https://orcid.org/0000-0002-9692-7577
Figures & data
Table 1 Bacterial Strains and Plasmids
Table 2 Primers Used for RT-PCR and CRISPRi System
Figure 1 An arabinose-inducible CRISPRi system for tunable repression of PA0715 expression in P. aeruginosa.
Notes: In the presence of glucose, the arabinose promoter does not work properly, resulting in the suppression of downstream dcas9 gene expression when the target gene is normally expressed. Conversely, in the presence of arabinose, the arabinose promoter is activated, initiating the dcas9 gene expression product and eventually interfering with the normal expression of the target gene.
Abbreviation: RNAP, RNA polymerase.
Figure 2 PA0715 and its putative expression product.
Notes: (A) The phylogenetic tree of the 17 proteins was constructed with the PA0715 product as its center. (B) The structural domain located at amino acids 40–255 is a member of the reverse transcriptase family, suggesting that PA0715 may be an RNA-directed DNA polymerase that catalyzes the synthesis of DNA single strands from RNA templates. The domain prediction of PA0715 product is based on “CDD tools” of “Conserved Domains” under NCBI database. (C) Structural comparison of the product of the PA0715 gene with known reverse transcriptase family proteins. The structural prediction of two kinds of protein is based on SWISS-MODEL database. (D) A stack represents each amino acid site in the sequence. The total height of the stack symbolizes the conservativeness of the amino acids at that position.
Figure 3 Effect of PA0715 suppression on the proliferation and activity of PAO1 strains.
Notes: (A) The inhibition efficiency of CRISPRi targeting PA0715 was verified by qRT-PCR. (B) Growth curves of the strains under different conditions. (C) Swimming, swarming and twitching of different strains in culture medium. Reduced swimming viability was manifested by a decrease in the size of the circular colonies. Swarming motility is characterized by the formation of characteristic irregular branches. The size of the twitching zone was observed to exhibit irregular patterns after staining with Coomassie Blue. (D) Maximum diameter of PAO1 strains in (C) that formed different shapes of motility zones on different media. n = 3,*p < 0.05,**p < 0.01, ***p < 0.001 vs the PAO1-pdsgRNA-Ara group using Student’s t-test. a:PAO1-pdsgRNA-Ara(PA0715-silent mutants); b:PAO1-pdsgRNA-Glu(control1); c:PAO1-Ara(control2); d:PAO1-Glu(control3); e:PAO1(control4).
Figure 4 Changes in pyocyanin production and biofilm formation due to PA0715 inhibition.
Notes: (A) Quantification of pyocyanin in the supernatant of PAO1 strain cultures after chloroform and 0.2 M HCL extraction. (B) Production of biofilm formed by the PAO1 strain on a solid surface determined by crystalline violet staining of the biofilm. (C) The crystalline violet in (B) was dissolved in 95% ethanol, and the absorbance was measured at 590 nm. The OD590 value represents the absolute amount of biofilm formation. (D) Quantification of live bacteria in biofilms. Live bacteria were quantified by detecting colony-forming units of PAO1 with the same OD value. n =3, *p < 0.05,**p < 0.01, ***p < 0.001, nsp > 0.05 vs the PAO1-pdsgRNA-Ara group using Student’s t-test.
Table 3 MIC of the Four Antibiotics Against PAO1 Strains Under Different Conditions (μg/mL)
Table 4 Expression of Part Genes Related to Motility and Attach-Ment, Biofilm Formation, Antibiotic Resistance and Susceptibility and Oxidative Phosphorylation
Figure 5 SEMimages of PA0715 being or not being inhibited by PAO1 cells.
Notes: The surface of PAO1 cells of all samples showed relatively normal morphology, but a few cells showed depression and roughness. The arrows show the depression and roughness on the cell surface. Scale bar: 5 μm. (A) PAO1-pdsgRNA-Ara(PA0715-silent mutants); (B) PAO1-pdsgRNA-Glu(control1); (C) PAO1-Ara(control2); (D) PAO1-Glu(control3); (E) PAO1(control4).
Figure 6 Survival of Galleria mellonella inoculated with PA0715-suppressed or control strains.
Notes: (A) Survival curve graphs of the G. mellonella at a strain concentration of 103 CFU/mL. (B) Survival corresponding to the 12th and 48th hour of infection. Larvae first exhibit melanization at the injection site during infection that gradually spreads throughout the body until death. Ten larvae per group were used for the infection tests. a:PAO1-pdsgRNA-Ara(PA0715-silent mutants); b:PAO1-pdsgRNA-Glu(control1); c:PAO1-Ara(control2); d:PAO1-Glu(control3); e:PAO1(control4).
Figure 7 Volcano map of genes regulated by PA0715, and functional classification based on the Gene Ontology (GO) database (http://www.geneontology.org).
Notes: (A) Volcano plots of significantly differentially expressed genes are shown. The abscissa in the volcano map represents the logarithm of gene differential expression multiple in PA0715 strain when PA0715 was inhibited or not. The vertical axis represents the negative logarithm of the false discovery rate of each gene. Each dot represents a gene, with red dots representing upregulated genes, green dots representing downregulated genes, and blue dots representing unchanged genes. (B) Functional classification of all PA0715-dependent genes according to the GO database. (C) Functional classification of PA0715-dependent upregulated genes according to the GO database. (D) Functional classification of PA0715-dependent downregulated genes according to the GO database. The bar chart for each category of enrichment pathways gives the absolute number of all significantly different genes up- or downregulated by PA0715.
Figure 8 KEGG functional annotation and RT-qPCR validation of differential genes in PAO1 strains with or without PA0715 being repressed.
Notes: (A) KEGG functional classification of all significantly differential genes. (B) Functional classification of the 900 upregulated differential genes. (C) Functional classification of the 857 downregulated differential genes. The bubble color indicates the significance of the enriched KEGG pathway. Enrichment factor indicates the ratio of the number of differential genes annotated as KEGG pathway categories to the number of all genes annotated as that category. (D) Six significant genes were used to validate the accuracy of RNA-seq results.
