Comparative genomics and DNA methylation analysis of Pseudomonas aeruginosa clinical isolate PA3 by single-molecule real-time sequencing reveals new targets for antimicrobials

Abstract

Introduction: Pseudomonas aeruginosa (P.aeruginosa) is an important opportunistic pathogen with broad environmental adaptability and complex drug resistance. Single-molecule real-time (SMRT) sequencing technique has longer read-length sequences, more accuracy, and the ability to identify epigenetic DNA alterations.

Methods: This study applied SMRT technology to sequence a clinical strain P. aeruginosa PA3 to obtain its genome sequence and methylation modification information. Genomic, comparative, pan-genomic, and epigenetic analyses of PA3 were conducted.

Results: General genome annotations of PA3 were discovered, as well as information about virulence factors, regulatory proteins (RPs), secreted proteins, type II toxin-antitoxin (TA) pairs, and genomic islands. A genome-wide comparison revealed that PA3 was comparable to other P. aeruginosa strains in terms of identity, but varied in areas of horizontal gene transfer (HGT). Phylogenetic analysis showed that PA3 was closely related to P. aeruginosa 60503 and P. aeruginosa 8380. P. aeruginosa's pan-genome consists of a core genome of roughly 4,300 genes and an accessory genome of at least 5,500 genes. The results of the epigenetic analysis identified one main methylation sites, N6-methyladenosine (m6A) and 1 motif (CATNNNNNNNTCCT/AGGANNNNNNNATG). 16 meaningful methylated sites were picked. Among these, purH, phaZ, and lexA are of great significance playing an important role in the drug resistance and biological environment adaptability of PA3, and the targeting of these genes may benefit further antibacterial studies.

Disucssion: This study provided a detailed visualization and DNA methylation information of the PA3 genome and set a foundation for subsequent research into the molecular mechanism of DNA methyltransferase-controlled P. aeruginosa pathogenicity.

Keywords: DNA methylation analysis; Pseudomonas aeruginosa; SMRT; antibacterial; comparative genome analysis; epigenetics.

Figure 1

Genome-wide circular map of P. aeruginosa PA3. PA3 Genome Visualization Circle Diagram. In the outermost region, protein-coding regions (CDS) are indicated in red. From outside to inside, the outermost loop shows the CDS on the negative strand, followed by the CDS on the positive strand, the Comprehensive Antibiotic Resistance Database (CARD) resistance gene cite ds DNA phage sequence, MGEs, HGT region, putative MTase, GC content (black), and GC bias (purple/green).

Figure 2

Regulatory proteins of P. aeruginosa PA3. (A) Distribution of TCS in the PA3 genome. (B) Detailed number and species of TCS proteins encoded by PA3. (C) Distribution of TFs in the PA3 genome. (D) Detailed number and species of TFs encoded by PA3. (E) Distribution of ODP in the PA3 genome. (F) Detailed quantities and types of ODPs encoded by PA3.

Figure 3

Secretory proteins of P. aeruginosa PA3. PA3 contains 576 Sec/SPI [“standard” secretory signal peptides transported by the Sec translocon and cleaved by Signal Peptidase I (Lep)], 215 Sec/SPII [lipoprotein signal peptides transported by the Sec translocon and cleaved by Signal Peptidase II (Lsp)], 30 Tat/SPI [Tat signal peptides transported by the Tat translocon and cleaved by Signal Peptidase I (Lep)], 4 Tat/SPII [Tat lipoprotein signal peptides transported by the Tat translocon and cleaved by Signal Peptidase II (Lsp)], 1 Sec/SPIII [Pilin and pilin-like signal peptides transported by the Sec translocon and cleaved by Signal Peptidase III (PilD/PibD)].

Figure 4

Type II T–A loci and genomic islands of P. aeruginosa PA3. (A) Distribution of 15 pairs of type II T–A loci in the PA3 genome. (B) Genomic islands in the PA3 genome. From outside to inside are GC content (red) and GC bias (purple/green) and three methods for predicting gene islands, IslandPath-DIMOB, IslandPick, SIGI-HMM (Single Island Genetic Island Hidden Markov Model).

Figure 5

BLAST comparison of the complete genome of P. aeruginosa PA3 with 19 other P. aeruginosa species. Figure legends of the 20 P. aeruginosa strains are shown from innermost to outermost. Identity is indicated by color.

Figure 6

Phylogenetic tree of P. aeruginosa based on 16S rRNA. Phylogenetic relationships of 126 strains of P. aeruginosa (showing only topological structure). The two subgroups are shown in red and blue branches, respectively. The 20 comparative P. aeruginosa strains in Figure 4 are shown in bold yellow font.

Figure 7

Phylogenetic tree of P. aeruginosa based on 16S rRNA. (A) Venn diagram of five P. aeruginosa strains showing the number of core genes, accessory genes, and their unique genes. (B) Pan- and core-genomes of 20 strains of P. aeruginosa with complete genomes.

Figure 8

The overview of 16 m6A methylation sites and their genes in P. aeruginosa PA3. The three tracks include 10 m6A sites on the positive strand, 6 sites on the negative strand, and their corresponding 16 genes.