SUMMARY The monobactams, exemplified by the natural product sulfazecin, are the only class of β-lactam antibiotics not inactivated by metallo-β-lactamases, which confer bacteria with extended-spectrum β-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs. Biochemical examination of the adenylation (A) domains provided evidence that L-2,3-diaminopropionate, not L-serine as previously thought, is the direct source of the β-lactam ring of sulfazecin. ATP/PPi exchange assay also revealed an unusual substrate selectivity shift of one A domain when expressed with or without the immediately upstream condensation domain. Gene inactivation analysis defined a cluster of 13 open reading frames sufficient for sulfazecin production, precursor synthesis, self-resistance, and regulation. The identification of a key intermediate supported a proposed NRPS-mediated mechanism of sulfazecin biosynthesis and β-lactam ring formation distinct from the nocardicins, another NRPS-derived subclass of monocyclic β-lactam. These findings will serve as the basis for further biosynthetic research and potential engineering of these important antibiotics.

The N-sulfonated monocyclic β-lactam ring characteristic of the monobactams confers resistance to zinc metallo-β-lactamases and affords the most effective class to combat carbapenem-resistant enterobacteria (CRE). Here we report unprecedented non-ribosomal peptide synthetase activities where an assembled tripeptide is N-sulfonated in trans prior to direct synthesis of the β-lactam ring in a non-canonical, cysteine-containing thioesterase domain. This means of azetidinone synthesis is distinct from the three others known in nature.

We have produced draft whole-genome sequences for two bacterial strains reported to produce the bulgecins as well as NRPS-derived monobactam β-lactam antibiotics. We propose classification of ATCC 31363 as Paraburkholderia acidophila. We further reaffirm that ATCC 31433 (Burkholderia ubonensis subsp. mesacidophila) is a taxonomically distinct producer of bulgecins with notable gene regions shared with Paraburkholderia acidophila. We use RAST multiple-gene comparison and MASH distancing with published genomes to order the draft contigs and identify unique gene regions for characterization. Forty-eight natural-product gene clusters are presented from PATRIC (RASTtk) and antiSMASH annotations. We present evidence that the 10 genes that follow the sulfazecin and isosulfazecin pathways in both species are likely involved in bulgecin A biosynthesis.

Chad Dunkel is a graduate student in Biological and Agricultural Engineering at the University of Idaho. Chad has also been an active member of the University of Idaho's Industrial Assessment Center (IAC) for approximately 3 years. Through the IAC program Chad has conducted energy assessments on 25 regional manufacturing facilities. Chad is currently conducting research in energy savings via implementation of variable frequency drive blower motor controls for adjusting dissolved oxygen levels in waste water aeration basins.Mr. Ryan A Oliver, BP Ryan Oliver earned his BS degrees in Electrical Engineering and Philosophy at the University of Idaho. While a student he participated in the Department of Energy funded Industrial Assessment Centre program. Now a resident of Alaska, Ryan works for BP as a Discipline Engineer and On-site Process Safety Specialist at the Central Gas Facility in Prudhoe Bay.Dr. Tao Xing P.E., University of Idaho, Moscow Dr. Tao Xing obtained his Ph.D. from Purdue University Mechanical Engineering in 2002. He is also a licensed U.S. Professional Engineer (P.E.). His research interests cover a broad range of thermal-fluid areas using theoretical and computational fluid dynamics (CFD), including renewable energy (wind turbines), multi-phase flows, free-surface flows, ship hydrodynamics, quantitative verification and validation, heating, ventilation, and air-conditioning system. His teaching interests focus on integration of simulation technology into engineering courses and laboratories, developing effective formative and summative evaluation methods, and developing innovative teaching modules toward achieving ABET learning outcomes. Dr. Herbert L. Hess, University of Idaho, MoscowHerb Hess is Professor of Electrical Engineering at the University of Idaho, where he teaches subjects in He received the PhD Degree from the University of Wisconsin-Madison in 1993. His research and teaching interests are in power electronics, electric machines and drives, electrical power systems, and analog/mixed signal electronics. He has taught senior capstone design since 1985 at several universities.Dr. Steven W. Beyerlein, University of Idaho, Moscow Dr. Beyerlein has taught at the University of Idaho for the last 27 years. He is coordinator of the college of engineering inter-disciplinary capstone design course. He is also a co-PI on a DOE sponsored Industrial Assessment Center program in which several of the student authors have been involved. Dr. Beyerlein has been active in research projects involving engine testing, engine heat release modeling, design of curricula for active , design pedagogy, and assessment of professional skills. Collaboration between Senior Design Students and Campus Facilities Staff in Creating a Viable Cogeneration Design for the Campus Wood-Fired Boiler AbstractAn experimental project was created within our inter-disciplinary product realization capstone course to bring together students and staff from our campus steam plant to explore the feasibility of an economical design for ...

No abstract