Comparative genomics of Pseudomonas fluorescens subclade III strains from human lungs

Abstract

Background: While the taxonomy and genomics of environmental strains from the P. fluorescens species-complex has been reported, little is known about P. fluorescens strains from clinical samples. In this report, we provide the first genomic analysis of P. fluorescens strains in which human vs. environmental isolates are compared.

Results: Seven P. fluorescens strains were isolated from respiratory samples from cystic fibrosis (CF) patients. The clinical strains could grow at a higher temperature (>34 °C) than has been reported for environmental strains. Draft genomes were generated for all of the clinical strains, and multi-locus sequence analysis placed them within subclade III of the P. fluorescens species-complex. All strains encoded type- II, -III, -IV, and -VI secretion systems, as well as the widespread colonization island (WCI). This is the first description of a WCI in P. fluorescens strains. All strains also encoded a complete I2/PfiT locus and showed evidence of horizontal gene transfer. The clinical strains were found to differ from the environmental strains in the number of genes involved in metal resistance, which may be a possible adaptation to chronic antibiotic exposure in the CF lung.

Conclusions: This is the largest comparative genomics analysis of P. fluorescens subclade III strains to date and includes the first clinical isolates. At a global level, the clinical P. fluorescens subclade III strains were largely indistinguishable from environmental P. fluorescens subclade III strains, supporting the idea that identifying strains as 'environmental' vs 'clinical' is not a phenotypic trait. Rather, strains within P. fluorescens subclade III will colonize and persist in any niche that provides the requirements necessary for growth.

Fig. 1

Phylogenetic tree of the P. fluorescens strains in this study. Multi-locus sequence analysis of the following eight housekeeping genes was used to infer the phylogenetic tree: dnaE (DNA polymerase III alpha subuit); ppsA (phosphoenolpyruvate synthase); recA (Recombinase A); rpoB (RNA polymerase subunit beta); gyrB (DNA gyrase subunit B); guaA (GMP synthease); mutL (DNA mismatch repair protein); pyrC (pyrimidine biosynthetic enzyme dihydroorotase) and acsA (acetyl-CoA synthetase). The concatenated sequences were aligned with MAFFT as described in the materials and methods. Clinical strains are highlighted in red [80, 81]

Fig. 2

Phenogram based on average nucleotide identity (ANI) between subclade III strains. Phenogram created based on a similarity matrix of the average nucleotide identity (ANI) between subclade III strains. Clustering performed using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis

Fig. 3

The pan, accessory and core genomes in subclade III strains. Each of the 11795 COGS was analyzed to determine how many genomes encoded that particular COG (1–11, x-axis). Then the number of COGS encoded by only one genome, two genomes, etc. was determined (y-axis). The pan genome contains the accessory and core. The average number of COGs per genome is 5592 (range: 5332–6123). The pan genome and its compartments was calculated using the COGtriganle clustering algorithm in GET_HOMOLOGUES [82, 83]

Fig. 4

The czc gene cluster in environmental and clinical subclade III strains. A. The total number of genes annotated as belonging to the czcA gene cluster in environmental and clinical subclade III strains. B. The percent G + C content of the czcA gene homologues. Open circles correspond to the G + C content found across the entire genome. Open circles correspond to the G + C content found across the entire genome. Arrows indicate the czcA homologs enclosed in boxes in Fig. 5

Fig. 5

Neighborhood-joining phylogenetic tree based on the amino acid sequence of czcA homologues. czcA homologues from clinical strains are highlighted in red; homologs from environmental strains are highlighted in green; homologues from other Pseudomonas spp. are highlighted in blue. Amino acid sequences of the proteins expressed from czcA homologues discovered in RAST [78]. All other bacteria species are publically available on RAST. The amino acid sequences were aligned with MAFFT [80, 81]

Fig. 6

Alignment of the PfiT amino acid sequences from subclade III strains. Amino acid sequence of the PifT protein aligned using MUSCLE [87]. Published sequence of PfiT used as reference (labeled PfiT) [27]. Only amino acids differing from reference are shown and colored. Percent nucleotide consensus match visualized in green underneath alignment. Alignment visualized with DNAstar MegAligPro

Fig. 7

Genes that encode the Type-II, III, IV, VI and WCI secretion system in subclade III strains. Genes annotated using the RAST pipeline [77]. Full gene names listed in Additional file 7: Table S6