Data Description: Photo Documentation of the North Campus Open Space Restoration Project

This document describes the content, purpose, methods and uses of the photo documentation data set for the UC Santa Barbara North Campus Open Space (NCOS) Restoration Project. Links to the photos and map are provided

Transposon and deletion mutagenesis of genes involved in perchlorate reduction in Azospira suillum PS.

UnlabelledAlthough much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism.ImportanceAlthough perchlorate is generated naturally in the environment, groundwater contamination is largely a result of industrial activity. Bacteria capable of respiring perchlorate and remediating contaminated water have been isolated, but relatively little is known about the biochemistry and genetics of this process. Here we used two complementary approaches to identify genes involved in perchlorate reduction. Most of these genes are located on a genomic island, which is potentially capable of moving between organisms. Some of the genes identified are known to be directly involved in the metabolism of perchlorate, but other new genes likely regulate the metabolism in response to environmental signals. This work has uncovered new questions about the regulation, energetics, and evolution of perchlorate reduction but also presents the tools to address them

Structure and evolution of chlorate reduction composite transposons.

UnlabelledThe genes for chlorate reduction in six bacterial strains were analyzed in order to gain insight into the metabolism. A newly isolated chlorate-reducing bacterium (Shewanella algae ACDC) and three previously isolated strains (Ideonella dechloratans, Pseudomonas sp. strain PK, and Dechloromarinus chlorophilus NSS) were genome sequenced and compared to published sequences (Alicycliphilus denitrificans BC plasmid pALIDE01 and Pseudomonas chloritidismutans AW-1). De novo assembly of genomes failed to join regions adjacent to genes involved in chlorate reduction, suggesting the presence of repeat regions. Using a bioinformatics approach and finishing PCRs to connect fragmented contigs, we discovered that chlorate reduction genes are flanked by insertion sequences, forming composite transposons in all four newly sequenced strains. These insertion sequences delineate regions with the potential to move horizontally and define a set of genes that may be important for chlorate reduction. In addition to core metabolic components, we have highlighted several such genes through comparative analysis and visualization. Phylogenetic analysis places chlorate reductase within a functionally diverse clade of type II dimethyl sulfoxide (DMSO) reductases, part of a larger family of enzymes with reactivity toward chlorate. Nucleotide-level forensics of regions surrounding chlorite dismutase (cld), as well as its phylogenetic clustering in a betaproteobacterial Cld clade, indicate that cld has been mobilized at least once from a perchlorate reducer to build chlorate respiration.ImportanceGenome sequencing has identified, for the first time, chlorate reduction composite transposons. These transposons are constructed with flanking insertion sequences that differ in type and orientation between organisms, indicating that this mobile element has formed multiple times and is important for dissemination. Apart from core metabolic enzymes, very little is known about the genetic factors involved in chlorate reduction. Comparative analysis has identified several genes that may also be important, but the relative absence of accessory genes suggests that this mobile metabolism relies on host systems for electron transport, regulation, and cofactor synthesis. Phylogenetic analysis of Cld and ClrA provides support for the hypothesis that chlorate reduction was built multiple times from type II dimethyl sulfoxide (DMSO) reductases and cld. In at least one case, cld has been coopted from a perchlorate reduction island for this purpose. This work is a significant step toward understanding the genetics and evolution of chlorate reduction

North Campus Open Space Restoration Project As-Built Grading and Hydrology Report

This report describes the as-built geomorphology and hydrology of the North Campus Open Space (NCOS) restoration project at the completion of the grading phase of the project. Topics covered include: a comparison of the predicted and as-built grading elevations, as-built cross-sections and thalwegs of the two main channels, and a comparison of the hydrology of the project site before and after the completion of the grading phase. A selection of photos of the project site taken before and after the completion of grading are provided at the end of the report. Ongoing project work not covered in this report includes: trail and bridge construction, planting and site maintenance, and the planned construction of a visitor interpretative plaza and maintenance equipment shed

North Campus Open Space Restoration Project Annual Monitoring Report: Year 2 (2019)

Born out of a vision shared by the local community, students, faculty, researchers and state and federal agencies, the North Campus Open Space (NCOS) restoration project is recreating more than 40 acres of estuarine and palustrine wetlands that historically comprised the upper portion of Devereux Slough that was filled in the mid-1960s to create the Ocean Meadows golf course. Led by the UC Santa Barbara Cheadle Center for Biodiversity and Ecological Restoration (CCBER) in collaboration with other UCSB departments, faculty, student and local community groups, contractors and government agencies, the project is also restoring more than 60 acres of upland habitats that include native grassland, coastal sage scrub, riparian, oak chaparral woodland, vernal pools and patches of annual wildflowers in clay and sandy soils. In addition to wetland and upland habitat restoration, the goals of the NCOS project include flood reduction, support for threatened and endangered species, public access and the provision of educational opportunities. Ancillary benefits of the project include carbon sequestration, preservation of local genotypes, and protection of adjacent ecological values and infrastructure through a design that integrates sea level rise considerations.Currently in its third year of implementation, the main planting phase of the project is approximately 90% complete, and the focus is now turning towards maintenance, continued monitoring, new research projects, and supplemental planting to add diversity, including special status species such as the Ventura marsh milk-vetch (Astragalus pycnostachys var. lanosissimus). This report describes the methods and results of monitoring for the first two years of the project, from vegetation and wildlife to wetland geomorphology, hydrology and water quality, carbon sequestration studies, community use and a detailed record of restoration efforts by type of worker, task and site location. This work documents the progress of the project and supports longer-term research and monitoring programs. Results from the second year of monitoring show substantial progress towards the project’s restoration goals, with many being met or exceeded

Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

UnlabelledDespite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth.ImportanceThe ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism

A pilot study of the S-MAP (Solutions for Medications Adherence Problems) intervention for older adults prescribed polypharmacy in primary care: Study protocol

Background: Adhering to multiple medications as prescribed is challenging for older patients (aged ≥ 65 years) and a difficult behaviour to improve. Previous interventions designed to address this have been largely complex in nature but have shown limited effectiveness and have rarely used theory in their design. It has been recognised that theory ('a systematic way of understanding events or situations') can guide intervention development and help researchers better understand how complex adherence interventions work. This pilot study aims to test a novel community pharmacy-based intervention that has been systematically developed using the Theoretical Domains Framework (12-domain version) of behaviour change. Methods: As part of a non-randomised pilot study, pharmacists in 12 community pharmacies across Northern Ireland (n = 6) and London, England (n = 6), will be trained to deliver the intervention to older patients who are prescribed ≥ 4 regular medicines and are non-adherent (self-reported). Ten patients will be recruited per pharmacy (n = 120) and offered up to four tailored one-to-one sessions, in the pharmacy or via telephone depending on their adherence, over a 3-4-month period. Guided by an electronic application (app) on iPads, the intervention content will be tailored to each patient's underlying reasons for non-adherence and mapped to the most appropriate solutions using established behaviour change techniques. This study will assess the feasibility of collecting data on the primary outcome of medication adherence (self-report and dispensing data) and secondary outcomes (health-related quality of life and unplanned hospitalisations). An embedded process evaluation will assess training fidelity for pharmacy staff, intervention fidelity, acceptability to patients and pharmacists and the intervention's mechanism of action. Process evaluation data will include audio-recordings of training workshops, intervention sessions, feedback interviews and patient surveys. Analysis will be largely descriptive. Discussion: Using pre-defined progression criteria, the findings from this pilot study will guide the decision whether to proceed to a cluster randomised controlled trial to test the effectiveness of the S-MAP intervention in comparison to usual care in community pharmacies. The study will also explore how the intervention components may work to bring about change in older patients' adherence behaviour and guide further refinement of the intervention and study procedures. Trial registration: This study is registered at ISRCTN: https://doi.org/10.1186/ISRCTN7383153