Thermolongibacillus Cihan Et Al

Genus

Firmicutes/Bacilli/Bacillales/Bacillaceae/ Thermolongibacillus Cihan 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 & Natural Sciences, Konya Food & Agriculture University, Konya, Turkey

Ther.mo.lon.gi.ba.cil’lus. Gr. adj. thermos hot; L. adj. Type species: Thermolongibacillus altinsuensis E265T, longus long; L. dim. n. bacillus small rod; N.L. masc. n. DSM 24979T, NCIMB 14850T Cihan et al. (2014)VP...... Thermolongibacillus long thermophilic rod. Thermolongibacillus is a genus in the phylum Fir- Gram-positive, motile rods, occurring singly, in pairs, or micutes,classBacilli, order Bacillales, and the family in long straight or slightly curved chains. Moderate alka- Bacillaceae. There are two species in the genus Thermo- lophile, growing in a pH range of 5.0–11.0; thermophile, longibacillus, T. altinsuensis and T. kozakliensis, isolated growing in a temperature range of 40–70∘C; halophile, from sediment and soil samples in different ther- tolerating up to 5.0% (w/v) NaCl. Catalase-weakly positive, mal hot springs, respectively. Members of this genus chemoorganotroph, grow aerobically, but not under anaer- are thermophilic (40–70∘C), halophilic (0–5.0% obic conditions. Young cells are 0.6–1.1 μm in width and NaCl), alkalophilic (pH 5.0–11.0), endospore form- 3.0–8.0 μm in length; cells in stationary and death phases ing, Gram-positive, aerobic, motile, straight rods. are 0.6–1.2 μm in width and 9.0–35.0 μm in length. Forming Cell morphologies are dependent on the species and extremely long cells after the late exponential growth phase appear as single, in pairs, or in long straight or slightly is a salient characteristic of this genus. Colony morphologies curved chains. Cells become long after stationary vary, depending on the species and the age of the culture. phase of growth. Endospores are terminally located, The dominant fatty acids are iso-C15:0,iso-C17:0,andC16:0. and their shapes vary from ellipsoidal to oval. They Iso-C15:0 constitutes more than 60% of all the fatty acid are chemoorganotrophs, showing variations in carbon contents. MK-7 is the dominant menaquinone. Contains low and energy metabolism, depending on the species. amount of meso-DPM with A1𝛄 or A1𝛄′type peptidoglycan

The dominant fatty acids are iso-C17:0,C16:0,and cross-linkages. Thermolongibacillus is a member of the phylum iso-C15:0. MK-7 is the dominant menaquinone. Their Firmicutes,classBacilli, order Bacillales, and family Bacillaceae. cell wall contains low amount of meso-diaminopimelate DNA G + C content (mol%): 43.5–44.8 (HPLC). (meso-DPM) and either A1γ or A1γ′-type peptidoglycan Type species: Thermolongibacillus altinsuensis Cihan et al. cross-linkages. They show <96.1% 16S rRNA gene (2014)VP. sequence similarities to all the other members of the Number of species with validated names: 2. family Bacillaceae. Family classification: The genus Thermolongibacillus is clas- DNA G + C content (mol%): 43.5–44.8 (HPLC). sified within the family Bacillaceae.

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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∘ConMI plates, respectively. (a) Young vegetative cells after 6 h; (b) vegetative and foresporal state of cells in a nonswollen sporangia after 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.)

7 μm 9 μm 3.5 μm E173aT 5 μm

(a) (b) (c)

18 μm 6 μm

E265T 4 μm 5 μm 6 μm

3 μm 18 μm 28 μm 2 μm

E173aT

(d) (e) (f)

15 μm 33 μm

E265T

1.5 μm

and exponential phases of growth. During the time between Further descriptive information the stationary and death phases (20–50 h of incubation), vegetative cells that have not sporulated become extremely Cell morphology and ultrastructure ...... long (9–35 μm), while their width remains almost the same (Figure 1e). When the cells are cultured on liquid media The genus Thermolongibacillus comprises two species with instead of MI plates, approximately half of the vegetative cells validly published names: Thermolongibacillus altinsuensis develop spores within 72 h, and the nonsporulated cells form (Cihan et al., 2014) and Thermolongibacillus kozakliensis filaments. Long cell formation can be observed in liquid (Cihan et al., 2014). Cells of this genus are described as cultures only after 7 days of incubation. The appearance Gram-positive, motile, spore-forming rods. When their morphological cell cycle is observed under phase-contrast of these long cells at the end of the exponential phase is microscope using cells grown on MI plates (Geobacillus ther- a defining characteristic of this genus, when compared to moglucosidans medium containing 1% soluble starch, pH 7.0, other thermophilic endospore-forming members of the fam- (Suzuki et al. 1976) during time periods for 6 h to 7 days ily Bacillaceae such as Aeribacillus, Anoxybacillus, Caldibacillus, (Figure 1), the cells are single, in pairs, or in long chains. Cell Geobacillus,andParageobacillus (Table 2). sizes show variation, depending on the incubation time. Cells Endospores in this genus are located terminally in the are 0.6–1.1 μm in width and 3.0–8.0 μminlengthinthelag cells without swollen sporangium. Sporulation starts after

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18–24 h of incubation on MI plates. However, in nutri- casein, acetate, pyruvate, succinate, benzoate, tryptone, pep- ent broth and MI broth, 50% sporulation can only be tone, yeast extract, and glycerol as sole carbon and energy observed after 72 h of incubation. Sizes of the free spores sources, but poorly oxidize butyrate, phenol, octane, and are 1.5–2 μm in length. Two members of this genus show dif- cyclohexane. They cannot grow on lactate, citrate, carbonate, ferent endospore-forming characteristics. Ellipsoidal to oval butanol, naphthalene, or naphthylamine. They are both neg- endospores occur in nonswollen sporangia of T. kozakliensis, ative for starch and gelatin utilization. Urea can be utilized whereas only ellipsoidal endospores occur in nonswollen only by T. altinsuensis. They are both nitrate reducers, positive sporangia of T. altinsuensis. for methyl red test, and negative for Voges–Proskauer test. Colonial and cultural characteristics They show different characteristics for acid production on ...... various carbon sources (Table 1). Both T. kozakliensis and Colony morphology of Thermolongibacillus shows variations, T. altinsuensis are negative for amylase, protease, lipase, depending on the species. Colonies of T. kozakliensis appear and α-glucosidase enzyme activities. They show sensitivity to as nonmucoid, cream color, opaque, circular, convex, and vancomycin, kanamycin, novobiocin, bacitracin, chloram- 1–3 mm in diameter with smooth surfaces on MI plates after phenicol, rifampicin, tetracycline, penicillin G, neomycin, 18–24 h of incubation at the optimum growth temperature and azithromycin. (60∘C). Actively growing T. altinsuensis’ colonies are ellipsoidal in shape, nonmucoid, light yellow in color, 2–6 mm Chemotaxonomic characteristics in width, and 4–10 mm in length with rough surface and ...... opaque centers with translucent edges on MI plates after The dominant cellular fatty acids in Thermolongibacillus 18–24 h of incubation at the optimum growth tempera- strains are iso-C15:0 (ranging from 60.68% to 63.94%), ture (55∘C). After the first cultivation following long-term iso-C (ranging from 12.50% to 12.74%), and C (rang- maintenance of T. altinsuensis, 60% of its colonies turn to 17:0 16:0 ing from 8.57% to 8.86%), and the iso-branched fatty acids cream color, circular, convex with entire edges, and 2–3 mm cover about 82–86% of all the cellular fatty acids. Other in diameter with smooth surfaces at 55∘C. Following two to three transfers, these round colonies turn ellipsoidal significant fatty acids include14:0 C (ranging from 3.97% in shape, become wider, and then form the final colony to 4.67%), anteiso-C15:0 (ranging from 2.29% to 3.21%), morphology of T. altinsuensis. 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- Nutrition and growth conditions > ...... tute about 9–14%. The higher iso-C15:0 ( 60%) and lower iso-C (∼12%) iso-branched fatty acid contents are the The two known members of this genus show differences in 17:0 differentiating points for the members of Thermolongibacil- their carbon and energy source preferences (Table 1). Both T. kozakliensis and T. altinsuensis can routinely be grown on lus from the other related thermophilic genera such as trypticase soy agar, MI agar, or nutrient agar. Strains are Geobacillus, Parageobacillus,andAnoxybacillus (Table 2). Q1 chemoorganotrophs and able to use a variety of different The polar lipids of Thermolongibacillus are diphos- carbon sources. T. altinsuensis is oxidase-positive and shows phatidylglycerol (DPG), phosphatidylglycerol (PG), phos- weak catalase activity, while T. kozakliensis is oxidase-negative phatidylethanolamine (PE), and two phospholipids (PL1 and catalase-weakly positive. They can grow aerobically, but and PL2). T. kozakliensis, however, contains minor amounts not in anaerobic conditions. Both species can grow at 40 of aminophospholipid (PN) and aminolipids (AL1 and AL2) ∘ ∘ and 70 C, with optimum growth at 60 CforT. kozakliensis in its cell membrane. and 55∘CforT. altinsuensis. They have a similar pH growth The major menaquinone of both T. kozakliensis and range from 5.0 to 11.0, with slightly different optima: pH 9.0 T. altinsuensis is MK-7, with percentages of 100% and 96%, for T. kozakliensis and pH 8.5 for T. altinsuensis. They show respectively. T. altinsuensis contains additional menaquinones varying salt tolerance. T. kozakliensis cangrowinanNaCl range of 0–1.5% (w/v), with an optimum of 0.5% (w/v), and in lower amounts: MK-6 (2%), MK-5 (1%), and MK-8 (1%). T. altinsuensis can tolerate up to 5.0% (w/v) NaCl, with an The strains of the genus contain relatively low amounts of ′ optimum of 3.0% (w/v). meso-Dpm in their cell walls. They also contain A1γ and A1γ

Growth on n-alkanes (C5 –C10) is not observed, but they cross-linkages in their peptidoglycan layers, which is different slowly oxidized octane (C8) in 7 days. Both species can utilize from the other genera of the family Bacillaceae (DSMZ, 2019).

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TABLE 1. Main salient morphologic, physiologic, phenotypic, chemotaxonomic, and genomic characteristics of the species T. kozakliensis and T. altinsuensis Characteristics T. kozakliensis E173aT T. altinsuensis E265T Colony morphology Circular, cream in color, 1–3 mm in Circular to ellipsoidal, cream to light yellow in diameter with entire edges color, 2–3 mm to 2–6 × 4–10 mm in diameter with entire to undulate edges Cell size in young cultures / after 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 stationary phases (μm) 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, phosphatidylethanolamine; 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, whereas negative 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 Ecology ......

Currently, no genome sequence is available for the members The species of the genus Thermolongibacillus were isolated of the Thermolongibacillus genus, and a comprehensive com- from different thermal hot springs. T. kozakliensis was isolated parative genomic study is yet to be performed in order to from a soil sample that had been collected beside Kozakli accomplish further genome annotations belonging to this thermal hot spring, and T. altinsuensis from a sediment sam- genus. However, the two known members of Thermolongibacil- ple that had been taken from Altinsu hot spring. These two lus genus, T. kozakliensis and T. altinsuensis, both contain hot spring sources have temperature range of 96–98∘C, a single extrachromosomal plasmid DNA varying in sizes 14.5 pH range of 6.8–7.5, and are both located in the Nevsehir and 15.5 kb, respectively. Province of Turkey’s Middle Anatolian Region (34∘43′E;

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Bergey’s Manual of Systematics of Archaea and Bacteria 5

Anoxybacillus -17:0, l ai -17:0 or i -15:0, 16:0 or -16:0 − − i 16:0, i / /

-15:0, 30–72 4.0–11.0 T, St MK-7 22 + E, Ov, C, S ND + + 0–5 2 i

Sulfobacillus k acids

-alicyclic

35–50 1.5–2.5 ND ND 6 ND O, S + − ND ND − ω

Thermobacillus j -17:0 16:0, ai

-16:0, 55–63 6.5–8.5 − E T, St 0–3 MK-7 2 − + − + i

Ureibacillus i -15:0, -17:0 i i

-16:0, 7.0–8.0 − 50–55 S T, St 0–5 MK-7 6 − + − + i

Caldibacillus f ) −

50–70 7.0–9.0 V( Ov T, C + − 0–2.5 MK-7 1 − + 16:0

Geobacillus ,f e, -16:0, -17:0 − − i i / / -15:0, 55–65 6.5–7.0 + E, C T, St 0–5 MK-7 21 4 + + + i

)

Brevibacillus − h -5:0 i / + -15:0 − i or -15:0, /

7.0 V( E ND 30–48 23 − + + + 0–4 MK-7 ai

Aneurinibacillus h -16:0 16:0, i

-15:0, 7.0 + E ND 37–55 8 − + − + 2–5 MK-7 i

Parageobacillus ,f g f, e, − 17:0 / 15:0, 16:0, 17:0, + i- i- i- ai- 37–80 6.0–9.0 4 − + E, C, Ov T, St, C + V, + 0–3 MK-7 16:0,

)

Bacillus − d − / / + − + /

7.0–9.5 379 7 V( E, C, S, B V + + 15–55 V MK-7 V, V

Aeribacillus c -17:0 -15:0, 16:0, i ai -15:0,

2 − + Ov T, St − + 30–70 5.0–10.5 0–1.5 MK-7 − ai

Caldalkalibacillus b -15:0, -17:0 ai i

-15:0, 2 − + S T + − 42–64 6.4–9.7 0–6 ND − i

Thermolongibacillus a -17:0 16:0, i + -15:0, 2 − + E, Ov T + W 5.0–11.0 0–5 − 40–70 MK-7 i Salient characteristics of the genera comprising thermophilic, endospore-forming rods † C) ∘ growth ( quinine fatty acids Species* Subspecies Shape** Position Swelling Characteristics Number of Gram reaction Spore Anaerobic Catalase Temperature pH NaCl (%) Main isoprenoid Major cellular TABLE 2.

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(f)

Anoxybacillus l -16:0, iso i ; ALs, and Ls

17:00 37.5–53.5 94.3–99.7 DPG, PE, PG,

Sulfobacillus k Pikuta et al. (2000). (l) Nazina et al. (2001); 46–57 95.2 ND

(e) Thermobacillus j -17:0, anteiso-C 57.5 ND ND ai ;

s; GL, glycolipids; L, lipids; APGL,

Ureibacillus i 15:00 98

36–41.5 > ND

Caldibacillus f -15:0, anteiso-C Claus and Berkeley (1986); ai GLs, and PLs ; (d) 49.9 − PE, APGL, Golovacheva and Karavaiko (1978); 17:00 (k)

,Sporeposition;T,Terminal;St,Subterminal;V,Variable;DPG,

†

Geobacillus ,f e, -17:0, iso-C i ; and AL1 (PAL, APGL, AL2 or GL1, and PL1-2) 99.3

42–55 DPG, PG, PE, ≥ 16:00

Brevibacillus h Touzel et al. (2000); 93.2 43–57 ND >

; 16:0, C Miñana-Galbis et al. (2010);

(j)

Aneurinibacillus (c) h 15:00 41–43 98.6 ND

-15:0, iso-C

i Parageobacillus ,f g f, e, Fortina et al. (2001); and AL1 98.7

(i)

42.1–44.4 DPG, PG, PE, <

Bacillus d 32–69 ND ND

Zhao et al. (2006), Zhao et al. (2008);

Aeribacillus c (b) Shida et al. (1996); (h) PGL, and GLs

39–41 DPG, PG, −

Caldalkalibacillus b ND − 45

Cihan et al. (2014);

Thermolongibacillus a (a) Aliyu et al. (2016); (g) and PL (PN and AL) DPG, PG, PE, 97.5 43.5–44.8 (continued) a, f C + . The dominant fatty acid and polar lipids were underlined if any. rRNA gene similarities (%) content (mol%) 16:00 Characteristics Polar lipids Intragenic 16S DNA G C diphosphatidylglycerol; PG, phosphatidylglycerol; PE,aminophosphoglycolipid; phosphatidylethanolamine; PAL, PL, phosphoaminolipid; PGL, phospholipids; phosphoglycolipids; PN, aminophospholipids; AL, aminolipid *Including synonyms; **Spore shape; E, Ellipsoidal; S, Spherical; Ov, Oval; C, Circular; B, Banana shaped; R, Round; Thedataweretakenfrom: TABLE 2. Coorevits et al. (2012);

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38∘38′N). Currently, no other strains related to the genus Within the family of Bacillaceae, T. altinsuensis and T. kozaklien- Thermolongibacillus are available on public databases based on sis show the lowest similarity to Natribacillus halophilus DSM 16S rRNA sequence similarity, indicating that this might be a 21771T with 84.9% and 83.2% similarities, respectively. rare taxon in the environment. Fingerprinting analyzes, screening the whole genome by PCRs for repetitive extragenic palindromic (rep) elements

Enrichments and isolation procedures such as BOX- and (GTG)5-PCR or internal transcribed sequences (ITS) between 16S and 23S rRNA genes, are The strains of Thermolongibacillus were isolated from hot present (Cihan et al., 2014). On the basis of the cumulative spring environments using an enrichment technique. They cluster analysis including three of these BOX-, (GTG)5-, and were isolated from collected samples on MI agar plates at ITS-PCR reactions, T. kozakliensis and T. altinsuensis cannot 60∘C with 24 h of aerobic incubation after an enrichment be differentiated from each other by means of their ITS culturing in MI broth containing 1% (w/v) soluble starch at fingerprinting profiles, but the rep-PCR patterns of the two 60∘C for 24 h with constant shaking at 250 rpm. Thermolongibacillus species are distinctive not only from each other, but also from the other related thermophilic Bacil- Maintenance procedures laceae members. In contrast to the members of the other thermophilic genera Aeribacillus and Caldibacillus, the species T. kozakliensis and T. altinsuensis can be maintained on trypti- from the genera Geobacillus (AL1 with PAL, APGL, AL2 or case soy agar, MI agar, or nutrient agar containing 3% agar GL1, and PL1-2) and Anoxybacillus (ALs and Ls) contained or in liquid cultures at 40–70∘C under aerobic conditions. major DPG, PG, and PE polar lipids in common with Ther- After 24 h of incubation on agar plates, sporulated cultures molongibacillus (PL, PN, and AL), but they all varied in other can be stored at +4∘C for more than 6 months. Strains can minor polar lipid contents. also be lyophilized or cryoprotected in trypticase soy broth or In addition, members from the genera Aeribacillus, MI broth cultures supplemented with 20% (w/v) glycerol at Aneurinibacillus, Anoxybacillus, Caldalkalibacillus, Caldibacillus, −80∘C for long-term maintenance. and Geobacillus contain iso-C15:0 as the major iso-branched fatty acid, but species belonging to the genus Thermolon- Procedures for testing special characteristics gibacillus are unique as their cell membranes contain very high amount of iso-branched iso-C15:0 and iso-C17:0 fatty acids, In the members of this genus, the colony morphology and cell exceeding 70% of their total fatty acid profiles. structure changes depend on the culturing conditions such Another distinctive characteristic for the members of as incubation time and the media used. Formation of long the genus Thermolongibacillus is the tendency to form long μ chains (9.0–35.0 μm in length) after a prolonged incubation chains of cell morphologies up to 35 m that can be observed between the late exponential and death growth phases as period is the discriminative feature for the members of the summarized in Table 2. genus Thermolongibacillus (Figure 1). Differentiation of the species within the genus Differentiation of the genus Thermolongibacillus Thermolongibacillus from other genera The two known members of this genus, T. kozakliensis and The phylogenetic analysis of the 16S rRNA gene sequences T. altinsuensis, share 97.3% 16S rRNA gene sequence iden- between the genus Thermolongibacillus and the other 74 tity. They display high sequence similarities to some other genera within the family Bacillaceae revealed that Thermolon- thermophilic endospore-forming species from the genera gibacillus is phylogenetically most related to five thermophilic Caldibacillus (<92.0%), Geobacillus (<93.9), and Parageobacil- endospore-forming genera belonging to Aeribacillus, Anoxy- lus (<96.2). In addition to a low 16S rRNA gene sequence bacillus, Caldibacillus, Geobacillus, and Parageobacillus,and identity, DNA–DNA hybridization analysis is also used when formed a subcluster with Caldibacillus, Geobacillus,andPa- differentiating closely related species (Stackebrandt et al., rageobacillus genera among these thermophilic members 2002). The DNA reassociation value between these two (Figure 2). The 16S rRNA gene sequences of T. altin- Thermolongibacillus speciesis55± 4.7% (mean ± SD), a value suensis and T. kozakliensis have highest sequence similarity below the ad hoc committee recommended 70% threshold (96.1–94.2%) to species from the genus Parageobacillus. to 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. kozakliensis 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 were conducted using MEGA X software in MS Windows environment (Tamura et al., 2004). Bar indicates 0.05 substitutions per nucleotide position. Escherichia coli ATCC 11775T is used as an outgroup in the tree. (Adapted from Saitou and Nei (1987) and Tamura et al. (2004).) 85% Alkalilactibacillus ikkensis DSM 19937 (EU281853) 88% Natronobacillus azotifigens 24KS-1 (EU143681) Paraliobacillus ryukyuensis DSM 15140 (AB087828) 87% 88% Saliterribacillus persicus DSM 27696 (QPJJ01000028) 88% Amphibacillus xylanus DSM 6626 (AP012050)

88% Pelagirhabdus alkalitolerans S5 (jgi.1102424) 88% Halolactibacillus halophilus DSM 17073 (jgi.1085781) 86% Streptohalobacillus salinus DSM 22440 (FJ746578) Gracilibacillus halotolerans DSM 11805 (AF036922) 88% 85% Pseudogracilibacillus auburnensis DSM 28556 (KJ490639) 88% Sinibacillus soli GD05 (KC404830) 87% Jilinibacillus soli 12 (HQ693527) Cerasibacillus quisquiliarum DSM 15825 (AB107894) 88% 88% Lentibacillus salicampi DSM 16425 (AY057394) 87% Virgibacillus pantothenticus DSM 26 (LGTO01000003) 88% Oceanobacillus iheyensis DSM 14371 (BA000028) 88% Ornithinibacillus bavariensis DSM 15681 (Y13066) 88% 81% Paucisalibacillus globulus DSM 18846 (AM114102) Aquibacillus halophilus B6B (HQ433456) 84% Sediminibacillus halophilus DSM 18088 (AM905297) Terribacillus saccharophilus DSM 21619 (AB243845) 88% Pontibacillus chungwhensis DSM 16287 (AY553296) 88% 84% Salinibacillus aidingensis 25-7 (AY321436) 86% Salirhabdus euzebyi DSM 19612 (AM292417) MeIghiribacillus thermohalophilus DSM 25894 (KC845574) 88% DSM 16966 (AJ717299) 88% Thalassobacillus devorans 88% Halobacillus halophilus DSM 2266 (HE717023) 87% Salimicrobium album DSM 20748 (X90834) Allobacillus halotolerans B3A (FJ347755) Halalkalibacillus halophilus DSM 18494 (AB264529) 84% Alkalibacillus haloalkaliphilus DSM 5271 (AJ238041) 87% DSM 21622 (BBCD01000052) 83% Piscibacillus salipiscarius DSM 13259 (AJ238042) 88% Filobacillus milosensis 91% 87% Aquisalibacillus elongatus DSM 18090 (AM911047) 86% Tenuibacillus multivorans CGMCC 1.3442 (jgi.1076194) 85% Anaerobacillus arseniciselenatis DSM 15340 (AJ865469) 86% Fermentibacillus polygoni IEB3 (LC054227) 85% Desertibacillus haloalkaliphilus KJ1-10-99 (KC989945) 85% Polygonibacilllus indicireducens In2-9 (LC054224) 84% Paralkalibacillus indicireducens Bps-1 (LC197841) 86% Fictibacillus barbaricus DSM 14730 (AJ422145) Swionibacillus sediminis BW11-2 (KY635836) 87% Compostibacillus humi DX-3 (JX274434) 91% 87% 88% Pueribacillus theae T8 (MG725951) Aureibacillus halotolerans DSM 28697 (KJ620986) 87% Falsibacillus pallidus DSM 25281 (QQAY01000036) 88% Pradoshia eiseniae EAG3 (PKOZ01000039) 87% Bacillus subtilis subsp. subtilis DSM 10 (ABQL01000001) DSM 25058 (HE577175) 87% Domibacillus robiginosus 87% Jeotgalibacillus alimentarius DSM 18867 (JXRQ01000005) Camelliibacillus cellulosilyticus THG-YT1 (MG786604) 81% Salisediminibacterium halotolerans DSM 26530 (EU581836) Texcoconibacillus texcoconensis DSM 24696 (JN571119) 85% 86% Geomicrobium halophilum DSM 21769 (AB449106) 89% 87% Natribacillus halophilus DSM 21771 (AB449109) 85% Salsuginibacillus kocurii DSM 18087 (AM492160) 88% Alkalicoccus halolimnae DSM 29191 (KX618877) 88% 88% Salipaludibacillus aurantiacus S9 (FOGT01000034) Thalassorhabdus alkalitolerans G27 (MF781072) DSM 20408 (NPFA01000042) 88% 87% Marinococcus halophilus Sinobaca qinghaiensis DSM 17008 (DQ168584) 88% Aliibacillus thermotolerans DSM 101851 (KT999394) 88% Alteribacillus bidgolensis DSM-25260 (jgi.1071278) 86% Salibacterium halotolerans S7 (LN812017) 83% 86% Anoxybacillus pushchinoensis DSM 12423 (jgi.1042845) Aeribacillus pallidus DSM 3670 (CP017703) Caldibacillus debilis DSM 16016 (AJ564616) 88% 81% Thermolongibacillus altinsuensis DSM 24979 (FJ429590) 87% Thermolongibacillus kozakliensis DSM 24978 (FJ430056) 85% Parageobacillus caldoxylosilyticus DSM 12041 (BAWO01000028) 87% Parageobacillus toebil DSM 14590 (BDAQ01000034) 87% Parageobacillus thermoglucosidasius DSM 2542 (BAWP01000055) 87% Geobacillus stearothermophilus DSM 22 (AB271757) 83% Caldalkalibacillus thermarum HA6 (AY753654) Microaerobacter geothermalis DSM 22679 (FN552009) 87% Desulfuribacillus alkaliarsenatis DSM 24608 (HM046584) 87% Tepidibacillus fermentans DSM 23802 (KC242245) 86% Vulcanibacillus modesticaldus DSM 14931 (AM050346) Escherichia coli ATCC 11775 (X80725) 0.050

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G + C contents for these two species are also different: 44.8 as a subcluster with these five thermophilic genera in the mol% for T. kozakliensis and 43.5 mol% for T. altinsuensis depicted Bacillaceae phylogenetic tree, and show 97.3% 16S (Cihan et al., 2014). rRNA gene sequence homology only to each other. T. altin-

These two species also show distinct (GTG)5-PCR and suensis has 96.1% sequence similarity to Parageobacillus toebii

BOX-PCR patterns. In (GTG)5-PCR analysis, three discrim- DSM 14590, 95.2% to Parageobacillus caldoxylosilyticus, 94.8% inative DNA bands of 2,641, 2,034, and 1,338 bp are only to Parageobacillus thermoglucosidasius DSM 2542T, 93.8% to found in T. kozakliensis,whereasT. altinsuensis has only Geobacillus stearothermophilus DSM22T, 93.5% to Aeribacillus one differentiative DNA band of 1,360 bp, not present in pallidus DSM 3670T, and 91.2% to Caldibacillus debilis DSM T. kozakliensis. Similarly, in BOX-PCR analyses, a DNA band of 16016T. T. kozakliensis’s similarities to these species are 94.2, 1,670 bp for T. kozakliensis and DNA bands of 648 and 490 bp 93.4, 93.5, 91.9, 91.4, and 89.8%, respectively. for T. altinsuensis are discriminative. However, ITS-PCR cannot be used as these two species have similar ITS fin- List of species of the genus Thermolongibacillus gerprinting profiles (Cihan et al., 2014; Koc et al., 2015). Thermolongibacillus species show variations in their spore Thermolongibacillus altinsuensis shapes as well as in cell and colony morphologies and minor Cihan et al. (2014)VP differences in their cell membrane and genomic contents as ...... displayed in Table 1. Some physiological features can also be al.tin.su.en’sis. N.L. masc. adj. altinsuensis pertaining to used to differentiate these species (Table 1). T. kozakliensis the isolation habitat, Altinsu hot spring located in Kozakli is oxidase-negative and has a lower salt tolerance (0–1.5% Province of Nevsehir in the Middle Anatolian Region of NaCl) compared to T. altinsuensis (0–5.0%). There are also Turkey (Cappadocia area). variations in their growth optima. The optimum growth tem- Cells are Gram-positive, motile, straight rods, and occur perature for T. kozakliensis is 60∘C and that of T. altinsuensis singly. The cell size varies, depending on the culture age. is 55∘C. Similarly, although the pH growth range for these During the early- and mid-logarithmic phase, cells were two species is the same for T. kozakliensis and T. altinsuensis, single and 0.7–1.1 by 3.5–8.0 μm in size; between stationary their optimum pH values are 9.0 and 8.5, respectively. Other and death phases, cells form chains and become strikingly metabolic functions in their carbon and energy metabolism longer 0.8–1.2 by 9.0–35.0 μm. Free spores are 1.5–2 μmin can also be used as differentiating factors as detailed in size, and they vary from ellipsoidal to oval. Colony morphol- Table 1. T. kozakliensis is negative for urea utilization, whereas ogy changes with the incubation period. Actively growing T. altinsuensis can utilize urea. On Sabouraud dextrose, colonies are ellipsoidal, light yellow, flat with undulate edges, T. kozakliensis can be grown, whereas, T. altinsuensis cannot be nonmucoid, with a rough surface, and are 2–6 × 4–10 mm. grown. These two species also display variations in their acid Aged colonies are circular, cream in color, 2–3 mm in diam- production capabilities when supplied with different sugars eter, and convex with entire edges with a smooth surface. (Table 1). Thermophilic. The optimum growth temperature is 55∘C ∘ Taxonomic comments with 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% The phylogenetic relationship between the two species of (w/v). Weakly positive for catalase and oxidase. Cells are the genus Thermolongibacillus and all the members from the chemoorganotrophs and capable of growing on a wide other related genera belonging to the family Bacillaceae is range of carbohydrates. Can utilize casein and urea and given in Figure 2, based on their 16S rRNA gene sequences. give a positive methyl red test. Positive for nitrate to nitrite At least one type species from the 74 genera within the reduction, but cannot produce gas from nitrate. Negative family Bacillaceae was included (a total of 78 species) to for starch, citrate, tyrosine, and gelatin utilization; growth the phylogenetic analysis. Genus Thermolongibacillus is most on Sabouraud dextrose; Voges–Proskauer test; and indole related with the five thermophilic endospore-forming gen- and H2S production. Amylase-, α-glucosidase-, protease-, and era according to their 16S rRNA gene sequences including lipase-negative. Harbors a single plasmid that gives a band ≤ ≤ Parageobacillus ( 96.1), Geobacillus ( 95.0), Anoxybacillus at 15.5 kb. Predominant fatty acids are iso-C15:0 (63.94%), ≤ ≤ ≤ ( 94.8), Aeribacillus ( 93.5%), and Caldibacillus ( 91.2). iso-C17:0,andC16:0. Cell membrane contains the polar lipids T. altinsuensis and T. kozakliensis display lower similarities to DPG, PG, PE, and two PLs. The major menaquinone is MK-7 all the other type strains within the Bacillaceae family, branch in addition to minor amounts of MK-6, MK-5, and MK-8.

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The type strain is Thermolongibacillus altinsuensis (E265 = 11B4240003. The authors are also grateful to Salih Gökhan DSM 24979 = NCIMB 14850), isolated from a sediment sam- Çöleri for his contributions to the sample collection studies ple of Altinsu hot spring in Kozakli province of Nevsehir, in harsh thermal environments. Turkey (Cappadocia Area). DNA G + C content (mol%): 43.5 (HPLC). Type strain: E265 = DSM 24979 = NCIMB 14850.

Thermolongibacillus kozakliensis References Cihan et al. (2014)VP ...... Aliyu H, Lebre P, Blom J, Cowan D, & De Maayer P (2016) ko.zak.li.en’sis. N.L. masc. adj. kozakliensis referring to the Phylogenomic re-assessment of the thermophilic genus isolation habitat, Kozakli Municipality hot spring located in Geobacillus. Syst Appl Microbiol 39: 527–533. Kozakli Province of Nevsehir in the Middle Anatolian Region Cihan AC, Koc M, Ozcan B, Tekin N, & Cokmus C (2014) of Turkey (Cappadocia area). Thermolongibacillus altinsuensis gen. nov., sp. nov. and Ther- Cells are Gram-positive, motile, straight, slim rods, gener- molongibacillus kozakliensis sp. nov., aerobic, thermophilic, ally occurring singly. In the early logarithmic growth phase, long bacilli isolated from hot springs. Int J Syst Evol Micro- cells occur singly, approximately 0.6–1.0 by 3.0–8.0 mm. biol 64: 187–197. Form long chains and elongate to 0.6–1.1 by 9.0–32.0 mm between stationary and death phases. Free spores are ellip- Claus D & Berkeley CW (1986) Genus Bacillus Cohn 1872. soidal and 1.5–2 mm in length. Colonies are cream color, In Bergey’s Manual of Systematic Bacteriology,vol.2,PHA opaque, circular and convex, nonmucoid, have smooth sur- Sneath, NS Mair, ME Sharpe, & JG Holt (Eds). Williams faces with regular margins along their edges, and 1–3 mm & Wilkins, Baltimore; pp 1105–1139. in diameter. Thermophilic. Optimum growth tempera- Coorevits A, Dinsdale AE, Halket G, Lebbe L, de Vos P, van ∘ ∘ ture is 60 C with a growth range of 40–70 C; pH at 9.0 Landschoot A et al. (2012) Taxonomic revision of the with a range of 5.0–11.0; and at 0.5% (w/v) NaCl with genus Geobacillus: Emendation of Geobacillus, G. stearother- a range of 0–1.5% (w/v). Catalase-weakly positive and mophilus, G. jurassicus, G. toebii, G. thermodenitrificans and oxidase-negative. Cells are chemoorganotrophs, capable of G. thermoglucosidans (nom. corrig., formerly ’thermoglucosi- growing on a wide range of carbohydrates. Can utilize casein, dasius’); transfer of Bacillus thermantarcticus. Int J Syst Evol grow on Sabouraud dextrose, and positive for methyl red Microbiol 62: 1470–1485. test. Amylase-, α-glucosidase-, protease-, and lipase-negative. Fortina MG, Pukall R, Schumann P, Mora D, Parini C, Man- Positive for nitrate to nitrite reduction, but cannot produce achini PL et al. (2001) Ureibacillus gen. nov., a new genus gas from nitrate. Negative for starch, citrate, tyrosine, gelatin, to accommodate Bacillus thermosphaericus (Andersson and urea utilization; Voges–Proskauer test; and indole et al. 1995), emendation of Ureibacillus thermosphaericus and H2S production. Hydrolyzes sugars. Harbors a single plasmid that gives a band at 14.5 kb. Contains major fatty and description of Ureibacillus terrenus sp. nov. Int J Syst Evol Microbiol 51: 447–455. acids of iso-C15:0 (60.68%), iso-C17:0,andC16:0. The major menaquinone is MK-7. Predominant polar lipids are DPG, Golovacheva RS & Karavaiko GI (1978) A new genus of ther- PG, PE, and two PLs with minor amounts of PN and two ALs. mophilic spore-forming bacteria. Sulfobacillus Microbiol ThetypestrainisThermolongibacillus kozakliensis (E173a = (English Transl Mikrobiol) 47: 658–664. DSM 24978 = NCIMB 14849), isolated from a soil sample of DSMZ (2019) Nomenclature of Peptidoglycan Structures Kozakli Municipality Thermal hot spring located in Kozakli of Cross-Linkage Types A and B. German Collection of Province of Nevsehir, Turkey (Cappadocia Area). Microorganisms and Cell Cultures: Peptidoglycan. https:// DNA G + C content (mol%): 44.8 (HPLC). www.dsmz.de/collection/catalogue/microorganisms/ Type strain: E173a = DSM 24978 = NCIMB 14849. special-groups-of-organisms/peptidoglycans (accessed 30 June 2019). Acknowledgments Koc M, Cokmus C, & Cihan AC (2015) The genotypic diver- This research was supported by the Scientific Research sity and lipase production of some thermophilic bacilli Project Office of Ankara University with the project number from different genera. Braz J Microbiol 46: 1065–1076.

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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. Queries in Chapter 1 Q1. Please check and confirm whether the identified layout is fine for Table2.

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