Resume

• 2007

  Doctor of Philosophy (PhD)

  Microbiology

  The Ohio State University

• 2002

  Bachelor of Science (BS)

  Microbiology

  Indiana University Bloomington

  Bachelor of Arts (BA)

  Biochemistry

  Indiana University Bloomington

• Molecular Cloning

  Enzyme Assays

  Western Blotting

  Biology

  Microbial Biochemistry

  Biochemistry

  Science

  qRT PCR

  PCR

  Protein Purification

  Microbial Physiology

  Protein Expression

  Microbial Genetics

  RT-PCR

  Microbiology

  Molecular Biology

  RNA isolation

  Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate

  Birgit Alber

  Marie Asao

  Kathrin Schneider

  Journal of Bacteriology

  The anoxygenic phototroph Rhodobacter sphaeroides uses 3-hydroxypropionate as a sole carbon source for growth. Previously

  we showed that the gene (RSP_1434) known as acuI

  which encodes a protein of the medium-chain dehydrogenase/reductase (MDR) superfamily

  was involved in 3-hydroxypropionate assimilation via the reductive conversion to propionyl-coenzyme A (CoA). Based on these results

  we speculated that acuI encoded acrylyl-CoA reductase. In this work

  we characterize the in vitro enzyme activity of purified

  recombinant AcuI using a coupled spectrophotometric assay. AcuI from R. sphaeroides catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Two other members of the MDR012 family within the MDR superfamily

  the products of SPO_1914 from Ruegeria pomeroyi and yhdH from Escherichia coli

  were shown to also be part of this new class of NADPH-dependent acrylyl-CoA reductases. The activities of the three enzymes were characterized by an extremely low Km for acrylyl-CoA (<3 μM) and turnover numbers of 45 to 80 s(-1). These homodimeric enzymes were highly specific for NADPH (Km = 18 to 33 μM)

  with catalytic efficiencies of more than 10-fold higher for NADPH than for NADH. The introduction of codon-optimized SPO_1914 or yhdH into a ΔacuI::kan mutant of R. sphaeroides on a plasmid complemented 3-hydroxypropionate-dependent growth. However

  in their native hosts

  SPO_1914 and yhdH are believed to function in the metabolism of substrates other than 3-hydroxypropionate

  where acrylyl-CoA is an intermediate. Complementation of the ΔacuI::kan mutant phenotype by crotonyl-CoA carboxylase/reductase from R. sphaeroides was attributed to the fact that the enzyme also uses acrylyl-CoA as a substrate.

  Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate

  Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars.

  Birgit E. Alber

  Propionyl coenzyme A (propionyl-CoA) assimilation by Rhodobacter sphaeroides proceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway

  propionyl-CoA carboxylase (PCC)

  was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway

  acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of the pccB gene

  which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation of pccB transcript levels and

  consequently

  PCC activity: growth substrate-dependent regulation was lost when pccR was inactivated by an in-frame deletion. In the pccR mutant

  lacZ expression from a 215-bp plasmid-borne pccB upstream fragment including 27 bp of the pccB coding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR

  together with homologs from other organisms

  formed a distinct clade within the family of short-chain fatty acyl coenzyme A regulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR).

  Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

  John A. Gerlt

  Steven C. Almo

  Yury Patskovsky

  We describe a general integrated bioinformatic and experimental strategy to discover the in vitro enzymatic activities and in vivo functions (metabolic pathways) of uncharacterized enzymes discovered in microbial genome projects using the ligand specificities of the solute binding proteins (SBPs) for ABC transporters. Using differential scanning fluorimetry

  we determined that the SBP for an ABC transporter encoded by the genome of Mycobacterium smegmatis is stabilized by d-threitol. Using sequence similarity networks and genome neighborhood networks to guide selection of target proteins for pathway enzymes

  we applied both in vitro and in vivo experimental approaches to discover novel pathways for catabolism of d-threitol

  l-threitol

  and erythritol.

  A General Strategy for the Discovery of Metabolic Pathways: D-Threitol

  L-Threitol

  and Erythritol Utilization in Mycobacterium smegmatis

  Michael

  Carter

  Drury University

  The Ohio State University

  University of Illinois at Urbana-Champaign

  Salisbury University

  As a member of the Enzyme Function Initiative

  I worked toward better characterizing the physiological and biochemical roles of proteins encoded by poorly and/or misannotated genes throughout bacterial genomes.

  Postdoctoral Researcher

  Urbana-Champaign

  Illinois Area

  University of Illinois at Urbana-Champaign

  Salisbury

  Maryland

  Assistant Professor Of Biology

  Salisbury University

  Springfield

  Missouri Area

  I engaged undergraduate students in Biology

  Molecular Biology

  Biochemistry

  and Genetics education and research. Students who researched with me developed a comprehensive knowledge of bacterial cellular physiology as we linked genetics to metabolic pathways

  deleting genes via homologous recombination

  purifying and investigating the kinetics of their purified proteins

  and the regulation of their expression to metabolic pathways.

  Visiting Assistant Professor of Biology

  Drury University

  Explored the regulation of acetate and propionate assimilation in purple nonsulfur bacteria and its links to central carbon metabolism.\n\nTeaching Assistant for:\n-Introductory Biology (Energy Transformations and Development)\n-Introductory Microbiology\n-General Microbiology\n-Microbial Genetics\n\nResearch Techniques:\n-Molecular Biology and Molecular Cloning\n-RNA Isolation\n-qRT PCR\n-Endpoint RT PCR\n-Spectrophotometric Enzyme Assays\n-Radioactive Enzyme Assays\n-Microbial Genetics\n-Immunoblotting\n-Protein Purification

  Graduate Teaching and Research Assistant

  Columbus

  Ohio Area

  The Ohio State University