New dioxygenase from metagenomic library from Brazilian soil: insights into antibiotic resistance and bioremediation. - Related Documents




#
Rank
Similarity
Title + Abs.
Year
PMID
012345
24701.0000New dioxygenase from metagenomic library from Brazilian soil: insights into antibiotic resistance and bioremediation. OBJECTIVES: Putative new dioxygenases were identified in a metagenomic β-lactam-resistance screening and, given their key role on aromatic metabolism, we raise the hypothesis that these enzymes maybe concomitantly related to antibiotic resistance and aromatic degradation. RESULTS: ORFs of three putative dioxygenases were isolated from resistant metagenomic clones. One of them, CRB2(1), was subcloned into pET24a expression vector and subjected to downstream phenotypic and bioinformatics analyses that demonstrated the "dual effect" of our metagenomic dioxygenase, on antibiotic and aromatic resistance. Furthermore, initial characterization assays strongly suggests that CRB2(1) open-reading frame is an extradiol-dioxygenase, most probably a bicupin domain gentisate 1,2-dioxygenase. This observation is, to our knowledge, the first description of a metagenomic dioxygenase and its action on β-lactam resistance. CONCLUSION: Unraveling the diversity of antibiotic resistance elements on the environment could not only identify new genes and mechanisms in which bacteria can resist to antibiotics, but also contribute to biotechnology processes, such as in bioremediation.201525994583
17410.9994Resistance to Arsenite and Arsenate in Saccharomyces cerevisiae Arises through the Subtelomeric Expansion of a Cluster of Yeast Genes. Arsenic is one of the most prevalent toxic elements in the environment, and its toxicity affects every organism. Arsenic resistance has mainly been observed in microorganisms, and, in bacteria, it has been associated with the presence of the Ars operon. In Saccharomyces cerevisiae, three genes confer arsenic resistance: ARR1, ARR2, and ARR3. Unlike bacteria, in which the presence of the Ars genes confers per se resistance to arsenic, most of the S. cerevisiae isolates present the three ARR genes, regardless of whether the strain is resistant or sensitive to arsenic. To assess the genetic features that make natural S. cerevisiae strains resistant to arsenic, we used a combination of comparative genomic hybridization, whole-genome sequencing, and transcriptomics profiling with microarray analyses. We observed that both the presence and the genomic location of multiple copies of the whole cluster of ARR genes were central to the escape from subtelomeric silencing and the acquisition of resistance to arsenic. As a result of the repositioning, the ARR genes were expressed even in the absence of arsenic. In addition to their relevance in improving our understanding of the mechanism of arsenic resistance in yeast, these results provide evidence for a new cluster of functionally related genes that are independently duplicated and translocated.202235805774
436820.9994Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase. BACKGROUND: The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects. RESULTS: Phylogenetic analysis of aligned arsC sequences shows broad similarities to the established 16S rRNA phylogeny, with separation of bacterial, archaeal, and subsequently eukaryotic arsC genes. However, inconsistencies between arsC and 16S rRNA are apparent for some taxa. Cyanobacteria and some of the gamma-Proteobacteria appear to possess arsC genes that are similar to those of Low GC Gram-positive Bacteria, and other isolated taxa possess arsC genes that would not be expected based on known evolutionary relationships. There is no clear separation of plasmid-borne and chromosomal arsC genes, although a number of the Enterobacteriales (gamma-Proteobacteria) possess similar plasmid-encoded arsC sequences. CONCLUSION: The overall phylogeny of the arsenate reductases suggests a single, early origin of the arsC gene and subsequent sequence divergence to give the distinct arsC classes that exist today. Discrepancies between 16S rRNA and arsC phylogenies support the role of horizontal gene transfer (HGT) in the evolution of arsenate reductases, with a number of instances of HGT early in bacterial arsC evolution. Plasmid-borne arsC genes are not monophyletic suggesting multiple cases of chromosomal-plasmid exchange and subsequent HGT. Overall, arsC phylogeny is complex and is likely the result of a number of evolutionary mechanisms.200312877744
445430.9994Functional verification of computationally predicted qnr genes. BACKGROUND: The quinolone resistance (qnr) genes are widely distributed among bacteria. We recently developed and applied probabilistic models to identify tentative novel qnr genes in large public collections of DNA sequence data including fragmented metagenomes. FINDINGS: By using inducible recombinant expressions systems the functionality of four identified qnr candidates were evaluated in Escherichia coli. Expression of several known qnr genes as well as two novel candidates provided fluoroquinolone resistance that increased with elevated inducer concentrations. The two novel, functionally verified qnr genes are termed Vfuqnr and assembled qnr 1. Co-expression of two qnr genes suggested non-synergistic action. CONCLUSION: The combination of a computational model and recombinant expression systems provides opportunities to explore and identify novel antibiotic resistance genes in both genomic and metagenomic datasets.201324257207
27140.9993Challenging the concept of bacteria subsisting on antibiotics. Antibiotic resistance concerns have been compounded by a report that soil bacteria can catabolise antibiotics, i.e. break down and use them as a sole carbon source. To date this has not been verified or reproduced, therefore in this study soil bacteria were screened to verify and reproduce this hypothesis. Survival in high concentrations of antibiotics was initially observed; however, on further analysis these bacteria either did not degrade the antibiotics or they used an intrinsic resistance mechanism (β-lactamases) to degrade the β-lactams, as demonstrated by high-performance liquid chromatography. These results did not verify or reproduce the hypothesis that bacteria subsist on antibiotics or catabolise antibiotics as previously reported. This study identified that bacteria with a catabolising phenotype did not degrade streptomycin or trimethoprim and therefore could not utilise the antibiotics as a nutrient source. Therefore, we conclude that soil bacteria do not catabolise antibiotics.201323507409
932750.9993Detection of the merA gene and its expression in the environment. Bacterial transformation of mercury in the environment has received much attention owing to the toxicity of both the ionic form and organomercurial compounds. Bacterial resistance to mercury and the role of bacteria in mercury cycling have been widely studied. The genes specifying the required functions for resistance to mercury are organized on the mer operon. Gene probing methodologies have been used for several years to detect specific gene sequences in the environment that are homologous to cloned mer genes. While mer genes have been detected in a wide variety of environments, less is known about the expression of these genes under environmental conditions. We combined new methodologies for recovering specific gene mRNA transcripts and mercury detection with a previously described method for determining biological potential for mercury volatilization to examine the effect of mercury concentrations and nutrient availability on rates of mercury volatilization and merA transcription. Levels of merA-specific transcripts and Hg(II) volatilization were influenced more by microbial activity (as manipulated by nutrient additions) than by the concentration of total mercury. The detection of merA-specific transcripts in some samples that did not reduce Hg(II) suggests that rates of mercury volatilization in the environment may not always be proportional to merA transcription.19968849424
26760.9993Molecular characterization of resistance-nodulation-division transporters from solvent- and drug-resistant bacteria in petroleum-contaminated soil. PCR assays for analyzing resistance-nodulation-division transporters from solvent- and drug-resistant bacteria in soil were developed. Sequence analysis of amplicons showed that the PCR successfully retrieved transporter gene fragments from soil. Most of the genes retrieved from petroleum-contaminated soils formed a cluster (cluster PCS) that was distantly related to known transporter genes. Competitive PCR showed that the abundance of PCS genes is increased in petroleum-contaminated soil.200515640241
634570.9993Transfer RNA gene numbers may not be completely responsible for the codon usage bias in asparagine, isoleucine, phenylalanine, and tyrosine in the high expression genes in bacteria. It is generally believed that the effect of translational selection on codon usage bias is related to the number of transfer RNA genes in bacteria, which is more with respect to the high expression genes than the whole genome. Keeping this in the background, we analyzed codon usage bias with respect to asparagine, isoleucine, phenylalanine, and tyrosine amino acids. Analysis was done in seventeen bacteria with the available gene expression data and information about the tRNA gene number. In most of the bacteria, it was observed that codon usage bias and tRNA gene number were not in agreement, which was unexpected. We extended the study further to 199 bacteria, limiting to the codon usage bias in the two highly expressed genes rpoB and rpoC which encode the RNA polymerase subunits β and β', respectively. In concordance with the result in the high expression genes, codon usage bias in rpoB and rpoC genes was also found to not be in agreement with tRNA gene number in many of these bacteria. Our study indicates that tRNA gene numbers may not be the sole determining factor for translational selection of codon usage bias in bacterial genomes.201223053196
381080.9993The Effect of the Presence and Absence of DNA Repair Genes on the Rate and Pattern of Mutation in Bacteria. Bacteria lose and gain repair genes as they evolve. Here, we investigate the consequences of gain and loss of 11 DNA repair genes across a broad range of bacteria. Using synonymous polymorphisms from bacteria and a set of 50 phylogenetically independent contrasts, we find no evidence that the presence or absence of these 11 genes affects either the overall level of diversity or the pattern of mutation. Using phylogenetic generalized linear squares yields a similar conclusion. It seems likely that the lack of an effect is due to variation in the genetic background and the environment which obscures any effects that the presence or absence of individual genes might have.202439376054
436790.9993Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon. Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistance to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the mer operon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram-negative and Gram-positive bacteria from both natural and clinical environments. The presence of mer genes in bacteria from sediment cores suggest that mer is an ancient system. Analysis of DNA sequences from mer operons and genes has revealed genetic variation both in operon structure and between individual genes from different mer operons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non-functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic mer operon and for the Tn21-related family of mer operons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect mer is a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.19979167257
8387100.9993Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.201728189581
6346110.9993Identification of unknown acid-resistant genes of oral microbiotas in patients with dental caries using metagenomics analysis. Acid resistance is critical for the survival of bacteria in the dental caries oral micro-environment. However, there are few acid-resistant genes of microbiomes obtained through traditional molecular biology experimental techniques. This study aims to try macrogenomics technologies to efficiently identify acid-resistant genes in oral microbes of patients with dental caries. Total DNA was extracted from oral microbiota obtained from thirty dental caries patients and subjected to high-throughput sequencing. This data was used to build a metagenomic library, which was compared to the sequences of two Streptococcus mutant known acid-resistant genes, danK and uvrA, using a BLAST search. A total of 19 and 35 unknown gene sequences showed similarities with S. mutans uvrA and dnaK in the metagenomic library, respectively. Two unknown genes, mo-dnaK and mo-uvrA, were selected for primer design and bioinformatic analysis based on their sequences. Bioinformatics analysis predicted them encoding of a human heat-shock protein (HSP) 70 and an ATP-dependent DNA repair enzyme, respectively, closely related with the acid resistance mechanism. After cloning, these genes were transferred into competent Escherichia coli for acid resistance experiments. E. coli transformed with both genes demonstrated acid resistance, while the survival rate of E. coli transformed with mo-uvrA was significantly higher in an acidic environment (pH = 3). Through this experiment we found that identify unknown acid-resistant genes in oral microbes of patients with caries by establishing a metagenomic library is very efficient. Our results provide an insight into the mechanisms and pathogenesis of dental caries for their treatment without affecting oral probiotics.202133675438
171120.9992Codon usage bias reveals genomic adaptations to environmental conditions in an acidophilic consortium. The analysis of codon usage bias has been widely used to characterize different communities of microorganisms. In this context, the aim of this work was to study the codon usage bias in a natural consortium of five acidophilic bacteria used for biomining. The codon usage bias of the consortium was contrasted with genes from an alternative collection of acidophilic reference strains and metagenome samples. Results indicate that acidophilic bacteria preferentially have low codon usage bias, consistent with both their capacity to live in a wide range of habitats and their slow growth rate, a characteristic probably acquired independently from their phylogenetic relationships. In addition, the analysis showed significant differences in the unique sets of genes from the autotrophic species of the consortium in relation to other acidophilic organisms, principally in genes which code for proteins involved in metal and oxidative stress resistance. The lower values of codon usage bias obtained in this unique set of genes suggest higher transcriptional adaptation to living in extreme conditions, which was probably acquired as a measure for resisting the elevated metal conditions present in the mine.201829742107
8678130.9992Metagenomics-Guided Discovery of Potential Bacterial Metallothionein Genes from the Soil Microbiome That Confer Cu and/or Cd Resistance. Metallothionein (MT) genes are valuable genetic materials for developing metal bioremediation tools. Currently, a limited number of prokaryotic MTs have been experimentally identified, which necessitates the expansion of bacterial MT diversity. In this study, we conducted a metagenomics-guided analysis for the discovery of potential bacterial MT genes from the soil microbiome. More specifically, we combined resistance gene enrichment through diversity loss, metagenomic mining with a dedicated MT database, evolutionary trace analysis, DNA chemical synthesis, and functional genomic validation to identify novel MTs. Results showed that Cu stress induced a compositional change in the soil microbiome, with an enrichment of metal-resistant bacteria in soils with higher Cu concentrations. Shotgun metagenomic sequencing was performed to obtain the gene pool of environmental DNA (eDNA), which was subjected to a local BLAST search against an MT database for detecting putative MT genes. Evolutional trace analysis led to the identification of 27 potential MTs with conserved cysteine/histidine motifs different from those of known prokaryotic MTs. Following chemical synthesis of these 27 potential MT genes and heterologous expression in Escherichia coli, six of them were found to improve the hosts' growth substantially and enhanced the hosts' sorption of Cu, Cd, and Zn, among which MT5 led to a 13.7-fold increase in Cd accumulation. Furthermore, four of them restored Cu and/or Cd resistance in two metal-sensitive E. coli strains.IMPORTANCE The metagenomics-guided procedure developed here bypasses the difficulties encountered in classic PCR-based approaches and led to the discovery of novel MT genes, which may be useful in developing bioremediation tools. The procedure used here expands our knowledge on the diversity of bacterial MTs in the environment and may also be applicable to identify other functional genes from eDNA.202032111593
6339140.9992Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment. Microorganisms that thrive in acidic environments are endowed with specialized molecular mechanisms to survive under this extremely harsh condition. In this work, we performed functional screening of six metagenomic libraries from planktonic and rhizosphere microbial communities of the Tinto River, an extremely acidic environment, to identify genes involved in acid resistance. This approach has revealed 15 different genes conferring acid resistance to Escherichia coli, most of which encoding putative proteins of unknown function or previously described proteins not known to be related to acid resistance. Moreover, we were able to assign function to one unknown and three hypothetical proteins. Among the recovered genes were the ClpXP protease, the transcriptional repressor LexA and nucleic acid-binding proteins such as an RNA-binding protein, HU and Dps. Furthermore, nine of the retrieved genes were cloned and expressed in Pseudomonas putida and Bacillus subtilis and, remarkably, most of them were able to expand the capability of these bacteria to survive under severe acid stress. From this set of genes, four presented a broad-host range as they enhance the acid resistance of the three different organisms tested. These results expand our knowledge about the different strategies used by microorganisms to survive under extremely acid conditions.201323145860
477150.9992Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage. Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.200717675438
9268160.9992The expression of integron arrays is shaped by the translation rate of cassettes. Integrons are key elements in the rise and spread of multidrug resistance in Gram-negative bacteria. These genetic platforms capture cassettes containing promoterless genes and stockpile them in arrays of variable length. In the current integron model, expression of cassettes is granted by the P(c) promoter in the platform and is assumed to decrease as a function of its distance. Here we explored this model using a large collection of 136 antibiotic resistance cassettes and show the effect of distance is in fact negligible. Instead, cassettes have a strong impact in the expression of downstream genes because their translation rate affects the stability of the whole polycistronic mRNA molecule. Hence, cassettes with reduced translation rates decrease the expression and resistance phenotype of cassettes downstream. Our data puts forward an integron model in which expression is contingent on the translation of cassettes upstream, rather than on the distance to the P(c).202439455579
8384170.9992In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors. Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut.201425408687
6335180.9992Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli. The activation of unrecognized antibiotic resistance genes in the bacterial cell can give rise to antibiotic resistance without the need for major mutations or horizontal gene transfer. We hypothesize that bacteria harbor an extensive array of diverse cryptic genes that can be activated in response to antibiotics via adaptive resistance. To test this hypothesis, we developed a plasmid assay to randomly manipulate gene copy numbers in Escherichia coli cells and identify genes that conferred resistance when amplified. We then tested for cryptic resistance to 18 antibiotics and identified genes conferring resistance. E. coli could become resistant to 50% of the antibiotics tested, including chloramphenicol, d-cycloserine, polymyxin B, and 6 beta-lactam antibiotics, following this manipulation. Known antibiotic resistance genes comprised 13% of the total identified genes, where 87% were unclassified (cryptic) antibiotic resistance genes. These unclassified genes encoded cell membrane proteins, stress response/DNA repair proteins, transporters, and miscellaneous or hypothetical proteins. Stress response/DNA repair genes have a broad antibiotic resistance potential, as this gene class, in aggregate, conferred cryptic resistance to nearly all resistance-positive antibiotics. We found that antibiotics that are hydrophilic, those that are amphipathic, and those that inhibit the cytoplasmic membrane or cell wall biosynthesis were more likely to induce cryptic resistance in E. coli. This study reveals a diversity of cryptic genes that confer an antibiotic resistance phenotype when present in high copy number. Thus, our assay can identify potential novel resistance genes while also describing which antibiotics are prone to induce cryptic antibiotic resistance in E. coli. IMPORTANCE Predicting where new antibiotic resistance genes will rise is a challenge and is especially important when new antibiotics are developed. Adaptive resistance allows sensitive bacterial cells to become transiently resistant to antibiotics. This provides an opportune time for cells to develop more efficient resistance mechanisms, such as tolerance and permanent resistance to higher antibiotic concentrations. The biochemical diversity harbored within bacterial genomes may lead to the presence of genes that could confer resistance when timely activated. Therefore, it is crucial to understand adaptive resistance to identify potential resistance genes and prolong antibiotics. Here, we investigate cryptic resistance, an adaptive resistance mechanism, and identify unknown (cryptic) antibiotic resistance genes that confer resistance when amplified in a laboratory strain of E. coli. We also pinpoint antibiotic characteristics that are likely to induce cryptic resistance. This study may help detect novel antibiotic resistance genes and provide the foundation to help develop more effective antibiotics.202134756069
3824190.9992Screening for novel antibiotic resistance genes. Knowledge of novel antibiotic resistance genes aids in the understanding of how antibiotics function and how bacteria fight them. This knowledge also allows future generations of an antibiotic or antibiotic group to be altered to allow the greatest efficacy. The method described here is very simple in theory. The bacterial strains are screened for antibiotic resistance. Cultures of the strain are grown, and DNA is extracted. A partial digest of the extraction is cloned into Escherichia coli, and the transformants are plated on selective media. Any colony that grows will possess the antibiotic resistance gene and can be further examined. In actual practice, however, this technique can be complicated. The detailed protocol will need to be optimized for each bacterial strain, vector, and cell line chosen.201020830570