Sp. Nov., a Facultative Intracellular Bacterium

中国科技论文在线 http://www.paper.edu.cn ARTICLE IN PRESS

Systematic and Applied Microbiology 30 (2007) 207–212 www.elsevier.de/syapm

Phenylobacterium zucineum sp. nov., a facultative intracellular bacterium isolated from a human erythroleukemia cell line K562$ Kun Zhanga,1, Weidong Hana,1, Rong Zhangb, Xiaoli Xua, Qiangrong Pana, Xun Hua,Ã aCancer Institute, The Second Afﬁliated Hospital, Zhejiang University Medical School, 88 Jiefang Road, Hangzhou 310009, PR China bDepartment of Clinical Microbiology, The Second Afﬁliated Hospital, Zhejiang University Medical School, 88 Jiefang Road, Hangzhou 310009, PR China

Received 11 July 2006

Abstract

A bacterial strain HLK1T was isolated from the human erythroleukemia cell line K562. This bacterium is a Gram-negative rod, motile with a polar ﬂagellum. It is strictly aerobic, nonfermentative, and oxidase and catalase positive. Its optimal growth occurs at 37 1C at pH between 6.5 and 7.5. Phylogenetically, although it shares 98% similarity with the 16S rRNA of Phenylobacterium lituiforme, the DNA–DNA hybridization value between the two species is only 43%. HLK1T has a DNA G+C content of 71.270.2 mol%. It is a facultative intracellular organism and may have pathogenic relevance with humans and mammals. On the basis of the phylogenetic and phenotypic characterization, strain HLK1T is proposed to be classiﬁed in the genus Phenylobacterium,asP. zucineum sp. nov. The type strain is HLK1T ( ¼ CGMCC 1.3786T,DSM¼ 18354). r 2006 Elsevier GmbH. All rights reserved.

Keywords: Phenylobacterium zucineum; Intracellular organism; Leukemia cell line

Introduction were observed on the blood agar plated with the cells but not with the medium. It is interesting to note that the K562 is a human leukemia cell line that is used in the K562 cells in the culture grow healthily with perfect laboratories all over the world. During karyotyping K562 morphology without any signs of bacterial contamina- using Giemsa staining, to our surprise, we repeatedly tion. It would be quite unusual if the cells indeed carry observed rod-bacteria-like structures within some of the intracellular bacteria, because most of the known cells. We then checked if the culture was contaminated by intracellular microbes in human cells eventually damage bacteria, as this can occur in cell culture. We collected the and kill host cells after invasion [5]. On the basis of these medium and cells from the same culture and plated them observations, we hypothesized that the K562 cells may separately onto the blood agar plate. Bacterial colonies carry an as yet unidentiﬁed intracellular bacterial species.

$The GenBank accession numbers for the 16S rRNA gene and DNA gyrase subunit B (gyrB) sequence of P. zucineum HLK1T, and Isolation the gyrB sequence for P. lituiforme are AY628697, DQ311666, and DQ311665, respectively. ÃCorresponding author. Tel.: +86 571 87783656. K562 cells were cultured in RPMI-1640 com- E-mail address: [email protected] (X. Hu). plete culture medium as described by us previously 1These authors contribute equally to this work. [17,18]. About 1 million cells were plated onto blood

转载中国科技论文在线 http://www.paper.edu.cn ARTICLE IN PRESS 208 K. Zhang et al. / Systematic and Applied Microbiology 30 (2007) 207–212

agar (5% sheep erythrocytes) and incubated in a 16S rRNA, gyrB sequencing, and DNA–DNA humidified incubator at 37 1C for 3 days until bacterial hybridization colonies were observable. A single colony was picked to re-plate onto blood agar to purify the strain, and the About 0.3 mg of purified DNA was subjected to PCR purified strain was then grown in LB (Luria–Bertani) amplification of 16S rRNA or gyrB gene as described broth and labeled HLK1T. previously [2,20,22]. The PCR product (1387 bp for 16S rRNA or 1277 bp for gyrB) was then subcloned into pGEM-T-Easy vector (Promega, Madison, MI, USA) Morphology and physiological characteristics according to the manufacturer’s instruction, and sequenced using ABI Prism 377 DNA Sequencer. For DNA-hybridization assay, genomic DNA was extracted The morphology of HLK1T was examined by scan- from cell pellets of HLK1T or P. lituiforme Fail3T ning and transmission electron microscope. The motility according to Marmur [15]. DNA–DNA hybridization of cells was determined by phase-contrast microscopy. was carried out as described previously [4] with the Spore-forming assay was performed according to the modifications by Huss et al. [7] and Escara and Hutton methods reported previously [10]. The bacterium was [6], by using a model 2600 spectrophotometer equipped studied by standard conventional biochemical methods with a model 2527-R thermoprogrammer and plotter [16] and the API 20NE test kit (BioMerieux, Marcy (Gilford Instrument Laboratories, Inc., Oberlin, Ohio). l’Etoile, France) by strictly following the manufacturer’s DNA renaturation rates were computed with the instructions. HLK1T is a Gram-negative rod with TRANSFER.BAS program [8]. varying sizes of 0.3–0.5 mm in diameter and 0.5–2 mm The 16S rRNA sequence of HLK1T was compared in length (Supplementary Fig. 1). HLK1T is motile with with the known sequences in the GenBank/EMBL/ a polar flagellum (Supplementary Fig. 1). The growth DDBJ databases. The Blast results listed a closest match and biochemical features of this strain are described in with the 16S rRNA of species in the genus Phenylo- the section of description of Phenylobacterium zucineum bacterium bacteria. The phylogenetic relationships sp. nov. between the isolated strain and closely related bacteria For comparison purposes, we evaluated the ability of were determined using MEGA2 software [19]. Wood- strain HLK1T and type strain of P. lituiforme strain T sholea maritima was taken as an outgroup. A distance Fail3 (DSM 14363 T) to utilize L-phenylalanine as a matrix was generated under the assumptions of Kimura single carbon source. Growth was determined over a 2-parameter [11]. A dendrogram was inferred from this 96 h incubation period at 37 1C by measuring growth at matrix using the neighbor-joining method. Bootstrap OD and the formation of tyrosine using the modi- 600 replicates were performed to estimate the node relia- fied method of Lowry et al. [13].HLK1T could use bility of the dendrogram obtained. The bootstrap values L-phenylalanine as the sole carbon source, similar to the were obtained from 1000 trees generated with the strain of P. lituiforme Fail3T in a RouF’s minimal MEGA2 software package. The phylogenetic relation- medium containing either 0.06% or 0.006% yeast ship of HLK1T with the other bacteria is illustrated in extract [9]. Fig. 1. The phylogenetic tree was also confirmed by using the Fitch–Margoliash, maximum-likelihood, and maximum-parsimony methods. G+C content of DNA Strain HLK1T shares 95.0%, 95.6%, 95.9%, and 98.1% identity of the 16S rRNA sequences with Genomic DNA of HLK1T was prepared using a P. immobile (Y18216), P. falsum (AJ717391), P. koreense bacterial genomic DNA purification kit (V-Gene Bio- (AB166881) and P. lituiforme (AY534887). Therefore, tech., Hangzhou, China) according to the manufac- HLK1T could be categorized into the genus Phenylo- turer’s instructions and the G+C content of the DNA bacterium based on the identity of the 16S rRNA. was determined by HPLC [14], using a reverse-phase HLK1T is strikingly similar to Fail3T with 98.1% TM Atlantis dC18 column attached to HEWLETT Series identity of 16S rRNA sequence. In order to confirm if 1100 HPLC system. The DNA G+C content of HLK1T HLK1T is the same species as Fail3T or not, we was 71.270.2 mol%. In this assay, we used Escherchia performed DNA–DNA hybridization. The DNA hybri- coli K-12 strain as a reference. The DNA G+C% dization value between the two species is only 43%, content of E. coli K-12 strain was 49.670.3, con- which clearly separates HLK1T from Fail3T. In addition, sistent with the previous results [3]. Recently, we HLK1T is distinct from Fail3T in the following aspects. have started a whole genome sequencing project for Firstly, HLK1T shares low (89%) similarity of gyrB HLK1T, and the G+C content based on the 100% with Fail3T; secondly, HLK1T is a facultative intracel- completed whole genomic sequence (4.2 megabases) is lular organism, whereas the other is not; thirdly, 70.9 mol%. they display significantly different NaCl tolerance; 中国科技论文在线 http://www.paper.edu.cn ARTICLE IN PRESS K. Zhang et al. / Systematic and Applied Microbiology 30 (2007) 207–212 209

Fig. 1. Phylogenetic relationship of P. zucineum and related species within the family Caulobacteraceae, based on 16S rRNA sequences. The tree was created by the neighbor-joining method; percentage numbers at nodes are levels of bootstrap support from 1000 resampled data sets. Solid circles indicate that the corresponding nodes were also recovered in Fitch–Margoliash, maximum likelihood and maximum-parsimony trees. Woodsholea maritima (accession number AJ578476) was used as the outgroup. Scale bar represents 1 nucleotide substitution per 100 nucleotides.

fourthly, while HLK1T is obligately aerobic, Fail3T is which were isolated from subsurface aquifer, alkaline facultatively anaerobic. Further differences are listed in groundwater, soil, and activated sludge of a wastewater Table 1. The results, therefore, indicated that these 2 treatment plant, respectively. To date, no evidence strains are the distinct species but with close phylogenetic demonstrated that these microbes have any association relationship. with eukaryotic cells. HLK1T, therefore, represents an only species so far in the genus Phenylobacterium that can infect and survive in human cells, and may have The invasion of Hela cells by HLK1T and Fail3T pathogenic relevance to human.

We used electron and confocal microscopy to examine the internalization of the bacteria into the cells (Fig. 2 Emended description of Phenylobacterium and Supplementary Fig. 2). With the increment of the incubation time, the numbers of cells with internalized Lingens et al. 1985 bacteria increased. At 24 h after infection, 100% cells T The properties of the genus are those given by Lingens were observed to have intracellular HLK1 , confirming T et al. [12], Kanso and Patel [9], Tiago et al. [21] and that HLK1 is a facultative intracellular bacterium. On T Aslam [1]. It has to be expanded as follows: the other hand, Fail3 was not observed within Hela The DNA base ratio of Phenylobacterium is cells under the same experimental condition using 64–72 mol% G+C. Isolated from soil, water, or transmission EM technologies. P. zucineum In order to confirm that the observed bacteria in Hela eukaryotes. is the only species which is T isolated from eukaryotes. cells were HLK1 , the cells were plated onto blood agar to isolate the bacteria. We compared the bacterial colonies with those of the original HLK1T, both of which demonstrated the same colony features. Further, Description of P. zucineum sp. nov. we randomly picked colonies isolated from Hela cells subjected for 16S rRNA sequencing, which confirmed P. zucineum (N.L. arbitrary n. zuci, acronym of the the identity of HLK1T. Zhejiang University Cancer Institute; N.L. neut. adj. The genus Phenylobacterium was firstly described by zucineum, belonging to the Zhejiang University Cancer Lingens et al. [12] and at present comprises four species, Institute). P. lituiforme (FaiI3T) [9], P. falsum (AC49T) [21], Strain HLK1T is a facultative intracellular organism. P. immobile (ET) [12],andP. koreense (Slu-01T) [1], HLK1T grows at 25–47 1C, optimally at 37 1C and does 中国科技论文在线 http://www.paper.edu.cn ARTICLE IN PRESS 210 K. Zhang et al. / Systematic and Applied Microbiology 30 (2007) 207–212

Table 1. Comparison of selected characteristics of P. zucineum HLK1T with the type strains of P. lituiforme (FaiI3T), P. falsum (AC49T), P. immobile (ET) and P. koreense (Slu-01T)

Characteristics HLK1T P. lituiformea P. falsumb P. immobilec P. koreensed

Isolation source K562 cell Water from Alkaline Soil Activated sludge subsurface aquifer groundwater Pigmentation Gray Yellow to pale- Colorless Colorless Colorless yellow Motility + + – – – Flagella Single, polar Single, polar None None None O2 requirement Obligate aerobes Facultative Obligate aerobes Obligate aerobes Obligate aerobes anaerobe Optimum growth 37 40–41 30–35 28–30 28–30 temperature (1C) pH growth range 6–9, optimal at 6–9, optimal at 6–10, optimal at 6.5–8, optimal at 6.5–8.5, optimal at 6.5–7.5 6–6.5 7.5–8 6.8–7 7–8 NaCl growth range Optimal at 1% Optimal at 0% Optimal at 0% Optimal at 0% Optimal at 0% No growth at 6% No growth at 1% No growth at 8% No growth at 1% No growth at 3% Urease (+) + b-Galactosidase – ND + ND – Oxidase + – + (+) + Hydrolyze gelatin (+) + Esculin hydrolysis (+) – ND Nitrate reduction + + + Utilization of Glucose + + – – – Sucrose – + – – – Acetate – + ND – + Alanine – ND + ND – Citrate – + – – – L-Phenylalanine + + – + + Facultative + ND ND ND intracellular G+C content 71.270.2 66.5 66.9 65.5 68.1 (mol%)

+, positive; (+), positive, weak or delayed response; –, negative; ND, not deﬁned. aData from Kanso and Patel [9]. bData from Tiago [21]. cData from Lingens et al. [12]. dData from Aslam et al. [1].

Fig. 2. The representative electron micrograph of intracellular HLK1T in Hela cells. Cells were incubated with HLK1T with MOI of 1:200 for 24 h and collected for electron microscopic examination as described in Materials and methods. Bar, 1 mm. 中国科技论文在线 http://www.paper.edu.cn ARTICLE IN PRESS K. Zhang et al. / Systematic and Applied Microbiology 30 (2007) 207–212 211

not grow at 4 1C. It grows well in nutrient broth and (Department of Infectious Diseases, Center for Mole- MacConkey agar but not on Salmonella–Shigella Agar. cular Microbiology and Infection, Imperial College of It grows in nutrient broth with 0–4% (optimally at 1%) Technology, Science, and Medicine, United Kingdom) NaCl, but not with 6% NaCl. Cells cultured in LB broth for critical reading of the manuscript. or in heart-infusion broth occur singly, in pairs, or in short chains. HLK1T has no hemolytic activities on blood agar. Colonies on blood agar are grayish, punctate, circular and convex. The surface of the colony Appendix A. Supplementary materials is rough, dry and opaque. The whole colony could be moved entirely. The online version of this article contains additional HLK1T is a Gram-negative rod with a size of supplementary data. Please visit doi:10.1016/j.syapm. 0.3–0.5 mm 0.5–2 mm. The non-spore forming cells are 2006.07.002. motile with a unipolar flagellum. Biochemically, Positive reactions are observed for catalase, oxidase, nitrate reduction, assimilation of References glucose, suberate, 3-hydroxybutyrate, L-proline and L- phenylalanine. The strain is weakly positive for esculin [1] Z. Aslam, W.T. Im, L.N. Ten, S.T. Lee, P. koreense sp. nov., isolated from South Korea, Int. J. Syst. Evol. and gelatin hydrolysis. It possesses weak urease activity, Microbiol. 55 (2005) 2001–2005. and gives a delayed positive result for oxidization of [2] B.L. Beaman, L. Beaman, Nocardia species: host–parasite glucose, maltose, fructose and D-galactose. The strain relationships, Clin. Microbiol. Rev. 7 (1994) 213–264. yields negative reactions in tests for DNase, arginine [3] F.R. Blattner, G. Plunkett, C.A. Bloch, N.T. Perna, V. dihydrolase, lysine decarboxylase, b-galactosidase, or- Burland, M. Riley, J. Collado-Vides, J.D. Glasner, C.K. nithine decarboxylase, and test of hydrogen disulfide Rode, G.F. Mayhew, J. Gregor, N.W. Davis, H.A. (H2S) in Kligler iron agar, indole production, methyl Kirkpatrick, M.A. Goeden, D.J. Rose, B. Mau, Y. Shao, red, Voges–Proskauer. It does not utilize arabinose, The complete genome sequence of Escherichia coli K-12, mannose, mannitol, N-acetyl glucosamine, maltose, Science 277 (1997) 1453–1474. gluconate, adipate, malate, citrate, phenyl-acetate, [4] J. De Ley, H. Cattoir, A. Reynaerts, The quantitative caprate, rhamnose, ribose, inositol, sucrose, itacomic measurement of DNA hybridization from renaturation rates, Eur. J. Biochem. 12 (1970) 133–142. acid, suberate, malonate, acetates, D-lactate, L-alanine, [5] D.A. Drevets, P.J. Leenen, R.A. Greenfield, Invasion of salicin, melibiose, L-fucose, D-sorbieirte, propionate, the central nervous system by intracellular bacteria, Clin. valerate, histidine, 5-keto-gluconate, glycogen, 4-hydro- Microbiol. Rev. 17 (2004) 323–347. xybenzoate, 2-keto gluconate, p-hydroxybenzoate, [6] J.F. Escara, J.R. Hutton, Thermal stability and renatura- L-serine. It can neither oxidize nor ferment glucose tion of DNA in dimethyl sulfoxide solutions: acceleration D-xylose, lactose, sucrose, arabinose, cellobiose, rham- of the renaturation rate, Biopolymers 19 (1980) nose, mannitol, sorbitol, and salicin. 1315–1327. The DNA G+C content is 7171 mol%. The type [7] V.A.R. Huss, H. Festl, K.-H. Schleifer, Studies on the strain, HLK1T (CGMCC 1.3786T, DSM 18354), was spectrophotometric determination of DNA hybridization isolated from a human erythroleukemia cell line K562. from renaturation rates, Syst. Appl. Microbiol. 4 (1983) 184–192. [8] K.-D. Jahnke, Basic computer program for evaluation of spectroscopic DNA renaturation data from GILFORD Acknowledgments system 2600 spectrometer on a PC/XT/AT type personal computer, J. Microbiol. Methods 15 (1992) 61–73. This investigation was supported in part by Cheung [9] S. Kanso, B.K. Patel, P. lituiforme sp. nov., a moderately Kong Scholars Programme (National Ministry of thermophilic bacterium from a subsurface aquifer, and Education, China, and Li Ka Shing Foundation, Hong emended description of the genus Phenylobacterium, Int. Kong) to XH, Zhejiang Natural Sciences Foundation J. Syst. Evol. Microbiol. 54 (2004) 2141–2146. Grant, China (R204204) to XH, and the Bureau For [10] S. Kanso, B.K. Patel, Microvirga subterranea gen. nov., Sciences and Technologies Grant (2005E10006), Zhe- sp. nov., a moderate thermophile from a deep subsurface jiang, China, to XH. We are indebted to Prof. J. Euze´by Australian thermal aquifer, Int. J. Syst. Evol. Microbiol. (Ecole National Veterinaire, Toulouse, France) and Prof. 53 (2003) 401–406. [11] M. Kimura, A simple method for estimating evolu- Hans G. Truper (Institut fur Mikrobiologie & Biotech- ¨ ¨ tionary rates of base substitutions through comparative nologie, Universitaet Bonn, Germany) for the new studies of nucleotide sequences, J. Mol. Evol. 16 (1980) organism’s nomenclature. We thank Dr. X. Dong and 111–120. Mr. C. Wu (Institute of Microbiology, Chinese Academy [12] F. Lingens, R. Blecher, H. Blecher, F. Blobel, J. Eberspa of Sciences, Beijing, China) for technical assistance cher, C. Fro hner, H. Go risch, G. Layh, P. immobile gen. of DNA–DNA hybridization, and Dr. C. Tang nov., sp. nov., a Gram-negative bacterium that degrades 中国科技论文在线 http://www.paper.edu.cn ARTICLE IN PRESS 212 K. Zhang et al. / Systematic and Applied Microbiology 30 (2007) 207–212

the herbicide chloridazon, Int. J. Syst. Bacteriol. 35 (1985) P-glycoprotein, Cancer Chemother. Pharmacol. (2005) 26–39. 1–8. [13] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, [19] S. Kumar, K. Tamura, I.B. Jakobsen, M. Nei, MEGA2: Protein measurement with the Folin phenol reagent, molecular evolutionary genetics analysis software, Bioin- J. Biol. Chem. 193 (1951) 265–275. formatics 17 (2001) 1244–1245. [14] M. Mesbah, U. Premachandran, W.B. Whitman, Precise [20] R.K. Saiki, D.H. Gelfand, S. Stoffel, S.J. Scharf, measurement of the G+C content of deoxyribonucleic R. Higuchi, G.T. Horn, K.B. Mullis, H.A. Erlich, acid by high-performance liquid chromatography, Int. J. Primer-directed enzymatic ampliﬁcation of DNA with a Syst. Bacteriol. 39 (1989) 159–167. thermostable DNA polymerase, Science 239 (1988) [15] J. Marmur, A procedure for the isolation of deoxyribo- 487–491. nucleic acid from micro-organisms, J. Mol. Biol. 3 (1961) [21] I. Tiago, V. Mendes, C. Pires, P.V. Morais, A. Verssimo, 208–218. P. falsum sp. nov., an Alphaproteobacterium isolated from [16] P.R. Murray, E.J. Baro, J.H. Jorgensen, M.A. Pfaller, a nonsaline alkaline groundwater, and emended descrip- R.H.E. Yolken (Eds.), Manual of Clinical Micro- tion of the genus Phenylobacterium, Syst. Appl. Micro- biology, eighth ed., ASM Press, Washington, DC, 2003. biol. 28 (2005) 295–302. [17] Q. Pan, T. Wang, Q. Lu, X. Hu, Schisandrin B – a novel [22] S. Yamamoto, S. Harayama, PCR ampliﬁcation and inhibitor of P-glycoprotein, Biochem. Biophys. Res. direct sequencing of gyrB genes with universal primers Commun. 335 (2005) 406–411. and their application to the detection and taxonomic [18] P. Qiangrong, T. Wang, Q. Lu, X. Hu, Dibenzocyclooc- analysis of Pseudomonas putida strains, Appl. Environ. tadiene lingnans: a class of novel inhibitors of Microbiol. 61 (1995) 3768.