RASGAP - Word Related Documents




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100.8381Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria. Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-nptII-TtrpA) and mTn5-Gm (containing Ptac/PaacCI-aacCI-TtrpA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-Ptac-nptII-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCI-aacCI-TtrpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other gram-negative bacteria.199910411257
977310.8325Cross resistance emergence to polymyxins in Acinetobacter baumannii exposed in vitro to an antimicrobial peptide. Multidrug-resistant bacteria are a growing public health concern. Antimicrobial peptides (AMPs) are proposed alternatives to classical antibiotics towards infections caused by resistant bacteria. TAT-RasGAP(317-326) is an AMP able to target Gram-negative bacteria and is especially efficient towards Acinetobacter baumannii. In this study, we performed in vitro resistance selection on several A. baumannii strains, in order to determine to which extent these bacteria can develop resistance to TAT-RasGAP(317-326). A. baumannii rapidly developed resistance to TAT-RasGAP(317-326) and subsequently, in approximately half of the cases, cross-resistance to last-resort polypeptidic antibiotics polymyxins. Cross-resistant isolates predominantly bore mutations in the pmrAB operon, involved in modulation of lipopolysaccharides' charge at the bacterial surface, similarly to polymyxin-resistant clinical isolates. We thus show here that contact of A. baumannii with an AMP structurally different from polymyxins can induce unexpected cross-resistance towards them. This indicates that precautions must be taken for the clinical application of AMPs.202540442488
999520.8316Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon. Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect β-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5α. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5α was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization.201020729070
54130.8306A Teleost Bactericidal Permeability-Increasing Protein Kills Gram-Negative Bacteria, Modulates Innate Immune Response, and Enhances Resistance against Bacterial and Viral Infection. Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense.201627105425
74640.8300Novel antimicrobial 3-phenyl-4-phenoxypyrazole derivatives target cell wall lipid intermediates with low mammalian cytotoxicity. The growing crisis of antimicrobial resistance (AMR) underscores the critical need for innovative antimicrobial discoveries. Novel antibiotics targeting the bacterial cell wall remain an attractive area of research, due to their conservation and essentiality in bacteria and their absence in eukaryotic cells. Antibiotics targeting lipid II are of special interest due to the reduced potential for target modification of lipid components and their surface accessibility to inhibitors. In this study, we identified 3-phenyl-4-phenoxypyrazole analogues named PYO12 and PYO12a with bactericidal activity against gram-positive bacteria and low cytotoxicity for different types of mammalian cells. Gram-negative bacteria were resistant to PYO12 activity through extrusion of this compound via efflux pumps. Exposure to PYO12 induces expression of genes involved in resistance to antimicrobials targeting the cell wall, suggesting that PYO12 acts via binding to lipid II or other lipid intermediates involved in peptidoglycan or teichoic acid biosynthesis. Antagonism of PYO12 antibacterial activity by undecaprenyl-pyrophosphate supports the idea that PYO12 may bind to the lipid moiety of lipid II blocking the shuttling of peptidoglycan precursors across the cytoplasmic membrane. These findings open opportunities to further develop these compounds as antibiotics targeting bacterial cell wall synthesis.202541083642
600850.8300Photopolymerized keratin-PGLa hydrogels for antibiotic resistance reversal and enhancement of infectious wound healing. Infectious wounds have become serious challenges for both treatment and management in clinical practice, so development of new antibiotics has been considered an increasingly difficult task. Here, we report the design and synthesis of keratin 31 (K31)-peptide glycine-leucine-amide (PGLa) photopolymerized hydrogels to rescue the antibiotic activity of antibiotics for infectious wound healing promotion. K31-PGLa displayed an outstanding synergistic effect with commercial antibiotics against drug-resistant bacteria by down-regulating the synthesis genes of efflux pump. Furthermore, the photopolymerized K31-PGLa/PEGDA hydrogels effectively suppressed drug-resistant bacteria growth and enhanced skin wound closure in murine. This study provided a promising alternative strategy for infectious wound treatment.202337810750
32960.8299Effect of NlpE overproduction on multidrug resistance in Escherichia coli. NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In this study, we report that overproduction of NlpE increases multidrug and copper resistance through activation of the genes encoding the AcrD and MdtABC multidrug efflux pumps in Escherichia coli.201020211889
843470.8299A potent and selective antimicrobial poly(amidoamine) dendrimer conjugate with LED209 targeting QseC receptor to inhibit the virulence genes of gram negative bacteria. The pandemic of multidrug-resistant Gram negative bacteria (GNB) is a worldwide healthcare concern, and very few antibiotics are being explored to match the clinical challenge. Recently, amino-terminated poly(amidoamine) (PAMAM) dendrimers have shown potential to function as broad antimicrobial agents. However, PAMAM displays a generation dependent cytotoxicity to mammalian cells and low selectivity on bacterial cells, which limits PAMAM to be developed as an antibacterial agent for systemic administration. We conjugated G3 PAMAM with LED209, a specific inhibitor of quorum sensor QseC of GNB, to generate a multifunctional agent PAMAM-LED209. Intriguingly, PAMAM-LED209 showed higher selectivity on GNB and lower cytotoxicity to mammalian cells, yet remained strong antibacterial activity. PAMAM-LED209 also inhibited virulence gene expression of GNB, and did not induce antibiotic-resistance. The present work firstly demonstrated that PAMAM-LED209 conjugate had a highly selective anti-GNB activity and low cytotoxicity, which offered a feasible strategy for combating multidrug-resistant GNB infections. FROM THE CLINICAL EDITOR: This research team demonstrated that a novel PAMAM-LED209 conjugate had highly selective activity against Gram-negative bacteria, coupled with low cytotoxicity, offering a potential strategy for combating multidrug-resistant infections.201525461286
10980.8297Identification of two putative ATP-cassette genes in Encephalitozoon intestinalis. Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis. It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica. The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene. We report here two complete sequences EiABC1 and EiABC2, encoding different ATP-binding cassette genes from E. intestinalis, including a P-gp.200111730796
53290.8295Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.199910514571
6363100.8292The effect of tetronasin and monensin on fermentation, microbial numbers and the development of ionophore-resistant bacteria in the rumen. The Gram-negative rumen bacteria Fibrobacter succinogenes S85, Prevotella ruminicola M384 and Veillonella parvula L59 were grown in media containing successively increasing concentrations of the ionophores, monensin and tetronasin. All three species became more resistant to the ionophore with which they were grown. Increased resistance to one ionophore caused increased resistance to the other, and cross-resistance to another ionophore--lasalocid--and an antibiotic--avoparcin. Recovery of tetronasin-resistant bacteria from the rumen of monensin-fed sheep increased and vice versa, indicating that similar cross-resistance occurred in vivo.19938407673
368110.8288Construction and complementation of in-frame deletions of the essential Escherichia coli thymidylate kinase gene. This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the kanamycin resistance gene kka1, encoding amino glycoside 3'-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.200616461678
393120.8287Antibiotic marker modifications of lambda Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains. The Red recombinase system of bacteriophage Lambda has been used to inactivate chromosomal genes in bacteria using PCR products. In this study, we describe the replacement of the ampicillin resistance marker of helper plasmids pKD46 and pCP20 by a gentamicin resistance gene to disrupt chromosomal genes and then to eliminate FRT flanked resistance gene in multiple antibiotic-resistant Salmonella enterica strains.200818619499
115130.8286Genome mining reveals uncommon alkylpyrones as type III PKS products from myxobacteria. Type III polyketide synthases (PKSs) are comparatively small homodimeric enzymes affording natural products with diverse structures and functions. While type III PKS biosynthetic pathways have been studied thoroughly in plants, their counterparts from bacteria and fungi are to date scarcely characterized. This gap is exemplified by myxobacteria from which no type III PKS-derived small molecule has previously been isolated. In this study, we conducted a genomic survey of myxobacterial type III PKSs and report the identification of uncommon alkylpyrones as the products of type III PKS biosynthesis from the myxobacterial model strain Myxococcus xanthus DK1622 through a self-resistance-guided screening approach focusing on genes encoding pentapetide repeat proteins, proficient to confer resistance to topoisomerase inhibitors. Using promoter-induced gene expression in the native host as well as heterologous expression of biosynthetic type III PKS genes, sufficient amounts of material could be obtained for structural elucidation and bioactivity testing, revealing potent topoisomerase activity in vitro.201930506464
650140.8283Lipoplexes to Deliver Oligonucleotides in Gram-Positive and Gram-Negative Bacteria: Towards Treatment of Blood Infections. Bacterial resistance to antibiotics threatens the ability to treat life-threatening bloodstream infections. Oligonucleotides (ONs) composed of nucleic acid mimics (NAMs) able to inhibit essential genes can become an alternative to traditional antibiotics, as long as they are safely transported in human serum upon intravenous administration and they are carried across the multilayered bacterial envelopes, impermeable to ONs. In this study, fusogenic liposomes were considered to transport the ONs and promote their internalization in clinically relevant bacteria. Locked nucleic acids and 2'-OMethyl RNA were evaluated as model NAMs and formulated into DOTAP-DOPE liposomes followed by post-PEGylation. Our data showed a complexation stability between the post-PEGylated liposomes and the ONs of over 82%, during 24 h in native human serum, as determined by fluorescence correlation spectroscopy. Quantification by a lipid-mixing assay showed that liposomes, with and without post-PEGylation, fused with all bacteria tested. Such fusion promoted the delivery of a fraction of the ONs into the bacterial cytosol, as observed by fluorescence in situ hybridization and bacterial fractionation. In short, we demonstrated for the first time that liposomes can safely transport ONs in human serum and intracellularly deliver them in both Gram-negative and -positive bacteria, which holds promise towards the treatment of bloodstream infections.202134210111
225150.8282Mechanisms of bactericidal action and resistance of polymyxins for Gram-positive bacteria. Polymyxins are cationic antimicrobial peptides used as the last-line therapy to treat multidrug-resistant Gram-negative bacterial infections. The bactericidal activity of polymyxins against Gram-negative bacteria relies on the electrostatic interaction between the positively charged polymyxins and the negatively charged lipid A of lipopolysaccharide (LPS). Given that Gram-positive bacteria lack an LPS-containing outer membrane, it is generally acknowledged that polymyxins are less active against Gram-positive bacteria. However, Gram-positive bacteria produce negatively charged teichoic acids, which may act as the target of polymyxins. More and more studies suggest that polymyxins have potential as a treatment for Gram-positive bacterial infection. This mini-review discusses recent advances in the mechanism of the antibacterial activity and resistance of polymyxins in Gram-positive bacteria.Key Points• Teichoic acids play a key role in the action of polymyxins on Gram-positive bacteria.• Polymyxin kills Gram-positive bacteria by disrupting cell surface and oxidative damage.• Modification of teichoic acids and phospholipids contributes to polymyxin resistance in Gram-positive bacteria.• Polymyxins have potential as a treatment for Gram-positive bacterial infection.202032157424
534160.8280Plasmid shuttle vector with two insertionally inactivable markers for coryneform bacteria. A new shuttle vector pCEM500 replicating in Escherichia coli and in Brevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Kmr/Gmr from plasmid pSa of Gram-negative bacteria and Smr/Spr from plasmid pCG4 of Corynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained in B. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.19902148164
506170.8277A kiss of death--proteasome-mediated membrane fusion and programmed cell death in plant defense against bacterial infection. Eukaryotes have evolved various means for controlled and organized cellular destruction, known as programmed cell death (PCD). In plants, PCD is a crucial regulatory mechanism in multiple physiological processes, including terminal differentiation, senescence, and disease resistance. In this issue of Genes & Development, Hatsugai and colleagues (pp. 2496-2506) demonstrate a novel plant defense strategy to trigger bacteria-induced PCD, involving proteasome-dependent tonoplast and plasma membrane fusion followed by discharge of vacuolar antimicrobial and death-inducing contents into the apoplast.200919884251
355180.8277Evolution of multiple-antibiotic-resistance plasmids mediated by transposable plasmid deoxyribonucleic acid sequences. Two plasmid deoxyribonucleic acid sequences mediating multiple antibiotic resistance transposed in vivo between coexisting plasmids in clinical isolates of Serratia marcescens. This event resulted in the evolution of a transferable multiresistance plasmid. Both sequences, designated in Tn1699 and Tn1700, were flanked by inverted deoxyribonucleic acid repetitions and could transpose between replicons independently of the Excherichia coli recA gene function. Tn1699 and Tn1700 mediated ampicillin, carbenicillin, kanamycin, and gentamicin resistance but differed in the type of gentamicin-acetyltransferase enzymes that they encoded. The structural genes for these enzymes share a great deal of polynucleotide sequence similarity despite their phenotypic differences. The transposition of Tn1699 and Tn1700 to coresident transferable plasmids has contributed to the dissemination of antibiotic resistance among other gram-negative bacteria. These organisms have recently caused nosocomial infections in epidemic proportions.1979387747
119190.8277Heterologous Expression Reveals Ancient Properties of Tei3—A VanS Ortholog from the Teicoplanin Producer Actinoplanes teichomyceticus. Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticus—the producer of the last-resort-drug teicoplanin—has been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein combining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A. teichomyceticus was hereby proposed.202236555354