Plasmid Research Articles

Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that blaTEM gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of blaTEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4g/0.5g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of blaTEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and blaTEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of blaTEM gene in P/T-R isolates, generated by three different genetic events: (1) IS26-mediated duplication of the blaTEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying blaTEM, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.

Protein expression systems combining a flavonoid-inducible promoter and T7 RNA polymerase in Bacillus subtilis.

Recombinant protein production must be tightly controlled when overproduction adversely affects the host bacteria. We developed a flavonoid-inducible T7 expression system in Bacillus subtilis using the qdoI promoter to control the T7 RNA polymerase gene (T7 pol). Using the egfp reporter gene controlled by the T7 promoter in a multicopy plasmid, we confirmed that this expression system is tightly regulated by flavonoids, such as quercetin and fisetin. Altering the qdoI promoter for T7 pol control to its hybrid derivative increased the expression level by 6.6-fold at maximum values upon induction. However, faint expression leakage was observed under a noninducing condition. Therefore, the two expression systems with the original qdoI promoter and the hybrid construct can be used selectively, depending on the high control accuracy or production yield required.

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump.

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward β-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.

Insertion Sequences Determine Plasmid Adaptation to New Bacterial Hosts.

Plasmids facilitate the vertical and horizontal spread of antimicrobial resistance genes between bacteria. The host range and adaptation of plasmids to new hosts determine their impact on the spread of resistance. In this work, we explore the mechanisms driving plasmid adaptation to novel hosts in experimental evolution. Using the small multicopy plasmid pB1000, usually found in Pasteurellaceae, we studied its adaptation to a host from a different bacterial family, Escherichia coli. We observed two different mechanisms of adaptation. One mechanism is single nucleotide polymorphisms (SNPs) in the origin of replication (oriV) of the plasmid, which increase the copy number in E. coli cells, elevating the stability, and resistance profile. The second mechanism consists of two insertion sequences (ISs), IS1 and IS10, which decrease the fitness cost of the plasmid by disrupting an uncharacterized gene on pB1000 that is harmful to E. coli. Both mechanisms increase the stability of pB1000 independently, but only their combination allows long-term maintenance. Crucially, we show that the mechanisms have a different impact on the host range of the plasmid. SNPs in oriV prevent the replication in the original host, resulting in a shift of the host range. In contrast, the introduction of ISs either shifts or expands the host range, depending on the IS. While IS1 leads to expansion, IS10 cannot be reintroduced into the original host. This study gives new insights into the relevance of ISs in plasmid-host adaptation to understand the success in spreading resistance. IMPORTANCE ColE1-like plasmids are small, mobilizable plasmids that can be found across at least four orders of Gammaproteobacteria and are strongly associated with antimicrobial resistance genes. Plasmid pB1000 carries the gene blaROB-1, conferring high-level resistance to penicillins and cefaclor. pB1000 has been described in various species of the family Pasteurellaceae, for example, in Haemophilus influenzae, which can cause diseases such as otitis media, meningitis, and pneumonia. To understand the resistance spread through horizontal transfer, it is essential to study the mechanisms of plasmid adaptation to novel hosts. In this work we identify that a gene from pB1000, which encodes a peptide that is toxic for E. coli, and the low plasmid copy number (PCN) of pB1000 in E. coli cells are essential targets in the described plasmid-host adaptation and therefore limit the spread of pB1000-encoded blaROB-1. Furthermore, we show how the interplay of two adaptation mechanisms leads to successful plasmid maintenance in a different bacterial family.

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo.

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) and are also thought to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and little is known about their functional activities and target specificities in vivo. Here, we describe five pAgos from mesophilic bacteria that act as programmable DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmids and chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum, can induce DNA interference between homologous sequences resulting in targeted processing of multicopy plasmid and genomic elements. EmaAgo also protects bacteria from bacteriophage infection, by loading phage-derived guide DNAs and decreasing phage DNA content and phage titers. Thus, the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

Liquid chromatography-mass spectrometry analyses of polyprenyl phosphates in Escherichia coli cells in which genes for isoprenoid synthesis are amplified

Overproduction of isopentenyl diphosphate by the amplification of the genes for the methylerythritol 4-phosphate pathway, dxs and dxr, is known to be deleterious for the growth of Escherichia coli. We hypothesized that overproduction of one of the endogenous isoprenoids, in addition to isopentenyl diphosphate itself, might be the cause of the reported reduced growth rate and attempted to identify the causative agent. In order to analyze polyprenyl phosphates, they were methylated by the reaction with diazomethane. The resulting dimethyl esters of polyprenyl phosphates with carbon numbers from 40 to 60 were quantitated by high-performance liquid chromatography-mass spectrometric analysis detecting ion peaks of the sodium ion adducts. The E.coli was transformed by a multi-copy plasmid carrying both the dxs and dxr genes. Amplification of dxs and dxr significantly increased the levels of polyprenyl phosphates and 2-octaprenylphenol. The levels of Z,E-mixed polyprenyl phosphates with carbon numbers of 50-60 in the strain in which ispB was co-amplified with dxs and dxr were lower than those in the control strain where only dxs and dxr were amplified. The levels of (all-E)-octaprenyl phosphate and 2-octaprenylphenol in the strains in which ispU/rth or crtE was co-amplified with dxs and dxr were lower than those in the control strain. Although the increase in the level of each isoprenoid intermediate was blocked, the growth rates of these strains were not restored. Neither polyprenyl phosphates nor 2-octaprenylphenol can be determined to be the cause of the growth rate reduction seen with dxs and dxr amplification.

CsgI (YccT) Is a Novel Inhibitor of Curli Fimbriae Formation in Escherichia coli Preventing CsgA Polymerization and Curli Gene Expression.

Curli fimbriae are amyloids-found in bacteria (Escherichia coli)-that are involved in solid-surface adhesion and bacterial aggregation during biofilm formation. The curli protein CsgA is coded by a csgBAC operon gene, and the transcription factor CsgD is essential to induce its curli protein expression. However, the complete mechanism underlying curli fimbriae formation requires elucidation. Herein, we noted that curli fimbriae formation was inhibited by yccT-i.e., a gene that encodes a periplasmic protein of unknown function regulated by CsgD. Furthermore, curli fimbriae formation was strongly repressed by CsgD overexpression caused by a multicopy plasmid in BW25113-the non-cellulose-producing strain. YccT deficiency prevented these CsgD effects. YccT overexpression led to intracellular YccT accumulation and reduced CsgA expression. These effects were addressed by deleting the N-terminal signal peptide of YccT. Localization, gene expression, and phenotypic analyses revealed that YccT-dependent inhibition of curli fimbriae formation and curli protein expression was mediated by the two-component regulatory system EnvZ/OmpR. Purified YccT inhibited CsgA polymerization; however, no intracytoplasmic interaction between YccT and CsgA was detected. Thus, YccT-renamed CsgI (curli synthesis inhibitor)-is a novel inhibitor of curli fimbriae formation and has a dual role as an OmpR phosphorylation modulator and CsgA polymerization inhibitor.

Elimination of enzymes catalysis compartmentalization enhancing taxadiene production in Saccharomyces cerevisiae.

Taxadiene is an important precursor in taxol biosynthesis pathway, but its biosynthesis in eukaryotic cell factories is limited, which seriously hinders the biosynthesis of taxol. In this study, it is found that there was the catalysis compartmentalization between two key exogenous enzymes of geranylgeranyl pyrophosphate synthase and taxadiene synthase (TS) for taxadiene synthesis progress, due to their different subcellular localization. Firstly, the enzyme-catalysis compartmentalization was overcome by means of the intracellular relocation strategies of taxadiene synthase, including N-terminal truncation of taxadiene synthase and enzyme fusion of GGPPS-TS. With the help of two strategies for enzyme relocation, the taxadiene yield was increased by 21% and 54% respectively, among them the GGPPS-TS fusion enzyme is more effective. Further, the expression of GGPPS-TS fusion enzyme was improved via the multi-copy plasmid, resulting that the taxadiene titer was increased by 38% to 21.8mg/L at shake-flask level. Finally, the maximum taxadiene titer of 184.2mg/L was achieved by optimization of the fed-batch fermentation conditions in 3L bioreactor, which is the highest reported titer of taxadiene biosynthesis accomplished in eukaryotic microbes. This study provides a successful example for improving biosynthesis of complex natural products by solving the critical problem of multistep enzymes catalysis compartmentalization.

Segregational instability of multicopy plasmids: A population genetics approach.

Plasmids are extra-chromosomal genetic elements that encode a wide variety of phenotypes and can be maintained in bacterial populations through vertical and horizontal transmission, thus increasing bacterial adaptation to hostile environmental conditions like those imposed by antimicrobial substances. To circumvent the segregational instability resulting from randomly distributing plasmids between daughter cells upon division, nontransmissible plasmids tend to be carried in multiple copies per cell, with the added benefit of exhibiting increased gene dosage and resistance levels. But carrying multiple copies also results in a high metabolic burden to the bacterial host, therefore reducing the overall fitness of the population. This trade-off poses an existential question for plasmids: What is the optimal plasmid copy number? In this manuscript, we address this question by postulating and analyzing a population genetics model to evaluate the interaction between selective pressure, the number of plasmid copies carried by each cell, and the metabolic burden associated with plasmid bearing in the absence of selection for plasmid-encoded traits. Parameter values of the model were estimated experimentally using Escherichia coli K12 carrying a multicopy plasmid encoding for a fluorescent protein and bla TEM-1, a gene conferring resistance to β-lactam antibiotics. By numerically determining the optimal plasmid copy number for constant and fluctuating selection regimes, we show that plasmid copy number is a highly optimized evolutionary trait that depends on the rate of environmental fluctuation and balances the benefit between increased stability in the absence of selection with the burden associated with carrying multiple copies of the plasmid.

Microcin MccI47 selectively inhibits enteric bacteria and reduces carbapenem-resistant Klebsiella pneumoniae colonization in vivo when administered via an engineered live biotherapeutic

ABSTRACT The gastrointestinal (GI) tract is the reservoir for multidrug resistant (MDR) pathogens, specifically carbapenem-resistant (CR) Klebsiella pneumoniae and other Enterobacteriaceae, which often lead to the spread of antimicrobial resistance genes, severe extraintestinal infections, and lethal outcomes. Selective GI decolonization has been proposed as a new strategy for preventing transmission to other body sites and minimizing spreading to susceptible individuals. Here, we purify the to-date uncharacterized class IIb microcin I47 (MccI47) and demonstrate potent inhibition of numerous Enterobacteriaceae, including multidrug-resistant clinical isolates, in vitro at concentrations resembling those of commonly prescribed antibiotics. We then genetically modify the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to produce MccI47 from a stable multicopy plasmid by using MccI47 toxin production in a counterselection mechanism to engineer one of the native EcN plasmids, which renders provisions for inducible expression and plasmid selection unnecessary. We then test the clinical relevance of the MccI47-producing engineered EcN in a murine CR K. pneumoniae colonization model and demonstrate significant MccI47-dependent reduction of CR K. pneumoniae abundance after seven days of daily oral live biotherapeutic administration without disruption of the resident microbiota. This study provides the first demonstration of MccI47 as a potent antimicrobial against certain Enterobacteriaceae, and its ability to significantly reduce the abundance of CR K. pneumoniae in a preclinical animal model, when delivered from an engineered live biotherapeutic product. This study serves as the foundational step toward the use of engineered live biotherapeutic products aimed at the selective removal of MDR pathogens from the GI tract.

The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function.

Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.

Pyoluteorin and 2,4-diacetylphloroglucinol are major contributors to Pseudomonas protegens Pf-5 biocontrol against Botrytis cinerea in cannabis.

Pseudomonas protegens Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen Botrytis cinerea causing gray mold disease in Cannabis sativa crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against B. cinerea in the phyllosphere of C. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genes pltD or phlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. In vitro confrontation assays revealed that deletion mutant ∆pltD inhibited B. cinerea growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆phlA inhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered in vitro inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆pltD and ∆phlA significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities in planta similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of P. protegens Pf-5 against B. cinerea in the cannabis phyllosphere.

Characterisation of the triclosan efflux pump TriABC and its regulator TriR in Agrobacterium tumefaciens C58

TriR serves as a repressor for a resistance-nodulation-cell division (RND) efflux pump TriABC involved in triclosan (TCS) resistance in Agrobacterium tumefaciens. The triR gene is transcribed divergently from the triABC operon. TriR specifically bound to the triR-triA intergenic region, at an imperfect 10 bp inverted repeat, 5′-TTGACTAttC-GgtTAGTCAA-3′ (TriR box), that was revealed by DNase I footprinting and electrophoretic mobility shift assay. TCS treatment appeared to up-regulate triR and triABC expression, via preventing TriR binding to the triR-triA intergenic region. Promoter-lacZ fusions and β-galactosidase activity assay further demonstrated TriR-mediated repression of triABC and triR autoregulation. Site-directed mutagenesis confirmed the identified TriR box is essential for TriR repression. A. tumefaciens mutant strains disrupting either triR or triA were constructed to determine their biological functions. The triA mutant showed hypersensitivity to TCS and sodium dodecyl sulfate (SDS), whereas the triR mutant was hyper-resistant, compared to wild-type. In addition to TCS and SDS, overproduction of TriABC from a multi-copy plasmid conferred enhanced resistance to a quaternary ammonium compound, benzalkonium chloride. Molecular modelling was able to predict the model of TriR and docking simulations were able to anticipate plausible binding interactions between TriR and TCS ligand.

Synthetic Saccharomyces cerevisiae tolerate and degrade highly pollutant complex hydrocarbon mixture

Fungal laccase (Lac) has become a very useful biocatalyst in different industries, bio-refineries and, most importantly, bioremediation. Many reports have also linked hydrocarbon tolerance and degradation by various microorganisms with Lac secretion. In this study, Trametes trogii Lac (Ttlcc1) was engineered into Saccharomyces cerevisiae strain CEN.PK2–1 C under the constitutive GPD promoter (pGPD) for multi-fold synthesis with efficient hydrocarbon tolerance and degradation. Protein expression in heterologous hosts is strictly strain-specific, it can also be influenced by the synthetic design and culture conditions. We compared synthetic designs with different shuttle vectors for the yeast strains and investigated the best culture conditions by varying the pH, temperature, carbon, nitrogen sources, and CuSO4 amount. Two S. cerevisiae strains were built in this study: byMM935 and byMM938. They carry the transcription unit pGPD-Ttlcc1-CYC1t either inside the pRSII406 integrative plasmid (byMM935) or the pRSII426 multicopy plasmid (byMM938). The performance of these two synthetic strains were studied by comparing them to the wild-type strain (byMM584). Both byMM935 and byMM938 showed significant response to different carbon sources (glucose, galactose, lactose, maltose, and sucrose), nitrogen sources (NH4Cl, NH4NO3, KNO3, malt extract, peptone, and yeast extract), and solid state fermentation of different plant biomasses (bagasse, banana peels, corn cob, mandarin peels, and peanut shells). They performed best in optimized growth conditions with specific carbon and nitrogen sources, and a preferred pH in the range 3.5–4.5, temperature between 30 and 40 0C, and 1 mM CuSO4. In optimized yeast-growth medium, strain byMM935 showed the highest laccase activities of 1.621 ± 0.063 U/mL at 64 h, whereas byMM938 gave its highest activity (1.417 ± 0.055 U/mL) at 48 h. In this work, we established, by using Bushnell Hass synthetic medium, that the new Ttlcc1-yeast strains tolerated extreme pH and complex hydrocarbon mixture (CHM) toxicity. They degraded 60–90% of the key components in CHM within 48 h, including poly-cyclic aromatic hydrocarbons, alkyl indenes, alkyl tetralines, alkyl benzenes, alkyl biphenyls, and BTEX (Benzene, Toluene, Ethylbenzene, and Xylenes). This is the first report on the hydrocarbon degradation potential of a Ttlcc1-yeast. Compared to the native organism, such synthetic strains are better suited for meeting growing demands and have potentials for application in large-scale in situ bioremediation of hydrocarbon-polluted sites.

Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae.

Horizontal gene transfer can trigger rapid shifts in bacterial evolution. Driven by a variety of mobile genetic elements-in particular bacteriophages and plasmids-the ability to share genes within and across species underpins the exceptional adaptability of bacteria. Nevertheless, invasive mobile genetic elements can also present grave risks to the host; bacteria have therefore evolved a vast array of defences against these elements1. Here we identify two plasmid defence systems conserved in the Vibrio cholerae El Tor strains responsible for the ongoing seventh cholera pandemic2-4. These systems, termed DdmABC and DdmDE, are encoded on two major pathogenicity islands that are a hallmark of current pandemic strains. We show that the modules cooperate to rapidly eliminate small multicopy plasmids by degradation. Moreover, the DdmABC system is widespread and can defend against bacteriophage infection by triggering cell suicide (abortive infection, or Abi). Notably, we go on to show that, through an Abi-like mechanism, DdmABC increases the burden of large low-copy-number conjugative plasmids, including a broad-host IncC multidrug resistance plasmid, which creates a fitness disadvantage that counterselects against plasmid-carrying cells. Our results answer the long-standing question of why plasmids, although abundant in environmental strains, are rare in pandemic strains; have implications for understanding the dissemination of antibiotic resistance plasmids; and provide insights into how the interplay between two defence systems has shapedthe evolution of the most successful lineage of pandemic V. cholerae.

Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA

SummaryArgonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.

Intra- and interpopulation transposition of mobile genetic elements driven by antibiotic selection.

The spread of genes encoding antibiotic resistance is often mediated by horizontal gene transfer (HGT). Many of these genes are associated with transposons, a type of mobile genetic element that can translocate between the chromosome and plasmids. It is widely accepted that the translocation of antibiotic resistance genes onto plasmids potentiates their spread by HGT. However, it is unclear how this process is modulated by environmental factors, especially antibiotic treatment. To address this issue, we asked whether antibiotic exposure would select for the transposition of resistance genes from chromosomes onto plasmids and, if so, whether antibiotic concentration could tune the distribution of resistance genes between chromosomes and plasmids. We addressed these questions by analysing the transposition dynamics of synthetic and natural transposons that encode resistance to different antibiotics. We found that stronger antibiotic selection leads to a higher fraction of cells carrying the resistance on plasmids because the increased copy number of resistance genes on multicopy plasmids leads to higher expression of those genes and thus higher cell survival when facing antibiotic selection. Once they have transposed to plasmids, antibiotic resistance genes are primed for rapid spread by HGT. Our results provide quantitative evidence for a mechanism by which antibiotic selection accelerates the spread of antibiotic resistance in microbial communities.

Developing Multi-Copy Chromosomal Integration Strategies for Heterologous Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae.

Caffeic acid, a plant-sourced phenolic compound, has a variety of biological activities, such as antioxidant and antimicrobial properties. The caffeic acid biosynthetic pathway was initially constructed in S. cerevisiae, using codon-optimized TAL (coTAL, encoding tyrosine ammonia lyase) from Rhodobacter capsulatus, coC3H (encoding p-coumaric acid 3-hydroxylase) and coCPR1 (encoding cytochrome P450 reductase 1) from Arabidopsis thaliana in 2 μ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of coTAL via delta integration with the POT1 gene (encoding triose phosphate isomerase) as selection marker and episomal expression of coC3H, coCPR1 using the episomal plasmid pLC-c3 were combined, and caffeic acid production was proved to be improved. Next, the delta and rDNA multi-copy integration methods were applied to integrate the genes coC3H and coCPR1 into the chromosome of high p-coumaric acid yielding strain QT3-20. The strain D9 constructed via delta integration outperformed the other strains, leading to 50-fold increased caffeic acid production in optimized rich media compared with the initial construct. The intercomparison between three alternative multi-copy strategies for de novo synthesis of caffeic acid in S. cerevisiae suggested that delta-integration was effective in improving caffeic acid productivity, providing a promising strategy for the production of valuable bio-based chemicals in recombinant S. cerevisiae.

Characterization of essential elements for improved episomal expressions in Kluyveromyces marxianus.

Kluyveromyces marxianus is a promising cell factory for producing heterologous proteins. Essential components including partition element, copy number control element, promoter, and terminator in multicopy plasmids applicable for K. marxianus are not extensively characterized. The pKD1 is an endogenous multicopy plasmid identified in K. lactis, and the pKD1-based plasmid is the only multicopy plasmid successfully applied in K. marxianus. The circular plasmid pKD1 contains three major open reading frames, namely A, B, and C. Here, we showed that the A gene was responsible for maintaining the high-copy number of pKD1-based plasmid in K. marxianus. Deletion of the B or C gene impaired the stable propagation of pKD1-based plasmid in K. marxianus, and this defect could not be rescued by the trans expression of B and C genes. In a quantitative analysis of a series of promoters and terminators, AFT1 promoter from Pichia pastoris, OM45 promoter and INU1 terminator from K. marxianus, and a set of synthetic terminators, supported high-level expressions. A combination of AFT1 or OM45 promoter with INU1 terminator in a pKD1-based plasmid achieved high-level expressions of four different heterologous proteins. Our study provides valuable elements for high-level episomal expressions in K. marxianus.

Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation.

Chitosanase hydrolyzes β-(1,4)-linked glycosidic bonds are used in chitosan chains to release oligosaccharide mixtures. Here, we cloned and expressed a cold-adapted chitosanase (CDA, Genbank: MW094131) using multi-copy expression plasmids (CDA1/2/3/4) in Pichia pastoris. We identified elevated CDA expression levels in multi-copy strains, with strain PCDA4 selected for high-density fermentation and enzyme-activity studies. The high-density fermentation approach generated a CDA yield of 20014.8 U/mL, with temperature and pH optimization experiments revealing the highest CDA activity at 20 °C and 5.0, respectively. CDA was stable at 10 °C and 20 °C. Thus, CDA could be used at low temperatures. CDA was then displayed on P. pastoris using multi-copy expression plasmids. Then, multi-copy strains were constructed and labelled as PCDA(1-3)-AGα1. Further studies showed that the expression of CDA(1-3)-AGα1 in multi-copy strains was increased, and that strain PCDA3-AGα1 was chosen for high-density fermentation and enzyme activity studies. By using a multi-copy expression and high-density fermentation approach, we observed CDA-AGα1 expression yields of 102415 U/g dry cell weight. These data showed that the displayed CDA exhibited improved thermostability and was more stable over wider temperature and pH ranges than free CDA. In addition, displayed CDA could be reused. Thus, the data showed that displaying enzymes on P. pastoris may have applications in industrial settings.