HeLa cells expressing GFP alone or GFP-Rab protein fusions (green), were infected with Red- (blue)

<p><b>Copyright information:</b></p><p>Taken from " Trafficking is Defined by Continuous Dynamic Interactions with the Endolysosomal System"</p><p></p><p>Traffic (Copenhagen, Denmark) 2007;8(3):212-225.</p><p>Published online 15 Jan 2007</p><p>PMCID:PMC2063589.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> Dextran-647 (red) was added 2 h p.i. to load the endocytic pathway. Live-cell imaging was initiated 1 h after addition of dextran-647. A single confocal plane is shown (D, overlay) to illustrate the presence of dextran-647 (B) in SCVs containing Red- (C) that are outlined by GFP-Rab7 wt (A). Scale bar = 5 μm. E) Quantification of dextran-647 and V-ATPase positive SCVs. SCVs containing dextran-647 (gray bars) were scored in GFP-expressing cells and expressed as percentage of dextran–647 positive SCVs in mock-transfected cells (set to 100%). For quantification of V-ATPase accumulation on the SCV, transfected cells (black bars) were infected with Red- then fixed and processed for immunofluorescence 1 h p.i. V-ATPase was detected using mouse monoclonal antibodies followed by Cy5-conjugated donkey α-mouse antibodies. SCVs staining positive for V-ATPase were counted in GFP-expressing cells and expressed as percentage of V-ATPase positive SCVs in mock-transfected cells (set to 100%). Values are the mean ± S.D. of three independent experiments ( ≥ 50 in each experiment). Asterisk indicates significant difference from all other conditions (p < 0.05, , Tukey’s test)

To load lysosomes HeLa (A, B and D), or C2BBe1 (C), cells were preincubated with dextran-647 (red) o/n, then chased in dextran-free media for 3 h before infection with Red- (blue)

<p><b>Copyright information:</b></p><p>Taken from " Trafficking is Defined by Continuous Dynamic Interactions with the Endolysosomal System"</p><p></p><p>Traffic (Copenhagen, Denmark) 2007;8(3):212-225.</p><p>Published online 15 Jan 2007</p><p>PMCID:PMC2063589.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> After infection, cells were incubated with dextran-488 (green) to load endosomes and live-cell imaging was initiated at 3 h p.i. A) A single confocal plane showing co-localization of dextran-647 and dextran-488 in a single SCV (arrow). B) A projection of 36 focal planes with YZ (red line) and XZ (blue line) side views to show co-localization of dextrans. C) A single confocal plane from the center of Red--infected C2BBe1 epithelial cells, showing co-localization of dextran-647 and dextran-488 in individual SCVs (arrows). D) Selected frames from a time-lapse series showing delivery of dextran-488 (top) from preloaded lysosomes to an SCV (arrows). Image acquisition started 15 min p.i. (00:00). Scale bars = 5 μm

To load lysosomes, HeLa cells were preincubated with dextran-488 o/n, then chased in dextran-free media for 3 h before infection with Red-

<p><b>Copyright information:</b></p><p>Taken from " Trafficking is Defined by Continuous Dynamic Interactions with the Endolysosomal System"</p><p></p><p>Traffic (Copenhagen, Denmark) 2007;8(3):212-225.</p><p>Published online 15 Jan 2007</p><p>PMCID:PMC2063589.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> To follow endocytic access to the SCV dextran-488 was added various times after infection. Cells were then observed by confocal microscopy. Images of dextran-488 and Red- were overlaid and the number of SCVs containing dextran-488 were counted. A) Time course of SCV interaction with terminal lysosomes (filled bars) and incoming endocytic traffic (open bars). B) To assess the effect of microtubule depolymerization or V-ATPase inhibition on marker acquisition 20 μM nocodazole or 0.5 μM bafilomycin was added, respectively, 1 h before infection and maintained throughout the experiment. Live-cell imaging was initiated at 2 h p.i. and SCVs containing dextran-488 were counted. Values are the mean ± SD of three independent experiments ( ≥ 60 in each experiment). Asterisk indicates significant difference from control-treated cells (p < 0.05, , Tukey’s test)

Oligonucleotides used in this study^a.

a<p>Synthetic restriction sites are underlined.</p

The role of the dIR in OspC synthesis mediated by relaxation of DNA supercoiling.

<p>Immunoblot analysis of whole-cell lysates from strains grown to late log phase at 23°C in 10 ng ml⁻¹ coumermycin A₁ (Cou) (+) or in DMSO as a solvent control (−). Membranes were probed with antibodies against OspC (upper panel) or FlaB (lower panel).</p

The <i>ospC</i> operator and mutagenesis strategy.

<p>(A) <i>ospC</i> operator mutations are linked to the kanamycin resistance cassette (<i>flgBp-aphI</i>). The sequence upstream of the <i>ospC</i> gene in <i>B. burgdorferi</i> strain 297 is shown (WT) with the dIR (solid arrows) and the overlapping pIR (dashed arrows). The nucleotides that have been changed are marked in bold. The strain nomenclature is as follows: dIR⁺ has the nucleotide sequence changed but the complementarity of the inverted repeats maintained, and dIR⁻ has the distal inverted repeat disrupted but the complementarity of the proximal IR intact. (B) PCR of genomic DNA from <i>ospC</i> operator mutants (lane 1, WT with <i>flgBp</i>-<i>aphI</i> cassette; lane 2, dIR⁺; lane 3, dIR⁻; and lane 4, no template control) using primers primers kanR 488R (a) and ospC D1572R+AgeI (b) to determine the orientation of the <i>flgBp</i>-<i>aphI</i> antibiotic resistance cassette.</p

Table_3_The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi.XLSX

<p>The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by Rel_Bbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that Rel_Bbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were Rel_Bbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. Rel_Bbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.</p

<i>ospC</i> expression induced by relaxation of supercoiling is dependent on the dIR of the operator.

<p>qRT-PCR analyses of <i>flaB</i> (gray bars) and <i>ospC</i> (black bars) mRNA levels from strains grown to late log phase at 23°C in 10 ng ml⁻¹ coumermycin A₁ (Cou) or in DMSO as a solvent control. Values represent the mean and error bars the SE of the mean from three independent experiments. * = <i>P</i><0.05 by an unpaired <i>t</i>-test.</p

The dIR is required for OspC synthesis regulated by temperature.

<p>(A) Immunoblot analysis of whole-cell lysates from strains grown at 23°C and then temperature shifted to 34°C and grown to late log phase. The wild-type parental strain (297 WT) and the strain with a wild-type <i>ospC</i> operator linked to the antibiotic resistance cassette (WT) are controls. (B) Immunoblot analysis of whole-cell lysates from strains grown at 34°C and then temperature shifted to 23°C and grown to late log phase. Membranes were probed with antibodies against OspC (upper panels) or FlaB (lower panels).</p

Table_1_The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi.xlsx

<p>The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by Rel_Bbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that Rel_Bbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were Rel_Bbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. Rel_Bbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.</p