Journal: bioRxiv

Article Title: CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

doi: 10.1101/2022.03.10.483831

Figure Lengend Snippet: Cas12a targeting does not elicit abortive infection or prevent the rise of phage esapers. (A) Growth curves of P. aeruginosa PAO1 tn7::LbCas12a strain expressing targeting or non-targeting crRNAs infected with ϕDMS3 at multiplicities of infection (MOI) indicated in legend. (B) Phage plaque assays with ten-fold serial dilutions of wildtype or mutated ϕ JBD30 phage. Bacterial clearance (black) indicates phage replication. Sequences of the targeted phage gene show mutated nucleotides (magenta) in phages able to escape Cas12a targeting. PAM, protospacer adjacent motif (teal); WT, wildtype phage; ESC, escaper phage.

Article Snippet: To generate the PAO1 tn7::LbCas12a strain (Supplementary Figure S1), LbCas12a was first amplified from pMBP-LbCas12a (Addgene #113431) and cloned into pUC18-mini-Tn7T-lac. pUC18-Tn7T-LbCas12a was co-electroporated with pTNS3 plasmid into the P. aeruginosa PAO1 strain according to previously published protocols ( , ).

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

doi: 10.1101/2022.03.10.483831

Figure Lengend Snippet: Cas12a does not detectably cis-cleave invasive ssDNA in bacteria. (A) Conjugation efficiency for plasmid cis-targeting in P. aeruginosa PAO1 strain. During conjugation, the donor is nicked and the leading strand (LDS) is transferred as ssDNA to the wildtype (-LbCas12a) or LbCas12a-expressing (+LbCas12a) recipient cell via the mating pilus. LbCas12a was co-expressed with crRNAs complementary to a DMS3 protospacer cloned into the leading (LDS) or lagging (LGS) strand, with (+) or without (-) the correct protospacer adjacent motif (PAM). T/D indicates the ratio of transconjugants to donors. Data are presented as mean±SEM (n = 3). LDS, complementary protospacer is on leading strand; LGS, complementary protospacer is on lagging strand; NP, no protospacer on plasmid. (B) Plaque assay for phage cis-targeting in E. coli BW25113 F’ strain. Bacterial lawns were infected with dsDNA λ vir or with M13, which enters and leaves the cell as ssDNA but replicates via a dsDNA intermediate. LbCas12a was co-expressed in bacteria with crRNA complementary to M13 positive strand (without PAM) or λ vir (with PAM) during phage infection. Bacterial clearance (black) indicates phage replication. (C) In vitro LbCas12a cleavage assay on dsDNA template encoding DMS3 protospacer or M13 ssDNA.

Article Snippet: To generate the PAO1 tn7::LbCas12a strain (Supplementary Figure S1), LbCas12a was first amplified from pMBP-LbCas12a (Addgene #113431) and cloned into pUC18-mini-Tn7T-lac. pUC18-Tn7T-LbCas12a was co-electroporated with pTNS3 plasmid into the P. aeruginosa PAO1 strain according to previously published protocols ( , ).

Techniques: Bacteria, Conjugation Assay, Plasmid Preparation, Expressing, Clone Assay, Plaque Assay, Infection, In Vitro, Cleavage Assay

Journal: bioRxiv

Article Title: CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

doi: 10.1101/2022.03.10.483831

Figure Lengend Snippet: Cas12a does not trans-cleave invasive ssDNA in bacteria. (A) Right: Schematic for plasmid coconjugation and cleavage in P. aeruginosa PAO1 strain. Two plasmids were conjugated simultaneously from different donors into the same recipient cell. LbCas12a was co-expressed in the recipient cell with crRNA complementary to pTarget but not pTrans. A parallel co-conjugation mating using pTrans and pTarget(-PS) (identical to pTarget but lacking the protospacer and PAM) was carried out as a control. Conjugation efficiency was assessed using antibiotic selection specific to each plasmid and represented as the transconjugant to donor ratio (T/D). Data are presented as mean ± SEM ( n = 3); ** p =0.0036 by unpaired, two-sided Student’s t test; ns, not significant ( p >0.05) (B) Right: Schematic for phage co-infection in E. coli. Bacterial lawns of E. coli BW25113 F’ strain co-expressing LbCas12a and λ-targeting or non-targeting crRNA were singly infected or co-infected with ssDNA phage M13 and dsDNA phage λvir. Strains were infected with either low titer (low), high titer (high), or ten-fold serial dilutions (serial) of phage as indicated.

Article Snippet: To generate the PAO1 tn7::LbCas12a strain (Supplementary Figure S1), LbCas12a was first amplified from pMBP-LbCas12a (Addgene #113431) and cloned into pUC18-mini-Tn7T-lac. pUC18-Tn7T-LbCas12a was co-electroporated with pTNS3 plasmid into the P. aeruginosa PAO1 strain according to previously published protocols ( , ).

Techniques: Bacteria, Plasmid Preparation, Conjugation Assay, Control, Selection, Infection, Expressing

Journal: Frontiers in Microbiology

Article Title: Pseudomonas aeruginosa Swarmer Cells Adaptation Toward UVc Radiations

doi: 10.3389/fmicb.2019.00556

Figure Lengend Snippet: (A) Photographs of Petri dishes showing the effects of UVc exposure on swarmer cells of P. aeruginosa for the case of (T5) 5 min of treatment and (T15) 15 min of treatment. Note that (UT) represents Petri dishes of untreated samples. The UVc radiation of strength I = 4 mW/cm 2 is switched on after the colony was allowed to grow for time t = 18 h. After the UVc radiation is switched off, the bacterial colony was allowed to recover overnight ( t = 36 h). Plates were photographed against a black background so that zones of bacterial colonization appear white and uncolonized agar appears black. The zone of migration is marked by a disrupted line red circle in (UT) photograph, the zone of narrowing is also marked by a disrupted line red circle in (T5), and the red square in T15 shows some emergent colonies in the zone of regression. (B) Colony diameter of swarming colonies before (UT) and after exposure to UVc radiations for 5 (T5) and 15 (T15) min. ( ∗ ) shows significant differences.

Article Snippet: Pseudomonas aeruginosa PAO1 was supplied by Pasteur Institute (Tunisia).

Techniques: Migration

Journal: Frontiers in Microbiology

Article Title: Pseudomonas aeruginosa Swarmer Cells Adaptation Toward UVc Radiations

doi: 10.3389/fmicb.2019.00556

Figure Lengend Snippet: Fatty acid proportions (%) of saturated (SFA), unsaturated (UFA), and cyclic fatty acids (CFA) in planktonic and swarmer cells of Pseudomonas aeruginosa. Different letters mean significant difference ( P < 0.05) for each class of fatty acids. ( ∗ ) shows significant difference ( P < 0.05).

Article Snippet: Pseudomonas aeruginosa PAO1 was supplied by Pasteur Institute (Tunisia).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Pseudomonas aeruginosa Swarmer Cells Adaptation Toward UVc Radiations

doi: 10.3389/fmicb.2019.00556

Figure Lengend Snippet: Electron microscopic images of P. aeruginosa swarmer cells removed from the swarm edge and observed at the population (A) and at the single-cell level (B) . Bars: A = 300 μm; B = 100 μm. In A , the arrows show the waved outer membrane with some emanating blebs.

Article Snippet: Pseudomonas aeruginosa PAO1 was supplied by Pasteur Institute (Tunisia).

Techniques: Membrane

Journal: Frontiers in Microbiology

Article Title: Pseudomonas aeruginosa Swarmer Cells Adaptation Toward UVc Radiations

doi: 10.3389/fmicb.2019.00556

Figure Lengend Snippet: TEM micrograph of a full swarming area profile, after 5 min of exposure, in thin section (A) and at higher resolution (B) . Electron microscopic images of P. aeruginosa swarmer cell with an excess of S-layer at the both ends of the bacterial cell (C) . (A) Bars = 500 μm; (B) bars = 200 μm; (C) bars = 300 μm. The micrograph reveals extensive heterogeneity in terms of cell distribution and physiological status. Black star indicates ghost cells with empty envelope structure and damaged cells with condensed cytoplasmic material (black arrowheads) are visible. Double arrow shows cellular dilation in some cells after shorter exposure. Black arrow indicates membrane separation and white arrow indicates early disintegration. Arrows indicate S-layer.

Article Snippet: Pseudomonas aeruginosa PAO1 was supplied by Pasteur Institute (Tunisia).

Techniques: Membrane

Journal: Genetics

Article Title: Fitness Is Strongly Influenced by Rare Mutations of Large Effect in a Microbial Mutation Accumulation Experiment

doi: 10.1534/genetics.114.163147

Figure Lengend Snippet: The distribution of indel mutations in proteins. Comparison between the observed and expected position of frameshifts in coding regions. Proteins were divided into three equal pieces and we counted the number of frameshifts (overlapping with homopolymeric tracts) that fell in each section. Expected frequencies were computed by counting the number of homopolymeric tracts in the P. aeruginosa PAO1 proteome that fall in each section. The differences between observed and expected values were statistically significant for the N-terminal third of proteins (one-sided exact binomial test: P = 0.037).

Article Snippet: A list of P. aeruginosa PAO1 genes with annotated Clusters of Orthologous Groups (COGs) categories ( Tatusov et al. 2000 ) was downloaded from the National Center for Biotechnology Information.

Techniques: Comparison