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1.
Figure 8

Figure 8. RsmZ restores pigment production in a letA mutant strain. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

Wilde type Legionella pneumophila and a letA mutant strain carrying prsmZ activate pigment production in PE growth phase, in contrast to a letA mutant strain.

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.
2.
Figure 7

Figure 7. Electron microscopy of PE phase Legionella pneumophila wild type, ΔletA, ΔrsmZ and ΔrsmYZ. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

In contrast to the letA mutant, mutants in the small ncRNAs synthesize flagella in PE-growth phase. A) wild type, B) ΔrsmZ, C) ΔrsmYZ, D) ΔletA.

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.
3.
Figure 6

Figure 6. Sodium sensitivity is repressed and pigment production is activated by LetA, RsmY and RsmZ. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

A) PE phase ΔrsmY, ΔrsmZ, ΔrsmYZ and ΔletA mutants of strain Paris are not sensitive to NaCl as compared to PE phase wild type strain Paris. B) RsmZ, RsmY, RsmYZ and LetA activate pigment production in Legionella

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.
4.
Figure 4

Figure 4. RsmY and RsmZ bind CsrA in vitro. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

Electromobility shift assay with 100nM of RNA combined with varying concentrations of purified CsrA-His. A) RsmZ RNA and recombinant CsrA in 6% Blue Native Bis-Tris/Tricine Gel. Lane 1: no CsrA, 0.1 µM control RNA; lane 2: 2.0 µM CsrA + 0.1 µM control RNA, lane 3: 5.0 µM CsrA + 0.1 µM control RNA, lane 4: no CsrA, 0.1 µM RsmZ, lane 5: 0.5 µM CsrA + 0.1 µM RsmZ; lane 6: 1.0 µM CsrA+ 0.1 µM RsmZ; lane 7: 2.0 µM CsrA+ 0.1 µM RsmZ; lane 8: 5.0 µM CsrA+ 0.1 µM RsmZ; lane 9: 1µg CsrA (silver-stained). B) RsmY RNA and recombinant CsrA in 6% Blue Native Bis-Tris/Tricine Gel. Lane 1: no CsrA, 0.1 µM control RNA; lane 2: 2.0 µM CsrA + 0.1 µM control RNA, lane 3: 5.0 µM CsrA + 0.1 µM control RNA, lane 4: no CsrA, 0.1 µM RsmY, lane 5: 0.5 µM CsrA + 0.1 µM RsmY; lane 6: 1.0 µM CsrA+ 0.1 µM RsmY; lane 7: 2.0 µM CsrA+ 0.1 µM RsmY; lane 8: 5.0 µM CsrA+ 0.1 µM RsmY; lane 9: 1µg CsrA (silver-stained)

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.
5.
Figure 1

Figure 1. Two small ncRNAs, named RsmY and RsmZ are located in intergenic regions. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

A) Sequence of the small RNAs and the flanking regions; Blue letters, sequence of the small RNA. Red letters, LetA binding site; yellow boxes, GGA motifs representing the CsrA binding sites; arrows indicate flanking genes in the L. pneumophila strains Paris genome. B) Predicted structure of the RsmY and RsmZ of Legionella pneumophila predicted by the program Mfold (http://mfold.burnet.edu.au/). The typical GGA (arrows) motifs are located in the loop regions

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.
6.
Figure 3

Figure 3. RsmY and RsmZ of L. pneumophila induce glycogen accumulation and biofilm formation in E. coli. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

A) Escherichia coli wild-type (BL21) containing the empty plasmid pTS10 and E. coli producing L. pneumophila RsmY (prsmY) or RsmZ (prsmZ), respectively, were streaked onto Kornberg medium containing 1% glucose and 100 mg.ml-1 Ampicillin, then stained with iodine vapour to visualize glycogen accumulation. B) Quantification of glycogen accumulation expressed in µg equivalent glucose per unit of OD600 measures and biofilm formation of E. coli carrying RsmY and RsmZ of L. pneumophila as judged from OD570 measures after solubilization of cristal violet.

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.
7.
Figure 2

Figure 2. LetA binds specifically upstream of rsmY and rsmZ. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

Electrophoretic Mobility Shift Assay with Strep-tag purified LetA protein and 35 bp fragment of HPLC-purified rsmY and rsmZ oligonucleotides containing the predicted LetA binding site and a 35 bp control DNA fragment without LetA binding site each with a biotin tag at the 5’ends A) Identified LetA-binding site and the L. pneumophila consensus sequence as compared to the E. coli consensus binding site for UvrY. B) Lane 1–4: 50ng control DNA; Lane 5–8: 50ng 35bp DNA fragment upstream rsmY containing the predicted LetA binding site, incubated with 0, 2, 4, 6 µM LetA; Lane 9–12: 50ng 35bp DNA fragment upstream rsmZ containing the predicted LetA binding site incubated with 0, 2, 4, 6 µM LetA, respectively.

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.
8.
Figure 9

Figure 9. Model of the regulatory network governing differentiation of L. pneumophila from replicative/non virulent to transmissive/virulent. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

During the transmissive phase, amino acid starvation triggers the ppGpp synthetase RelA and fatty acid starvation triggers the ppGpp synthetase/hydrolase SpoT to produce the alarmone (p)ppGpp. (p)ppGpp may be sensed by the sensor kinase LetS which then phosphorylates LetA. Phosphorylated LetA binds upstream of the small ncRNAs RsmY and RsmZ and activates their transcription. CsrA that is bound near the ribosomal binding site of its mRNAs targets (e.g. probably LqsR, FleQ and Hfq) inhibits their translation. The presence of RsmY and RsmZ titrate CsrA away from its targets, which then enables translation of the mRNAs, leading to expression of transmissive phenotypes. In contrast flagella biosynthesis does not seem to be dependent on the RsmYZ-CsrA-pathway but is controlled by the LetA/LetS TCS, probably via LetE repression. Furthermore, LetA/LetS influences directly or indirectly ci-di-GMP levels which also may influence motility.

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.
9.
Figure 5

Figure 5. Intracellular replication of L. pneumophila in Acanthamoeba castellanii and THP-1 macrophages is dependent on functional RsmY and RsmZ. From: Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

A) A. castellani infection; Black, wild type L. pneumophila strain Paris; light blue, ΔrsmY L. pneumophila strain Paris; blue, ΔrsmZ L. pneumophila strain Paris; dark blue, ΔrsmYZ L. pneumophila strain Paris; green ΔletA L. pneumophila strain Paris. B) A. castellani infection; Black, wild type L. pneumophila strain Paris; light blue, ΔrsmZ L. pneumophila strain Paris carrying the empty plasmid pMMB; red, ΔrsmZ L. pneumophila strain Paris carrying plasmid prsmZ with an IPTG inducible promoter; For the complementation experiment all strains were grown in presence of IPTG. C) Infection of THP-1 human macrophages with Legionella pneumophila Paris wild type and ΔrsmYZ L. pneumophila strain Paris. The number of viable bacteria within amoebae was evaluated by the standard plate count assay. Results are expressed as Log10 ratio CFU Tn/T0. Each time point represents the mean of +/− SD of two independent experiments. Infections were performed at 37°C.

Tobias Sahr, et al. Mol Microbiol. ;72(3):741-762.

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