Mark McIntosh, Ph.D
Professor and Chairman
Director of DNA Core Facility
Veterinary Pathobiology and Molecular Microbiology & Immunology
Phone: (573) 882-8989
Fields of Interest
- Bacterial Pathogenesis
- Ph.D. 1978, University of Texas at Austin
Iron-mediated regulation of gene expresssion; bacterial membrane transport processes using energy-driven receptors; molecular biology of mycoplasmas.
The McIntosh laboratory is studying the processes and regulation of iron assimilationin bacteria. Microbes secrete iron chelating molecules (siderophores) to solubilize iron for metabolic needs. The biosynthesis and transport of siderophores are regulated at the molecular level by intracellular iron concentrations.
Using the Escherichia coli siderophore enterobactin as a model, we are investigating a complex gene system (ent-biosynthesis; fep-transport; fes-iron removal), composed of six separate transcripts emanating from three tightly iron-controlled bidirectional promoter/operator regions.
Current studies that focus on regulation of the expression of these genes at the molecular level include: (1) characterization of mutations affecting cis (iron-controlled promoters and operators) and trans (the Fur repressor) components of the regulatory process; (2) protein-DNA interactions that mediate this process; and (3) transcriptional and post-transcriptional regulatory phenomena that link this control to other bacterial metabolic stress responses including oxygen stress and carbon starvation.
The point of entry for ferric enterobactin involves specific recognition of the ferrated siderophone at the outer membrane by the receptor FepA. We have investigated the structural and functional topology of the outer membrane receptor FepA, a prototype for TonB dependent, energy-driven membrane channels. Using epitope insertions and PCR-generated random mutations, we have identified key amino acid residues in FepA that define its binding domains for the ligands enterobactin and colicin B, transport pathways through the membrane channel, and its interactions with TonB.
Figure: Taq DNA polymerase was used in a standard PCR reaction to create random mutations in the N-terminal plug domain of FepA (Zhou et al., 1991). The PCR product was then used to create a pool of plasmid from which mutant fepA alleles could be selected with colicin B. The figure above maps the mutations generated using this strategy on the FepA crystal structure (Buchanan et al., 1999). Several of the b-strands of the barrel domain (shown in red) have been removed to reveal the plug domain. The plug domain is shown in blue and the mutations in yellow. These mutations have helped us further define regions of the plug necessary for ligand binding, ligand transport, and interaction with the periplasmic energy transducer, TonB. Buchanan, SK, et al. 1999. Nature Structural Biology. 6:5 6-63. Zhou, YH, et al. 1991. Nucleic Acids Research. 19:6052. Barnard, TJ, et al. 2001. Mol. Microbiol. 41:526.
We have recently identified a cytoplasmic membrane protein, EntS, that is required for efficient export of enterobactin from the cells. EntS is a 12-TMS protein related to the major facilitator class of membrane exporters. TLC and HPLC analysis of culture supernatants from an EntS null-mutant reveal exported enterobactin breakdown products resulting from ferric enterobactin esterase (Fes) activity on the non-secreted siderophore.
Figure: Topological Predictions of P43: (A) The predictedopological model for the 12-TMS MFS class P43 protein in E.coli. Locations of conserved motifs for MFS class proteins are denoted. (B) A generic model of the 12-TMS MFS family with the known conserved domains represented by black circles (•) [Adapted from Putman et al., 2000]. (C) A representation of the E.coli chromosomal enterobactin gene cluster showing the gene encoding P43, ybdA. Arrows indicate the direction of transcription. (D) Alignment of the amino acid sequence of P43 with the consensus sequence (CON) reported for 12-TMS MFS proteins and the E.coli tetracycline pump, TetA.
Figure: HPLC Analysis of Enterobactin Secretion in entS Mutants: Strains were grown for 4 hours to an OD 600=0.7 under high (+Fe) or low (+Dip) iron conditions in 10 ml MOPS minimal medium. Enterobactin and enterobactin breakdown products were extracted from culture supernatants with ethyl acetate, dried, and resuspended in 50 ml of methanol and separated by a 10-50% gradient of acetonitrile and water with 0.1% TFA, then detected at 220 nm. Panels show individual chrom atograms os enterobactin and DHBA standards (panels 1 and 2), media blanks (panel 3 and 4), extract representative of growth under high iron condition (panel 5, +Fe), and extracts grown under low iron conditions (panels 6-8, +Dip). Detected enterobactin peaks (E) and DHBA marker peaks (D) are labeled as defined by the standards in panels 1 and 2. Numbers (1-5) mark enterovactin breakdown product peaks indentified by mass spectrometry where present [1: monomer, 2: dimer, 3: trimer, 4 and 5: unk nown]. In panels where an enterobactin peak was detected, retention time, peak height (in mV) and area under the peak (in mV) and area under the peak (in my V/sec) were calculated.
We also investigate genetic elements involved in genomic variation in mycoplasmas. These studies are aimed at:
- examination of the structural organization and expression of selected genes from the mycoplasma genome, unique because of its relatively high A-T content (73%) and its unusual genetic code (the utilization of the opal codon UGA to code for tryptophan);
- the characterization of gene sequences that transpose or amplify within the mycoplasma genome, creating genetic variability that may play a role in the immunopathology of the disease;
- the development of an expression system for efficient production of mycoplasma proteins in an E. coli host;
- the distribution and function of families of specific mycoplasma proteins, (including surface proteins and restriction-modification enzymes) that may be involved in host cell interactions important to these pathogens.
We have identified a 16-kb gene island from M. hyopneumoniae encoding eight struct ural genes and two copies of IS1221, and shown that these sequences are also distributed among various strains of two other mycoplasma species. We are investigating the mechanisms controlling the interspecies transfer of these sequences.
- Calcutt MJ, Foecking MF, Heidari MB, and MA McIntosh. Complete Genome Sequence of Mycoplasma flocculare Strain Ms42T (ATCC 27399T). Genome Announc. 2015 Mar 12;3(2). pii: e00124-15. doi: 10.1128/genomeA.00124-15. PMID: 25767245
- McGreevy JW, Hakim CH, McIntosh MA, and D Duan.Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy.Dis Model Mech. 2015 Mar;8(3):195-213. Review. PMID: 25740330
- Kodippili K, Vince L, Shin JH, Yue Y, Morris GE, McIntosh MA, and D Duan. Characterization of 65 epitope-specific dystrophin monoclonal antibodies in canine and murine models of duchenne muscular dystrophy by immunostaining and western blot. PLoS One. 2014 Feb 7;9(2):e88280. doi: 10.1371/journal.pone.0088280. eCollection 2014. PMID: 24516626
- Rai DK, Schafer EA, Singh K, McIntosh MA, Sarafianos SG, and E Rieder.Repeated exposure to 5D9, an inhibitor of 3D polymerase, effectively limits the replication of foot-and-mouth disease virus in host cells. Antiviral Res. 2013 Jun;98(3):380-5. doi: 10.1016/j.antiviral.2013.03.022. Epub 2013 Apr 8. PMID: 23578728
- Durk RC, Singh K, Cornelison CA, Rai DK, Matzek KB, Leslie MD, Schafer E, Marchand B, Adedeji A, Michailidis E, Dorst CA, Moran J, Pautler C, Rodriguez LL, McIntosh MA, Rieder E, and SG Sarafianos. Inhibitors of foot and mouth disease virus targeting a novel pocket of the RNA-dependent RNA polymerase. PLoS One. 2010 Dec 21;5(12):e15049. doi: 10.1371/journal.pone.0015049. PMID: 21203539
- Bachman SL, Sporn E, Furrer JL, Astudillo JA, Calaluce R, McIntosh MA, Miedema BW, and K Thaler.Colonic sterilization for natural orifice translumenal endoscopic surgery (NOTES) procedures: a comparison of two decontamination protocols. Surg Endosc. 2009 Aug;23(8):1854-9. doi: 10.1007/s00464-008-0295-0. Epub 2009 Jan 1. PMID: 19118416
- Hook-Barnard IG, TJ Brickman, and MA McIntosh. 2007. Identification of an AU-rich Translational Enhancer within the Escherichia coli fepB Leader RNA. J. Bacteriol. 189:4028-4037.
- Endsley, J.J., J.L. Furrer, M.A. Endsley, M.A. McIntosh, A.C. Maue, W.R. Waters, D.R. Lee, and D.M. Estes. 2004 Characterization of bovine homologues of granulysis and NK-lysin. J. Immun. 173:2607-2614.
- Lavrrar JL and MA McIntosh. 2002. The architecture of a Fur binding site - a comparative analysis. J. Bacteriol. 185:2194-2202.
- Lavrrar JL, CA Christoffersen, and MA McIntosh. 2002. Fur-DNA interactions in the bidirectional fepDGC-entS promoter region in Escherichia coli. J. Mol. Biol. 322:983-995.
- Furrer JL, DN Sanders, I Hook-Barnard, and MA McIntosh. 2002. Export of the siderophore enterobactin in Escherichia coli: involvement of a 43-kDa membrane exporter. Mol. Microbiol. 44(5):1225-1234.
- Barnard TJ, ME Watson, Jr., and MA McIntosh. 2001. Mutations in the Escherichia coli receptor FepA reveal residues involved in ligand binding and transport. Mol. Microbiol. 41:527-536.
- Christoffersen, CA, TJ Brickman, I Hook-Barnard, and MA McIntosh. 2001. Regulatory architecture of the iron-regulated fepD-ybdA bidirectional promoter region in Escherichia coli. J. Bacteriol. 183:2059-2070.
- Larsen, R.A., D. Foster-Hartnett, M.A. McIntosh, K. Postle. 1997. Regions of Escherichia coli TonB and FepA proteins essential for in vivo physical interactions. J Bacteriol 179 (10):3213-3221.
- Zheng, J. and M.A. McIntosh. 1995. Characterization of IS 1221 from Mycoplasma hyorhinis: expression of its putative transposase in Escherichia coli incorporates a ribosomal frameshift mechanism. Mol. Microbiol. 16(4):669-685.
- Armstrong, S.K. and M.A. McIntosh. 1995. Epitope insertions define functional and topological features of the Escherichia coli ferric enterobactin receptor. J. Biol. Chem. 270:2483-2488.