thermolongibacillus - science.ankara.edu.tr

Genus

Firmicutes/Bacilli/Bacillales/Bacillaceae/

ThermolongibacillusCihan et al. (2014)VP

..........................................................................................................................................................................................

Arzu Coleri Cihan, Department of Biology, Faculty of Science, Ankara University, Ankara, Turkey

Kivanc Bilecen and Cumhur Cokmus, Department of Molecular Biology & Genetics, Faculty of Agriculture & NaturalSciences, Konya Food & Agriculture University, Konya, Turkey

Ther.mo.lon.gi.ba.cil’lus. Gr. adj. thermos hot; L. adj.longus long; L. dim. n. bacillus small rod; N.L. masc. n.Thermolongibacillus long thermophilic rod.

Thermolongibacillus is a genus in the phylum Fir-micutes, class Bacilli, order Bacillales, and the familyBacillaceae. There are two species in the genus Thermo-longibacillus, T. altinsuensis and T. kozakliensis, isolatedfrom sediment and soil samples in different ther-mal hot springs, respectively. Members of this genusare thermophilic (40–70∘C), halophilic (0–5.0%NaCl), alkalophilic (pH 5.0–11.0), endospore form-ing, Gram-positive, aerobic, motile, straight rods.Cell morphologies are dependent on the species andappear as single, in pairs, or in long straight or slightlycurved chains. Cells become long after stationaryphase of growth. Endospores are terminally located,and their shapes vary from ellipsoidal to oval. Theyare chemoorganotrophs, showing variations in carbonand energy metabolism, depending on the species.The dominant fatty acids are iso-C17:0, C16:0, andiso-C15:0. MK-7 is the dominant menaquinone. Theircell wall contains low amount of meso-diaminopimelate(meso-DPM) and either A1γ or A1γ′-type peptidoglycancross-linkages. They show <96.1% 16S rRNA genesequence similarities to all the other members of thefamily Bacillaceae.

DNA G+C content (mol%): 43.5–44.8 (HPLC).

Type species: Thermolongibacillus altinsuensis E265T,DSM 24979T, NCIMB 14850T Cihan et al. (2014)VP...................................................................................

Gram-positive, motile rods, occurring singly, in pairs, or

in long straight or slightly curved chains. Moderate alka-

lophile, growing in a pH range of 5.0–11.0; thermophile,

growing in a temperature range of 40–70∘C; halophile,

tolerating up to 5.0% (w/v) NaCl. Catalase-weakly positive,

chemoorganotroph, grow aerobically, but not under anaer-

obic conditions. Young cells are 0.6–1.1 μm in width and

3.0–8.0 μm in length; cells in stationary and death phases

are 0.6–1.2 μm in width and 9.0–35.0 μm in length. Forming

extremely long cells after the late exponential growth phase

is a salient characteristic of this genus. Colony morphologies

vary, depending on the species and the age of the culture.

The dominant fatty acids are iso-C15:0, iso-C17:0, and C16:0.

Iso-C15:0 constitutes more than 60% of all the fatty acid

contents. MK-7 is the dominant menaquinone. Contains low

amount of meso-DPM with A1𝛄 or A1𝛄′type peptidoglycan

cross-linkages. Thermolongibacillus is a member of the phylum

Firmicutes, class Bacilli, order Bacillales, and family Bacillaceae.

DNA G+C content (mol%): 43.5–44.8 (HPLC).

Type species: Thermolongibacillus altinsuensis Cihan et al.

(2014)VP.

Number of species with validated names: 2.

Family classification: The genus Thermolongibacillus is clas-

sified within the family Bacillaceae.

......................................................................................................................................................................................................

Bergey’s Manual of Systematics of Archaea and Bacteria, Online © 2015 Bergey’s Manual Trust. This article is © 2019 Bergey’s Manual Trust.

DOI: 10.1002/9781118960608.gbm01604. Published by John Wiley & Sons, Inc., in association with Bergey’s Manual Trust.

2 Bergey’s Manual of Systematics of Archaea and Bacteria

FIGURE 1. Phase-contrast micrographs of the strains T. kozakliensis E173aT and T. altinsuensis E265T grown at 60 and 55∘C on MIplates, respectively. (a) Young vegetative cells after 6 h; (b) vegetative and foresporal state of cells in a nonswollen sporangiaafter 18 h; (c) and (d) sporulating cells after 24 h; (e) thin and extremely long cells after 48 h; (f) free spores after 72 h. Bar,5 μm. (Cihan et al. (2014). Reproduced with permission of Microbiology Society.)

E173aT

E265T

E173aT

E265T

(a) (b) (c)

(d) (e) (f)

5 μm

4 μm

18 μm

15 μm 33 μm

1.5 μm

3 μm28 μm 2 μm

5 μm

6 μm

6 μm

18 μm

9 μm

7 μm

3.5 μm

Further descriptive information

Cell morphology and ultrastructure...................................................................................

The genus Thermolongibacillus comprises two species withvalidly published names: Thermolongibacillus altinsuensis(Cihan et al., 2014) and Thermolongibacillus kozakliensis(Cihan et al., 2014). Cells of this genus are described asGram-positive, motile, spore-forming rods. When theirmorphological cell cycle is observed under phase-contrastmicroscope using cells grown on MI plates (Geobacillus ther-moglucosidans medium containing 1% soluble starch, pH 7.0,(Suzuki et al. 1976) during time periods for 6 h to 7 days(Figure 1), the cells are single, in pairs, or in long chains. Cellsizes show variation, depending on the incubation time. Cellsare 0.6–1.1 μm in width and 3.0–8.0 μm in length in the lag

and exponential phases of growth. During the time between

the stationary and death phases (20–50 h of incubation),

vegetative cells that have not sporulated become extremely

long (9–35 μm), while their width remains almost the same

(Figure 1e). When the cells are cultured on liquid media

instead of MI plates, approximately half of the vegetative cells

develop spores within 72 h, and the nonsporulated cells form

filaments. Long cell formation can be observed in liquid

cultures only after 7 days of incubation. The appearance

of these long cells at the end of the exponential phase is

a defining characteristic of this genus, when compared to

other thermophilic endospore-forming members of the fam-

ily Bacillaceae such as Aeribacillus, Anoxybacillus, Caldibacillus,

Geobacillus, and Parageobacillus (Table 2).

Endospores in this genus are located terminally in the

cells without swollen sporangium. Sporulation starts after

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This article is © 2019 Bergey’s Manual Trust. Published by John Wiley & Sons, Inc., in association with Bergey’s Manual Trust.

Bergey’s Manual of Systematics of Archaea and Bacteria 3

18–24 h of incubation on MI plates. However, in nutri-ent broth and MI broth, 50% sporulation can only beobserved after 72 h of incubation. Sizes of the free sporesare 1.5–2 μm in length. Two members of this genus show dif-ferent endospore-forming characteristics. Ellipsoidal to ovalendospores occur in nonswollen sporangia of T. kozakliensis,whereas only ellipsoidal endospores occur in nonswollensporangia of T. altinsuensis.

Colonial and cultural characteristics...................................................................................

Colony morphology of Thermolongibacillus shows variations,depending on the species. Colonies of T. kozakliensis appearas nonmucoid, cream color, opaque, circular, convex, and1–3 mm in diameter with smooth surfaces on MI plates after18–24 h of incubation at the optimum growth temperature(60∘C). Actively growing T. altinsuensis’ colonies are ellip-soidal in shape, nonmucoid, light yellow in color, 2–6 mmin width, and 4–10 mm in length with rough surface andopaque centers with translucent edges on MI plates after18–24 h of incubation at the optimum growth tempera-ture (55∘C). After the first cultivation following long-termmaintenance of T. altinsuensis, 60% of its colonies turn tocream color, circular, convex with entire edges, and 2–3 mmin diameter with smooth surfaces at 55∘C. Following twoto three transfers, these round colonies turn ellipsoidalin shape, become wider, and then form the final colonymorphology of T. altinsuensis.

Nutrition and growth conditions...................................................................................

The two known members of this genus show differences intheir carbon and energy source preferences (Table 1). BothT. kozakliensis and T. altinsuensis can routinely be grown ontrypticase soy agar, MI agar, or nutrient agar. Strains arechemoorganotrophs and able to use a variety of differentcarbon sources. T. altinsuensis is oxidase-positive and showsweak catalase activity, while T. kozakliensis is oxidase-negativeand catalase-weakly positive. They can grow aerobically, butnot in anaerobic conditions. Both species can grow at 40and 70∘C, with optimum growth at 60∘C for T. kozakliensisand 55∘C for T. altinsuensis. They have a similar pH growthrange from 5.0 to 11.0, with slightly different optima: pH 9.0for T. kozakliensis and pH 8.5 for T. altinsuensis. They showvarying salt tolerance. T. kozakliensis can grow in an NaClrange of 0–1.5% (w/v), with an optimum of 0.5% (w/v), andT. altinsuensis can tolerate up to 5.0% (w/v) NaCl, with anoptimum of 3.0% (w/v).

Growth on n-alkanes (C5 –C10) is not observed, but theyslowly oxidized octane (C8) in 7 days. Both species can utilize

casein, acetate, pyruvate, succinate, benzoate, tryptone, pep-

tone, yeast extract, and glycerol as sole carbon and energy

sources, but poorly oxidize butyrate, phenol, octane, and

cyclohexane. They cannot grow on lactate, citrate, carbonate,

butanol, naphthalene, or naphthylamine. They are both neg-

ative for starch and gelatin utilization. Urea can be utilized

only by T. altinsuensis. They are both nitrate reducers, positive

for methyl red test, and negative for Voges–Proskauer test.

They show different characteristics for acid production on

various carbon sources (Table 1). Both T. kozakliensis and

T. altinsuensis are negative for amylase, protease, lipase,

and α-glucosidase enzyme activities. They show sensitivity to

vancomycin, kanamycin, novobiocin, bacitracin, chloram-

phenicol, rifampicin, tetracycline, penicillin G, neomycin,

and azithromycin.

Chemotaxonomic characteristics...................................................................................

The dominant cellular fatty acids in Thermolongibacillus

strains are iso-C15:0 (ranging from 60.68% to 63.94%),

iso-C17:0 (ranging from 12.50% to 12.74%), and C16:0 (rang-

ing from 8.57% to 8.86%), and the iso-branched fatty acids

cover about 82–86% of all the cellular fatty acids. Other

significant fatty acids include C14:0 (ranging from 3.97%

to 4.67%), anteiso-C15:0 (ranging from 2.29% to 3.21%),

iso-C16:0 (ranging from 1.05% to 2.38%), and anteiso-C17:0

(ranging from 2.01% to 3.52%). These latter groups consti-

tute about 9–14%. The higher iso-C15:0 (>60%) and lower

iso-C17:0 (∼12%) iso-branched fatty acid contents are the

differentiating points for the members of Thermolongibacil-

lus from the other related thermophilic genera such as

Geobacillus, Parageobacillus, and Anoxybacillus (Table 2). Q1

The polar lipids of Thermolongibacillus are diphos-

phatidylglycerol (DPG), phosphatidylglycerol (PG), phos-

phatidylethanolamine (PE), and two phospholipids (PL1

and PL2). T. kozakliensis, however, contains minor amounts

of aminophospholipid (PN) and aminolipids (AL1 and AL2)

in its cell membrane.

The major menaquinone of both T. kozakliensis and

T. altinsuensis is MK-7, with percentages of 100% and 96%,

respectively. T. altinsuensis contains additional menaquinones

in lower amounts: MK-6 (2%), MK-5 (1%), and MK-8 (1%).

The strains of the genus contain relatively low amounts of

meso-Dpm in their cell walls. They also contain A1γ and A1γ′

cross-linkages in their peptidoglycan layers, which is different

from the other genera of the family Bacillaceae (DSMZ, 2019).

......................................................................................................................................................................................................



TABLE 1. Main salient morphologic, physiologic, phenotypic, chemotaxonomic, and genomic characteristics of thespecies T. kozakliensis and T. altinsuensis

Characteristics T. kozakliensis E173aT T. altinsuensis E265T

Colony morphology Circular, cream in color, 1–3 mm indiameter with entire edges

Circular to ellipsoidal, cream to light yellow incolor, 2–3 mm to 2–6× 4–10 mm in diameterwith entire to undulate edges

Cell size in young cultures / afterstationary phases (μm)

0.6–1.0 by 3.0–8.0/0.6–1.1 by 9.0–32.0 0.7–1.1 by 3.5–8.0/0.8–1.2 by 9.0–35.0

Spore shape Ellipsoidal, oval Ellipsoidal

Free spores (μm) 1.5–2.0 1.5–2.0

Oxidase − ⊥

Temperature requirement (∘C) (opt) 40–70 (60) 40–70 (55)

pH requirement (opt) 5.0–11.0 (9.0) 5.0–11.0 (8.5)

Tolerance to NaCl (%) (opt) 0–1.5 (0.5) 0–5 (3)

Utilization of

Urea − +Growth on Sabouraud Dextrose + −Acid production from

Lactose ⊥ +Glucose + −D-(+)-Galactose − +Sucrose + −D-Sorbitol − +L-Arabinose ⊥ +Raffinose − +Ribose + −

Major fatty acids Iso-C15:0 (63.94%), iso-C17:0, C16:0 Iso-C15:0 (60.68%), iso-C17:0, C16:0

Menaquinone MK-7 (100%) MK-7 (96%), MK-6 (2%), MK-5 (1%), MK-8(1%)

Predominant polar lipids DPG, PG, PE, PLs, PN, and ALs DPG, PG, PE, and two PLs

DNA G+C content, Tm 44.8 43.5

Plasmid Single (14.5 kb) Single (15.5 kb)

Intragenic 16S rRNA gene similarities(%)

97.5

⊥Weakly positive. The predominant polar lipids are underlined. DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phos-phatidylethanolamine; PL, phospholipids; PN, aminophospholipids; AL, aminolipids. Both species are positive for Gram reaction, motility,can both produce terminal spores; grow aerobically; weakly positive for catalase activity, also positive for acid production from maltose,D-fructose, D-(+)-xylose, D-(+)-mannose and D-(−)-mannitol, casein hydrolysis, Methyl red test, and reduction of nitrate to nitrite, whereasnegative reaction for the citrate, tyrosine, starch, gelatin, and trehalose utilization; H2S production in TSI; Voges–Proskauer (pH 6.9);and indole tests; negative for gas production from glucose and nitrate, and cannot produce amylase, protease, lipase, and α-glucosidase(Cihan et al., 2014).

Genome features...................................................................................

Currently, no genome sequence is available for the members

of the Thermolongibacillus genus, and a comprehensive com-

parative genomic study is yet to be performed in order to

accomplish further genome annotations belonging to this

genus. However, the two known members of Thermolongibacil-lus genus, T. kozakliensis and T. altinsuensis, both contain

single extrachromosomal plasmid DNA varying in sizes 14.5

and 15.5 kb, respectively.

Ecology...................................................................................

The species of the genus Thermolongibacillus were isolated

from different thermal hot springs. T. kozakliensis was isolated

from a soil sample that had been collected beside Kozakli

thermal hot spring, and T. altinsuensis from a sediment sam-

ple that had been taken from Altinsu hot spring. These two

hot spring sources have temperature range of 96–98∘C, a

pH range of 6.8–7.5, and are both located in the Nevsehir

Province of Turkey’s Middle Anatolian Region (34∘43′E;

......................................................................................................................................................................................................



TA

BL

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gro

ds

Ch

arac

teri

stic

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Caldalkalibacillusb

Aeribacillusc

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Aneurinibacillush

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Geobacilluse,f

Caldibacillusf

Ureibacillusi

Thermobacillusj

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Anoxybacillusl

Num

ber

of

Spec

ies*

22

237

94

823

211

62

622

Subs

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es−

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Tem

pera

ture

(∘C

)40

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0–

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9.0

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......................................................................................................................................................................................................



TA

BL

E2.

(con

tinue

d)

Ch

arac

teri

stic

s

Thermolongibacillusa

Caldalkalibacillusb

Aeribacillusc

Bacillusd

Parageobacilluse,f,g

Aneurinibacillush

Brevibacillush

Geobacilluse,f

Caldibacillusf

Ureibacillusi

Thermobacillusj

Sulfobacillusk

Anoxybacillusl

Pola

rlip

idsa,

fD

PG,P

G,P

E,

and

PL(P

Nan

dA

L)

ND

DPG

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nd

GL

s

ND

DPG

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dA

L1

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ND

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L,A

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L2

orG

L1,

and

PL1-

2)

PE,A

PGL

,G

Ls,

and

PLs

ND

ND

ND

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Ls,

and

Ls

Intr

agen

ic16

SrR

NA

gen

esi

mila

riti

es(%

)

97.5

−−

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<98

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>98

ND

95.2

94.3

–99

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DN

AG+

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nte

nt

(mol

%)

43.5

–44

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39–

4132

–69

42.1

–44

.441

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43–

5742

–55

49.9

36–

41.5

57.5

46–

5737

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53.5

Th

eda

taw

ere

take

nfr

om:(a

)C

ihan

etal

.(20

14);

(b)

Zh

aoet

al.(

2006

),Z

hao

etal

.(20

08);

(c)

Miñ

ana-

Gal

bis

etal

.(20

10);

(d)

Cla

usan

dB

erke

ley

(198

6);(e

)N

azin

aet

al.(

2001

);(f

)

Coo

revi

tset

al.(

2012

);(g

)A

liyu

etal

.(20

16);

(h)

Shid

aet

al.(

1996

);(i

)Fo

rtin

aet

al.(

2001

);(j

)To

uzel

etal

.(20

00);

(k)

Gol

ovac

hev

aan

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(197

8);(l

)Pi

kuta

etal

.(20

00).

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clud

ing

syn

onym

s;**

Spor

esh

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E,E

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anan

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min

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PG,

diph

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phos

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osph

atid

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han

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PL,

phos

phol

ipid

s;PN

,am

inop

hos

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ipid

s;A

L,

amin

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ids;

GL

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AL

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5:0,

iso-

C15

:00;1

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:00;i

-17:

0,is

o-C

17:0

0;a

i-15:

0,an

teis

o-C

15:0

0;a

i-17:

0,an

teis

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17:0

0;i

-16:

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any.

......................................................................................................................................................................................................



38∘38′N). Currently, no other strains related to the genus

Thermolongibacillus are available on public databases based on

16S rRNA sequence similarity, indicating that this might be a

rare taxon in the environment.

Enrichments and isolation procedures

The strains of Thermolongibacillus were isolated from hot

spring environments using an enrichment technique. They

were isolated from collected samples on MI agar plates at

60∘C with 24 h of aerobic incubation after an enrichment

culturing in MI broth containing 1% (w/v) soluble starch at

60∘C for 24 h with constant shaking at 250 rpm.

Maintenance procedures

T. kozakliensis and T. altinsuensis can be maintained on trypti-

case soy agar, MI agar, or nutrient agar containing 3% agar

or in liquid cultures at 40–70∘C under aerobic conditions.

After 24 h of incubation on agar plates, sporulated cultures

can be stored at +4∘C for more than 6 months. Strains can

also be lyophilized or cryoprotected in trypticase soy broth or

MI broth cultures supplemented with 20% (w/v) glycerol at

−80∘C for long-term maintenance.

Procedures for testing special characteristics

In the members of this genus, the colony morphology and cell

structure changes depend on the culturing conditions such

as incubation time and the media used. Formation of long

chains (9.0–35.0 μm in length) after a prolonged incubation

period is the discriminative feature for the members of the

genus Thermolongibacillus (Figure 1).

Differentiation of the genus Thermolongibacillusfrom other genera

The phylogenetic analysis of the 16S rRNA gene sequences

between the genus Thermolongibacillus and the other 74

genera within the family Bacillaceae revealed that Thermolon-

gibacillus is phylogenetically most related to five thermophilic

endospore-forming genera belonging to Aeribacillus, Anoxy-

bacillus, Caldibacillus, Geobacillus, and Parageobacillus, and

formed a subcluster with Caldibacillus, Geobacillus, and Pa-

rageobacillus genera among these thermophilic members

(Figure 2). The 16S rRNA gene sequences of T. altin-

suensis and T. kozakliensis have highest sequence similarity

(96.1–94.2%) to species from the genus Parageobacillus.

Within the family of Bacillaceae, T. altinsuensis and T. kozaklien-sis show the lowest similarity to Natribacillus halophilus DSM21771T with 84.9% and 83.2% similarities, respectively.

Fingerprinting analyzes, screening the whole genome byPCRs for repetitive extragenic palindromic (rep) elementssuch as BOX- and (GTG)5-PCR or internal transcribedsequences (ITS) between 16S and 23S rRNA genes, arepresent (Cihan et al., 2014). On the basis of the cumulativecluster analysis including three of these BOX-, (GTG)5-, andITS-PCR reactions, T. kozakliensis and T. altinsuensis cannotbe differentiated from each other by means of their ITSfingerprinting profiles, but the rep-PCR patterns of the twoThermolongibacillus species are distinctive not only from eachother, but also from the other related thermophilic Bacil-laceae members. In contrast to the members of the otherthermophilic genera Aeribacillus and Caldibacillus, the speciesfrom the genera Geobacillus (AL1 with PAL, APGL, AL2 orGL1, and PL1-2) and Anoxybacillus (ALs and Ls) containedmajor DPG, PG, and PE polar lipids in common with Ther-molongibacillus (PL, PN, and AL), but they all varied in otherminor polar lipid contents.

In addition, members from the genera Aeribacillus,Aneurinibacillus, Anoxybacillus, Caldalkalibacillus, Caldibacillus,and Geobacillus contain iso-C15:0 as the major iso-branchedfatty acid, but species belonging to the genus Thermolon-gibacillus are unique as their cell membranes contain veryhigh amount of iso-branched iso-C15:0 and iso-C17:0 fatty acids,exceeding 70% of their total fatty acid profiles.

Another distinctive characteristic for the members ofthe genus Thermolongibacillus is the tendency to form longchains of cell morphologies up to 35 μm that can be observedbetween the late exponential and death growth phases assummarized in Table 2.

Differentiation of the species within the genusThermolongibacillus

The two known members of this genus, T. kozakliensis andT. altinsuensis, share 97.3% 16S rRNA gene sequence iden-tity. They display high sequence similarities to some otherthermophilic endospore-forming species from the generaCaldibacillus (<92.0%), Geobacillus (<93.9), and Parageobacil-lus (<96.2). In addition to a low 16S rRNA gene sequenceidentity, DNA–DNA hybridization analysis is also used whendifferentiating closely related species (Stackebrandt et al.,2002). The DNA reassociation value between these twoThermolongibacillus species is 55± 4.7% (mean± SD), a valuebelow the ad hoc committee recommended 70% thresholdto define novel species (Cihan et al., 2014). Genomic DNA

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FIGURE 2. Neighbor-joining evolutionary distance phylogenetic tree based on the 16S rRNA gene sequences of strains T. koza-kliensis E173aT and T. altinsuensis E265T with all 74 representative members of the genera belonging to the family Bacillaceae.The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987), and the evolutionary dis-tances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004). Evolutionary analyses wereconducted using MEGA X software in MS Windows environment (Tamura et al., 2004). Bar indicates 0.05 substitutions pernucleotide position. Escherichia coli ATCC 11775T is used as an outgroup in the tree. (Adapted from Saitou and Nei (1987)and Tamura et al. (2004).)

Alkalilactibacillus ikkensis DSM 19937 (EU281853)

Natronobacillus azotifigens 24KS-1 (EU143681)

Paraliobacillus ryukyuensis DSM 15140 (AB087828)

Saliterribacillus persicus DSM 27696 (QPJJ01000028)

Amphibacillus xylanus DSM 6626 (AP012050)

Pelagirhabdus alkalitolerans S5 (jgi.1102424)

Halolactibacillus halophilus DSM 17073 (jgi.1085781)

Streptohalobacillus salinus DSM 22440 (FJ746578)

Gracilibacillus halotolerans DSM 11805 (AF036922)

Pseudogracilibacillus auburnensis DSM 28556 (KJ490639)

Sinibacillus soli GD05 (KC404830)

Jilinibacillus soli 12 (HQ693527)

Cerasibacillus quisquiliarum DSM 15825 (AB107894)

Lentibacillus salicampi DSM 16425 (AY057394)

Virgibacillus pantothenticus DSM 26 (LGTO01000003)

Oceanobacillus iheyensis DSM 14371 (BA000028)

Ornithinibacillus bavariensis DSM 15681 (Y13066)

Paucisalibacillus globulus DSM 18846 (AM114102)

Aquibacillus halophilus B6B (HQ433456)

Sediminibacillus halophilus DSM 18088 (AM905297)

Terribacillus saccharophilus DSM 21619 (AB243845)

Pontibacillus chungwhensis DSM 16287 (AY553296)

Salinibacillus aidingensis 25-7 (AY321436)

Salirhabdus euzebyi DSM 19612 (AM292417)

MeIghiribacillus thermohalophilus DSM 25894 (KC845574)

Thalassobacillus devorans DSM 16966 (AJ717299)

Halobacillus halophilus DSM 2266 (HE717023)

Salimicrobium album DSM 20748 (X90834)

Allobacillus halotolerans B3A (FJ347755)

Halalkalibacillus halophilus DSM 18494 (AB264529)

Alkalibacillus haloalkaliphilus DSM 5271 (AJ238041)

Piscibacillus salipiscarius DSM 21622 (BBCD01000052)

Filobacillus milosensis DSM 13259 (AJ238042)

Aquisalibacillus elongatus DSM 18090 (AM911047)

Tenuibacillus multivorans CGMCC 1.3442 (jgi.1076194)

Anaerobacillus arseniciselenatis DSM 15340 (AJ865469)

Fermentibacillus polygoni IEB3 (LC054227)

Desertibacillus haloalkaliphilus KJ1-10-99 (KC989945)

Polygonibacilllus indicireducens In2-9 (LC054224)

Paralkalibacillus indicireducens Bps-1 (LC197841)

Fictibacillus barbaricus DSM 14730 (AJ422145)

Swionibacillus sediminis BW11-2 (KY635836)

Compostibacillus humi DX-3 (JX274434)

Pueribacillus theae T8 (MG725951)

Aureibacillus halotolerans DSM 28697 (KJ620986)

Falsibacillus pallidus DSM 25281 (QQAY01000036)

Pradoshia eiseniae EAG3 (PKOZ01000039)

Bacillus subtilis subsp. subtilis DSM 10 (ABQL01000001)

Domibacillus robiginosus DSM 25058 (HE577175)

Jeotgalibacillus alimentarius DSM 18867 (JXRQ01000005)

Camelliibacillus cellulosilyticus THG-YT1 (MG786604)

Salisediminibacterium halotolerans DSM 26530 (EU581836)

Texcoconibacillus texcoconensis DSM 24696 (JN571119)

Geomicrobium halophilum DSM 21769 (AB449106)

Natribacillus halophilus DSM 21771 (AB449109)

Salsuginibacillus kocurii DSM 18087 (AM492160)

Alkalicoccus halolimnae DSM 29191 (KX618877)

Salipaludibacillus aurantiacus S9 (FOGT01000034)

Thalassorhabdus alkalitolerans G27 (MF781072)

Marinococcus halophilus DSM 20408 (NPFA01000042)

Sinobaca qinghaiensis DSM 17008 (DQ168584)

Aliibacillus thermotolerans DSM 101851 (KT999394)

Alteribacillus bidgolensis DSM-25260 (jgi.1071278)

Salibacterium halotolerans S7 (LN812017)

Anoxybacillus pushchinoensis DSM 12423 (jgi.1042845)

Aeribacillus pallidus DSM 3670 (CP017703)

Caldibacillus debilis DSM 16016 (AJ564616)

Parageobacillus caldoxylosilyticus DSM 12041 (BAWO01000028)

Parageobacillus toebil DSM 14590 (BDAQ01000034)

Parageobacillus thermoglucosidasius DSM 2542 (BAWP01000055)

Geobacillus stearothermophilus DSM 22 (AB271757)

Caldalkalibacillus thermarum HA6 (AY753654)

Microaerobacter geothermalis DSM 22679 (FN552009)

Desulfuribacillus alkaliarsenatis DSM 24608 (HM046584)

Tepidibacillus fermentans DSM 23802 (KC242245)

Vulcanibacillus modesticaldus DSM 14931 (AM050346)

Escherichia coli ATCC 11775 (X80725)

Thermolongibacillus altinsuensis DSM 24979 (FJ429590)

Thermolongibacillus kozakliensis DSM 24978 (FJ430056)

85%

88%

87%88%

88%

88%88%

86%

85%88%

88%88%

88%

88%88%

88%88%

88%88%

88%

87%

84%

87%

83%

88%

87%86%

86%85%

85%

85%

84%

86%

87%91%

91%

88%

88%87%

87%

87%87%

81%

85%

89%85%

87%86%

88%88%88%

88%

88%

88%

88%

87%

85%

87%

87%

83%

87%

87%86%

0.050

87%

81%

83%

86%

86%

87%

87%

84%86%

81%

84%

88%

87%

87%

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G+C contents for these two species are also different: 44.8mol% for T. kozakliensis and 43.5 mol% for T. altinsuensis(Cihan et al., 2014).

These two species also show distinct (GTG)5-PCR andBOX-PCR patterns. In (GTG)5-PCR analysis, three discrim-inative DNA bands of 2,641, 2,034, and 1,338 bp are onlyfound in T. kozakliensis, whereas T. altinsuensis has onlyone differentiative DNA band of 1,360 bp, not present inT. kozakliensis. Similarly, in BOX-PCR analyses, a DNA band of1,670 bp for T. kozakliensis and DNA bands of 648 and 490 bpfor T. altinsuensis are discriminative. However, ITS-PCRcannot be used as these two species have similar ITS fin-gerprinting profiles (Cihan et al., 2014; Koc et al., 2015).Thermolongibacillus species show variations in their sporeshapes as well as in cell and colony morphologies and minordifferences in their cell membrane and genomic contents asdisplayed in Table 1. Some physiological features can also beused to differentiate these species (Table 1). T. kozakliensisis oxidase-negative and has a lower salt tolerance (0–1.5%NaCl) compared to T. altinsuensis (0–5.0%). There are alsovariations in their growth optima. The optimum growth tem-perature for T. kozakliensis is 60∘C and that of T. altinsuensisis 55∘C. Similarly, although the pH growth range for thesetwo species is the same for T. kozakliensis and T. altinsuensis,their optimum pH values are 9.0 and 8.5, respectively. Othermetabolic functions in their carbon and energy metabolismcan also be used as differentiating factors as detailed inTable 1. T. kozakliensis is negative for urea utilization, whereasT. altinsuensis can utilize urea. On Sabouraud dextrose,T. kozakliensis can be grown, whereas, T. altinsuensis cannot begrown. These two species also display variations in their acidproduction capabilities when supplied with different sugars(Table 1).

Taxonomic comments

The phylogenetic relationship between the two species ofthe genus Thermolongibacillus and all the members from theother related genera belonging to the family Bacillaceae isgiven in Figure 2, based on their 16S rRNA gene sequences.At least one type species from the 74 genera within thefamily Bacillaceae was included (a total of 78 species) tothe phylogenetic analysis. Genus Thermolongibacillus is mostrelated with the five thermophilic endospore-forming gen-era according to their 16S rRNA gene sequences includingParageobacillus (≤96.1), Geobacillus (≤95.0), Anoxybacillus(≤94.8), Aeribacillus (≤93.5%), and Caldibacillus (≤91.2).T. altinsuensis and T. kozakliensis display lower similarities toall the other type strains within the Bacillaceae family, branch

as a subcluster with these five thermophilic genera in the

depicted Bacillaceae phylogenetic tree, and show 97.3% 16S

rRNA gene sequence homology only to each other. T. altin-suensis has 96.1% sequence similarity to Parageobacillus toebiiDSM 14590, 95.2% to Parageobacillus caldoxylosilyticus, 94.8%

to Parageobacillus thermoglucosidasius DSM 2542T, 93.8% to

Geobacillus stearothermophilus DSM22T, 93.5% to Aeribacilluspallidus DSM 3670T, and 91.2% to Caldibacillus debilis DSM

16016T. T. kozakliensis’s similarities to these species are 94.2,

93.4, 93.5, 91.9, 91.4, and 89.8%, respectively.

List of species of the genus Thermolongibacillus

Thermolongibacillus altinsuensisCihan et al. (2014)VP

...................................................................................

al.tin.su.en’sis. N.L. masc. adj. altinsuensis pertaining to

the isolation habitat, Altinsu hot spring located in Kozakli

Province of Nevsehir in the Middle Anatolian Region of

Turkey (Cappadocia area).

Cells are Gram-positive, motile, straight rods, and occur

singly. The cell size varies, depending on the culture age.

During the early- and mid-logarithmic phase, cells were

single and 0.7–1.1 by 3.5–8.0 μm in size; between stationary

and death phases, cells form chains and become strikingly

longer 0.8–1.2 by 9.0–35.0 μm. Free spores are 1.5–2 μm in

size, and they vary from ellipsoidal to oval. Colony morphol-

ogy changes with the incubation period. Actively growing

colonies are ellipsoidal, light yellow, flat with undulate edges,

nonmucoid, with a rough surface, and are 2–6× 4–10 mm.

Aged colonies are circular, cream in color, 2–3 mm in diam-

eter, and convex with entire edges with a smooth surface.

Thermophilic. The optimum growth temperature is 55∘Cwith a growth range of 40–70∘C; pH 8.5 with a range of

5.0–11.0; and at 3.0% (w/v) NaCl with a range of 0–5.0%

(w/v). Weakly positive for catalase and oxidase. Cells are

chemoorganotrophs and capable of growing on a wide

range of carbohydrates. Can utilize casein and urea and

give a positive methyl red test. Positive for nitrate to nitrite

reduction, but cannot produce gas from nitrate. Negative

for starch, citrate, tyrosine, and gelatin utilization; growth

on Sabouraud dextrose; Voges–Proskauer test; and indole

and H2S production. Amylase-, α-glucosidase-, protease-, and

lipase-negative. Harbors a single plasmid that gives a band

at 15.5 kb. Predominant fatty acids are iso-C15:0 (63.94%),

iso-C17:0, and C16:0. Cell membrane contains the polar lipids

DPG, PG, PE, and two PLs. The major menaquinone is MK-7

in addition to minor amounts of MK-6, MK-5, and MK-8.

......................................................................................................................................................................................................



The type strain is Thermolongibacillus altinsuensis (E265=DSM 24979=NCIMB 14850), isolated from a sediment sam-ple of Altinsu hot spring in Kozakli province of Nevsehir,Turkey (Cappadocia Area).

DNA G+C content (mol%): 43.5 (HPLC).Type strain: E265=DSM 24979=NCIMB 14850.

Thermolongibacillus kozakliensisCihan et al. (2014)VP

...................................................................................

ko.zak.li.en’sis. N.L. masc. adj. kozakliensis referring to theisolation habitat, Kozakli Municipality hot spring located inKozakli Province of Nevsehir in the Middle Anatolian Regionof Turkey (Cappadocia area).

Cells are Gram-positive, motile, straight, slim rods, gener-ally occurring singly. In the early logarithmic growth phase,cells occur singly, approximately 0.6–1.0 by 3.0–8.0 mm.Form long chains and elongate to 0.6–1.1 by 9.0–32.0 mmbetween stationary and death phases. Free spores are ellip-soidal and 1.5–2 mm in length. Colonies are cream color,opaque, circular and convex, nonmucoid, have smooth sur-faces with regular margins along their edges, and 1–3 mmin diameter. Thermophilic. Optimum growth tempera-ture is 60∘C with a growth range of 40–70∘C; pH at 9.0with a range of 5.0–11.0; and at 0.5% (w/v) NaCl witha range of 0–1.5% (w/v). Catalase-weakly positive andoxidase-negative. Cells are chemoorganotrophs, capable ofgrowing on a wide range of carbohydrates. Can utilize casein,grow on Sabouraud dextrose, and positive for methyl redtest. Amylase-, α-glucosidase-, protease-, and lipase-negative.Positive for nitrate to nitrite reduction, but cannot producegas from nitrate. Negative for starch, citrate, tyrosine, gelatin,and urea utilization; Voges–Proskauer test; and indoleand H2S production. Hydrolyzes sugars. Harbors a singleplasmid that gives a band at 14.5 kb. Contains major fattyacids of iso-C15:0 (60.68%), iso-C17:0, and C16:0. The majormenaquinone is MK-7. Predominant polar lipids are DPG,PG, PE, and two PLs with minor amounts of PN and two ALs.

The type strain is Thermolongibacillus kozakliensis (E173a=DSM 24978=NCIMB 14849), isolated from a soil sample ofKozakli Municipality Thermal hot spring located in KozakliProvince of Nevsehir, Turkey (Cappadocia Area).

DNA G+C content (mol%): 44.8 (HPLC).Type strain: E173a=DSM 24978=NCIMB 14849.

Acknowledgments

This research was supported by the Scientific ResearchProject Office of Ankara University with the project number

11B4240003. The authors are also grateful to Salih Gökhan

Çöleri for his contributions to the sample collection studies

in harsh thermal environments.

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Queries in Chapter 1

Q1. Please check and confirm whether the identified layout is fine for Table 2.

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