A versatile one-step CRISPR-Cas9 based approach to plasmid-curing

Abstract Background Plasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking. Results Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have constructed a universal plasmid-curing system (pFREE) and developed a one-step protocol and PCR procedure that allow for identification of plasmid-free clones within 24 h. While the context of the targeted replicons affects efficiency, we obtained curing efficiencies between 40 and 100% for the plasmids most widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both Escherichia coli and the promising cell factory chassis Pseudomonas putida. Conclusion As a fast and freely available plasmid-curing system, targeting virtually all vectors used for cloning and expression purposes, we believe that pFREE has the potential to eliminate the need for individualized vector suicide solutions in molecular biology. We envision the application of pFREE to be especially useful in methodologies involving multiple plasmids, used sequentially or simultaneously, which are becoming increasingly popular for genome editing or combinatorial pathway engineering

Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid–host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli. We use experimental evolution, mathematical modelling and population sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions of costly regions from the plasmid backbone, effectively expanding the host-range of the plasmid. Although these adaptations were also beneficial to plasmid persistence in a naïve K. pneumoniae host, they were never observed in this species, indicating that differential evolvability can limit opportunities of plasmid adaptation. While insertion sequences are well known to supply plasmids with adaptive traits, our findings suggest that they also play an important role in plasmid evolution by maintaining the plasticity necessary to alleviate plasmid–host constrains. Further, the observed evolutionary strategy consistently followed by all evolved E. coli lineages exposes a trade-off between horizontal and vertical transmission that may ultimately limit the dissemination potential of clinical multidrug resistance plasmids in these hosts

Diverse genetic error modes constrain large-scale bio-based production

The declining performance of scale-up bioreactor cultures is commonly attributed to phenotypic and physical heterogeneities. Here, the authors reveal multiple recurring intra-pathway error modes that limit engineered E. coli mevalonic acid production over time- and industrial-scale fermentations

Evolution of antibiotic resistance in biofilm and planktonic <i>P. aeruginosa</i> populations exposed to sub-inhibitory levels of ciprofloxacin

ABSTRACT The opportunistic Gram-negative pathogen Pseudomonas aeruginosa , known for its intrinsic and acquired antibiotic resistance, has a notorious ability to form biofilms, which often facilitate chronic infections. The evolutionary paths to antibiotic resistance have mainly been investigated in planktonic cultures and are less studied in biofilms. We experimentally evolved P. aeruginosa PAO1 colony biofilms and stationary-phase planktonic cultures for seven passages in the presence of subinhibitory levels (0.1 mg/liter) of ciprofloxacin (CIP) and performed a genotypic (whole-bacterial population sequencing) and phenotypic assessment of the populations. We observed a higher proportion of CIP resistance in the CIP-evolved biofilm populations than in planktonic populations exposed to the same drug concentrations. However, the MICs of ciprofloxacin were lower in CIP-resistant isolates selected from the biofilm population than the MICs of CIP-resistant isolates from the planktonic cultures. We found common evolutionary trajectories between the different lineages, with mutations in known CIP resistance determinants as well as growth condition-dependent adaptations. We observed a general trend toward a reduction in type IV-pilus-dependent motility (twitching) in CIP-evolved populations and a loss of virulence-associated traits in the populations evolved in the absence of antibiotic. In conclusion, our data indicate that biofilms facilitate the development of low-level mutational resistance, probably due to the lower effective drug exposure than in planktonic cultures. These results provide a framework for the selection process of resistant variants and the evolutionary mechanisms involved under the two different growth conditions. </jats:p

Genome Dynamics of Escherichia coli during Antibiotic Treatment: Transfer, Loss, and Persistence of Genetic Elements In situ of the Infant Gut

Elucidating the adaptive strategies and plasticity of bacterial genomes in situ is crucial for understanding the epidemiology and evolution of pathogens threatening human health. While much is known about the evolution of Escherichia coli in controlled laboratory environments, less effort has been made to elucidate the genome dynamics of E. coli in its native settings. Here, we follow the genome dynamics of co-existing E. coli lineages in situ of the infant gut during the first year of life. One E. coli lineage causes a urinary tract infection (UTI) and experiences several alterations of its genomic content during subsequent antibiotic treatment. Interestingly, all isolates of this uropathogenic E. coli strain carried a highly stable plasmid implicated in virulence of diverse pathogenic strains from all over the world. While virulence elements are certainly beneficial during infection scenarios, their role in gut colonization and pathogen persistence is poorly understood. We performed in vivo competitive fitness experiments to assess the role of this highly disseminated virulence plasmid in gut colonization, but found no evidence for a direct benefit of plasmid carriage. Through plasmid stability assays, we demonstrate that this plasmid is maintained in a parasitic manner, by strong first-line inheritance mechanisms, acting on the single-cell level, rather than providing a direct survival advantage in the gut. Investigating the ecology of endemic accessory genetic elements, in their pathogenic hosts and native environment, is of vital importance if we want to understand the evolution and persistence of highly virulent and drug resistant bacterial isolates