International Journal of Systematic and Evolutionary Microbiology (2015), 65, 147–153
DOI 10.1099/ijs.0.067652-0
Pseudochelatococcus lubricantis gen. nov., sp. nov. and Pseudochelatococcus contaminans sp. nov. from coolant lubricants Peter Ka¨mpfer,1 Stefanie P. Glaeser,1 Marco Gra¨ber,23 Andreas Rabenstein,2 Jan Kuever2 and Hans-Ju¨rgen Busse3 1
Correspondence
Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, Germany
Peter Ka¨mpfer [email protected]
2
Bremen Institute for Materials Testing, Department of Microbiology, Bremen, Germany
3
Institut fu¨r Bakteriologie, Mykologie und Hygiene, Veterina¨rmedizinische Universita¨t, A-1210 Wien, Austria
Two Gram-negative, rod-shaped, non-spore-forming bacteria, isolated from metal working fluids were investigated to determine their taxonomic positions. On the basis of 16S rRNA gene sequence phylogeny, both strains (MPA 1113T and MPA 1105T) formed a distinct cluster with 97.7 % sequence similarity between them, which was in the vicinity of members of the genera Methylobacterium, Camelimonas, Chelatococcus, Bosea, Salinarimonas and Microvirga to which they showed low sequence similarities (below 94 %). The predominant compounds in the polyamine pattern and in the quinone system of the two strains were spermidine and ubiquinone Q-10, respectively. The polar lipid profiles were composed of the major compounds: phosphatidylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine, major or moderate amounts of diphosphatidylglycerol, two unidentified glycolipids and three unidentified aminolipids. Several minor lipids were also detected. The major fatty acids were either C19 : 0 cyclo v8c or C18 : 1v7c. The results of fatty acid analysis and physiological and biochemical tests allowed both, the genotypic and phenotypic differentiation of the isolates from each other, while the chemotaxonomic traits allowed them to be differentiated from the most closely related genera. In summary, low 16S rRNA gene sequence similarities and marked differences in polar lipid profiles, as well as in polyamine patterns, is suggestive of a novel genus for which the name Pseudochelatococcus gen. nov. is proposed. MPA 1113T (5CCM 8528T5LMG 28286T5CIP 110802T) and MPA 1105T (5CCM 8527T5LMG 28285T) are proposed to be the type strains representing two novel species within the novel genus, Pseudochelatococcus gen. nov., for which the names Pseudochelatococcus lubricantis sp. nov. and Pseudochelatococcus contaminans sp. nov. are suggested, respectively.
Metal working fluids (coolant lubricants) are used in machining processes such as turning, milling, drilling and grinding. Among the functions of these technical fluids is the cooling of pieces of work and tools, providing lubrication, and removal of chips and swarf from the work zone. Water miscible metal working fluids in the form of emulsions or solutions are commonly colonized by bacteria and fungi (Ka¨mpfer et al., 2009a, b, c; Lodders & Ka¨mpfer, 2012). Microbial degradation of components of the metalworking fluids causes a range of problems from the discoloration of the fluid and evolution of
malodour to the loss of the quality of pieces of work and tool failure (Rabenstein et al., 2009). Moreover, high levels of bacterial and fungal contamination can lead to hygiene problems and pose a risk to the health of workers.
3Present address: vaxxinova GmbH, Cuxhaven, Germany.
DNA for 16S rRNA gene sequence analysis was extracted from pure cultures of both strains using a DNeasy Blood & Tissue kit (Qiagen). 16S rRNA gene fragments were amplified using the primers GM3F and GM4R as described
Abbreviations: The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strains MPA 1113T and MPA 1105T are KJ886939 and KJ886940.
067652 G 2015 IUMS
Printed in Great Britain
Two organisms, strains MPA 1105T and MPA 1113T, were isolated from metal working emulsions from tanks of machine tools at a factory in northern Germany using Plate Count agar (Merck KGaA,) and cultivation at 25 uC. Growth was also observed on Pseudomonas agar F (Merck KGaA,) and tryptone soy agar (TSA, Oxoid) at 25 uC.
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previously (Kuever et al., 2001). Sequence analysis was carried out with the sequencing primers 515f and 907rm by LGC Genomics GmbH, Germany. A first BLAST analysis in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) showed that, using a cultivation independent approach to metal working fluid aerosols, several environmental 16S rRNA gene sequences had been detected, which were identical to the 16S rRNA gene sequence of strain MPA 1105T, with one environmental 16S rRNA gene sequence identical to strain MPA 1113T (Perkins & Angenent, 2010). BLAST analysis against the type strain database using the EzTaxon server (http://www.ezbiocloud.net/eztaxon; Kim et al., 2012), however, showed that the 16S rRNA gene sequence similarities with the most closely related type strains of the genus Chelatococcus were below 94 % for both strains. Strains MPA 1113T and MPA 1105T formed beige-coloured colonies on nutrient agar (NA; Oxoid) at 37 uC. Subcultivation for further analyses was carried out on TSA at 28 uC for 48 h. Gram-staining was performed as described by Gerhardt et al. (1994). Cell morphology was observed under a Axioskop 40 Zeiss light microscope at 61000, with cells grown for 3 days at 28 uC on TSA. Phylogenetic analysis was based on nearly full-length 16S rRNA gene sequences of 1406 and 1409 nt for MPA 1113T and MPA 1105T covering 16S rRNA gene sequence positions 24 to 1491 and 22 to 1491 [according to the E. coli numbering given by Brosius et al. (1978)], respectively. Analysis was performed in ARB release 5.2 (Ludwig et al., 2004) and the ‘All-Species Living Tree’ project (LTP; Yarza et al., 2008) database release LTPs115 (March 2014). SINA version 1.2.11 was used to align sequences not included in the database, according to SILVA seed alignment (http://www.arb-silva. de; Pruesse et al., 2012), before sequences were implemented into the ARB database. Phylogenetic trees were reconstructed with the Maximum-likelihood method using RAxML v7.04 (Stamatakis, 2006) with GTR-GAMMA or PhyML (Guindon & Gascuel, 2003) and, the neighbour-joining method with the Jukes–Cantor correction (Jukes & Cantor, 1969) and rapid bootstrap analysis based on 100 resamplings. Phylogenetic trees were calculated based on nucleotide sequence positions 98 and 1449 of the 16S rRNA gene (E. coli numbering according to Brosius et al., 1978). Pairwise 16S rRNA gene sequence similarities were calculated with the ARB Neighbour joining tool without the use of an evolutionary model. The two new strains MPA 1113T and MPA 1105T shared 97.7 % sequence similarity to each other, but below 94 % sequence similarities to type strains of closely related genera, Chelatococcus (92.7–93.8 %), Camelimonas (91.5–92.8 %) and Bosea (91.5–92.7 %). Similarities to type strains of other genera were below 92 %. All phylogenetic trees showed that the two strains formed a distinct cluster, which changed position in the phylogenetic trees depending on the treeing method and algorithm used. In the maximumlikelihood tree, based on RAxML MPA 1113T and MPA 1105T, they were positioned closest to type strains of the 148
genera Chelatococcus and Camelimonas (Fig. 1). In contrast, in the PhyML based maximum-likelihood tree the cluster of the two strains was placed between clusters of the genera Methylobacterium and Bosea and in the Neighbourjoining tree between clusters of the genera Methylobacter, Microvirga and Salinarimonas (data not shown). The unclear phylogenetic placement of the cluster containing the two new strains among clusters of different genera belonging to different families of the Alphaproteobacteria, indicated that there was a problem in clearly assigning the two strains to a family. For quinone, polar lipid and polyamine analyses cells were grown on PYE medium (0.3 % w/v peptone from casein, 0.3 % w/v yeast extract, pH 7.2) at 28 uC. Polar lipids and quinones were extracted and analysed as described previously (Tindall, 1990a, b; Altenburger et al., 1996). Polyamines were extracted and analysed as described by Busse & Auling (1988). HPLC analyses were carried out using the equipment described by Stolz et al. (2007). The polar lipid profiles of strains MPA 1113T and MPA 1105T were similar, but showed a quite rare profile. Strain MPA 1113T contained the major lipids phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine, moderate amounts of two glycolipids (GL1, GL2) and an aminolipid (AL3), and minor amounts of four aminolipids (AL1, AL2, AL4, AL5), and five lipids that were only visible after total polar lipid staining (L1–L5) (Fig. 2a). Strain MPA 1105T differed in showing a polar lipid profile where the glycolipids GL1 and GL2, aminolipids AL1, AL2, AL3 and AL4 were also detected in major amounts, with the presence of another aminolipid (AL6) in moderate amounts and diphosphatidylglycerol present in minor amounts (Fig. 2b). The quinone system of strains MPA 1113T and MPA 1105T contained Q-10 (both 98.0 %) as the predominant ubiquinone. In addition, strain MPA 1113T contained Q-9 (1.3 %), Q-11 (0.7 %) and traces of Q-8, while strain MPA 1105T contained Q-11 (1.2 %), Q-9 (0.7 %) and Q-8 (0.2 %). In the polyamine pattern in both strains spermidine was predominant. Strain MPA 1113T contained per g dry weight: 58.1 mmol spermidine, 8.4 mmol putrescine, 0.6 mmol spermine, 0.5 mmol 1,3-diaminopropane, 0.3 mmol sym-homospermidine and traces of cadaverine (,0.1 mmol). Strain MPA 1105T contained per g dry weight: 34.2 mmol spermidine, 2.1 mmol spermine, 1.5 mmol putrescine, 1.5 mmol 1,3-diaminopropane, 0.4 mmol sym-norspermidine, 0.1 mmol sym-homospermidine and traces of cadaverine (,0.1 mmol). These chemotaxonomic traits agree well with traits already reported for members of the class Alphaproteobacteria but differ to some extent from other related genera of the Alphaproteobacteria (Table 1). Fatty acids analysis was performed as described by Ka¨mpfer & Kroppenstedt (1996). Total cell hydrolysates were generated from biomass after growth on TS agar for 48 h at 28 uC. We assessed the growth of the two strains and observed the colony sizes at 12 h intervals before selecting the time points for generating biomass. Growth (and colony expansion) could be clearly observed after 48 h of incubation International Journal of Systematic and Evolutionary Microbiology 65
Pseudochelatococcus lubricantis gen. nov., sp. nov. 100
10 %
99
72
100
8
100
73
100
45
Methylobacterium Microvirga
2
Salinarimonas
8
Bosea Camelimonas lactis M 2040T (FN430422) Camelimonas abortus UK34/07-5T (FR851926) Chelatococcus daeguensis K106T (EF584507) 100 Chelatococcus sambhunathii HT4T (DQ322070) Chelatococcus asaccharovorans TE2T (AJ294349) 100 Pseudochelatococcus lubricantis MPA 1113T (KJ886939) Pseudochelatocuccus contaminans MPA 1105T (KJ886940) 100 2 Rhodoblastus 100
Methylovirgula ligni DSM 19998T (FM252034) Methylocapsa acidiphila B2T (AJ278726) Methylocapsa aurea KYGT (FN433469) Methyloferula stellata AR4T (FR686343) 77
89
6
3
72
Beijerinckia Methylocella Methylorosula polaris V22T (EU586035)
6 Methylocystis Methylosinus sporium NCIMB 11126T (Y18946) 95 Methylosinus trichosporium OB3bT (Y18947) 98 5 Rhodoplanes 83 100 3 Blastochloris 100 7 100 88 100
Kaistia
Pseudolabrys taiwanensis CC-BB4T (DQ062742)
100 7 Labrys Aminobacter aminovorans DSM 7048T (AJ011759) Mesorhizobium loti LMG 6125T (X67229) Phyllobacterium myrsinacearum STM 948T (AY785315)
Fig. 1. Maximum-likelihood tree based on nearly full-length 16S rRNA gene sequences showing the phylogenetic positions of strains MPA 1113T and MPA 1105T among type strains of the closest related genera. Numbers at branch notes represent Bootstrap values (.70 %) based on 100 resamplings. Numbers in cluster-boxes represent the number of type strains included in the clusters. Aminobacter aminovorans DSM 7048T, Mesorhizobium loti LMG 6125T, and Phyllobacterium myrsinacearum STM 948T were used as outgroups. Bar, 10 % divergence.
compared with 60 h and 72 h of incubation. Significant differences were found between the fatty acid profiles of the two strains. MPA 1113T contained C18 : 1v7c (86 %) as the major fatty acid, followed by C19 : 0 cyclo v8c (4.9 %), summed feature 2 (comprising 3-OH C14 : 0/C16 : 0 Iso I, 4.8 %) and C16 : 0 (4.3 %). MPA 1105T contained C19 : 0 cyclo v8c (91.3 %) as the major fatty acid, followed by C18 : 1v7c (4.7 %) and minor amounts of summed feature 2 (comprising 3-OH C14 : 0/C16 : 0 Iso I, 2.0 %) and C16 : 0 (2.0 %). In contrast to the fatty acid profiles of those reported for species of the genera Camelimonas, Chelatococcus, Bosea, Salinarimonas and Microvirga, hydroxyl fatty acids were absent in the profiles of MPA 1113T and MPA 1105T (Table 1). Detailed physiological characterization of strains MPA 1113T and MPA 1105T was performed as described previously http://ijs.sgmjournals.org
(Ka¨mpfer et al., 1991). The presence of urease was determined as recommended by the manufacturer (Merck), according to Christensen (1946). Results of the physiological characterizations are given in the species description. The two strains could be differentiated from their utilization of a few of the carbon substrates tested. D-Fructose (weakly), adipate (weakly), azelate, DL-3-hydroxybutyrate, itaconate, suberate, L-alanine, b-alanine, D-histidine, L-aspartate and T L-proline, could only be assimilated by MPA 1113 . T Furthermore, MPA 1113 grew in the presence of 1–5 % (w/v) NaCl, but strain MPA 1105T only in the presence of 1–3 % (w/v) NaCl. For further genotypic characterization, high molecular mass genomic DNA was extracted using the method described by Pitcher et al. (1989). The genomic DNA G+C mol% content of strains MPA 1113T and MPA 1105T was 149
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(a)
L1
GL1
(b)
L1 GL1
DPG GL2
GL2 PME
AL1 AL2 AL3 AL4 AL5 L3
DPG
PME AL1 AL2
L4 PG
L5
PG
AL3 AL4
PC L2
AL6
AL5
L3 L5
L2
PC
L4
Fig. 2. Polar lipid profile of MPA 1113T (a) and MPA 1105T (b) after two-dimensional TLC and detection with molybdophosphoric acid. PME, phosphatidylmonomethylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PC, phosphatidylcholine; AL1–6, unidentified aminolipids; GL 1 and 2, unidentified glycolipids; L1–5, unidentified polar lipids.
determined as described previously (Urdiain et al., 2008) and was 58.2 (+/20.5) and 53.3 (+/20.1) mol% for MPA 1113T and MPA 1105T, respectively.
phosphatidylglycerol and phosphatidylcholine and moderate to major amounts of two glycolipids. Major fatty acids are C19 : 0 cyclo v8c and/or C18 : 1v7c.
DNA–DNA hybridization experiments were performed between the two strains, MPA 1113T and MPA 1105T, using the method described by Ziemke et al. (1998). The hybridization results gave a value of 49.2 % (37.1 %) (results of reciprocal analysis in parentheses), indicating that the two strains are representitives of two different species.
The G+C content of the DNA is different for different species of the genus, 58.2 (+/20.5) (type species) and 53.3 (+/20.1) mol%. The type species is Pseudochelatococcus lubricantis.
On the basis of the polyphasic approach we propose a novel genus with the name Pseudochelatococcus gen. nov. with two novel species, Pseudochelatococcus lubricantis sp. nov. and Pseudochelatococcus contaminans sp. nov., with type strains MPA 1113T and MPA 1105T, respectively.
Description of Pseudochelatococcus lubricantis sp. nov. Pseudochelatococcus lubricantis [lu.bri.can’tis. L. v. lubricare to lubricate; N.L. n. lubricans, -antis (from L. part. adj. lubricans) a lubricant; N.L. gen. lubricantis of/from a (coolant) lubricant].
Cells are non-motile, non-spore-forming rods (approx. 2 mm in length and 1 mm in width). Gram-stain-negative, oxidasepositive, showing an oxidative metabolism. Good growth occurs on R2A, TSA, PYE, and nutrient agar at 25–30 uC. Beige, translucent and shiny colonies with entire edges are formed within 24 h, with a diameter of approximately 2 mm. Predominant compound in the polyamine pattern is spermidine. The quinone system is ubiquinone Q-10. The polar lipid profile is composed of the major lipids phosphatidylmonomethylethanolamine, diphosphatidylglycerol,
Cell and colony morphology and growth characteristics are as given in the genus description. Growth is detected on TSA at temperatures ranging from 4 uC to 50 uC (weak). No growth is observed at 0 uC and 55 uC. Growth is also positive in TS broth at pH values ranging from 5.5–10.5. Growth is observed in TS broth containing (in addition) 1–5 % (w/v) NaCl. Beige, translucent and shiny colonies with entire edges are formed within 24 h, with a diameter of approximately 2 mm. The quinone system and polyamine pattern are in agreement with the genus description. In addition to the lipids listed in the genus description moderate amounts of an aminolipid (AL3) and minor amounts of four aminolipids (AL1, AL2, AL4, AL5) and five lipids only visible after total polar lipid staining (L1- L5) are present. The fatty acid profile is dominated by C18 : 1v7c and contains a minor amount of C19 : 0 cyclo v8c. Utilizes the following carbon
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International Journal of Systematic and Evolutionary Microbiology 65
Description of Pseudochelatococcus gen. nov. Pseudochelatococcus (Pseu.do.che.la.to.coc’cus. Gr. adj. pseudeˆs false; Chelatococcus a genus name; N.L. masc. n. Pseudochelatococcus the false Chelatococcus).
Pseudochelatococcus lubricantis gen. nov., sp. nov.
Table 1. Traits distinguishing Pseudochelatococcus gen. nov. from the related genera Camelimonas, Chelatococcus, Methylobacterium, Bosea, Salinarimonas and Microvirga Taxa: 1, Pseudochelatococcus; 2, Camelimonas, 3, Chelatococcus; 4, Methylobacterium; 5, Bosea; 6, Salinarimonas; 7, Microvirga. +, Positive; 2, negative; NR, not recorded; PUT, putrescine; SPD, spermidine; HSPD, sym-homospermindine; PE, phosphatidylethanolamine; PME, phosphatidylmonomethylethanolamine; GL 1 and 2, unidentified glycolipids. Characteristic
1
2
Hydroxyfatty acid present: C16 : 0 3-OH – – – – C18 : 1 2-OH – + C18 : 0 3-OH Major polyamine: PUT +/2 + SPD + + HSPD – – Polar lipids: PE – + PME + +/2 + – Glycolipids, GL1 and GL2 , present
3*
4D
5d
6§
7I
– + +
– – +/2
+ – –
– – +
– – +/2
+ + +
+/2 – +
NR
NR
NR
NR
NR
NR
NR
NR
NR
– + –
NR
+ – –
– + –
+ + –
NR NR
*Data from Auling et al. (1993), Yoon et al. (2008). DData from Busse & Auling (1988); Auling et al. (1991); Tani et al. (2012a, b); Weon et al. (2008); Veyisoglu et al. (2013). dDas et al. (1996). §Liu et al. (2010). IWeon et al. (2010); Ardley et al. (2012); Radl et al. (2014). For chromatographic motilities of glycolipids GL1 and GL2 see Fig. 2.
sources: D-fructose (weakly), acetate, propionate, 4-aminobutyrate, adipate (weakly), azelate, fumarate, glutarate, DL-3hydroxybutyrate, itaconate, DL-lactate, L-malate, pyruvate, suberate, L-alanine, b-alanine, D-histidine, L-aspartate and Lproline. Does not utilize D-glucose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose, D-galactose, gluconate, D-mannose, maltose, alpha-melibiose, L-rhamnose, D-ribose, salicin, sucrose, trehalose, D-xylose, adonitol, inositol, maltitol, D-mannitol, D-sorbitol, cis-aconitate, trans-aconitate, mesaconate, oxoglutarate, putrescine, L-leucine, L-ornithine, L-phenylalanine, L-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzate. Hydrolyses L-alanine-pNA (pNA5para-nitroanilide) and 2-deoxythymidine-59-pNP-phosphate and does not hydrolyse: aesculin, oNP-b-D-galactopyranoside (oNP5orthonitrophenyl-), pNP-b-D-glucuronide (pNP5para-nitrophenyl-), pNP-alpha-D-glucopyranoside, pNP-b-D-glucopyranoside, pNP-b-D-xyloside, and L-glutamate-gamma-3-carboxypNA. The type strain MPA 1113T (5CCM 8528T5LMG 28286T) was isolated from coolant lubricants. The G+C content of the genomic DNA of the type strain is 58.2 mol% (+/20.5). http://ijs.sgmjournals.org
Description of Pseudochelatococcus contaminans sp. nov. Pseudochelatococcus contaminans [con.ta’mi.nans. L. part. adj. contaminans contaminating, polluting, isolated as a contaminant of an industrial coolant lubricant]. Cell and colony morphology and growth characteristics are as given in the genus description. Growth is detected on TSA at temperatures ranging from 4 uC to 50 uC (weak). No growth is observed at 0 uC and 55 uC. Growth is also positive in TS broth at pH values ranging from 5.5–10.5. Growth is observed in TS broth containing (in addition) 1–3 % (w/v) NaCl. Beige, translucent and shiny colonies with entire edges are formed within 24 h, with a diameter of approximately 2 mm. The quinone system and polyamine pattern are in agreement with the genus description. In addition to the lipids listed in the genus description aminolipids AL1, AL2, AL3, AL4 and AL5 and five lipids only visible after total polar lipid staining (L1-L5) are detected in major amounts; another aminolipid (AL6) is detected in moderate amounts and diphosphatidylglycerol is present in minor amounts. The fatty acid profile is predominated by C19 : 0 cyclo v8c and contains minor amounts of C18 : 1v7c. Utilizes the following carbon sources: acetate, propionate, 4-aminobutyrate, fumarate, glutarate, DL-lactate, L-malate and pyruvate. Does not utilize D-glucose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose, D-fructose, D-galactose, gluconate, Dmannose, maltose, alpha-melibiose, L-rhamnose, D-ribose, salicin, sucrose, trehalose, D-xylose, adonitol, inositol, maltitol, D-mannitol, D-sorbitol, cis-aconitate, trans-aconitate, adipate, azelate, DL-3hydroxybutyrate, itaconate, mesaconate, oxoglutarate, putrescine, suberate, L-alanine, b-alanine, D-histidine, L-aspartate, L-proline, L-leucine, Lornithine, L-phenylalanine, L-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzoate. Hydrolyses: L-alanine-pNA (pNA 5para-nitroanilide) and 2-deoxythymidine-59-pNP-phosphate (weakly). Does not hydrolyse: aesculin, oNP-b-Dgalactopyranoside (oNP5ortho-nitrophenyl-), pNP-b-Dglucuronide (pNP5para-nitrophenyl-), pNP-alpha-D-glucopyranoside, pNP-b-D-glucopyranoside, pNP-b-D-xyloside and L-glutamate-gamma-3-carboxy-pNA. The type strain MPA 1105T (5CCM 8527T5LMG 28285T) was isolated from coolant lubricants. The G+C content of the genomic DNA of the type strain is 53.3 mol%(+/20.1).
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from a metal-working fluid. Int J Syst Evol Microbiol 59, 2464– 2467. Ka¨mpfer, P., Lodders, N., Warfolomeow, I., Falsen, E. & Busse, H. J. (2009b). Corynebacterium lubricantis sp. nov., isolated from a coolant
lubricant. Int J Syst Evol Microbiol 59, 1112–1115. Ka¨mpfer, P., Lodders, N., Warfolomeow, I. & Busse, H.-J. (2009c).
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http://ijs.sgmjournals.org
Ziemke, F., Ho¨fle, M. G., Lalucat, J. & Rossello´-Mora, R. (1998).
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DOI 10.1099/ijs.0.067652-0
Pseudochelatococcus lubricantis gen. nov., sp. nov. and Pseudochelatococcus contaminans sp. nov. from coolant lubricants Peter Ka¨mpfer,1 Stefanie P. Glaeser,1 Marco Gra¨ber,23 Andreas Rabenstein,2 Jan Kuever2 and Hans-Ju¨rgen Busse3 1
Correspondence
Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, Germany
Peter Ka¨mpfer [email protected]
2
Bremen Institute for Materials Testing, Department of Microbiology, Bremen, Germany
3
Institut fu¨r Bakteriologie, Mykologie und Hygiene, Veterina¨rmedizinische Universita¨t, A-1210 Wien, Austria
Two Gram-negative, rod-shaped, non-spore-forming bacteria, isolated from metal working fluids were investigated to determine their taxonomic positions. On the basis of 16S rRNA gene sequence phylogeny, both strains (MPA 1113T and MPA 1105T) formed a distinct cluster with 97.7 % sequence similarity between them, which was in the vicinity of members of the genera Methylobacterium, Camelimonas, Chelatococcus, Bosea, Salinarimonas and Microvirga to which they showed low sequence similarities (below 94 %). The predominant compounds in the polyamine pattern and in the quinone system of the two strains were spermidine and ubiquinone Q-10, respectively. The polar lipid profiles were composed of the major compounds: phosphatidylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine, major or moderate amounts of diphosphatidylglycerol, two unidentified glycolipids and three unidentified aminolipids. Several minor lipids were also detected. The major fatty acids were either C19 : 0 cyclo v8c or C18 : 1v7c. The results of fatty acid analysis and physiological and biochemical tests allowed both, the genotypic and phenotypic differentiation of the isolates from each other, while the chemotaxonomic traits allowed them to be differentiated from the most closely related genera. In summary, low 16S rRNA gene sequence similarities and marked differences in polar lipid profiles, as well as in polyamine patterns, is suggestive of a novel genus for which the name Pseudochelatococcus gen. nov. is proposed. MPA 1113T (5CCM 8528T5LMG 28286T5CIP 110802T) and MPA 1105T (5CCM 8527T5LMG 28285T) are proposed to be the type strains representing two novel species within the novel genus, Pseudochelatococcus gen. nov., for which the names Pseudochelatococcus lubricantis sp. nov. and Pseudochelatococcus contaminans sp. nov. are suggested, respectively.
Metal working fluids (coolant lubricants) are used in machining processes such as turning, milling, drilling and grinding. Among the functions of these technical fluids is the cooling of pieces of work and tools, providing lubrication, and removal of chips and swarf from the work zone. Water miscible metal working fluids in the form of emulsions or solutions are commonly colonized by bacteria and fungi (Ka¨mpfer et al., 2009a, b, c; Lodders & Ka¨mpfer, 2012). Microbial degradation of components of the metalworking fluids causes a range of problems from the discoloration of the fluid and evolution of
malodour to the loss of the quality of pieces of work and tool failure (Rabenstein et al., 2009). Moreover, high levels of bacterial and fungal contamination can lead to hygiene problems and pose a risk to the health of workers.
3Present address: vaxxinova GmbH, Cuxhaven, Germany.
DNA for 16S rRNA gene sequence analysis was extracted from pure cultures of both strains using a DNeasy Blood & Tissue kit (Qiagen). 16S rRNA gene fragments were amplified using the primers GM3F and GM4R as described
Abbreviations: The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strains MPA 1113T and MPA 1105T are KJ886939 and KJ886940.
067652 G 2015 IUMS
Printed in Great Britain
Two organisms, strains MPA 1105T and MPA 1113T, were isolated from metal working emulsions from tanks of machine tools at a factory in northern Germany using Plate Count agar (Merck KGaA,) and cultivation at 25 uC. Growth was also observed on Pseudomonas agar F (Merck KGaA,) and tryptone soy agar (TSA, Oxoid) at 25 uC.
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previously (Kuever et al., 2001). Sequence analysis was carried out with the sequencing primers 515f and 907rm by LGC Genomics GmbH, Germany. A first BLAST analysis in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) showed that, using a cultivation independent approach to metal working fluid aerosols, several environmental 16S rRNA gene sequences had been detected, which were identical to the 16S rRNA gene sequence of strain MPA 1105T, with one environmental 16S rRNA gene sequence identical to strain MPA 1113T (Perkins & Angenent, 2010). BLAST analysis against the type strain database using the EzTaxon server (http://www.ezbiocloud.net/eztaxon; Kim et al., 2012), however, showed that the 16S rRNA gene sequence similarities with the most closely related type strains of the genus Chelatococcus were below 94 % for both strains. Strains MPA 1113T and MPA 1105T formed beige-coloured colonies on nutrient agar (NA; Oxoid) at 37 uC. Subcultivation for further analyses was carried out on TSA at 28 uC for 48 h. Gram-staining was performed as described by Gerhardt et al. (1994). Cell morphology was observed under a Axioskop 40 Zeiss light microscope at 61000, with cells grown for 3 days at 28 uC on TSA. Phylogenetic analysis was based on nearly full-length 16S rRNA gene sequences of 1406 and 1409 nt for MPA 1113T and MPA 1105T covering 16S rRNA gene sequence positions 24 to 1491 and 22 to 1491 [according to the E. coli numbering given by Brosius et al. (1978)], respectively. Analysis was performed in ARB release 5.2 (Ludwig et al., 2004) and the ‘All-Species Living Tree’ project (LTP; Yarza et al., 2008) database release LTPs115 (March 2014). SINA version 1.2.11 was used to align sequences not included in the database, according to SILVA seed alignment (http://www.arb-silva. de; Pruesse et al., 2012), before sequences were implemented into the ARB database. Phylogenetic trees were reconstructed with the Maximum-likelihood method using RAxML v7.04 (Stamatakis, 2006) with GTR-GAMMA or PhyML (Guindon & Gascuel, 2003) and, the neighbour-joining method with the Jukes–Cantor correction (Jukes & Cantor, 1969) and rapid bootstrap analysis based on 100 resamplings. Phylogenetic trees were calculated based on nucleotide sequence positions 98 and 1449 of the 16S rRNA gene (E. coli numbering according to Brosius et al., 1978). Pairwise 16S rRNA gene sequence similarities were calculated with the ARB Neighbour joining tool without the use of an evolutionary model. The two new strains MPA 1113T and MPA 1105T shared 97.7 % sequence similarity to each other, but below 94 % sequence similarities to type strains of closely related genera, Chelatococcus (92.7–93.8 %), Camelimonas (91.5–92.8 %) and Bosea (91.5–92.7 %). Similarities to type strains of other genera were below 92 %. All phylogenetic trees showed that the two strains formed a distinct cluster, which changed position in the phylogenetic trees depending on the treeing method and algorithm used. In the maximumlikelihood tree, based on RAxML MPA 1113T and MPA 1105T, they were positioned closest to type strains of the 148
genera Chelatococcus and Camelimonas (Fig. 1). In contrast, in the PhyML based maximum-likelihood tree the cluster of the two strains was placed between clusters of the genera Methylobacterium and Bosea and in the Neighbourjoining tree between clusters of the genera Methylobacter, Microvirga and Salinarimonas (data not shown). The unclear phylogenetic placement of the cluster containing the two new strains among clusters of different genera belonging to different families of the Alphaproteobacteria, indicated that there was a problem in clearly assigning the two strains to a family. For quinone, polar lipid and polyamine analyses cells were grown on PYE medium (0.3 % w/v peptone from casein, 0.3 % w/v yeast extract, pH 7.2) at 28 uC. Polar lipids and quinones were extracted and analysed as described previously (Tindall, 1990a, b; Altenburger et al., 1996). Polyamines were extracted and analysed as described by Busse & Auling (1988). HPLC analyses were carried out using the equipment described by Stolz et al. (2007). The polar lipid profiles of strains MPA 1113T and MPA 1105T were similar, but showed a quite rare profile. Strain MPA 1113T contained the major lipids phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine, moderate amounts of two glycolipids (GL1, GL2) and an aminolipid (AL3), and minor amounts of four aminolipids (AL1, AL2, AL4, AL5), and five lipids that were only visible after total polar lipid staining (L1–L5) (Fig. 2a). Strain MPA 1105T differed in showing a polar lipid profile where the glycolipids GL1 and GL2, aminolipids AL1, AL2, AL3 and AL4 were also detected in major amounts, with the presence of another aminolipid (AL6) in moderate amounts and diphosphatidylglycerol present in minor amounts (Fig. 2b). The quinone system of strains MPA 1113T and MPA 1105T contained Q-10 (both 98.0 %) as the predominant ubiquinone. In addition, strain MPA 1113T contained Q-9 (1.3 %), Q-11 (0.7 %) and traces of Q-8, while strain MPA 1105T contained Q-11 (1.2 %), Q-9 (0.7 %) and Q-8 (0.2 %). In the polyamine pattern in both strains spermidine was predominant. Strain MPA 1113T contained per g dry weight: 58.1 mmol spermidine, 8.4 mmol putrescine, 0.6 mmol spermine, 0.5 mmol 1,3-diaminopropane, 0.3 mmol sym-homospermidine and traces of cadaverine (,0.1 mmol). Strain MPA 1105T contained per g dry weight: 34.2 mmol spermidine, 2.1 mmol spermine, 1.5 mmol putrescine, 1.5 mmol 1,3-diaminopropane, 0.4 mmol sym-norspermidine, 0.1 mmol sym-homospermidine and traces of cadaverine (,0.1 mmol). These chemotaxonomic traits agree well with traits already reported for members of the class Alphaproteobacteria but differ to some extent from other related genera of the Alphaproteobacteria (Table 1). Fatty acids analysis was performed as described by Ka¨mpfer & Kroppenstedt (1996). Total cell hydrolysates were generated from biomass after growth on TS agar for 48 h at 28 uC. We assessed the growth of the two strains and observed the colony sizes at 12 h intervals before selecting the time points for generating biomass. Growth (and colony expansion) could be clearly observed after 48 h of incubation International Journal of Systematic and Evolutionary Microbiology 65
Pseudochelatococcus lubricantis gen. nov., sp. nov. 100
10 %
99
72
100
8
100
73
100
45
Methylobacterium Microvirga
2
Salinarimonas
8
Bosea Camelimonas lactis M 2040T (FN430422) Camelimonas abortus UK34/07-5T (FR851926) Chelatococcus daeguensis K106T (EF584507) 100 Chelatococcus sambhunathii HT4T (DQ322070) Chelatococcus asaccharovorans TE2T (AJ294349) 100 Pseudochelatococcus lubricantis MPA 1113T (KJ886939) Pseudochelatocuccus contaminans MPA 1105T (KJ886940) 100 2 Rhodoblastus 100
Methylovirgula ligni DSM 19998T (FM252034) Methylocapsa acidiphila B2T (AJ278726) Methylocapsa aurea KYGT (FN433469) Methyloferula stellata AR4T (FR686343) 77
89
6
3
72
Beijerinckia Methylocella Methylorosula polaris V22T (EU586035)
6 Methylocystis Methylosinus sporium NCIMB 11126T (Y18946) 95 Methylosinus trichosporium OB3bT (Y18947) 98 5 Rhodoplanes 83 100 3 Blastochloris 100 7 100 88 100
Kaistia
Pseudolabrys taiwanensis CC-BB4T (DQ062742)
100 7 Labrys Aminobacter aminovorans DSM 7048T (AJ011759) Mesorhizobium loti LMG 6125T (X67229) Phyllobacterium myrsinacearum STM 948T (AY785315)
Fig. 1. Maximum-likelihood tree based on nearly full-length 16S rRNA gene sequences showing the phylogenetic positions of strains MPA 1113T and MPA 1105T among type strains of the closest related genera. Numbers at branch notes represent Bootstrap values (.70 %) based on 100 resamplings. Numbers in cluster-boxes represent the number of type strains included in the clusters. Aminobacter aminovorans DSM 7048T, Mesorhizobium loti LMG 6125T, and Phyllobacterium myrsinacearum STM 948T were used as outgroups. Bar, 10 % divergence.
compared with 60 h and 72 h of incubation. Significant differences were found between the fatty acid profiles of the two strains. MPA 1113T contained C18 : 1v7c (86 %) as the major fatty acid, followed by C19 : 0 cyclo v8c (4.9 %), summed feature 2 (comprising 3-OH C14 : 0/C16 : 0 Iso I, 4.8 %) and C16 : 0 (4.3 %). MPA 1105T contained C19 : 0 cyclo v8c (91.3 %) as the major fatty acid, followed by C18 : 1v7c (4.7 %) and minor amounts of summed feature 2 (comprising 3-OH C14 : 0/C16 : 0 Iso I, 2.0 %) and C16 : 0 (2.0 %). In contrast to the fatty acid profiles of those reported for species of the genera Camelimonas, Chelatococcus, Bosea, Salinarimonas and Microvirga, hydroxyl fatty acids were absent in the profiles of MPA 1113T and MPA 1105T (Table 1). Detailed physiological characterization of strains MPA 1113T and MPA 1105T was performed as described previously http://ijs.sgmjournals.org
(Ka¨mpfer et al., 1991). The presence of urease was determined as recommended by the manufacturer (Merck), according to Christensen (1946). Results of the physiological characterizations are given in the species description. The two strains could be differentiated from their utilization of a few of the carbon substrates tested. D-Fructose (weakly), adipate (weakly), azelate, DL-3-hydroxybutyrate, itaconate, suberate, L-alanine, b-alanine, D-histidine, L-aspartate and T L-proline, could only be assimilated by MPA 1113 . T Furthermore, MPA 1113 grew in the presence of 1–5 % (w/v) NaCl, but strain MPA 1105T only in the presence of 1–3 % (w/v) NaCl. For further genotypic characterization, high molecular mass genomic DNA was extracted using the method described by Pitcher et al. (1989). The genomic DNA G+C mol% content of strains MPA 1113T and MPA 1105T was 149
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(a)
L1
GL1
(b)
L1 GL1
DPG GL2
GL2 PME
AL1 AL2 AL3 AL4 AL5 L3
DPG
PME AL1 AL2
L4 PG
L5
PG
AL3 AL4
PC L2
AL6
AL5
L3 L5
L2
PC
L4
Fig. 2. Polar lipid profile of MPA 1113T (a) and MPA 1105T (b) after two-dimensional TLC and detection with molybdophosphoric acid. PME, phosphatidylmonomethylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PC, phosphatidylcholine; AL1–6, unidentified aminolipids; GL 1 and 2, unidentified glycolipids; L1–5, unidentified polar lipids.
determined as described previously (Urdiain et al., 2008) and was 58.2 (+/20.5) and 53.3 (+/20.1) mol% for MPA 1113T and MPA 1105T, respectively.
phosphatidylglycerol and phosphatidylcholine and moderate to major amounts of two glycolipids. Major fatty acids are C19 : 0 cyclo v8c and/or C18 : 1v7c.
DNA–DNA hybridization experiments were performed between the two strains, MPA 1113T and MPA 1105T, using the method described by Ziemke et al. (1998). The hybridization results gave a value of 49.2 % (37.1 %) (results of reciprocal analysis in parentheses), indicating that the two strains are representitives of two different species.
The G+C content of the DNA is different for different species of the genus, 58.2 (+/20.5) (type species) and 53.3 (+/20.1) mol%. The type species is Pseudochelatococcus lubricantis.
On the basis of the polyphasic approach we propose a novel genus with the name Pseudochelatococcus gen. nov. with two novel species, Pseudochelatococcus lubricantis sp. nov. and Pseudochelatococcus contaminans sp. nov., with type strains MPA 1113T and MPA 1105T, respectively.
Description of Pseudochelatococcus lubricantis sp. nov. Pseudochelatococcus lubricantis [lu.bri.can’tis. L. v. lubricare to lubricate; N.L. n. lubricans, -antis (from L. part. adj. lubricans) a lubricant; N.L. gen. lubricantis of/from a (coolant) lubricant].
Cells are non-motile, non-spore-forming rods (approx. 2 mm in length and 1 mm in width). Gram-stain-negative, oxidasepositive, showing an oxidative metabolism. Good growth occurs on R2A, TSA, PYE, and nutrient agar at 25–30 uC. Beige, translucent and shiny colonies with entire edges are formed within 24 h, with a diameter of approximately 2 mm. Predominant compound in the polyamine pattern is spermidine. The quinone system is ubiquinone Q-10. The polar lipid profile is composed of the major lipids phosphatidylmonomethylethanolamine, diphosphatidylglycerol,
Cell and colony morphology and growth characteristics are as given in the genus description. Growth is detected on TSA at temperatures ranging from 4 uC to 50 uC (weak). No growth is observed at 0 uC and 55 uC. Growth is also positive in TS broth at pH values ranging from 5.5–10.5. Growth is observed in TS broth containing (in addition) 1–5 % (w/v) NaCl. Beige, translucent and shiny colonies with entire edges are formed within 24 h, with a diameter of approximately 2 mm. The quinone system and polyamine pattern are in agreement with the genus description. In addition to the lipids listed in the genus description moderate amounts of an aminolipid (AL3) and minor amounts of four aminolipids (AL1, AL2, AL4, AL5) and five lipids only visible after total polar lipid staining (L1- L5) are present. The fatty acid profile is dominated by C18 : 1v7c and contains a minor amount of C19 : 0 cyclo v8c. Utilizes the following carbon
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Description of Pseudochelatococcus gen. nov. Pseudochelatococcus (Pseu.do.che.la.to.coc’cus. Gr. adj. pseudeˆs false; Chelatococcus a genus name; N.L. masc. n. Pseudochelatococcus the false Chelatococcus).
Pseudochelatococcus lubricantis gen. nov., sp. nov.
Table 1. Traits distinguishing Pseudochelatococcus gen. nov. from the related genera Camelimonas, Chelatococcus, Methylobacterium, Bosea, Salinarimonas and Microvirga Taxa: 1, Pseudochelatococcus; 2, Camelimonas, 3, Chelatococcus; 4, Methylobacterium; 5, Bosea; 6, Salinarimonas; 7, Microvirga. +, Positive; 2, negative; NR, not recorded; PUT, putrescine; SPD, spermidine; HSPD, sym-homospermindine; PE, phosphatidylethanolamine; PME, phosphatidylmonomethylethanolamine; GL 1 and 2, unidentified glycolipids. Characteristic
1
2
Hydroxyfatty acid present: C16 : 0 3-OH – – – – C18 : 1 2-OH – + C18 : 0 3-OH Major polyamine: PUT +/2 + SPD + + HSPD – – Polar lipids: PE – + PME + +/2 + – Glycolipids, GL1 and GL2 , present
3*
4D
5d
6§
7I
– + +
– – +/2
+ – –
– – +
– – +/2
+ + +
+/2 – +
NR
NR
NR
NR
NR
NR
NR
NR
NR
– + –
NR
+ – –
– + –
+ + –
NR NR
*Data from Auling et al. (1993), Yoon et al. (2008). DData from Busse & Auling (1988); Auling et al. (1991); Tani et al. (2012a, b); Weon et al. (2008); Veyisoglu et al. (2013). dDas et al. (1996). §Liu et al. (2010). IWeon et al. (2010); Ardley et al. (2012); Radl et al. (2014). For chromatographic motilities of glycolipids GL1 and GL2 see Fig. 2.
sources: D-fructose (weakly), acetate, propionate, 4-aminobutyrate, adipate (weakly), azelate, fumarate, glutarate, DL-3hydroxybutyrate, itaconate, DL-lactate, L-malate, pyruvate, suberate, L-alanine, b-alanine, D-histidine, L-aspartate and Lproline. Does not utilize D-glucose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose, D-galactose, gluconate, D-mannose, maltose, alpha-melibiose, L-rhamnose, D-ribose, salicin, sucrose, trehalose, D-xylose, adonitol, inositol, maltitol, D-mannitol, D-sorbitol, cis-aconitate, trans-aconitate, mesaconate, oxoglutarate, putrescine, L-leucine, L-ornithine, L-phenylalanine, L-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzate. Hydrolyses L-alanine-pNA (pNA5para-nitroanilide) and 2-deoxythymidine-59-pNP-phosphate and does not hydrolyse: aesculin, oNP-b-D-galactopyranoside (oNP5orthonitrophenyl-), pNP-b-D-glucuronide (pNP5para-nitrophenyl-), pNP-alpha-D-glucopyranoside, pNP-b-D-glucopyranoside, pNP-b-D-xyloside, and L-glutamate-gamma-3-carboxypNA. The type strain MPA 1113T (5CCM 8528T5LMG 28286T) was isolated from coolant lubricants. The G+C content of the genomic DNA of the type strain is 58.2 mol% (+/20.5). http://ijs.sgmjournals.org
Description of Pseudochelatococcus contaminans sp. nov. Pseudochelatococcus contaminans [con.ta’mi.nans. L. part. adj. contaminans contaminating, polluting, isolated as a contaminant of an industrial coolant lubricant]. Cell and colony morphology and growth characteristics are as given in the genus description. Growth is detected on TSA at temperatures ranging from 4 uC to 50 uC (weak). No growth is observed at 0 uC and 55 uC. Growth is also positive in TS broth at pH values ranging from 5.5–10.5. Growth is observed in TS broth containing (in addition) 1–3 % (w/v) NaCl. Beige, translucent and shiny colonies with entire edges are formed within 24 h, with a diameter of approximately 2 mm. The quinone system and polyamine pattern are in agreement with the genus description. In addition to the lipids listed in the genus description aminolipids AL1, AL2, AL3, AL4 and AL5 and five lipids only visible after total polar lipid staining (L1-L5) are detected in major amounts; another aminolipid (AL6) is detected in moderate amounts and diphosphatidylglycerol is present in minor amounts. The fatty acid profile is predominated by C19 : 0 cyclo v8c and contains minor amounts of C18 : 1v7c. Utilizes the following carbon sources: acetate, propionate, 4-aminobutyrate, fumarate, glutarate, DL-lactate, L-malate and pyruvate. Does not utilize D-glucose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose, D-fructose, D-galactose, gluconate, Dmannose, maltose, alpha-melibiose, L-rhamnose, D-ribose, salicin, sucrose, trehalose, D-xylose, adonitol, inositol, maltitol, D-mannitol, D-sorbitol, cis-aconitate, trans-aconitate, adipate, azelate, DL-3hydroxybutyrate, itaconate, mesaconate, oxoglutarate, putrescine, suberate, L-alanine, b-alanine, D-histidine, L-aspartate, L-proline, L-leucine, Lornithine, L-phenylalanine, L-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzoate. Hydrolyses: L-alanine-pNA (pNA 5para-nitroanilide) and 2-deoxythymidine-59-pNP-phosphate (weakly). Does not hydrolyse: aesculin, oNP-b-Dgalactopyranoside (oNP5ortho-nitrophenyl-), pNP-b-Dglucuronide (pNP5para-nitrophenyl-), pNP-alpha-D-glucopyranoside, pNP-b-D-glucopyranoside, pNP-b-D-xyloside and L-glutamate-gamma-3-carboxy-pNA. The type strain MPA 1105T (5CCM 8527T5LMG 28285T) was isolated from coolant lubricants. The G+C content of the genomic DNA of the type strain is 53.3 mol%(+/20.1).
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