
optimal conditions is 13.5 h. Displays activities of alkaline
and acid phosphatase, naphthol-AS-BI-phosphohydrolase,
esterase (C4), esterase lipase (C8), leucyl arylamidase and
gelatinase. Variable response for a-glucosidase. Shows no
activity of lipase (C14), valine arylamidase, cystine arylami-
dase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosi-
dase, b-glucuronidase, b-glucosidase, N-acetyl-b-
glucosaminidase, a-mannosidase or a-fucosidase. Shows
negative responses for reduction of nitrates, indole pro-
duction, fermentation of glucose, arginine dihydrolase,
lysine decarboxylase, ornithine decarboxylase, citrate utiliz-
ation, H2S production, tryptophan deaminase, acetoin pro-
duction and urease. Substrates used for growth include
cornmeal, gelatin, peptone, peptonized milk, soybean
flour (type not roasted), yeast extract, adonitol, cellulose,
L -arabinose, D -arabinose, D -mannitol, D -sorbitol, D -galac-
tose, D -glucose, lactose, maltose, D -mannose, melezitose,
raffinose, trehalose, dextrin, D -sorbitol, galactitol, inulin,
L -rhamnose, N-acetyl-D -glucosamine, laminarin, starch,
sucrose, trehalose, xylan, L -arginine, L -glutamic acid, L -
methionine, L -proline, alginate, CM-cellulose, polyethylene
glycol, lactate, propionate and pyruvate. Weak responses
are observed on glycerol, D -ribose, cellobiose, aesculin,
melibiose, b-cyclodextrin, DL -tryptophan, L -asparagine, L -
leucine, L -phenylalanine and L -serine. No growth occurs
on keratin, D -fructose, D -xylose, D -glucose 6-phosphate,
L -sorbose, D -glucosamine, glycine, L -alanine, L -histidine,
L -lysine, L -tyrosine, a-lipoic acid, 2,2-dimethyl succinate,
citrate, succinate, D -glucuronate, 4-aminobenzoate, abietic
acid, deoxycholic acid and diethanolamine. Grows in
1|R2A broth, 1/5 R2A broth, 1/10 R2A broth, 1/50 R2A
broth, 1|nutrient broth, 1/10 nutrient broth, 1/10 trypti-
case soy broth and 1/10 brain heart infusion broth. A weak
response is observed in 1/100 R2A broth. No growth is
observed in 1|trypticase soy broth or 1|brain heart
infusion broth.
The type strain is CB-286315 ( 5 DSM 29007 5 CECT
T T
8660T), isolated from a Mediterranean forest soil sampled
in the Sierra de Tejeda, Almijara and Alhama Natural Park,
Spain (36u 549 34.40 N 3u 519 52.99 W, 972 m above sea-
level). A miniature diffusion chamber system named the dif-
fusion sandwich system (DSS) was used to isolate the strain.
The DNA G+C content of the type strain is 67.0 mol%.
Description of Longimicrobiaceae fam. nov.
Longimicrobiaceae (Lon.gi.mi.cro.bi.a9ce.ae. N.L. neut. n.
Longimicrobium type genus of the family; suff. -aceae
ending to denote a family; N.L. neut. pl. n. Longimicrobia-
ceae the Longimicrobium family).
The description is the same as for the genus Longimicro-
bium. The type genus is Longimicrobium. The DNA G+C
content of the type strain of the type species of the type
genus is 67.0 mol%.
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Description of Longimicrobiales ord. nov.
Longimicrobiales (Lon.gi.mi.cro.bi.a9les. N.L. neut. n. Long-
imicrobium type genus of the order; suff. -ales ending to
denote an order; N.L. neut. pl. n. Longimicrobiales the
order of the genus Longimicrobium).
The description is the same as for the genus Longimicro-
bium. The type genus is Longimicrobium gen. nov. The
DNA G+C content of the type strain of the type species
of the type genus is 67.0 mol%.
Description of Longimicrobia classis nov.
Longimicrobia (Lon.gi.mi.cro9bi.a. N.L. neut. n. Longimi-
crobium type genus of the type order of the class; suff. -ia
ending to denote a class; N.L. neut. pl. n. Longimicrobia
the class of the order Longimicrobiales).
The delineation of the class is primarily determined on the
basis of phylogenetic analysis of 16S rRNA gene sequences
and is equivalent to group 3 of the phylum Gemmatimona-
detes proposed by Hanada & Sekiguchi (2014). The class
currently comprises the sole order Longimicrobiales,
which is also the type order.
Acknowledgements
This research was supported by the Non-Oriented Basic Research
Program of the Spanish Ministry of Science and Innovation (project
SAF2010-15010). The authors thank Professor Dr Aharon Oren and
Professor Dr Bernhard Schink for their advice on the nomenclature
of strain CB-286315T. The technical assistance of Dr Ignacio Gonzá-
lez, Mrs Cristina Carmona and Mr Bernabé Martos is appreciated. We
are grateful to the administration of the Sierra de Tejeda, Almijara
and Alhama Natural Park for permission to collect soil.
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