Browsing by Author "Douthit, Stephanie Ann"

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Characterization of enzymes that modify or degrade the Pseudomonas virulence factor, alginate
(Montana State University - Bozeman, College of Agriculture, 2004) Douthit, Stephanie Ann; Chairperson, Graduate Committee: Michael Franklin
Biosynthesis of the polysaccharide alginate is important for Pseudomonas aeruginosa to establish chronic pulmonary infections in Cystic Fibrosis patients. Alginate is a linear polymer of β 1-4 linked D-mannuronate (M) interspersed with its C-5 epimer, L-guluronate (G). Initially D-mannuronate residues are polymerized into the periplasm as polymannuronic acid. In the periplasm, some polymannuronate residues are converted to L-guluronate residues by the C-5 epimerase, AlgG. Alginate is further modified by the addition of O-acetyl groups to the D-mannuronate residues Algl, AlgJ, and AlgF. The focus of this research was to further characterize the alginate modifying enzymes, AlgG and AlgJ. We found that AlgG contains a repeating sequence that is characterized as a CArbohydrate-binding and Sugar Hydrolases (CASH) domain. Proteins containing this domain fold as right-handed β-helices (RHβH) and bind to long chain linear polysaccharides. AlgG was predicted to fold as a RHβH by the 3D-PSSM structural prediction program. RHβH models of AlgG predict that the identified 324-DPHD-327 motif lies in the long shallow groove that may accommodate alginate. Site-directed mutations of this motif disrupt enzymatic activity, but not structural integrity, suggesting that these mutations lie in the epimerase catalytic domain. Asparagines 362 and 367 are predicted to stack with other asparagine residues along the β-helix. Results obtained from site directed mutants of N362 or N367 suggest that these mutations disrupt asparagine stacking and protein stability. Original attempts to identify alginate binding motifs were made using phage display peptide libraries. This technique proved unsuccessful in identifying binding motifs in AlgG or AlgJ, as discussed in Chapter 3. However, we were able to characterize AlgG with structural modeling, and identified two potentially important motifs in AlgJ. Two putative guluronate specific lyases were also identified in P. aeruginosa, PA1167 and PA1784. Overexpression of PA1167 in mucoid strains FRD1 and FRD1153 results in a non-mucoid phenotype, suggesting this acts as an alginate lyase. The experiments also show the P. aeruginosa cannot use alginate as a carbon source. This research provides a greater understanding of carbohydrate/protein interactions between alginate modifying enzymes and alginate.
Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed ß-helix
(2005-06) Douthit, Stephanie Ann; Dlakic, Mensur; Ohman, Dennis E.; Franklin, Michael J.
The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of ß1-4-linked O-acetylated D-mannuronate (M) and L-guluronate (G), is important for its activity as a virulence factor. Alginate structure is mediated by AlgG, a periplasmic C-5 mannuronan epimerase. AlgG also plays a role in protecting alginate from degradation by the periplasmic alginate lyase AlgL. Here, we show that the C-terminal region of AlgG contains a right-handed ß-helix (RHßH) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RHßH of AlgG has a long shallow groove that may accommodate alginate, similar to protein/polysaccharide interactions of other CASH domain proteins. The shallow groove contains a 324-DPHD motif that is conserved among AlgG and the extracellular mannuronan epimerases of Azotobacter vinelandii. Point mutations in this motif disrupt mannuronan epimerase activity but have no effect on alginate secretion. The D324A mutation has a dominant negative phenotype, suggesting that the shallow groove in AlgG contains the catalytic face for epimerization. Other conserved motifs of the epimerases, 361-NNRSYEN and 381-NLVAYN, are predicted to lie on the opposite side of the RHßH from the catalytic center. Point mutations N362A, N367A, and V383A result in proteins that do not protect alginate from AlgL, suggesting that these mutant proteins are not properly folded or not inserted into the alginate biosynthetic scaffold. These motifs are likely involved in asparagine and hydrophobic stacking, required for structural integrity of RHßH proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RHßH protects alginate from degradation by AlgL by channeling the alginate polymer through the proposed alginate biosynthetic scaffold while epimerizing approximately every second D-mannuronate residue to L-guluronate along the epimerase catalytic face.
Evidence that the AlgI/AlgJ gene cassette, required for O-acetylation of Pseudomonas aeruginosa alginate, evolved by lateral gene transfer
(2004-07) Franklin, Michael J.; Douthit, Stephanie Ann; McClure, Marcella A.
Pseudomonas aeruginosa strains, isolated from chronically infected patients with cystic fibrosis, produce the O-acetylated extracellular polysaccharide, alginate, giving these strains a mucoid phenotype. O acetylation of alginate plays an important role in the ability of mucoid P. aeruginosa to form biofilms and to resist complement-mediated phagocytosis. The O-acetylation process is complex, requiring a protein with seven transmembrane domains (AlgI), a type II membrane protein (AlgJ), and a periplasmic protein (AlgF). The cellular localization of these proteins suggests a model wherein alginate is modified at the polymer level after the transport of O-acetyl groups to the periplasm. Here, we demonstrate that this mechanism for polysaccharide esterification may be common among bacteria, since AlgI homologs linked to type II membrane proteins are found in a variety of gram-positive and gram-negative bacteria. In some cases, genes for these homologs have been incorporated into polysaccharide biosynthetic operons other than for alginate biosynthesis. The phylogenies of AlgI do not correlate with the phylogeny of the host bacteria, based on 16S rRNA analysis. The algI homologs and the gene for their adjacent type II membrane protein present a mosaic pattern of gene arrangement, suggesting that individual components of the multigene cassette, as well as the entire cassette, evolved by lateral gene transfer. AlgJ and the other type II membrane proteins, although more diverged than AlgI, contain conserved motifs, including a motif surrounding a highly conserved histidine residue, which is required for alginate O-acetylation activity by AlgJ. The AlgI homologs also contain an ordered series of motifs that included conserved amino acid residues in the cytoplasmic domain CD-4; the transmembrane domains TM-C, TM-D, and TM-E; and the periplasmic domain PD-3. Site-directed mutagenesis studies were used to identify amino acids important for alginate O-acetylation activity, including those likely required for (i) the interaction of AlgI with the O-acetyl precursor in the cytoplasm, (ii) the export of the O-acetyl group across the cytoplasmic membrane, and (iii) the transfer of the O-acetyl group to a periplasmic protein or to alginate. These results indicate that AlgI belongs to a family of membrane proteins required for modification of polysaccharides and that a mechanism requiring an AlgI homolog and a type II membrane protein has evolved by lateral gene transfer for the esterification of many bacterial extracellular polysaccharides.