Promicromonospora Umidemergens Sp. Nov., Isolated from Moisture from Indoor Wall Material

This is an author manuscript that has been accepted for publication in International Journal of Systematic and Evolutionary Microbiology, copyright Society for General Microbiology, but has not been copy-edited, formatted or proofed. Cite this article as appearing in International Journal of Systematic and Evolutionary Microbiology. This version of the manuscript may not be duplicated or reproduced, other than for personal use or within the rule of ‘Fair Use of Copyrighted Materials’ (section 17, Title 17, US Code), without permission from the copyright owner, Society for General Microbiology. The Society for General Microbiology disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by any other parties. The final copy-edited, published article, which is the version of record, can be found at http://ijs.sgmjournals.org, and is freely available without a subscription 24 months after publication. First published in: Int J Syst Evol Microbiol, 2009. 60(3) 537-541. doi:10.1099/ijs.0.014142-0 Promicromonospora umidemergens sp. nov., isolated from moisture from indoor wall material

K. Martin,1 J. Scha¨fer2 and P. Ka¨mpfer2

Correspondence 1Institut Leibniz-Institut fu¨r Naturstoff-Forschung und Infektionsbiologie eV, Hans-Kno¨ll-Institut, P. Ka¨mpfer D-07745 Jena, Germany [email protected] 2Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, giessen.de Germany

A Gram-positive, yellow-pigmented, branched-hyphae-forming micro-organism, strain 09-Be- 007T, was isolated from the wall of an indoor environment. Based on 16S rRNA gene sequence similarity studies, strain 09-Be-007T belonged to the genus Promicromonospora. The novel isolate showed sequence similarities of 98.8 % to Promicromonospora aerolata V54AT, 98.9 % to Promicromonospora vindobonensis V45T, 98.1 % to Promicromonospora sukumoe DSM 44121T, 98.2 % to Promicromonospora kroppenstedtii RS16T, 96.7 % to Promicromonospora flava CC 0387T and 97.8 % to Promicromonospora citrea DSM 43110T, the type strain of the type species of this genus. Cell wall sugars of strain 09-Be-007T were galactose, rhamnose and glucose. The diagnostic diamino acid of the cell-wall peptidoglycan was lysine. The major

menaquinones detected were MK-9(H4) and MK-9(H6). Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. Major fatty acids were iso-

C15 : 0 and anteiso-C15 : 0; moderate amounts of anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 1 were also found. All these data supported the affiliation of strain 09-Be-007T to the genus Promicromonospora. DNA–DNA hybridization values and physiological and biochemical data enabled strain 09-Be-007T to be differentiated genotypically and phenotypically from the six recognized species of the genus Promicromonospora. For these reasons, strain 09-Be-007T represents a novel species, for which the name Promicromonospora umidemergens sp. nov. is proposed, with 09-Be-007T (5DSM 22081T5CCM 7634T) as the type strain.

The genus Promicromonospora currently harbours six Strain 09-Be-007T was enriched and recovered from a species: Promicromonospora citrea (Krasil’nikov et al., wall that was colonized with moulds. After extraction of 1961), Promicromonospora sukumoe (Takahashi et al., 1 g sample material by shaking for 15 min in 10 ml 0.9 % 1987), Promicromonospora aerolata and Promicro- NaCl solution containing 0.01 % (v/v) Tween 80, monospora vindobonensis (Busse et al., 2003), Promicro- aliquots of this suspension were spread on agar plates monospora kroppenstedtii (Alonso-Vega et al., 2008), and containing organic medium M79 agar (Prauser & Falta, Promicromonospora flava (Jiang et al., 2009). Two species 1968) and incubated for 2 weeks at 28 uC. The new strain originally described as members of the genus Promicro- was maintained on M79 agar and preserved at 280 uC monospora have been transferred to other genera: by mixing well-grown M79 broth cultures in a 1 : 1 Promicromonospora enterophila (Ja¨ger et al., 1983) was ratio with glycerol preservation medium (Salser, 1978) transferred to the genus Oerskovia as Oerskovia enterophila containing K2HPO4 (1.26 %), KH2PO4 (0.36 %), (Stackebrandt et al., 2002), and Promicromonospora MgSO4 .7H2O (0.01 %), sodium citrate (0.09 %), pachnodae (Cazemier et al., 2003) was reclassified as (NH4)2SO4 (0.18 %) and glycerol (8.80 %). Stock cultures Xylanimicrobium pachnodae (Stackebrandt & Schumann, of the isolate in liquid M79 supplemented with 5 % DMSO 2004). P. citrea, P. sukumoe and P. kroppenstedtii were were also maintained in the vapour phase of liquid isolated from soil samples, whereas P. aerolata and P. nitrogen. vindobonensis were isolated from the air of the medieval Morphological properties, Gram-staining and cell mor- Virgilkapelle in Vienna. P. flava was isolated from sediment phology were observed microscopically as described by from the Baltic Sea in Germany. Ka¨mpfer & Kroppenstedt (2004). Strain 09-Be-007T formed yellow-pigmented colonies with characteristic The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene wrinkly and shining surfaces. Branching septate hyphae, sequence of strain 09-Be-007T is FN293378. which grew on the agar surface and penetrated into the

K. Martin, J. Scha¨fer and P. Ka¨mpfer agar, were observed microscopically. Aerial mycelium was hydrolysates contained the diagnostic diamino acid not observed. In older cultures, hyphae broke up into non- lysine together with glutamic acid and alanine. Whole- motile fragments of various sizes. One-day-old cultures cell sugars, determined by TLC (Becker et al., 1965), stained Gram-positive. were galactose, rhamnose and glucose. The same composition was reported for P. vindobonensis and P. DNA isolation was performed with a commercial DNA aerolata. P. flava contained ribose in addition to these extraction kit (GenElute Plant Genomic DNA kit; Sigma) sugars, whereas P. citrea contained only galactose and after disruption of cells by a 1 min bead-beating step with P. kroppenstedtii contained galactose plus rhamnose 1 g Zirconia beads (0.1 mm diameter) at maximum speed. (Table 1). Multiple sequence alignment and analysis of the data were Menaquinones were extracted as described by Collins et al. performed using the software package MEGA version 4 (1977) and analysed by HPLC (Groth et al., 1996). In (Tamura et al., 2007), the ARB software package (version contrast to previously described species of the genus December 2007; Ludwig et al., 2004) and the correspond- Promicromonospora, which contain MK-9(H4)asthe ing SILVA SSURef 95 database (version July 2008; Pruesse predominant menaquinone, strain 09-Be-007T exhibited a et al., 2007). Genetic distance calculations (distance quinone system with a high amount of MK-9(H ) (48 %), options according to the Kimura-2 model) and clustering 6 followed by MK-9(H ) (36 %). Minor amounts of MK- were determined with the neighbour-joining method (Fig. 4 8(H4) (6 %) and MK-9(H2) (2 %) were also detected. 1) and the maximum-likelihood method with FASTDNAML Menaquinone MK-8(H ) has also been found in P. (Olsen et al., 1994; results not shown). Bootstrap values 4 kroppenstedtii and P. flava, but not in the other four were based on 1000 replications. The 16S rRNA gene species of the genus Promicromonospora. P. citrea contained sequence of strain 09-Be-007T was a continuous stretch of only one nonhydrated menaquinone with nine isoprenoid 1327 bp. units (MK-9). Sequence similarity calculations after pairwise local align- Polar lipids were extracted according to Minnikin et al. ment indicated that the closest relatives of strain 09-Be- (1979). Major lipids were diphosphatidylglycerol, phos- 007T were P. aerolata V54AT (98.8 % 16S rRNA gene phatidylglycerol, phosphatidylinositol, three unknown sequence similarity) and P. vindobonensis V45T (98.9 % glycolipids and one unknown phospholipid. The same similarity). complex pattern of phospholipids with the major compo- Bacterial biomass for chemotaxonomic investigations was nents and some unknown glyco- and phospholipids was prepared by cultivating strain 09-Be-007T for 24–48 h in also described for P. kroppenstedtii. Diphosphatidylglycerol shake flasks in liquid organic medium M79 at 180 r.p.m. is found in all species of the genus Promicromonospora, and 28 uC. For fatty acid analysis, cells were grown on except P. citrea, and phosphatidylglycerol has not been tryptic soy agar (TSA) at 28 uC for 48 h. reported for P. sukumoe (Table 1). Cell-wall amino acids were determined by TLC accord- The fatty acid profile (Table 2) of strain 09-Be-007T was ing to Schleifer & Kandler (1972). The peptidoglycan very similar to those of the other species with predominant

Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationships between strain 09-Be-007T, other members of the genus Promicromonospora and closely related taxa of the family Promicromonosporaceae. Numbers at the nodes are bootstrap percentages (1000 replications). All nodes of the Promicromonospora branch were also seen in the maximum-likelihood tree. Bar, 0.005 changes per nucleotide position.

Promicromonospora umidemergens sp. nov.

Table 1. Chemotaxonomic characteristics that differentiate the type strains of members of the genus Promicromonospora

Strains: 1, 09-Be-007T;2,P. vindobonensis V45T;3,P. aerolata V54AT;4,P. kroppenstedtii RS16T;5,P. citrea DSM 43110T;6,P. sukumoe DSM 44121T;7,P. flava CC 0387T. Data are from this study, Takahashi et al. (1987), Kalakoutskii et al. (1989), Busse et al. (2003) and Jiang et al. (2009).

Characteristic 1 2 3 4 5 6 7

Menaquinone MK-9(H6), 48; MK-9(H4), 92; MK-9(H4), 93; MK-9(H4), 64; MK-9 MK-9(H4), MK-9(H4), 86; composition MK-9(H4), 36; MK-9(H2), 4; MK-9(H2), 7 MK-9(H6), 15; MK-9, MK-8(H4), 7; (%) MK-8(H4), 6; MK-9(H6), 4 MK-8(H4), 10; MK-9(H2), MK-9(H2), 5; MK-9(H2), 2 MK-9(H2), 2 MK-9(H6) MK-9, 3 Polar lipids* PG, DPG, PI, DPG, PG, DPG, PG, 2 PG, DPG, PI, PG, PI, PI, DPG DPG, PG, unknown PL, unknown unknown GL, GL and PGL unidentified unknown GL, 3 unknown GL GL, PGL 2 unknown PGL, glucosamine- unknown PL unknown PL phospholipid Cell-wall Lys (Glu, Ala) Glu : Gly : Ala : Lys Glu : Gly : Ala : Lys Ala : Glu : Lys Lys-Ala (A3a) Lys (A3a) Lys (Ala) compositionD (0.98 : 0.69 : (1.05 : 0.43 : (2.3 : 2.0 : 1.0) 2.88 : 1.0) (A3a) 3.4 : 1.0) (A3a) (A4a) Cell-wall rha, gal, glu§ rha, gal, glu rha, gal, glu gal, rha gal rha, glu, rib§ rha, gal, glu, sugarsd rib§

*DPG, Diphosphatidylglycerol; GL, glycolipid; PG, Phosphatidylglycerol; PGL, phosphoglycolipid; PI, phosphatidylinositol; PL, phospholipid. DAla, Alanine; Glu, glutamic acid; Gly, glycine; Lys, lysine. Amino acids given in parentheses are minor components. dgal, Galactose; glu, glucose; rha, rhamnose; rib, ribose. §Whole-cell sugars.

amounts of iso-C15 : 0 (between 30 and 41 %) and anteiso- (16.3 %). It should be mentioned here that P. flava may C15 : 0 (between 34 and 52 %). The newly described species not belong to the genus Promicromonospora and should be P. flava is an exception, showing a higher proportion of studied again for its taxonomic allocation (see Tables 1 to 3 anteiso-C15 : 0 (57.2 %) and less than 20 % iso-C15 : 0 and Fig. 1).

Table 2. Fatty acid contents of strain 09-Be-007T and the type strains of all other species of the genus Promicromonospora grown under the same conditions

Strains: 1, 09-Be-007T;2,P. vindobonensis V45T;3,P. aerolata V54AT;4,P. kroppenstedtii RS16T;5,P. sukumoe DSM 44121T;6,P. citrea DSM 43110T;7,P. flava CC 0387T. All strains were grown on TSA for 48 h at 28 uC and analysed using the MIDI system (Ka¨mpfer & Kroppenstedt, 1996). 2, Not detected. Data are from this study except where indicated.

Fatty acid 1 2 3 4 5 6 7*

C13 : 0 2220.2 222 C14 : 0 2 1.5 0.8 1.8 1.3 0.6 2 iso-C14 : 0 0.5 2 0.3 1.5 0.9 1.2 2 iso-C15 : 1 G 7.0 2 0.4 0.5 3.4 22 anteiso-C15 : 1 A 2.5 2 0.4 1.2 4.4 22 iso-C15 : 0 38.6 36.7 30.8 39.7 36.6 40.6 16.3 anteiso-C15 : 0 34.8 46.5 51.1 43.2 38.0 38.9 57.2 C15 : 0 2221.0 1.6 1.0 2 C16 : 0 N alcohol 22220.4 22 iso-C16 : 0 4.8 2.2 2.2 4.4 3.9 4.0 2 C16 : 0 1.0 6.0 3.8 1.9 2.4 0.8 2 Summed feature 5D 2 2.0 1.4 2222

iso-C17 : 0 3.5 1.3 1.5 1.2 2.0 3.1 2 anteiso-C17 : 0 6.8 3.8 6.7 3.4 4.6 9.7 2 C18 : 1v9c 220.5 2222 C18 : 3v6,9,12c 22220.4 22

*Data from Jiang et al. (2009).

DSummed feature 5 contains C18 : 2v6,9c and/or anteiso-C18 : 0, which could not be separated by GLC with the MIDI system.

K. Martin, J. Scha¨fer and P. Ka¨mpfer

Table 3. Biochemical characteristics of type strains of members of the genus Promicromonospora

Strains: 1, 09-Be-007T;2,P. kroppenstedtii RS16T;3,P. vindobonensis V45T;4,P. aerolata V54AT;5,P. sukumoe IFO 14650T;6,P. citrea IFO 12397T; T 7, P. flava CC 0387 . +, Positive; (+), weakly positive; 2, negative; ND, not determined. Data are from this study except where indicated.

Characteristic 1 2 3 4 5 6 7*

Assimilation of: N-Acetyl-D-glucosamine ++D + (+) ++ ND Trehalose ++D + (+) ++ + Cellobiose ++D (+) +++ ND D-Fructose ++d (+) +++ 2 D-Glucose, D-xylose ++D + 2 ++ + Maltose ++d + 2 ++ 2 D-Mannose, salicin ++d (+) 2 + 2 ND D-Ribose, sucrose ++D (+) 2 + 2 + D-Sorbitol ++D (+) 2 + 22 L-Arabinose ++D 22++ ND L-Aspartate, L-histidine + 222++ ND Pyruvate ++(+)(+) + (+) ND L-Proline + (+) + (+) + (+) ND DL-3-Hydroxybutyrate, DL-lactate, L-malate ++(+)(+) ++ ND Fumarate ++(+) 2 ++ ND L-Alanine 2222+ (+) ND L-Rhamnose + 2 (+)(+) 2 + 2 Glutarate (+) 2 (+)(+) 22ND Propionate (+) 2 + 222ND Hydrolysis of:D§ ND pNP-phenylphosphonate, L-proline-pNA, L-alanine-pNA, +++2 ++ ND pNP-b-D-xylopyranoside, pNP-b-D-glucopyranoside Aesculin ++D (+) 2 ++ ND bis-pNP phosphate ++22++ ND pNP-b-D-galactopyranoside ++D 22(+)(+) ND

*Data from Jiang et al. (2009). DData for P. kroppenstedtii are congruent with those from Alonso-Vega et al. (2008). dData for P. kroppenstedtii are not congruent with those from Alonso-Vega et al. (2008). §pNA, p-Nitroanilide; pNP, p-nitrophenyl.

Although the 16S rRNA gene sequence similarity studies 007T showed low DNA–DNA hybridization with P. clearly supported the affiliation of strain 09-Be-007T to the aerolata V54AT (42.4 %, reciprocal 56 %), P. vindobonensis genus Promicromonospora, some chemotaxonomic markers V45T (42.2 %, reciprocal 46 %) and P. kroppenstedtii RS16T differed from those of previously described species of this (36 %, reciprocal 23.2 %). genus. The results of the comparative physiological characterization carried out using identical test conditions Description of Promicromonospora are given in Table 3 and in the species description, with umidemergens sp. nov. methods as described previously (Ka¨mpfer et al., 1991). Promicromonospora umidemergens (u.mi.de.mer9gens. L. The observed chemotaxonomic (Table 1) and physiological adj. umidus moist; L. part. adj. emergens emerging, rising; (Table 3) differences between the type strains of the N.L. part. adj. umidemergens rising moisture). recognized members of the genus Promicromonospora and strain 09-Be-007T clearly warrant the creation of a separate A Gram-stain-positive, aerobic, chemo-organotrophic species. bacterium. Forms yellow-pigmented colonies with characteristic wrinkly and shining surfaces. Possesses branched DNA–DNA hybridization experiments were performed septate hyphae that grow on the agar surface and penetrate with strain 09-Be-007T and P. aerolata V54AT, P. into the agar. Aerial mycelium is not observed. Hyphae vindobonensis V45T and P. kroppenstedtii RS16T using the break up into non-motile fragments of various sizes. The method described by Ziemke et al. (1998) with a minor major components of the quinone system are MK-9(H6) m variation in the nick translation step (2 g DNA was and MK-9(H4). The diagnostic diamino acid of the cell- labelled during a 3 h incubation at 15 uC). Strain 09-Be- wall peptidoglycan is lysine; glutamic acid and alanine are

Promicromonospora umidemergens sp. nov. also found. The polar lipid profile consists of dipho- 1961a, 107AL.InBergey’s Manual of Systematic Bacteriology, vol. 4, pp. sphatidylglycerol, phosphatidylglycerol, phosphatidylinosi- 2392–2395. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. tol, three unknown glycolipids and one unknown Baltimore: Williams & Wilkins. phospholipid as major lipids. Fatty acid composition is Ka¨mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty dominated by iso- and anteiso-branched fatty acids. Major acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005. fatty acids are iso-C15 : 0 and anteiso-C15 : 0; moderate Ka¨mpfer, P. & Kroppenstedt, R. M. (2004). Pseudonocardia benzeni- amounts of iso-C15 : 1, anteiso-C15 : 1, iso-C17 : 0 and ante- vorans sp. nov. Int J Syst Evol Microbiol 54, 749–751. iso-C17 : 0 are also found. Carbon source utilization patterns and differential characteristics of P. umidemergens and Ka¨mpfer, P., Steiof, M. & Dott, W. (1991). Microbiological characterization of a fuel-oil contaminated site including numerical related Promicromonospora species (determined under 21 identical conditions) are indicated in Table 3. identification of heterotrophic water and soil bacteria. Microb Ecol , 227–251. T T The type strain is 09-Be-007 (5DSM 22081 5CCM Krasil’nikov, N. A., Kalakoutskii, L. V. & Kirillova, N. F. (1961). A new 7634T), isolated by C. Trautmann, Berlin, Germany, from genus of Actinomycetales, Promicromonospora gen. nov. Bull Acad Sci the wall of a house with rising damp and colonized with USSR Ser Biol 1, 107–112. moulds. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Acknowledgements Res 32, 1363–1371. 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