Development of a plasmid stabilization system in Vibrio natriegens for the high production of 1,3-propanediol and 3-hydroxypropionate

Vibrio natriegens is a promising industrial chassis with a super-fast growth rate and high substrate uptake rates. V. natriegens was previously engineered to produce 1,3-propanediol (1,3-PDO) from glycerol by overexpressing the corresponding genes in a plasmid. However, antibiotic selection pressure for plasmid stability was not satisfactory and plasmid loss resulted in reduced productivity of the bioprocess. In this study, we developed an antibiotic-free plasmid stabilization system for V. natriegens. The system was achieved by shifting the glpD gene, one of the essential genes for glycerol degradation, from the chromosome to plasmid. With this system, engineered V. natriegens can stably maintain a large expression plasmid during the whole fed-batch fermentation and accumulated 69.5 g/L 1,3-PDO in 24 h, which was 23% higher than that based on antibiotic selection system. This system was also applied to engineering V. natriegens for the production of 3-hydroxypropionate (3-HP), enabling the engineered strain to accumulate 64.5 g/L 3-HP in 24 h, which was 30% higher than that based on antibiotic system. Overall, the developed strategy could be useful for engineering V. natriegens as a platform for the production of value-added chemicals from glycerol. Graphic Abstract

Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures

It is of major interest to ensure stable and performant microbial bioprocesses, therefore maintaining high strain robustness is one of the major future challenges in industrial microbiology. Strain robustness can be defined as the persistence of genotypic and/or phenotypic traits in a system. In this work, robustness of an engineered strain is defined as plasmid expression stability, cultivability, membrane integrity and macroscopic cell behavior and was assessed in response to implementations of sugar feeding strategies (pulses and continuous) and two plasmid stabilization systems (kanamycin resistance and Post-Segregational Killing hok/sok). Fed-batch bioreactor cultures, relevant mode to reach high cell densities and higher cell generation number, were implemented to investigate the robustness of C. necator engineered strains. Host cells bore a recombinant plasmid encoding for a plasmid expression level monitoring system, based on eGFP fluorescence quantified by flow cytometry. We first showed that well-controlled continuous feeding in comparison to a pulse-based feeding allowed a better carbon use for protein synthesis (avoiding organic acid excretion), a lower heterogeneity of the plasmid expression and a lower cell permeabilization. Moreover, the plasmid stabilization system Post-Segregational Killing hok/sok, an autonomous system independent on external addition of compounds, showed the best ability to maintain plasmid expression level stability insuring a greater population homogeneity in the culture. Therefore, in the case of engineered C. necator, the PSK system hok/sok appears to be a relevant and an efficient alternative to antibiotic resistance system for selection pressure, especially, in the case of bioprocess development for economic and environmental reasons.

A peptide of a type I toxin−antitoxin system inducesHelicobacter pylorimorphological transformation from spiral shape to coccoids

Toxin−antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins’ biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin−antitoxin system (AapA1−IsoA1) expressed from the chromosome of the human pathogenHelicobacter pylori. We show that expression of the AapA1 toxin inH. pyloricauses growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targetsH. pyloriinner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5′-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important inH. pyloriinfections refractory to treatment.

Identification of a Novel Plasmid Carrying mcr-4.3 in an Acinetobacter baumannii Strain in China

ABSTRACT Here, we identified mcr-4.3 in Acinetobacter baumannii, which had not been previously observed to carry an mcr gene. The mcr-4.3-harboring A. baumannii strain AB18PR065 was isolated from pig feces from a slaughterhouse in Guangdong Province of China. The mcr-4.3-carrying pAB18PR065 is 25,602 bp in size and could not be transferred in conjugation, transformation, and electroporation experiments, as we did not find any conjugation-related genes therein. pAB18PR065 harbors two copies of type II toxin-antitoxin systems, which are functional in plasmid stabilization and maintenance. pAB18PR065 shares similarity only with one recently identified plasmid, pAb-MCR4.3 (35,502 bp), from a clinical A. baumannii strain. It is likely that the emergence of pAb-MCR4.3 was due to the insertion of an 11,386-bp, ISAba19-based, composite transposon into pAB18PR065. These data indicate that mcr-4.3 was captured by an A. baumannii-original plasmid via horizontal gene transfer.

A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral-shape to coccoids

SummaryToxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which in type I systems is an antisense-RNA. While the regulatory mechanisms of these systems are mostly well-defined, the toxins’ biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori. We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by Cryo-EM. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or ATP concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression.Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.Significance StatementHelicobacter pylori, a gastric pathogen causing 800,000 deaths in the world annually, is encountered both in vitro and in patients as spiral-shaped bacteria and as round cells named coccoids. We discovered that the toxin from a chromosomal type I toxin-antitoxin system is targeting H. pylori membrane and acting as an effector of H. pylori morphological conversion to coccoids. We showed that these round cells maintain their membrane integrity and metabolism, strongly suggesting that they are viable dormant bacteria. Oxidative stress was identified as a signal inducing toxin expression and coccoid formation. Our findings reveal new insights into a form of dormancy of this bacterium that might be associated with H. pylori infections refractory to treatment.

Emerging patterns of plasmid-host coevolution that stabilize antibiotic resistance

ABSTRACTMultidrug resistant bacterial pathogens have become a serious global human health threat, and conjugative plasmids are important drivers of the rapid spread of resistance to last-resort antibiotics. Whereas antibiotics have been shown to select for adaptation of resistance plasmids to their new bacterial hosts, orvice versa, a general evolutionary mechanism has not yet emerged. Here we conducted an experimental evolution study aimed at determining general patterns of plasmid-bacteria evolution. Specifically, we found that a large conjugative resistance plasmid follows the same evolutionary trajectories as its non-conjugative mini-replicon in the same and other species. Furthermore, within a single host–plasmid pair three distinct patterns of adaptive evolution led to increased plasmid persistence: i) mutations in the replication protein gene (trfA1); ii) the acquisition by the resistance plasmid of a transposon from a co-residing plasmid encoding a putative toxin-antitoxin system; iii) a mutation in the host’s global transcriptional regulator genefur. Since each of these evolutionary solutions individually have been shown to increase plasmid persistence in other plasmid-host pairs, our work points towards common mechanisms of plasmid stabilization. These could become the targets of future alternative drug therapies to slow down the spread of antibiotic resistance.

Persistent, Toxin-Antitoxin System-Independent, Tetracycline Resistance-Encoding Plasmid from a Dairy Enterococcus faecium Isolate

ABSTRACTA tetracycline-resistant (Tetr) dairyEnterococcus faeciumisolate designated M7M2 was found to carry bothtet(M) andtet(L) genes on a 19.6-kb plasmid. After consecutive transfer in the absence of tetracycline, the resistance-encoding plasmid persisted in 99% of the progenies. DNA sequence analysis revealed that the 19.6-kb plasmid contained 28 open reading frames (ORFs), including atet(M)-tet(L)-mobgene cluster, as well as a 10.6-kb backbone highly homologous (99.9%) to the reported plasmid pRE25, but without an identified toxin-antitoxin (TA) plasmid stabilization system. The derived backbone plasmid without the Tetrdeterminants exhibited a 100% retention rate in the presence of acridine orange, suggesting the presence of a TA-independent plasmid stabilization mechanism, with its impact on the persistence of a broad spectrum of resistance-encoding traits still to be elucidated. Thetet(M)-tet(L) gene cluster from M7M2 was functional and transmissible and led to acquired resistance inEnterococcus faecalisOG1RF by electroporation and inStreptococcus mutansUA159 by natural transformation. Southern hybridization showed that both thetet(M) andtet(L) genes were integrated into the chromosome ofS. mutansUA159, while the whole plasmid was transferred to and retained inE. faecalisOG1RF. Quantitative real-time reverse transcription-PCR (RT-PCR) indicated tetracycline-induced transcription of both thetet(M) andtet(L) genes of pM7M2. The results indicated that multiple mechanisms might have contributed to the persistence of antibiotic resistance-encoding genes and that the plasmids pM7M2, pIP816, and pRE25 are likely correlated evolutionarily.