Journal: bioRxiv

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1101/2024.05.24.595836

Figure Lengend Snippet: a. Overview of the CRISPR adaptation process, highlighting key known host factors. b . Schematic of the CRISPRi adaptation host factor screen. c . Binned coverage plot of sgRNAs across the E. coli genome. sgRNA occupancy was calculated as the difference between the normalised (post/pre-screen) binned sgRNA counts per base of the experimental (+dCas9) and paired control (–dCas9) conditions. Regions of the genome with high (“enriched”) sgRNA coverage are interpreted to be genomic loci that positively regulate CRISPR adaptation; regions of the genome with low (or negative, i.e., “depleted”) sgRNA coverage are interpreted to be genomic loci that negatively regulate CRISPR adaptation. The highest-ranking regions with attributable genes are labelled; other labelled loci are the Ori and Ter regions, the murA gene, and the CRISPR-II array. n = 9 biological replicates. d . Volcano plot showing log2 fold change for each sgRNA versus adjusted –log10 p-values ( n = 9 biological replicates). The horizontal dashed line represents an adjusted p-value of 0.05; the vertical lines represent log2 fold changes of –0.75 and 0.75. Genes targeted by sgRNAs differentially enriched that were selected for individual validation are coloured in pink. e . Top: deep-sequencing based measurement of the rates of new spacer acquisition in Keio knockouts harbouring pSCL565, after growth for 48h in liquid culture without induction of Cas1-Cas2 expression. Acquisition rates are shown relative to the wild-type parental strain. Open circles represent biological replicates ( n ≥ 3), bars are the mean (one-way ANOVA effect of strain P <0.0001; Sidak’s corrected multiple comparisons for wild-type vs. knockouts, Δ pcnB P=0.00217, Δ sspA P=0.000102, polA ΔKlenow P<0.0001; others ns). Bottom: representative agarose gel for the data shown. Expansions of the CRISPR array can be seen as higher sized bands above the parental array length. Additional statistical details in Supplemental Table 1 .

Article Snippet: E. coli K-12 MG1655 and LC-E75 (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: CRISPR, Control, Biomarker Discovery, Sequencing, Expressing, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1101/2024.05.24.595836

Figure Lengend Snippet: a. Prespacer substrates for CRISPR adaptation arise from a variety of sources. b. Breakdown of normalised spacer count (total number of new spacers / number of CRISPR arrays sequenced) according to spacer origin ( E. coli or plasmid) and strain of interest. c. Breakdown of percent of spacer attributable to each spacer origin ( E. coli or plasmid) and strain of interest. d. Motifs in the 15bp up- and downstream of the newly acquired spacer in its source location. e-f: Binned coverage plot of newly acquired spacer across the E. coli genome (outer, purple) and pSCL565 plasmid (inner, tan) for the wild-type strain ( e ) and derivatives ( f-h ). See for the full set. i. qPCR-based measurement of the relative copy number of pSCL565 Ori and cas1 sequences in the wild-type and polA ΔKlenow mutant. Open circles represent biological replicates ( n ≥ 3), bars are the mean (one-way ANOVA effect of strain and target P <0.0001; Sidak’s corrected multiple comparisons for wild-type vs. Δ sspA , CDF ori copy number P<0.0001, cas1 copy number P<0.0001). Additional statistical details in Supplemental Table 1 .

Article Snippet: E. coli K-12 MG1655 and LC-E75 (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: CRISPR, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1101/2024.05.24.595836

Figure Lengend Snippet: Binned coverage plot of newly acquired spacer across the E. coli genome (left) and pSCL565 plasmid (right) for strains selected for individual validation. a-i : wild-type, Δ pcnB , Δ sspA , Δ uraA , Δ omsF , polA ΔKlenow, Δ rclR , Δ yeaO and Δ ompC . Wild-type is E. coli BW25113, parental strain to the Keio collection; all other strains besides polA ΔKlenow are from the Keio collection. polA ΔKlenow was constructed as described previously .

Article Snippet: E. coli K-12 MG1655 and LC-E75 (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: Plasmid Preparation, Biomarker Discovery, Construct

Journal: bioRxiv

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1101/2024.05.24.595836

Figure Lengend Snippet: a . sspAB operon, proteins and function. Bottom left: crystal structure of an SspA dimer (blue) in complex with E. coli RNAP-promoter open complex, showing the conserved SspA PHP 84–86 residues (red) interacting with RNAP (pink) and α (purple) (PDB 7DY6 ). Top right: crystal structure of SspB escorting an SsrA-tagged substrate being delivered to the ClpXP protease complex (PDB 8ET3 65 ). b . Schematic of the sspAB operon of WT, Δ sspA :: kan R and Δ sspB :: kan R strains. kan R : kanamycin resistance cassette. c . Deep-sequencing based measurement of the rates of new spacer acquisition in strains harbouring pSCL565 and, in the case of the Δ sspA :: kan R , either an empty plasmid or a low (∼5) copy plasmid encoding the sspAB operon, after growth for 48h in liquid culture. Adaptation rates are shown relative to the wild-type parental strain. Open circles represent biological replicates ( n ≥ 3), bars are the mean. Horizontal dashed line represents the mean rate of spacer acquisition in the wild-type strain (one-way ANOVA effect of strain P <0.0001; Sidak’s corrected multiple comparisons for wild-type vs. knockouts, Δ sspA P<0.0001, Δ sspB P=0.109807; Δ sspA vs. Δ sspB P<0.0001). d . Schematic of the sspAB operon variant rescue plasmids. All plasmids are low (∼5) copy, and encode variants of the sspAB operon under its native regulation. Frameshift mutants of SspA (AN – >AQ – GCC|AAC>GC T | CAA |C) and SspB (PR – >PS – CCA|CGT>CCA| T CG |T) encode sequences with single base insertions to cause protein translation to terminate early. The SspA PHP 84–86 >AAA 84–86 mutant is RNAP-binding deficient and thus does not enable the shift in promoter use (α σ α S ) . A single sspA rescue plasmid yielded no transformants into the Δ sspA :: kan R strain over multiple attempts. e . Top: deep-sequencing based measurement of the rates of new spacer acquisition in strains harbouring pSCL565 and, in the case of the Δ sspA :: kan R , either an empty plasmid or a low (∼5) copy plasmid encoding variants of the sspAB operon as described in d ., after growth for 48h in liquid culture. Adaptation rates are shown relative to the wild-type parental strain. Open circles represent biological replicates ( n ≥ 3), bars are the mean. Horizontal dashed line represents the mean rate of spacer acquisition in the wild-type strain (one-way ANOVA effect of strain P <0.0001; Sidak’s corrected multiple comparisons for wild-type vs. knockouts, Δ sspA P<0.0001, Δ sspA + empty plasmid P<0.0001, Δ sspA + sspAB rescue P = 1, Δ sspA + sspA * (PHP84-86>AAA84-86) & sspB rescue P<0.0001; Δ sspA vs. rescues, Δ sspA + empty vector P=0.997758, Δ sspA + sspA * (PHP84-86>AAA84-86) & sspB P=0.334315, Δ sspA + sspAB P<0.0001, Δ sspA + sspB P=0.892991, Δ sspA + sspA * & sspB * (frameshifted) P=1). Bottom: representative agarose gel for the data shown. Expansions of the CRISPR array can be seen as higher sized bands above the parental array length. Additional statistical details in Supplemental Table 1.

Article Snippet: E. coli K-12 MG1655 and LC-E75 (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: Sequencing, Plasmid Preparation, Variant Assay, Mutagenesis, Binding Assay, Agarose Gel Electrophoresis, CRISPR

Journal: bioRxiv

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1101/2024.05.24.595836

Figure Lengend Snippet: a . Model for SspA-mediated regulation of CRISPR-Cas defence. Phage infection triggers upregulation of SspA , which in turn induces a global transcriptional shift towards 0 S -regulated promoters. This results in H-NS downregulation , , induction of CRISPR-Cas mediated defence through de-repression Cas gene expression , , leading to increased rates of CRISPR adaptation and interference. b . Schematic of the sspAB and hns operons of WT, Δ sspA :: FRT, Δ hns :: FRT and Δ sspA :: FRT Δ hns :: FRT strains. FRT : flippase recognition target, a scar left after the removal of resistance cassettes. c . Schematic of the CRISPR interference-mediated defence assays in pre-immunised E. coli strains. Top: schematic of the CRISPR-I immunisation (defence) plasmids. All plasmids are low (∼5) copy, and encode an E. coli CRISPR-I array with a first spacer encoding either a Target (complementary to the α genome , ), or a Non-Target (NT) spacer. Bottom: The experimental strains were electroporated with either the T or NT plasmid, and infected to varying titres of αvir. Note that the strains encode a complete endogenous E. coli Type I-E CRISPR-Cas system. d . Representative plaque assays of αvir on experimental strains (described above) pre-immunised with either T or NT defence plasmids. Strains were infected with αvir and grown on plates at 30°C for 16h. Full plaque assay plates for n = 3 biological replicates in . e . Efficiency of plating of αvir on experimental strains. Open circles represent biological replicates ( n ≥ 3) of individual plaque assays, bars are the mean (one-way ANOVA effect of strain P =0.033454; Sidak’s corrected multiple comparisons for wild-type vs. knockouts, Δ sspA P=0.181757, Δ hns P=0.043319, ΔsspA Δhns P = 0.043316; for Δ hns vs. Δ sspA Δhns P=1). f. Anti-phage defence and growth in overnight liquid culture of experimental strains, post αvir infection (MOI: 0.1). Hue around solid line (mean) represents the standard deviation across 3 biological replicates.

Article Snippet: E. coli K-12 MG1655 and LC-E75 (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: CRISPR, Infection, Gene Expression, Plasmid Preparation, Plaque Assay, Standard Deviation

Journal: bioRxiv

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1101/2024.05.24.595836

Figure Lengend Snippet: a . Deep-sequencing based measurement of the rates of new spacer acquisition in strains pre-immunised with either a T or NT defence plasmid, harvested 3h post λvir infection in liquid culture and growth at 30°C. Open circles represent biological replicates ( n ≥ 3), bars are the mean (one-way ANOVA effect of strain P < <0.0001; Sidak’s corrected multiple comparisons for wild-type +T vs. knockouts +T, Δ sspA P=082553, Δ hns P<0.0001, Δ sspA Δ hns P=0.999999; Δ sspA +T vs. knockouts + T, Δ hns P<0.0001, Δ sspA Δ hns P=0.154762; Δ hns + T vs. Δ hns +NT P<0.0001; Δ hns + T vs. Δ sspA Δ hns +T P<0.0001). b. Breakdown of normalised spacer count (total number of new spacers / number of CRISPR arrays sequenced) according to spacer origin ( E. coli , lambda or plasmid) and strain of interest. c. Binned coverage plot of Δ hns + T newly acquired spacers across the lambda genome (outer, purple). The location of the T immunisation spacer is shown on the lambda genome; “missing in /\vir” indicates a genomic region missing in our strain of /\vir. d . Percent of spacers acquired that are on the same strand as the T immunisation spacer, according to the spacer source ( E. coli or lambda). e . Schematic of the sspAB and hns operonic rescue plasmids. All plasmids are low (∼5) copy, and encode either 1. The sspAB operon, 2. The hns operon, or 3. both, under their native regulation. f . Schematic of the CRISPR adaptation assays in wild-type, sspA and/or hns mutant strains. Strains were electroporated with pSCL565 and rescue plasmids 1., 2., or 3. (see e .), and assessed for their ability to acquire new spacers into the endogenous CRISPR I array. g . PCR-based detection of new spacer acquisition into the CRISPR I array of wild-type, of WT, Δ sspA :: FRT, Δ hns :: FRT and Δ sspA :: FRT Δ hns :: FRT strains harbouring pSCL565 and rescue plasmids 1., 2., or 3. (see e .), after growth for 48h in liquid culture. Open circles represent biological replicates ( n ≥ 3), bars are the mean. Horizontal dashed line represents the mean rate of spacer acquisition in the wild-type strain (one-way ANOVA effect of strain P < <0.0001; Sidak’s corrected multiple comparisons for wild-type vs. knockouts, Δ sspA P<0.0001, Δ hns P<0.0001, Δ sspA Δ hns P<0.0001; Δ sspA vs. knockouts, Δ hns P=0.714182, Δ sspA Δ hns P=0.002269, Δ sspA + sspAB rescue P<0.0001; Δ hns vs. knockouts, Δ sspA Δ hns P<0.0001, Δ hns + hns rescue P<0.0001; Δ sspA Δ hns vs. Δ sspA Δ hns + sspA & hns rescues P<0.0001). h . PCR-based detection of new spacer acquisition into the CRISPR I array of WT, Δ sspA :: FRT, Δ hns :: FRT , Δ sspA :: FRT Δcas3-Cascade::Cm R or Δ hns :: FRT Δcas3-Cascade::Cm R strains harbouring pSCL565 after growth for 48h in liquid culture. Open circles represent biological replicates ( n ≥ 3), bars are the mean (one-way ANOVA effect of strain P <0.0001; Sidak’s corrected multiple comparisons for wild-type vs. knockouts, Δ sspA P<0.0001, Δ hns P<0.0001, Δ sspA Δ cas3-cascade P<0.0001, Δ hns Δ cas3-cascade P=0.125466; Δ sspA vs. Δ hns P=0.004161; Δ sspA vs. Δ sspA Δ cas3 - cascade P=0.310715; Δ hns vs. Δ hns Δ cas3-cascade P<0.0001; Δ sspA Δ cas3 - cascade vs. Δ hns Δ cas3 - cascade P<0.0001). Horizontal dashed line represents the mean rate of spacer acquisition in the wild-type strain. Additional statistical details in Supplemental Table 1 .

Article Snippet: E. coli K-12 MG1655 and LC-E75 (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: Sequencing, Plasmid Preparation, Infection, CRISPR, Mutagenesis

Journal: bioRxiv

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1101/2024.05.24.595836

Figure Lengend Snippet: Distribution of newly acquired spacers in Δ hns +T and Δ sspA Δ hns +T strains upon lambda infection. a . Binned coverage plot of Δ hns + T newly acquired spacers across the E. coli genome (outer, purple). b . Binned coverage plot of Δ sspA Δ hns + T newly acquired spacers across the lambda genome (outer, purple).

Article Snippet: E. coli K-12 MG1655 and LC-E75 (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: Infection

Journal: International Journal of Biochemistry and Molecular Biology

Article Title: Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

doi:

Figure Lengend Snippet: Analysis of thiol content in E. coli cells. Analysis of the thiol content of wild type and gsp, yjfC, and ygiC gene knockout E. coli strains grown in LB media to stationary phase under anaerobic conditions was performed using HPLC. Labeled peaks represent DTNB derivatives of G-Sp and GSH. Peak immediately followed GSH (14 min) corresponds to DTNB derivative of γ-glutamylcysteine. Peaks at 18 min and 21 min are 2-nitro-5-thiobenzoate and the excess of DTNB correspondently.

Article Snippet: Materials E. coli K-12 genomic DNA was from ATCC (Manassas).

Techniques: Gene Knockout, Labeling

Journal: Nanomedicine : nanotechnology, biology, and medicine

Article Title: Synergic antibacterial coatings combining titanium nanocolumns and tellurium nanorods.

doi: 10.1016/j.nano.2018.12.009

Figure Lengend Snippet: Fig. 6: E. coli colonies on the different samples after 16h. N=3. *p<0.005 versus control, **p<0.01 versus control.

Article Snippet: For the sake of comparison, the sputtered Ti thin films as well as commercial Ti disks from Goodfellow (thickness: 0.5 mm, code: 303-115-36) were also used: no significant differences between them were observed, so their results were averaged and jointly labeled D. Escherichia coli (strain K-12 HB101; Bio-Rad, Hercules, CA) and Staphylococcus aureus (subsp. aureus Rosenbach, ATCC® 12600TM; ATCC, Manassas, VA) bacteria were used.

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase

doi: 10.1074/jbc.RA119.010999

Figure Lengend Snippet: Incorporation of nitroTyr into CaM expressed in E. coli. A, the human CaM construct with C-terminal His6 tag bearing either the native Tyr codon or an amber stop codon at amino acid position 99 or position 138 was co-transformed with a plasmid expressing an Methanocaldococcus jannaschii tyrosyl-tRNA synthetase/tRNACUA pair engineered to efficiently incorporate nitroTyr. B, WT– and nitroTyr–CaM was expressed in autoinduction medium with or without nitroTyr supplementation, purified using the C-terminal His6 tag, and analyzed by 15% SDS-PAGE gel. C, nitroTyr incorporation into CaM was determined here by Western blotting with a primary antibody against nitroTyr. D, electrospray ionization mass spectrometry confirms the quantitative incorporation of nitroTyr into CaM because all measured pure protein masses match their expected molecular masses.

Article Snippet: E. coli DH10B was transformed with pBad–CaM–(99 or 138 TAG) and pDule–nitroTyr–5B (Addgene plasmid no. 85498) ( 30 ).

Techniques: Construct, Transformation Assay, Plasmid Preparation, Expressing, Purification, SDS Page, Western Blot, Mass Spectrometry