Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

BACKGROUND: The metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics. RESULTS: Site-directed mutations were generated of amino acids previously predicted to be important in substrate binding. Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying K(m) and k(cat) values for the different enzymes and substrates and that no direct correlation between K(m) and the effect of the mutation on substrate binding could be drawn. Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding. CONCLUSIONS: The data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-β-lactamase L1. The results in this work also show that K(m) values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction. This work represents the first experimental testing of one of the computational models of the metallo-β-lactamases

The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis

The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic framework for these data. Updated monthly, these services are made available through the RDP-II website (http://rdp.cme.msu.edu/). RDP-II release 9.21 (August 2004) contains 101 632 bacterial small subunit rRNA gene sequences in aligned and annotated format. High-throughput tools for initial taxonomic placement, identification of related sequences, probe and primer testing, data navigation and subalignment download are provided. The RDP-II email address for questions or comments is [email protected]

Genomic encyclopedia of bacterial and archaeal type strains, phase III : the genomes of soil and plant-associated and newly described type strains

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project to sequence about 250 bacterial and archaeal genomes of elevated phylogenetic diversity. Herein, we propose to extend this approach to type strains of prokaryotes associated with soil or plants and their close relatives as well as type strains from newly described species. Understanding the microbiology of soil and plants is critical to many DOE mission areas, such as biofuel production from biomass, biogeochemistry, and carbon cycling. We are also targeting type strains of novel species while they are being described. Since 2006, about 630 new species have been described per year, many of which are closely aligned to DOE areas of interest in soil, agriculture, degradation of pollutants, biofuel production, biogeochemical transformation, and biodiversity

The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data

Substantial new features have been implemented at the Ribosomal Database Project in response to the increased importance of high-throughput rRNA sequence analysis in microbial ecology and related disciplines. The most important changes include quality analysis, including chimera detection, for all available rRNA sequences and the introduction of myRDP Space, a new web component designed to help researchers place their own data in context with the RDP's data. In addition, new video tutorials describe how to use RDP features. Details about RDP data and analytical functions can be found at the RDP-II website ()

Genomic encyclopedia of bacterial and archaeal type strains, phase III : the genomes of soil and plant-associated and newly described type strains

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project to sequence about 250 bacterial and archaeal genomes of elevated phylogenetic diversity. Herein, we propose to extend this approach to type strains of prokaryotes associated with soil or plants and their close relatives as well as type strains from newly described species. Understanding the microbiology of soil and plants is critical to many DOE mission areas, such as biofuel production from biomass, biogeochemistry, and carbon cycling. We are also targeting type strains of novel species while they are being described. Since 2006, about 630 new species have been described per year, many of which are closely aligned to DOE areas of interest in soil, agriculture, degradation of pollutants, biofuel production, biogeochemical transformation, and biodiversity

Chapter: Landcare on the Poverty-Protection Interface in an Asian Watershed

Serious methodological and policy hurdles constrain effective natural resource management that alleviates poverty while protecting environmental services in tropical watersheds. We review the development of an approach to integrate biodiversity conservation and agroforestry development through the active involvement of communities and their local governments near the Kitanglad Range Natural Park in the Manupali watershed, central Mindanao, the Philippines. Agroforestry innovations were developed to suit the biophysical and socioeconomic conditions of the buffer zone. These included practices for tree farming, and conservation farming for annual cropping on slopes. Institutional innovations improved resource management, resulting in an effective social contract to protect the natural biodiversity o f the park. Fruit and timber tree production dramatically increased, re-establishing tree cover in the buffer zone. Natural vegetative contour strips were installed on several hundred sloping farms. Soil erosion and runoff declined, while the buffer strips increased maize yields by an average of 0.5 t/ha on hill-slope farms. The scientific knowledge base guided the development and implementation of a natural resource management plan for the municipality of Lantapan. A dynamic grassroots movement o f farmer-led Landcare groups evolved in the villages near the park boundary, which had significant impact on conservation in both the natural and managed ecosystems. Encroachment in the natural park was reduced 95% in three years. The local Landcare groups also restored stream corridor vegetation. This integrated approach has been recognized as a national model for local natural resource and watershed management in the Philippines. Currently, the collaborating institutions are evolving a negotiation support system to resolve the interactions between the three management domains: the park, the ancestral domain claim, and the municipalities. This integrated systems approach operated effectively with highly constrained funding, suggesting that commitment and impact may best be stimulated by a “drip-feed” approach rather than by large, externally funded efforts

Overview of mathematical approaches used to model bacterial chemotaxis I: the single cell

Mathematical modeling of bacterial chemotaxis systems has been influential and insightful in helping to understand experimental observations. We provide here a comprehensive overview of the range of mathematical approaches used for modeling, within a single bacterium, chemotactic processes caused by changes to external gradients in its environment. Specific areas of the bacterial system which have been studied and modeled are discussed in detail, including the modeling of adaptation in response to attractant gradients, the intracellular phosphorylation cascade, membrane receptor clustering, and spatial modeling of intracellular protein signal transduction. The importance of producing robust models that address adaptation, gain, and sensitivity are also discussed. This review highlights that while mathematical modeling has aided in understanding bacterial chemotaxis on the individual cell scale and guiding experimental design, no single model succeeds in robustly describing all of the basic elements of the cell. We conclude by discussing the importance of this and the future of modeling in this area

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

The Remote Learning Experience at Portland State University in Spring 2020

It is an endeavor to understand what we have and will learn about the impact of remote instruction on faculty, students and relevant academic support teams. Simply put: We want to learn from an experiment foisted upon us by a health crisis. We have engaged in an incredibly innovative response. And now, we ask what have we learned? How might we improve? And, most importantly, are there implications from this experiment for the future of instruction at PSU and throughout higher education? The project was organized around two stages in the Spring 2020 term. Stage One: Out of the Gate: Reflections and Lessons Learned (First half of the term) Stage Two: Reaching the Finish Line: Lessons Learned and Recommendations for moving forward (Second half of the term). The project began the week of April 20 and continued through June 12. The original plan called for the following participants: (a) ten undergraduate students to put together a group of 8-10 other students to discuss the questions posed in the study; (b) Three graduate students who would assemble 5-7 fellow graduate students; (c) Three tenured or tenure-track faculty, two non-tenure-track faculty and three adjunct faculty, each of whom would form a chat group of 5-7 other faculty to discuss the questions posed in the study. In addition, Judith Ramaley put together a chat group of a dozen student support unit leaders to explore how each unit adjusted as the university moved quickly to remote learning and remote work and then, in a second round, what lessons each had learned throughout the spring term about ways to support students and assist faculty members who were also seeking to help their students