Suicide Vector Research Articles

Construction and characterization of a modA gene mutant strain of Klebsiella pneumoniae

To construct a mutant strain of Klebsiella pneumoniae NTUH- K2044 with modA gene deletion and its complementary strain and explore the role of modA gene in modulating anaerobic nitrate respiratory growth and phenotypes of K. pneumoniae. The modA deletion mutant K. pneumoniae strain was constructed by homologous recombination using the suicide vector pKO3-Km. To obtain the complementary strain C-modA, the whole sequence fragment containing the promoter, open reading frame and terminator regions of modA was cloned into pGEM-T-easy and electrically transformed into the modA deletion mutant. The NTUH-K2044 wild-type strain, modA gene deletion mutant and complementary strain were compared by measuring in vitro anaerobic nitrate respiration growth, competitiveness index, biofilm quantification, mucoviscosity assay and morphological measurement using Image J. The modA deletion mutant strain ΔmodA and the complementary strain C-modA were successfully constructed. The modA gene knockout strain showed inhibited anaerobic nitrate respiratory growth compared with the wild- type and C-modA strains with significantly weakened competitiveness, reduced capacity of biofilm synthesis during anaerobiosis, and lowered mucoviscosity under anaerobic conditions. The ΔmodA strain showed a spherical morphology in anaerobic conditions as compared with the normal short rod-like morphology of K. pneumoniae, with also distinctly shorter length than the wild-type and C-modA strains. The molybdate transport system encoding gene modA is associated with the pathogenic capacity of K. pneumoniae by modulating its anaerobic nitrate respiration, competitiveness, biofilm formation, hypermucoviscous phenotype and morphology.

A Novel System to Selective Tagging of Sinorhizobium fredii Symbiotic Plasmids.

Conventional systems used to tag and transfer symbiotic plasmids (pSyms) of rhizobial strains are based in mutagenesis with transposons. In those processes, numerous clones must be analyzed to find one of them with the transposon inserted in the pSym. Following this strategy, the insertion might interrupt a gene that can affect the symbiotic phenotype of the bacteria tagged. Here, we have developed a new system based in homologous recombination that generates Sinorhizobium fredii strains with pSyms tagged by the insertion of a suicide vector which harbor a truncated copy of S. fredii HH103 nodZ gene, a mob site, and a kanamycin-resistant gene. When it is introduced by conjugation in a S. fredii strain, the vector integrates in pSym by only one recombination event. This pSym tagged can be transferred in matting experiments to other strains in the presence of a helper plasmid. Following this method, we have tagged several strains and transferred their pSyms to a recipient strain demonstrating the potential of this new system.

Effects and molecular mechanism of sugar transporter ESA_RS15745 on desiccation resistance, motility, and biofilm formation of Cronobacter sakazakii.

Cronobacter sakazakii, an important Gram-negative foodborne pathogen, can cause neonatal meningitis and sepsis with high rates of infection and death. Gene ESA_RS15745 encodes a sugar transporter protein, which is not only essential for osmotic pressure maintenance during bacterial growth and reproduction but also associated with their desiccation tolerance, motility, and biofilm formation. Here, a mutant strain of ESA_RS15745 (ΔESA_RS15745) and the complementation strain (cpESA_RS15745) were constructed using a suicide vector knockout and gene complementation. ΔESA_RS15745 was found to have a decrease in its ability to transport maltose and trehalose and resist desiccation, whereas an increase in the ability of motility and biofilm formation, implying that ESA_RS15745 may positively regulate sugar transport and desiccation tolerance and negatively regulate motility and biofilm formation. To further investigate the molecular mechanisms underlying the function of related genes, RNA-seq was performed to explore the differentially expressed genes in the mutants. RNA-seq results showed the upregulation of 114 genes (mainly including those regulating chemotaxis and flagellar motility) and the downregulation of 22 genes (mainly including those regulating sugar transport). qRT-PCR analysis supported the RNA-seq results and showed that ESA_RS15745 may influence the dehydration tolerance though decreasing the intracellular trehalose content and negatively regulate the motility though the chemotactic signaling pathway. In addition, the biofilm formation of C. sakazakii should also be speculated to negatively regulate by ESA_RS15745 by consuming the extracellular carbohydrates concentration and then downregulating the intracellular cyclic diguanosine monophosphate. This study offers a reference for comprehending the molecular mechanism of gene ESA_RS15745 in C. sakazakii.

Transforming the untransformable with knockout minicircles: High-efficiency transformation and vector-free allelic exchange knockout in the fish pathogen Photobacterium damselae.

Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange via homologous recombination is a generic method of targeted gene editing in bacteria. However, generally applicable protocols are lacking, and suboptimal approaches are often used for nonstandard but epidemiologically important species. Photobacterium damselae subsp. piscicida (Pdp) is a primary pathogen of fish in aquaculture and has been considered hard to transform since the mid-1990s. Consequently, conjugative transfer of RK2/RP4 suicide vectors from Escherichiacoli S17-1/SM10 donor strains, a system prone to off-target mutagenesis, was used to deliver the allelic exchange DNA in previous studies. Here we have achieved efficient electrotransformation in Pdp using a salt-free highly concentrated sucrose solution, which performs as a hypertonic wash buffer, cryoprotectant, and electroporation buffer. High-efficiency transformation has enabled vector-free mutagenesis for which we have employed circular minimalistic constructs (knockout minicircles) containing only allelic exchange essentials that were generated by Gibson assembly. Preparation of competent cells using sucrose and electroporation/integration of minicircles had virtually no detectable off-target promutagenic effect. In contrast, a downstream sacB selection apparently induced several large deletions via mobilization of transposable elements. Electroporation of minicircles into sucrose-treated cells is a versatile broadly applicable approach that may facilitate allelic exchange in a wide range of microbial species. The method permitted inactivation of a primary virulence factor unique to Pdp, apoptogenic toxin AIP56, demonstrating the efficacy of minicircles for difficult KO targets located on the high copy number of small plasmids.

Construction and biological characterization of a Proteus mirabilis strain with modABC gene deletion

To construct a modABC gene knockout strain of Proteus mirabilis and explore the effect of modABC gene deletion on biological characteristics of Proteus mirabilis. Fusion PCR was used to obtain the fusion gene of modABC and the kanamycin-resistant gene Kn, which was ligated with the suicide vector pCVD442 and transduced into Proteus mirabilis. The modABC gene knockout strain of Proteus mirabilis was obtained after homologous recombination with the suicide vector. PCR and Sanger sequencing were used to identify genomic deletion of modABC gene in the genetically modified strain. The concentration of molybdate in the wild-type and gene knockout strains was determined using inductively coupled plasma mass spectrometry (ICP-MS), and their survival ability in LB medium was compared under both aerobic and anaerobic conditions. PCR and sanger sequencing confirmed genomic deletion of modABC gene in the obtained Proteus mirabilis strain. The concentration of intracellular molybdenum in the modABC gene knockout strain was 1.22 mg/kg, significantly lower than that in the wild-type strain (1.46 mg/kg, P < 0.001). Under the aerobic condition, the modABC gene knockout strain grown in LB medium showed no significant changes in survival ability compared with the wild-type strain, but its proliferation rate decreased significantly under the anaerobic condition and also when cultured in nitrate-containing LB medium under anaerobic condition. Homologous recombination with the suicide vector can be used for modABC gene knockout in Proteus mirabilis. modABC gene participates in molybdate uptake and is associated with anaerobic growth of Proteus mirabilis in the presence of nitrate.

Transformation and Mutagenesis of Chlamydia trachomatis and C. muridarum Utilizing pKW Vector

A gene deletion by allelic exchange via homologous recombination from a bacterial genome represents a valuable genetic tool for studying a role(s) of determinants involved in various aspects of pathogenesis. Due to chlamydial obligate intracellular lifestyle and comparatively low transformation rate, the mutagenesis of Chlamydia utilizes types of suicide vectors that have to be maintained and propagated by the bacteria throughout several rounds of their intracellular developmental cycle. These deletion constructs must be lost by chlamydiae once null mutant formation is achieved. pKW is a small, 5.45 bp, pUC19-derived vector, which has been recently successfully employed for the generation of deletion mutants in C. trachomatis, serovariant D, and C. muridarum. This vector contains both, E. coli as well as chlamydial species-specific plasmid origins of replication, allowing for its propagation by both bacterial genera under a selective pressure. However, once the selective antibiotic is removed from culture, chlamydiae rapidly lose pKW, and the subsequent reintroduction of the selective antibiotic back to chlamydiae-infected cells results efficiently in the selection of generated deletion mutants. Protocols provided here describe in detail the preparation of pKW deletion constructs for C. trachomatis and C. muridarum applicable for chlamydial transformation and the production of null mutant in non-essential genes. Protocols provided here, are describing in detail methods for assembly of the pKW shuttle vector and generation of deletion mutants in C. trachomatis and C. muridarum. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assembly of pKW shuttle vector Basic Protocol 2: Generation of a deletion mutant in C. trachomatis, serovars D and L2 and, Chlamydia muridarum Support Protocol: Transformation of C. trachomatis, serovars B.

Quorum sensing inhibition through site-directed mutation by deletion PCR

Quorum sensing induces biofilms and virulence factors that are adverse industrially and medically. Nowadays, quorum sensing inhibitions focus on signal analogs or signal degradation, but these methods have several downsides, which are temporal and affected by several environmental factors. In this research, we used deletion PCR to perform site-directed mutagenesis on the quorum sensing pathway gene and then analyzed its effects on quorum sensing. Serratia fonticola DSM 4576 strain was utilized as the research strain, and the gram-negative bacteria's universal quorum sensing pathway, which is conducted by acyl-homoserine lactone (AHL), was analyzed. The structure and active site of the AHL synthase enzyme encoded by S. fonticola DSM 4576's luxI-type gene were predicted. The gene's partial section solely encodes the enzyme's active site. By using sequence and ligation-independent cloning, the obtained mutagenic gene was cloned into the suicide vector pEX18Ap. The recombinant vector was used to transform wild-type S. fonticola DSM 4576 strains, and the mutants were determined through two-step selections and PCR genotyping. The gene expression level and biofilm formation were quantitatively analyzed through RT-PCR and biofilm assay, and no significant difference was noted in the gene expression between wild types and mutants. However, when mutants were compared to wildtypes, there was a significant decrease in biofilm formation as a result of quorum sensing induced bioreaction. Thus, we propose a quorum sensing inhibitory technique based on enzyme mutation on the quorum sensing pathway, and we proved the feasibility of enzyme active site's site-directed mutation through deletion PCR.

Transcriptomic Changes and satP Gene Function Analysis in Pasteurella multocida with Different Levels of Resistance to Enrofloxacin

Pasteurella multocida (Pm) is one of the major pathogens of bovine respiratory disease (BRD), which can develop drug resistance to many of the commonly used antibiotics. Our earlier research group found that with clinical use of enrofloxacin, Pm was more likely to develop drug resistance to enrofloxacin. In order to better understand the resistance mechanism of Pm to enrofloxacin, we isolated PmS and PmR strains with the same PFGE typing in vitro, and artificially induced PmR to obtain the highly resistant phenotype, PmHR. Then transcriptome sequencing of clinically isolated sensitive strains, resistant and highly drug-resistant strains, treated with enrofloxacin at sub-inhibitory concentrations, were performed. The satP gene, of which the expression changed significantly with the increase in drug resistance, was screened. In order to further confirm the function of this gene, we constructed a satP deletion (ΔPm) strain using suicide vector plasmid pRE112, and constructed the C-Pm strain using pBBR1-MCS, and further analyzed the function of the satP gene. Through a continuously induced resistance test, it was found that the resistance rate of ΔPm was obviously lower than that of Pm in vitro. MDK99, agar diffusion and mutation frequency experiments showed significantly lower tolerance of ΔPm than the wild-type strains. The pathogenicity of ΔPm and Pm was measured by an acute pathogenicity test in mice, and it was found that the pathogenicity of ΔPm was reduced by about 400 times. Therefore, this study found that the satP gene was related to the tolerance and pathogenicity of Pm, and may be used as a target of enrofloxacin synergistic effect.

Treponema pallidum subsp. pallidum with an Artificially impaired TprK antigenic variation system is attenuated in the Rabbit model of syphilis.

The TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum's ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen's surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted β-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis. A suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DCKO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DCKO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DCKO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DCKO strain remained uninfected. These data further support the critical role of TprK in T. pallidum virulence and persistence during infection.

Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid

Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication.Key points• Improved vector for homologous recombination in R. opacus.• Mycolic acid is important for survival of R. opacus like it is for Mycobacterium.• Similar conditional suicide plasmids may be constructed for other bacteria.Graphical abstract

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu.

Pseudonocardia species are emerging as important microorganisms of global concern with unique and increasingly significant ecological roles and represent a prominent source of bioactive natural products, but genetic engineering of these organisms for biotechnological applications is greatly hindered due to the limitation of efficient genetic manipulation tools. In this regard, we report here the establishment of an efficient genetic manipulation system for a newly isolated strain, Pseudonocardia alni Shahu, based on plasmid conjugal transfer from Escherichia coli to Pseudonocardia. Conjugants were yielded upon determining the optimal ratio between the donor and recipient cells, and designed genome modifications were efficiently accomplished, including exogenous gene integration based on an integrative plasmid and chromosomal stretch removal by homologous recombination using a suicidal non-replicating vector. Collectively, this work has made the P. alni Shahu accessible for genetic engineering, and provided an important reference for developing genetic manipulation methods in other rare actinomycetes.

Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Function Characterization of Endogenous Plasmids in Cronobacter sakazakii and Identification of p-Coumaric Acid as Plasmid-Curing Agent.

Virulence traits and antibiotic resistance are frequently provided by genes located on plasmids. However, experimental verification of the functions of these genes is often lacking due to a lack of related experimental technology. In the present study, an integrated suicide vector was used to efficiently and specifically delete a bacterial endogenous plasmid in Cronobacter sakazakii. The pESA3 plasmid was removed from C. sakazakii BAA-894, and we confirmed that this plasmid contributes to the invasion and virulence of this strain. In addition, the pGW1 plasmid was expunged from C. sakazakii GZcsf-1, and we confirmed that this plasmid confers multidrug resistance. We further screened plasmid-curing agents and found that p-coumaric acid had a remarkable effect on the curing of pESA3 and pGW1 at sub-inhibitory concentrations. Our study investigated the contribution of endogenous plasmids pESA3 and pGW1 by constructing plasmid-cured strains using suicide vectors and suggested that p-coumaric acid can be a safe and effective plasmid-curing agent for C. sakazakii.

Broad Purpose Vector for Site-Directed Insertional Mutagenesis in Bifidobacterium breve.

Members of the genus Bifidobacterium are notoriously recalcitrant to genetic manipulation due to their extensive and variable repertoire of Restriction-Modification (R-M) systems. Non-replicating plasmids are currently employed to achieve insertional mutagenesis in Bifidobacterium. One of the limitations of using such insertion vectors is the presence within their sequence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain. For this reason, vectors have been developed with the aim of methylating and protecting the vector using a methylase-positive Escherichia coli strain, in some cases containing a cloned bifidobacterial methylase. Here, we present a mutagenesis approach based on a modified and synthetically produced version of the suicide vector pORI28 (named pFREM28), where all known restriction sites targeted by Bifidobacterium breve R-M systems were removed by base substitution (thus preserving the codon usage). After validating the integrity of the erythromycin marker, the vector was successfully employed to target an α-galactosidase gene responsible for raffinose metabolism, an alcohol dehydrogenase gene responsible for mannitol utilization and a gene encoding a priming glycosyltransferase responsible for exopolysaccharides (EPS) production in B. breve. The advantage of using this modified approach is the reduction of the amount of time, effort and resources required to generate site-directed mutants in B. breve and a similar approach may be employed to target other (bifido)bacterial species.

Improvement of the attenuated mutant strain of Brucella melitensis Rev1 as a potential vaccine candidate

Vaccination is the best approach to prevent brucellosis. The existing vaccines such as Rev1 are associated with disadvantages such as abortion in pregnant animals. They also induce antibodies to the O-polysaccharide which may complicate diagnosis. Therefore, it is necessary to generate an appropriate vaccine. The aim of this study is the improvement of the available mutant strain of Brucella melitensis Rev1, in which the prosamine synthetize coding sequence from the O-PS region has been replaced with kanR coding sequence, via the replacement of kanR with GFP reporter encoding sequence by using homologous recombination. The syntactic deletion cassette, containing the GFP encoding sequence flanked with the upstream and downstream sequences of kanR encoding sequence from Rev1 mutant strain, was cloned into pBluescriptIISK(-) vector by SacI and KpnI, and the suicide vector pBluescriptSK-∆kanR/gfp was electroporated into mutant strain. The constructs were confirmed by using PCR, restriction mapping and DNA sequencing. The loss of kanR function in transfected cells was confirmed by using the GFP phenotyping with flow cytometry and fluorescence microscopy, and following the phenotypic characterization of the mutant strain on selective media and the genomic DNA analysis with specific primers. This kanR-free Rev1 mutant strain lack the O-polysaccharide and have a reduced ability to enter and survive in the host cell. Therefore, it could be evaluated as a potential vaccine candidate if eliciting protective immunity.

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria.

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.

Loss of U1498 methylation in 16S rRNA by RsmE methyltransferase associates its role with aminoglycoside resistance in mycobacteria

ObjectivesModulation of methylation pattern through mutations in ribosomal methyltransferases is a key mechanism of bacterial drug resistance. However, RsmG (GidB), which specifically methylates G527 in 16S rRNA, remains the only conserved methyltransferase known to be associated with low-level drug resistance in mycobacterial isolates. The mycobacterial RsmE homologue methylates U1498 in 16S rRNA in a highly specific manner. U1498 lies in the vicinity of the binding site for various aminoglycosides in the ribosome. However, the association of methylation at U1498 with altered drug response remains poorly understood. MethodsA deletion mutant of the RsmE homologue in Mycobacterium smegmatis was generated by a suicidal vector strategy and drug susceptibility assays were performed on wild-type, knockout and complemented strains with varying concentrations of ribosomal- and non-ribosomal-targeting drugs. ResultsDeletion of the RsmE homologue of M. smegmatis led to an at least two-fold increase in the minimum inhibitory concentrations (MICs) of aminoglycosides that bind in the decoding centre proximal to U1498 in the 30S subunit. The change in MICs was highly specific and reproducible and did not show any cross-resistance to other drug classes. Surprisingly, Rv2372c, the RsmE homologue of Mycobacterium tuberculosis, has the largest number of mutations among conserved ribosomal methyltransferases, after gidB, highlighting the role of mutations in RsmE methyltransferase as a key emerging mechanism of resistance in clinical strains. ConclusionWe present the first evidence of an association of methylation of U1498 in 16S rRNA with development of low-level resistance in mycobacteria that must be tackled in a timely manner.

A Glutamine Insertion at Codon 432 of RpoB Confers Rifampicin Resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is an infectious respiratory disease caused by Mycobacterium tuberculosis and one of the top 10 causes of death worldwide. Treating TB is challenging; successful treatment requires a long course of multiple antibiotics. Rifampicin (RIF) is a first-line drug for treating TB, and the development of RIF-resistant M. tuberculosis makes treatment even more difficult. To determine the mechanism of RIF resistance in these strains, we searched for novel mutations by sequencing. Four isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and unique mutations encoding RpoB G158R, RpoB V168A, RpoB S188P, and RpoB Q432insQ, respectively. To evaluate their correlation with RIF resistance, plasmids carrying rpoB genes encoding these mutant proteins were transfected into the H37Rv reference strain. The plasmid complementation of RpoB indicated that G158R, V168A, and S188P did not affect the MIC of RIF. However, the MIC of RIF was increased in H37Rv carrying RpoB Q432insQ. To confirm the correlation between RIF resistance and Q432insQ, we cloned an rpoB fragment carrying the insertion (encoding RpoB Q432insQ) into H37Rv by homologous recombination using a suicide vector. All replacement mutants expressing RpoB Q432insQ were resistant to RIF (MIC > 1 mg/L). These results indicate that RpoB Q432insQ causes RIF resistance in M. tuberculosis.

Development of a Method for Simultaneous Generation of Multiple Genetic Modification in Salmonella enterica Serovar Typhimurium

To comprehensively analyze bacterial gene function, it is important to simultaneously generate multiple genetic modifications within the target gene. However, current genetic engineering approaches, which mainly use suicide vector- or λ red homologous recombination-based systems, are tedious and technically difficult to perform. Here, we developed a flexible and easy method to simultaneously construct multiple modifications at the same locus on the Salmonella enterica serovar Typhimurium chromosome. The method combines an efficient seamless assembly system in vitro, red homologous recombination in vivo, and counterselection marker sacB. To test this method, with the seamless assembly system, various modification fragments for target genes cpxR, cpxA, and acrB were rapidly and efficiently constructed in vitro. sacBKan cassettes generated via polymerase chain reaction were inserted into the target loci in the genome of Salmonella Typhimurium strain CVCC541. The resulting pKD46-containing kanamycin-resistant recombinants were selected and used as intermediate strains. Multiple target gene modifications were then carried out simultaneously via allelic exchange using various homologous recombinogenic DNA fragments to replace the sacBKan cassettes in the chromosomes of the intermediate strains. Using this method, we successfully carried out site-directed mutagenesis, seamless deletion, and 3 × FLAG tagging of the target genes. This method can be used in any bacterial species that supports sacB gene activity and λ red-mediated recombination, allowing in-depth functional analysis of bacterial genes.

Efficient dual-negative selection for bacterial genome editing

BackgroundGene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering.ResultsHere, we established a time-effective variant of consecutive single-crossovers. This method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in-frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Adapted versions enabled efficient gene editing also in Pseudomonas aeruginosa and multi-drug resistant (MDR) Escherichia coli clinical isolates.ConclusionsOur method is time-effective and allows facile manipulation of multiple bacterial species including MDR clinical isolates. We anticipate that this method might be broadly applicable to additional bacterial species, including those for which recombineering has been difficult to implement.