SGM Journals
Actinobacteria

Leucobacter aerolatus sp. nov., from the air of a duck barn

E. Martin, N. Lodders, U. Jäckel, P. Schumann, P. Kämpfer

  • 1Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany
  • 2Bundesanstalt für Arbeitschutz und Arbeitsmedizin, D-10317 Berlin, Germany
  • 3Deutsche Sammlung von Mikroorganismen und Zellkulturen, D-38124 Braunschweig, Germany
  • Correspondence
    Peter Kämpfer
    peter.kaempfer{at}umwelt.uni-giessen.de

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2838–2842 · https://doi.org/10.1099/ijs.0.021303-0

    View at publisher PubMed

    Abstract

    A Gram-positive, non-spore-forming actinobacterium (Sj 10T) was isolated on tryptone soy agar from the air of a duck barn after filter sampling. Based on 16S rRNA gene sequence similarity studies, strain Sj 10T was shown to belong to the genus Leucobacter and was closely related to Leucobacter chromiireducens subsp. chromiireducens L-1T (97.8 %), Leucobacter tardus DSM 19811T (97.3 %) and Leucobacter luti RF6T (97.3 %). The peptidoglycan of strain Sj 10T contained 2,4-diaminobutyric acid in combination with a lower amount of lysine as diagnostic diamino acids. In addition, threonine, glycine, alanine and glutamic acid were found. Menaquinone MK-11 was the major respiratory quinone; MK-12 and MK-10 were detected in minor amounts. The polar lipid pattern consisted of phosphatidylglycerol, diphosphatidylglycerol and one unknown component each of a phospholipid, glycolipid and aminoglycolipid. Strain Sj 10T contained the major fatty acids anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0, like other members of the genus Leucobacter. Results of DNA–DNA hybridization, physiological and biochemical tests enabled strain Sj 10T to be differentiated genotypically and phenotypically from the most closely related Leucobacter species. Strain Sj 10T represents a novel species of the genus Leucobacter, for which the name Leucobacter aerolatus sp. nov. is proposed, with Sj 10T (=DSM 22806T =CCM 7705T) as the type strain.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Sj 10T is FN597581.

    • Fig. S1, which shows a two-dimensional TLC of polar lipids of strain Sj 10T, is available with the online version of this paper.

    The genus Leucobacter (family Microbacteriaceae) was proposed by Takeuchi et al. (1996) and at the time of writing comprises nine recognized species: Leucobacter komagatae (Takeuchi et al., 1996); Leucobacter chromiireducens (Morais et al., 2004; Muir & Tan, 2007); Leucobacter aridicollis (Morais et al., 2004); Leucobacter albus (Lin et al., 2004); Leucobacter alluvii and Leucobacter luti (Morais et al., 2006); Leucobacter iarius (Somvanshi et al., 2007); Leucobacter tardus (Behrendt et al., 2008); and Leucobacter chironomi (Halpern et al. 2009). The species Leucobacter chromiireducens has been divided into the subspecies Leucobacter chromiireducens subsp. solipictus and Leucobacter chromiireducens subsp. chromiireducens (Muir & Tan, 2007). All species of the genus are characterized by 2,4-diaminobutyric acid (DAB) as the diagnostic diamino acid in the peptidoglycan. However, differences in the amounts of other amino acids in the peptidoglycan have been reported.

    Strain Sj 10T was isolated after bioaerosol sampling in a German duck barn on tryptone soy agar (TSA) at 25 °C. The duck barn accommodated 2500 ducks, each 2 months old. To determine the culturable diversity of airborne bacteria in the duck barn, bioaerosols were collected according to the German standard procedure (VDI 4252-Blatt 2) on gelatin filters (diameter 8.0 cm) by sampling 500 l air at a sampling rate of 30 l min−1. The gelatin filters were dissolved and decimally diluted in sterile 0.9 % NaCl (up to 10−8). The cultivation approach was performed on various media including TSA plates by inoculating 0.1 ml of each dilution in triplicate and incubating plates at 25 °C. Strain Sj 10T was isolated from a TSA plate inoculated from the second decimal dilution.

    Morphological properties, Gram-staining and cell morphology were observed microscopically as described by Kämpfer & Kroppenstedt (2004). Isolation of the DNA was performed with a commercial DNA extraction kit (GenElute Plant Genomic DNA kit; Sigma-Aldrich) after mixing cells with 1 g Zirconia beads (diameter 0.1 mm) and disruption for 1 min at maximum speed on a vortexer (Vortex-Genie 2; Carl Roth). The 16S rRNA gene was analysed as described by Kämpfer et al. (2003). Phylogenetic analysis was performed using the arb software package (Strunk et al., 2000; Ludwig et al., 2004) and the corresponding SILVA SSURef 95 database (version July 2008; Pruesse et al., 2007) as well as the software package mega version 4.0 (Tamura et al., 2007) after multiple alignment of data by clustal_x (Thompson et al., 1997). The sequenced length of the 16S rRNA gene of strain Sj 10T was 1397 bp. Nucleotide sequence distances were calculated (distance options according to the Kimura-2 model; Kimura, 1980) using the software package mega version 4.0 (Tamura et al., 2007). Sequence similarity calculations after neighbour-joining analysis showed that the most similar sequences to that of strain Sj 10T were those of L. chromiireducens subsp. chromiireducens L-1T (97.8 %), L. tardus DSM 19811T (97.3 %) and L. luti RF6T (97.3 %). Similarities to strains of other species of the genus Leucobacter were <97.3 %. A tree based on the maximum-likelihood algorithm is shown in Fig. 1⇓.

    Figure image not available in archive
    Fig. 1.

    Phylogenetic analysis based on 16S rRNA gene sequences available from EMBL (accession numbers are given in parentheses). The phylogenetic tree was reconstructed using the arb software package (version December 2007; Ludwig et al., 2004) and the corresponding SILVA SSURef 95 database (version July 2008; Pruesse et al., 2007). Tree building was performed using the maximum-likelihood method with fastDNAml (Olsen et al., 1994). Bar, 0.10 nt substitutions per nucleotide position.

    The peptidoglycan was isolated after disruption of the cells by shaking with glass beads and subsequent trypsin digestion, according to the method of Schleifer (1985). The amino acids and peptides in the cell wall hydrolysates were analysed by two-dimensional ascending TLC (2D-TLC) on cellulose plates by using previously described solvent systems (Schleifer, 1985). The molar ratios of the amino acids were determined by GC (GC 14A; Shimadzu) and GC-MS (320-MS Quadrupole; Varian) of N-heptafluorobutyryl amino acid isobutyl esters (MacKenzie, 1987; Groth et al., 1996).

    Polar lipids of strain Sj 10T were extracted according to Minnikin et al. (1979) and analysed by 2D-TLC (Collins et al., 1980). Menaquinone analysis was carried out by HPLC as described by Groth et al. (1996). Fatty acid analysis was performed according to Kämpfer & Kroppenstedt (1996).

    The fatty acid profile of strain Sj 10T was very similar to those of closely related species (Table 1⇓). The major fatty acids were anteiso-C15 : 0 (59.0 %), iso-C16 : 0 (19.7 %) and anteiso-C17 : 0 (10.6 %).

    Table 1.

    Major fatty acid composition (%) of type strains of species of the genus Leucobacter

    Strains: 1, Sj 10T; 2, L. tardus DSM 19811T; 3, L. chromiireducens subsp. chromiireducens L-1T; 4, L. luti RF6T. Data for all strains are from this study. All strains were grown on TSA medium for 48 h at 28 °C. −, Not detected.

    The hydrolysate (4 M HCl, 100 °C, 16 h) of the purified peptidoglycan of strain Sj 10T contained the amino acids 2,4-DAB, lysine, alanine, glycine, threonine and glutamic acid in a molar ratio of approximately 0.7 : 0.2 : 2.1 : 0.3 : 0.8 : 1.0. All amino acids were identified by 2D-TLC, GC and characteristic MS fragment ions of their N-heptafluorobutyryl amino acid isobutyl esters. 4-Aminobutyric acid, which was found in other species of the genus Leucobacter, could not be detected in strain Sj 10T. Likewise, leucine, isoleucine and other amino acids, which occur in proteins but are not constituents of the peptidoglycan (Schleifer & Kandler, 1972), could not be detected in the hydrolysate; the cell wall preparation appeared to be free of contaminating proteins. Dinitrophenylation according to Schleifer (1985) revealed that alanine represents the N-terminus of the interpeptide bridge. Strain Sj 10T differed from L. komagatae, L. albus and L. chironomi (Takeuchi et al., 1996; Lin et al., 2004; Halpern et al., 2009) in the absence of 4-aminobutyric acid, and from L. albus, L. aridicollis, L. komagatae and L. tardus (Lin et al., 2004; Morais et al., 2004; Takeuchi et al., 1996; Behrendt et al., 2008) in the presence of threonine. The peptidoglycan of strain Sj 10T was similar in its composition to those of L. chromiireducens subsp. chromiireducens, L. luti, L. alluvii and L. iarius (Morais et al., 2004, 2006; Somvanshi et al., 2007), but differed in the partial substitution of DAB by lysine. The variation in the cell wall composition within the genus Leucobacter is very interesting. A detailed analysis of genes coding for these differences will perhaps reveal the evolutionary background of these changes.

    Strain Sj 10T contained MK-11 (83 %) as major menaquinone, like all other species of the genus Leucobacter. MK-12 (11 %) and MK-10 (4 %) were detected in minor amounts. The identified polar lipid components of strain Sj 10T, namely phosphatidylglycerol and diphosphatidylglycerol, are also reported to occur in other Leucobacter species (Takeuchi et al., 1996; Somvanshi et al., 2007; Muir & Tan, 2007). In addition, the strain contained one unknown component each of a phospholipid, glycolipid and aminoglycolipid (Supplementary Fig. S1, available in IJSEM Online).

    Results of the comparative physiological characterization, carried out under identical test conditions with methods as described previously (Kämpfer et al., 1991), are given in Table 2⇓ and the species description. Several tests could be used to differentiate strain Sj 10T from the other Leucobacter type strains.

    Table 2.

    Physiological characteristics of the type strains of species of the genus Leucobacter

    Strains: 1, Sj 10T; 2, L. tardus DSM 19811T; 3, L. chromiireducens subsp. chromiireducens L-1T; 4, L. luti RF6T. +, Positive; −, negative; (+), weakly positive. All strains were positive for utilization of 4-aminobutyrate and pyruvate. All strains were negative for utilization of aesculin, ortho-nitrophenyl-β-d-galactopyranoside, pNP-β-d-glucuronide, pNP-α-d-glucopyranoside, pNP-β-d-glucopyranoside, pNP-β-d-xylopyranoside, bis-pNP-phosphate, pNP-phenyl-phosphonate, l-arabinose, p-arbutin, cellobiose, gluconate, d-mannose, α-melibiose, sucrose, salicin, d-xylose, adonitol, maltitol, adipate, azelate, dl-3-hydroxybutyrate, itaconate, mesaconate, suberate, l-leucine, l-phenylalanine, l-serine, l-tryptophan, 4-hydroxybenzoate and phenylacetate.

    DNA–DNA hybridization experiments were performed with Sj 10T and the type strains of the most closely related species of the genus Leucobacter on the basis of the method given by Ziemke et al. (1998). Strain Sj 10T showed low DNA–DNA similarities to L. tardus DSM 19811T (15.8 %, reciprocal 17.5 %), L. luti RF6T (36.2 %, reciprocal 30.0 %) and L. chromiireducens subsp. chromiireducens L-1T (32.1 %, reciprocal 31.3 %). These differences and the observed physiological and chemotaxonomic differences between these type strains (Tables 1⇑ and 2⇑) clearly warrant the creation of a separate species.

    Description of Leucobacter aerolatus sp. nov.

    Leucobacter aerolatus (ae.ro.la′tus. Gr. n. aer air; L. part. adj. latus -a -um carried; N.L. masc. part. adj. aerolatus airborne).

    Cells are non-motile, non-spore-forming rods (approx. 2 μm in length). Gram-positive, oxidase-positive (weak reaction), with aerobic respiration. Good growth occurs after 3 days of incubation on TSA and nutrient agar at 25–30 °C. On TSA, colonies are white and shiny with a diameter of approximately 2 mm. The peptidoglycan contains the amino acids 2,4-DAB, lysine, threonine, glycine, alanine and glutamic acid. The quinone system comprises MK-11 as major component and minor amounts of MK-12 and MK-10. The polar lipid profile contains phosphatidylglycerol, diphosphatidylglycerol and one unknown component each of a phospholipid, glycolipid and aminoglycolipid. Major fatty acids are anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. Minor amounts of an unknown fatty acid (14.263) are also detected (Table 1⇑). The following carbon sources are utilized: N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactose, d-glucose, l-rhamnose, d-ribose, trehalose, myo-inositol, putrescine, 4-aminobutyrate, dl-lactate, pyruvate, l-alanine, l-aspartate, l-histidine, l-ornithine and l-proline. Results are weakly positive for maltose and oxoglutarate. The following carbon sources are not utilized: l-arabinose, p-arbutin, cellobiose, d-fructose, gluconate, d-mannose, α-melibiose, sucrose, salicin, d-xylose, adonitol, maltitol, d-mannitol, d-sorbitol, acetate, propionate, cis-aconitate, trans-aconitate, adipate, citrate, fumarate, glutarate, dl-3-hydroxybutyrate, itaconate, l-malate, mesaconate, suberate, β-alanine, l-leucine, l-phenylalanine, l-serine, l-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzoate and phenylacetate. On the basis of the method described by Kämpfer et al. (1991) the following compounds are hydrolysed: 2-deoxythymidine-5′-para-nitrophenyl (pNP)-phosphate, l-alanine-para-nitroanilide (pNA) and l-proline-pNA. The following compounds are not hydrolysed: aesculin, pNP-β-d-galactopyranoside, pNP-β-d-glucuronide, pNP-α-d-glucopyranoside, pNP-β-d-glucopyranoside, pNP-β-d-xylopyranoside, bis-pNP-phosphate, pNP-phenylphosphonate, pNP-phosphorylcholine and l-glutamate-γ-3-carboxy-pNA.

    The type strain is Sj 10T (=DSM 22806T =CCM 7705T), isolated from the air of a duck barn in Germany.

    Acknowledgments

    We are grateful to Gundula Will and Anika Wasner (DSMZ) for excellent technical assistance and Jean Euzéby for support with the nomenclature.

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