Proximity-Ligation Metagenomic Sequence Analysis Reveals That the Antibiotic Resistome Makes Significant Transitions During Municipal Wastewater Treatment.

Shotgun and proximity-ligation metagenomic sequencing were used to generate thousands of metagenome assembled genomes (MAGs) from the untreated wastewater, activated sludge bioreactors, and anaerobic digesters from two full-scale municipal wastewater treatment facilities. Analysis of the antibiotic resistance genes (ARGs) in the pool of contigs from the shotgun metagenomic sequences revealed significantly different relative abundances and types of ARGs in the untreated wastewaster compared to the activated sludge bioreactors or the anaerobic digesters (p < 0.05).

Specifications Grading Is an Effective Approach to Teaching Biochemistry.

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.

Novel class 1 integron harboring antibiotic resistance genes in wastewater-derived bacteria as revealed by functional metagenomics.

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.

Identification of a Novel Plasmid-Borne Gentamicin Resistance Gene in Nontyphoidal Isolated from Retail Turkey.

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.

ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli.

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.

The introduction of metagenomics into an undergraduate biochemistry laboratory course yielded a predicted reductase that decreases triclosan susceptibility in Escherichia coli.

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.

Summer workshop in metagenomics: one week plus eight students equals gigabases of cloned DNA.

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.

Metagenomic analysis of apple orchard soil reveals antibiotic resistance genes encoding predicted bifunctional proteins.

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.

Genome-wide hierarchy of replication origin usage in Saccharomyces cerevisiae.

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.

Dual roles for Mcm10 in DNA replication initiation and silencing at the mating-type loci.

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.

Mcm1 promotes replication initiation by binding specific elements at replication origins.

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.

Mcm7, a subunit of the presumptive MCM helicase, modulates its own expression in conjunction with Mcm1.

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.

Mcm1 binds replication origins.

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.