Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality.

Plasmids of the ColE1 family are among the most frequently used plasmids in molecular biology. They were adopted early in the field for many biotechnology applications, and as model systems to study plasmid biology. The mechanism of replication of ColE1 plasmids is well understood, involving the interaction between a plasmid-encoded sense-antisense gene pair (RNAI and RNAII). Because of its mechanism of replication, bacterial cells cannot maintain two different plasmids with the same origin, with one being rapidly lost from the population — a process known as plasmid incompatibility. While mutations in the regulatory genes RNAI and RNAII have been reported to make colE1 plasmids more compatible, there has been no attempt to engineer compatible colE1 origins, which can be used for multi-plasmid applications and that can bypass design constrains created by the current limited plasmid origin repertoire available. Here, we show that by targeting sequence diversity to the loop regions of RNAI (and RNAII), it is possible to select new viable colE1 origins that are compatible with the wild-type one. We demonstrate origin compatibility is not simply determined by sequence divergence in the loops, and that pairwise compatibility is not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality. New characterised plasmid origins increase flexibility and accessible complexity of design for challenging synthetic biology applications where biological circuits can be dispersed between multiple independent genetic elements.

pKBuS13, a KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 833, Carrying Tn4401bInserted into an Xer Site-Specific Recombination Locus

ABSTRACTHere, we report the first detection of aKlebsiella pneumoniaecarbapenemase 2 (KPC-2)-producingKlebsiella pneumoniaestrain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. ItsblaKPCdeterminant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::blaKPC-2transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401bdetected a region that did not have significant matches in databases other than the genome of a carbapenem-resistantEscherichia colistrain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence oftragenes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported.

Resistome Analysis of Enterobacter cloacae CY01, an Extensively Drug-Resistant Strain Producing VIM-1 Metallo-β-Lactamase from China

ABSTRACTResistome analysis of clinical VIM-1-producingEnterobacter cloacaestrain CY01 from China revealed the presence of multiple resistance determinants. Two resistance plasmids were identified in CY01. The pCY-VIM plasmid was 14 kb in size and possessed a replicase gene (repA), a gene cluster encoding the partitioning function (parABC), and a carbapenemase gene (blaVIM-1). Another 5.9-kb plasmid, pCY-MdT, with anaac(6′)-Ibgene, was very closely related (13 nucleotide differences) to pMdT1, a ColE1 plasmid carryingaac(6′)-Ib-cr4.

Prevalence of ColE1-Like Plasmids and Kanamycin Resistance Genes in Salmonella enterica Serovars

ABSTRACT Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kanr) phenotypes, 102 Kanr Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kanr Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3′)-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group.