Fodinicola Acaciae Sp. Nov., an Endophytic Actinomycete Isolated from the Roots of Acacia Mangium Willd

Fodinicola acaciae sp. nov., an endophytic actinomycete isolated from the roots of Acacia mangium Willd. and its genome analysis

Huyền Thị Thanh Phạm1,2, Wipawadee Suwannapan1, Wilaiwan Koomsiri1,2, Yuki

Inahashi,3 Akira Také,4 Atsuko Matsumoto,3 Arinthip Thamchaipenet1,2*

1Department of Genetics, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900,

Thailand

2Omics Center for Agriculture, Bioresources, Food and Health, Kasetsart University (OmiKU),

Bangkok 10900, Thailand

3Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-

8641, Japan

4Department of Microbiology, Kitasato University School of Medicine, 1-5-1 Kitasato, Minami- ku, Sagamihara, Kanagawa 252-0374, Japan

*Author for correspondence:

Arinthip Thamchaipenet

Tel: +66-2-562-5444 ext. 646703; Fax: +66-2-579-5528; E-mail: [email protected]

Table S1. Cultural characteristics of strain GKU 173T and Fodinicola feengrottensis HKI 0501T at 28 oC for 21 days

Media Characteristics GKU 173T HKI 0501T ISP 2 Growth Abundant Poor Aerial mycelium Greyish red, white Orange Substrate mycelium Wrinkled, brownish red Orange Soluble pigment Deep red None ISP 3 Growth Abundant Good Aerial mycelium White White, pale orange Substrate mycelium Pastel red Wrinkled, orange Soluble pigment Greyish red None ISP 4 Growth Abundant, flat Moderate Aerial mycelium White Pale orange Substrate mycelium Light orange Wrinkled, pale orange Soluble pigment None (clear zone) None ISP 5 Growth Abundant, flat Poor Aerial mycelium White Sparse white, orange Substrate mycelium Pale orange to pastel red Pale orange Soluble pigment None none ISP 6 Growth Good Moderate Aerial mycelium Creamy, pale orange Creamy, light yellow Substrate mycelium Wrinkled, pale orange Wrinkled, light orange Soluble pigment None None ISP 7 Growth Abundant, flat Poor Aerial mycelium White Orange Substrate mycelium Light orange to pale orange Pale orange Soluble pigment None None Bennett’s agar Growth Abundant Abundant Aerial mycelium White with brownish gray spots Pale orange Substrate mycelium Wrinkled, deep red Wrinkled, pale orange Soluble pigment Deep red None Organic Growth Abundant Good medium 79 Aerial mycelium Short white, pastel red Pale orange, creamy Substrate mycelium Wrinkled, orange-red to high red Wrinkled, pale orange Soluble pigment High red None MS Growth Abundant Abundant Aerial mycelium White Pale orange Substrate mycelium Violet Brown Pale orange Soluble pigment Violet Brown None

(a) (b)

Figure S1. PGP-traits of strain GKU 173T. (a) Phosphate solubilization on Pikowskaya’s agar supplemented with 2 % tricalcium phosphate. (b) Siderophore production on CAS agar.

T 99 Frankia asymbiotica M16386 (KY744730) 99 Frankia saprophytica CN3T (MF960832) Frankia inefficax EuI1cT (KX695197) Frankia canadensis ARgP5T (L40612) Frankia elaeagni BMG5.12T (KB893676) Frankia discariae BCU110501T (KB891152) 70 Frankia irregularis G2T (MH145366) Frankia casuarinae CcI3T (CP000249) 56 Frankia alni ACN14AT (CT573213) 67 83 Frankia torreyi CpI1T (GU296536) Frankia coriariae BMG5.1T (JWIO01000013) 79 Acidothermus cellulolyticus ATCC 43068T (AJ007290) Jatrophihabitans fulvus PB158T (KF056992) Jatrophihabitans endophyticus S9-650T (JQ346802) 99 Jatrophihabitans telluris N237T (LC323105) 51 96 Jatrophihabitans soli KIS75-12T (KP017569) 96 Jatrophihabitans huperziae CPCC 204076T (KR184574) Jiangella alkaliphila DSM 45079T (LT629791) Jiangella mangrovi 3SM4-07T (AB976540) Jiangella rhizosphaerae NEAU-YY265T (MH144586) 97 100 Jiangella anatolica GTF31T (POTW01000167) Jiangella gansuensis DSM 44835T (KI911782) Jiangella endophytica KE2-3T (LC032128) Jiangella alba DSM 45237T (FJ157186) Jiangella muralis DSM 45357T (LFXL01000071) GKU 173T 92 Fodinicola feengrottensis HKI 0501T (EF490376) Cryptosporangium aurantiacum DSM 46144T (jgi.1107629) Cryptosporangium mongoliense MN08-A0264T (AB531472) Cryptosporangium japonicum IFO 15966T (D85466) T 62 Cryptosporangium phraense A-T 5661 (MH071352) T 64 Cryptosporangium arvum KK073874 (KK073874) Cryptosporangium cibodasense LIPI11-2-Ac046T (LC025527) Cryptosporangium eucalypti EURKPP3H10T (LC085570) Cryptosporangium minutisporangium IFO 15962T (AB037007) Streptosporangium roseum DSM43021T (X89947)

0.01

Figure S2. Maximum-likelihood phylogenetic tree on the basis of 16S rRNA gene sequences of strain GKU 173T and related species. Streptosporangium roseum DSM43021T was used as an outgroup. Numbers at nodes refer to bootstrap values (based on 1000 replicates; only values >50

% are shown). Bar, 0.01 nucleotide substitutions per site.

T 99 Frankia asymbiotica M16386 (KY744730) 99 Frankia saprophytica CN3T (MF960832) Frankia inefficax EuI1cT (KX695197) Frankia discariae BCU110501T (KB891152) T 77 Frankia elaeagni BMG5.12 (KB893676) Frankia irregularis G2T (MH145366) 62 Frankia canadensis ARgP5T (L40612) T 79 Frankia alni ACN14A (CT573213) Frankia torreyi CpI1T (GU296536) 56 Frankia casuarinae CcI3T (CP000249) Frankia coriariae BMG5.1T (JWIO01000013) 70 Acidothermus cellulolyticus ATCC 43068T (AJ007290) Jatrophihabitans fulvus PB158T (KF056992) Jatrophihabitans endophyticus S9-650T (JQ346802) 99 Jatrophihabitans telluris N237T (LC323105) 61 90 T 60 Jatrophihabitans soli KIS75-12 (KP017569) 97 Jatrophihabitans huperziae CPCC 204076T (KR184574) Jiangella alkaliphila DSM 45079T (LT629791) 56 Jiangella mangrovi 3SM4-07T (AB976540) Jiangella rhizosphaerae NEAU-YY265T (MH144586) 98 100 Jiangella gansuensis DSM 44835T (KI911782) Jiangella anatolica GTF31T (POTW01000167) Jiangella endophytica KE2-3T (LC032128) Jiangella alba DSM 45237T (FJ157186) 78 Jiangella muralis DSM 45357T (LFXL01000071) GKU 173T 95 Fodinicola feengrottensis HKI 0501T (EF490376) Cryptosporangium mongoliense MN08-A0264T (AB531472) Cryptosporangium aurantiacum DSM 46144T (jgi.1107629) 83 Cryptosporangium cibodasense LIPI11-2-Ac046T (LC025527) T 58 Cryptosporangium japonicum IFO 15966 (D85466) Cryptosporangium arvum KK073874T (KK073874) 64 Cryptosporangium eucalypti EURKPP3H10T (LC085570) Cryptosporangium minutisporangium IFO 15962T (AB037007) Cryptosporangium phraense A-T 5661T (MH071352) Streptosporangium roseum DSM43021T (X89947)

Figure S3. Maximum-parsimony phylogenetic tree on the basis of 16S rRNA gene sequences of strain GKU 173T and related species. Streptosporangium roseum DSM43021T was used as an outgroup. Numbers at nodes refer to bootstrap values (based on 1000 replicates; only values >50

% are shown).

L-lys GlcN Mur DAP GKU 173T Gly D-Glu D-Ala Ile

Figure S4. Analysis of the cell wall peptidoglycan by one dimensional TLC on cellulose plate.

Abbreviation: D-ala, D-alanine, DAP, diaminopimelic acid, GlcN, glucosamine, D-glu, D- glutamic acid, Gly, glycine, Ile, isoleucine, L-lys, L-lysine, Mur, muramic acid

a b

DPG DPG

PE dimension

2 2 Unknown PC

PI PI

st st 1 dimension 1 dimension

c d

dimension

2 2

st st 1 dimension 1 dimension

Figure S5. Phospholipids analysis separated by two-dimensional TLC. First and second dimension conditions are chloroform:methanol:water (65:25:4) and chloroform:acetic acid:methanol:water (40:7.5:6:2), respectively. (a) Phospholipid standard sprayed with phosphomolybdic acid reagent, (b) phospholipids of strain GKU 173T sprayed with phosphomolybdic acid reagent, (c) PE standard sprayed with ninhydrin reagent, (d) phospholipids of strain GKU 173T sprayed with ninhydrin reagent. Abbreviation: DPG, diphosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol 1 2 3

Figure S6. Phospholipids analysis separated by one-dimensional TLC and sprayed with dragendorff reagent. Lane 1, phosphatidylcholine (PC) standard; Lane 2, mixture of PC and phospholipids of strain GKU 173T; Lane 3, phospholipids of strain GKU 173T.