Susan K. De Long

Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.

A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols.

Fusel alcohols are natural products of amino acid catabolism in the yeast Saccharomyces cerevisiae that cause morphological changes similar to those seen during pseudohyphal growth. We have discovered that certain of these alcohols, including butanol and isoamyl alcohol, bring about a rapid inhibition of translation at the initiation step. This inhibition is strain specific and is not explained by previously described translational control pathways. Using genetic mapping, we have identified a proline to serine allelic variation at amino acid 180 of the GCD1 gene product as the genetic locus that allows translational regulation upon butanol addition. Gcd1p forms part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2‐GDP to eIF2‐GTP. This represents one of the key limiting steps of translation initiation and we provide evidence that fusel alcohols target eIF2B in order to bring about translational regulation.

Prokaryotic suppression subtractive hybridization PCR cDNA subtraction, a targeted method to identify differentially expressed genes.

ABSTRACT Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.

Microbial community acclimation enhances waste hydrolysis rates under elevated ammonia and salinity conditions

Hydrolysis rates under potentially inhibitory concentrations of ammonia and salinity were investigated for two model feedstocks (manure and food waste). Rates were determined under a range of ammonia and salinity concentrations (1.0-10.0 g TAN [total ammonia nitrogen] L(-1) and 3.9-20.0 g sodium L(-1)) with unacclimated and acclimated microbial inocula. Microbial community changes as a function of acclimation and feedstock were also investigated. Using unacclimated inocula, hydrolysis was found to be severely inhibited for elevated ammonia and salinity (~4 to 10-fold, respectively) or hydrolysis was not detected. However, for inocula acclimated over 2-4 months, statistically significant inhibition generally was not detectable. Molecular analyses demonstrated that microbial community composition changed during acclimation, and bacterial communities under elevated ammonia were distinct from communities under elevated salinity. Feedstock source also had a major influence on bacterial community structure.

qPCR assays to quantify genes and gene expression associated with microbial perchlorate reduction

Quantitative PCR (qPCR) assays targeting cld (developed in this work) and pcrA (previously described) were used to quantify these perchlorate-related genes in a perchlorate-reducing enrichment culture. Transcript copies were quantified in perchlorate-reducing Rhodocyclaceae strain JDS4. Oxygen and nitrate inhibited expression of cld and pcrA.

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress.

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.

Impact of inoculum sources on biotransformation of pharmaceuticals and personal care products

Limited knowledge of optimal microbial community composition for PPCP biotreatment, and of the microbial phylotypes that drive biotransformation within mixed microbial communities, has hindered the rational design and operation of effective and reliable biological PPCP treatment technologies. Herein, bacterial community composition was investigated as an isolated variable within batch biofilm reactors via comparison of PPCP removals for three distinct inocula. Inocula pre-acclimated to model PPCPs were derived from activated sludge (AS), ditch sediment historically-impacted by wastewater treatment plant effluent (Sd), and material from laboratory-scale soil aquifer treatment (SAT) columns. PPCP removals were found to be substantially higher for AS- and Sd-derived inocula compared to the SAT-derived inocula despite comparable biomass. Removal patterns differed among the 6 model compounds examined (diclofenac, 5-fluorouracil, gabapentin, gemfibrozil, ibuprofen, and triclosan) indicating differences in biotransformation mechanisms. Sphingomonas, Beijerinckia, Methylophilus, and unknown Cytophagaceae were linked with successful PPCP biodegradation via next-generation sequencing of 16S rRNA genes over time. Results indicate the criticality of applying engineering approaches to control bacterial community compositions in biotreatment systems.

Investigation of pathogen disinfection and regrowth in a simple graywater recycling system for toilet flushing

AbstractGraywater treatment systems must inactivate pathogens, prevent regrowth, be low cost, and be simple to operate to support their widespread adoption for alleviating water stress. A treatment system comprised only of filtration and disinfection could meet these constraints. To investigate pathogen disinfection and regrowth in such a system with minimal organic matter removal, herein three disinfectants (chlorine, ultraviolet irradiation, and ozone) were tested in combination with three filter types (coarse, sand, and cartridge) for inactivation of pathogens in graywater from the showers and hand washbasins of 14 student residences. Graywater was spiked with bacterial and viral pathogens or surrogates post-filtration. Chlorination post-filtration achieved log reductions greater than 7.1, 8.0, and 7.4 for Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, respectively, and 3.8 for MS2 bacteriophage. UV was similarly effective, but would not prevent regrowth without a disinfectant resid...

Development of a Screening Assay for Surrogate Markers of Chk1 Inhibitor-Induced Cell Cycle Release

Chk1 is a key regulator of the S and G2/M checkpoints and is activated following DNA damage by agents such as the topoisomerase I inhibitor camptothecin (CPT). It has been proposed that Chk1 inhibitors used in combination with such a DNA damaging agent to treat tumors would potentiate cytotoxicity and increase the therapeutic index, particularly in tumors lacking functional p53. The aim of this study was to determine whether gene expression analysis could be used to inform lead optimization of a novel series of Chk1 inhibitors. The candidate small-molecule Chk1 inhibitors were used in combination with CPT to identify potential markers of functional Chk1 inhibition, as well as resulting cell cycle progression, using cDNA-based microarrays. Differential expression of several of these putative marker genes was further validated by RT-PCR for use as a medium-throughput assay. In the presence of DNA damage, Chk1 inhibitors altered CPT-dependent effects on the expression of cell cycle and DNA repair genes in a manner consistent with a Chk1-specific mechanism of action. Furthermore, differential expression of selected marker genes, cyclin E2, EGR1, and DDIT3, was dose dependent for Chk1 inhibition. RT-PCR results for these genes following treatment with a panel of Chk1 inhibitors showed a strong correlation between marker gene response and the ability of each compound to abrogate cell cycle arrest in situ following CPT-induced DNA damage. These results demonstrate the utility of global expression analysis to identify surrogate markers, providing an alternative method for rapid compound characterization to support advancement decisions in early drug discovery.

Enhanced anaerobic digestion performance via combined solids- and leachate-based hydrolysis reactor inoculation

Suboptimal conditions in anaerobic digesters (e.g., presence of common inhibitors ammonia and salinity) limit waste hydrolysis and lead to unstable performance and process failures. Application of inhibitor-tolerant inocula improves hydrolysis, but approaches are needed to establish and maintain these desired waste-hydrolyzing bacteria in high-solids reactors. Herein, performance was compared for leach bed reactors (LBRs) seeded with unacclimated or acclimated inoculum (0-60% by mass) at start-up and over long-term operation. High quantities of inoculum (∼60%) increase waste hydrolysis and are beneficial at start-up or when inhibitors are increasing. After start-up (∼112days) with high inoculum quantities, leachate recirculation leads to accumulation of inhibitor-tolerant hydrolyzing bacteria in leachate. During long-term operation, low inoculum quantities (∼10%) effectively increase waste hydrolysis relative to without solids-derived inoculum. Molecular analyses indicated that combining digested solids with leachate-based inoculum doubles quantities of Bacteria contacting waste over a batch and supplies additional desirable phylotypes Bacteriodes and Clostridia.