Bacterial purification and DNA extraction
R. felis (strain California 2) was cultivated on XTC cells growing on RPMI with 5% fetal bovine serum, supplemented with 5 mM L-glutamine. The purification of the bacteria was performed by different steps. First, the bacteria were treated in the presence of 1% trypsine in K36 buffer for 1 h at 37 °C, then centrifuged and digested by DNAseI for 1 h at 37 °C to reduce the eukaryotic DNA contamination. The sample was loaded on a renograffin gradient and the bands of the purified bacteria were washed in K36, treated again by DNAseI. After inactivation with EDTA (50 mM), the bacteria were resuspended in TE, dispatched in 150-μl tubes and stored at −80 °C. Depending on this initial concentration, one or two tubes were diluted in 1 ml of TNE (10 mM Tris [pH 7.5], 150 mM NaCl, 2 mM EDTA) and incubated for 5 h at 37 °C in the presence of lysozyme (2 mg/ml). Lysis was performed for 2 h at 37 °C by adding 1% SDS and RNAseI (25 μg/ml). Overnight treatment with 1 mg/ml of proteinase K followed at 37 °C. After three phenol–chloroform extractions and alcoholic precipitation, the DNA was resuspended in 30 μl of TE and its concentration was estimated by agarose gel electrophoresis.
Pulsed-field agarose gel electrophoresis
The concentrated bacterial suspension was included in 1% (vol/vol) Incert agarose gel blocks (BMA, Rockland, Maryland, United States). The agarose blocks were digested by Proteinase K (1 mg/ml) (Eurobio Laboratories, Paris, France) in 1% lauroylsarcosine and 0.5 M EDTA (pH 8) (Sigma-Aldrich, St. Louis, Missouri, United States) for 24 h at 50 °C. Fresh Proteinase K was then added and the incubation was continued for 24 h. The blocks were then washed twice in TE (pH 7.6) for 30 min at room temperature. Proteinase K inactivation was performed through incubation in a 4% phenylmethylsulfonyl fluoride (MBI Fermentas, Burlington, Canada) solution for 1 h at 50 °C. This inactivation step was carried out twice. The blocks were then washed two to three times in TE and stored in 0.5 M EDTA (pH 8) at 4 °C. Before restriction enzyme digestion, the agarose blocks were equilibrated twice with TE for 15 min. Digestion was carried out for 4 h, then fresh enzyme was added and the incubation was continued overnight. The digested agarose blocks and molecular-weight markers (Low Range PFG Marker, Lambda Ladder PFG Marker [New England Biolabs, Beverly, Massachusetts, United States]) were equilibrated in 0.5× TBE (50 mM Tris, 50 mM boric acid, 1 mM EDTA).
Each agarose block was laid in a 1% PFEG agarose (Sigma-Aldrich) solution in 0.5× TBE. Pulsed-field gel electrophoresis was carried out on a CHEF-DR II device (Bio-Rad, Hercules, California, United States) under different electrophoresis conditions. The 1% agarose gel was run at 200 V using ramped pulse times from 1 to 5 s for 10 h to observe the pattern of small DNA fragments (2–48 kb). The migration was taking place under the following two consecutive conditions: (i) a ramping time from 3 to 10 s at 200 V for 12 h, with the pattern representative for 48- to 242-kb fragments, then (ii) a ramping time from 20 to 40 s at 180 V for 15 h, with the pattern representative for 145- to 610-kb fragments.
Shotgun of R. felis genome and sequencing strategy
Three shotgun genomic libraries were constructed by mechanical shearing of the genomic DNA using a Hydroshear device (GeneMachine, http://genome.nhgri.nih.gov/genemachine/
). DNA fragments were blunt-ended using T4 DNA polymerase (New England Biolabs) and ligated to the BstXI adapter. Fragments of 3, 4.5, and 7 kb were separated on a preparative agarose gel (FMC BioProducts, Rockland, Maryland, United States), extracted with Qiaquick kit (Qiagen, Valencia, California, United States), and ligated into pCDNA2.1 (Invitrogen, Carlsbad, California, United States) for the two smaller inserts and into pCNS (a low copy number vector; C. R., unpublished data) for the largest one. DNA cloning was performed using electrocompetent E. coli
DH10B Electromax cells (Invitrogen). Plasmid DNAs were purified and pools of 96 clones were analyzed by gel electrophoresis to validate the libraries. DNA sequencing of insert ends was carried out using Big Dye 3.1 terminator chemistry on an automated capillary ABI3700 sequencer (Applied Biosystems, Foster City, California, United States).
Sequences were analyzed and assembled into contigs using Phred, Phrap, and Consed software [51
] taking all sequences into account. Sequences were considered valid when at least 75% of the nucleotides had a Phred score of more than 20. The finishing of the genome sequencing included only additional directed reactions that were performed on an ABI3100 sequencer. Two circular plasmid molecules of 63 and 38 kbp, respectively, were identified from the assembled sequences. On the chromosome, three small regions of 41, 155, and 64 bp failed by dropping of sequence. A number of parameters (DMSO, glycerol, hybridization, and elongation temperature) were tested one by one or were combined to sequence over these gaps. We finally succeeded with the association of another type of chemistry, D
-rhodamine with 2 M betaine. We designed and used 420 primers (i) to close the sequencing gaps by walking either on shotgun subclones or on the chromosome and (ii) to improve sequence regions of low quality.
The integrity of the assembly was validated by comparing the restriction patterns obtained by pulsed-field gel electrophoresis with those deduced from the electronic consensus sequence. The selection of restriction enzymes was based on rare sites. We analyzed single digests of R. felis DNA. The main restriction enzymes used for these studies were ApaI, AfeI, FspI, and SbfI. This comparative study confirmed the predicted length of the R. felis DNA fragments.
The structures for pRF and pRFδ plasmids were controlled by specific primer amplifications (see Figure S1
). Three PCRs were performed and the amplification results were in agreement with the expected hypothesis. These PCR results validate the two distinct plasmid forms (62.8 and 39 kbp, respectively). Meanwhile, a Southern blot was performed through a pulsed-field electrophoresis gel. Uncut genomic R. felis
DNA and R. felis
DNA digested by the restriction enzyme PvuI (corresponding to a unique site in the pRF-specific region) were analyzed. These blocks of DNA were loaded twice onto the gel with the molecular-weight markers: Lambda Marker (Bio-Rad) and Low Range PFG Marker (New England Biolabs) as described above, with a pulse time from 1 to 5 s for 12 h at 180 V. The gel was treated and transferred onto Hybond N+ (Amersham Biosciences, Little Chalfont, United Kingdom) with a vacuum blot. The DNA was fixed by heating for 2 h at 80 °C, and the membrane was cut into two pieces. Two probes were derived from two PCR products. The first, pRFh–pRFi (726 bp), was designed within the pRF-specific insert, and the second, pRFa–pRFg (251 bp), was designed to encompass the deletion site of the pRFδ. These two probes were labeled with dCTP32
and hybridized at 65 °C for 17 h on each membrane. Membranes were washed three times in 1× SSC and 0.1% SDS at 65 °C. The exposure time ranged from 6 h to overnight at −80 °C on ECL film. The hybridizations were clearly established on R. felis
digested by PvuI and led to one signal with the pRFh–pRFi probe and two signals for the two plasmid structures with the pRFa–pRFg probe at a predicted molecular weight compatible with our prediction (see Figure S2
We tested 30 samples of fleas naturally infected by R. felis obtained from different geographic areas (Algeria [11 fleas], France [15 fleas], and New Zealand [four fleas]) with three pairs of primers: (i) primers designed in the traD gene (pRF37F1/R1), (ii) primers in the pRF plasmid (pRFe–pRFf), and (iii) primers in the pRFδ plasmid (pRFa–pRFg). We confirmed positive PCR products of (i) 196 bp, (ii) 208 bp, and (iii) 251 bp for all the 30 cases.
We predicted protein-coding genes (ORFs) using SelfID [52
] as previously described [8
]. tRNA genes were identified using tRNAscan-SE [53
]. Database searches were performed using BLAST programs [54
] against Swiss-Prot/TrEMBL [55
], the NCBI CDD database [56
], and SMART [57
]. The number of transposases, ankyrin/TPR-containing genes, autotransporter domains, and integrases were computed using PSI-BLAST with NCBI/CDD entries related to those domains with an E
-value threshold of 10−5
. Repeated DNA sequences were identified with the use of RepeatFinder [58
], by ignoring the sequence similarity between pRF and pRFδ. To identify Rickettsia
palindromic elements, we used hidden Markov models [59
] based on the previously identified RPE sequences [60
By taking advantage of genome colinearity, we identified orthologous relationships of genes in R. felis, R. conorii, R. sibirica, R. prowazekii
, and R. typhi
with the use of Genomeview (S. Audic, unpublished software). Based on the gene orthology, we defined R. felis
–specific ORFs, which were of one of the following three classes: Class I ORFs exhibiting no homologous ORFs in the other four Rickettsia
genomes; Class II ORFs exhibiting homologous ORFs but no orthologous ORFs in the other four Rickettsia
genomes; and Class III ORFs exhibiting orthologous ORFs in some or all of the other four Rickettsia
, all of which exhibit degraded (split or fragmented) genes relative to the R. felis
ORF. Plasmid-encoded ORFs were by definition classified into Class I or II. A gene composed of more than one ORF was defined as “split gene.” A gene composed of a single ORF whose length is shorter than 50% of the longest ortholog was defined as a “fragmented” ORF. We used T-Coffee [61
] and MEGA [62
] for multiple sequence alignment and phylogenetic tree analyses, respectively. The analyses of horizontal gene transfer were performed by BLAST search against the Swiss-Prot/TrEMBL nonredundant database, excluding rickettsial sequences, as well as by methods based on nucleotide composition bias [63
]. We obtained the minimum number of inversions to associate a pair of Rickettsia
genomes using GRAPPA release 2.0 [65
Ultrastructural characterization of pili by electronic microscopy
R. felis cells were carefully collected from the supernatant of XTC cells infected for 5 d and grown at 28 °C. Following centrifugation (400 g, 10 min), bacteria were fixed for 1 h at 4 °C in glutaraldehyde (2.5% in phosphate-buffered saline [PBS]). Cells were then washed in PBS and placed on a carbon–formvar-coated 400-mesh copper grid (Electron Microscopy Sciences, Hatfield, Pennsylvania, United States) for 15 min then negatively stained with 2% phosphotungstic acid for 10 s, before analysis by electron microscopy (Philips Morgagni 268D, Philips Electronics, Eindhoven, the Netherlands).
Estimation of β-lactamase activity
To evaluate the level of β-lactamase activity, 104R. felis cells grown on XTC cells and then sonicated were mixed with amoxicillin to a final concentration of 20 μg/ml, and incubated for 2 h at 28 °C. The concentration of amoxicillin was measured in the R. felis + amoxicillin suspension as well as in a suspension of XTC cells without bacteria + amoxicillin, before and after incubation, using high-performance liquid chromatography. In addition, the minimum inhibitory concentrations of these four suspensions were estimated by growth inhibition of a Micrococcus luteus strain.
RNA extraction and RT-PCR
Approximately 6.5 × 105 bacteria were used to infect one 25-cm3 flask of confluent XTC cells maintained at 28 °C. Infected cells were harvested 48 h later, centrifuged (12,000 g, 10 min), and pellets were immediately frozen in liquid nitrogen before being stored at −80 °C. Total RNA was isolated by using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. At the end of the extraction procedure, all samples were treated with RNase-Free DNase Set (Qiagen) for 30 min. The concentration and quality of isolated RNA were determined with the Agilent 2100 bioanalyzer (Agilent Technologies, Englewood, New Jersey, United States). Aliquots of the DNase-treated total RNA samples were stored at −80 °C until use. RT-PCR was performed from 2 μl of RNA (25 μl final reaction volume) with the Superscript One-Step RT-PCR with Platinum Taq (Invitrogen). Possible DNA contamination was assessed with the Expand high-fidelity polymerase (Roche, Basel, Switzerland). Cycling conditions were 30 min at 50 °C, 5 min at 95 °C, and 40 cycles at 30 s at 95 °C, 30s at 50 °C, and 1 min at 72 °C, followed by a final extension cycle of 7 min at 72 °C. The RT-PCRs were conducted on the PTC-100 thermocycler (Bio-Rad). Amplification products were run on 2% (wt/vol) agarose gels, and the DNA was stained with ethidium bromide. The size of the PCR product was determined by comparison with DNA molecular-weight marker VI (Boehringer Ingelheim, Ingelheim, Germany).
Detection of F-actin and immunofluorescence staining
Vero cells grown to semiconfluence on glass coverslips were infected with R. felis for 24–48 h at 28 °C in a humidified CO2 incubator (5% CO2). Infected cells were then fixed for 1 h at 4 °C with formaldehyde (3% wt/vol in PBS supplemented with 1 mM MgCl2 and 1 mM CaCl2), washed three times in PBS, and then made permeable with 0.2% Triton X-100 in PBS for 1 min. After three washings in PBS, the coverslips were incubated for 1 h with a monoclonal anti–R. felis antibody. Bacteria were visualized by staining with anti-mouse-Alexa 594 antibody (1:300) and F-actin with FITC-phalloidin (1:250). The coverslips were mounted using Fluoprep (BioMérieux, Marcy-l'Etoile, France) and were examined with a confocal laser scanning microscope using a 100× oil immersion objective lens.
Human blood (10 ml) was centrifuged (1,500 g, 10 min), and after three PBS washings, erythrocytes were resuspended in 20 ml of PBS. This suspension (100 μl) was mixed with 800 μl of PBS and 100 μl of rickettsial suspension (106, 105, and 104 bacteria, respectively). In some experiments, rickettsiae were incubated for 1 h at 35 °C in the presence of 2 mM DTT. Complete hemolysis was determined by adding 900 μl of H2O to erythrocytes, and spontaneous hemolysis corresponded to control without bacteria. Following 3 h of incubation at 35 °C, the samples were fixed using paraformaldehyde (0.3% final concentration) and centrifuged. Hemoglobin release was estimated by measurement of the optical density of the supernatant at 545 nm. This experiment was performed in duplicate.
The sequences of the primers for PCR and RT-PCR are provided in Table S3