RESUMO
Specifications grading is a relatively recent approach to assessing student learning. In this approach, students make progress toward completion of a course by demonstrating mastery of specific skills or material. The assessment tools are short, frequent exercises that can be attempted multiple times until mastered. This contrasts with the traditional, exam-based assessment of student learning. There are multiple benefits to the specifications grading-based strategy, including reduced test anxiety, better knowledge retention, and increased flexibility. In this study, specifications grading was implemented into an upper-level biochemistry course at a private, liberal arts university. The student cohort consisted almost exclusively of junior and senior biochemistry, biology, and chemistry majors. Students earned points for demonstrating mastery on each of 12 short quizzes in addition to points earned from laboratory exercises and on the cumulative final exam. Student attitudes were assessed using three surveys that were administered at the beginning, middle, and end of the course. The survey results indicated that the students had overall favorable opinions of the specifications grading approach and its use in this course. A comparison of student performance on the quizzes to their performance on the final exam showed that the students learned and retained the course material. Combining the survey and performance data, we demonstrated that the students' perceptions of their learning correlated well with their performances on the specifications grading tools. Together, these results indicated that specifications grading is an effective approach to assessing student learning and to maintaining student enthusiasm in an upper-level biochemistry course.
RESUMO
Combatting antibiotic resistance is critical to our ability to treat infectious diseases. Here, we identified and characterized diverse antimicrobial resistance genes, including potentially mobile elements, from synthetic wastewater treatment microcosms exposed to the antibacterial agent triclosan. After seven weeks of exposure, the microcosms were subjected to functional metagenomic selection across 13 antimicrobials. This was achieved by cloning the combined genetic material from the microcosms, introducing this genetic library into E. coli, and selecting for clones that grew on media supplemented with one of the 13 antimicrobials. We recovered resistant clones capable of growth on media supplemented with a single antimicrobial, yielding 13 clones conferring resistance to at least one antimicrobial agent. Antibiotic susceptibility analysis revealed resistance ranging from 4 to >50 fold more resistant, while one clone showed resistance to multiple antibiotics. Using both Sanger and SMRT sequencing, we identified the predicted active gene(s) on each clone. One clone that conferred resistance to tetracycline contained a gene encoding a novel tetA-type efflux pump that was named TetA(62). Three clones contained predicted active genes on class 1 integrons. One integron had a previously unreported genetic arrangement and was named In1875. This study demonstrated the diversity and potential for spread of resistance genes present in human-impacted environments.
Assuntos
Integrons , Águas Residuárias , Antibacterianos/farmacologia , Bactérias/genética , Resistência Microbiana a Medicamentos , Farmacorresistência Bacteriana Múltipla , Escherichia coli/genética , Humanos , Integrons/genética , Metagenômica , PlasmídeosRESUMO
The spread of antibiotic-resistant bacteria presents a global health challenge. Efficient surveillance of bacteria harboring antibiotic resistance genes (ARGs) is a critical aspect to controlling the spread. Increased access to microbial genomic data from many diverse populations informs this surveillance but only when functional ARGs are identifiable within the data set. Current, homology-based approaches are effective at identifying the majority of ARGs within given clinical and nonclinical data sets for several pathogens, yet there are still some whose identities remain elusive. By coupling phenotypic profiling with genotypic data, these unknown ARGs can be identified to strengthen homology-based searches. To prove the efficacy and feasibility of this approach, a published data set from the U.S. National Antimicrobial Resistance Monitoring System (NARMS), for which the phenotypic and genotypic data of 640 Salmonella isolates are available, was subjected to this analysis. Six isolates recovered from the NARMS retail meat program between 2011 and 2013 were identified previously as phenotypically resistant to gentamicin but contained no known gentamicin resistance gene. Using the phenotypic and genotypic data, a comparative genomics approach was employed to identify the gene responsible for the observed resistance in all six of the isolates. This gene, grdA, is harbored on a 9,016-bp plasmid that is transferrable to Escherichia coli, confers gentamicin resistance to E. coli, and has never before been reported to confer gentamicin resistance. Bioinformatic analysis of the encoded protein suggests an ATP binding motif. This work demonstrates the advantages associated with coupling genomics technologies with phenotypic data for novel ARG identification.
Assuntos
Farmacorresistência Bacteriana Múltipla , Gentamicinas , Perus/microbiologia , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Gentamicinas/farmacologia , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Salmonella/genéticaRESUMO
BACKGROUND/AIMS: We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in Escherichia coli. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. METHODS: ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into E. coli, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. RESULTS: Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into E. coli, whereas the mutant remained susceptible to triclosan. Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. CONCLUSION: ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.
Assuntos
Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Oxirredutases/metabolismo , Triclosan/farmacologia , Sequência de Aminoácidos , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Mutação , Homologia de Sequência de AminoácidosRESUMO
Streptomycin is commonly used to control fire blight disease on apple trees. Although the practice has incited controversy, little is known about its nontarget effects in the environment. We investigated the impact of aerial application of streptomycin on nontarget bacterial communities in soil beneath streptomycin-treated and untreated trees in a commercial apple orchard. Soil samples were collected in two consecutive years at 4 or 10 days before spraying streptomycin and 8 or 9 days after the final spray. Three sources of microbial DNA were profiled using tag-pyrosequencing of 16S rRNA genes: uncultured bacteria from the soil (culture independent) and bacteria cultured on unamended or streptomycin-amended (15 µg/ml) media. Multivariate tests for differences in community structure, Shannon diversity, and Pielou's evenness test results showed no evidence of community response to streptomycin. The results indicate that use of streptomycin for disease management has minimal, if any, immediate effect on apple orchard soil bacterial communities. This study contributes to the profile of an agroecosystem in which antibiotic use for disease prevention appears to have minimal consequences for nontarget bacteria.
Assuntos
Agricultura/métodos , Malus/microbiologia , Microbiota/efeitos dos fármacos , Controle de Pragas/métodos , Microbiologia do Solo , Estreptomicina/farmacologia , Análise de Variância , Microbiota/genética , Análise Multivariada , RNA Ribossômico 16S/genética , Estreptomicina/efeitos adversos , WisconsinRESUMO
Traditional undergraduate science classes often include a laboratory component aimed at enabling the students to experience the classroom topics firsthand. Typically, these experiments are chosen because they have known outcomes that will clearly demonstrate particular aspects of scientific theory. While this approach has its benefits in skill development and concept reinforcement, the lack of novelty inherent in repeating experiments that have been repeated for many years does not accurately convey the feeling of true scientific discovery to the students. In this work, we have designed and implemented a series of experiments into an undergraduate biochemistry curriculum that incorporates the opportunity for scientific discovery, while simultaneously creating an environment for learning routine laboratory techniques. Through this set of experiments, students enrolled in the course were successful in identifying and beginning to characterize an unknown bacterial gene that confers increased tolerance to triclosan on its host.
Assuntos
Bioquímica/educação , Escherichia coli/efeitos dos fármacos , Laboratórios , Metagenômica/educação , Oxirredutases/genética , Triclosan/farmacologia , Universidades , Antibacterianos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Pesquisa/educaçãoRESUMO
We designed a week-long laboratory workshop in metagenomics for a cohort of undergraduate student researchers. During this course, students learned and utilized molecular biology and microbiology techniques to construct a metagenomic library from Puerto Rican soil. Pre-and postworkshop assessments indicated student learning gains in technical knowledge, skills, and confidence in a research environment. Postworkshop construction of additional libraries demonstrated retention of research techniques by the students.
RESUMO
To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibiotic-resistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a beta-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional beta-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a beta-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance.
Assuntos
DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli , Malus/crescimento & desenvolvimento , Metagenômica/métodos , Proteínas , Solo/análise , Acetiltransferases/genética , Acetiltransferases/metabolismo , Antibacterianos/farmacologia , Cloranfenicol/farmacologia , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Canamicina/farmacologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Proteínas/genética , Proteínas/metabolismo , Análise de Sequência de DNA , Transposases/genética , Transposases/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Antibiotic-resistant pathogens are profoundly important to human health, but the environmental reservoirs of resistance determinants are poorly understood. The origins of antibiotic resistance in the environment is relevant to human health because of the increasing importance of zoonotic diseases as well as the need for predicting emerging resistant pathogens. This Review explores the presence and spread of antibiotic resistance in non-agricultural, non-clinical environments and demonstrates the need for more intensive investigation on this subject.
Assuntos
Farmacorresistência Bacteriana/genética , Genes Bacterianos , Antibacterianos/uso terapêutico , Meio Ambiente , Monitoramento Ambiental , Bactérias Gram-Negativas/genética , Humanos , Cinética , Seleção Genética , Movimentos da Água , VentoRESUMO
BACKGROUND: In analyzing the stability of DNA replication origins in Saccharomyces cerevisiae we faced the question whether one set of sequences is significantly enriched in the number and/or the quality of the matches of a particular position weight matrix relative to another set. RESULTS: We present SADMAMA, a computational solution to a address this problem. SADMAMA implements two types of statistical tests to answer this question: one type is based on simplified models, while the other relies on bootstrapping, and as such might be preferable to users who are averse to such models. The bootstrap approach incorporates a novel "site-protected" resampling procedure which solves a problem we identify with naive resampling. CONCLUSION: SADMAMA's utility is demonstrated here by offering a plausible explanation to the differential ARS activity observed in our previous mcm1-1 mutant experiments 1, by suggesting the relevance of multiple weak ACS matches to efficient replication origin function in Saccharomyces cerevisiae, and by suggesting an explanation to the observed negative effect FKH2 has on chromatin silencing 2. SADMAMA is available for download from http://www.cs.cornell.edu/~keich/.
Assuntos
DNA Fúngico/genética , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Interpretação Estatística de Dados , Variação Genética/genética , Modelos Genéticos , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Replication origins in a genome are inherently different in their base sequence and in their response to temporal and cell cycle regulation signals for DNA replication. To investigate the chromosomal determinants that influence the efficiency of initiation of DNA replication genome-wide, we made use of a reverse strategy originally used for the isolation of replication initiation mutants in Saccharomyces cerevisiae. In yeast, replication origins isolated from chromosomes support the autonomous replication of plasmids. These replication origins, whether in the context of a chromosome or a plasmid, will initiate efficiently in wild-type cells but show a dramatically contrasted efficiency of activation in mutants defective in the early steps of replication initiation. Serial passages of a genomic library of autonomously replicating sequences (ARSs) in such a mutant allowed us to select for constitutively active ARSs. We found a hierarchy of preferential initiation of ARSs that correlates with local transcription patterns. This preferential usage is enhanced in mutants defective in the assembly of the prereplication complex (pre-RC) but not in mutants defective in the activation of the pre-RC. Our findings are consistent with an interference of local transcription with the assembly of the pre-RC at a majority of replication origins.
Assuntos
Genoma Fúngico , Origem de Replicação/fisiologia , Saccharomyces cerevisiae/genética , DNA Fúngico , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Proteína 1 de Manutenção de Minicromossomo/genética , Modelos Biológicos , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/fisiologia , Plasmídeos/isolamento & purificação , Transcrição GênicaRESUMO
Recent studies linking DNA replication proteins to transcriptional silencing suggest that some of the same mechanisms that facilitate the initiation of replication at origins might be involved in establishing repressed chromatin at silencer elements. Our ongoing studies of several mutants of the replication initiation factor Mcm10 of budding yeast revealed an associated defect in the production of mating type pheromones. This observation prompted us to look more directly at the effect of MCM10 mutations on the expression of a reporter gene in the mating type locus and to assay for physical interactions between Mcm10 and known silencing factors. Our findings, that Mcm10 mutants disrupt mating loci silencing and that Mcm10 interacts with Sir2 and Sir3, suggest that Mcm10 also plays an essential, and separable role in transcriptional silencing.
Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , Genes Fúngicos Tipo Acasalamento/genética , Proteínas de Saccharomyces cerevisiae/genética , Elementos Silenciadores Transcricionais/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona , Genes Reporter/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteínas de Manutenção de Minicromossomo , Mutação/genética , Feromônios/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2 , Sirtuínas/genética , Sirtuínas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Minichromosome maintenance protein 1 (Mcm1) is required for efficient replication of autonomously replicating sequence (ARS)-containing plasmids in yeast cells. Reduced DNA binding activity in the Mcm1-1 mutant protein (P97L) results in selective initiation of a subset of replication origins and causes instability of ARS-containing plasmids. This plasmid instability in the mcm1-1 mutant can be overcome for a subset of ARSs by the inclusion of flanking sequences. Previous work showed that Mcm1 binds sequences flanking the minimal functional domains of ARSs. Here, we dissected two conserved telomeric X ARSs, ARS120 (XARS6L) and ARS131a (XARS7R), that replicate with different efficiencies in the mcm1-1 mutant. We found that additional Mcm1 binding sites in the C domain of ARS120 that are missing in ARS131a are responsible for efficient replication of ARS120 in the mcm1-1 mutant. Mutating a conserved Mcm1 binding site in the C domain diminished replication efficiency in ARS120 in wild-type cells, and increasing the number of Mcm1 binding sites stimulated replication efficiency. Our results suggest that threshold occupancy of Mcm1 in the C domain of telomeric ARSs is required for efficient initiation. We propose that origin usage in Saccharomyces cerevisiae may be regulated by the occupancy of Mcm1 at replication origins.
Assuntos
Replicação do DNA , DNA/metabolismo , Proteína 1 de Manutenção de Minicromossomo/metabolismo , Origem de Replicação , Sequência de Bases , Sítios de Ligação , Cromossomos Fúngicos , Pegada de DNA , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The Saccharomyces cerevisiae Mcm7 protein is a subunit of the presumed heteromeric MCM helicase that melts origin DNA and unwinds replication forks. Previous work showed that Mcm1 binds constitutively to the MCM7 promoter and regulates MCM7 expression. Here, we identify Mcm7 as a novel cofactor of Mcm1 in the regulation of MCM7 expression. Transcription of MCM7 is increased in the mcm7-1 mutant and decreased in the mcm1-1 mutant, suggesting that Mcm7 modulates its own expression in conjunction with Mcm1. Indeed, Mcm7 stimulates Mcm1 binding to the early cell cycle box upstream of the promoters of MCM7 as well as CDC6 and MCM5. Whereas Mcm1 binds these promoters constitutively, Mcm7 is recruited during late M phase, consistent with Mcm7 playing a direct role in modulating the periodic expression of early cell cycle genes. The multiple roles of Mcm7 in replication initiation, replication elongation, and autoregulation parallel those of the oncoprotein, the large T-antigen of the SV40 virus.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Proteínas de Ligação a DNA/biossíntese , Proteína 1 de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/biossíntese , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Antígenos Transformantes de Poliomavirus/metabolismo , Sequência de Bases , Northern Blotting , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Eletroforese em Gel de Poliacrilamida , Formaldeído/farmacologia , Proteína 1 de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo , Mitose , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , TemperaturaRESUMO
Mcm1 is an essential protein required for the efficient replication of minichromosomes and the transcriptional regulation of early cell cycle genes in Saccharomyces cerevisiae. In this study, we report that Mcm1 is an abundant protein that associates globally with chromatin in a punctate pattern. We show that Mcm1 is localized at replication origins and plays an important role in the initiation of DNA synthesis at a chromosomal replication origin in vivo. Using purified Mcm1 protein, we show that Mcm1 binds cooperatively to multiple sites at autonomously replicating sequences. These results suggest that, in addition to its role as a transcription factor for the expression of replication genes, Mcm1 may influence the local structure of replication origins by direct binding.