I have identified four distinct classes of mutants involved in either lipoic acid biosynthesis or... more I have identified four distinct classes of mutants involved in either lipoic acid biosynthesis or the covalent attachment of lipoic acid to proteins in E. coli. Two of these classes of mutants were isolated as Tn1000dKn insertion mutants and both classes map to min 14.5 on the E. coli chromosome. The other two classes of mutants were isolated as spontaneous selenolipoic acid resistant (slr) mutants. Selenolipoic acid was shown to be a potent inhibitor of wild type E. coli.The Tn1000dKn mutations defined two genes, lipA and lipB, involved in the production of lipoylated proteins. Both the lipA and lipB genes were sequenced. The deduced amino acid sequence of the lipA gene showed some similarity to biotin synthase. It is thus likely that lipA encodes a sulfur insertion enzyme analogous to biotin synthase and that the two enzymes share a common sulfur donor. The slr-7 allele (maps to min 15.25) was shown to be a suppressor of lipA150::Tn1000dKn indicating that the slr-7 gene also plays a role in lipoic acid biosynthesis.The amino acid sequence of the lipB gene did not share significant amino acid similarity to any protein in the Genebank Data Base. However, several experiments support the conclusion that the lipB gene is required for the covalent attachment of endogenously synthesized lipoic acid to protein. First, analysis of the lipoic acid content of a lipB null mutant grown under lipoate deficient conditions revealed that lipB mutants possessed functional, but attenuated lipoic acid biosynthetic activity. Secondly, the lipoic acid auxotrophic phenotype of a lipB null mutant could be suppressed by overexpression of the lut encoded lipoyl ligase (T. Morris, manuscript in preparation). The primary function of the lut encoded ligase is to covalently attach exogenous sources of lipoic acid to protein. Finally, I have shown that a lut lipB double mutant does not synthesize any detectable lipoyl-proteins.I have shown that slr-1 is an allele of lut. Not only is the slr-1 mutant defective for lipoyl ligase activity, it is also 100 fold more resistant to selenolipoic acid than an isogenic wild type strain. Moreover, the slr-1 lipoyl ligase is able to distinguish between lipoic acid and selenolipoic acid indicating that slr-1 mutant is a specificity mutant.U of I OnlyETDs are only available to UIUC Users without author permissio
Identification of the Gene Encoding Lipoate-Protein Ligase A of Escherichia coli MOLECULAR CLONIN... more Identification of the Gene Encoding Lipoate-Protein Ligase A of Escherichia coli MOLECULAR CLONING AND CHARACTERIZATION OF THE 1plA GENE AND GENE PRODUCT*
Biotination of proteins is a post-translational modification that requires a folded acceptor doma... more Biotination of proteins is a post-translational modification that requires a folded acceptor domain. We previously showed that an acceptor domain fused to the carboxyl terminus of several cytosolic proteins results in biotinated fusion proteins in vivo. We now show that proteins encoded by translational gene fusions of two periplasmic proteins, alkaline phosphatase and TEM beta-lactamase, to carboxyl-terminal biotin-accepting sequences are biotinated and exported by Escherichia coli. Expression of the alkaline phosphatase fusion protein in wild type strains resulted in inefficient biotination of the fusion product. This result was due to the rapid export of the acceptor protein before biotination could occur since a very large increase in biotinated fusion protein levels was observed in strains lacking the SecB chaperone protein. The beta-lactamase fusion protein was biotinated but was only stable in strains lacking the DegP periplasmic protease. Both biotinated fusion proteins accumulated in the culture medium in strains possessing defective outer membranes. These results indicate that the export machinery can accommodate both a post-translational modification and a protein domain previously folded into its mature conformation in vivo.
Growth of Shigella spp. in the presence of the bile salt deoxycholate or chenodeoxycholate enhanc... more Growth of Shigella spp. in the presence of the bile salt deoxycholate or chenodeoxycholate enhanced the bacterial invasion of HeLa cells. Growth in the presence of other structurally similar bile salts or detergents had little or no effect. Deoxycholate-enhanced invasion was not observed when bacteria were exposed to deoxycholate at low temperatures or when chloramphenicol was added to the growth medium, indicating that bacterial growth and protein synthesis are required. Increased invasion is associated with the presence of an intact Shigella virulence plasmid and is correlated with increased secretion of a set of proteins, including the Ipa proteins, to the outer membrane and into the growth medium. The increased invasion induced by the bile salts appears to be due to increased adherence. The enhanced adherence was specific to Shigella spp., since the enteroinvasive Escherichia coli strains tested did not exhibit the effect in response to growth in bile salts.
Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vi... more Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vibriobactin is structurally similar to enterobactin, the siderophore produced by Escherichia coli, and both organisms produce 2,3-dihydroxybenzoic acid (DHBA) as an intermediate in siderophore biosynthesis. To isolate and characterize V. cholerae genes involved in vibriobactin biosynthesis, we constructed a genomic cosmid bank of V. cholerae DNA and isolated clones that complemented mutations in E. coli enterobactin biosynthesis genes. V. cholerae homologs of entA, entB, entC, entD, and entE were identified on overlapping cosmid clones. Our data indicate that the vibriobactin genes are clustered, like the E. coli enterobactin genes, but the organization of the genes within these clusters is different. In this paper, we present the organization and sequences of genes involved in the synthesis and activation of DHBA. In addition, a V. cholerae strain with a chromosomal mutation in vibA was ...
Background/Question/Methods Microbes play a key role in promoting a healthy ecosystem in grasslan... more Background/Question/Methods Microbes play a key role in promoting a healthy ecosystem in grassland prairies. However, the factors governing the structure of rhizosphere microbial communities are not fully understood. In this study, we compared the bacterial and fungal community composition in the rhizosphere of a native grass (Andropogon gerardii) and a non-native grass (Sorghum halepense) from two North Texas prairies with dramatically different soil textures. Soils were analyzed for pH, organic matter, moisture, total carbon, total nitrogen, and clay and sand percentages. Microbial community fingerprints were generated using terminal restriction fragment length polymorphism (TRFLP) analysis. The overall structure of bacterial and fungal communities between the prairie sites and grass species were compared by analyzing binary data using the Additive Main Effects and Multiplicative Interaction (AMMI) model. This technique combines analysis of variance to first partition the variatio...
Proceedings of the National Academy of Sciences, 1994
Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-keto... more Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-ketoacid dehydrogenase enzyme complexes. We report the chemical synthesis and biological properties of lipoic acid analogs in which one or both sulfur atoms were replaced by selenium. Replacement of either the C-6 or the C-8 sulfur atom with selenium results in lipoic acid derivatives with apparently unaltered biological properties. However, simultaneous replacement of both sulfur atoms gave an analog (selenolipoic acid) that inhibited growth of wild-type Escherichia coli when present in minimal glucose medium at 50 ng/ml. This growth inhibition was reversed by the addition of either excess lipoic acid or acetate plus succinate. Labeling experiments with [75Se]selenolipoic acid showed that this compound was efficiently incorporated into the alpha-ketoacid dehydrogenase complexes of growing cells. Spontaneously arising selenolipoic acid-resistant (slr) mutants were isolated. Two of these isol...
Genomics is not only essential for students to understand biology but also provides unprecedented... more Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning appr...
Biochemistry and Molecular Biology Education, 2001
Two degenerate primers are described that can be used to amplify a 340 bp fragment of the ureC ge... more Two degenerate primers are described that can be used to amplify a 340 bp fragment of the ureC gene from a variety of urease producing bacteria. A series of experiments are outlined that enable students to develop restriction maps of the urease gene clusters from different bacteria. Students learn a variety of molecular biology techniques including polymerase chain reaction, agarose gel electrophoresis, and Southern blotting. This project allows for multiple experimental outcomes using the same two PCR primers.
Biochemistry and Molecular Biology Education, 2013
We used the Integrated Microbial Genomes Annotation Collaboration Toolkit as a framework to incor... more We used the Integrated Microbial Genomes Annotation Collaboration Toolkit as a framework to incorporate microbial genomics research into a microbiology and biochemistry course in a way that promoted student learning of bioinformatics and research skills and emphasized teamwork and collaboration as evidenced through multiple assessment mechanisms. Student teams in microbiology used bioinformatics tools to identify and characterize gene products from Mucilaginibacter paludis necessary for the synthesis of specific amino acids and then designed and carried out growth experiments to determine if the organism could indeed synthesize the amino acids. Students in biochemistry worked to characterize one of the amino acid biosynthetic pathways reconstructed by a previous microbiology class through amplification and cloning of the M. paludis genes and complementation analysis of Escherichia coli mutants.
I have identified four distinct classes of mutants involved in either lipoic acid biosynthesis or... more I have identified four distinct classes of mutants involved in either lipoic acid biosynthesis or the covalent attachment of lipoic acid to proteins in E. coli. Two of these classes of mutants were isolated as Tn1000dKn insertion mutants and both classes map to min 14.5 on the E. coli chromosome. The other two classes of mutants were isolated as spontaneous selenolipoic acid resistant (slr) mutants. Selenolipoic acid was shown to be a potent inhibitor of wild type E. coli.The Tn1000dKn mutations defined two genes, lipA and lipB, involved in the production of lipoylated proteins. Both the lipA and lipB genes were sequenced. The deduced amino acid sequence of the lipA gene showed some similarity to biotin synthase. It is thus likely that lipA encodes a sulfur insertion enzyme analogous to biotin synthase and that the two enzymes share a common sulfur donor. The slr-7 allele (maps to min 15.25) was shown to be a suppressor of lipA150::Tn1000dKn indicating that the slr-7 gene also plays a role in lipoic acid biosynthesis.The amino acid sequence of the lipB gene did not share significant amino acid similarity to any protein in the Genebank Data Base. However, several experiments support the conclusion that the lipB gene is required for the covalent attachment of endogenously synthesized lipoic acid to protein. First, analysis of the lipoic acid content of a lipB null mutant grown under lipoate deficient conditions revealed that lipB mutants possessed functional, but attenuated lipoic acid biosynthetic activity. Secondly, the lipoic acid auxotrophic phenotype of a lipB null mutant could be suppressed by overexpression of the lut encoded lipoyl ligase (T. Morris, manuscript in preparation). The primary function of the lut encoded ligase is to covalently attach exogenous sources of lipoic acid to protein. Finally, I have shown that a lut lipB double mutant does not synthesize any detectable lipoyl-proteins.I have shown that slr-1 is an allele of lut. Not only is the slr-1 mutant defective for lipoyl ligase activity, it is also 100 fold more resistant to selenolipoic acid than an isogenic wild type strain. Moreover, the slr-1 lipoyl ligase is able to distinguish between lipoic acid and selenolipoic acid indicating that slr-1 mutant is a specificity mutant.U of I OnlyETDs are only available to UIUC Users without author permissio
Identification of the Gene Encoding Lipoate-Protein Ligase A of Escherichia coli MOLECULAR CLONIN... more Identification of the Gene Encoding Lipoate-Protein Ligase A of Escherichia coli MOLECULAR CLONING AND CHARACTERIZATION OF THE 1plA GENE AND GENE PRODUCT*
Biotination of proteins is a post-translational modification that requires a folded acceptor doma... more Biotination of proteins is a post-translational modification that requires a folded acceptor domain. We previously showed that an acceptor domain fused to the carboxyl terminus of several cytosolic proteins results in biotinated fusion proteins in vivo. We now show that proteins encoded by translational gene fusions of two periplasmic proteins, alkaline phosphatase and TEM beta-lactamase, to carboxyl-terminal biotin-accepting sequences are biotinated and exported by Escherichia coli. Expression of the alkaline phosphatase fusion protein in wild type strains resulted in inefficient biotination of the fusion product. This result was due to the rapid export of the acceptor protein before biotination could occur since a very large increase in biotinated fusion protein levels was observed in strains lacking the SecB chaperone protein. The beta-lactamase fusion protein was biotinated but was only stable in strains lacking the DegP periplasmic protease. Both biotinated fusion proteins accumulated in the culture medium in strains possessing defective outer membranes. These results indicate that the export machinery can accommodate both a post-translational modification and a protein domain previously folded into its mature conformation in vivo.
Growth of Shigella spp. in the presence of the bile salt deoxycholate or chenodeoxycholate enhanc... more Growth of Shigella spp. in the presence of the bile salt deoxycholate or chenodeoxycholate enhanced the bacterial invasion of HeLa cells. Growth in the presence of other structurally similar bile salts or detergents had little or no effect. Deoxycholate-enhanced invasion was not observed when bacteria were exposed to deoxycholate at low temperatures or when chloramphenicol was added to the growth medium, indicating that bacterial growth and protein synthesis are required. Increased invasion is associated with the presence of an intact Shigella virulence plasmid and is correlated with increased secretion of a set of proteins, including the Ipa proteins, to the outer membrane and into the growth medium. The increased invasion induced by the bile salts appears to be due to increased adherence. The enhanced adherence was specific to Shigella spp., since the enteroinvasive Escherichia coli strains tested did not exhibit the effect in response to growth in bile salts.
Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vi... more Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vibriobactin is structurally similar to enterobactin, the siderophore produced by Escherichia coli, and both organisms produce 2,3-dihydroxybenzoic acid (DHBA) as an intermediate in siderophore biosynthesis. To isolate and characterize V. cholerae genes involved in vibriobactin biosynthesis, we constructed a genomic cosmid bank of V. cholerae DNA and isolated clones that complemented mutations in E. coli enterobactin biosynthesis genes. V. cholerae homologs of entA, entB, entC, entD, and entE were identified on overlapping cosmid clones. Our data indicate that the vibriobactin genes are clustered, like the E. coli enterobactin genes, but the organization of the genes within these clusters is different. In this paper, we present the organization and sequences of genes involved in the synthesis and activation of DHBA. In addition, a V. cholerae strain with a chromosomal mutation in vibA was ...
Background/Question/Methods Microbes play a key role in promoting a healthy ecosystem in grasslan... more Background/Question/Methods Microbes play a key role in promoting a healthy ecosystem in grassland prairies. However, the factors governing the structure of rhizosphere microbial communities are not fully understood. In this study, we compared the bacterial and fungal community composition in the rhizosphere of a native grass (Andropogon gerardii) and a non-native grass (Sorghum halepense) from two North Texas prairies with dramatically different soil textures. Soils were analyzed for pH, organic matter, moisture, total carbon, total nitrogen, and clay and sand percentages. Microbial community fingerprints were generated using terminal restriction fragment length polymorphism (TRFLP) analysis. The overall structure of bacterial and fungal communities between the prairie sites and grass species were compared by analyzing binary data using the Additive Main Effects and Multiplicative Interaction (AMMI) model. This technique combines analysis of variance to first partition the variatio...
Proceedings of the National Academy of Sciences, 1994
Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-keto... more Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-ketoacid dehydrogenase enzyme complexes. We report the chemical synthesis and biological properties of lipoic acid analogs in which one or both sulfur atoms were replaced by selenium. Replacement of either the C-6 or the C-8 sulfur atom with selenium results in lipoic acid derivatives with apparently unaltered biological properties. However, simultaneous replacement of both sulfur atoms gave an analog (selenolipoic acid) that inhibited growth of wild-type Escherichia coli when present in minimal glucose medium at 50 ng/ml. This growth inhibition was reversed by the addition of either excess lipoic acid or acetate plus succinate. Labeling experiments with [75Se]selenolipoic acid showed that this compound was efficiently incorporated into the alpha-ketoacid dehydrogenase complexes of growing cells. Spontaneously arising selenolipoic acid-resistant (slr) mutants were isolated. Two of these isol...
Genomics is not only essential for students to understand biology but also provides unprecedented... more Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning appr...
Biochemistry and Molecular Biology Education, 2001
Two degenerate primers are described that can be used to amplify a 340 bp fragment of the ureC ge... more Two degenerate primers are described that can be used to amplify a 340 bp fragment of the ureC gene from a variety of urease producing bacteria. A series of experiments are outlined that enable students to develop restriction maps of the urease gene clusters from different bacteria. Students learn a variety of molecular biology techniques including polymerase chain reaction, agarose gel electrophoresis, and Southern blotting. This project allows for multiple experimental outcomes using the same two PCR primers.
Biochemistry and Molecular Biology Education, 2013
We used the Integrated Microbial Genomes Annotation Collaboration Toolkit as a framework to incor... more We used the Integrated Microbial Genomes Annotation Collaboration Toolkit as a framework to incorporate microbial genomics research into a microbiology and biochemistry course in a way that promoted student learning of bioinformatics and research skills and emphasized teamwork and collaboration as evidenced through multiple assessment mechanisms. Student teams in microbiology used bioinformatics tools to identify and characterize gene products from Mucilaginibacter paludis necessary for the synthesis of specific amino acids and then designed and carried out growth experiments to determine if the organism could indeed synthesize the amino acids. Students in biochemistry worked to characterize one of the amino acid biosynthetic pathways reconstructed by a previous microbiology class through amplification and cloning of the M. paludis genes and complementation analysis of Escherichia coli mutants.
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