Pseudomonas stutzeri is a species complex with extremely broad phenotypic and genotypic diversity. However, very little is known about its diversity, taxonomy and phylogeny at the genomic scale. To address these issues, we systematically and comprehensively defined the taxonomy and nomenclature for this species complex and explored its genetic diversity using hundreds of sequenced genomes. By combining average nucleotide identity (ANI) evaluation and phylogenetic inference approaches, we identified 123 P. stutzeri complex genomes covering at least six well-defined species among all sequenced Pseudomonas genomes; of these, 25 genomes represented novel members of this species complex. ANI values of ≥∼95% and digital DNA-DNA hybridization (dDDH) values of ≥∼60% in combination with phylogenomic analysis consistently and robustly supported the division of these strains into 27 genomovars (most likely species to some extent), comprising 16 known and 11 unknown genomovars. We revealed that 12 strains had mistaken taxonomic assignments, while 16 strains without species names can be assigned to the species level within the species complex. We observed an open pan-genome of the P. stutzeri complex comprising 13,261 gene families, among which approximately 45% gene families do not match any sequence present in the COG database, and a large proportion of accessory genes. The genome contents experienced extensive genetic gain and loss events, which may be one of the major mechanisms driving diversification within this species complex. Surprisingly, we found that the ectoine biosynthesis gene cluster (ect) was present in all genomes of P. stutzeri species complex strains but distributed at very low frequency (43 out of 9548) in other Pseudomonas genomes, suggesting a possible origin of the ancestors of P. stutzeri species complex in high-osmolarity environments. Collectively, our study highlights the potential of using whole-genome sequences to re-evaluate the current definition of the P. stutzeri complex, shedding new light on its genomic diversity and evolutionary history.

Mercury (Hg) pollution poses human health and environmental risks worldwide, as it can have toxic effects and causes selective pressure that facilitates the spread of antibiotic resistant genes (ARGs) among microbes. More and more studies have revealed that numerous Hg-related genes (HRGs) can help to resist and transform Hg. In the present study, we systematically analyzed the HRG distribution, abundance, organization, and their co-distribution with ARGs, using 18,731 publicly available plasmid genomes isolated from a Gammaproteobacteria host. Our results revealed that there were many Hg-resistant (mer) operon genes but they were not extensively distributed across plasmids, with only 9.20% of plasmids harboring HRGs. Additionally, no hgcAB genes (which methylate Hg to create methylmercury) were identified in any of the analyzed plasmids. The host source significantly influenced the number of HRGs harbored by plasmids; plasmids isolated from humans and animals harbored a significantly smaller number of HRGs than plasmids isolated from the wastewater and sludge. HRG clusters displayed an extremely high organizational diversity (88 HRG cluster types), though incidences of more than half of the HRG cluster types was <5. This indicates the frequent rearrangement among HRGs in plasmids. The 1368 plasmids harboring both HRGs and ARGs, were dominated by Klebsiella, followed by Escherichia, Salmonella, and Enterobacter. The tightness of the HRG and ARG co-distribution in plasmids was affected by the host sources but not by pathogenicity. HRGs were more likely to co-occur with specific ARG classes (sulfonamide, macrolide-lincosamide-streptogramin, and aminoglycoside resistance genes). Collectively, our results reveal the distribution characteristics of HRGs in plasmids, and they have important implications for further understanding the environmental risks caused by the spread of ARGs through the plasmid-mediated co-transfer of ARGs and HRGs.