TOL Plasmid pWW0 Transposons Facilitate Adaptation of Pseudomonas putida for Growth on the New Carbon Source m-Cresol.

Performance of the pBHR1 mobilization protein MobV and its role in stable plasmid expression in Rhodopseudomonas palustris CGA009

This study investigates the effects of pBHR1’s native mobilization protein, MobV, on the retention of pBBR1 origin plasmids in R. palustris, and provides design principles for constructing stable, high-performing vectors in non-model gram-negative hosts.

Broad adaptations of plasmid-carrying Escherichia coli and Klebsiella pneumoniae to water as a reservoir

The spread of antimicrobial-resistant Enterobacterales is a major One Health issue, with aquatic environments increasingly recognized as potential reservoirs. However, data on bacterial adaptation to water is limited. We examined the survival and transcriptomic adaptation of Escherichia coli and Klebsiella pneumoniae, including plasmid-cured variants (PCVs), during 14 days in sterilized tap and river water. The strains belonged to sequence types (ST)648 and ST307, representing international high-risk clonal lineages. Viable cell counts of wild-type strains and PCVs remained stable in both water types. We used RNA sequencing, followed by functional analysis of the differentially expressed genes. Considerable transcriptomic changes occurred, especially in K. pneumoniae, with extensive regulation of genes related to inorganic ion transport, and coenzyme and nutrient transport and metabolism. Adaptational differences between wild-type strains and PCVs highlighted plasmid-associated effects. These findings demonstrate strain-specific adaptive responses to aquatic environments and underline the context-dependent influence of plasmids in shaping adaptation.

A Conditional Plasmid System for Markerless Gene Deletion in Genetically Recalcitrant Fusobacterium nucleatum subsp. animalis.

This chapter provides a detailed, step-by-step protocol for implementing a conditional plasmid system that enables efficient, markerless gene deletion in FNA strains and provides a powerful and adaptable tool for advancing genetic studies in this genetically recalcitrant subspecies.

Genomic insights into the bioremediation potential of Paenibacillus taichungensis strain 53b: a multi-resistant plant growth-promoting bacterium

Identification of essential genes for conjugative transfer in antimicrobial resistance-associated pELF-type linear plasmids of opportunistic pathogen Enterococcus faecium

The pELF-type linear plasmid is a critical mobile genetic element responsible for the dissemination of various antimicrobial resistance (AMR) genes, most notably vancomycin resistance in Enterococcus faecium, which is a leading cause of hospital outbreaks worldwide. Despite their crucial role in the expansion of AMR, the molecular mechanisms underlying the conjugative transfer of these linear plasmids remain poorly understood. In this study, the transfer (tra) region of pELF2, a representative vanA-harboring linear plasmid was characterized. Transcriptomic data suggested that the FtsK/VirD4-type adenosine triphosphatase is encoded within a multi-gene operon. By developing a genetic manipulation framework for E. faecium, an extensive mutational analysis of the tra region was performed and the following three essential genes were identified: traCB4 (a putative VirB4 analog), traDD4 (a VirD4-like coupling protein), and traGB6 (a putative VirB6 analog). These genes are indispensable for conjugative transfer. Reporter assays experimentally confirmed the presence of a functional promoter upstream of the identified tra genes. We confirmed that these genes are highly conserved among pELF-type plasmid sequences deposited in public database. The study findings revealed that pELF-type plasmids utilize highly minimized conjugation machinery, which is similar to unusual systems previously identified in other gram-positive bacteria, such as Streptomyces. This study provides the first molecular insights into the transmission of these clinically important linear plasmids in enterococci and lays a foundation for understanding the dissemination of resistance determinants mediated by atypical mobile genetic elements.

The metal-uptake-deficient Escherichia coli strain GR536 contains the ϕ80 prophage

Abstract We report the genome sequence of Escherichia coli GR536, a previously constructed metal-uptake-deficient strain derived from E. coli W3110. Growth of GR536 in an iron-restricted liquid medium resulted in apparent lysis during the early exponential growth phase. This effect was exacerbated in cells transformed with pBAD30, a commonly used arabinose-inducible expression vector. Whole-genome sequencing confirmed the expected gene disruptions (entC, feoABC, mntH, zupT::cat and fecABCDE::kan). However, comparison to E. coli W3110 identified the presence of the ϕ80 prophage (46.16 kbp) and cryptic prophage CPZ-55 (6.763 kbp), as well as the absence of cryptic prophage e14 (15.193 kbp). We also identified 9 IS-element deletions, 3 IS-element insertions, 7 other deletions or insertions and 74 candidate individual nucleotide changes. The growth defect in GR536 correlated with lysis due to the production of ϕ80 virions as determined by the inability of isolated phage to infect an E. coli strain lacking the phage receptor (∆fhuA) and the BamHI digestion pattern of the purified phage DNA. We further determined that the ϕ80-dependent lysis in GR536 is exacerbated by the presence of the chloramphenicol- and kanamycin-resistance markers introduced during construction of GR536 and the pBAD30 plasmid multiple cloning site. Removal of the markers (E. coli GR536*) and disruption of the pBAD30 multiple cloning site generated a strain that showed a 104-fold reduction in ϕ80 production. Furthermore, construction of a W3110 lysogen containing the ϕ80 prophage and comparison with GR536* grown under the same conditions showed a ~104-fold higher level of phage production by the parent strain, indicating that phage-dependent lysis was not increased by the deletion of the metal-uptake genes and thus independent of iron availability. These observations clarify growth conditions that limit the effects of ϕ80-dependent lysis when using GR536 to identify metal-uptake genes by complementation, specifically, removal of the antibiotic resistance markers and the avoidance of using intact pBAD30 as a negative control.