Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa

Abstract

Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C and D, three genes for LPS biosynthesis and had reduced antibiotic resistance genes when compared with clinical strains. Genes belonging to proteases, bacterial exporters and DNA stabilization were found to be under strong positive selection, thus facilitating pathogenicity and survival of the outliers. The outliers had the complete operon for the production of vibrioferrin, a siderophore present in plant growth promoting bacteria. The competence to acquire multidrug resistance and new virulence factors makes these strains a potential threat. However, we identified major regulatory hubs that can be used as drug targets against both the classical and outlier groups.

Keywords: Pseudomonas aeruginosa; drug targets; environmental genomics; outliers; positive selection.

FIGURE 1

General genomic attributes of Pseudomonas aeruginosa CR1. (A) Circular map of chromosome 1 of CR1 strain. From outside to inside Ring 1: COG categories of ORFs in the positive strand. Ring 2: COG categories of ORFs in the negative strand. Ring 3: Regions of Genomic plasticity starting from zero bp mark in a clockwise direction. Ring 4: BLASTn alignment (expected threshold = 1e-220) between CR1 and previously sequenced outlier PA7 (teal); Ring 5: PAO1 (aqua); Ring 6: UCBPP-PA14 (orange). Rings 7 and 8: GC content and GC skew, respectively. (B) Circular map of plasmid pCR1. From outside to inside Rings 1, 2, 3, and 4 depicts COG categories of ORFs of the positive strand, genes on positive strand, genes on negative strand and COG categories of ORFs of negative strand, respectively. Rings 5 and 6 represents GC content and GC skew, respectively.

FIGURE 2

Phylogenetic analysis of P. aeruginosa strains using pan-genome matrix. The consensus pan-genome matrix was generated using the intersection of pan genes predicted by COG and OMCL algorithms to construct a parsimony tree using PARS from the PHYLIP. Three major clusters represented by PAO1 (Blue), UCBPP-PA14 (Green), and PA7 (Maroon) were observed. The outlier clade represented by PA7 was split into 2 clades one with a representative genome of PA7 (Maroon) and second by strain CR1 (red).

FIGURE 3

Estimation of core and pan-genome of P. aeruginosa strains with Tettelin fit (blue: pangenome; red: core-genome). (A,B) Represents the estimate of the pan and core genome of 14 outlier strains; (C,D) represents the estimate of core and pan-genome of 64 P. aeruginosa strains included in the current study and (E,F) represents estimate of the core and pan-genome curves for 78 genomes. The x-axis represents the number of genomes (g) while the y-axis represents the core genome and pan-genome size (number of genes). The equation for estimating pan and core-genome size according to Tettelin fit are given with the respected graphs.

FIGURE 4

Comparative functional analysis based on COG categories between core and accessory genes in (A) outlier strains (n = 14) and (B) classical strains (n = 64). COG-based binning of core genes and accessory genes. The abscissa denotes different COG functional categories. The ordinate denotes the number of genes in each COG category. Four COG functional categories (RNA processing and modification, Chromatin structure and dynamics, Extracellular structures, and Cytoskeleton) including only one or without homologs in the COG collection are not displayed. Significant enrichment of gene occurrence in the individual category is marked by asterisks (^∗p < 0.05, ^∗∗p < 0.001; FDR < 10% Chi-square test).

FIGURE 5

Functional analysis of Pseudomonas outlier strains. (A) The amino acid sequences of all the 14 genomes were assigned KO number by KAAS server. The protein families were then mapped for a minimal set of pathways using a parsimony approach. The heat map was constructed based on most abundant top 50 subsystems using Pearson correlation. (B) The ORF’s were mapped against the COG database using rpsblast. For both the heatmaps rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage.

FIGURE 6

PCA analysis on pathogenic potential of P. aeruginosa outlier strains. The PCA- analysis was performed on the four parameters namely number of pathogenic and non-pathogenic families annotated, probability of being a pathogen and input proteome percentage among the 14 strains. PA7 along with WH-SGI-V-07370, WH-SGI-V-07064, and WH-SGI-V-07072 clustered in a different quadrant from the other strains showing high pathogenicity.

FIGURE 7

Protein–Protein Interaction network of (A) Core genes of outlier strains (n = 14) and (B) Core genes of classical strains (n = 64). The circles represent protein hubs and line represent edges. The radius of major regulatory hubs decreases as the number of interactions decreases. The topological properties of these networks depicting the correlation coefficient values (r²). (C) Node degree distribution, (D) average clustering coefficient, (E) average neighborhood connectivity. All these properties follow the power distribution and show the nature of the scale-free network and hierarchical organization.