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678800.9296Release and Constancy of an Antibiotic Resistance Gene in Seawater under Grazing Stress by Ciliates and Heterotrophic Nanoflagellates. Extracellular DNA (exDNA) is released from bacterial cells through various processes. The antibiotic resistance genes (ARGs) coded on exDNA may be horizontally transferred among bacterial communities by natural transformation. We quantitated the released/leaked tetracycline resistance gene, tet(M) over time under grazing stress by ciliates and heterotrophic nanoflagellates (HNFs), and found that extracellular tet(M) (ex-tetM) increased with bacterial grazing. Separate microcosms containing tet(M)-possessing bacteria with ciliates or HNFs were prepared. The copy number of ex-tetM in seawater in the ciliate microcosm rapidly increased until 3 d after the incubation, whereas that in the HNF microcosm showed a slower increase until 20 d. The copy number of ex-tetM was stable in both cases throughout the incubation period, suggesting that extracellular ARGs are preserved in the environment, even in the presence of grazers. Additionally, ARGs in bacterial cells were constant in the presence of grazers. These results suggest that ARGs are not rapidly extinguished in a marine environment under grazing stress.201728592722
874010.9198Nitrite reductase activity of sulphate-reducing bacteria prevents their inhibition by nitrate-reducing, sulphide-oxidizing bacteria. Sulphate-reducing bacteria (SRB) can be inhibited by nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB), despite the fact that these two groups are interdependent in many anaerobic environments. Practical applications of this inhibition include the reduction of sulphide concentrations in oil fields by nitrate injection. The NR-SOB Thiomicrospira sp. strain CVO was found to oxidize up to 15 mM sulphide, considerably more than three other NR-SOB strains that were tested. Sulphide oxidation increased the environmental redox potential (Eh) from -400 to +100 mV and gave 0.6 nitrite per nitrate reduced. Within the genus Desulfovibrio, strains Lac3 and Lac6 were inhibited by strain CVO and nitrate for the duration of the experiment, whereas inhibition of strains Lac15 and D. vulgaris Hildenborough was transient. The latter had very high nitrite reductase (Nrf) activity. Southern blotting with D. vulgaris nrf genes as a probe indicated the absence of homologous nrf genes from strains Lac3 and Lac6 and their presence in strain Lac15. With respect to SRB from other genera, inhibition of the known nitrite reducer Desulfobulbus propionicus by strain CVO and nitrate was transient, whereas inhibition of Desulfobacterium autotrophicum and Desulfobacter postgatei was long-lasting. The results indicate that inhibition of SRB by NR-SOB is caused by nitrite production. Nrf-containing SRB can overcome this inhibition by further reducing nitrite to ammonia, preventing a stalling of the favourable metabolic interactions between these two bacterial groups. Nrf, which is widely distributed in SRB, can thus be regarded as a resistance factor that prevents the inhibition of dissimilatory sulphate reduction by nitrite.200312823193
678720.9190Impact of chlorine disinfection on intracellular and extracellular antimicrobial resistance genes in wastewater treatment and water reclamation. Wastewater treatment plants and water reclamation facilities are reservoirs of antimicrobial resistance genes (ARGs). These ARGs are not limited solely to intracellular DNA (inARGs) but include extracellular DNA (exARGs) present in wastewater. The release of exARGs from cells can be exacerbated by treatment processes, including chlorine disinfection, which disrupts bacterial cells. Given the potential for exARGs to drive horizontal gene transfer and contribute to the proliferation of antimicrobial resistance, it is imperative to recognize these fractions as emerging environmental pollutants. In this study, we conducted a comprehensive year-long assessment of both inARGs and exARGs, further differentiating between dissolved exARGs (Dis_exARGs) and exARGs adsorbed onto particulate matter (Ads_exARGs), within a full-scale wastewater treatment and water reclamation facility. The results revealed that Ads_exARGs comprised up to 30 % of the total ARGs in raw sewage with high biomass content. Generally, treatments at low and high doses of chlorine increased the abundance of Dis_exARGs and Ads_exARGs. The fate of ARG levels that varied depending on the type of ARGs suggested variations in the susceptibility of the host bacteria to chlorination. Moreover, co-occurrence of several potential opportunistic pathogenic bacteria and ARGs were observed. Therefore, we propose higher doses of chlorination as a prerequisite for the effective removal of inARGs and exARGs.202439067603
782830.9180Simultaneous elimination of antibiotic-resistant bacteria and antibiotic resistance genes by different Fe-N co-doped biochars activating peroxymonosulfate: The key role of pyridine-N and Fe-N sites. The coexistence of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in the environment poses a potential threat to public health. In our study, we have developed a novel advanced oxidation process for simultaneously removing ARGs and ARB by two types of iron and nitrogen-doped biochar derived from rice straw (FeN-RBC) and sludge (FeN-SBC). All viable ARB (approximately 10(8) CFU mL(-1)) was inactivated in the FeN-RBC/ peroxymonosulfate (PMS) system within 40 min and did not regrow after 48 h even in real water samples. Flow cytometry identified 96.7 % of dead cells in the FeN-RBC/PMS system, which verified the complete inactivation of ARB. Thorough disinfection of ARB was associated with the disruption of cell membranes and intracellular enzymes related to the antioxidant system. Whereas live bacteria (approximately 200 CFU mL(-1)) remained after FeN-SBC/PMS treatment. Intracellular and extracellular ARGs (tetA and tetB) were efficiently degraded in the FeN-RBC/PMS system. The production of active species, primarily •OH, SO(4)(•-) and Fe (IV), as well as electron transfer, were essential to the effective disinfection of FeN-RBC/PMS. In comparison with FeN-SBC, the better catalytic performance of FeN-RBC was mainly ascribed to its higher amount of pyridine-N and Fe(0), and more reactive active sites (such as CO group and Fe-N sites). Density functional theory calculations indicated the greater adsorption energy and Bader charge, more stable Fe-O bond, more easily broken OO bond in FeN-RBC/PMS, which demonstrated the stronger electron transfer capacity between FeN-RBC and PMS. To encapsulate, our study provided an efficient and dependable method for the simultaneous elimination of ARGs and ARB in water.202438669989
842140.9178Dynamic stepwise opening of integron attC DNA hairpins by SSB prevents toxicity and ensures functionality. Biologically functional DNA hairpins are found in archaea, prokaryotes and eukaryotes, playing essential roles in various DNA transactions. However, during DNA replication, hairpin formation can stall the polymerase and is therefore prevented by the single-stranded DNA binding protein (SSB). Here, we address the question how hairpins maintain their functional secondary structure despite SSB's presence. As a model hairpin, we used the recombinogenic form of the attC site, essential for capturing antibiotic-resistance genes in the integrons of bacteria. We found that attC hairpins have a conserved high GC-content near their apical loop that creates a dynamic equilibrium between attC fully opened by SSB and a partially structured attC-6-SSB complex. This complex is recognized by the recombinase IntI, which extrudes the hairpin upon binding while displacing SSB. We anticipate that this intriguing regulation mechanism using a base pair distribution to balance hairpin structure formation and genetic stability is key to the dissemination of antibiotic resistance genes among bacteria and might be conserved among other functional hairpins.201728985409
865950.9178Phage phylogeny, molecular signaling, and auxiliary antimicrobial resistance in aerobic and anaerobic membrane bioreactors. Phage emit communication signals that inform their lytic and lysogenic life cycles. However, little is known regarding the abundance and diversity of the genes associated with phage communication systems in wastewater treatment microbial communities. This study focused on phage communities within two distinct biochemical wastewater environments, specifically aerobic membrane bioreactors (AeMBRs) and anaerobic membrane bioreactors (AnMBRs) exposed to varying antibiotic concentrations. Metagenomic data from the bench-scale systems were analyzed to explore phage phylogeny, life cycles, and genetic capacity for antimicrobial resistance and quorum sensing. Two dominant phage families, Schitoviridae and Peduoviridae, exhibited redox-dependent dynamics. Schitoviridae prevailed in anaerobic conditions, while Peduoviridae dominated in aerobic conditions. Notably, the abundance of lytic and lysogenic proteins varied across conditions, suggesting the coexistence of both life cycles. Furthermore, the presence of antibiotic resistance genes (ARGs) within viral contigs highlighted the potential for phage to transfer ARGs in AeMBRs. Finally, quorum sensing genes in the virome of AeMBRs indicated possible molecular signaling between phage and bacteria. Overall, this study provides insights into the dynamics of viral communities across varied redox conditions in MBRs. These findings shed light on phage life cycles, and auxiliary genetic capacity such as antibiotic resistance and bacterial quorum sensing within wastewater treatment microbial communities.202438677036
30960.9176Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost. Plasmids promote adaptation of bacteria by facilitating horizontal transfer of diverse genes, notably those conferring antibiotic resistance. Some plasmids, like those of the incompatibility group IncP-1, are known to replicate and persist in a broad range of bacteria. We investigated a poorly understood exception, the IncP-1β plasmid pBP136 from a clinical Bordetella pertussis isolate, which quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution, we found that the inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid persistence in E. coli. The gene inactivation caused alterations in the plasmid regulatory system, including decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. This is consistent with our findings that Upf31 represses its own transcription. It also caused secondary transcriptional changes in many chromosomal genes. In silico analyses predicted that Upf31 interacts with the plasmid regulator KorB at its C-terminal dimerization domain (CTD). We showed experimentally that adding the CTD of upf31/pBP136 to the naturally truncated upf31 allele of the stable IncP-1β archetype R751 results in plasmid destabilization in E. coli. Moreover, mutagenesis showed that upf31 alleles encoded on nearly half of the sequenced IncP-1β plasmids also possess this destabilization phenotype. While Upf31 might be beneficial in many hosts, we show that in E. coli some alleles have harmful effects that can be rapidly alleviated with a single mutation. Thus, broad-host-range plasmid adaptation to new hosts can involve fine-tuning their transcriptional circuitry through evolutionary changes in a single gene.202540138356
673270.9175Assessment of Bioavailability of Biochar-Sorbed Tetracycline to Escherichia coli for Activation of Antibiotic Resistance Genes. Human overuse and misuse of antibiotics have caused the wide dissemination of antibiotics in the environment, which has promoted the development and proliferation of antibiotic resistance genes (ARGs) in soils. Biochar (BC) with strong sorption affinity to many antibiotics is considered to sequester antibiotics and hence mitigate their impacts to bacterial communities in soils. However, little is known about whether BC-sorbed antibiotics are bioavailable and exert selective pressure on soil bacteria. In this study, we probed the bioavailability of tetracycline sorbed by BCs prepared from rice-, wheat-, maize-, and bean-straw feedstock using Escherichia coli MC4100/pTGM bioreporter strain. The results revealed that BC-sorbed tetracycline was still bioavailable to the E. coli attached to BC surfaces. Tetracycline sorbed by BCs prepared at 400 °C (BC400) demonstrated a higher bioavailability to bacteria compared to that sorbed by BCs prepared at 500 °C (BC500). Tetracycline could be sorbed primarily in the small pores of BC500 where bacteria could not access due to the size exclusion to bacteria. In contrast, tetracycline could be sorbed mainly on BC400 surfaces where bacteria could conveniently access tetracycline. Increasing the ambient humidity apparently enhanced the bioavailability of BC400-sorbed tetracycline. BC500-sorbed tetracycline exposed to varying levels of ambient humidity showed no significant changes in bioavailability, indicating that water could not effectively mobilize tetracycline from BC500 pores to surfaces where bacteria could access tetracycline. The results from this study suggest that BCs prepared at a higher pyrolysis temperature could be more effective to sequester tetracycline and mitigate the selective pressure on soil bacteria.202032786566
842380.9175Horizontal Gene Transfer From Bacteria and Plants to the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis. Arbuscular mycorrhizal fungi (AMF) belong to Glomeromycotina, and are mutualistic symbionts of many land plants. Associated bacteria accompany AMF during their lifecycle to establish a robust tripartite association consisting of fungi, plants and bacteria. Physical association among this trinity provides possibilities for the exchange of genetic materials. However, very few horizontal gene transfer (HGT) from bacteria or plants to AMF has been reported yet. In this study, we complement existing algorithms by developing a new pipeline, Blast2hgt, to efficiently screen for putative horizontally derived genes from a whole genome. Genome analyses of the glomeromycete Rhizophagus irregularis identified 19 fungal genes that had been transferred between fungi and bacteria/plants, of which seven were obtained from bacteria. Another 18 R. irregularis genes were found to be recently acquired from either plants or bacteria. In the R. irregularis genome, gene duplication has contributed to the expansion of three foreign genes. Importantly, more than half of the R. irregularis foreign genes were expressed in various transcriptomic experiments, suggesting that these genes are functional in R. irregularis. Functional annotation and available evidence showed that these acquired genes may participate in diverse but fundamental biological processes such as regulation of gene expression, mitosis and signal transduction. Our study suggests that horizontal gene influx through endosymbiosis is a source of new functions for R. irregularis, and HGT might have played a role in the evolution and symbiotic adaptation of this arbuscular mycorrhizal fungus.201829887874
638790.9174Insights into the Evolutionary and Ecological Roles of Bathyarchaeia in Arsenic Detoxification. Arsenic (As) is a prevalent toxic element, posing significant risks to organisms, including microbes. While microbial arsenic detoxification has been extensively studied in bacteria, archaeal mechanisms remain understudied. Here, we investigated arsenic resistance genes in Bathyarchaeia, one of the most abundant archaeal lineages on Earth. Comprehensive genomic analysis of 318 Bathyarchaeia representatives revealed a widespread distribution of arsenic resistance genes, with 60% of genomes harboring genes for arsenate reduction (arsR1 and arsC2), arsenite methylation (arsM), and arsenic transport (acr3, arsP, and arsB). Phylogenetic analysis revealed that these genes are widely distributed across 14 archaeal phyla, including Asgardarchaeota, Thermoproteota, and Thermoplasmatota, with close evolutionary relationships among these archaeal lineages. In situ investigation of sediment columns and laboratory microcosm experiments demonstrated a strong positive correlation between Bathyarchaeia abundance and arsenic concentrations, suggesting their adaptation to arsenic-rich environments. Molecular dating analysis placed the emergence of Bathyarchaeia at approximately 3.01 billion years ago, with the evolution of their arsenic resistance mechanisms closely tracking major geological events, including the Great Oxidation Event (2.4-2.1 Gya), Huronian Glaciation (2.29-2.25 Gya), and Cryogenian Glaciation (∼700 Mya). Our findings highlight the critical role of Archaea in the arsenic cycle and provide insights into the evolutionary history of arsenic resistance associated with paleogeochemical changes in Bathyarchaeia.202540921195
7935100.9173Removal of antibiotic resistance genes by Cl(2)-UV process: Direct UV damage outweighs free radicals in effectiveness. Antibiotic resistance genes (ARGs) pose significant environmental health problems and have become a major global concern. This study investigated the efficacy and mechanism of the Cl(2)-UV process (chlorine followed by UV irradiation) for removing ARGs in various forms. The Cl(2)-UV process caused irreversible damage to nearly all ARB at typical disinfectant dosages. In solutions containing only extracellular ARGs (eARGs), the Cl₂-UV process achieved over 99.0 % degradation of eARGs. When both eARGs and intracellular ARGs (iARGs) were present, the process reached a 97.2 % removal rate for iARGs. While the abundance of eARGs initially increased due to the release of iARGs from lysed cells during pre-chlorination, subsequent UV irradiation rapidly degraded the released eARGs, restoring their abundance to near-initial levels by the end of the Cl₂-UV process. Analysis of the roles in degrading eARGs and iARGs during the Cl(2)-UV process revealed that UV, rather than free radicals, was the dominant factor causing ARG damage. Pre-chlorination enhanced direct UV damage to eARGs and iARGs by altering plasmid conformation and promoting efficient damage to high UV-absorbing cellular components. Furthermore, no further natural transformation of residual ARGs occurred following the Cl(2)-UV treatment. This study demonstrated strong evidence for the effectiveness of the Cl(2)-UV process in controlling antibiotic resistance.202540048777
194110.9173Possible Role of CHAD Proteins in Copper Resistance. Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.202438399813
41120.9172Rice WRKY13 regulates cross talk between abiotic and biotic stress signaling pathways by selective binding to different cis-elements. Plants use a complex signal transduction network to regulate their adaptation to the ever-changing environment. Rice (Oryza sativa) WRKY13 plays a vital role in the cross talk between abiotic and biotic stress signaling pathways by suppressing abiotic stress resistance and activating disease resistance. However, it is not clear how WRKY13 directly regulates this cross talk. Here, we show that WRKY13 is a transcriptional repressor. During the rice responses to drought stress and bacterial infection, WRKY13 selectively bound to certain site- and sequence-specific cis-elements on the promoters of SNAC1 (for STRESS RESPONSIVE NO APICAL MERISTEM, ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR1/2, CUP-SHAPED COTYLEDON), the overexpression of which increases drought resistance, and WRKY45-1, the knockout of which increases both bacterial disease and drought resistance. WRKY13 also bound to two cis-elements of its native promoter to autoregulate the balance of its gene expression in different physiological activities. WRKY13 was induced in leaf vascular tissue, where bacteria proliferate, during infection, and in guard cells, where the transcriptional factor SNAC1 enhances drought resistance, during both bacterial infection and drought stress. These results suggest that WRKY13 regulates the antagonistic cross talk between drought and disease resistance pathways by directly suppressing SNAC1 and WRKY45-1 and autoregulating its own expression via site- and sequence-specific cis-elements on the promoters of these genes in vascular tissue where bacteria proliferate and guard cells where the transcriptional factor SNAC1 mediates drought resistance by promoting stomatal closure.201324130197
7737130.9172Distinctive signatures of pathogenic and antibiotic resistant potentials in the hadal microbiome. BACKGROUND: Hadal zone of the deep-sea trenches accommodates microbial life under extreme energy limitations and environmental conditions, such as low temperature, high pressure, and low organic matter down to 11,000 m below sea level. However, microbial pathogenicity, resistance, and adaptation therein remain unknown. Here we used culture-independent metagenomic approaches to explore the virulence and antibiotic resistance in the hadal microbiota of the Mariana Trench. RESULTS: The results indicate that the 10,898 m Challenger Deep bottom sediment harbored prosperous microbiota with contrasting signatures of virulence factors and antibiotic resistance, compared with the neighboring but shallower 6038 m steep wall site and the more nearshore 5856 m Pacific basin site. Virulence genes including several famous large translocating virulence genes (e.g., botulinum neurotoxins, tetanus neurotoxin, and Clostridium difficile toxins) were uniquely detected in the trench bottom. However, the shallower and more nearshore site sediment had a higher abundance and richer diversity of known antibiotic resistance genes (ARGs), especially for those clinically relevant ones (e.g., fosX, sul1, and TEM-family extended-spectrum beta-lactamases), revealing resistance selection under anthropogenic stresses. Further analysis of mobilome (i.e., the collection of mobile genetic elements, MGEs) suggests horizontal gene transfer mediated by phage and integrase as the major mechanism for the evolution of Mariana Trench sediment bacteria. Notably, contig-level co-occurring and taxonomic analysis shows emerging evidence for substantial co-selection of virulence genes and ARGs in taxonomically diverse bacteria in the hadal sediment, especially for the Challenger Deep bottom where mobilized ARGs and virulence genes are favorably enriched in largely unexplored bacteria. CONCLUSIONS: This study reports the landscape of virulence factors, antibiotic resistome, and mobilome in the sediment and seawater microbiota residing hadal environment of the deepest ocean bottom on earth. Our work unravels the contrasting and unique features of virulence genes, ARGs, and MGEs in the Mariana Trench bottom, providing new insights into the eco-environmental and biological processes underlying microbial pathogenicity, resistance, and adaptative evolution in the hadal environment.202235468809
614140.9171Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival. Ehrlichia chaffeensis and Anaplasma phagocytophilum are agents of human monocytic and granulocytic ehrlichioses, respectively. They are extremely sensitive to mechanical stress and are pleomorphic gram-negative bacteria. Membrane incorporation of cholesterol from the eukaryotic host is known to be essential for other fragile and pleomorphic bacteria and mycoplasmas that lack a cell wall. Thus, we tested whether cholesterol is required for E. chaffeensis and A. phagocytophilum. Using a freeze fracture technique and biochemical analysis, these bacteria were found to contain significant levels of membrane cholesterol. These bacteria lack genes for cholesterol biosynthesis or modification. However, host cell-free bacteria had the ability to take up directly exogenous cholesterol or NBD-cholesterol, a fluorescent cholesterol derivative. Treatment of the bacteria with cholesterol extraction reagent methyl-beta-cyclodextrin caused their ultrastructural changes. Furthermore, pretreatment of the bacteria with methyl-beta-cyclodextrin or NBD-cholesterol deprived these bacteria of the ability to infect leukocytes, thus killing these obligate intracellular bacteria. Analysis of E. chaffeensis and A. phagocytophilum genome sequences revealed that these bacteria lack all genes for the biosynthesis of lipid A and most genes for the biosynthesis of peptidoglycan, which confer structural strength to gram-negative bacteria. Taken together, these results suggest that human ehrlichiosis agents became cholesterol dependent due to the loss of these genes. As the first report of gram-negative bacteria incorporating cholesterol for survival, these findings offer insight into the unique nature of their parasitism and imply that cholesterol is important in the control of human ehrlichioses.200312933880
538150.9171The biochemical and genetic basis for high frequency thiomethyl galactoside resistance in lambda,lambdadg lysogens of Escherichia coli. In a culture of Escherichia coli K12 gal (lambdadg), cells which form large colonies on agar plates containing galactose and thiomethyl beta-D-galactoside (TMG) appear at high frequency. These clones are resistant to growth inhibition by TMG on galactose minimal medium. Biochemical studies of the steady-state levels of galactokinase and UDPgalactose 4-epimerase suggest that the resistant clones have extra copies of the genes for the galactose-metabolizing enzymes. The mutation for TMG resistance is not located in either the bacterial or the bacteriophage genome, but is probably due to an aberrant association between cell and prophage DNA. Mapping the TMG-resistant characteristic by phage P1 indicates that TMG-resistant bacteria posses at least two GAL+ OPERONS, ONE OF WHICH IS COTRANSDUCIBLe with bio+. In addition, TMG-resistant bacteria behave like lambdadg polylysogens when challenged with the phage lambdaI90c17. From these genetic experiments we conclude that TMG-resistant bacteria arise by duplication of the lambdadg prophage. Finally, gal+ bacteria which carry a single, additional, lambdadg prophage are TMG-resistant. TMG resistance is probably a gal+ gene dosage effect.1978344832
343160.9171Cloning and molecular characterization of three arylamine N-acetyltransferase genes from Bacillus anthracis: identification of unusual enzymatic properties and their contribution to sulfamethoxazole resistance. The arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that catalyze the N-acetylation of arylamines and their N-hydroxylated metabolites. These enzymes play a key role in detoxication of numerous drugs and xenobiotics. We report here the cloning, functional expression, and characterization of three new NAT genes (termed banatA, banatB, and banatC) from the pathogen Bacillus anthracis. The sequences of the corresponding proteins are approximately 30% identical with those of characterized eukaryotic and prokaryotic NAT enzymes, and the proteins were recognized by an anti-NAT antibody. The three genes were endogenously expressed in B. anthracis, and NAT activity was found in cell extracts. The three NAT homologues exhibited distinct structural and enzymatic properties, some of which have not previously been observed with other NAT enzymes. Recombinant BanatC displayed strong NAT activity toward several prototypic NAT substrates, including the sulfonamide antibiotic sulfamethoxazole (SMX). As opposed to BanatC, BanatB also had acetyl-CoA (AcCoA) and p-nitrophenyl acetate (PNPA) hydrolysis activity in the absence of arylamine substrates, indicating that it may act as an AcCoA hydrolase. BanatA was devoid of NAT or AcCoA/PNPA hydrolysis activities, suggesting that it may be a new bacterial NAT-like protein with unknown function. Expression of BanatC in Escherichia coli afforded higher-than-normal resistance to SMX in the recombinant bacteria, whereas an inactive mutant of the enzyme did not. These data indicate that BanatC could contribute to the resistance of B. anthracis to SMX.200717511472
7836170.9170Efficient Degradation of Intracellular Antibiotic Resistance Genes by Photosensitized Erythrosine-Produced (1)O(2). Intracellular antibiotic resistance genes (iARGs) constitute the important part of wastewater ARGs and need to be efficiently removed. However, due to the dual protection of intracellular DNA by bacterial membranes and the cytoplasm, present disinfection technologies are largely inefficient in iARG degradation. Herein, we for the first time found that erythrosine (ERY, an edible dye) could efficiently degrade iARGs by producing abundant (1)O(2) under visible light. Seven log antibiotic-resistant bacteria were inactivated within only 1.5 min, and 6 log iARGs were completely degraded within 40 min by photosensitized ERY (5.0 mg/L). A linear relationship was established between ARG degradation rate constants and (1)O(2) concentrations in the ERY photosensitizing system. Surprisingly, a 3.2-fold faster degradation of iARGs than extracellular ARGs was observed, which was attributed to the unique indirect oxidation of iARGs induced by (1)O(2). Furthermore, ERY photosensitizing was effective for iARG degradation in real wastewater and other photosensitizers (including Rose Bengal and Phloxine B) of high (1)O(2) yields could also achieve efficient iARG degradation. The findings increase our knowledge of the iARG degradation preference by (1)O(2) and provide a new strategy of developing technologies with high (1)O(2) yield, like ERY photosensitizing, for efficient iARG removal.202337531556
6906180.9170Insights into the mobility and bacterial hosts of antibiotic resistance genes under dinotefuran selection pressure in aerobic granular sludge based on metagenomic binning and functional modules. Dinotefuran (DIN) is toxic to non-target organisms and accelerates the evolution of antibiotic resistance, which poses a problem for the stable operation of the activated sludge process in wastewater treatment plants (WWTPs). However, the emergence and the transfer mechanism of antibiotic resistance genes (ARGs) in activated sludge systems under DIN stress remains unclear. Thus, in the study, the potential impact of DIN on ARGs and virulence factor genes (VFGs) in aerobic granular sludge (AGS) was investigated in depth using metagenomic binning and functional modules. It was found that DIN stress increased the total abundance of ARGs, mobile genetic elements (MGEs), and VFGs in the AGS system, with the highest abundance of fabG (4.6%), tnpA (55.6%) and LPS (39.0%), respectively. The proliferation of the enteric pathogens Salmonella enterica and Escherichia coli in the system indicates that DIN induces exposure of harmless bacteria to the infected environment. The genera Nitrospira (1169 ARG subtypes) and Dechloromonas (663 ARG subtypes) were identified as the potentially antibiotic-resistant bacteria carrying the most ARGs and MGEs in the metagenome-assembled genomes. Co-localization patterns of some ARGs, MGEs, and the SOS response-related gene lexA were observed on metagenome-assembled contigs under high levels of DIN exposure, suggesting DIN stimulated ROS production (101.8% increase over control), altered cell membrane permeability, and increased the potential for horizontal gene transfer (HGT). Furthermore, the DNA damage caused by DIN in AGS led to the activation of the antioxidant system and the SOS repair response, which in turn promoted the expression of the type IV secretion system and HGT through the flagellar channel. This study extends the previously unappreciated DIN understanding of the spread and associated risks of ARGs and VFGs in the AGS system of WWTPs. It elucidates how DIN facilitates HGT, offering a scientific basis for controlling emerging contaminant-induced resistance.202539798650
129190.9170Evidence for vital role of endo-β-N-acetylglucosaminidase in the resistance of Arthrobacter protophormiae RKJ100 towards elevated concentrations of o-nitrobenzoate. Arthrobacter protophormiae RKJ100 was previously characterized for its ability to tolerate extremely high concentrations of o-nitrobenzoate (ONB), a toxic xenobiotic environmental pollutant. The physiological responses of strain RKJ100 to ≥30 mM ONB indicated towards a resistance mechanism manifested via alteration of cell morphology and cell wall structure. In this study, we aim to characterize gene(s) involved in the resistance of strain RKJ100 towards extreme concentrations (i.e. 150 mM) of ONB. Transposon mutagenesis was carried out to generate a mutant library of strain RKJ100, which was then screened for ONB-sensitive mutants. A sensitive mutant was defined and selected as one that could not tolerate ≥30 mM ONB. Molecular and biochemical characterization of this mutant showed that the disruption of endo-β-N-acetylglucosaminidase (ENGase) gene caused the sensitivity. ENGase is an important enzyme for oligosaccharide processing and cell wall recycling in bacteria, fungi, plants and animals. Previous reports have already indicated several possible roles of this enzyme in cellular homeostasis. Results presented here provide the first evidence for its involvement in bacterial resistance towards extreme concentrations of a toxic xenobiotic compound and also suggest that strain RKJ100 employs ENGase as an important component in osmotic shock response for resisting extreme concentrations of ONB.201424562786