No two clones are alike: characterization of heterologous subpopulations in a transgenic cell line of the model diatom Phaeodactylum tricornutum.

BACKGROUND: Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom. RESULTS: Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein (eGFP). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system. CONCLUSIONS: Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications.

Increased copy number couples the evolution of plasmid horizontal transmission and plasmid-encoded antibiotic resistance.

Conjugative plasmids are mobile elements that spread horizontally between bacterial hosts and often confer adaptive phenotypes, including antimicrobial resistance (AMR). Theory suggests that opportunities for horizontal transmission favor plasmids with higher transfer rates, whereas selection for plasmid carriage favors less-mobile plasmids. However, little is known about the mechanisms leading to variation in transmission rates in natural plasmids or the resultant effects on their bacterial host. We investigated the evolution of AMR plasmids confronted with different immigration rates of susceptible hosts. Plasmid RP4 did not evolve in response to the manipulations, but plasmid R1 rapidly evolved up to 1,000-fold increased transfer rates in the presence of susceptible hosts. Most evolved plasmids also conferred on their hosts the ability to grow at high concentrations of antibiotics. This was because plasmids evolved greater copy numbers as a function of mutations in the copA gene controlling plasmid replication, causing both higher transfer rates and AMR. Reciprocally, plasmids with increased conjugation rates also evolved when selecting for high levels of AMR, despite the absence of susceptible hosts. Such correlated selection between plasmid transfer and AMR could increase the spread of AMR within populations and communities.

Conjugative plasmid-encoded toxin-antitoxin system PrpT/PrpA directly controls plasmid copy number.

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis.

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia pseudotuberculosis Infection.

Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Yersinia pseudotuberculosis upregulates the virulence plasmid copy number (PCN) during infection and that the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the present study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer's patches and cecum during the clonal invasive phase of the infection, while the highest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues and will be readily applicable to other infection models.

Enhanced production of 5-hydroxytryptophan through the regulation of L-tryptophan biosynthetic pathway.

5-Hydroxytryptophan (5-HTP) is the precursor of the neurotransmitter serotonin and has been used for the treatment of various diseases such as depression, insomnia, chronic headaches, and binge eating associated obesity. The production of 5-HTP had been achieved in our previous report, by the development of a recombinant strain containing two plasmids for biosynthesis of L-tryptophan (L-trp) and subsequent hydroxylation. In this study, the L-trp biosynthetic pathway was further integrated into the E. coli genome, and the promoter strength of 3-deoxy-7-phosphoheptulonate synthase, which catalyzes the first step of L-trp biosynthesis, was engineered to increase the production of L-trp. Hence, the 5-HTP production could be manipulated by the regulation of copy number of L-trp hydroxylation plasmid. Finally, the 5-HTP production was increased to 1.61 g/L in the shaking flasks, which was 24% improvement comparing to the original producing strain, while the content of residual L-trp was successfully reduced from 1.66 to 0.2 g/L, which is beneficial for the downstream separation and purification. Our work shall promote feasible progresses for the industrial production of 5-HTP.

The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms.

This work assesses the effect of chemical induction with isopropyl ß-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.

Integrating after CEN Excision (ICE) Plasmids: Combining the ease of yeast recombination cloning with the stability of genomic integration.

Yeast recombination cloning is a straightforward and powerful method for recombining a plasmid backbone with a specific DNA fragment. However, the utility of yeast recombination cloning is limited by the requirement for the backbone to contain an CEN/ARS element, which allows for the recombined plasmids to propagate. Although yeast CEN/ARS plasmids are often suitable for further studies, we demonstrate here that they can vary considerably in copy number from cell to cell and from colony to colony. Variation in plasmid copy number can pose an unacceptable and often unacknowledged source of phenotypic variation. If expression levels are critical to experimentation, then constructs generated with yeast recombination cloning must be subcloned into integrating plasmids, a step that often abrogates the utility of recombination cloning. Accordingly, we have designed a vector that can be used for yeast recombination cloning but can be converted into the integrating version of the resulting vector without an additional subcloning. We call these "ICE" vectors, for "Integrating after CEN Excision." The ICE series was created by introducing a "rare-cutter" NotI-flanked CEN/ARS element into the multiple cloning sites of the pRS series yeast integration plasmids. Upon recovery from yeast, the CEN/ARS is excised by NotI digest and subsequently religated without need for purification or transfer to new conditions. Excision by this approach takes ~3 hr, allowing this refinement in the same time frame as standard recombination cloning.

Experimental determination of codon usage-dependent selective pressure on high copy-number genes in Saccharomyces cerevisiae.

One of the central hypotheses in the theory of codon usage evolution is that in highly expressed genes, particular codon usage patterns arise because they facilitate efficient gene expression and are thus selected for in evolution. Here, we use plasmid copy number assays and growth rate measurements to explore details of the relationship between codon usage, gene expression level, and selective pressure in Saccharomyces cerevisiae. We find that when high expression levels are required, optimal codon usage is beneficial and provides a fitness advantage, consistent with evolutionary theory. However, when high expression levels are not required, optimal codon usage is surprisingly and strongly selected against. We show that this selection acts at the level of protein synthesis, and we exclude a number of molecular mechanisms as the source for this negative selective pressure including nutrient and ribosome limitations and proteotoxicity effects. These findings deepen our understanding of the evolution of codon usage bias, as well as the design of recombinant protein expression systems.

Multi-level engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalysts for the production of C9 chemicals from oleic acid.

Whole-cell biotransformation is one of the promising alternative approaches to microbial fermentation for producing high-value chemicals. Baeyer-Villiger monooxygenase (BVMO)-based Escherichia coli biocatalysts have been engineered to produce industrially relevant C9 chemicals, such as n-nonanoic acid and 9-hydroxynonanoic acid, from a renewable long-chain fatty acid. The key enzyme in the biotransformation pathway (i.e., BVMO from Pseudomonans putida KT2440) was first engineered, using structure modeling-based design, to improve oxidative and thermal stabilities. Using a stable and tunable plasmid (STAPL) system, E. coli host cells were engineered to have increased plasmid stability and homogeneity of the recombinant E. coli population, as well as to optimize the level of BVMO expression. Multi-level engineering of the key enzyme in host cells, allowed recombinant E. coli expressing a fatty acid double-bond hydratase, a long-chain secondary alcohol dehydrogenase, and the engineered BVMO from P. putida KT2440 (i.e., E6BVMO_C302L/M340L), to ultimately produce C9 chemicals (i.e., n-nonanoic acid and 9-hydroxynonanoic acid) from oleic acid, with a yield of up to 6 mmoL/g dry cells. This yield was 2.4-fold greater than the yield in the control strain before engineering. Therefore, this study will contribute to the development of improved processes for the biosynthesis of industrially relevant medium chain fatty acids via whole-cell biocatalysis.

Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number.

Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.

Dynamics in Copy Numbers of Five Plasmids of a Dairy Lactococcus lactis Strain under Dairy-Related Conditions Including Near-Zero Growth Rates.

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-borne genes and the activity of the corresponding proteins are severely affected by changes in the numbers of plasmid copies. We studied the impact of growth rate on the dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected, and these varied in size (3 to 39 kb), in replication mechanism (theta or rolling circle), and in putative (dairy-associated) functions. The copy numbers ranged from 1.5 to 40.5, and the copy number of theta-type replicating plasmids was negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h-1 to 0.6 h-1), the copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates, showing that the plasmid replication rate was strictly controlled. One low-copy-number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations, reflected in a complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation, or the presence of citrate (maximum 2.2-fold), signifying the stability in copy number of the plasmids.IMPORTANCELactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for the growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation, oligopeptide uptake, and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-borne genes, it is important to know the factors that influence the plasmid copy numbers. We monitored the plasmid copy numbers of L. lactis at near-zero growth rates, characteristic for cheese ripening. Moreover, we analyzed the effects of pH, nutrient limitation, and the presence of citrate. This showed that the plasmid copy numbers were stable, giving insight into plasmid copy number dynamics in dairy fermentations.

Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum.

Beyond its traditional role as an L-amino acid producer, Corynebacterium glutamicum has recently received significant attention regarding its use in the production of various biochemicals and recombinant proteins. However, despite these attributes, limitations in genetic tools are still hampering the engineering of C. glutamicum for use in more potential hosts. Here, we engineered a C. glutamicum via adaptive laboratory evolution to enhance the production of recombinant proteins. During the continuous cultivation, C. glutamicum producing enhanced green fluorescent proteins was screened using high-speed flow cytometer, and in the end, we successfully isolated an evolved strain with a fluorescence intensity 4.5-fold higher than that of the original strain. Extensive analysis of the evolved strain confirmed that the plasmid prepared from the evolved strain contains the nonsense mutation in the parB locus, which mutation contributed to increasing the copy number of plasmid by approximately 10-fold compared to that of the wild type. To validate the usefulness of the high-copy-number plasmid, we examined the secretory production of endoxylanase and the bioconversion of xylose to xylonate using xylonate dehydrogenase. In the fed-batch cultivation, the use of the high-copy-number plasmid led to 1.4-fold increase in the production of endoxylanase (~ 1.54 g/L in culture medium) without cell growth retardation comparing cultivation with cells harboring original plasmid. The expression of xylonate dehydrogenase in the high-copy-number plasmid also improved the bioconversion into xylonic acid by approximately 1.5-fold compared to the original plasmid.

Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration.

The CEN/ARS-based low-copy plasmids and 2 µ-based high-copy plasmids have been broadly used for both fundamental studies and practical applications in Saccharomyces cerevisiae. However, the relative low copy numbers and narrow dynamic range limit their applications in many cases. In this study, the expression level of the selection marker proteins was engineered to increase the plasmid copy numbers. A series of plasmids with step-wise increased copy numbers were constructed. The copy number of the plasmids with engineered dominant markers (5-100 copies per cell) showed a positive correlation with the concentration of antibiotics supplemented to the growth media. Based on this finding, we developed a simple yet highly efficient strategy, named Pathway Optimization by Tuning Antibiotic Concentrations (POTAC) to rapidly balance the flux of multi-gene pathways at the DNA level in S. cerevisiae. As proof of concept, POTAC was used to optimize the lycopene and n-butanol biosynthetic pathways, increasing the production of lycopene and n-butanol by 10- and 100-fold, respectively. Additionally, multiplex genome integration with controllable copy numbers was attempted by combining the engineered dominant markers with the CRISPR/Cas9 system. Biotechnol. Bioeng. 2016;113: 2462-2473. © 2016 Wiley Periodicals, Inc.

Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR.

BACKGROUND: Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Constant modification has brought forth countless plasmid vectors whose characteristics in terms of average plasmid copy number (PCN) and stability are rarely known. The crucial factor determining the PCN is the replication system; most replication systems in use today belong to a small number of different classes and are available through repositories like the Standard European Vector Architecture (SEVA). RESULTS: In this study, the PCN was determined in a set of seven SEVA-based expression plasmids only differing in the replication system. The average PCN for all constructs was determined by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, respectively. CONCLUSIONS: The average PCN determined here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct molecular evidence that the average PCN does not represent the true number of plasmid molecules in individual cells.

Antisense-RNA mediated control of plasmid replication - pIP501 revisited.

Over the past decade, a wealth of small noncoding RNAs (sRNAs) have been discovered in the genomes of almost all bacterial species, where they constitute the most abundant class of posttranscriptional regulators. These sRNAs are key-players in prokaryotic metabolism, stress response and virulence. However, the first bona-fide antisense RNAs had been found already in 1981 in plasmids, where they regulate replication or maintenance. Antisense RNAs involved in plasmid replication control - meanwhile investigated in depth for almost 35 years - employ a variety of mechanisms of action: They regulate primer maturation, inhibit translation of essential replication initiator proteins (Rep proteins) as well as leader peptides or the formation of activator pseudoknots required for efficient rep translation. Alternatively they attenuate transcription or translation of rep mRNAs. Some antisense RNAs collaborate with transcriptional repressors to ensure proper copy-number control. Here, I summarize our knowledge on replication control of the broad-host range plasmid pIP501 that was originally isolated from Streptococcus agalactiae. Plasmid pIP501 uses two copy number-control elements, RNAIII, a cis-encoded antisense RNA, and transcriptional repressor CopR. RNA III mediates transcription attenuation, a rather widespread concept that found its culmination in the recent discovery of riboswitches. A peculiarity of pIP501 is the unusual stability of RNA III, which requires a second function of CopR: CopR does not only repress transcription from the essential repR promoter, but also prevents convergent transcription between rep mRNA and RNAIII, thereby indirectly increasing the amount of RNAIII. The concerted action of these two control elements is necessary to prevent plasmid loss at dangerously low copy numbers.

The complete nucleotide sequence of the multi-drug resistance-encoding IncL/M plasmid pACM1.

The 89,977 bp nucleotide sequence of pACM1, isolated from a 1993 outbreak strain of cephalosporin-resistant Klebsiella oxytoca, has been completed and assigned GenBank accession number KJ541681. The plasmid has a single 31,842 bp mosaic multi-drug resistance-encoding (MDR) region comprising the mer resistance module of Tn1696, two integrons with a total of seven cassettes, one complete copy each of IS1R and IS26, and the bla(SHV-5)-carrying Tn2003 (with defective IS26 termini), all within a Tn1721-like element inserted into the mucB gene of the IncL/M plasmid backbone. The Tn1721-Tn1696 combination resembles sequence found in the chromosomal MDR islands of some Acinetobacter baumannii isolates. Among the completely sequenced IncL/M resistance plasmids, the Tn1721-based MDR region is unique, but data from older studies suggest that this type of plasmid was widespread in the 1990s. Since resistance gene dosage is affected by plasmid copy number (PCN), we used a relatively simple new "efficiency-corrected" qPCR assay to measure the PCN of pACM1. There are approximately three copies per chromosome in an Escherichia coli DH5α host, and two in the original Klebsiella oxytoca isolate. We could not find similar PCN data for other medically important plasmids for comparison. The study of this plasmid property and its effect on resistance levels should be facilitated in the future by the availability of qPCR instruments and complete genome sequences.

Porin deficiency or plasmid copy number increase mediated carbapenem-resistant Escherichia coli resistance evolution.

This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the blaNDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 µg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 µg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.

Temperatures above 37°C increase virulence of a convergent Klebsiella pneumoniae sequence type 307 strain.

Background: Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Methods: Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Results: Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. Conclusion: This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

Escherichia coli cells evade inducible parE toxin expression by reducing plasmid copy number.

Plasmids play important roles in microbial ecosystems, serving as carriers of antibiotic resistance and virulence. In the laboratory, they are essential tools for genetic manipulation and recombinant protein expression. We uncovered an intriguing survival phenotype in a fraction of the bacterial population while using plasmid-mediated arabinose-inducible gene expression to monitor the production of toxic ParE proteins. This phenotype was not correlated with changes to the plasmid sequence and could not be rescued by increasing arabinose uptake. Instead, survival correlates with a marked reduction in plasmid copy number (PCN). Reduced PCN is reproducible, not a function of the pre-existing population, and can be sequentially enriched by continual passage with induction. The reduction in PCN appears to allow mitigation of toxicity from the expression of ParE proteins while balancing the need to maintain a threshold PCN to withstand selection conditions. This indicates an adaptive cellular response to stressful conditions, likely by altering the regulation of plasmid replication. Furthermore, this survival mechanism appears to not be limited to a specific bacterial strain of Escherichia coli or ParE toxin family member, suggesting a generalized response. Finally, bacterial whole genome sequencing indicated an N845S residue substitution in DNA polymerase I, which correlates with the observed reduction in PCN and has been previously reported to impact plasmid replication. Further understanding this molecular mechanism has broader implications for this adaptive response of the dynamics of plasmid-mediated gene expression, microbial adaptation, and genetic engineering methodologies. IMPORTANCE: This research has increased our understanding of how bacteria respond to the pressure from plasmid-borne toxic genes, such as those found in toxin-antitoxin systems. Surprisingly, we found that bacteria survived toxic ParE protein expression by reducing the number of these plasmids in the cells. This discovery reveals another way in which bacteria can balance toxin expression with antibiotic selection to attenuate the effects of deleterious genes. This insight is not only valuable for understanding bacterial survival strategies but may also influence the development of better tools in biotechnology, where plasmids are often used to study the functional roles of genes.