Simkania Negevensis Strain ZT: Growth, Antigenic and Genome Characteristics
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International Journal of Systematic Bacteriology (1 999), 49,815-820 Printed in Great Britain Simkania negevensis strain ZT: growth, antigenic and genome characteristics Simona Kahane,' Karin D. E. Everett,2t Nina Kimmell and Maureen G. Friedmad Author for correspondence : Maureen G. Friedman. Tel : + 972 7 640 0867. Fax : + 972 7 627 62 15 e-mail : [email protected] 1 Department of Virology, Simkania negevensis is the type species of Simkaniaceae, a recently proposed Faculty of Health Sciences, family in the order Chlamydiales. In the current study, growth, antigenic and Ben Gurion University of the Negev, Beer Sheva, genomic characteristics of this intracellular bacterium were investigated and Israel compared to those of members of the family Chlamydiaceae. Growth of the * Avian and Swine organism, as assessed by infectivity assays, reached a plateau in 2-3 d Respiratory Diseases although by light microscopy the cytopathic effect on the host cells increased Research Unit, National for 12 or more days after infection. 5. negevensis growth was unaffected by Animal Disease Center, Agricultural Research sulfadiazine. Cells infected by 5. negevensis strain ZT were not recognized by Service, US Department of either of two monoclonal antibodies specific for Chlamydiaceae LPS and Agriculture, Ames, IA several specific Chlamydiaceae ompA primers were unable to PCR amplify a 5. 50010, USA negevensis gene. The 5. negevensis genome contained one copy of the ribosomal operon. The genome size of 5. negevensis strain ZT was determined by PFGE to be 1.7 Mbp, and the G+C content was 42.5 molo/o. These data, taken together with other published data, are consistent with the proposal that 5. negevensis belongs to a distinct family in the order Chlamydiales. Keywords : Chlamydiales, Simkaniaceae, rDNA copy number, genome size, Chlamydia INTRODUCTION Amann et al., 1997; Everett & Andersen, 1997; Pettersson et al., 1997; Everett et al., 1999). Simkania negevensis, ' previously characterized as the While it is evident that S. negevensis shares a common chlamydia-like micro-organism Z, ' is related to other ancestor with the Chlamydiaceae, a well-studied family Chlamydiales species in the order based on its rDNA in the Chlamydiales, S. negevensis differs from sequence and intracellular, chlamydia-like develop- Chlamydiaceae et al., et al., members of the in that it is resistant to mental cycle (Kahane 1993, 1995; Everett ampicillin, penicillin G and cyclosporin (Kahane et al., 1999). The family Simkaniaceae currently includes Chlamydia ZT, Simkania 1993). Antibody prepared against GroEL only strain which is the type strain of cross-reacts with S. negevensis GroEL, but no other negevensis, and the description of the family is derived ZT serological cross-reaction between these bacteria has from current information about the strain as been documented (Kahane et al., 1993). In addition, compared with other families in the Chlamydiales. The S. nege- the extrachromosomal plasmid that is present in some full-length 16s and 23s rRNA sequences of Chlamydiaceae strains is absent from S. negevensis vensis strain ZT are 80-87 % identical to those of other et al., Chlamydiaceae Chlamydiales et al., (Kahane 1993). In contrast to members of the (Everett 1999). and all known bacteria, S. negevensis has a group I Phylogenetic analyses of the ribosomal sequences have 23s S. et al., intron in the rRNA (K. D. E. Everett, Kahane, been published previously (Kahane 1995; R. M. Bush & M. G. Friedman, unpublished results). S. negevensis was isolated as a culture contaminant .. .. , .. , . , . , .. , . , . , .. , . , . , . , ,, . , , +Present address: Department of Medical Microbiology and Parasit- from human and simian Cells. s. negevensis is Similar to ology, College of Veterinary Medicine, University of Georgia, Athens, GA other chlamydia1 species in that it is a probable 30602-7371, USA. aetiologic agent of human disease. It has been impli- Abbreviations: EB, elementary body; IFU, infectious-centre-forming unit. cated in cases of bronchiolitis in infants (Kahane et al., The GenBank accession number for the 235 rRNA sequence of Sirnkania 1998) and is associated serologically with community- negevensis strain ZT is U68460 acquired pneumonia in adults (Lieberman et al., 1997). 00932 0 1999 IUMS 81 5 S. Kahane and others There is evidence that human exposure to S. negevensis with chromogen (Savyon Diagnostics), and slides were is widespread (Kahane et al., 1996; Friedman et al., examined for stained cells by light microscopy. FITC-stained 1999). The purpose of this study was to provide an slides were examined with a Zeiss 9901 fluorescence micro- extended description of the family Simkaniaceae by scope. determining additional growth, antigenic and genomic DNA base composition. The DNA G+C content of the S. characteristics of the type strain of S. negevensis. negevensis genome was determined by the thermal de- naturation method (Stanton et al., 1997). DNA was pre- METHODS pared carefully from EBs to minimize shearing. Renografin- purified EBs were suspended in 30 mM Tris, 10 mM EDTA, Bacterial strains. The bacterial strains used in these studies 50 mM DTT, pH 9.0, and lysed for 1 h at 37 "C. Nonidet P- were S. negevensis strain ZT (ATCC VR 1471*) (Kahane et 40 was then added to 1 %, along with 0.5 mg DNase-free al., 1993), Chlamydophila pneumoniae strain TW- 183T RNase ml-' (Boehringer Mannheim), and incubation was (Washington Research Foundation), C. trachomatis strain continued for 1 h at 37 "C. Proteinase K (GibcoBRL Life D/UW-3/CX (H. D. Caldwell, NIAID Rocky Mountain Technologies) was added to 0.2 mg ml-' and the lysate was Laboratory, Hamilton, MT, USA) and strain L2/434/BU incubated overnight at 37 "C. The DNA was then se- (Dr. R. Cevenini, Department of Microbiology, University quentially extracted with TE buffer (10 mM Tris, 10 mM of Bologna, Italy), Chlamydia muridarum strain MoPnT (H. EDTA, pH 8.0) saturated with phenol, phenol/chloro- D. Caldwell), Chlamydia suis strain R22 (NADC), and form/isoamyl alcohol (24: 24: 1, by vol.) and chloroform, Chlamydophila psittaci strain NJl and strain GD (from and finally dialysed extensively at 4 "C against 1 x SSC NADC) (Everett & Andersen, 1997). buffer (0.15 M NaCl, 15 mM sodium citrate, pH 7.0). Con- Growth characteristics of 5. negevensis. S. negevensis strain trol DNA was prepared from Renografin-purified EBs of C. ZTwas grown in Vero cells (ATCC CCL 81) obtained from psittaci strains NJ1 and GD using CsCl gradient isolation the American Type Culture Collection (ATCC, Manassas, and ultracentrifugation. Melting temperatures were deter- VA, USA) in RPMT medium (Biological Industries) con- mined using the Beckman DU 650 Spectrometer, the taining 15 YOfoetal bovine serum (FBS), 1 % glucose, 100 Beckman High Performance Temperature Controller, and units penicillin ml-', 100 pg gentamicin ml-', 160 pg vanco- software that calculated the first derivative and the 2-point mycin ml-l and 1 pg cycloheximide ml-'. To test for mean (Beckman). The S. negevensis G + C content was the sulfadiazine resistance, some experiments were duplicated in mean of three experiments. the presence of 2-100 pg sulfadiazine ml-' (Sigma). In some Analysis of the genome size of 5. negevensis strain ZT, by experiments, glucose-free medium was used for the growth PFGE. S. negevensis was grown in Vero cells and EBs were of cells infected by strain ZT.Glucose-free medium consisted purified by the Renografin method as described above. Prior of special order preparations of RPMI containing no glucose to the Renografin gradient step, cellular DNA was removed (Biological Industries) that were supplemented with dialysed by treatment with bovine pancreatic DNase (Amersham ; FBS (1 5 YO)and with antibiotics as above. Whole cells were 300 U ml-' containing 5 mM MgSO,, 30 min, 37 "C). Agar- removed from the flasks with glass beads, and after mild ose blocks for pulsed field gel electrophoresis experiments sonication, elementary bodies (EBs) that served as inoculum were prepared by mixing equal volumes of 2 Oh low-melting- for growth curves and DNA preparation were purified using point (50 "C) agarose (Bio-Rad) and purified S. negevensis Renografin (Solvay Animal Health) by the method of EBs (approx. 1 pg in each plug). After overnight incubation Caldwell et al. (1981). For growth curves, monolayers of of the agarose blocks at room temperature with 30 mM Tris, Vero cells in 25 cm2flasks were inoculated with S. negevensis 10 mM EDTA, 50 mM DTT, pH 9-0, RNA was digested EBs at an m.0.i. of 0.5. At various times after infection, cells with DNase-free RNase in TE buffer. Proteinase K digestion were scraped into the medium with 1 mm diameter glass was carried out overnight in TE buffer and was followed by beads, and the suspension was frozen at - 70 "C with 50 YO extensive washing of the plugs in TE buffer at room FBS. Infectious-centre-forming units (IFUs) were deter- temperature for several days. Control plugs containing DNA mined for each time point by titration in 10-fold dilutions on from C. muridarum MoPnT, C. suis R22 and C. trachomatis Vero cell monolayers plated in 96-well cell culture plates. D/UW-3/CX were prepared similarly, except that pellets of Detection of IFUs was done with an immunoperoxidase Chlamydia and Chlamydia-infected cells were prepared as assay using rabbit antisera raised against S. negevensis described in Everett et al. (1999). (Kahane et al., 1998). The plugs were inserted into a 1 % (w/v) agarose gel Reaction of 5. negevensis with Chlamydiaceae group-specific x x mAbs. Monolayers of Vero cells were infected with S. (1 2.5 12.5 1-2 cm) and electrophoresed at 150 V, negevensis strain ZT, Chlamydia trachomatis strain L2/434/ 14 "C using the CHEF-DR Drive Module, Pulsewave BU, or C. pneumoniae strain TW-183T. Fully intact mono- 760, and power supply model 200/2.0 (Bio-Rad) with layers showing clear evidence of chlamydial inclusions were recirculating buffer containing 22.5 mM Tris-borate, released by light trypsinization after 5 d (S.