Plasmid Loss Research Articles

A growth-coupling strategy for improving the stability of terpenoid bioproduction in Escherichia coli

BackgroundAchieving cost-competitiveness remains challenging for industrial biomanufacturing. With whole-cell biocatalysis, inefficiency presents when individual cells vary in their production levels. The problem exacerbates when the basis for such production heterogeneity is heritable. Here, evolution selects for the low- and non-producers, as they have lowered/abolished the cost of bioproduction to fitness. With the scale of population expansion required for industrial bioproduction, the asymmetrical enrichment can be severe enough to compromise the performance, and hence commercial viability of the bioprocess. Clearly, addressing production heterogeneity is crucial, especially in improving the stability of bioproduction across the cell generations. In this respect, we designed a growth-coupling strategy for terpenoid bioproduction in Escherichia coli. By knocking out the native 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) gene and introducing the heterologous mevalonate pathway, we created a chassis that relies solely on the latter for synthesis of all terpenoids. We hypothesise that the need to sustain the biosynthesis of endogenous life-sustaining terpenoids will impose a minimum level of productivity, which concomitantly improves the bioproduction of our target terpenoid.ResultsFollowing the confirmation of lethality of a dxr knockout, we challenged the strains with a continuous plasmid-based bioproduction of linalool. The Δdxr strain achieved an improved productivity profile in the first three days post-inoculation when compared to the parental strain. Productivity of the Δdxr strain remained observable near the end of 12 days, and after a disruption in nutrient and oxygen supply in a separate run. Unlike the parental strain, the Δdxr strain did not evolve the same deleterious mutations in the mevalonate pathway, nor a viable subgroup that had lost its resistance to the antibiotic selection pressure (a plausible plasmid loss event). We believe that this divergence in the evolution trajectories is indicative of a successful growth-coupling.ConclusionWe have demonstrated a proof of concept of a growth-coupling strategy that improves the performance, and stability of terpenoid bioproduction across cell generations. The strategy is relatively broad in scope, and easy to implement in the background as a ‘fail-safe’ against a fall in productivity below the imposed minimum. We thus believe this work will find widespread utility in our collective effort towards industrial bioproduction.

Genomic Analysis and Virulence Assessment of Hypervirulent Klebsiella pneumoniae K16-ST660 in Severe Cervical Necrotizing Fasciitis

ObjectiveTo investigate the source of infection in a patient with recurrent severe neck infections caused by Klebsiella pneumoniae and to analyze the virulence of isolates obtained from different sites of the patient. MethodsWe collected preoperative neck abscess puncture fluid, intraoperative neck drainage fluid, sputum, intestinal fecal specimens, and blood samples from a patient who visited Wuxi Second People's Hospital twice between 2017 and 2018. We conducted isolation, identification, drug sensitivity tests, and string tests on the isolates. Capsule serotyping and virulence gene analysis were performed using PCR. The genetic relationship of different isolates was assessed by Multilocus Sequence Typing and virulence was evaluated using the Galleria mellonella infection model. Additionally, whole-genome sequencing was used to analyze the chromosomal and plasmid genes of one isolate. ResultsKlebsiella pneumoniae was detected in the sputum and fecal specimens from both hospitalizations, as well as the preoperative ultrasound-guided puncture fluid and intraoperative drainage fluid from the first hospitalization, resulting in six isolates. These isolates were all K16 serotype, positive in the string test, and identified as ST660 by Multilocus Sequence Typing, indicating they belonged to the same clone. Virulence gene analysis showed that wcaG, iucB, iroNB, rmpA, rmpA2, Aer, kfuBC, ureA, fimH, mrkD, uge, and peg344 were positive, while allS, cf29a, and Wzy_K1 were negative. In the Galleria mellonella virulence assay, the lethality of different isolates was dose-dependent. The K16 group showed significantly higher larval mortality compared to other control groups (including K1, K2, K5, K20, and K57 groups). Genome sequencing revealed that plasmid p17388 carried numerous virulence genes and insertion sequences, particularly ISKPN74, and showed high homology with other Klebsiella plasmids. ConclusionThis study is the first to report severe cervical necrotizing fasciitis caused by the K16-ST660 Klebsiella pneumoniae Isolate. The high virulence of these isolates was confirmed by the Galleria mellonella virulence assay and the detection of numerous virulence genes. In-depth analysis of plasmid p17388 suggests that ISKPN74 may enhance stable integration of the plasmid into the bacterial chromosome through recombinases and transposases, thereby reducing the likelihood of plasmid loss and increasing bacterial virulence. Additionally, IS5 family insertion sequences may carry extra promoters or enhancers that, when inserted upstream of mucoviscosity-associated genes such as rmpA, may increase the transcription levels of downstream genes. This ISKPN74-mediated integration or insertion reveals a complex genetic mechanism that may contribute to the severity of infections caused by ST660 isolates. Our findings offer new insights into the virulence and structure of ST660-K16 Klebsiella pneumoniae, suggesting that further investigation into the specific mechanisms by which these insertion sequences enhance virulence could aid in developing novel infection management strategies.

Modified Tn7 transposon vectors for controlled chromosomal gene expression.

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.IMPORTANCEChromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology.

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Different Roles of Dioxin-Catabolic Plasmids in Growth, Biofilm Formation, and Metabolism of Rhodococcus sp. Strain p52.

Microorganisms harbor catabolic plasmids to tackle refractory organic pollutants, which is crucial for bioremediation and ecosystem health. Understanding the impacts of plasmids on hosts provides insights into the behavior and adaptation of degrading bacteria in the environment. Here, we examined alterations in the physiological properties and gene expression profiles of Rhodococcus sp. strain p52 after losing two conjugative dioxin-catabolic megaplasmids (pDF01 and pDF02). The growth of strain p52 accelerated after pDF01 loss, while it decelerated after pDF02 loss. During dibenzofuran degradation, the expression levels of dibenzofuran catabolic genes on pDF01 were higher compared to those on pDF02; accordingly, pDF01 loss markedly slowed dibenzofuran degradation. It was suggested that pDF01 is more beneficial to strain p52 under dibenzofuran exposure. Moreover, plasmid loss decreased biofilm formation, especially after pDF02 loss. Transcriptome profiling revealed different pathways enriched in upregulated and downregulated genes after pDF01 and pDF02 loss, indicating different adaptation mechanisms. Based on the transcriptional activity variation, pDF01 played roles in transcription and anabolic processes, while pDF02 profoundly influenced energy production and cellular defense. This study enhances our knowledge of the impacts of degradative plasmids on native hosts and the adaptation mechanisms of hosts, contributing to the application of plasmid-mediated bioremediation in contaminated environments.

Plasmid-Stabilizing Strains for Antibiotic-Free Chemical Fermentation.

Plasmid-mediated antibiotic-free fermentation holds significant industrial potential. However, the requirements for host elements and energy during plasmid inheritance often cause cell burden, leading to plasmid loss and reduced production. The stable maintenance of plasmids is primarily achieved through a complex mechanism, making it challenging to rationally design plasmid-stabilizing strains and characterize the associated genetic factors. In this study, we introduced a fluorescence-based high-throughput method and successfully screened plasmid-stabilizing strains from the genomic fragment-deletion strains of Escherichia coli MG1655 and Bacillus subtilis 168. The application of EcΔ50 in antibiotic-free fermentation increased the alanine titer 2.9 times. The enhanced plasmid stability in EcΔ50 was attributed to the coordinated deletion of genes involved in plasmid segregation and replication control, leading to improved plasmid maintenance and increased plasmid copy number. The increased plasmid stability of BsΔ44 was due to the deletion of the phage SPP1 surface receptor gene yueB, resulting in minimized sporulation, improved plasmid segregational stability and host adaptation. Antibiotic-free fermentation results showed that strain BsΔyueB exhibited a 61.99% higher acetoin titer compared to strain Bs168, reaching 3.96 g/L. When used for the fermentation of the downstream product, 2,3-butanediol, strain BsΔyueB achieved an 80.63% higher titer than Bs168, reaching 14.94 g/L using rich carbon and nitrogen feedstocks. Overall, our work provided a plasmid-stabilizing chassis for E. coli and B. subtilis, highlighting their potential for antibiotic-free fermentation of valuable products and metabolic engineering applications.

Mobile genetic element-driven genomic changes in a community-associated methicillin-resistant Staphylococcus aureus clone during its transmission in a regional community outbreak in Japan.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are now a public health concern in both community and healthcare settings worldwide. We previously identified a suspected case of a maternity clinic-centred outbreak of CA-MRSA skin infection in a regional community in Japan by PFGE-based analysis. In this study, we performed genome sequence-based analyses of 151 CA-MRSA isolates, which included not only outbreak-related isolates that we previously defined based on identical or similar PFGE patterns but also other isolates obtained during the same period in the same region. Our analysis accurately defined 133 isolates as outbreak-related isolates, collectively called the TDC clone. They belonged to a CA-MRSA lineage in clonal complex (CC) 30, known as the South West Pacific (SWP) clone. A high-resolution phylogenetic analysis of these isolates combined with their epidemiological data revealed that the TDC clone was already present and circulating in the region before the outbreak was recognized, and only the isolates belonging to two sublineages (named SL4 and SL5) were directly involved in the outbreak. Long persistence in patients/carriers and frequent intrahousehold transmission of the TDC clone were also revealed by this analysis. Moreover, by systematic analyses of the genome changes that occurred in this CA-MRSA clone during transmission in the community, we revealed that most variations were associated with mobile genetic elements (MGEs). Variant PFGE types were generated by alterations of prophages and genomic islands or insertion sequence (IS)-mediated insertion of a plasmid or a sequence of unknown origin. Dynamic changes in plasmid content, which were linked to changes in antimicrobial resistance profiles in specific isolates, were generated by frequent gain and loss of plasmids, most of which were self-transmissible or mobilizable. The introduction of IS256 by a plasmid (named pTDC02) into sublineage SL5 led to SL5-specific amplification of IS256, and amplified IS256 copies were involved in some of the structural changes of chromosomes and plasmids and generated variations in the repertoire of virulence-related genes in limited isolates. These data revealed how CA-MRSA genomes change during transmission in the community and how MGEs are involved in this process.

Dependence of post-segregational killing mediated by Type II restriction-modification systems on the lifetime of restriction endonuclease effective activity.

Plasmid-borne Type II restriction-modification (RM) systems mediate post-segregational killing (PSK). PSK is thought to be caused by the dilution of restriction and modification enzymes during cell division, resulting in accumulation of unmethylated DNA recognition sites and their cleavage by restriction endonucleases. PSK is the likely reason for stabilization of plasmids carrying RM systems in the absence of selection for plasmid maintenance. In this study, we developed a CRISPR interference-based method to eliminate RM-carrying plasmids and study PSK-related phenomena with minimal perturbation to the Escherichia coli host. Plasmids carrying the EcoRV, Eco29kI, and EcoRI RM systems were highly stable, and their loss resulted in SOS response and PSK. In contrast, plasmids carrying the Esp1396I system were poorly stabilized; their loss led to a temporary cessation of growth, followed by full recovery. We demonstrate that this unusual behavior is due to a limited lifetime of the Esp1396I restriction endonuclease activity, which, upon Esp1396I plasmid loss, disappears approximately after two cycles of cell division, i.e., before unmethylated sites appear in significant numbers. Our results indicate that whenever PSK induced by a loss of RM systems, and, possibly, other toxin-antitoxin systems, is considered, the lifetimes of individual system components and the growth rate of host cells shall be taken in account. Mathematical modeling shows, that unlike the situation with classical toxin-antitoxin systems, RM system-mediated PSK is possible when the lifetimes of restriction endonuclease and methyltransferase activities are similar, as long as the toxic restriction endonuclease activity persists for more than two chromosome replication cycles.IMPORTANCEIt is widely accepted that many Type II restriction-modification (RM) systems mediate post-segregational killing (PSK) if plasmids that encode them are lost. In this study, we harnessed an inducible CRISPR-Cas system to remove RM plasmids from Escherichia coli cells to study PSK while minimally perturbing cell physiology. We demonstrate that PSK depends on restriction endonuclease activity lifetime and is not observed when it is less than two replication cycles. We present a mathematical model that explains experimental data and shows that unlike the case of toxin-antitoxin-mediated PSK, the loss of an RM system induced PSK even when the RM enzymes have identical lifetimes.

The JMU-SalVac-System: A Novel, Versatile Approach to Oral Live Vaccine Development.

Salmonella enterica Serovar Typhi Ty21a (Ty21a) is the only licensed oral vaccine against typhoid fever. Due to its excellent safety profile, it has been used as a promising vector strain for the expression of heterologous antigens for mucosal immunization. As the efficacy of any bacterial live vector vaccine correlates with its ability to express and present sufficient antigen, the genes for antigen expression are traditionally located on plasmids with antibiotic resistance genes for stabilization. However, for use in humans, antibiotic selection of plasmids is not applicable, leading to segregational loss of the antigen-producing plasmid. Therefore, we developed an oral Ty21a-based vaccine platform technology, the JMU-SalVac-system (Julius-Maximilians-Universität Würzburg) in which the antigen delivery plasmids (pSalVac-plasmid-series) are stabilized by a ΔtyrS/tyrS+-based balanced-lethal system (BLS). The system is made up of the chromosomal knockout of the essential tyrosyl-tRNA-synthetase gene (tyrS) and the in trans complementation of tyrS on the pSalVac-plasmid. Further novel functional features of the pSalVac-plasmids are the presence of two different expression cassettes for the expression of protein antigens. In this study, we present the construction of vaccine strains with BLS plasmids for antigen expression. The expression of cytosolic and secreted mRFP and cholera toxin subunit B (CTB) proteins as model antigens is used to demonstrate the versatility of the approach. As proof of concept, we show the induction of previously described in vivo inducible promoters cloned into pSalVac-plasmids during infection of primary macrophages and demonstrate the expression of model vaccine antigens in these relevant human target cells. Therefore, antigen delivery strains developed with the JMU-SalVac technology are promising, safe and stable vaccine strains to be used against mucosal infections in humans.

The histone-like nucleoid-structuring protein encoded by the plasmid pMBL6842 regulates both plasmid stability and host physiology of Pseudoalteromonas rubra SCSIO 6842

Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host metabolism, creating a competitive advantage for plasmid-free cells. Plasmid stability hinges on efficient replication and partition mechanisms. While plasmids commonly encode histone-like nucleoid-structuring (H-NS) family proteins, the precise influence of plasmid-encoded H-NS proteins on stability remains elusive. In this study, we examined the conjugative plasmid pMBL6842, harboring the hns gene, and observed its positive regulation of parAB transcription, critical for plasmid segregation. Deletion of hns led to rapid plasmid loss, which was remedied by hns complementation. Further investigations unveiled adverse effects of hns overexpression on the bacterial host. Transcriptome analysis revealed hns's role in regulating numerous bacterial genes, impacting both host growth and swimming motility in the presence of the hns gene. Therefore, our study unveils the multifaceted roles of H-NS in both plasmid stability and host physiology, underscoring its biological significance and paving the way for future inquiries into the involvement of H-NS in horizontal gene transfer events.

Construction of a grpE-based plasmid addiction system in Escherichia coli and its application in phloroglucinol biosynthesis.

Biotechnical processes in Escherichia coli often operate with artificial plasmids. However, these bioprocesses frequently encounter plasmid loss. To ensure stable expression of heterologous genes in E. coli BL21(DE3), a novel plasmid addiction system (PAS) was developed. This PAS employed an essential gene grpE encoding a cochaperone in the DnaK-DnaJ-GrpE chaperone system as the selection marker, which represented a chromosomal ΔgrpE mutant harboring episomal expression plasmids that carry supplementary grpE alleles to restore the deficiency. To demonstrate the feasibility of this system, it was implemented in phloroglucinol (PG) biosynthesis, manifesting improved host tolerance to PG and increased PG production. Specifically, PG titer significantly improved from 0.78±0.02 to 1.34±0.04 g l-1, representing a 71.8% increase in shake-flask fermentation. In fed-batch fermentation, the titer increased from 3.71±0.11 to 4.54±0.10 g l-1, showing a 22.4% increase. RNA sequencing and transcriptome analysis revealed that the improvements were attributed to grpE overexpression and upregulation of various protective chaperones and the biotin acetyl-CoA carboxylase ligase coding gene birA. This novel PAS could be regarded as a typical example of nonanabolite- and nonmetabolite-related PAS. It effectively promoted plasmid maintenance in the host, improved tolerance to PG, and increased the titer of this compound.

The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans

BackgroundBacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxyphenylacetate (3-HPA).MethodsThe functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC–MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability.ResultsThe exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds.ConclusionsThe long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

Engineering Compositionally Uniform Yeast Whole-Cell Biocatalysts with Maximized Surface Enzyme Density for Cellulosic Biofuel Production.

In recent decades, whole-cell biocatalysis has played an increasingly important role in the food, pharmaceutical, and energy sector. One promising application is the use of ethanologenic yeast displaying minicellulosomes on the cell surface to combine cellulose hydrolysis and fermentation into a single step for consolidated bioprocessing. However, cellulosic ethanol production using existing yeast whole-cell biocatalysts (yWCBs) has not reached industrial feasibility due to their inefficient cellulose hydrolysis. As prior studies have demonstrated enzyme density on the yWCB surface to be one of the most important parameters for enhancing cellulose hydrolysis, we sought to maximize this parameter at both the population and single-cell levels in yWCBs displaying tetrafunctional minicellulosomes. At the population level, enzyme density is limited by the presence of a nondisplay population constituting 25-50% of all cells. In this study, we identified the cause to be plasmid loss and successfully eliminated the nondisplay population to generate compositionally uniform yWCBs. At the single-cell level, we demonstrate that enzyme density is limited by molecular crowding, which hinders minicellulosome assembly. By adjusting the integrated gene copy number, we obtained yWCBs of tunable enzyme display levels. This tunability allowed us to avoid the crowding-limited regime and achieve a maximum enzyme density per cell. As a result, the best strain showed a cellulose-to-ethanol yield of 4.92 g/g, corresponding to 96% of the theoretical maximum and near-complete conversion (∼96%) of the starting cellulose (1% PASC). Our holistic engineering strategy that combines a population and single-cell level approach is broadly applicable to enhance the WCB performance in other biocatalytic cascade schemes.

Spread and evolution of blaKPC-plasmid between Serratia marcescens and Klebsiella pneumoniae

ObjectivesblaKPC-carrying Enterobacterales have post great challenges to global healthcare systems. In this study, we reported the evolution and spread of blaKPC between Serratia marcescens and Klebsiella pneumoniae. MethodsFour S. marcescens and one K. pneumoniae strains were isolated from the sputum samples of the patient. Antimicrobial susceptibility tests and whole genome sequencing were performed to investigate the phenotype & genotype of strains. Conjugation assays, cloning experiment and kinetic parameters measuring were performed to explore the spread and antimicrobial resistance mechanisms. ResultsThe evolution and transmission of blaKPC-2 occurred during the treatment of ceftazidime-avibactam and trimethoprim-sulfamethoxazole. Analysis of the antimicrobial susceptibility and genetic profiles of the clinical strains showed that blaKPC-2 evolved into blaKPC-71 and blaKPC-44, together with resistance to ceftazidime-avibactam and carbapenems susceptibility recovery under antimicrobial pressure. Cloning and expression of blaKPC-44 & blaKPC-71 in E. coli DH5α showed that KPC-44 and KPC-71 resulted in a 64∼128-fold increase in the MIC value for ceftazidime-avibactam. Meanwhile, the kinetic assays also showed that the enzyme activity of KPC-44 and KPC-71 towards carbapenems was destroyed and couldn't be inhibited by avibactam. Based on the conjugation assay and whole genome sequence analyses, we provided evolutionary insights into the transmission pathway trace of blaKPC-bearing plasmids between S. marcescens and K. pneumoniae. ConclusionsMixed-species co-infection is one of the risk factors leading to the spread of plasmids carrying carbapenem-resistant genes, and increased surveillance of multidrug-resistant Enterobacterales is urgently needed.

Plasmid-free cheater cells commonly evolve during laboratory growth.

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.

RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae

ABSTRACT Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid- carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

Single-cell evidence for plasmid addiction mediated by toxin-antitoxin systems.

Toxin-antitoxin (TA) systems are small selfish genetic modules that increase vertical stability of their replicons. They have long been thought to stabilize plasmids by killing cells that fail to inherit a plasmid copy through a phenomenon called post-segregational killing (PSK) or addiction. While this model has been widely accepted, no direct observation of PSK was reported in the literature. Here, we devised a system that enables visualization of plasmid loss and PSK at the single-cell level using meganuclease-driven plasmid curing. Using the ccdsystem, we show that cells deprived of a ccd-encoding plasmid show hallmarks of DNA damage, i.e. filamentation and induction of the SOS response. Activation of ccd triggered cell death in most plasmid-free segregants, although some intoxicated cells were able to resume growth, showing that PSK-induced damage can be repaired in a SOS-dependent manner. Damage induced by ccd activates resident lambdoid prophages, which potentiate the killing effect of ccd. The loss of a model plasmid containing TA systems encoding toxins presenting various molecular mechanisms induced different morphological changes, growth arrest and loss of viability. Our experimental setup enables further studies of TA-induced phenotypes and suggests that PSK is a general mechanism for plasmid stabilization by TA systems.

Method for plasmid-based antibiotic-free fermentation

BackgroundAntibiotic-based plasmid selection and maintenance is a core tool in molecular biology; however, while convenient, this strategy has numerous drawbacks for biological manufacturing. Overuse of antibiotics and antibiotic resistance genes (ARG) contributes to the development of antimicrobial resistance, which is a growing threat to modern medicine. Antibiotics themselves are costly and therefore often omitted in fermentations, leading to plasmid loss and a corresponding loss in product yield. Furthermore, constitutive expression of a plasmid-encoded antibiotic resistance gene imposes a significant metabolic burden on the cells. For many fermentation products (e.g., in nutrition and medicine), the use of antibiotic resistance genes is subject to strict regulations and should be avoided. We present a method for plasmid selection and maintenance with stringent selection pressure that is independent of antibiotics and ARG. Furthermore, it can be used without any restrictions regarding culture medium and temperature.ResultsThe developed method involves modification of a bacterial strain such that an essential gene is expressed genomically under the control of an inducible promoter. A copy of the same essential gene with the endogenous promoter is supplied on a plasmid for selection. In the absence of the inducer for the genomic copy of the essential gene, cells rely on expression of the plasmid-encoded gene copy, leading to tight selection for plasmid maintenance. Induction of the genomic copy of the essential gene enables the engineered strain to be propagated in the absence of a plasmid. Here, we describe the genetic setup and demonstrate long-term, tight selection for plasmid maintenance with a variety of different plasmids and E. coli strains.ConclusionsThis method facilitates plasmid-based fermentations by eliminating the need for antibiotic selection and improving plasmid maintenance.

Mechanism for transmission and pathogenesis of carbapenem-resistant Enterobacterales harboring the carbapenemase IMP and clinical countermeasures.

Carbapenem-resistant Enterobacterales (CRE) are some of the most important pathogens causing infections, which can be challenging to treat. We identified four blaIMP-carrying CRE isolates and collected clinical data. The transferability and stability of the plasmid were verified by conjugation, successive passaging, and plasmid elimination assays. The IncC blaIMP-4-carrying pIMP4-ECL42 plasmid was successfully transferred into the recipient strain, and the high expression of traD may have facilitated the conjugation transfer of the plasmid. Interestingly, the plasmid showed strong stability in clinical isolates. Whole-genome sequencing was performed on all isolates. We assessed the sequence similarity of blaIMP -harboring plasmid from our institution and compared it to plasmids for which sequence data are publicly available. We found that four blaIMP-carrying CRE belonged to four different sequence types. The checkerboard technique and time-kill assays were used to investigate the best antimicrobial therapies for blaIMP-carrying CRE. The time-kill assay showed that the imipenem of 1× minimum inhibitory concentration (MIC) alone had the bactericidal or bacteriostatic effect against IMP-producing strains at 4-12 h in vitro. Moreover, the combination of tigecycline (0.5/1/2 × MIC) and imipenem (0.5/1 × MIC) showed a bactericidal effect against the blaIMP-26-carrying CRECL60 strain.IMPORTANCECarbapenem-resistant Enterobacterales (CRE) are an urgent public health threat, and infections caused by these microorganisms are often associated with high mortality and limited treatment options. This study aimed to determine the clinical features, molecular characteristics, and plasmid transmissible mechanisms of blaIMP carriage as well as to provide a potential treatment option. Here, we demonstrated that conjugated transfer of the IncC blaIMP-4-carrying plasmid promotes plasmid stability, so inhibition of conjugated transfer and enhanced plasmid loss may be potential ways to suppress the persistence of this plasmid. The imipenem alone or tigecycline-imipenem combination showed a good bactericidal effect against IMP-producing strains. In particular, our study revealed that imipenem alone or tigecycline-imipenem combination may be a potential therapeutic option for patients who are infected with IMP-producing strains. Our study supports further trials of appropriate antibiotics to determine optimal treatment and emphasizes the importance of continued monitoring of IMP-producing strains in the future.

Learning by selective plasmid loss for intracellular synthetic classifiers

We propose a learning mechanism for intracellular synthetic genetic classifiers based on the selective elimination (curing) of plasmids bearing parts of the classifier circuit. Our focus is on a two-input, two-plasmid classifier scheme designed to solve a simple proof-of-concept learning problem. The problem is formulated in terms of Boolean variables, and the learning process boils down to selecting the classification rule from three options, given a set of training examples. We begin with a Boolean description of the classifier circuit, demonstrating how it implements the required learning algorithm. We then transition to a continuous steady-state model and establish conditions on its parameters to ensure that the learning process and the classifier output correspond to the Boolean description, at least approximately. The approach to intracellular classifier learning presented here essentially relies on two key prerequisites: (i) compatibility among the plasmids constituting the classifier, such that they have independent or weakly interacting copy number control systems, and (ii) conditional elimination mechanism in each plasmid triggered by a signal from the gene network. The feasibility of this approach is supported by recent experimental findings on engineering compatible pairs and triplets of plasmids and controlled selective plasmid curing. While learning by plasmid loss has certain limitations in universality, we anticipate that it provides greater persistence of a trained classifier to internal and external fluctuations and to degradation over time, as compared to alternative intracellular learning mechanisms outlined in the literature, such as based on gene network dynamics or on variable copy numbers of plasmids sharing a common copy number control system.