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999500.9432Direct 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
786810.9364A double-quenching paperclip ECL biosensing platform for ultrasensitive detection of antibiotic resistance genes (mecA) based on Ti(3)C(2) MXene-Au NPs as a coreactant accelerator. The global spread of environmental biological pollutants, such as antibiotic-resistant bacteria and their antibiotic resistance genes (ARGs), has emerged as a critical public health concern. It is imperative to address this pressing issue due to its potential implications for public health. Herein, a DNA paperclip probe with double-quenching function of target cyclic cleavage was proposed, and an electrochemiluminescence (ECL) biosensing platform was constructed using Ti(3)C(2) MXene in-situ reduction growth of Au NPs (TCM-Au) as a coreactant accelerator, and applied to the sensitive detection of ARGs. Thanks to the excellent catalytic performance, large surface area and Au-S affinity of TCM-Au, the ECL performance of CdS QDs have been significantly improved. By cleverly utilizing the negative charge of the paperclip nucleic acid probe and its modification group, double-quenching of the ECL signal was achieved. This innovative approach, combined with target cyclic amplification, facilitated specific and sensitive detection of the mecA gene. This biosensing platform manifested highly selective and sensitive determination of mecA genes in the range of 10 fM to 100 nM and a low detection limit of 2.7 fM. The credible detectability and anti-interference were demonstrated in Yangtze river and Aeration tank outlet, indicating its promising application toward pollution monitoring of ARGs.202337666010
871920.9353Genomics Insights into Pseudomonas sp. CG01: An Antarctic Cadmium-Resistant Strain Capable of Biosynthesizing CdS Nanoparticles Using Methionine as S-Source. Here, we present the draft genome sequence of Pseudomonas sp. GC01, a cadmium-resistant Antarctic bacterium capable of biosynthesizing CdS fluorescent nanoparticles (quantum dots, QDs) employing a unique mechanism involving the production of methanethiol (MeSH) from methionine (Met). To explore the molecular/metabolic components involved in QDs biosynthesis, we conducted a comparative genomic analysis, searching for the genes related to cadmium resistance and sulfur metabolic pathways. The genome of Pseudomonas sp. GC01 has a 4,706,645 bp size with a 58.61% G+C content. Pseudomonas sp. GC01 possesses five genes related to cadmium transport/resistance, with three P-type ATPases (cadA, zntA, and pbrA) involved in Cd-secretion that could contribute to the extracellular biosynthesis of CdS QDs. Furthermore, it exhibits genes involved in sulfate assimilation, cysteine/methionine synthesis, and volatile sulfur compounds catabolic pathways. Regarding MeSH production from Met, Pseudomonas sp. GC01 lacks the genes E4.4.1.11 and megL for MeSH generation. Interestingly, despite the absence of these genes, Pseudomonas sp. GC01 produces high levels of MeSH. This is probably associated with the metC gene that also produces MeSH from Met in bacteria. This work is the first report of the potential genes involved in Cd resistance, sulfur metabolism, and the process of MeSH-dependent CdS QDs bioproduction in Pseudomonas spp. strains.202133514061
130.9299Constructs 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
872140.9296Chromium metabolism characteristics of coexpression of ChrA and ChrT gene. OBJECTIVE: Serratia sp. S2 is a wild strain with chromium resistance and reduction ability. Chromium(VI) metabolic-protein-coding gene ChrA and ChrT were cloned from Serratia sp. S2, and ligated with prokaryotic expression vectors pET-28a (+) and transformed into E. coli BL21 to construct ChrA, ChrT and ChrAT engineered bacteria. By studying the characteristics of Cr(VI) metabolism in engineered bacteria, the function and mechanism of the sole expression and coexpression of ChrA and ChrT genes were studied. METHODS: Using Serratia sp. S2 genome as template, ChrA and ChrT genes were amplified by PCR, and prokaryotic expression vectors was ligated to form the recombinant plasmid pET-28a (+)-ChrA, pET-28a (+)-ChrT and pET-28a (+)-ChrAT, and transformed into E. coli BL21 to construct ChrA, ChrT, ChrAT engineered bacteria. The growth curve, tolerance, and reduction of Cr(VI), the distribution of intracellular and extracellular Cr, activity of chromium reductase and intracellular oxidative stress in engineered bacteria were measured to explore the metabolic characteristics of Cr(VI) in ChrA, ChrT, ChrAT engineered bacteria. RESULTS: ChrA, ChrT and ChrAT engineered bacteria were successfully constructed by gene recombination technology. The tolerance to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrA > ChrT > Control (P < 0.05), and the reduction ability to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrT > ChrA (P < 0.05). The chromium distribution experiments confirmed that Cr(VI) and Cr(III) were the main valence states. Effect of electron donors on chromium reductase activity was NADPH > NADH > non-NAD(P)H (P < 0.05). The activity of chromium reductase increased significantly with NAD(P)H (P < 0.05). The Glutathione and NPSH (Non-protein Sulfhydryl) levels of ChrA, ChrAT engineered bacteria increased significantly (P < 0.05) under the condition of Cr(VI), but there was no significant difference in the indexes of ChrT engineered bacteria (P > 0.05). CONCLUSION: ChrAT engineered bacteria possesses resistance and reduction abilities of Cr(VI). ChrA protein endows the strain with the ability to resist Cr(VI). ChrT protein reduces Cr(VI) to Cr(III) by using NAD(P)H as electronic donor. The reduction process promotes the production of GSH, GSSG and NPSH to maintain the intracellular reduction state, which further improves the Cr(VI) tolerance and reduction ability of ChrAT engineered bacteria.202032768747
80250.9295YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved in furfural tolerance. Previous results have demonstrated that the silencing of adjacent genes encoding NADPH-dependent furfural oxidoreductases (yqhD dkgA) is responsible for increased furfural tolerance in an E. coli strain EMFR9 [Miller et al., Appl Environ Microbiol 75:4315-4323, 2009]. This gene silencing is now reported to result from the spontaneous insertion of an IS10 into the coding region of yqhC, an upstream gene. YqhC shares homology with transcriptional regulators belonging to the AraC/XylS family and was shown to act as a positive regulator of the adjacent operon encoding YqhD and DkgA. Regulation was demonstrated by constructing a chromosomal deletion of yqhC, a firefly luciferase reporter plasmid for yqhC, and by a direct comparison of furfural resistance and NADPH-dependent furfural reductase activity. Closely related bacteria contain yqhC, yqhD, and dkgA orthologs in the same arrangement as in E. coli LY180. Orthologs of yqhC are also present in more distantly related Gram-negative bacteria. Disruption of yqhC offers a useful approach to increase furfural tolerance in bacteria.201120676725
787060.9287Hierarchical Bi(2)O(2)CO(3) wrapped with modified graphene oxide for adsorption-enhanced photocatalytic inactivation of antibiotic resistant bacteria and resistance genes. There is growing pressure for wastewater treatment plants to mitigate the discharge of antibiotic resistant bacteria (ARB) and extracellular resistance genes (eARGs), which requires technological innovation. Here, hierarchical Bi(2)O(2)CO(3) microspheres were wrapped with nitrogen-doped, reduced graphene oxide (NRGO) for enhanced inactivation of multidrug-resistant E. coli NDM-1 and degradation of the plasmid-encoded ARG (bla(NDM-1)) in secondary effluent. The NRGO shell enhanced reactive oxygen species (ROS) generation (•OH and H(2)O(2)) by about three-fold, which was ascribed to broadened light absorption region (red-shifted up to 459 nm) and decreased electron-transfer time (from 55.3 to 19.8 ns). Wrapping enhanced E. coli adsorption near photocatalytic sites to minimize ROS scavenging by background constituents, which contributed to the NRGO-wrapped microspheres significantly outperforming commercial TiO(2) photocatalyst. ROS scavenger tests indicated that wrapping also changed the primary inactivation pathway, with photogenerated electron holes and surface-attached hydroxyl radicals becoming the predominant oxidizing species with wrapped microspheres, versus free ROS (e.g., •OH, H(2)O(2) and •O(2)(-)) for bare microspheres. Formation of resistance plasmid-composited microsphere complexes, primary due to the π-π stacking and hydrogen bonding between the shell and nucleotides, also minimized ROS scavenging and kept free plasmid concentrations below 10(2) copies/mL. As proof-of-concept, this work offers promising insight into the utilization of NRGO-wrapped microspheres for mitigating antibiotic resistance propagation in the environment.202032679343
9270.9282Quantitative trait loci for partial resistance to Pseudomonas syringae pv. maculicola in Arabidopsis thaliana. Segregation of partial resistance to Pseudomonas syringae pv. maculicola (Psm) ES4326 was studied in the recombinant inbred population created from accessions (ecotypes) Columbia (Col-4), the more susceptible parent, and Landsberg (Ler-0). Plants were spray inoculated with lux-transformed bacteria in experiments to measure susceptibility. The amount of disease produced on a range of Col × Ler lines by spray inoculation was highly correlated with that produced by pressure infiltration of bacteria into the apoplast. Quantitative trait locus (QTL) analysis identified four loci that contributed to partial resistance: QRpsJIC-1.1, QRpsJIC-2.1, QRpsJIC-3.1 and QRpsJIC-5.1 on chromosomes 1, 2, 3 and 5, respectively. QRpsJIC-3.1, located 8.45 cM from the top of the consensus genetic map of chromosome 3, had a large, approximately additive effect on partial resistance, explaining 50% of the genetic variation in this population. Fine mapping narrowed the region within which this QTL was located to 62 genes. A list of candidate genes included several major classes of resistance gene.201323724899
39280.9281Stable Tagging of Rhizobium meliloti with the Firefly Luciferase Gene for Environmental Monitoring. A system for stable tagging of gram-negative bacteria with the firefly luciferase gene, luc, is described. A previously constructed fusion constitutively expressing luc from the lambdap(R) promoter was used. Stable integration into the bacterial genome was achieved by use of mini-Tn5 delivery vectors. The procedure developed was applied for tagging of representative gram-negative bacteria, such as Escherichia coli, Rhizobium meliloti, Pseudomonas putida, and Agrobacterium tumefaciens. The system permitted the detection of tagged R. meliloti in the presence of more than 10 CFU per plate without the use of any selective markers (such as antibiotic resistance genes). No significant differences in growth rates or soil survival were found between the marked strain and the wild-type strain. Studies of bioluminescent R. meliloti also revealed a good correlation between cell biomass and bioluminescence. The firefly luciferase tagging system is an easy, safe, and sensitive method for the detection and enumeration of bacteria in the environment.199316349015
872590.9279CuO nanoparticles facilitate soybean suppression of Fusarium root rot by regulating antioxidant enzymes, isoflavone genes, and rhizosphere microbiome. BACKGROUND: Fusarium root rot is a widespread soil-borne disease severely impacting soybean yield and quality. Compared to traditional fertilizers' biological and environmental toxicity, CuO nanoparticles (NPs) hold promise for disease control in a low dose and high efficiency manner. METHODS: We conducted both greenhouse and field experiments, employing enzymatic assays, elemental analysis, qRT-PCR, and microbial sequencing (16S rRNA, ITS) to explore the potential of CuO NPs for sustainable controlling Fusarium-induced soybean disease. RESULTS: Greenhouse experiments showed that foliar spraying of CuO NPs (10, 100, and 500 mg L(-1)) promoted soybean growth more effectively than EDTA-CuNa(2) at the same dose, though 500 CuO NPs caused mild phytotoxicity. CuO NPs effectively controlled root rot, while EDTA-CuNa(2) worsened the disease severity by 0.85-34.04 %. CuO NPs exhibited more substantial antimicrobial effects, inhibiting F. oxysporum mycelial growth and spore germination by 5.04-17.55 % and 10.24-14.41 %, respectively. 100 mg L(-1) CuO NPs was the optimal concentration for balancing soybean growth and disease resistance. Additionally, CuO NPs boosted antioxidant enzyme activity (CAT, POD, and SOD) in leaves and roots, aiding in ROS clearance during pathogen invasion. Compared to the pathogen control, 100 mg L(-1) CuO NPs upregulated the relative expression of seven isoflavone-related genes (Gm4CL, GmCHS8, GmCHR, GmCHI1a, GmIFS1, GmUGT1, and GmMYB176) by 1.18-4.51 fold, thereby enhancing soybean disease resistance in place of progesterone-receptor (PR) genes. Field trials revealed that CuO NPs' high leaf-to-root translocation modulated soybean rhizosphere microecology. Compared to the pathogen control, 100 mg L(-1) CuO NPs increased nitrogen-fixing bacteria (Rhizobium, Azospirillum, Azotobacter) and restored disease-resistant bacteria (Pseudomonas, Burkholderia) and fungi (Trichoderma, Penicillium) to healthy levels. Furthermore, 100 mg L(-1) CuO NPs increased beneficial bacteria (Pedosphaeraceae, Xanthobacteraceae, SCI84, etc.) and fungi (Trichoderma, Curvularia, Hypocreales, etc.), which negatively correlated with F. oxysporum, while recruiting functional microbes to enhance soybean yield. CONCLUSION: 100 mg L(-1) CuO NPs effectively promoting soybean growth and providing strong resistance against root rot disease by improving antioxidant enzyme activity, regulating the relative expression of isoflavone-related genes, increasing beneficial bacteria and fungi and restoring disease-resistant. Our findings suggest that CuO NPs offer an environmentally sustainable strategy for managing soybean disease, with great potential for green production.202540096759
6197100.9277Structure and location of tellurium deposited in Escherichia coli cells harbouring tellurite resistance plasmids. The plasmids RP4Ter and pHH1508a, which belong to the P and HII incompatibility groups, respectively, confer resistance to potassium tellurite (K2TeO3) on Escherichia coli. The genes for tellurite resistance were cloned from each plasmid onto the vector pUC8 to create pDT1366 and pD1364, respectively. Unstained, unfixed bacteria carrying these plasmids contained black intracellular deposits when grown on media containing tellurite. Thin sections of these bacteria fixed with glutaraldehyde were prepared and examined by electron microscopy. The black deposits were located inside the cell and were frequently associated with the inner membrane of the bacterium. Bacteria containing pDT1366 or pDT1364, and therefore a higher gene dosage of the Ter determinant, contained more black deposits, but had a decreased resistance, as measured by the minimum inhibitory concentration using the agar dilution method. Using the technique of electron spectroscopic imaging, the black intracellular deposits were shown to contain predominantly reduced metallic tellurium, and significant amounts of oxygen or carbon, thereby confirming earlier results using X-ray diffraction analysis of whole cells.19883042886
391110.9277New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Three types of new variants of the broad-host-range transposon Tn5 are described. (i) Tn5-mob derivatives with the new selective resistance (R) markers GmR, SpR and TcR facilitate the efficient mobilization of replicons within a wide range of Gram-negative bacteria. (ii) Promoter probe transposons carry the promoterless reporter genes lacZ, nptII, or luc, and NmR, GmR or TcR as selective markers. These transposons can be used to generate transcriptional fusions upon insertion, thus facilitating accurate determinations of gene expression. (iii) Tn5-P-out derivatives carry the npt- or tac-promoter reading out from the transposon, and TcR, NmR or GmR genes. These variants allow the constitutive expression of downstream genes. The new Tn5 variants are available on mobilizable Escherichia coli vectors suitable as suicidal carriers for transposon mutagenesis of non-E. coli recipients and some on a phage lambda mutant to be used for transposon mutagenesis in E. coli.19892551782
8431120.9277A quaternary ammonium salt grafted tannin-based flocculant boosts the conjugative transfer of plasmid-born antibiotic resistance genes: The nonnegligible side of their flocculation-sterilization properties. This study developed dual-function tannin-based flocculants, namely tannin-graft-acrylamide-diallyl dimethyl ammonium chloride (TGCC-A/TGCC-C), endowed with enhanced flocculation-sterilization properties. The impacts of these flocculants on proliferation and transformation of antibiotic resistance genes (ARGs) among bacteria during the flocculation-deposition process were examined. TGCC-A/TGCC-C exhibited remarkable flocculation capacities towards both Escherichia coli and Staphylococcus aureus, encompassing a logarithmic range of initial cell density (10(8)-10(9) CFU/mL) and a broad pH spectrum (pH 2-11). The grafted quaternary ammonium salt groups played pivotal parts in flocculation through charge neutralization and bridging mechanisms, concurrently contributing to sterilization by disrupting cellular membranes. The correlation between flocculation and sterilization entails a sequential progression, where an excess of TGCC, initially employed for flocculation, is subsequently consumed for sterilization purposes. The frequencies of ARGs conjugative transfer were enhanced in bacterial flocs across all TGCC treatments, stemming from augmented bacterial aggregation and cell membrane permeability, elicited stress response, and up-regulated genes encoding plasmid transfer. These findings underscore the indispensable role of flocculation-sterilization effects in mediating the propagation of ARGs, consequently providing substantial support for the scientific evaluation of the environmental risks associated with flocculants in the context of ARGs dissemination during the treatment of raw water featuring high bacterial density.202337619725
335130.9276Construction and characterization of a replication-competent retroviral shuttle vector plasmid. We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.200211799171
320140.9276Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector. To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.200212084480
102150.9275Paradoxical behaviour of pKM101; inhibition of uvr-independent crosslink repair in Escherichia coli by muc gene products. In strains of Escherichia coli deficient in excision repair (uvrA or uvrB), plasmid pKM101 muc+ but not pGW219 mucB::Tn5 enhanced resistance to angelicin monoadducts but reduced resistance to 8-methoxy-psoralen interstrand DNA crosslinks. Thermally induced recA-441 (= tif-1) bacteria showed an additional resistance to crosslinks that was blocked by pKM101. Plasmid-borne muc+ genes also conferred some additional sensitivity to gamma-radiation and it is suggested that a repair step susceptible to inhibition by muc+ gene products and possibly involving double-strand breaks may be involved after both ionizing radiation damage and psoralen crosslinks.19853883148
8184160.9274Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria. The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.202032523110
6352170.9274Cloning and characterization of grpE in Acetobacter pasteurianus NBRC 3283. The grpE gene in Acetobacter pasteurianus NBRC 3283 was cloned and characterized, to elucidate the mechanism underlying the resistance of acetic acid bacteria to the stressors existing during acetic acid fermentation. This gene was found to be located in tandem with two related genes, appearing on the genome in the order grpE-dnaK-dnaJ. A sigma(32)-type promoter sequence was found in the upstream region of grpE. The relative transcription levels of grpE, dnaK, and dnaJ mRNA were in the ratio of approximately 1:2:0.1, and the genes were transcribed as grpE-dnaK, dnaK, and dnaJ. The transcription level of grpE was elevated by heat shock and treatment with ethanol. Co-overexpression of GrpE with DnaK/J in cells resulted in improved growth compared to the single overexpression of DnaK/J in high temperature or ethanol-containing conditions, suggesting that GrpE acts cooperatively with DnaK/J for expressing resistance to those stressors considered to exist during acetic acid fermentation. Our findings indicate that GrpE is closely associated with adaptation to stressors in A. pasteurianus and may play an important role in acetic acid fermentation.201020129077
131180.9274Characterization of Two Highly Arsenic-Resistant Caulobacteraceae Strains of Brevundimonas nasdae: Discovery of a New Arsenic Resistance Determinant. Arsenic (As), distributed widely in the natural environment, is a toxic substance which can severely impair the normal functions in living cells. Research on the genetic determinants conferring functions in arsenic resistance and metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. More and more new arsenic resistance (ars) determinants have been identified to be conferring resistance to diverse arsenic compounds and encoded in ars operons. There is a hazard in mobilizing arsenic during gold-mining activities due to gold- and arsenic-bearing minerals coexisting. In this study, we isolated 8 gold enrichment strains from the Zijin gold and copper mine (Longyan, Fujian Province, China) wastewater treatment site soil, at an altitude of 192 m. We identified two Brevundimonas nasdae strains, Au-Bre29 and Au-Bre30, among these eight strains, having a high minimum inhibitory concentration (MIC) for As(III). These two strains contained the same ars operons but displayed differences regarding secretion of extra-polymeric substances (EPS) upon arsenite (As(III)) stress. B. nasdae Au-Bre29 contained one extra plasmid but without harboring any additional ars genes compared to B. nasdae Au-Bre30. We optimized the growth conditions for strains Au-Bre29 and Au-Bre30. Au-Bre30 was able to tolerate both a lower pH and slightly higher concentrations of NaCl. We also identified folE, a folate synthesis gene, in the ars operon of these two strains. In most organisms, folate synthesis begins with a FolE (GTP-Cyclohydrolase I)-type enzyme, and the corresponding gene is typically designated folE (in bacteria) or gch1 (in mammals). Heterologous expression of folE, cloned from B. nasdae Au-Bre30, in the arsenic-hypersensitive strain Escherichia coli AW3110, conferred resistance to As(III), arsenate (As(V)), trivalent roxarsone (Rox(III)), pentavalent roxarsone (Rox(V)), trivalent antimonite (Sb(III)), and pentavalent antimonate (Sb(V)), indicating that folate biosynthesis is a target of arsenite toxicity and increased production of folate confers increased resistance to oxyanions. Genes encoding Acr3 and ArsH were shown to confer resistance to As(III), Rox(III), Sb(III), and Sb(V), and ArsH also conferred resistance to As(V). Acr3 did not confer resistance to As(V) and Rox(V), while ArsH did not confer resistance to Rox(V).202235628430
7860190.9274Enhanced removal of antibiotic-resistant bacteria and resistance genes by three-dimensional electrochemical process using MgFe(2)O(4)-loaded biochar as both particle electrode and catalyst for peroxymonosulfate activation. In this study, MgFe(2)O(4)-loaded biochar (MFBC) was used as a three-dimensional particle electrode to active peroxymonosulfate (EC/MFBC/PMS) for the removal of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). The results demonstrated that, under the conditions of 1.0 mM PMS concentration, 0.4 g/L material dosage, 5 V voltage intensity, and MFBC preparation temperature of 600 °C, the EC/MFBC600/PMS system achieved complete inactivation of E. coli DH5α within 5 min and the intracellular sul1 was reduced by 81.5 % after 30 min of the treatment. Compared to EC and PMS alone treatments, the conjugation transfer frequency of sul1 rapidly declined by 92.9 % within 2 min. The cell membrane, proteins, lipids, as well as intracellular and extracellular ARGs in E. coli DH5α were severely damaged by free radicals in solution and intracellular reactive oxygen species (ROS). Furthermore, up-regulation was observed in genes associated with oxidative stress, SOS response and cell membrane permeability in E. coli DH5α, however, no significant changes were observed in functional genes related to gene conjugation and transfer mechanisms. This study would contribute to the underlying of PMS activation by three-dimensional particle electrode, and provide novel insights into the mechanism of ARB inactivation and ARGs degradation under PMS advanced oxidation treatment.202439197284