Clonal isolation of the <i>E. chaffeensis</i> mCherry and GFP mutants.

<p>Host cell-free organisms recovered from nearly 90% infected culture flask were used to make 10-fold serial dilutions and cultured in DH82 cultures. The culture flasks resulting in <i>E. chaffeensis</i> growth from the highest dilution were used to isolate genomic DNA and for assessing the Himar1 insertions by performing insertion-specific PCRs. Lanes 1–8, genomic DNA from mixed populations of mutants prior to cloning by serial dilution was used as the template for PCRs targeting to the 8 Himar genomic insertion sites (as listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>); Lanes 1c–8c, as in lanes 1–8, but using DNAs recovered after serial dilution cloning of the organisms; Lanes with the letter N refer to no template DNA controls for each of the PCRs and lane M contained the 1 kb+ DNA molecular weight marker.</p

Transcriptional analysis of the transposon mutants with insertions within the protein coding regions of Ech_0379, Ech_0601, Ech_0660 and Ech_0230 genes.

<p>Total RNA isolated from wild type <i>E. chaffeensis</i> grown in DH82 culture (lanes 1, 2, 9, 10, 17,18, 25, and 26) and AAE2 tick cell culture (lanes 3, 4, 11, 12, 19, 20, 27, and 28) and the mCherry mutant culture derived (lanes 5,6, 29, and 30) and GFPuv first transformation (lanes 13 and 14) and second transformation (lanes 21 and 22) cultures grown in DH82 cultures was assessed by RT-PCR for evaluating the gene expression. PCR products resolved in lanes 1–8 included primers targeted to the Ech_0379 and lanes 9–16 contained primer set for the gene Ech_0601, lanes 17–24 contained primers targeted to Ech_0660, and lanes 25–32 had primers targeted to Ech_0230. (Lanes 2, 4, 6, 10, 12, 14, 18, 20, 22, 26, and 28 did not include reverse transcriptase; lanes 7, 15, 23, and 31 served as the positive controls as they included the wild type genomic DNA; and lanes 8, 16, 24, and 32 contained no template and served as negative controls.).</p

Expression of GFPuv and mCherry in transformed <i>E. chaffeensis</i>.

<p>Himar1 transposon mutants of <i>E. chaffeensis</i> were generated following electroporation of the constructs into the cell-free organisms recovered from infected ISE6 tick cells and re-cultured in macrophage and tick cells. A drop of culture suspension under a cover slip was imaged under uv-illumination using an Olympus BX61 spinning disk confocal microscope and a Qfire digital camera. Panels A and B represent <i>E. chaffeensis</i> Arkansas isolate expressing mCherry and GFPuv, respectively. Bars = 10 µm.</p

Nested PCR verification of the <i>E. chaffeensis</i> infection with clonal population mutants in deer blood.

*<p>Clonal population mutants 2 (mutation in the noncoding region between the genes Ech_0284 and Ech_0285) and 9 (mutation in the gene Ech_0660) as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>.</p

Schematic representation of the preparation of <i>E. chaffeensis</i> transposon mutants and <i>in vivo</i> screening to identify genes important for the pathogen's growth in deer and tick hosts.

<p>The protocol involves recovering <i>in vitro</i> culture-derived <i>E. chaffeensis</i> organisms, subjecting them to transposon mutagenesis, reinfecting to naïve host cells, selecting the mutants in culture resisting to antibiotic clearance, infecting a natural host (white-tailed deer), acquisition feding of ticks on the deer and finally assessing the mutants survived in deer and ticks.</p

Basic Local Alignment Search Tool (BLAST) homology search analysis of Ech_0379.

<p>The translated protein coding sequence of Ech_0379 was subjected to BLAST analysis by searching against protein databases available through the National Center for Biotechnology Information (NCBI) web site. The homologies having significant sequence similarities with identities above 30% and positives above 50% to a sequence motif in the Ech_0379 were identified and presented. Three significant hits were presented in the Figure.</p

Validation of transposon insertions in the <i>E. chaffeensis</i> genome.

<p>The insertion sites in the <i>E. chaffeensis</i> genome were verified by PCR with primers designed to bind to the genomic region upstream of the insertion sites (forward primer) and to the inserted DNA (spectinomycin resistance gene) (reverse primer). Product sizes for all 9 insertions are different and the predicted size amplicons were observed only in PCRs containing the mutant genomic DNAs as the templates. Lanes 1–9 represent different insertion targets (as listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>) amplified from the mutant genomic DNAs of mCherry mutants (lanes 1–5), and GFPuv culture DNAs from the first (lanes 6–8) and second (lane 9) transformation experiments; N, no template control; D, wild type <i>E. chaffeensis</i> DNA used as the template; M, 1 kb+ DNA molecular weight marker.</p

Nested PCR verification of the <i>E. chaffeensis</i> infection status and for the transposon insertion sites in ticks^* fed on infected deer.

*<p>Ten ticks were assessed and three of which tested negative for the pathogen.</p>**<p>Genomic insertion sites as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>.</p

A cartoon illustration of the <i>E. chaffeensis</i> genomic locations mapped for the transposon mutants.

<p><i>E. chaffeensis</i> genomic DNA from three independent transformations with mCherry (one transformation) and GFPuv (two transformations) Himar1 transposon plasmids was used to determine the integration locations by inverse PCRs and ST-PCRs followed by DNA sequence analysis. Genomic locations of the insertion sites and the genes at or near the insertions, as per the whole genome data (GenBank # CP000236.1), were presented. The gene expression data assessed by RT-PCR were also included in the figure (E, expressed gene; m, in macrophage culture; t, in tick cell culture; No, gene not expressed). The insertions in mCherry transformants are shown as solid red bars, and insertions in GFPuv transformants are depicted as solid green bars (dark green bar, the first GFPuv transformants; light green bar, the second GFPuv transformant).</p

Nested PCR verification of the <i>E. chaffeensis</i> infection status and for the transposon insertion sites assessed in deer blood DNA^#.

#<p>Second infection experiment;</p>**<p>Genomic insertion sites as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>.</p