TAXONOMIC DESCRIPTION Dunlap et al., Int J Syst Evol Microbiol 2017;67:2720–2725 DOI 10.1099/ijsem.0.002007

Bacillus swezeyi sp. nov. and haynesii sp. nov., isolated from desert soil

Christopher A. Dunlap,1,* David A. Schisler,1 Elizabeth B. Perry,2† Nora Connor,2‡ Frederick M. Cohan2 and Alejandro P. Rooney1

Abstract Two isolates of Gram-reaction-positive, facultatively anaerobic, motile, rod-shaped, endospore-forming were identified during a survey of the diversity of strains belonging to the genus Bacillus deposited in the Agriculture Research Service Culture Collection. These strains were originally isolated from soil in Evolution Canyon III (Israel) in a survey of ecological diversification. Phylogenetic analysis of the 16S rRNA gene of strains NRRL B-41294T and NRRL B-41327T determined they were closely related to members of the Bacillus licheniformis clade. The genome of each strain was sequenced, and further analysis indicated that the strains represented unique species based on in silico DNA–DNA hybridization analyses. A phylogenomic analysis revealed that NRRL B-41294T and NRRL B-41327T were closely related to the group that includes B. licheniformis. In phenotypic characterization, both NRRL B-41294T and NRRL B-41327T were found to grow at temperatures of between 15 and 60 C and tolerated up to 12 % NaCl (w/v). The predominant cellular fatty acids

were anteiso-C15 : 0 and iso-C15 : 0, and peptidoglycan from cell walls contained meso-diaminopimelic acid. The DNA G+C content was 45.7 and 44.3 mol% for NRRL B-41327T and NRRL B-41294T, respectively. Furthermore, each strain had a unique carbon utilization pattern that distinguished it from its nearest phylogenetic neighbours. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that these strains represent two novel species within the genus Bacillus, for which the name Bacillus swezeyi sp. nov. is proposed, with type strain NRRL B-41294T (=CCUG 70177T), and the name Bacillus haynesii sp. nov. is proposed, with type strain NRRL B-41327T (=CCUG 70178T).

During a survey of the biodiversity of the strains belonging authors or any culture collection [6]. Regardless, the 16S to the genus Bacillus in the Agricultural Research Service rRNA gene sequence of strain NRRL B-41327T was found Culture Collection (NRRL), we identified two strains to share the highest identity (99.7 %) with the type strains of (NRRL B-41327T and NRRL B-41294T) that appeared to be B. sonorensis (99.7 %) and B. paralicheniformis (99.7 %), and phylogenetically unique, based on 16S rRNA gene sequen- strain NRRL B-41294T was found to share similarly high ces. As part of a study incorporating ecology into bacterial levels of nucleotide sequence identity with the type strains systematics [1], these strains were isolated from separate of B. sonorensis (99.2 %) and B. licheniformis (99.1 %). – soil samples (1 3 cm depth) taken in Evolution Canyon III, Because past studies of species of the genus Bacillus have located in the southern Negev Desert, at Nahal Shaharut, shown that multilocus sequence analysis (MLSA) [7] and Isreal. Both strains shared a close relationship with strains phylogenomic analysis [4, 8–13] can better differentiate spe- in the Bacillus licheniformis clade, which includes B. licheni- cies at the molecular level than analysis of 16S rRNA gene formis [2], Bacillus paralicheniformis [3], Bacillus glycinifer- sequence data alone, we produced draft genome sequences mentans [4] and Bacillus sonorensis [5]. Although it is also a of NRRL B-41327T and NRRL B-41294T to confirm their member of this clade, Bacillus aerius was not included since taxonomic identities. DNA–DNA relatedness (in silico) the type strain is no longer available from the original between these strains and the type strains of the nearest

Author affiliations: 1United States Department of Agriculture, Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, Peoria, IL, USA; 2Department of Biology, Wesleyan University, Middletown, CT, USA. *Correspondence: Christopher A. Dunlap, [email protected] Keywords: desert; soil. Abbreviation: DDH, DNA-DNA hybridization. †Present address: Department of Biostatistics, Yale University, New Haven, CT, USA. ‡Present address: Department of Computer Science, University of Colorado, USA. The GenBank/EMBL/DDBJ accession numbers for the draft genome sequences of Bacillus haynesii NRRL B-41327T and Bacillus swezeyi NRRL B- 41294T are MRBL00000000 and MRBK00000000, respectively. The accession numbers for the 16S rRNA gene sequences of Bacillus haynesii NRRL B-41327T and Bacillus swezeyi NRRL B-41294T are MRBL01000076 and MRBK01000096, respectively. Two supplementary figures are available with the online Supplementary Material.

002007 Downloaded from www.microbiologyresearch.org by IP: 52.0.234.912720 On: Wed, 17 Jul 2019 07:41:21 Dunlap et al., Int J Syst Evol Microbiol 2017;67:2720–2725 phylogenetic neighbours showed that these strains represent Becton Dickinson) diluted 1 : 9 with water containing 50 mg À1 two novel species in this clade. We subsequently character- MnSO4 l using cells grown in 10 ml of broth in 50 ml con- ized these strains using a polyphasic taxonomic approach ical tubes incubated at 28 C and 200 r.p.m. for 48 h. Cells and compared them with the type strains of closely related containing endospores were heat fixed and observed using species of the genus Bacillus. On the basis of the data gath- phase contrast microscopy. Strain NRRL B-41294T pro- ered in this study, strains NRRL B-41294T and NRRL B- duced central–paracentral, ellipsoidal endospores with 41327T should be recognized as representatives of two novel unswollen sporangia (Fig. S1, available in the online Supple- species of the genus Bacillus, for which the names Bacillus mentary Material). Colonies formed on TGY were creamy swezeyi sp. nov. and Bacillus haynesii sp. nov. are proposed. white, mucoid, translucent and raised, and were 3–4 mm in Below we describe the results of our analyses. diameter after 2 days of incubation at 37 C. Rods were found to be 0.8±0.1 µm in diameter and 2.8±0.4 µm in Strains NRRL B-41294T and NRRL B-41327T grew well on length when cultured in 1/10 TSB. Strain NRRL B-41327T nutrient agar (NA; Difco), Luria–Bertani agar (LB; Difco), produced central-paracentral, ellipsoidal endospores with trypticase soy agar (TSA; Difco), Reasoner’s 2A agar (R2A)  unswollen sporangia (Fig. S2). Colonies formed on TGY and tryptone glucose yeast extract agar (TGY) at 30 C. We were creamy white, mucoid, translucent and raised, and tested the temperature range for growth at 4, 10, 15, 25, 30,  were 3–4 mm in diameter after 2 days of incubation at 37, 50, 55, 60 and 65 C on R2A agar by allowing plates to  37 C. Rods measured 0.7±0.1 µm in diameter and 2.1 incubate for up to 14 days. The pH range for growth was ±0.2 µm in length when cultured in 1/10 TSB. determined from pH 3.0 to pH 11.0 for 3 days in TGY broth buffered with acetate buffer (pH 3.0–6.0), Tris/HCl buffer Carbon source utilization was tested using the OmniLog (pH 7.0–9.0) or carbonate buffer (pH 10.0–11.0), and Data Collection system (Biolog). Strains NRRL B-41327T adjusted with HCl or NaOH [14], at 28 C. NaCl tolerance and NRRL B-41294T were cultured overnight on Biolog uni- was investigated by using TGY broth supplemented with 0– versal growth plates and prepared according to the manu- 20 % (w/v) NaCl, in 2 % increments, at 28 C for 3 days. facturer’s instructions for the GEN III MicroPlate test panel Both strains were able to grow on NA, LB, R2A, TSA and using protocol A (Biolog) at 33 C. An OmniLog Data TGY agar. Both strains grew at a pH range of 5.0–10.0 and a Collection instrument (Biolog) was used to collect data in temperature range of 15–60 C. They grew with a range of 15 min increments for 22 h. The experiment was conducted NaCl concentrations from 0 to 12 % (w/v). The optimum in triplicate. The metabolic characteristics of the two novel temperature and pH for growth were 37 C and pH 7.0, strains and the closely related strains provided some distin- respectively, based on optical densities. Growth under guishing phenotypes (Table 1). Cells of strains NRRL B- anaerobic conditions was determined on anaerobic agar 41294T and NRRL B-41327T could be distinguished from (Difco) at 30 C using a GasPak jar (Merck) in an atmo- closely related strains based on carbon utilization assays. In sphere of <1 % O2, 13 % CO2 and 86 % N2 for 7 days [15]. traditional metabolic assays, the two strains utilized citrate Both strains were able to grow under these conditions and, and hydrolysed starch, gelatin and casein. They produced therefore, are considered facultative anaerobes. Spore acid from glucose, but no gas. Catalase and oxidase activities morphology was determined in tryptic soy broth (TSB; were examined using 3 % (v/v) hydrogen peroxide solution

Table 1. Summary of distinguishing metabolic phenotypic properties of B. swezeyi sp. nov. and B. haynesii sp. nov. Strains: 1, B. swezeyi sp. nov. NRRL B-41294T; 2, B. haynesii sp. nov. NRRL B-41327T; 3, B. glycinifermentans GO-13T; 4, B. sonorensis NRRL B-23154T; 5, B. licheniformis ATCC 14580T; 6, B. paralicheniformis KJ-16T.

Biolog GEN III 1 2 3 4 5 6

Stachyose À + ÀÀÀÀ Raffinose À + ÀÀÀÀ Melibiose À + ÀÀÀÀ D-Galactose + + À + ÀÀ D-Sorbitol + + À + + + D-Gluconic acid + + À + + + myo-Inositol + + + + ÀÀ L-Alanine + + À + À + D-Galacturonic acid À + À + ÀÀ Mucic acid + + À + ÀÀ D-Saccharic acid + ÀÀ + ÀÀ Formic acid À + ÀÀÀÀ Lincomycin + ÀÀÀÀ + Nalidixic acid ÀÀÀ + ÀÀ

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Table 2. Fatty acid profiles of B. swezeyi sp. nov., B. haynesii sp. nov. and closely related type strains Strains: 1, B. haynesii sp. nov. NRRL B-41327T; 2, B. swezeyi sp. nov. NRRL B-41294T; 3, B. paralicheniformis KJ-16T; 4, B. licheniformis ATCC 14580T; 5, B. sonorensis NRRL B-23154T; 6, B. glycinifermentans GO-13T. Only fatty acids representing greater than 0.6 % of the total fatty acids are shown.

Fatty acid 1 2 3 4 5 6

iso-C14 : 0 1.5 1.1 0.6 0.9 0.8 0.5

iso-C15 : 0 35.8 34.5 31.5 29.2 36.2 28.9

anteiso-C15 : 0 41.6 40.1 37.7 39.7 38.9 36.9

iso-C16 : 0 2.7 2.6 2.7 3.6 2.4 3 – C16 : 1!11c 1.3 1.3 0.6 0.6 0.6

C16 : 0 2.3 3.3 5.3 3.5 3.3 4.1 – iso-C17 : 1!10c 1.3 1.2 0.4 0.5 0.4

iso-C17 : 0 5.7 6.3 8. 9 8.2 7.1 11.1

anteiso-C17 : 0 7.8 8.3 11.3 12.5 8.9 14.7 and 1 % (w/v) tetramethyl-p-phenylenediamine dihydro- protocol for extraction and production of fatty acid methyl chloride (Difco), respectively. Both strains were catalase esters. The total cellular fatty acid profiles showed a large positive and oxidase negative. amount of branched fatty acids; the major components (>5.0 %) were anteiso-C , iso-C , iso-C , anteiso- Next, we characterized chemotaxonomic properties of the 15 : 0 15 : 0 17 : 0 C and C . The profiles of the close relatives B. two novel strains. Determination of cell-wall diamino acid 17 : 0 16 : 0 licheniformis, B. paralicheniformis, B. glycinifermentans and was performed according to the method described by Sta- B. sonorensis were included for comparison (Table 2). The neck and Roberts [16]. The cell-wall peptidoglycan of both fatty acid profiles did not appear to be distinguishing. strains contained meso-diaminopimelic acid. Total cellular fatty acid content was measured using the MIDI protocol DNA extraction for genome sequencing was performed on (Microbial Identification) and analysed on an Agilent 7890 pelleted bacterial biomass cultured in TGY liquid medium gas chromatograph. The strains were grown for 24 h at using a Qiacube instrument with a QIAmp DNA mini QIA- 28 C on TSA and prepared using the standard MIDI cube kit (Qiagen). The genomic DNA was prepared for

T 100 B. subtilis subsp. inaquosorum KCTC 13429 (AMXN01) T 0.050 100 B. subtilis subsp. spizizenii TU-B-10 (CP002905) T 100 B. subtilis subsp. subtilis 168 (AL009126) 100 T B. tequilensis KCTC 13622 (AYTO01) T 100 B. vallismortis str DV1-F-3 (AFSH01) T B. mojavensis KCTC 3706 (AFSI01) 100 T 100 B. axarquiensis NRRL B-41617 (LPVD01) T 100 B. atrophaeus NRRL NRS-213 (LSBB01) T B. nakamurai NRRL B-41091 (LSAZ01) T B. velezensis NRRL B-41580 (LLZC01) 100 T B. siamensis KCTC 13613 (AJVF01) 100 T 100 B. amyloliquefaciens DSM 7 (FN597644) T 100 B. glycinifermentans GO-13 (LECW01) T B. sonorensis KCTC 13918 (AYTN01)

T 100 B. swezeyi B-41294 (MRBK01)** T B. paralicheniformis KJ-16 (LBMN02) 100 T 100 B. licheniformis DSM 13 (CP000002) T 100 B. haynesii NRRL NRRL B-41327 (MRBL01)**

Fig. 1. Phylogenetic tree reconstructed from the core genomes of type strains of species from the B. subtilis group (350 genes). Boot- strap values >50 %, based on 1500 pseudoreplicates, are indicated at branch points. Bacillus cereus was used as an outgroup, and only the relevant part of the tree is presented. Bar, 0.05 nucleotide substitutions per site. ** Designates subject strains.

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94 B. licheniformis BL1202 (CP017247) B. licheniformis BT1BCT2 (AZSY01) 70 B. licheniformis 5NAP23 (JYBQ01) 100 B. licheniformis SRCM100115 (LYUH01) 0.02 B. licheniformis CG-B52 (AVEZ01) 74 42 B. licheniformis 3F-3 (JFYM01) 100 B. licheniformis CGMCC 3963 (AMWQ01) 100 54 B. licheniformis HRBL-15TD (CP014781) B. licheniformis VSD4 (MLKN01) T 100 B. licheniformis DSM 13 (AE017333) 28 B. licheniformis GB2 (JYGX01) B. licheniformis 10-1-A (AJLV01) 100 66 B. licheniformis LMG 17339 (AZSY01) 100 B. licheniformis 5-2-D (AJLW01) 100 B. licheniformis F1-1 (AZSL01) 100 B. licheniformis WX-02 (CP012100) 100 B. licheniformis F2-1 (AZSM01) Bacillus haynesii S 16 (AZYP01) ** 100 T Bacillus haynesii NRRL B-41327 (MRBL01)** B. paralicheniformis APC1576 (LXPD01) B. paralicheniformis LMG 6934 (AZSZ01) B. paralicheniformis ATCC 9945a (CP005965) 100100 B. paralicheniformis LMG 7559 (AZSX01) 100 NCIMB 8874 (MBGK01) 100 B. paralicheniformis T B. paralicheniformis KJ-16 (LBMN02) 100 B. paralicheniformis S127 (LFIM01) 100 100 B. paralicheniformis ATCC 12759 (JMPZ01) 100 B. paralicheniformis BL-09 (CP010524) B. paralicheniformis G-1 (AZSK01) 100 B. paralicheniformis SB47 (ATNR01) 100 B. paralicheniformis CPSM8 (ANNR01) 100 B. paralicheniformis MSP5.4 (JXAP01) T Bacillus swezeyi NRRL B-41294 (MRBK01)** 100 Bacillus swezeyi NRRL B-41282 (MTJL01)**

Fig. 2. Phylogenetic unrooted neighbour-joining tree reconstructed from the core genomes of all the genomes in Genbank closely related to B. licheniformis (2868 genes). Bootstrap values >50 %, based on 1500 pseudoreplicates, are indicated at branch points. Bar, 0.02 nucleotide substitutions per site. ** Designates subject strains. sequencing using a Nextera XT library preparation kit fol- bootstrap support for internal branches were obtained from lowing the manufacturer’s suggested protocols. The libraries 1500 pseudoreplicates. The results of the phylogenomic prepared were sequenced using a MiSeq DNA sequencer analysis using a core genome (350 genes) alignment of with the MiSeq V3 2Â300 sequencing kit. The resulting NRRL B-41327T, NRRL B-41294T and all type strains of spe- reads were quality trimmed to the Q30 confidence level. The cies in the Bacillus subtilis group are shown in Fig. 1. The draft genome was assembled using CLCbio Genomics type strains most closely related to NRRL B-41327T and Workbench 9.5 (Qiagen) using default parameters. NRRL B-41294T were B. licheniformis DSM 13T, B. parali- cheniformis KJ-16T, B. sonorensis KCTC 13918T and B. gylci- Genome comparisons and alignments for phylogenetic trees nifermentans GO-13T. To provide a more taxonomically were made using BIGSdb software [17]. The phylogenetic comprehensive analysis, all available genomes of B. licheni- tree was reconstructed using MEGA 7.09 software [18]. Neigh- formis and B. paralicheniformis were included in a second – bour-joining trees were reconstructed using the Tamura phylogenomic analysis, which yielded a core genome of 2868 Nei model [19] with a gamma correction (alpha value=0.5) genes. The results of the analysis showed that the strains with complete deletion. This model was chosen on the basis formed four distinct clades corresponding to the species B. of the likelihood test implemented in MEGA 7.09. Measures of licheniformis and B. paralicheniformis, a clade consisting of

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Table 3. Genome comparisons of B. swezeyi sp. nov. and B. haynesii sp. nov. with closely related species Regression-based pairwise DDH values are reported in the upper right half of the matrix. Average nucleotide identities are reported in the lower left half of the matrix.

Strain 1 2 3 4 5 6 7

1. B. haynesii sp. nov. NRRL B-41327T 91.5 26.7 64.4 61.6 23.8 24.5 2. B. haynesii sp. nov. S 16 99.0 26.7 64.0 61.6 24.0 24.6 3. B. swezeyi sp. nov. NRRL B-41294T 83.6 83.7 26.4 26.1 25.2 26.2 4. B. licheniformis DSM 13T 95.6 95.6 83.3 58.4 23.7 24.5 5. B. paralicheniformis KJ-16T 95.3 95.3 83.1 94.8 24.1 24.4 6. B. glycinifermentans GO-13T 81.1 83.7 82.2 81.0 81.2 29.9 7. B. sonorensis KCTC 13918T 81.5 81.4 83.1 81.8 81.6 85.5

NRRL B-41294T and a closely related strain (NRRL B- and his service to the scientific community as the 41282), and a clade consisting of strain NRRL B-41237 and a first curator of the Agricultural Research Service (NRRL) closely related strain (B. licheniformis S 16) (Fig. 2). Strain Culture Collection]. NRRL B-41282 was originally isolated from the same soil T Cells are Gram-reaction-positive, facultatively anerobic, location as NRRL B-41294 and appears to be clonal. B. motile, endospore-forming rods that arere 0.6–0.9 µm in licheniformis S 16 was originally isolated from a soil sample diameter and 1.8–2.5 µm in length when cultured in 1/10 in Pakistan, and no additional information is available. The TSB. Central–paracentral, ellipsoidal endospores are analysis also identified six B. paralicheniformis strains (LMG observed with unswollen sporangia. Colonies formed on 6934, LMG 7559, S127, SB47, CPSM8 and MSP5.4) that TGY are creamy white, mucoid, translucent, raised and are were incorrectly labelled as B. licheniformis or Bacillus sp. 3–4 mm in diameter after 2 days of incubation at 37 C. Able The B. licheniformis strain BT1BCT2 was accessioned into to grow on NA, LB, R2A, TSA and TGY agar. Cells can be GenBank as Bacillus sp. readily distinguished from those of closely related strains In silico DNA–DNA hybridization (DDH) was determined based on utilization of various carbon sources: stachyose, online at http://ggdc.dsmz.de/distcalc2.php using the raffinose, melibiose and D-galacturonic acid. Grows at pH –  Genome-to-Genome Distance Calculation (GGDC) version 5.5 10 and at temperatures of from 15 to 60 C. Has a NaCl tolerance of 0–12 % (w/v). The optimum temperature and 2.1 as described by Meier-Kolthoff et al. [20]. The estimated  in silico DDH values were calculated using formula two at pH for growth are 37 C and pH 7.0, respectively. The cells the GGDC website, originally described by Auch et al. [21] are catalase-positive and oxidase-negative. The cell-wall and updated by Meier-Kolthoff et al. [20]. Formula two of peptidoglycan contains meso-diaminopimelic acid. The pri- the GGDC is the only function appropriate to analyse draft mary fatty acids are anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0, ante- genomes, which is the case for most of the genomes in this iso-C17 : 0 and C16 : 0. Can hydrolyse starch, gelatin and study [20]. DNA relatedness of NRRL B-41327T, NRRL B- casein, and can utilize citrate. Produces acid from glucose, 41294T and their most closely related species is shown in but does not produce gas. T Table 3. DNA relatedness of NRRL B-41327 and strain S The type strain is NRRL B-41327T (=CCUG 70178T) and 16 was found to be 91.5 %, confirming that they are conspe- was isolated from soil in Evolution Canyon III in the South- cific. The in silico DDH relatedness between NRRL B- ern Negev desert in Israel. The DNA G+C content of the T 41327 and the type strains of neighbouring species varied type strain is 45.7 mol%. from 23.8 to 64.4 %, which is below the threshold of 70 % for species delineation [22]. Average nucleotide identities DESCRIPTION OF BACILLUS SWEZEYI were calculated using the EzBioCloud web service [23] and SP. NOV. are also reported in Table 3. Bacillus swezeyi [swe¢zy.i. N.L. gen. masc. n. swezeyi of Swe- Based upon the consensus of phylogenomic and phenotypic zey, named for James L. Swezey of the US Department of analyses, we conclude that strains NRRL B-41294T and T Agriculture for his contributions to bacterial taxonomy and NRRL B-41327 represent novel species within the genus his service to the scientific community as a long-time pat- Bacillus. ents curator and general collections manager of the Agricul- tural Research Service (NRRL) Culture Collection]. DESCRIPTION OF BACILLUS HAYNESII SP. NOV. Cells are Gram-reaction-positive, facultatively anerobic, motile, endospore-forming rods that are 0.7–0.9 µm in Bacillus haynesii [hay.nes¢i.i. N.L. gen. masc. n. haynesii of diameter and 2.2–3.7 µm in length when cultured in 1/10 Haynes, named for William C. Haynes of the US Depart- TSB. Central–paracentral ellipsoidal endospores are ment of Agriculture for his contributions to bacterial observed with unswollen sporangia. Colonies formed on

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TGY are creamy white, mucoid, translucent, raised and are complex and description of Bacillus subtilis subsp. inaquosorum 3–4 mm in diameter after 2 days of incubation at 37 C. Able subsp. nov. Int J Syst Evol Microbiol 2009;59:2429–2436. to grow on NA, LB, R2A, TSA and TGY agar. Cells may be 8. Dunlap CA. Phylogenomic analysis shows that ‘Bacillus vanillea’ is a later heterotypic synonym of Bacillus siamensis. Int J Syst Evol distinguished from those of closely related strains based on Microbiol 2015;65:3507–3510. utilization of various carbon sources: D-galactose, myo-ino- 9. Dunlap CA, Bowman MJ, Schisler DA, Rooney AP. Genome sitol, mucic acid and D-saccharic acid. Grows at pH 5.5–10 analysis shows Bacillus axarquiensis is not a later heterotypic  and at temperatures of from 15 to 60 C. They have a NaCl synonym of Bacillus mojavensis; reclassification of Bacillus mal- tolerance of 0–12 % (w/v). The optimum temperature and acitensis and Brevibacterium halotolerans as heterotypic syno-  nyms of Bacillus axarquiensis. Int J Syst Evol Microbiol 2016;66: pH for growth are 37 C and pH 7.0, respectively. The cells 2438–2443. are catalase-positive and oxidase-negative. The cell-wall 10. Dunlap CA, Kim SJ, Kwon SW, Rooney AP. Phylogenomic analysis peptidoglycan contains meso-diaminopimelic acid. The pri- shows that Bacillus amyloliquefaciens subsp. plantarum is a later mary fatty acids are anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0, ante- heterotypic synonym of Bacillus methylotrophicus. Int J Syst Evol – iso-C17 : 0 and C16 : 0. Can hydrolyse starch, gelatin and Microbiol 2015;65:2104 2109. casein and utilize citrate. Produces acid from glucose, but 11. Dunlap CA, Kim SJ, Kwon SW, Rooney AP. 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