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. $The GenBank accession numbers for the 16S rRNA gene and DNA gyrase subunit B (gyrB) sequence of P. zucineum HLK1T, and Isolation the gyrB sequence for P. lituiforme are AY628697, DQ311666, and DQ311665, respectively. ÃCorresponding author. Tel.: +86 571 87783656. K562 cells were cultured in RPMI-1640 com- E-mail address: [email protected] (X. Hu). plete culture medium as described by us previously 1These authors contribute equally to this work. [17,18]. About 1 million cells were plated onto blood 0723-2020/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2006.07.002 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.