In-depth genome and pan-genome analysis of a metal-resistant bacterium Pseudomonas parafulva OS-1

Abstract

A metal-resistant bacterium Pseudomonas parafulva OS-1 was isolated from waste-contaminated soil in Ranchi City, India. The isolated strain OS-1 showed its growth at 25-45°C, pH 5.0-9.0, and in the presence of ZnSO₄ (upto 5 mM). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain OS-1 belonged to the genus Pseudomonas and was most closely related to parafulva species. To unravel the genomic features, we sequenced the complete genome of P. parafulva OS-1 using Illumina HiSeq 4,000 sequencing platform. The results of average nucleotide identity (ANI) analysis indicated the closest similarity of OS-1 to P. parafulva PRS09-11288 and P. parafulva DTSP2. The metabolic potential of P. parafulva OS-1 based on Clusters of Othologous Genes (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated a high number of genes related to stress protection, metal resistance, and multiple drug-efflux, etc., which is relatively rare in P. parafulva strains. Compared with other parafulva strains, P. parafulva OS-1 was found to have the unique β-lactam resistance and type VI secretion system (T6SS) gene. Additionally, its genomes encode various CAZymes such as glycoside hydrolases and other genes associated with lignocellulose breakdown, suggesting that strain OS-1 have strong biomass degradation potential. The presence of genomic complexity in the OS-1 genome indicates that horizontal gene transfer (HGT) might happen during evolution. Therefore, genomic and comparative genome analysis of parafulva strains is valuable for further understanding the mechanism of resistance to metal stress and opens a perspective to exploit a newly isolated bacterium for biotechnological applications.

Keywords: Pseudomonas parafulva; carbohydrate active enzymes; genome; pan-genome; virulence.

Figure 1

Classification of the cluster of orthologus (COG) functional annotation of P. parafulva OS-1 genome. Colored bars indicate a number of genes assigned to each COG functional category.

Figure 2

Circular genome map of P. parafulva OS-1 compared to other type strains. The percentage similarity is represented by different color codes. The information is read from outer circle to inner as follows: genome size, genes on the forward strand, genes on the reverse strand, t-RNA, r-RNA, GC content, GC skew + and - COG names: Information storage and processing [J] Translation, ribosomal structure and biogenesis [A] RNA processing and modification [K] Transcription [L] Replication, recombination and repair [B] Chromatin structure and dynamics, cellular processes and signaling [D] Cell cycle control, cell division, chromosome partitioning [Y] Nuclear structure [V] Defense mechanisms [T] Signal transduction mechanisms [M] Cell wall/membrane/envelope biogenesis [N] Cell motility [Z] Cytoskeleton [W] Extracellular structures [U] Intracellular trafficking, secretion, and vesicular transport [O] Post-translational modification, protein turnover, chaperones metabolism [C] Energy production and conversion [G] Carbohydrate transport and metabolism [E] Amino acid transport and metabolism [F] Nucleotide transport and metabolism [H] Coenzyme transport and metabolism [I] Lipid transport and metabolism [P] Inorganic ion transport and metabolism [Q] Secondary metabolites biosynthesis, transport and catabolism poorly characterized [R] General function prediction only [S] Function unknown.

Figure 3

Average nucleotide identity (ANI) matrix showing genomic relatedness performing multi-genome comparison among P. parafulva strains from NCBI Database.

Figure 4

The distribution of various CAZymes like carbohydrate-binding modules (CBMs), glycoside hydrolases (GHs), glycosyl transferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and auxiliary activities (AAs) in P. parafulva OS-1, and its comparison to other P. parafulva strains.

Figure 5

Graph of the pan-genome distribution of P. parafulva genomes; (A) Bar plot of pangenome distribution; (B) Circular plot of pan genome: innermost circle (in light pink) represents core genome. The 2nd innermost circle (in dark orange) represents the gene in the shell, whereas 2nd outermost circle (in yellow) represents the soft core and the outermost circle (in red) represents cloud genes that are unique or present in a maximum of two genomes; (C) Comparison between the core-genome and pan-genome tree. Normalized Robinson-Foulds (nRF) and normalized matching cluster (nMC) scores were used to measure the congruence of the two trees.

Figure 6

The heatmap (COG functional) represents the distribution of the functional abundance of differentially enriched metabolic functions in P. parafulva genomes. Heatmap showing the normalized relative abundance of the clusters of orthologous groups (COG) categories enriched in the protein-coding genes in the selected P. parafulva genomes. The strains and COG categories were clustered using the Manhattan distance. The color scale represents the relative abundance of gene content for each category, normalized by the sample mean.