Tips for efficiently maintaining pET expression plasmids.

pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible using either a Tn3.1-type genetic fragment (which encodes a ß-lactamase and confers resistance to ß-lactam antibiotics) or a Tn903.1-type genetic fragment (which encodes an aminoglycoside-3'-phosphotransferase and confers resistance aminoglycoside antibiotics). Herein we have investigated how efficiently pET plasmids are maintained using these two fragments. The study reveals that pET plasmids are efficiently maintained with both Tn3.1 and Tn903.1 genetic fragments prior to the induction of recombinant protein production, and over short induction times (i.e., 2 h). However, over longer induction times (i.e., 20 h), the efficiency of plasmid maintenance depends on the host strain used, and the type of antibiotic selection cassette used. Based on our collective observations, we have 2 general tips for efficiently maintaining pET plasmids during recombinant production experiments. Tip #1: Use a strain with lowered levels of the T7 RNA polymerase, such as C41(DE3). pET plasmids will be efficiently maintained over long induction times with both the Tn3.1 and Tn903.1 genetic fragments, regardless of whether antibiotics are present during cultivation. Tip #2: If a strain with higher levels of T7 RNA polymerase strain is necessary, such as BL21(DE3)), keep induction times short or use a plasmid containing a Tn903.1-type fragment and select with kanamycin.

Tn3-like structures co-harboring of blaCTX-M-65, blaTEM-1 and blaOXA-10 in the plasmids of two Escherichia coli ST1508 strains originating from dairy cattle in China.

The purpose of this study was to determine the level of horizontal transmission of the blaCTX-M-65 gene and the role of its associated mobile genetic elements (MGEs) in the bovine-derived Escherichia coli. After PCR identification, two plasmids carrying blaCTX-M-65 were successfully transferred to the recipient E. coli J53 Azr through conjugation assays and subsequently selected for Whole-Genome sequencing (WGS) analysis. The resistance profiles of these two positive strains and their transconjugants were also determined through antimicrobial susceptibility tests. Whole genome data were acquired using both the PacBio sequencing platform and the Illumina data platform. The annotated results were then submitted to the Genbank database for accession number recording. For comparison, the genetic environment of plasmids carrying the resistance gene blaCTX-M-65 was mapped using the Easyfig software. WGS analysis revealed Tn3-like composite transposons bearing blaCTX-M-65, blaTEM-1, and blaOXA-10 in the IncHI2-type plasmids of these two E. coli ST1508 strains. A phylogenetic tree was generated from all 48 assembled E. coli isolates blaCTX-M-65, blaTEM-1, and blaOXA-10 from the NCBI Pathogen Detection database with our two isolates, showing the relationships and the contribution of SNPs to the diversity between genetic samples. This study suggests that the transmissibility of blaCTX-M-65 on Tn3-like composite transposons contributes to an increased risk of its transmission in E. coli derived from dairy cattle.

Molecular Characteristics and Genetic Analysis of Extensively Drug-Resistant Isolates with different Tn3 Mobile Genetic Elements.

Extensively drug-resistant (XDR) bacteria are the main caues for causing clinical infectious diseases. Our aim was to distinguish the present molecular epidemiological situation of XDR Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli isolates recovered from local hospitals in Changzhou. Antibiotic susceptibility and phenotypic analysis, multilocus sequence typing and Pulsed Field Gel Electrophoresis were performed to trace these isolates. Resistant phenotype and gene analysis from 29 XDR strains demonstrated that they mainly included TEM, CTX-M-1/2, OXA-48, and KPC products. A. baumannii strains belonged to sequence type (ST) ST224, and carrying the blaCTX-M-2/TEM gene. The quinolone genes aac(6')-ib-cr and qnrB were carrying only in A. baumannii and E.coli. Three (2.3%) of these strains were found to contain the blaNDM-1 or blaNDM-5 gene. A new genotype of K. pneumoniae was found as ST2639. Epidemic characteristics of the XDR clones showed that antibiotic resistance genes distributed unevenly in different wards in Changzhou's local hospitals. With the sequencing of blaNDM carrying isolates, the plasmids often carrying a highly conservative Tn3-relavent mobile genetic element. The especially coupled insert sequence ISKox3 may be a distinctive resistance gene transfer loci. The genotypic diversity variation of XDRs suggested that tracking and isolating the sources of antibiotic resistance especially MBL-encoding genes such as blaNDM-will help manage the risk of infection by these XDRs.

Genetic Factors Associated with Enhanced blaKPC Expression in Tn3/Tn4401 Chimeras.

The expression of the blaKPC gene plays a key role in carbapenem resistance in Enterobacteriaceae However, the genetic regulators of the blaKPC gene have not been completely elucidated, especially the genes in Tn3-Tn4401 chimeras. Two novel Tn3-Tn4401 chimera isoforms were characterized in our hospital, isoform A (CTA), which harbors a 121-bp deletion containing the PX promoter and was present in 22.6% (54/239) of isolates, and isoform C (CTC), which harbors a 624-bp insertion and a P1 promoter deletion and was present in only 1 isolate. The carbapenem MICs of both isoforms were 2-fold or more higher than those of the wild type (Tn3-Tn4401 chimera, CTB), and blaKPC was most highly expressed in CTA. Bioinformatics and 5' rapid amplification of cDNA ends (5' RACE) experiments indicated a novel strong putative promoter, PY, at the 3' end of the ISKpn8 gene. PY mutation nearly abrogated blaKPC expression (P < 0.01) and restored carbapenem susceptibility in all 3 isoforms. Although the mutation of PX or P1 halved blaKPC expression in CTB (P < 0.05), PX deletion caused a 68% increase in blaKPC expression (P = 0.037) in CTA. The level of blaKPC mRNA in CTC was 8-fold higher than that in InCTC, which harbors P1 (P = 0.011). These results suggest that PY is a core promoter of the blaKPC gene in the chimeras and that the deletion of the PX and P1 promoters enhanced gene expression in CTA and CTC, respectively.

Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly.

The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

Comparative Analysis of blaKPC Expression in Tn4401 Transposons and the Tn3-Tn4401 Chimera.

Two basic structures that carry the blaKPC gene, the Tn4401 transposon and the Tn3-Tn4401 chimera, have been identified within and outside China. However, the different blaKPC expression levels and promoter activities of these two structures are not completely understood. We constructed Tn4401a, Tn4401b, and Tn3-Tn4401 chimera recombinants and found that the imipenem (IPM) and meropenem (MEM) MICs for the Escherichia coli transformants carrying the chimera were 2-fold higher than for those carrying Tn4401b but 2-fold lower than for those carrying Tn4401a In addition to the promoter P1, we characterized a novel potential promoter sequence (PX) in the chimera using 5' rapid amplification of cDNA ends (5' RACE), of which the -35 and -10 sequences were TTCAAA and TGAGACAAT, respectively. Although mutation of P1, P2, or PX significantly downregulated blaKPC mRNA expression in each structure (P < 0.05), the P2 mutation resulted in 2- and 3-fold greater decreases than the P1 mutation in Tn4401a and Tn4401b, respectively. Similarly, despite no significant difference in the PX and P1 mutations in the chimera, the carbapenem MIC and Klebsiella pneumoniae carbapenemase (KPC) production resulting from P2 mutations were significantly lower than those of P1 (P < 0.01) in the Tn4401 transposons. These studies indicate that the Tn3-Tn4401 chimera contains a novel potential blaKPC promoter, PX, and that its carbapenem resistance falls in between those of Tn4401a and Tn4401b.

Genomic Characterization of Carbapenemase-Producing Klebsiella pneumoniae with Chromosomally Carried blaNDM-1.

We report here Klebsiella pneumoniae strains carrying chromosomal blaNDM-1 in Thailand. The genomes of these two isolates include a 160-kbp insertion containing blaNDM-1, which is almost identical to that in the IncHI1B-like plasmid. Further analysis indicated that IS5-mediated intermolecular transposition and Tn3 transposase-mediated homologous recombination resulted in the integration of blaNDM-1 into the chromosome from an IncHI1B-like plasmid. The spread of this type of carbapenem-resistant Enterobacteriaceae may threaten public health and warrants further monitoring.

The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity.

Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4+ T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial.

Involvement of Tn3 transposon in formation and transmission of hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

IMPORTANCE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to most common antibiotics, becoming the most important and prevalent nosocomial opportunity pathogen. Besides, K. pneumoniae can also cause severe community-acquired infections, such as primary liver abscess and endophthalmitis. These pathogens are commonly referred to as hvKp. CRKP and hvKp have evolved separately, each occupying its own clonal lineage and exhibiting a variety of properties. Our study provides important insights into the evolutionary events related to the arousal of virulence and drug resistance in K. pneumoniae through plasmid transmission, mediated by Tn3 transposon. Our study also provides evidence that multiple mechanisms contribute to the successful transfer of non-conjugative virulence plasmid, and the involvement of transposons enhances the efficiency. A good knowledge of its transmission mechanisms is fundamental to finding effective strategies to combat these threatening pathogens. Transposons are widely present in bacteria, spreading resistance and virulence genes between the environment and humans. Therefore, emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens should be highly valued.

Toxin-Antitoxin Gene Pairs Found in Tn3 Family Transposons Appear To Be an Integral Part of the Transposition Module.

Much of the diversity of prokaryotic genomes is contributed by the tightly controlled recombination activity of transposons (Tns). The Tn3 family is arguably one of the most widespread transposon families. Members carry a large range of passenger genes incorporated into their structures. Family members undergo replicative transposition using a DDE transposase to generate a cointegrate structure which is then resolved by site-specific recombination between specific DNA sequences (res) on each of the two Tn copies in the cointegrate. These sites also carry promoters controlling expression of the recombinase and transposase. We report here that a number of Tn3 members encode a type II toxin-antitoxin (TA) system, typically composed of a stable toxin and a labile antitoxin that binds the toxin and inhibits its lethal activity. This system serves to improve plasmid maintenance in a bacterial population and, until recently, was believed to be associated with bacterial persistence. At least six different TA gene pairs are associated with various Tn3 members. Our data suggest that several independent acquisition events have occurred. In contrast to most Tn3 family passenger genes, which are generally located away from the transposition module, the TA gene pairs abut the res site upstream of the resolvase genes. Although their role when part of Tn3 family transposons is unclear, this finding suggests a potential role for the embedded TA in stabilizing the associated transposon with the possibility that TA expression is coupled to expression of transposase and resolvase during the transposition process itself.IMPORTANCE Transposable elements (TEs) are important in genetic diversification due to their recombination properties and their ability to promote horizontal gene transfer. Over the last decades, much effort has been made to understand TE transposition mechanisms and their impact on prokaryotic genomes. For example, the Tn3 family is ubiquitous in bacteria, molding their host genomes by the paste-and-copy mechanism. In addition to the transposition module, Tn3 members often carry additional passenger genes (e.g., conferring antibiotic or heavy metal resistance and virulence), and three were previously known to carry a toxin-antitoxin (TA) system often associated with plasmid maintenance; however, the role of TA systems within the Tn3 family is unknown. The genetic context of TA systems in Tn3 members suggests that they may play a regulatory role in ensuring stable invasion of these Tns during transposition.

First report of Klebsiella quasipneumoniae harboring blaKPC-2 in Saudi Arabia.

Background: Nosocomial infections caused by multi-drug resistant Enterobacteriaceae are a global public health threat that ought to be promptly identified, reported, and addressed accurately. Many carbapenem-resistant Enterobacteriaceae-associated genes have been identified in Saudi Arabia but not the endemic Klebsiella pneumoniae carbapenemases (KPCs), which are encoded by blaKPC-type genes. KPCs are known for their exceptional spreading potential. Methods: We collected n = 286 multi-drug resistant (MDR) Klebsiella spp. isolates as part of screening for resistant patterns from a tertiary hospital in Saudi Arabia between 2014 and 2018. Antimicrobial susceptibility testing was carried out using both VITEK II and the broth microdilution of all collected isolates. Detection of resistance-conferring genes was carried out using Illumina whole-genome shotgun sequencing and PacBio SMRT sequencing protocols. Results: A Carbapenem-resistant Enterobacteriaceae (CRE) Klebsiella quasipneumoniae subsp. similipneumoniae strain was identified as a novel ST-3510 carrying a blaKPC-2 carbapenemase encoding gene. The isolate, designated as NGKPC-421, was obtained from shotgun Whole Genome Sequencing (WGS) surveillance of 286 MDR Klebsiella spp. clinical isolates. The NGKPC-421 isolate was collected from a septic patient in late 2017 and was initially misidentified as K. pneumoniae. The sequencing and assembly of the NGKPC-421 genome resulted in the identification of a putative ~ 39.4 kb IncX6 plasmid harboring a blaKPC-2 gene, flanked by transposable elements (ISKpn6-blaKPC-2-ISKpn27). Conclusion: This is the first identification of a KPC-2-producing CRE in the Gulf region. The impact on this finding is of major concern to the public health in Saudi Arabia, considering that it is the religious epicenter with a continuous mass influx of pilgrims from across the world. Our study strongly highlights the importance of implementing rapid sequencing-based technologies in clinical microbiology for precise taxonomic classification and monitoring of antimicrobial resistance patterns.

Improved pullulan production by a mutant of Aureobasidium melanogenum TN3-1 from a natural honey and capsule shell preparation.

Aureobasidium melanogenum TN3-1 isolated from a natural honey was a highly genome-duplicated yeast-like fungal strain and a very high pullulan producer. In this study, simultaneous removal of both duplicated AMY1 genes encoding α-amylase and duplicated PKS1 genes responsible for melanin biosynthesis in A. melanogenum TN3-1 rendered a mutant AMY-PKS-11 to transform 140.0 g/L of glucose to produce 103.50 g/L of pigment-free pullulan with molecular weight (Mw) of 3.2 × 105 g/mol. α-Amylase activity produced by the mutant AMY-PKS-11 and expression of the AMY1 genes and PKS genes in it was reduced, but expression of various genes responsible for pullulan biosynthesis in the mutant AMY-PKS-11 was up-regulated. The produced pullulan was used to make the capsule shells successfully and the prepared pullulan capsule shells had various advantages such as high strength, good oxygen barrier properties, raw materials availability, tightness, lightness and high water resistance and may be suitable for all the consumers. Therefore, the prepared capsule shells had highly potential applications in food and pharmaceutical industries.

Transposons and pathogenicity in Xanthomonas: acquisition of murein lytic transglycosylases by TnXax1 enhances Xanthomonas citri subsp. citri 306 virulence and fitness.

Xanthomonas citri subsp. citri 306 (XccA) is the causal agent of type A citrus canker (CC), one of the most significant citriculture diseases. Murein lytic transglycosylases (LT), potentially involved in XccA pathogenicity, are enzymes responsible for peptidoglycan structure assembly, remodeling and degradation. They directly impact cell wall expansion during bacterial growth, septum division allowing cell separation, cell wall remodeling allowing flagellar assembly, bacterial conjugation, muropeptide recycling, and secretion system assembly, in particular the Type 3 Secretion System involved in bacterial virulence, which play a fundamental role in XccA pathogenicity. Information about the XccA LT arsenal is patchy: little is known about family diversity, their exact role or their connection to virulence in this bacterium. Among the LTs with possible involvement in virulence, two paralogue open reading frames (ORFs) (one on the chromosome and one in plasmid pXAC64) are passenger genes of the Tn3 family transposon TnXax1, known to play a significant role in the evolution and emergence of pathogenicity in Xanthomonadales and to carry a variety of virulence determinants. This study addresses LT diversity in the XccA genome and examines the role of plasmid and chromosomal TnXax1 LT passenger genes using site-directed deletion mutagenesis and functional characterization. We identified 13 XccA LTs: 12 belong to families 1A, 1B, 1C, 1D (two copies), 1F, 1G, 3A, 3B (two copies), 5A, 6A and one which is non-categorized. The non-categorized LT is exclusive to the Xanthomonas genus and related to the 3B family but contains an additional domain linked to carbohydrate metabolism. The categorized LTs are probably involved in cell wall remodeling to allow insertion of type 3, 4 and 6 secretion systems, flagellum assembly, division and recycling of cell wall and degradation and control of peptidoglycan production. The TnXax1 passenger LT genes (3B family) are not essential to XccA or for CC development but are implicated in peptidoglycan metabolism, directly impacting bacterial fitness and CC symptom enhancement in susceptible hosts (e.g., Citrus sinensis). This underlines the role of TnXax1 as a virulence and pathogenicity-propagating agent in XccA and suggests that LT acquisition by horizontal gene transfer mediated by TnXax1 may improve bacterial fitness, conferring adaptive advantages to the plant-pathogen interaction process.

Characterization of genetic determinants involved in antibiotic resistance in Aeromonas spp. and fecal coliforms isolated from different aquatic environments.

Aeromonas spp. and fecal coliforms, two abundant and cultivable bacterial populations that can be found in water ecosystems, might substantially contribute to the spread of antibiotic resistance. We investigated the presence and spread of transposons (elements that can move from one location to another in the genome), integrons (structures able to capture and incorporate gene cassettes) and resistance plasmids in strains isolated from polluted and unpolluted water. We recovered 231 Aeromonas and 250 fecal coliforms from water samplings with different degrees of pollution (hospital sewage, activated sludge of a wastewater treatment plant, river water before and after treatment and water from an alpine lake). Sixteen Aeromonas spp. and 22 fecal coliforms carried intI, coding for the site-specific integrase of class 1 integrons, while 22 Aeromonas spp. and 14 fecal coliforms carried tnpA, the transposase gene of the Tn3-family of replicative transposons. The majority of intI and tnpA-positive strains were phenotypically resistant to at least four antibiotics. Integrons and transposons were mainly located on mobilizable plasmids. Our results did not detect common mobile structures in the two populations and therefore relativize the role played by Aeromonas spp. as vectors of antimicrobial resistance determinants between water and commensal gut bacteria.

Lipid- and polyion complex-based micelles as agonist platforms for TNFR superfamily receptors.

Receptor clustering is important for signaling among the therapeutically relevant TNFR superfamily of receptors. In nature, this clustering is driven by trimeric ligands often presented in large numbers as cell surface proteins. Molecules capable of driving similar levels of clustering could make good agonists and hold therapeutic value. However, recapitulating such extensive clustering using typical biotherapeutic formats, such as antibodies, is difficult. Consequently, generating effective agonists of TNFR superfamily receptors is challenging. Toward addressing this challenge we have used lipid- and polyion complex-based micelles as platforms for presenting receptor-binding biologics in a multivalent format that facilitates receptor clustering and imparts strong agonist activity. We show that receptor-binding scFvs or small antibody mimetics that have no agonist activity on their own can be transformed into potent agonists through multivalent presentation on a micelle surface and that the activity of already active multivalent agonists can be enhanced. Using this strategy, we generated potent agonists against two different TNFR superfamily receptors and mouse tumor model studies demonstrate that these micellar agonists have therapeutic efficacy in vivo. Due to its ease of implementation and applicability independent of agonist molecular format, we anticipate that this strategy could be useful for developing agonists to a variety of receptors that rely on clustering to signal.

Diversity and role of plasmids in adaptation of bacteria inhabiting the Lubin copper mine in Poland, an environment rich in heavy metals.

The Lubin underground mine, is one of three mining divisions in the Lubin-Glogow Copper District in Lower Silesia province (Poland). It is the source of polymetallic ore that is rich in copper, silver and several heavy metals. Black shale is also significantly enriched in fossil organic matter in the form of long-chain hydrocarbons, polycyclic aromatic hydrocarbons, organic acids, esters, thiophenes and metalloporphyrins. Biological analyses have revealed that this environment is inhabited by extremophilic bacteria and fungi. Kupfershiefer black shale and samples of water, bottom and mineral sediments from the underground (below 600 m) Lubin mine were taken and 20 bacterial strains were isolated and characterized. All exhibited multi-resistant and hypertolerant phenotypes to heavy metals. We analyzed the plasmidome of these strains in order to evaluate the diversity and role of mobile DNA in adaptation to the harsh conditions of the mine environment. Experimental and bioinformatic analyses of 11 extrachromosomal replicons were performed. Three plasmids, including a broad-host-range replicon containing a Tn3 family transposon, carried genes conferring resistance to arsenic, cadmium, cobalt, mercury and zinc. Functional analysis revealed that the resistance modules exhibit host specificity, i.e., they may increase or decrease tolerance to toxic ions depending on the host strain. The other identified replicons showed diverse features. Among them we identified a catabolic plasmid encoding enzymes involved in the utilization of histidine and vanillate, a putative plasmid-like prophage carrying genes responsible for NAD biosynthesis, and two repABC-type plasmids containing virulence-associated genes. These findings provide an unique molecular insight into the pool of extrachromosomal replicons and highlight their role in the biology and adaptation of extremophilic bacteria inhabiting terrestrial deep subsurface.

Autonomous and non-autonomous Tn3-family transposons and their role in the evolution of mobile genetic elements.

The Tn3 family of transposons includes diverse elements that encode homologous transposases and contain conserved terminal inverted repeat sequences (IRs). The recent identification of non-autonomous elements, named TIMEs (Tn3-derived Inverted-repeat Miniature Elements), has shed new light on the diversity and evolution of this transposon family. A common feature of TIMEs and other members of this family is their ability to mobilize genomic DNA for transposition as part of composite transposons. These elements significantly influence the structure and properties of plasmids and other mobile genetic elements (MGEs). They may contain and move by transposition (i) plasmid replication systems, (ii) toxin-antitoxin systems and (iii) site-specific recombination modules that can resolve plasmid multimers. Some Tn3 family elements may also transfer large segments of chromosomal DNA into plasmids, which increases the pool of mobile DNA that can take part in horizontal gene transfer.