Effect of leader mutations and the presence of L20 on the transcription profile

<p><b>Copyright information:</b></p><p>Taken from "Ribosomal protein L20 controls expression of the operon via a transcription attenuation mechanism"</p><p></p><p>Nucleic Acids Research 2007;35(5):1578-1588.</p><p>Published online 8 Feb 2007</p><p>PMCID:PMC1865079.</p><p>© 2007 The Author(s).</p> Single round transcription assays were performed on PCR templates comprising the promoter and 5′ noncoding region of using RNA polymerase. RT and T on the left side indicate read-through or prematurely terminated leader transcripts. The size of the marker fragments in bases is indicated. Numbers at the bottom indicate the percentage of read-through transcripts (%RT = (RT/(T + RT) × 100). () Templates were wild type (wt) or contained the A, B or A + B mutations depicted in . () Where indicated proteins were added in 50-fold molar excess over the template during the elongation phase of the single-round transcription assay. The L20 (Bs L20) and L17 (Bs L17) r-proteins were native, L20 (Ec L20) only contained the C-terminal half of the protein

RNase probing of the leader mRNA structure in the absence or presence of L20 protein

<p><b>Copyright information:</b></p><p>Taken from "Ribosomal protein L20 controls expression of the operon via a transcription attenuation mechanism"</p><p></p><p>Nucleic Acids Research 2007;35(5):1578-1588.</p><p>Published online 8 Feb 2007</p><p>PMCID:PMC1865079.</p><p>© 2007 The Author(s).</p> An leader transcript (nts 1–168) was subjected to cleavage by RNases V1 or T1. Where indicated purified L20 protein was added prior to RNase cleavage. () Cleavages by RNase V1 and RNase T1 are shown on the leader mRNA structure. Colors indicate the change in cleavage efficiency observed in the presence of L20: black (no change), red (increase), blue (decrease). The numbers next to the symbols locate the corresponding cleavages on the gels (boxes). Shaded and encircled nucleotides correspond to positions conserved at the L20-binding site on 23S rRNA as shown in . () Cleavages on an unlabeled transcript were detected by primer extension with labeled oligonucleotide HP697 (positions 168–141 on leader). () Cleavages on a 5′ labeled transcript were analyzed directly

Effect of L20 overproduction on the transcription profile of the operon

<p><b>Copyright information:</b></p><p>Taken from "Ribosomal protein L20 controls expression of the operon via a transcription attenuation mechanism"</p><p></p><p>Nucleic Acids Research 2007;35(5):1578-1588.</p><p>Published online 8 Feb 2007</p><p>PMCID:PMC1865079.</p><p>© 2007 The Author(s).</p> Northern analysis of total RNA of a wild-type strain harboring plasmid pHML17 carrying an IPTG inducible copy of the (L20) gene was performed using two different probes. () RNA was separated on a 0.8% agarose gel and probed with an specific probe including the entire leader sequence (see Materials and methodssection). () RNA separated on a 8% polyacrylamide gel was probed with oligonucleotide HP1080 complementary to positions +8 to +34 of the leader. Where indicated IPTG (1 mM) was added to mid-log cultures for 20 min prior to isolation of the RNA. RT = read-through transcript, T = terminated transcript. The positions of the molecular size markers are indicated. Percentages of read-through transcripts are indicated below the gel

Purification of the nuclease responsible for the cleavage in mRNA leader

<p><b>Copyright information:</b></p><p>Taken from "Ribonucleases J1 and J2: two novel endoribonucleases in with functional homology to RNase E"</p><p>Nucleic Acids Research 2005;33(7):2141-2152.</p><p>Published online 14 Apr 2005</p><p>PMCID:PMC1079966.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () The leader was transcribed using plasmid pHMS17 linearized by ClaI as template and RNA polymerase (see Materials and Methods). The transcripts, one prematurely terminated at the leader terminator and a read-through transcript, were incubated in 50 μl post-transcriptional assays without the addition of proteins, with 15 μg of a ribosomal high salt wash (HSW) or with the most active enriched protein fraction eluting from a Superdex 200 (S 200) column. Scissors indicate the processed transcript. () Mapping of the processing site shown in (A) in the specifier domain. Primer extension was performed with P-labelled oligonucleotide HP238 on mRNA synthesized in the presence of HSW. Arrows indicate the processing sites within the specifier domain, referred to as cleavage 1 (). A sequencing ladder was run in parallel using the same primer. () Purification of the endoribonucleolytic activity responsible for cleavage in the specifier domain. The most active fractions from the heparin (Hep) and S 200 columns were separated on a 15% SDS–polyacrylamide gel and silver stained. Molecular weight marker proteins (M) were run in the first lane. () Cleavage of the leader transcript by purified YkqC and YmfA proteins. mRNA was prepared as described in (A) and incubated in 50 μl post-transcriptional assays without proteins, with 15 μg of a HSW or with 200 ng of the purified proteins, YmfA, YkqC and a mixture of YmfA and YkqC. The processed transcripts are indicated by scissors and a number (1: cleavage in the AG-box; 2: cleavage near the T-box, see and ). () Cleavage of a leader transcript generated with T7 RNA polymerase from a PCR fragment (see Materials and Methods) by purified YkqC and YmfA proteins. The fully synthesized mRNA was incubated in 10 μl post-transcriptional assays without any proteins or with 200 ng of the purified proteins, YmfA and YkqC. The processed transcripts are indicated by scissors

Sensibility of the YkqC () and YmfA () processing activity to the 5′ end phosphorylation state of the substrate

<p><b>Copyright information:</b></p><p>Taken from "Ribonucleases J1 and J2: two novel endoribonucleases in with functional homology to RNase E"</p><p>Nucleic Acids Research 2005;33(7):2141-2152.</p><p>Published online 14 Apr 2005</p><p>PMCID:PMC1079966.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Equal amounts of P uniformly labelled tri- (lanes 1–7) and monophosphorylated (lanes 8–14) leader transcripts were used as substrates in 50 μl post-transcriptional processing assays with 1 μg of YkqC or YmfA. Samples (5 μl) were taken at 0 min (lanes 1 and 8), 1 min (lanes 2 and 9), 2.5 min (lanes 3 and 10), 5 min (lanes 4 and 11), 10 min (lanes 5 and 12), 15 min (lanes 6 and 13) and 20 min (lanes 7 and 14) after addition of purified YkqC (A) or YmfA (B), directly diluted with 2.5 μl of 3× gel loading buffer to stop the reaction and run on a 5% polyacrylamide gel. The processed transcript is indicated by scissors and a number (1: cleavage in the AG-box; scissor 2: cleavage near the T-box)

Microscopic observation of internalized <i>S</i>. <i>aureus</i>.

<p>Internalization of <i>S</i>. <i>aureus</i> N305 as observed by transmission electron microscopy. <i>S</i>. <i>aureus</i> N305 (at an MOI of 100:1) was incubated for 2 h with bMEC either alone (A, B) or in the presence of <i>L</i>. <i>casei</i> BL23 wt (C, D) or <i>srtA2</i> mutant (E, F) strains, at an MOI of 2,000:1.</p

Impact of <i>L</i>. <i>casei</i> BL380 (BL23 <i>bnaG</i>) on <i>S</i>. <i>aureus</i> internalization into bMEC.

<p>Internalization rates of <i>S</i>. <i>aureus</i> N305 after 2 h of interaction with bMEC and co-incubation with <i>L</i>. <i>casei</i> BL23 and <i>L</i>. <i>casei</i> BL380 (<i>bnaG</i>) at an MOI of 2,000:1. <i>S</i>. <i>aureus</i> was used at an MOI of 100:1. The internalization assay of <i>S</i>. <i>aureus</i> alone was used as a reference. Internalization rates were then defined as the internalized <i>S</i>. <i>aureus</i> population in the presence of the different <i>L</i>. <i>casei</i> strains relative to the internalized <i>S</i>. <i>aureus</i> population of the reference experiment. Data are presented as means ± standard deviations. Each experiment was done in triplicate, and differences between groups were compared using one-way ANOVA with Bonferroni's Multiple Comparison Test. *: P < 0.05.</p

Cell surface proteome of <i>L</i>. <i>casei</i> BL23 wt and <i>srtA2</i> mutant as determined by enzymatic shaving.

<p>Cell surface proteome of <i>L</i>. <i>casei</i> BL23 wt and <i>srtA2</i> mutant as determined by enzymatic shaving.</p

Contribution of sortase SrtA2 to <i>Lactobacillus casei</i> BL23 inhibition of <i>Staphylococcus aureus</i> internalization into bovine mammary epithelial cells - Fig 4

<p><b>Internalization of <i>L</i>. <i>casei</i> BL23 wt (A) and <i>srtA2</i> mutant (B) strains as observed by transmission electron microscopy</b>. Degradation vesicles (white arrows) were observed in a greater proportion in cells containing mutant <i>srtA2</i>.</p

Resistance of <i>L</i>. <i>casei</i> BL23 wt and <i>srtA2</i> strains to H₂O₂.

<p>Resistance of <i>L</i>. <i>casei</i> BL23 wt (●, ○) and <i>srtA2</i> (■, □) strains to H₂O₂ was evaluated in the stationary phase of growth of <i>L</i>. <i>casei</i> (24h—MRS). The residual population was evaluated at 0, 10, 20 and 30 min after exposure to 0.25% (●, ■) and 0.5% H₂O₂ (○, □). Data are presented as means ± standard deviations. Each experiment was done in triplicate, and differences between groups were compared using Student’s t-test. *: P < 0.05.</p