Antonie van Leeuwenhoek (2015) 107:1633–1638 DOI 10.1007/s10482-015-0434-2
SHORT COMMUNICATION
Chryseobacterium limigenitum sp. nov., isolated from dehydrated sludge Peter Ka¨mpfer • Janja Trcˇek • Barbara Skok Andrej Sˇorgo • Stefanie P. Glaeser
•
Received: 16 February 2015 / Accepted: 20 March 2015 / Published online: 27 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract An intense yellow pigmented strain (SUR2T) isolated from dehydrated activated sludge was studied in detail to clarify its taxonomic assignment. Cells of the isolate showed a rod-shaped morphology and stained Gram-negative. Comparative 16S rRNA gene sequence analysis revealed highest similarities to the type strains of Chryseobacterium polytrichastri YG4-6T (98.6 %), Chryseobacterium aahli T68FT (97.9 %), Chryseobacterium daeguense K105T and Chryseobacterium gregarium DSM 79109T (both 97.4 %). 16S rRNA genesequence similarities to all other Chryseobacterium species were below 97.3 %. The fatty acid analysis of strain SUR2T revealed a Chryseobacterium typical
profile composed mainly of the fatty acids C15:0 iso, C15:0 iso 2-OH, C17:1 iso x9c, and C17:0 iso 3-OH. DNA– DNA hybridizations with the type strains of C. polytrichastri, C. aahli, C. daeguense and C. gregarium resulted in values below 70 %. Differentiating biochemical and chemotaxonomic properties showed differences to the most closely related species and suggest that the isolate SUR2T represents a novel species, for which the name Chryseobacterium limigenitum sp. nov. (type strain SUR2T = ZIM B1019T = CCM 8594T = LMG 28734T) is proposed. Keywords Chryseobacterium limigenitum Taxonomy Dehydrated sludge Municipal sewage plant
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of strain SUR2T is LN811705.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0434-2) contains supplementary material, which is available to authorized users. P. Ka¨mpfer (&) S. P. Glaeser Institut fu¨r Angewandte Mikrobiologie, Justus-LiebigUniversita¨t Giessen, Heinrich-Buff-Ring 26–32, 35392 Giessen, Germany e-mail: [email protected] J. Trcˇek B. Skok A. Sˇorgo Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Maribor, Slovenia J. Trcˇek Faculty of Chemistry and Chemical Engineering, University of Maribor, Maribor, Slovenia
The genus Chryseobacterium was described 20 years ago by Vandamme et al. (1994) now contains a large number of species, some of which were isolated from waste water, sewage and contaminated soil: Chryseobacterium daeguense was isolated from waste water of a textile dye work company in Korea (Yoon et al. 2007), Chryseobacterium caeni from a nickelcomplexed cyanide-degrading bioreactor (Quan et al. 2007), Chryseobacterium defluvii from sewage of a sequence batch reactor (Ka¨mpfer et al. 2003), Chryseobacterium flavum from polluted soil (Zhou et al. 2007), Chryseobacterium humi and Chryseobacterium palustre from metal contaminated soil (Pires et al.
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2007) and Chryseobacterium hungaricum from hydrogen-contaminated soil (Szoboszlay et al. 2008). Strain SUR2T was isolated from dehydrated sludge of the municipal sewage plant situated at Dogosˇe near Maribor in Slovenia. The strain was initially isolated on nutrient agar (NA, Sigma-Aldrich) at 30 °C and also further maintained and subcultivated on this agar at 30 °C for 48 h. Analyses of the 16S rRNA gene sequence, the fatty acid methyl ester composition of whole cell hydrolysates, and further biochemical and physiological features were conducted to characterize the strain. In addition, DNA–DNA hybridizations were performed with type strains of those species most closely related on the basis of 16S rRNA gene sequence similarities, among them Chryseobacterium polytrichastri DSM 26899T (obtained from DSMZ), Chryseobacterium aahli LMG 27338T (obtained from LMG), Chryseobacterium gregarium DSM 19109T and Chryseobacterium daeguense K105T (both obtained from Undine Behrendt, Germany). The cultural and morphological characteristics were recorded from cultures grown on tryptic soy agar (TSA). Gram staining was performed according to Gerhardt et al. (1994) and the motility test was done under a light microscope with cells grown for 3 days in tryptic soy broth (TSB; Oxoid) at 30 °C. Temperature-dependent growth was tested at 4, 11, 30, 36, 40 and 45 °C on NA. NaCl tolerance was investigated at different concentrations of NaCl (0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 (w/v) %) in TSB. pH dependent growth was tested in TSB adjusted with HCl and NaOH to pH values between 4.0 and 12.0. Strain SUR2T showed a Gram-negative staining behaviour and formed visible (diameter about 2 mm) yellow colonies within 48 h at 30 °C. The isolate did not grow below 4 °C and above 37 °C. All strains grew very slowly at 36 °C and at a NaCl concentration of 1–3 % (w/v). Colonies showed a translucent glistening appearance with entire edges. A yellow pigment of the flexirubin type (KOH method according to Reichenbach 1989) was produced on NA. Oxidase activity was positive using the Oxidase reagent (bioMe´rieux) according to the instructions of the manufacturer. Cells of the strain were non-motile rods (approx. 1 lm wide and 2 lm long). No spores were observed. All strains grew well on NA, brain heart infusion agar, R2A agar and TSA, but not on MacConkey agar (Oxoid).
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The strain was physiologically/biochemically characterized using the 96-well plate test system (Ka¨mpfer et al. 1991) and by some additional biochemical tests: production of hydrogen sulphide using the lead acetate paper and triple-sugar-iron methods, indole reaction with Ehrlich’s and Kovacs’ reagents, activity of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, DNase (Oxoid CM321; supplemented with 0.01 % toluidine blue), b-galactosidase (ONPG), and urease on Christensen’s urea agar (Ka¨mpfer 1990), hydrolysis of casein, gelatin (plate method), starch, and tyrosine (Smibert and Krieg 1994). Similar to many other Chryseobacterium species, the strain utilized very few carbon sources, but was able to hydrolyze some chromogenic substrates. The biochemical/physiological data are given in Table 1 and in the species description. The analysis of the cellular fatty acid profiles was performed as described elsewhere (Ka¨mpfer and Kroppenstedt 1996) using a HP gas chromatograph HP 6890 with a Sherlock MIDI software version 2.11 and the TSBA peak naming table version 4.1. Prior to fatty acid extraction the strains were cultured on TS agar at 28 °C for 48 h. The results showed a Chryseobacterium-typical profile for SUR2T with the following most abundant fatty acids: C15:0 iso, C17:0 iso 3-OH, C17:1 isox9c and C15:0 iso 2-OH (which was detected as summed feature C15:0 iso 2-OH/C16:1 x7c, but as shown in several studies before, could be clearly Table 1 Comparison of characteristics of strain SUR2T with closely related Chryseobacterium species Characteristic
1
2
3
4
5
Sucrose
-
?*
?*
?
-
Arabinose
-
-*
-*
?
-
Salicin
-
(?)*
?*
-
-
Trehalose
-
?*
-*
-*
(?)
D-Xylose
-
?*
-*
-*
-
Acid production from
Strains/species 1, Chryseobacterium sp. SUR2T; 2, 3, Chryseobacterium polytrichastri DSM 26899T; Chryseobacterium aahli LMG 27338T; 4, Chryseobacterium gregarium DSM 19109T; 5, Chryseobacterium daeguense K105T. All data are from this study. ?, Positive; (?), weakly positive; -, negative. All strains produced acid from D-glucose and maltose and not from D-ribose. All strains were positive for digestion of casein and esculin hydrolysis * Results in agreement with those reported by Chen et al. (2015)
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identified as C15:0 iso 2-OH (Vandamme et al. 1994; Montero-Calasanz et al. 2013). Slight differences were found in comparison with the profiles of the type strains of the most closely related Chryseobacterium species (Table 2). Phylogenetic analysis was based on nearly fulllength 16S rRNA gene sequences. The 16S rRNA gene fragment of strain SUR2T obtained by sequence analysis was a continuous stretch of 1452 nucleotides spanning gene positions 17 to 1492 (according to Escherichia coli numbering published by Brosius et al. 1978). Pairwise sequence similarities to closest related type strains were obtained using the EzTaxon type strain database (Kim
Table 2 Long-chain fatty acid composition of strain SUR2T and closest related Chryseobacterium species Fatty acid C13:0 iso Unknown 13.565*
1 0.7
2
3
4
1.2
5 1.8
8.7
3.7
8.6
9.2
12.2
37.3
32.9
38.0
41.0
41.2
C15:0 iso 3-OH
2.6
3.6
2.4
2.7
3.1
C15:0 anteiso
5.4
3.7
2.9
8.9 1.0
C15:0 iso C15:1 iso F
C16:0
1.1
2.8
5.8
C16:0 3-OH
1.4
5.0
3.1
C16:0 iso 3-OH
1.4
1.4
Unknown 16.582*
1.1
C17:0 2-OH
1.2
1.5
C17:0 iso 3-OH
13.7
16.6
10.7
12.9
14.7
C17:1 iso x9c
16.1
8.5
1.9
13.1
20.6
8.5
17.8
24.3
8.2
8.1
5.3 2.0
C17:0 iso C17:0 anteiso
C18:1 x9c Summed feature 4
Taxa are listed as 1, Chryseobacterium sp. SUR2T; 2, 3, Chryseobacterium polytrichastri DSM 26899T; T Chryseobacterium aahli LMG 27338 ; 4, Chryseobacterium gregarium DSM 19109T; 5, Chryseobacterium daeguense K105T. All data are from this study * Unknown fatty acid; numbers indicate equivalent chain length Fatty acids that could not be separated by GC using the microbial identification system (Microbial ID) software were considered summed features. Summed feature four contains C15:0 iso 2-OH and/or C16:1 x7t. As shown in several studies, summed feature C15:0 iso 2-OH/C16:1 x7c could be clearly identified as C15:0 iso 2-OH (Vandamme et al. 1994; MonteroCalasanz et al. 2013)
et al. 2012). Phylogenetic trees were constructed using ARB release 5.2 (Ludwig et al. 2004) and the ‘‘AllSpecies Living Tree’’ Project (LTP; Yarza et al. 2008) database release (LTPs119, November 2014). Several phylogenetic trees were calculated including all type strains of Chryseobacterium species and type strains of Elizabethkingia species as outgroup. The trees were based on gene sequence positions 96–1394 (according to the E. coli numbering published by Brosius et al. 1978) which were covered by the sequenced 16S rRNA gene fragments of all included type strains. Phylogenetic trees were constructed with the maximum-likelihood method using RAxML v7.04 (Stamatakis 2006) with GTR-GAMMA and rapid bootstrap analysis (100 resamplings) and the maximum-parsimony method using DNAPARS version 3.6 (Felsenstein 2005). Trees are based on 100 resamplings (bootstrap analysis, Felsenstein 1985). To reduce the size of the final tree shown in the manuscript, type strains not directly clustering with SUR2T were removed from the tree without changing the overall tree topology (Fig. 1). Strain SUR2T shared with type strains of four Chryseobacterium species more than 97.4 % 16S rRNA gene sequence similarity, C. polytrichastri DSM 26899T, C. aahli LMG 27338T, C. gregarium DSM 19109T, and C. daeguense K105T and clustered with those species in the phylogenetic trees (Fig. 1). The strain showed a distinct clustering ([70 % bootstrap support) with C. polytrichastri DSM 26899T which however shared less than 98.7 % 16S rRNA gene sequence similarity with SUR2T. Clustering to the other species was not supported by high bootstrap values independently of the applied treeing method (Fig. 1). For further genotypic analysis high molecular weight genomic DNA was extracted as described by Pitcher and Saunders (1989). DNA–DNA hybridisation (DDH) experiments were performed with strain SUR2Tand the type strains of the four most closely related Chryseobacterium species according to the method of Ziemke et al. (1998) (except that for nick translation 2 lg of DNA were labelled during 3 h of incubation at 15 °C). Strain SUR2T showed a moderate DNA–DNA similarity to C. polytrichastri DSM 26899T (66 %, reciprocal 41 %), and low DNA–DNA similarities to C. aahli LMG 27338T (51 %), C. gregarium DSM 19109T (19 %) and C. daeguense K105T (30 %). On the basis of the results of this polyphasic study, it is concluded that strain SUR2T represents a novel
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Antonie van Leeuwenhoek (2015) 107:1633–1638 85
Chryseobacterium balustinum LMG 8329T (AY468447) Chryseobacterium piscium LMG 23089T (AM040439) Chryseobacterium scophthalmum LMG 13028T (AJ271009) Chryseobacterium piscicola VQ−6316sT (EU869190) 97 Chryseobacterium greenlandense UMB 34T (FJ932652) Chryseobacterium aquaticum 10−46T (AM748690) Chryseobacterium xinjiangense TSBY−67T (DQ166169) Chryseobacterium soli JS6−6T (EF591302) Chryseobacterium soldanellicola PSD1−4T (AY883415) Chryseobacterium piperi CTMT (EU999735) Chryseobacterium ginsenosidimutans THG 15T (GU138380) 91 "Chryseobacterium polytrichastri" YG4−6T (KC560018) Chryseobacterium limigenitum SUR2T (LN811705) Chryseobacterium yeoncheonense DCY67T (JX141782) 97 Chryseobacterium aahli T68F.09.P.LAT.MA.H.Kidney.DT (JX287893) Chryseobacterium gregarium DSM 19109T (AM773820) Chryseobacterium rigui CJ16T (JQ071497) Chryseobacterium taihuense THMBM1T (JQ283114) Chryseobacterium taeanense PHA3−4T (AY883416) 83 Chryseobacterium taichungense CC−TWGS1−8T (AJ843132) Chryseobacterium gambrini 5−1St1aT (AM232810) Chryseobacterium daecheongense CPW406T (AJ457206) Chryseobacterium wanjuense R2A10−2T (DQ256729) Chryseobacterium defluvii B2T (AJ309324) Chryseobacterium hispalense AG13T (EU336941) 98 Chryseobacterium takakiae AG1−2T (KC560016) Chryseobacterium profundimaris DY46T (KF434119) Chryseobacterium taiwanense BCRC 17412T (DQ318789) Chryseobacterium camelliae THG−C4−1T (JX843771) Chryseobacterium daeguense K105T (EF076759) Chryseobacterium hagamense RHA2−9T (DQ673672) Elizabethkingia meningoseptica ATCC 13253T (AJ704540) 100 Elizabethkingia anophelis 2.5T (EF426425) 86
0.1 Fig. 1 Maximum-parsimony tree based on nearly full-length 16S rRNA gene sequences showing the phylogenetic affiliation of strain SUR2T to closest related type strains of the genus Chryseobacterium. The tree was calculated in ARB using RAxML (GTR-GAMMA and rapid bootstrap analysis) and based on 16S rRNA gene sequence positions 96–1394 (according to E. coli numbering, Brosius et al. 1978). Numbers at nodes represent bootstraps [70 %. All type strains of the
genus Chryseobacterium were included in the analysis. Less related type strains were removed after tree construction without changing the overall tree topology. Circle at nodes that showed also a high bootstrap support in the maximum-likelihood analysis. Larger circle represent those nodes which were also supported by bootstrap values [70 %. Elizabethkingia type strains were used as outgroups. Bar 0.1 substitutions per sequence position
species, for which the name Chryseobacterium limigenitum sp. nov. is proposed.
48 h on nutrient agar, brain heart infusion agar, tryptic soy agar, and R2A agar at 4–36 °C. No growth occurs on MacConkey agar (Oxoid) at 28 °C. Unable to grow below 10 °C and above 37 °C. Cells grow in the presence of 1.0–2.0 % NaCl as an additional ingredient of nutrient agar. Colonies on nutrient agar produce a yellow colour and appear circular, translucent and glistening with entire edges. The yellow pigment of the flexirubin type is nondiffusible and non-fluorescent. Acid is produced from D-glucose, maltose and D-trehalose (weak). No acid is produced from sucrose, L-arabinose, adonitol, D-arabitol, dulcitol, erythritol, i-inositol, lactose, D-mannitol, D-melibiose, a-methyl-
Description of Chryseobacterium limigenitum sp. nov. Chryseobacterium limigenitum (li.mi.ge’ni.tum. L. masc. n. limus mud; L. neut. perf. part. genitum born, generated; N.L. neut. adj. limigenitum mud-borne). Cells show a Gram-negative staining behaviour. They are non-motile, non-spore forming rods, approx. 1 lm in width and 2 lm in length. Aerobic, oxidase positive, catalase positive. Good growth occurs after
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Antonie van Leeuwenhoek (2015) 107:1633–1638 D-glucoside, raffinose, L-rhamnose, salicin, D-sorbitol and
Urease activity, and hydrolysis of casein, gelatin, starch, DNA and tyrosine is positive, while indole production, hydrogen-sulphide production, activity of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, and b-galactosidase are negative. The following compounds are weakly utilized as a sole source of carbon: D-glucose and maltose. The following compounds are not utilized as a sole source of carbon: acetate, propionate, N-acetylgalactosamine, N-acetylglucosamine, L-arabinose, D-cellobiose, Dgalactose, gluconate, D-mannose, D-fructose, glycerol, D-mannitol, maltitol, a-D-melibiose, L-rhamnose, Dribose, salicin, D-sucrose, D-xylose, adonitol, i-inositol, D-sorbitol, putrescine, cis-aconitate, transaconitate, 4-aminobutyrate, adipate, azelate, fumarate, glutarate, DL-3-hydroxybutyrate, itaconate, DL-lactate, 2-oxoglutarate, pyruvate, suberate, citrate, mesaconate, L-alanine, b-alanine, L-ornithine, L-phenylalanine, L-serine, L-aspartate, L-histidine, L-leucine, L-proline, L-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzoate and phenylacetate. The chromogenic substrates p-nitrophenyl-a-Dglucopyranoside, p-nitrophenyl-b-D-glucopyranoside (weak), bis-p-nitrophenyl-phosphate, bis-p-nitrophenyl-phenyl-phosphonate, bis-p-nitrophenyl-phosphoryl-choline, 2-deoxythymidine-20 -p-nitrophenylphosphate, L-alanine-p-nitroanilide, c-L-glutamate-pnitroanilide, and L-proline-p-nitroanilide are hydrolysed. p-nitrophenyl-b-D-galactopyranoside, p-nitrophenyl-b-D-xylopyranoside and p-nitrophenyl-b-Dglucuronide are not hydrolysed. The major cellular fatty acids are C15:0 iso, C15:0 iso 2-OH, and C17:0 iso 3-OH. The type strain is SUR2T (= ZIM B1019T = CCM 8594T = LMG 28734) isolated from dehydrated sludge of the municipal sewage plant situated at Dogosˇe near Maribor in Slovenia.
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References
D-xylose.
Acknowledgments We thank Gundula Will, Katja Grebing and Maria Sowinsky for excellent technical assistance, Prof. Aharon Oren for his advice concerning the specific epithet and researchers involved in project Construction of pilot reactor for drying and disinfection of sludges from communal and industrial sewage plant, supported by Gorenje—Surovina d. o. o., Maribor, Slovenia and grant 4300-8/2013-22 from European Regional Development fund, in the frame of which Chryseobacterium limigenitum SUR2T was isolated.
Brosius J, Palmer ML, Kennedy PJ, Noller HF (1978) Complete nucleotide-sequence of a 16S ribosomal-RNA gene from Escherichia coli. PNAS 75:4801–4805 Chen XY, Zhao R, Chen ZL, Liu L, Li XD, Li YH (2015) Chryseobacterium polytrichastri sp. nov., isolated from a moss (Polytrichastrum formosum), and emended description of the genus Chryseobacterium. Antonie van Leeuwenhoek 107(2):403–410 Felsenstein J (1985) Confidence limits of phylogenies: an approach using the bootstrap. Evolution 39:783–791 Felsenstein J (2005) PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author, Department of Genome Sciences, University of Washington, Seattle Gerhardt P, Murray RGE, Wood WA, Krieg NR (1994) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC Ka¨mpfer P (1990) Evaluation of the Titertek-Enterobac-automated system (TTE-AS) for identification of members of the family Enterobacteriaceae. Zentralbl Bakteriol 273:164–172 Ka¨mpfer P, Kroppenstedt RM (1996) Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42:989–1005 Ka¨mpfer P, Steiof M, Dott W (1991) Microbiological characterisation of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria. Microb Ecol 21:227–251 Ka¨mpfer P, Dreyer U, Neef A, Dott W, Busse HJ (2003) Chryseobacterium defluvii sp. nov., isolated from wastewater. Int J Syst Evol Microbiol 53:93–97 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (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 Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar BA, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T, Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nucleic Acid Res 32:1363–1371 Montero-Calasanz MD, Go¨ker M, Rohde M, Spro¨er C, Schumann P, Busse H-J, Schmid M, Tindall BJ, Klenk HP, Camacho M (2013) Chryseobacterium hispalense sp. nov., a plant growth-promoting bacterium isolated from a rainwater pond in an olive plant nursery and emendation of the species Chryseobacterium defluvii, Chryseobacterium indologenes, Chryseobacterium wanjuense and Chryseobacterium gregarium. Int J Syst Evol Microbiol 63:4386–4395 Pires C, Carvalho MF, De Marco P, Magan N, Castro PM (2007) Chryseobacterium palustre sp. nov. and Chryseobacterium humi sp. nov., isolated from industrially contaminated sediments. Int J Syst Evol Microbiol 60:402–407
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1638 Pitcher DG, Saunders NA (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8:151–156 Quan ZX, Kim KK, Kim MK, Jin L, Lee ST (2007) Chryseobacterium caeni sp. nov., isolated from bioreactor sludge. Int J Syst Evol Microbiol 57:141–145 Reichenbach H (1989) The order cytophagales leadbetter 1974, 99AL. In: Staley JT, Bryant MP, Pfennig N, Holt JC (eds) Bergey’s manual of systematic bacteriology, vol 3. Williams & Wilkins, Baltimore, pp 2011–2073 Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 607–654 Stamatakis A (2006) RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690 Szoboszlay S, Atze´l B, Kukolya J, To´th EM, Ma´rialigeti K, Schumann P, Kriszt B (2008) Chryseobacterium hungaricum sp. nov., isolated from hydrocarbon-contaminated soil. Int J Syst Evol Microbiol 58:2748–2754
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SHORT COMMUNICATION
Chryseobacterium limigenitum sp. nov., isolated from dehydrated sludge Peter Ka¨mpfer • Janja Trcˇek • Barbara Skok Andrej Sˇorgo • Stefanie P. Glaeser
•
Received: 16 February 2015 / Accepted: 20 March 2015 / Published online: 27 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract An intense yellow pigmented strain (SUR2T) isolated from dehydrated activated sludge was studied in detail to clarify its taxonomic assignment. Cells of the isolate showed a rod-shaped morphology and stained Gram-negative. Comparative 16S rRNA gene sequence analysis revealed highest similarities to the type strains of Chryseobacterium polytrichastri YG4-6T (98.6 %), Chryseobacterium aahli T68FT (97.9 %), Chryseobacterium daeguense K105T and Chryseobacterium gregarium DSM 79109T (both 97.4 %). 16S rRNA genesequence similarities to all other Chryseobacterium species were below 97.3 %. The fatty acid analysis of strain SUR2T revealed a Chryseobacterium typical
profile composed mainly of the fatty acids C15:0 iso, C15:0 iso 2-OH, C17:1 iso x9c, and C17:0 iso 3-OH. DNA– DNA hybridizations with the type strains of C. polytrichastri, C. aahli, C. daeguense and C. gregarium resulted in values below 70 %. Differentiating biochemical and chemotaxonomic properties showed differences to the most closely related species and suggest that the isolate SUR2T represents a novel species, for which the name Chryseobacterium limigenitum sp. nov. (type strain SUR2T = ZIM B1019T = CCM 8594T = LMG 28734T) is proposed. Keywords Chryseobacterium limigenitum Taxonomy Dehydrated sludge Municipal sewage plant
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of strain SUR2T is LN811705.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0434-2) contains supplementary material, which is available to authorized users. P. Ka¨mpfer (&) S. P. Glaeser Institut fu¨r Angewandte Mikrobiologie, Justus-LiebigUniversita¨t Giessen, Heinrich-Buff-Ring 26–32, 35392 Giessen, Germany e-mail: [email protected] J. Trcˇek B. Skok A. Sˇorgo Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Maribor, Slovenia J. Trcˇek Faculty of Chemistry and Chemical Engineering, University of Maribor, Maribor, Slovenia
The genus Chryseobacterium was described 20 years ago by Vandamme et al. (1994) now contains a large number of species, some of which were isolated from waste water, sewage and contaminated soil: Chryseobacterium daeguense was isolated from waste water of a textile dye work company in Korea (Yoon et al. 2007), Chryseobacterium caeni from a nickelcomplexed cyanide-degrading bioreactor (Quan et al. 2007), Chryseobacterium defluvii from sewage of a sequence batch reactor (Ka¨mpfer et al. 2003), Chryseobacterium flavum from polluted soil (Zhou et al. 2007), Chryseobacterium humi and Chryseobacterium palustre from metal contaminated soil (Pires et al.
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2007) and Chryseobacterium hungaricum from hydrogen-contaminated soil (Szoboszlay et al. 2008). Strain SUR2T was isolated from dehydrated sludge of the municipal sewage plant situated at Dogosˇe near Maribor in Slovenia. The strain was initially isolated on nutrient agar (NA, Sigma-Aldrich) at 30 °C and also further maintained and subcultivated on this agar at 30 °C for 48 h. Analyses of the 16S rRNA gene sequence, the fatty acid methyl ester composition of whole cell hydrolysates, and further biochemical and physiological features were conducted to characterize the strain. In addition, DNA–DNA hybridizations were performed with type strains of those species most closely related on the basis of 16S rRNA gene sequence similarities, among them Chryseobacterium polytrichastri DSM 26899T (obtained from DSMZ), Chryseobacterium aahli LMG 27338T (obtained from LMG), Chryseobacterium gregarium DSM 19109T and Chryseobacterium daeguense K105T (both obtained from Undine Behrendt, Germany). The cultural and morphological characteristics were recorded from cultures grown on tryptic soy agar (TSA). Gram staining was performed according to Gerhardt et al. (1994) and the motility test was done under a light microscope with cells grown for 3 days in tryptic soy broth (TSB; Oxoid) at 30 °C. Temperature-dependent growth was tested at 4, 11, 30, 36, 40 and 45 °C on NA. NaCl tolerance was investigated at different concentrations of NaCl (0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 (w/v) %) in TSB. pH dependent growth was tested in TSB adjusted with HCl and NaOH to pH values between 4.0 and 12.0. Strain SUR2T showed a Gram-negative staining behaviour and formed visible (diameter about 2 mm) yellow colonies within 48 h at 30 °C. The isolate did not grow below 4 °C and above 37 °C. All strains grew very slowly at 36 °C and at a NaCl concentration of 1–3 % (w/v). Colonies showed a translucent glistening appearance with entire edges. A yellow pigment of the flexirubin type (KOH method according to Reichenbach 1989) was produced on NA. Oxidase activity was positive using the Oxidase reagent (bioMe´rieux) according to the instructions of the manufacturer. Cells of the strain were non-motile rods (approx. 1 lm wide and 2 lm long). No spores were observed. All strains grew well on NA, brain heart infusion agar, R2A agar and TSA, but not on MacConkey agar (Oxoid).
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The strain was physiologically/biochemically characterized using the 96-well plate test system (Ka¨mpfer et al. 1991) and by some additional biochemical tests: production of hydrogen sulphide using the lead acetate paper and triple-sugar-iron methods, indole reaction with Ehrlich’s and Kovacs’ reagents, activity of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, DNase (Oxoid CM321; supplemented with 0.01 % toluidine blue), b-galactosidase (ONPG), and urease on Christensen’s urea agar (Ka¨mpfer 1990), hydrolysis of casein, gelatin (plate method), starch, and tyrosine (Smibert and Krieg 1994). Similar to many other Chryseobacterium species, the strain utilized very few carbon sources, but was able to hydrolyze some chromogenic substrates. The biochemical/physiological data are given in Table 1 and in the species description. The analysis of the cellular fatty acid profiles was performed as described elsewhere (Ka¨mpfer and Kroppenstedt 1996) using a HP gas chromatograph HP 6890 with a Sherlock MIDI software version 2.11 and the TSBA peak naming table version 4.1. Prior to fatty acid extraction the strains were cultured on TS agar at 28 °C for 48 h. The results showed a Chryseobacterium-typical profile for SUR2T with the following most abundant fatty acids: C15:0 iso, C17:0 iso 3-OH, C17:1 isox9c and C15:0 iso 2-OH (which was detected as summed feature C15:0 iso 2-OH/C16:1 x7c, but as shown in several studies before, could be clearly Table 1 Comparison of characteristics of strain SUR2T with closely related Chryseobacterium species Characteristic
1
2
3
4
5
Sucrose
-
?*
?*
?
-
Arabinose
-
-*
-*
?
-
Salicin
-
(?)*
?*
-
-
Trehalose
-
?*
-*
-*
(?)
D-Xylose
-
?*
-*
-*
-
Acid production from
Strains/species 1, Chryseobacterium sp. SUR2T; 2, 3, Chryseobacterium polytrichastri DSM 26899T; Chryseobacterium aahli LMG 27338T; 4, Chryseobacterium gregarium DSM 19109T; 5, Chryseobacterium daeguense K105T. All data are from this study. ?, Positive; (?), weakly positive; -, negative. All strains produced acid from D-glucose and maltose and not from D-ribose. All strains were positive for digestion of casein and esculin hydrolysis * Results in agreement with those reported by Chen et al. (2015)
Antonie van Leeuwenhoek (2015) 107:1633–1638
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identified as C15:0 iso 2-OH (Vandamme et al. 1994; Montero-Calasanz et al. 2013). Slight differences were found in comparison with the profiles of the type strains of the most closely related Chryseobacterium species (Table 2). Phylogenetic analysis was based on nearly fulllength 16S rRNA gene sequences. The 16S rRNA gene fragment of strain SUR2T obtained by sequence analysis was a continuous stretch of 1452 nucleotides spanning gene positions 17 to 1492 (according to Escherichia coli numbering published by Brosius et al. 1978). Pairwise sequence similarities to closest related type strains were obtained using the EzTaxon type strain database (Kim
Table 2 Long-chain fatty acid composition of strain SUR2T and closest related Chryseobacterium species Fatty acid C13:0 iso Unknown 13.565*
1 0.7
2
3
4
1.2
5 1.8
8.7
3.7
8.6
9.2
12.2
37.3
32.9
38.0
41.0
41.2
C15:0 iso 3-OH
2.6
3.6
2.4
2.7
3.1
C15:0 anteiso
5.4
3.7
2.9
8.9 1.0
C15:0 iso C15:1 iso F
C16:0
1.1
2.8
5.8
C16:0 3-OH
1.4
5.0
3.1
C16:0 iso 3-OH
1.4
1.4
Unknown 16.582*
1.1
C17:0 2-OH
1.2
1.5
C17:0 iso 3-OH
13.7
16.6
10.7
12.9
14.7
C17:1 iso x9c
16.1
8.5
1.9
13.1
20.6
8.5
17.8
24.3
8.2
8.1
5.3 2.0
C17:0 iso C17:0 anteiso
C18:1 x9c Summed feature 4
Taxa are listed as 1, Chryseobacterium sp. SUR2T; 2, 3, Chryseobacterium polytrichastri DSM 26899T; T Chryseobacterium aahli LMG 27338 ; 4, Chryseobacterium gregarium DSM 19109T; 5, Chryseobacterium daeguense K105T. All data are from this study * Unknown fatty acid; numbers indicate equivalent chain length Fatty acids that could not be separated by GC using the microbial identification system (Microbial ID) software were considered summed features. Summed feature four contains C15:0 iso 2-OH and/or C16:1 x7t. As shown in several studies, summed feature C15:0 iso 2-OH/C16:1 x7c could be clearly identified as C15:0 iso 2-OH (Vandamme et al. 1994; MonteroCalasanz et al. 2013)
et al. 2012). Phylogenetic trees were constructed using ARB release 5.2 (Ludwig et al. 2004) and the ‘‘AllSpecies Living Tree’’ Project (LTP; Yarza et al. 2008) database release (LTPs119, November 2014). Several phylogenetic trees were calculated including all type strains of Chryseobacterium species and type strains of Elizabethkingia species as outgroup. The trees were based on gene sequence positions 96–1394 (according to the E. coli numbering published by Brosius et al. 1978) which were covered by the sequenced 16S rRNA gene fragments of all included type strains. Phylogenetic trees were constructed with the maximum-likelihood method using RAxML v7.04 (Stamatakis 2006) with GTR-GAMMA and rapid bootstrap analysis (100 resamplings) and the maximum-parsimony method using DNAPARS version 3.6 (Felsenstein 2005). Trees are based on 100 resamplings (bootstrap analysis, Felsenstein 1985). To reduce the size of the final tree shown in the manuscript, type strains not directly clustering with SUR2T were removed from the tree without changing the overall tree topology (Fig. 1). Strain SUR2T shared with type strains of four Chryseobacterium species more than 97.4 % 16S rRNA gene sequence similarity, C. polytrichastri DSM 26899T, C. aahli LMG 27338T, C. gregarium DSM 19109T, and C. daeguense K105T and clustered with those species in the phylogenetic trees (Fig. 1). The strain showed a distinct clustering ([70 % bootstrap support) with C. polytrichastri DSM 26899T which however shared less than 98.7 % 16S rRNA gene sequence similarity with SUR2T. Clustering to the other species was not supported by high bootstrap values independently of the applied treeing method (Fig. 1). For further genotypic analysis high molecular weight genomic DNA was extracted as described by Pitcher and Saunders (1989). DNA–DNA hybridisation (DDH) experiments were performed with strain SUR2Tand the type strains of the four most closely related Chryseobacterium species according to the method of Ziemke et al. (1998) (except that for nick translation 2 lg of DNA were labelled during 3 h of incubation at 15 °C). Strain SUR2T showed a moderate DNA–DNA similarity to C. polytrichastri DSM 26899T (66 %, reciprocal 41 %), and low DNA–DNA similarities to C. aahli LMG 27338T (51 %), C. gregarium DSM 19109T (19 %) and C. daeguense K105T (30 %). On the basis of the results of this polyphasic study, it is concluded that strain SUR2T represents a novel
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Antonie van Leeuwenhoek (2015) 107:1633–1638 85
Chryseobacterium balustinum LMG 8329T (AY468447) Chryseobacterium piscium LMG 23089T (AM040439) Chryseobacterium scophthalmum LMG 13028T (AJ271009) Chryseobacterium piscicola VQ−6316sT (EU869190) 97 Chryseobacterium greenlandense UMB 34T (FJ932652) Chryseobacterium aquaticum 10−46T (AM748690) Chryseobacterium xinjiangense TSBY−67T (DQ166169) Chryseobacterium soli JS6−6T (EF591302) Chryseobacterium soldanellicola PSD1−4T (AY883415) Chryseobacterium piperi CTMT (EU999735) Chryseobacterium ginsenosidimutans THG 15T (GU138380) 91 "Chryseobacterium polytrichastri" YG4−6T (KC560018) Chryseobacterium limigenitum SUR2T (LN811705) Chryseobacterium yeoncheonense DCY67T (JX141782) 97 Chryseobacterium aahli T68F.09.P.LAT.MA.H.Kidney.DT (JX287893) Chryseobacterium gregarium DSM 19109T (AM773820) Chryseobacterium rigui CJ16T (JQ071497) Chryseobacterium taihuense THMBM1T (JQ283114) Chryseobacterium taeanense PHA3−4T (AY883416) 83 Chryseobacterium taichungense CC−TWGS1−8T (AJ843132) Chryseobacterium gambrini 5−1St1aT (AM232810) Chryseobacterium daecheongense CPW406T (AJ457206) Chryseobacterium wanjuense R2A10−2T (DQ256729) Chryseobacterium defluvii B2T (AJ309324) Chryseobacterium hispalense AG13T (EU336941) 98 Chryseobacterium takakiae AG1−2T (KC560016) Chryseobacterium profundimaris DY46T (KF434119) Chryseobacterium taiwanense BCRC 17412T (DQ318789) Chryseobacterium camelliae THG−C4−1T (JX843771) Chryseobacterium daeguense K105T (EF076759) Chryseobacterium hagamense RHA2−9T (DQ673672) Elizabethkingia meningoseptica ATCC 13253T (AJ704540) 100 Elizabethkingia anophelis 2.5T (EF426425) 86
0.1 Fig. 1 Maximum-parsimony tree based on nearly full-length 16S rRNA gene sequences showing the phylogenetic affiliation of strain SUR2T to closest related type strains of the genus Chryseobacterium. The tree was calculated in ARB using RAxML (GTR-GAMMA and rapid bootstrap analysis) and based on 16S rRNA gene sequence positions 96–1394 (according to E. coli numbering, Brosius et al. 1978). Numbers at nodes represent bootstraps [70 %. All type strains of the
genus Chryseobacterium were included in the analysis. Less related type strains were removed after tree construction without changing the overall tree topology. Circle at nodes that showed also a high bootstrap support in the maximum-likelihood analysis. Larger circle represent those nodes which were also supported by bootstrap values [70 %. Elizabethkingia type strains were used as outgroups. Bar 0.1 substitutions per sequence position
species, for which the name Chryseobacterium limigenitum sp. nov. is proposed.
48 h on nutrient agar, brain heart infusion agar, tryptic soy agar, and R2A agar at 4–36 °C. No growth occurs on MacConkey agar (Oxoid) at 28 °C. Unable to grow below 10 °C and above 37 °C. Cells grow in the presence of 1.0–2.0 % NaCl as an additional ingredient of nutrient agar. Colonies on nutrient agar produce a yellow colour and appear circular, translucent and glistening with entire edges. The yellow pigment of the flexirubin type is nondiffusible and non-fluorescent. Acid is produced from D-glucose, maltose and D-trehalose (weak). No acid is produced from sucrose, L-arabinose, adonitol, D-arabitol, dulcitol, erythritol, i-inositol, lactose, D-mannitol, D-melibiose, a-methyl-
Description of Chryseobacterium limigenitum sp. nov. Chryseobacterium limigenitum (li.mi.ge’ni.tum. L. masc. n. limus mud; L. neut. perf. part. genitum born, generated; N.L. neut. adj. limigenitum mud-borne). Cells show a Gram-negative staining behaviour. They are non-motile, non-spore forming rods, approx. 1 lm in width and 2 lm in length. Aerobic, oxidase positive, catalase positive. Good growth occurs after
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Antonie van Leeuwenhoek (2015) 107:1633–1638 D-glucoside, raffinose, L-rhamnose, salicin, D-sorbitol and
Urease activity, and hydrolysis of casein, gelatin, starch, DNA and tyrosine is positive, while indole production, hydrogen-sulphide production, activity of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, and b-galactosidase are negative. The following compounds are weakly utilized as a sole source of carbon: D-glucose and maltose. The following compounds are not utilized as a sole source of carbon: acetate, propionate, N-acetylgalactosamine, N-acetylglucosamine, L-arabinose, D-cellobiose, Dgalactose, gluconate, D-mannose, D-fructose, glycerol, D-mannitol, maltitol, a-D-melibiose, L-rhamnose, Dribose, salicin, D-sucrose, D-xylose, adonitol, i-inositol, D-sorbitol, putrescine, cis-aconitate, transaconitate, 4-aminobutyrate, adipate, azelate, fumarate, glutarate, DL-3-hydroxybutyrate, itaconate, DL-lactate, 2-oxoglutarate, pyruvate, suberate, citrate, mesaconate, L-alanine, b-alanine, L-ornithine, L-phenylalanine, L-serine, L-aspartate, L-histidine, L-leucine, L-proline, L-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzoate and phenylacetate. The chromogenic substrates p-nitrophenyl-a-Dglucopyranoside, p-nitrophenyl-b-D-glucopyranoside (weak), bis-p-nitrophenyl-phosphate, bis-p-nitrophenyl-phenyl-phosphonate, bis-p-nitrophenyl-phosphoryl-choline, 2-deoxythymidine-20 -p-nitrophenylphosphate, L-alanine-p-nitroanilide, c-L-glutamate-pnitroanilide, and L-proline-p-nitroanilide are hydrolysed. p-nitrophenyl-b-D-galactopyranoside, p-nitrophenyl-b-D-xylopyranoside and p-nitrophenyl-b-Dglucuronide are not hydrolysed. The major cellular fatty acids are C15:0 iso, C15:0 iso 2-OH, and C17:0 iso 3-OH. The type strain is SUR2T (= ZIM B1019T = CCM 8594T = LMG 28734) isolated from dehydrated sludge of the municipal sewage plant situated at Dogosˇe near Maribor in Slovenia.
1637
References
D-xylose.
Acknowledgments We thank Gundula Will, Katja Grebing and Maria Sowinsky for excellent technical assistance, Prof. Aharon Oren for his advice concerning the specific epithet and researchers involved in project Construction of pilot reactor for drying and disinfection of sludges from communal and industrial sewage plant, supported by Gorenje—Surovina d. o. o., Maribor, Slovenia and grant 4300-8/2013-22 from European Regional Development fund, in the frame of which Chryseobacterium limigenitum SUR2T was isolated.
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1638 Pitcher DG, Saunders NA (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8:151–156 Quan ZX, Kim KK, Kim MK, Jin L, Lee ST (2007) Chryseobacterium caeni sp. nov., isolated from bioreactor sludge. Int J Syst Evol Microbiol 57:141–145 Reichenbach H (1989) The order cytophagales leadbetter 1974, 99AL. In: Staley JT, Bryant MP, Pfennig N, Holt JC (eds) Bergey’s manual of systematic bacteriology, vol 3. Williams & Wilkins, Baltimore, pp 2011–2073 Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 607–654 Stamatakis A (2006) RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690 Szoboszlay S, Atze´l B, Kukolya J, To´th EM, Ma´rialigeti K, Schumann P, Kriszt B (2008) Chryseobacterium hungaricum sp. nov., isolated from hydrocarbon-contaminated soil. Int J Syst Evol Microbiol 58:2748–2754
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