Comparative sequence analysis of IS50/Tn5 transposase

Author Posting. © American Society for Microbiology, 2004. This article is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Journal of Bacteriology 186 (2004): 8240-8247, doi:10.1128/JB.186.24.8240-8247.2004.Comparative sequence analysis of IS50 transposase-related protein sequences in conjunction with known structural, biochemical, and genetic data was used to determine domains and residues that play key roles in IS50 transposase function. BLAST and ClustalW analyses have been used to find and analyze six complete protein sequences that are related to the IS50 transposase. The protein sequence identity of these six homologs ranged from 25 to 55% in comparison to the IS50 transposase. Homologous motifs were found associated with each of the three catalytic residues. Residues that play roles in transposase-DNA binding, protein autoregulation, and DNA hairpin formation were also found to be conserved in addition to other residues of unknown function. On the other hand, some homologous sequences did not appear to be competent to encode the inhibitor regulatory protein. The results were also used to compare the IS50 transposase with the more distantly related transposase encoded by IS10.J.A. was supported by a grant from the National Science Foundation (MCB0084089) administered by W.S.R. S.R.B. held a National Research Council Research Associateship Award. W.S.R. is the Evelyn Mercer Professor of Biochemistry and Molecular Biology. Additional thanks are given to the NASA Astrobiology Institute (Cooperative Agreement NNA04CC04A to Mitchell L. Sogin) and the W. M. Keck Ecological and Evolutionary Genetics Facility within the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution at the M.B.L. Molecular graphics images were produced by using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (supported by NIH grant P41 RR-01081)

The Png1–Rad23 complex regulates glycoprotein turnover

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1–Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways

Evolution of the metabolic and regulatory networks associated with oxygen availability in two phytopathogenic enterobacteria

<p>Abstract</p> <p>Background</p> <p><it>Dickeya dadantii </it>and <it>Pectobacterium atrosepticum </it>are phytopathogenic enterobacteria capable of facultative anaerobic growth in a wide range of O₂concentrations found in plant and natural environments. The transcriptional response to O₂remains under-explored for these and other phytopathogenic enterobacteria although it has been well characterized for animal-associated genera including <it>Escherichia coli </it>and <it>Salmonella enterica</it>. Knowledge of the extent of conservation of the transcriptional response across orthologous genes in more distantly related species is useful to identify rates and patterns of regulon evolution. Evolutionary events such as loss and acquisition of genes by lateral transfer events along each evolutionary branch results in lineage-specific genes, some of which may have been subsequently incorporated into the O₂-responsive stimulon. Here we present a comparison of transcriptional profiles measured using densely tiled oligonucleotide arrays for two phytopathogens, <it>Dickeya dadantii </it>3937 and <it>Pectobacterium atrosepticum </it>SCRI1043, grown to mid-log phase in MOPS minimal medium (0.1% glucose) with and without O₂.</p> <p>Results</p> <p>More than 7% of the genes of each phytopathogen are differentially expressed with greater than 3-fold changes under anaerobic conditions. In addition to anaerobic metabolism genes, the O₂responsive stimulon includes a variety of virulence and pathogenicity-genes. Few of these genes overlap with orthologous genes in the anaerobic stimulon of <it>E. coli</it>. We define these as the conserved core, in which the transcriptional pattern as well as genetic architecture are well preserved. This conserved core includes previously described anaerobic metabolic pathways such as fermentation. Other components of the anaerobic stimulon show variation in genetic content, genome architecture and regulation. Notably formate metabolism, nitrate/nitrite metabolism, and fermentative butanediol production, differ between <it>E. coli </it>and the phytopathogens. Surprisingly, the overlap of the anaerobic stimulon between the phytopathogens is also relatively small considering that they are closely related, occupy similar niches and employ similar strategies to cause disease. There are cases of interesting divergences in the pattern of transcription of genes between <it>Dickeya </it>and <it>Pectobacterium </it>for virulence-associated subsystems including the type VI secretion system (T6SS), suggesting that fine-tuning of the stimulon impacts interaction with plants or competing microbes.</p> <p>Conclusions</p> <p>The small number of genes (an even smaller number if we consider operons) comprising the conserved core transcriptional response to O₂limitation demonstrates the extent of regulatory divergence prevalent in the Enterobacteriaceae. Our orthology-driven comparative transcriptomics approach indicates that the adaptive response in the eneterobacteria is a result of interaction of core (regulators) and lineage-specific (structural and regulatory) genes. Our subsystems based approach reveals that similar phenotypic outcomes are sometimes achieved by each organism using different genes and regulatory strategies.</p

Recommendations for Effective Integration of Ethics and Responsible Conduct of Research (E/RCR) Education into Course-Based Undergraduate Research Experiences: A Meeting Report.

Advancement of the scientific enterprise relies on individuals conducting research in an ethical and responsible manner. Educating emergent scholars in the principles of ethics/responsible conduct of research (E/RCR) is therefore critical to ensuring such advancement. The recent impetus to include authentic research opportunities as part of the undergraduate curriculum, via course-based undergraduate research experiences (CUREs), has been shown to increase cognitive and noncognitive student outcomes. Because of these important benefits, CUREs are becoming more common and often constitute the first research experience for many students. However, despite the importance of E/RCR in the research process, we know of few efforts to incorporate E/RCR education into CUREs. The Ethics Network for Course-based Opportunities in Undergraduate Research (ENCOUR) was created to address this concern and promote the integration of E/RCR within CUREs in the biological sciences and related disciplines. During the inaugural ENCOUR meeting, a four-pronged approach was used to develop guidelines for the effective integration of E/RCR in CUREs. This approach included: 1) defining appropriate student learning objectives; 2) identifying relevant curriculum; 3) identifying relevant assessments; and 4) defining key aspects of professional development for CURE facilitators. Meeting outcomes, including the aforementioned E/RCR guidelines, are described herein

Citizen science reveals widespread negative effects of roads on amphibian distributions

Landscape structure is important for shaping the abundance and distribution of amphibians, but prior studies of landscape effects have been species or ecosystem-specific. Using a large-scale, citizen science-generated database, we examined the effects of habitat composition, road disturbance, and habitat split (i.e. the isolation of wetland from forest by intervening land use) on the distribution and richness of frogs and toads in the eastern and central United States. Undergraduates from nine biology and environmental science courses collated occupancy data and characterized landscape structure at 1617 sampling locations from the North American Amphibian Monitoring Program. Our analysis revealed that anuran species richness and individual species distributions were consistently constrained by both road density and traffic volume. In contrast, developed land around wetlands had small, or even positive effects on anuran species richness and distributions after controlling for road effects. Effects of upland habitat composition varied among species, and habitat split had only weak effects on species richness or individual species distributions. Mechanisms underlying road effects on amphibians involve direct mortality, behavioral barriers to movement, and reduction in the quality of roadside habitats. Our results suggest that the negative effects of roads on amphibians occur across broad geographic regions, affecting even common species, and they underscore the importance of developing effective strategies to mitigate the impacts of roads on amphibian populations

Spinal Cord Injury Causes Sustained Disruption of the Blood-Testis Barrier in the Rat

There is a high incidence of infertility in males following traumatic spinal cord injury (SCI). Quality of semen is frequently poor in these patients, but the pathophysiological mechanism(s) causing this are not known. Blood-testis barrier (BTB) integrity following SCI has not previously been examined. The objective of this study was to characterize the effects of spinal contusion injury on the BTB in the rat. 63 adult, male Sprague Dawley rats received SCI (n = 28), laminectomy only (n = 7) or served as uninjured, age-matched controls (n = 28). Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), BTB permeability to the vascular contrast agent gadopentate dimeglumine (Gd) was assessed at either 72 hours-, or 10 months post-SCI. DCE-MRI data revealed that BTB permeability to Gd was greater than controls at both 72 h and 10 mo post-SCI. Histological evaluation of testis tissue showed increased BTB permeability to immunoglobulin G at both 72 hours- and 10 months post-SCI, compared to age-matched sham-operated and uninjured controls. Tight junctional integrity within the seminiferous epithelium was assessed; at 72 hours post-SCI, decreased expression of the tight junction protein occludin was observed. Presence of inflammation in the testes was also examined. High expression of the proinflammatory cytokine interleukin-1 beta was detected in testis tissue. CD68+ immune cell infiltrate and mast cells were also detected within the seminiferous epithelium of both acute and chronic SCI groups but not in controls. In addition, extensive germ cell apoptosis was observed at 72 h post-SCI. Based on these results, we conclude that SCI is followed by compromised BTB integrity by as early as 72 hours post-injury in rats and is accompanied by a substantial immune response within the testis. Furthermore, our results indicate that the BTB remains compromised and testis immune cell infiltration persists for months after the initial injury

Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis

In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes. [Supplemental material is available online at www.genome.org and is supplied as an extended table enumerating genes lost in two multiple round deletion strains (Δ20-1 and Δ20-4). These data are summarized in Table 1.