Curr Microbiol DOI 10.1007/s00284-015-0820-z

Leucobacter humi sp. nov., Isolated from Forest Soil Jihee Her1 • Sang-Seob Lee1

Received: 7 January 2015 / Accepted: 1 March 2015 Ó Springer Science+Business Media New York 2015

Abstract A novel bacterial strain, designated Re6T, was isolated from forest soil collected in Campbell University, North Carolina. The cells are aerobic, Gram-positive, nonmotile, and rod shaped. Growth occurred at 4–42 °C (optimum, 25 °C), pH 6–9 (optimum, pH 6), and in 0–3 % NaCl (w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Re6T belonged to the genus Leucobacter and showed the highest 16S rRNA gene sequence similarities with Leucobacter iarius JCM 14736T (98.3 %), Leucobacter luti JCM 14920T (97.9 %), Leucobacter komagatae JCM 9414T (97.8 %), and Leucobacter denitrificans KACC 14055T (97.7 %). Strain Re6T contained anteiso-C15:0 (45.2 %), iso-C16:0 (17.1 %), and anteiso-C17:0 (32.6 %) as the major cellular fatty acids; MK-11 as the major respiratory quinone; L-diaminobutyric acid as the diagnostic diamine acid in cell wall peptidoglycan; and diphosphatidylglycerol and phosphatidylglycerol as the main polar lipids. The DNA G?C content of the strain Re6T was 66.6 mol%. DNA–DNA hybridization results showed similarity values less than 50 % for DNA samples from the most closely related type strains of L. iarius, L. luti, and L. komagatae. On the basis of the data from the polyphasic analysis, strain Re6T is considered to The NCBI GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Re6T is KC818288.

Electronic supplementary material The online version of this article (doi:10.1007/s00284-015-0820-z) contains supplementary material, which is available to authorized users. & Sang-Seob Lee [email protected] 1

Department of Life Science, Graduate School of Kyonggi University, 94-6 Iui-dong Yeongtong-gu, Suwon 433-760, Republic of Korea

be a representative novel species in the genus Leucobacter, for which the name Leucobacter humi sp. nov. is proposed. The type strain is Re6T (KEMC 7301-006T = JCM 18638T).

Introduction The Genus Leucobacter belonged to family Microbacteriaceae was described aerobic, Gram-positive, non-motile, non-sporulating, and rod-shape bacteria with 2, 4-diaminobutyric acid (DAB) in their cell wall peptidoglycan. The species of genus Leucobacter have been isolated from many kinds of ecological niche such as soil (Leucobacter albus) [13], air of a duck barn (Leucobacter aerolatus) [15], bamboo extract (Leucobacter margaritifomis) [12], activated sludge (Leucobacter luti) [19], river sediment (Leucobacter alluvii) [19], a traditional Korean fermented seafood made of flatfish gajami-sikhae (Leucobacter celer) [24], dye waste water (Leucobacter kyeonggiensis) [8], chromium contaminated environment (Leucobacter chromiireducens and Leucobacter aridicollis) [18], and chironomid egg mass that was sampled in a waste stabilization pond in northern Israel (Leucobacter chironomii) [6]. In this study, we have isolated an aerobic, Gram-positive, non-motile, and rod-shaped bacterial strain Re6T from soil collected from forest soil in Campbell University (GPS: N 35° 240 3200 , W 78° 440 4600 ), North Carolina. On the basis of 16S rRNA gene sequence analysis, strain Re6T was considered to be a Leucobacter-like strain. Strain Re6T was subjected to polyphasic taxonomic investigation based on morphological, physiological, and chemotaxonomic characteristics. The results indicated that the strain Re6T

123

J. Her, S.-S. Lee: Leucobacter humi sp. nov., Isolated from Forest Soil

should be designated as a novel species in the genus Leucobacter.

Materials and Methods Isolation of Bacterial Strain and Culture Conditions Strain Re6T was originally isolated from forest soil collected in Campbell University, North Carolina. 1 g of the soil was inoculated with 100 ml of 1/10 diluted nutrient broth (Difco) and incubated aerobically in the shaking incubator (150 rpm, 28 °C) for 3 days. After the enrichment, 100 ll of the aliquot was spread onto a 1/10 diluted nutrient agar (NA, Difco) plate and incubated at 28 °C for 2 days. Single colonies on these plates were selected by transferring them onto new plates of half-strength TSA (Difco) agar (pH 5.0) and subjecting them to an additional incubation for 3 days at 20 °C. The purified cells were tentatively identified using the partial 16S rRNA gene sequences using the EzTaxon-e server (http://eztaxon-e. ezbiocloud.net/) [9]. The reference type strains, Leucobacter iarius JCM14736T, L. luti JCM14920T, Leucobacter komagatae JCM9414T, Leucobacter denitrificans KACC14055T, Leucobacter chromiiresistens JCM17813T, L. aridicollis JCM13321T, L. alluvii JCM14919T, L. albus JCM12888T, L. aerolatus JCM17972T, and Leucobacter chiromiireducens subsp. chromiireducens JCM13222T were obtained from the Korean Agricultural Culture Collection (KACC), the Korean Collection for Type Cultures (KCTC), and Korea Environmental Microorganisms Collection (KEMC). All the strains were maintained and cultivated on NA plates, aerobically, unless otherwise mentioned. Phenotypic and Biochemical Characteristics Cell morphology and motility were observed with a Nikon light microscope (91000 magnification), with the cells being allowed to grow for 2 days at 28 °C, on nutrient agar. Gram reaction was performed according to the classic Gram procedure described by Doetsch [2]. Growth at different temperatures (4, 10, 25, 37, and 42 °C) was assessed on nutrient broth (Difco) for 3 days. Growth at various pH levels (4, 5, 6, 7, 8, and 9) was assessed in nutrient broth at 25 °C. The API 20NE, API ID32GN, and API ZYM microtest systems were employed according to the recommendations of the manufacturer (bioMe´rieux) for studying carbon source utilization and the enzyme activities of the strains.

123

16S rRNA Sequencing and Phylogenetic Analysis The 16S rRNA gene of strain Re6T was amplified using genomic DNA using the universal bacterial primer set, 27F (50 -AGAGTTTGATCMTGGCTCAG-3) and 1492R (50 TACGGYTACCTTGTTACGACTT-30 ) [28]. The full sequence of the 16S rRNA gene was compiled with SeqMan software (DNASTAR Inc.). The 16S rRNA gene sequences of related taxa were obtained from GenBank and edited with the BioEdit program [5]. Multiple alignments were performed with the CLUSTAL X program [27]. Pairwise distances for the neighbor-joining algorithm [21] were calculated according to Kimura two-parameter model [7], and the phylogenetic tree was constructed in the MEGA 6 program [11]. Bootstrap analysis with 1000 replicates was also conducted to obtain confidence levels for the branches [4]. Min-mini heuristic method with a search factor of one was applied in maximum-parsimony analysis in the MEGA 6 program, and maximum-likelihood analysis was performed with phylip 3.69. Chemotaxonomic Characteristics In order to perform the fatty acid methyl ester analysis, cells were allowed to grow on TSA for 2 days at 28 °C, and then two 10 ll blue loops of well-grown cells were harvested. Fatty acid methyl esters were prepared, separated, and identified with the Sherlock Microbial Identification System (TSBA6, Version 6.0; MIDI), produced by MIDI, Inc., Newark, DE, USA [22]. Polar lipids were extracted according to the procedures described by Minnikin [16] and identified by two-dimensional TLC followed by spraying with appropriate detection reagents. For TLC development, first mobile phase was chloroform/ methanol/water (65:25:4, v/v/v) and second mobile phase was chloroform/methanol/acetic acid/water (80:12:15:4, v/v/v/v). Total lipid profile was detected by spraying with molybdophosphoric acid solution (Sigma-Aldrich; St. Louis, Mo) followed by heating at 150 °C; aminolipids (AL) by spraying with 0.2 % (w/v) ninhydrin solution followed by heating at 105 °C for 10 min. Isoprenoid quinone was extracted with chloroform/methanol (2:1, v/v), purified via thin-layer chromatography, evaporated under vacuum conditions, and re-extracted in n-hexane:water (1:1 v/v). The crude n-hexane quinone solution was purified and subsequently analyzed by HPLC, as previously described [1]. The cell wall composition was analyzed using thin-layer chromatography (TLC) on cellulose sheets [10, 23] instead of paper chromatography. The standard amino acids such as 2,4-diaminobutyric acid

J. Her, S.-S. Lee: Leucobacter humi sp. nov., Isolated from Forest Soil Table 1 Comparison of phenotypic characters between strain Re6T and the type strains of closely related Leucobacter species Characteristic

1

2

3

4

5

6

7

8

9

10

11

Colony color

White, creamy

White

Cream

Whitish brown

White

Yellow

Cream

Cream

White

White, shiny

Yellow

Colony shape

Circular

Circular

Circular

Circular

Circular

Irregular

Round

Circular

Smooth

Smooth

Circular, smooth, glistening,

Growth Temperature (optimum) pH range (optimum)

25

30

25

30

30

30

28

28

30

30

25–32

6

7

8

7

6

7–

7–8

8

7

8

7 ?

Enzyme activity (API ZYM) Alkaline phosphatase

w

-

?

w

-

-

?

-

?

-

Esterase (C4)

w

-

?

?

-

?

-

?

?

w

-

Esterase (C8)

?

-

w

?

?

w

-

-

w

?

?

Lipase (C14)

w

-

-

-

?

-

?

-

?

-

? ?

Leucine arylamidase

?

w

?

?

-

-

-

?

?

?

Valine arylamidase

w

-

-

w

?

?

w

-

-

?

-

Cystine arylamidase Trypsin

w -

w -

?

w -

w -

? ?

-

?

-

? -

?

Acid phosphatase

?

?

?

?

w

?

?

-

?

-

-

Naphtol-AS-BIphosphohydrolase

?

?

?

w

w

-

?

?

w

?

?

b-Glucosidase

?

?

-

-

-

-

?

-

?

-

-

Assimilation (API ID32GN & 20 NE) Nitrate reduction to N2

-

-

-

-

?

-

?

-

?

?

-

Production of acid from glucose

-

-

?

?

-

?

?

-

?

-

-

Arginine dihydrolase

-

?

?

-

w

?

-

-

-

?

w

b-Glucosidase (Esculin hydrolysis)

?

?

w

?

-

?

w

-

?

-

?

Protease (gelatin hydrolysis)

-

-

-

?

-

?

?

w

?

-

-

b-Galactosidase (PNPG)

-

-

?

-

-

-

?

-

-

?

w

D-Glucose

w

-

?

?

-

-

-

-

?

w

?

D-Mannitol

-

-

?

-

?

w

?

-

-

?

?

N-acetyl-Dglucosamine

?

?

-

-

?

-

-

-

?

-

-

D-Maltose

-

-

-

?

?

w

-

w

-

?

-

Gluconate

-

-

-

-

-

-

?

-

?

-

?

Caprate

-

-

?

-

?

-

-

-

-

?

-

Citrate

?

?

?

w

?

?

-

-

?

-

-

L-Rhamnose

-

?

-

-

-

-

-

-

?

?

-

N-acetyl-Dglucosamine

?

?

-

-

-

-

?

?

-

?

-

D-Ribose

?

-

-

w

-

?

?

?

-

?

-

myo-Inositol

?

-

-

-

-

-

-

?

?

?

-

Acetate

?

?

?

?

-

?

-

-

-

-

-

D, L-Lactate

?

-

? ?

-

-

-

? ?

? -

? w

? ?

? ?

L-Alanine

123

J. Her, S.-S. Lee: Leucobacter humi sp. nov., Isolated from Forest Soil Table 1 continued Characteristic

1

2

3

4

5

6

7

8

9

10

11 -

3-Hydroxybenzoate

?

?

-

w

-

w

-

w

-

-

D-Glucose

w

-

?

-

-

-

-

-

w

?

-

Salicin D-Sorbitol

? -

?

-

w

-

-

?

w

?

-

-

Citrate

?

-

?

-

-

-

w

?

?

-

-

L-Histidine

?

?

?

?

?

w

?

?

-

?

?

4-Hydroxybenzoate

-

-

?

w

-

w

?

?

?

-

-

L-Proline

?

?

?

?

-

?

-

?

w

?

?

Assimilation (API 50CH) Glycerol

w

?

-

w

-

-

?

w

?

-

?

Erythritol

-

?

-

-

-

?

?

-

-

?

?

D-Arabinose

-

-

-

-

-

-

?

-

-

?

-

L-Arabinose

-

-

-

?

-

-

?

-

?

-

-

D-Ribose

w

?

-

-

-

-

-

-

?

?

-

D-Galactose

-

-

-

-

-

w

-

-

-

?

-

D-Glucose

-

-

?

?

-

?

-

-

-

?

-

D-Fructose

?

?

-

-

-

-

?

-

-

-

-

D-Mannose L-Sorbose

-

w w

-

-

-

? ?

? ?

-

-

w

-

L-Rhamnose

-

?

?

-

-

-

-

-

-

?

-

Dulcitol

-

w

?

-

-

?

-

-

?

-

Inositol

-

?

?

-

-

-

-

-

-

?

-

N-acetylglucosamine

?

?

-

-

-

-

-

-

-

?

-

Arbutin

-

-

?

-

-

?

?

-

?

-

-

Esculin ferric citrate

?

?

?

-

-

?

-

-

-

?

-

Salicin

-

w

-

-

-

-

-

-

-

-

-

Glycogen

-

-

?

w

-

?

?

-

?

?

-

Xylitol

-

?

-

-

-

?

-

-

-

-

-

5-Ketogluconate (potassium)

w

?

-

-

?

-

-

-

-

?

?

Taxa: 1, strain Re6T; 2, Leucobacter iarius JCM14736T; 3, Leucobacter luti JCM14920T; 4, Leucobacter komagatae JCM9414T; 5, Leucobacter denitrificans M1T8B10T; 6, Leucobacter chromiiresistens JCM17813T; 7, Leucobacter aridicollis JCM13321T; 8, Leucobacter alluvii JCM14919T; 9, Leucobacter albus JCM12888T; 10, Leucobacter aerolatus JCM17972T; 11, Leucobacter chiromiireducens subsp. chromiireducens JCM13222T All the data were obtained in this study. All the strains are Gram-positive, aerobic, non-spore forming, and rod-shaped bacterium. ? positive reaction, - negative reaction, w weakly positive reaction

(L-DAB), L-alanine, D-alanine, D-serine, D-glutamic acid, glycine, D-glucosamine, L-aspartic acid, and D-aspartic acid were used to compare the amino acid composition. DNA Base Content and DNA–DNA Hybridization For the determination of the DNA G?C content, the genomic DNA of the strain Re-6T was extracted by following Marmur method [14], and concentration of

123

extracted DNA was over 1000 ng/ml. After extracting, DNA was enzymatically degraded into nucleosides. The nucleosides were analyzed by HPLC as described previously [16, 17]. DNA–DNA hybridization between strain Re6T and type strains of the genus Leucobacter were estimated by hybridization at 51.6 °C with photobiotin-labeled DNA probes [3] and carried out reciprocally with five replications per sample.

J. Her, S.-S. Lee: Leucobacter humi sp. nov., Isolated from Forest Soil Microbacterium esteraromaticum DSM 8609T (Y17231) 53

Microbacterium suwonense M1T8B9 T (GQ246683) Microbacterium arabinogalactanolyticum IFO 14344 T (AB004715) Microbacterium testaceum DSM 20166T (X77445)

99

Microbacterium resistens DMMZ 1710 T (Y14699) Microbacterium oleivorans DSM 16091 T (AJ698725)

67

Microbacterium schleiferi IFO 15075 T (AB004723)

98

Microbacterium hydrocarbonoxydans DSM 16089 T (AJ698726) Microbacterium phyllosphaerae DSM 13468 T (AJ277840) Microbacterium paraoxydans CF36T (AJ491806) Microbacterium luteolum IFO 15074 T (AB004718)

73

Microbacterium saperdae IFO 15038 T (AB004719)

84

Microbacterium oxydans DSM 20578 T (Y17227) 85

Microbacterium liquefaciens DSM 20638 T (X77444) Leucobacter kyeonggiensis F3-P9T (JQ039895)

83 67

Leucobacter celer NAL101T (GQ504012) Leucobacter chironomi DSM 19883 T (ATXU01000010)

66

Leucobacter chromiiresistens JG 31T (AGCW01000231) 100

Leucobacter alluvii RB10T (AM072820)

Leucobacter denitrificans M1T8B10 T (GQ246672) Leucobacter komagatae IFO 15245 T (AB007419)

55

Leucobacter aridicollis CIP 108388 T (AJ781047)

90 66

Leucobacter albus IAM 14851 T (AB012594)

Leucobacter humi Re 6T (KC818288) 57 100

Leucobacter iarius 40T (AM040493) Leucobacter luti RF6T (AM072819) Leucobacter margaritiformis A23T (JN038197) Leucobacter tardus K 70/01T (AM940158) Leucobacter chromiireducens subsp. chromiireducens L-1T (AJ781046)

100

Leucobacter chromiireducens subsp. solipictus TAN 31504 T (DQ845457) Leucobacter exalbidus K-540BT (AB514037) 97 78

Leucobacter aerolatus Sj10T (FN597581) Leucobacter salsicius M1-8T (AOCN01000008) Arthrobacter globiformis NBRC 12137 T (M23411)

0.01

Fig. 1 Phylogenetic tree of strains Re6T and related Leucobacter type strains based on 16S rRNA gene sequence comparisons. Neighbor-joining method conducted using the software BioEdit was

used. The sequences used for the comparative study are included in parentheses. Bar 1 % sequence dissimilarity. Arthrobacter globiformis NBRC 12137T was used as an out-group

Results and Discussion

Cells are Gram-positive, aerobic, non-motile, non-sporulating, and rod-shaped bacterium. Growth was observed at 4–42 °C (optimum at 25 °C) but only weakly grown at 10 and 42 °C. Strain Re6T was grown in the range of pH 6–9 with optimal growth at pH 6 and tolerance up to 3 % of NaCl (w/v), with optimal growth at 0 % NaCl (w/v).

Morphological and Physiological Characteristics Strain Re6T cultured on nutrient agar plate (Difco) at 28 °C was pale white colored, circular, and convex colonies.

123

J. Her, S.-S. Lee: Leucobacter humi sp. nov., Isolated from Forest Soil Table 2 Cellular fatty acid profiles of strain Re6T and related Leucobacter species

Fatty acids

1

2

3

4

5

6

7

8

9

-

-

10

11

Tr

Tr

Straight-chain 14:0 16:0

Tr 2.2

-

2.1

0.1

0.35

Tr

4.6

2.22

8.0

1.94

1.2

3.70

10.80

2.1

5.6

6.8

4.3

Saturated 14:0 iso 15:0 iso

Tr

Tr

Tr

1.6

3.7

15:0-Anteiso

45.2

54.7

16:0 iso

17.1

14.5

17:0 iso

Tr

Tr

-

17:0-Anteiso

32.6

24.6

19.2

T

Tr

Tr

Tr

Tr

1.0

Tr

1.8

Tr

Tr

11.4

6.0

9.7

-

-

63.2

49.51

40.8

52.8

58.6

59.0

56.8

58.7

9.2

15.45

15.1

14.2

11.8

11.7

23.2

20.1

14.0

1.6

2.7

Tr

-

Tr

-

-

Tr

17.57

25.2

18.3

17.5

25.5

13.6

11.1

26.5

T

1.0 53.2

T

Taxa: 1, strain Re6 ; 2, Leucobacter iarius JCM14736 ; 3, Leucobacter luti JCM14920 ; 4, Leucobacter komagatae JCM9414T; 5, Leucobacter denitrificans M1T8B10T; 6, Leucobacter chromiiresistens JCM17813T; 7, Leucobacter aridicollis JCM13321T; 8, Leucobacter alluvii JCM14919T; 9, Leucobacter albus JCM12888T; 10, Leucobacter aerolatus JCM17972T; 11, Leucobacter chiromiireducens subsp. chromiireducens JCM13222T All data were obtained in this study. Cells were grown on TSA for 48 h at 28 °C. - not detected. Tr, trace less than 1.0 %

Phylogenetic Analysis The 16S rRNA gene sequence of strain Re6T was found to be a continuous stretch of 1452 nucleotides. According to the stretch analyzed, strain Re6T was determined to belong to the phylum Actinobacteria, the subclass Actinobacteridae, the suborder Micrococcineae, and the family Microbacteriaceae. The highest degrees of sequence similarities were found to be with L. iarius 40T (98.3 %), L. luti RF6T (97.9 %), and L. komagatae IFO 15245T (97.8 %). The other Leucobacter species showed 16S rRNA gene similarities to strain Re6T by 97.70–94.12 %. In the phylogenetic tree (Fig. 1), strain Re6T clearly belonged to the Leucobacter lineage, as evidenced by the high bootstrap value of 100. The result of phylogenetic tree using maximum-parsimony analysis and maximum-likelihood analysis also indicated that strain Re6T belonged to the genus Leucobacter lineage. Chemotaxonomic Characteristics Fig. 2 Two-dimensional thin-layer chromatogram of the polar lipids of strain Re6T. TLC plate stained with 5 % ethanolic molybdophosphoric acid. Abbreviations: DPG diphosphatidylglycerol, PE phosphatidylethanolamine, PG phosphatidylglycerol, L unknown lipid, L1–2 unknown amino lipids

Results regarding the physiological characteristics of strain Re6T are summarized in the species description, and the comparison of selective characteristics with related type strains is shown in Table 1.

123

The cellular fatty acid profiles of strain Re6T and the related Leucobacter type strains are shown in Table 2. The major cellular fatty acids of strain Re6T included anteisoC15:0 (45.2 %), iso-C16:0 (17.1 %), and anteiso- C17:0 (32.64 %), whereas the minor fatty acids were iso-C17:0 (0.48 %), iso-C15:0 (1.55 %), C16:0 (2.16 %), iso-C14:0 (0.65 %), and C14:0 (0.19 %). No significant differences in fatty acid profiles were found between different species in

J. Her, S.-S. Lee: Leucobacter humi sp. nov., Isolated from Forest Soil

the genus Leucobacter. The phenotypic and chemotaxonomic characters which differentiate strain Re6T from other members of genus Leucobacter are listed in Table 1. Strain Re6T showed the major amounts of phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) (Fig. 2) similar to other members of the Leucobacter [13, 15, 18, 19, 20, 25, 26]. It also contains five known AL. The major quinone of strain Re6T was menaquione MK-11, and a minor amount of MK-10 was also detected like in other species of Leucobacter. Genomic Characteristics The G?C content of the genomic DNA of the strain Re6T was 66.6 %. Strain Re6T demonstrated relatively low values of DNA–DNA relatedness with related species and detailed values of DNA–DNA hybridization are shown in Supplementary Table S1. These results indicated that strain Re6T does not belong to any known species of Leucobacter [28]. Taxonomic Conclusion The results of phenotypic and chemotaxonomic analysis clearly distinguishes the novel strain as a novel species within the genus Leucobacter, for which the name Leucobacter humi sp. nov. is proposed.

In the API ID32GN and 20NE strip, acetate, N-acetyl-Dglucosamine, myo-inositol, L-proline, D-ribose, L-alanine, 3-hydroxybenzoate, D-glucose, salicin, propionate, caprate, n-valerate, citrate, L-histidine are assimilated, but 2-ketogluconate (a), D, L-3-hydroxybutyrate, 4-hydroxybenzoic acid, L-arabinose, D-sorbitol, L-fucose, D-melibiose, Dmannitol, L-serine, glycogen, 5-ketogluconate, D, L-lactate, malonate, suberate, itaconate, D-maltose, D-sucrose, and Lrhamnose are not assimilated. The predominant quinone was MK11 and major cellular fatty acids were iso-C16:0, anteiso-C15:0, and anteisoC17:0. The cell wall amino acids were L-DAB, L-alanine, glycine, and D-glutamic acid. The polar lipid profile was phosphatidylglycerol (PG), diph osphatidylglycerol (DPG), and 5 kinds of unknown AL. DNA G?C content was 66.6 % as determined by HPLC. The type strain, Re6T (KEMC 7301-006T = JCM 18638T), was isolated from surface soil of Campbell University forest, North Carolina, in the United states. Acknowledgments This work was supported by Kyonggi University‘s Graduate Research Assistantship 2015, Korea national Environmental Microorganisms Bank (2010-0007473). This research was supported by a Grant (14CTAP-C078666-01) from infrastructure and transportation technology promotion research Program funded by Ministry of Land, Infrastructure, and Transport of Korean government.

References Description of Leucobacter humi sp. nov Leucobacter humi (L. gen. n. humi, of Earth, Soil) Cells were found to be Gram-positive, aerobic, non-sporulating, non-motile, and rod shape after they were grown on nutrient agar (MB cell) at 28 °C for 3 days. The colonies grown on a nutrient agar plate for 2 days are pale white colored, circular, and convex. Growth occurred in the range of 25–37 °C but was weak at 4 and 42 °C but the optimum growth temperature was 25 °C. Tolerance to NaCl was observed up to 3 % with optimal growth at 0 % NaCl (w/v). This strain also can grow in the pH range of 6-9 with optimal growth at pH 6. In the API ZYM and API 20NE strips, enzyme activities are positive for acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase (C4), esterase (C8), gelatinase, b-glucosidase (esculin hydrolysis), Valine arylamidase, Naphtol-AS-BI-Phosphohydrolase, lipase (C14), and leucine arylamidase. Enzyme activities are negative for arginine dihydrolase, N–acetyl-b-glucosaminidase, arginine dihydrolase, a-chymotrypsin, a-fucosidase, a-galactosidase, b-galactosidase (ONPG), b-galactosidase (PNPG), b-glucuronidase, a-mannosidase, trypsin, and urease.

1. Collins MD, Jones D (1981) Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol Rev 45:316–354 2. Doetsch RN (1981) Determinative methods of light microscopy. In: Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR, Phillips GH (eds) Manual of methods for general bacteriology. American Society for Microbiology, Washington, pp 21–33 3. Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric DNA– DNA hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Evol Microbiol 39:224–229 4. Felsenstein J (1985) Confidence limit on phylogenies: an approach using the bootstrap. Evolution 39:783–791 5. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98 6. Halpern M, Shaked T, Pukall R et al (2009) Leucobacter chironomi sp. nov., a chromate-resistant bacterium isolated from a chironomid egg mass. Int J Syst Evol Microbiol 59:665–670 7. Kimura M (1983) The neutral theory of molecular evolution. Cambridge University Press, Cambridge 8. Kim HJ, Lee SS (2011) Leucobacter kyeonggiensis sp. nov., a new species isolated from dye waste water. J Microbiol 49(6):1044–1049 9. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH et al (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721

123

J. Her, S.-S. Lee: Leucobacter humi sp. nov., Isolated from Forest Soil 10. Komagata K, Suzuki K (1987) Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19:161–207 11. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163 12. Lee JH, Lee SS (2012) Leucobacter margaritiformis sp. nov., isolated from bamboo extract. Curr Microbiol 64(5):441–448 13. Lin YC, Uemori K, de Briel DA et al (2004) Zimmermannella helvola gen. nov., sp. nov., Zimmermannella alba sp. nov., Zimmermannella bifida sp. nov., Zimmermannella faecalis sp. nov. and Leucobacter albus sp. nov., novel members of the family Microbacteriaceae. Int J Syst Evol Microbiol 54:1669–1676 14. Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro- organisms. J Mol Biol 8:201–218 15. Martin E, Lodders N, Ja¨ckel U, Schumann P, Ka¨mpfer P (2010) Leucobacter aerolatus sp. nov., from the air of a duck barn. Int J Syst Evol Microbiol 60(12):2838–2842 16. Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G?C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167 17. Minnikin DE, O’Donnell AG, Goodfellow M et al (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241 18. Morais PV, Francisco R, Branco R et al (2004) Leucobacter chromiireducens sp. nov, and Leucobacter aridicollis sp. nov., two new species isolated from a chromium contaminated environment. Syst Appl Microbiol 27:646–652 19. Morais PV, Paulo C, Francisco R et al (2006) Leucobacter luti sp. nov., and Leucobacter alluvii sp. nov., two new species of the

123

20.

21.

22. 23.

24.

25.

26.

27.

28.

genus Leucobacter isolated under chromium stress. Syst Appl Microbiol 29:414–421 Muir RE, Tan MW (2007) Leucobacter chromiireducens subsp. solipictus subsp. nov., a pigmented bacterium isolated from the nematode Caenorhabditis elegans, and emended description of L. chromiireducens. Int J Syst Evol Microbiol 57:2770–2776 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sasser M (1990). Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark: MIDI Inc Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477 Shin NR, Kim MS, Jung MJ et al (2011) Leucobacter celer sp. nov., isolated from Korean fermented seafood. Int J Syst Evol Microbiol 61:2353–2357 Somvanshi VS, Lang E, Schumann P et al (2007) Leucobacter iarius sp. nov., in the family Microbacteriaceae. Int J Syst Evol Microbiol 57:682–686 Takeuchi M, Weiss N, Schumann P et al (1996) Leucobacter komagatae gen. nov., sp. nov., a new aerobic Gram-positive, nonsporulating rod with 2,4-diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46:967–971 Thompson JD, Gibson TJ, Plewniak F et al (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882 Weisburg WG, Barns SM, Pelletier DA et al (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703

Leucobacter humi sp. nov., Isolated from Forest Soil.

A novel bacterial strain, designated Re6(T), was isolated from forest soil collected in Campbell University, North Carolina. The cells are aerobic, Gr...
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