Plasmid Transformation Research Articles

Effects of different resistance mechanisms on susceptibility to different classes of antibiotics in Klebsiella pneumoniae strains: a strategic system for the screening and activity testing of new antibiotics.

A strategic system for the screening and testing of new antibiotics was created to facilitate the development of antibiotics that are robustly effective against MDR bacteria. In-frame deletion, site-directed mutagenesis and plasmid transformation were used to generate genetically engineered strains with various resistance mechanisms from a fully susceptible clinical isolate of Klebsiella pneumoniae. Antimicrobial susceptibility testing and a mouse infection model were used to test antibiotics against these strains in vitro and in vivo, respectively. A total of 193 strains, including 29 strains with chromosome-mediated resistance, 33 strains with plasmid-mediated resistance and 131 strains with a combination of both resistance mechanisms were constructed; these strains covered resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, folate pathway inhibitors and other antibiotics. MICs for all strains were tested, and the effects of genetic modifications on increasing the MICs were assessed. Ceftazidime and cefotaxime were used to assess the correlation between antibacterial activities in vitro and in vivo. Against a K. pneumoniae strain with blaOXA-48, ceftazidime had a lower MIC (0.5 mg/L) than cefotaxime (2 mg/L). Ceftazidime had an ED50 of 30 mg/kg, and no mice survived treatment with the same dose of cefotaxime. A positive correlation was observed between these in vitro and in vivo results. The system developed here could reduce the considerable time required to evaluate the effectiveness of new antibiotics against MDR bacteria, particularly in the early stages of drug development. This system could also be expanded as new resistance mechanisms emerge.

Formulation of chitosan with the polyepitope HIV-1 protein candidate vaccine efficiently boosts cellular immune responses in mice.

Human immunodeficiency virus-1 (HIV-1) continues to be a major global public health issue and priority. Despite the variety of antiretroviral therapies, it seems that an effective vaccine against HIV-1 is still very necessary. An ideal HIV-1 vaccine should be able to elicit both humoral and cellular immunities. In this respect, polyepitope vaccines, incorporated from several conserved regions of HIV-1 proteins, have received much attention recently. Herein, the immunogenicity of the HIV-1 polyepitope protein-based candidate vaccines was evaluated in BALB/c mice. Following the plasmid (pET23a-HIV-1-tat/pol/gag/env) preparation and transformation, the recombinant protein expression was optimized in Escherichia coli BL21 (DE3) host cells. After the HIV-1-top4 protein purification, chitosan and alum adjuvants were added to the vaccines formulations to reinforce the immunogenicity of the candidate vaccines. Mice were subcutaneously immunized three times at 2-week intervals with the candidate vaccines and the elicitation of both humoral and cellular immune responses were investigated. Taken together, the results showed that chitosan adjuvanted candidate vaccine conferred a stronger immunogenicity and elicited higher cellular responses than other candidate vaccines (P<0.05). Thereby, it seems that co-utilizing of potent adjuvants with the HIV-1 polyepitope protein vaccines can help to open new avenues for strategies for HIV/AIDS vaccine design.

Complete Genome Sequence of Campylobacter jejuni subsp. jejuni ATCC 35925.

ABSTRACTHere, we report the complete genome sequence of Campylobacter jejuni ATCC 35925, an avian isolate from Sweden. The genome gives insight into the ATCC 35925 strain’s remarkable ability to tolerate copper and its permissiveness to plasmid transformation.

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis.

Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a Δdcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections.IMPORTANCE Staphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen.

Clonal Dissemination of OXA-232 Carbapenemase-Producing Klebsiella pneumoniae in Neonates.

Five OXA-232 carbapenemase-producing Klebsiella pneumoniae isolates, belonging to the pandemic clone sequence type 15 (ST15), were isolated from neonates and coproduced blaCTX-M-15 and blaSHV-1 genes. All isolates were resistant to ertapenem (MICs of >32 μg/ml) and meropenem (MICs of 4 to 8 μg/ml) and susceptible or intermediate to imipenem (MICs of 1 to 2 μg/ml). The blaOXA-232 gene was located on a ColE-type transformable plasmid of 6,141 bp. To the best of our knowledge, this is the first report of OXA-232 carbapenemase among clinical isolates in China.

Bacillus subtilis RecA with DprA-SsbA antagonizes RecX function during natural transformation.

Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA–SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA–SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA–SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA–SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA–SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.

Emergence of a multidrug-resistant Citrobacter freundii ST8 harboring an unusual VIM-4 gene cassette in Poland

ObjectivesThe growing incidence of multidrug-resistant (MDR) bacteria is an emerging challenge in modern medicine. The utility of carbapenems, which are considered ‘last-line’ agents, is being diminished by the growing incidence of various resistance mechanisms in Gram-negative bacteria. A molecular investigation was performed of an MDR carbapenem-resistant Citrobacter freundii of sequence type 8 (ST8) isolated from a hematology patient with acute myeloid leukemia. MethodsMultilocus sequence typing and analysis of the nucleotide sequence of the class I integron were performed using PCR and Sanger sequencing. Transformation of the resistance plasmid isolated following the alkaline lysis method was performed using chemically competent E. coli TOP10. ResultsMolecular analysis of the carbapenem-resistant C. freundii revealed the presence of the VIM-4 isoenzyme located on the ∼55-kb transferable resistance plasmid. Interestingly, the blaVIM-4 gene was inserted into an unusual gene cassette containing a 169-bp direct repeat of the 3′ segment of the blaVIM-4 gene. ConclusionsAll unusual gene cassettes containing VIM-DR (direct repeat) described thus far have been harbored by non-fermenters, i.e., Acinetobacter and Pseudomonas, underscoring the importance of resistance determinant mobility, which may go even beyond genus, family, and order boundaries. Great efforts need to be taken to explore pathways of resistance to ‘last-resort’ antimicrobials, especially among clinically relevant pathogens.

An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1

BackgroundPerennial ryegrass (Lolium perenne L.) is an important temperate grass used for turf and forage purposes. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient protoplast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system.ResultsIn this report, a highly efficient protoplast isolation (5.6 × 107 protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein–protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time.ConclusionThis efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcellular localization and protein–protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass.

Anti-Restriction Protein, KlcAHS, Promotes Dissemination of Carbapenem Resistance.

Carbapenemase-producing Klebsiella pneumoniae (KPC) has emerged and spread throughout the world. A retrospective analysis was performed on carbapenem-resistant K. pneumoniae isolated at our teaching hospital during the period 2009–2010, when the initial outbreak occurred. To determine the mechanism(s) that underlies the increased infectivity exhibited by KPC, Multilocus Sequence Typing (MLST) was conducted. A series of plasmids was also extracted, sequenced and analyzed. Concurrently, the complete sequences of blaKPC−2-harboring plasmids deposited in GenBank were summarized and aligned. The blaKPC−2 and KlcAHS genes in the carbapenem-resistant K. pneumoniae isolates were examined. E. coli strains, carrying different Type I Restriction and Modification (RM) systems, were selected to study the interaction between RM systems, anti-RM systems and horizontal gene transfer (HGT). The ST11 clone predominated among 102 carbapenem-resistant K. pneumoniae isolates, all harbored the blaKPC−2 gene; 98% contained the KlcAHS gene. KlcAHS was one of the core genes in the backbone region of most blaKPC−2 carrying plasmids. Type I RM systems in the host bacteria reduced the rate of pHS10842 plasmid transformation by 30- to 40-fold. Presence of the anti-restriction protein, KlcAHS, on the other hand, increased transformation efficiency by 3- to 6-fold. These results indicate that RM systems can significantly restrict HGT. In contrast, KlcAHS can disrupt the RM systems and promote HGT by transformation. These findings suggest that the anti-restriction protein, KlcAHS, represents a novel mechanism that facilitates the increased transfer of blaKPC-2 and KlcAHS-carrying plasmids among K. pneumoniae strains.

Occurrence of blaDHA-1 mediated cephalosporin resistance in Escherichia coli and their transcriptional response against cephalosporin stress: a report from India

BackgroundTreatment alternatives for DHA-1 harboring strains are challenging as it confers resistance to broad spectrum cephalosporins and may further limit treatment option when expressed at higher levels. Therefore, this study was designed to know the prevalence of DHA genes and analyse the transcription level of DHA-1 against different β-lactam stress.MethodsScreening of AmpC β-lactamase phenotypically by modified three dimensional extract method followed by Antimicrobial Susceptibility and MIC determination. Genotyping screening of β-lactamase genes was performed by PCR assay followed by their sequencing. The blaDHA-1 transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of blaDHA gene was performed by transformation and conjugation and plasmid incompatibility typing, DNA fingerprinting by enterobacterial repetitive intergenic consensus sequences PCR.Results16 DHA-1 genes were screened positive from 176 Escherichia coli isolates and primer extension analysis showed a significant increase in DHA-1 mRNA transcription in response to cefotaxime at 8 µg/ml (6.99 × 102 fold), ceftriaxone at 2 µg/ml (2.63 × 103 fold), ceftazidime at 8 µg/ml (7.06 × 103 fold) and cefoxitin at 4 µg/ml (3.60 × 104 fold) when compared with untreated strain. These transcription data were found significant when analyzed statistically using one way ANOVA. Four different ESBL genes were detected in 10 isolates which include CTX-M (n = 6), SHV (n = 4), TEM (n = 3) and OXA-10 (n = 1), whereas, carbapenemase gene (NDM) was detected only in one isolate. Other plasmid mediated AmpC β-lactamases CIT (n = 9), EBC (n = 2) were detected in nine isolates. All DHA-1 genes detected were encoded in plasmid and incompatibility typing from the transformants indicated that the plasmid encoding blaDHA-1 was carried mostly by the FIA and L/M Inc group.ConclusionThis study demonstrates the prevalence of DHA-1 gene in this region and highlights high transcription of DHA-1 when induced with different β-lactam antibiotics. Therefore, cephalosporin treatment must be restricted for the patients infected with pathogen expressing this resistance determinant.

Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation.

ABSTRACTBacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub “superspreaders,” releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications.

Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint

Toxic, mutagenic and carcinogenic heavy metals from tannery industries cause the pollution of agricultural environment and natural water sources. This study aims to isolate, investigate and identify naturally occurring bacteria capable of reducing and detoxifying heavy metals (Chromium, Cadmium and Lead) from tannery effluent. Three isolates were identified up to genus level based on their morphological, cultural, physiological and biochemical characteristics as Gemella sp., Micrococcus sp. and Hafnia sp. Among them Gemella sp. and Micrococcus sp. showed resistance to Lead (Pb), chromium (Cr) and cadmium (Cd), where Hafnia sp. showed sensitivity to cadmium (Cd). All isolates showed different MICs against the above heavy metals at different levels. Degrading potentiality was assessed using Atomic Absorption Spectrophotometer where Gemella sp. and Micrococcus sp. showed 55.16±0.06% and 36.55±0.01% reduction of Pb respectively. On the other hand, moderate degradation of Cd was shown by Gemella sp. (50.99±0.01%) and Micrococcus sp. (38.64±0.06%). Heavy metals degradation capacity of Gemella sp. and Micrococcus sp. might be plasmid mediated, which might be used for plasmid transformation to transfer heavy metal accumulation capability. Therefore, identification of three bacteria for their heavy metal resistance and biodegradation capacity might be a base study to develop the production of potential local bioremediation agents in toxic tannery effluent treatment technology.

Production of Protein Kinases in E. coli.

Recombinant protein expression is widely used to generate milligram quantities of protein kinases for crystallographic, enzymatic, or other biophysical assays in vitro. Expression in E. coli is fast, cheap, and reliable. Here I present a detailed protocol for the production of human Aurora-A kinase. I begin with transformation of a suitable plasmid into an expression strain of E. coli, followed by growth and harvesting of bacterial cell cultures. Finally, I describe the purification of Aurora-A to homogeneity using immobilized metal affinity and size exclusion chromatographies.

Visible Light-Responsive Platinum-Containing Titania Nanoparticle-Mediated Photocatalysis Induces Nucleotide Insertion, Deletion and Substitution Mutations.

Conventional photocatalysts are primarily stimulated using ultraviolet (UV) light to elicit reactive oxygen species and have wide applications in environmental and energy fields, including self-cleaning surfaces and sterilization. Because UV illumination is hazardous to humans, visible light-responsive photocatalysts (VLRPs) were discovered and are now applied to increase photocatalysis. However, fundamental questions regarding the ability of VLRPs to trigger DNA mutations and the mutation types it elicits remain elusive. Here, through plasmid transformation and β-galactosidase α-complementation analyses, we observed that visible light-responsive platinum-containing titania (TiO2) nanoparticle (NP)-mediated photocatalysis considerably reduces the number of Escherichia coli transformants. This suggests that such photocatalytic reactions cause DNA damage. DNA sequencing results demonstrated that the DNA damage comprises three mutation types, namely nucleotide insertion, deletion and substitution; this is the first study to report the types of mutations occurring after photocatalysis by TiO2-VLRPs. Our results may facilitate the development and appropriate use of new-generation TiO2 NPs for biomedical applications.

The involvement of the ceramide transporter in Alzheimer’s disease Investigating the binding properties of the ceramide transporter in the human brain by optimizing the splitubiquitin Yeast Two-Hybrid system

Background: Alzheimer’s disease (AD) is the most common form of dementia, characterized by aggregates of Amyloid-ß (Aβ) peptides. The ceramide transporter (CERTL ) exists in a complex with serum amyloid P component in the Aβ plaques of AD patients. The goal is to obtain more information about the protein binding activity of CERTL in the human brain. To be able to do so, the Yeast Two-Hybrid (Y2H) technique, that was used to confirm the results from a previously executed CERTL Y2H screening, had to be optimized. Technical problems were encountered with the plasmid purification of the yeast NMY51 using the EZNA plasmid DNA minikit I and with the transformation in competent E.coli cells. Initially it was believed that there was a problem with the EZNA plasmid DNA minikit I. In this report troubleshooting was performed to identify the problem of the plasmid purification technique that has been used. Methods: To examine the protein binding activity of CERTL a split-ubiquitin Y2H system was used. In this system proteins are attached to two parts of one complete ubiquitin molecule, called the split-ubiquitin. The middle region domain of CERTL (the bait) was attached to the C-terminal half of the split-ubiquitin and the transcription factor LexA-VP16. The possible interactor, a prey library containing human brain proteins was fused to the N-terminal half of the split-ubiquitin. The presence of an interaction between bait and prey will results in the re-assembly of the split-ubiquitin, which activates ubiquitin proteases. The transcription factor is released and reporter genes are activated.The results of a previously executed Y2H screening had to be confirmed by performing another Y2H screening. The new screening had to be carried out using glycerol stocks of the yeast NMY51, containing both bait and prey, from the earlier performed Y2H screening. To optimize prey plasmid purification from the yeast using the EZNA plasmid DNA minikit I, troubleshooting was performed on the technique. Next to this, other methods to isolate the plasmid DNA, a QIAprep spin miniprep kit protocol and a phenol extraction and ethanol precipitation, were performed. The failing results were possibly due to the yeast being stored as a glycerol stock. In order to correct for this, a freshly transformed yeast sample was made. Results: Extracting the plasmid from glycerol stocks from the yeast NMY51 did not succeed. Plasmid purification and transformation of the prey plasmid from the fresh transformed yeast in E.coli competent cells was successful. Conclusion: From the results it is now believed that storing the yeast NMY51 containing the prey plasmid in glycerol stocks causes the prey plasmid to integrate into the genomic DNA of the yeast. Performing a new Y2H screening would therefore only be possible on fresh transformed yeast NMY51 cells.

Natural Escherichia coli strains undergo cell-to-cell plasmid transformation

Horizontal gene transfer is a strong tool that allows bacteria to adapt to various environments. Although three conventional mechanisms of horizontal gene transfer (transformation, transduction, and conjugation) are well known, new variations of these mechanisms have also been observed. We recently reported that DNase-sensitive cell-to-cell transfer of nonconjugative plasmids occurs between laboratory strains of Escherichia coli in co-culture. We termed this phenomenon “cell-to-cell transformation.” In this report, we found that several combinations of Escherichia coli collection of reference (ECOR) strains, which were co-cultured in liquid media, resulted in DNase-sensitive cell-to-cell transfer of antibiotic resistance genes. Plasmid isolation of these new transformants demonstrated cell-to-cell plasmid transfer between the ECOR strains. Natural transformation experiments, using a combination of purified plasmid DNA and the same ECOR strains, revealed that cell-to-cell transformation occurs much more frequently than natural transformation under the same culture conditions. Thus, cell-to-cell transformation is both unique and effective. In conclusion, this study is the first to demonstrate cell-to-cell plasmid transformation in natural E. coli strains.

Development of a genetically engineered Escherichia coli strain for plasmid transformation in Corynebacterium glutamicum

Gene disruption and replacement in Corynebacterium glutamicum is dependent upon a high transformation efficiency. The cglIR-cgIIR restriction system is a major barrier to introduction of foreign DNA into Corynebacterium glutamicum cells. To improve the transformation efficiency of C. glutamicum, the cglIM gene encoding methyltransferase in the cglIR-cglIIR-cglIM restriction-modification system of C. glutamicum ATCC 13032 was chromosomally integrated and expressed in Escherichia coli, resulting in an engineered strain E. coli AU1. The electro-transformation experiments of C. glutamicum ATCC 13032 with the E. coli-C. glutamicum shuttle plasmid pAU4 showed that the transformation efficiency of C. glutamicum with pAU4 DNA extracted from E. coli TG1/pAU4 was 1.80±0.21×102cfu/μg plasmid DNA, while using pAU4 DNA extracted from E. coli AU1/pAU4, the transformation efficiency reached up to 5.22±0.33×106cfu/μg plasmid DNA. The results demonstrated that E. coli AU1 is able to confer the cglIM-specific DNA methylation pattern to its resident plasmid, which makes the plasmid resistant to the cglIR-cglIIR restriction and efficiently transferred into C. glutamicum. E. coli AU1 is a useful intermediate host for efficient transformation of C. glutamicum.

Use of a fluoride channel as a new selection marker for fission yeast plasmids and application to fast genome editing with CRISPR/Cas9.

Fission yeast is a powerful model organism that has provided insights into important cellular processes thanks to the ease of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in Schizosaccharomyces pombe. In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe. Copyright © 2016 John Wiley & Sons, Ltd.

High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP+ to NAD+. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD+, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

Imipenem represses CRISPR-Cas interference of DNA acquisition through H-NS stimulation in Klebsiella pneumoniae.

Analysis of the genome of Klebsiella pneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5′-TTN-3′ was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem.