Browsing by Author "Chen, Jiann-Shin"
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- Analysis of the Roles of the cwlD Operon Products during Sporulation in Bacillus subtilisGilmore, Meghan Elizabeth (Virginia Tech, 2000-11-15)CwlD has sequence similarities to N-acetyl muramoyl-L-alanine amidases, a class of enzymes known to cleave the bond between the peptide side chain and the N-acetyl muramic acid residue in cortex peptidoglycan formation during sporulation. A major difference between vegetative peptidoglycan and spore peptidoglycan is the presence of muramic-d -lactam (MAL) in spore peptidoglycan. It was previously determined that a cwlD null mutant does not contain muramic-d -lactam in the spore cortex peptidoglycan and the mutant spores were unable to complete germination. Therefore, it is believed that CwlD plays a role in MAL formation during sporulation. However, the specific role of the protein had not been demonstrated. It was also previously found that cwlD is in a two-gene operon with orf1. Orf1 is produced within the forespore with CwlD. The hypothesized role of Orf1 is to inhibit CwlD activity from within the forespore. Muramoyl-L-alanine amidase activity was demonstrated by CwlD in vivo. Therefore, CwlD is carrying out the first step of MAL synthesis, cleaving the peptide side chain while other enzymes are needed to complete MAL formation. Two different forms of CwlD were over-expressed, with and without the protein's signal peptide sequence. Both forms of the protein were purified and in both cases activity was undetectable. Antibodies specific for CwlD were obtained which can be used in future research as a tool to further characterize CwlD activity. A series of B. subtilis cwlD operon mutants were constructed altering the expression patterns of Orf1 and CwlD within the mother cell and forespore compartments. Various resistance properties and the germination ability of the mutant dormant spores were analyzed. It was determined that the absence of just Orf1 or Orf1 and CwlD from within the forespore has no effect on the phenotypes tested. Peptidoglycan from developing mutant forespores was extracted and analyzed throughout sporulation. Evidence was obtained demonstrating that the role of Orf1 is not to inhibit CwlD from within the forespore as hypothesized.
- Azotobacter vinelandii Nitrogenase: Effect of Amino-Acid Substitutions at the Alpha Gln-191 Residue of the MoFe Protein on Substrate Reduction and CO InhibitionVichitphan, Kanit (Virginia Tech, 2003-09-17)The FeMo cofactor is one of two types of prosthetic group found in the larger of the two nitrogenase component proteins, called the MoFe protein, and it is strongly implicated as the substrate binding and reduction site. The glutamine-191 residue in the Alpha-subunit of the MoFe protein of A. vinelandii nitrogenase was targeted for substitution because its side chain is involved in a hydrogen-bond network from one of the terminal carboxylates of the homocitrate component of FeMo cofactor through to the backbone NH of Alpha Gly-61, which is adjacent to Alpha Cys-62, which ligates to the P cluster (the second type of prosthetic group in the MoFe protein). A variety of altered MoFe proteins produced by the A. vinelandii mutant strains, namely the Alpha Pro-191, Alpha Ser-191, Alpha Thr-191, Alpha His-191, Alpha Glu-191, and Alpha Arg-191 altered MoFe proteins, have been purified to homogeneity and the catalytic properties of these altered MoFe proteins have been compared to those of wild type MoFe protein. Unlike wild type, the six altered MoFe proteins have decreased catalytic activity on substrate reduction and exhibited H2 evolution that was partially inhibited by added CO. Moreover, some of altered MoFe proteins with lower specific activity for the C2H4 production can produce C2H6 from C2H2. The results from the pH and activity studies indicate that the substitutions on the MoFe protein have an effect on the contribution of the responsible acid-base group(s) involved in proton transfer for H+- and C2H2-reduction. Furthermore, the inhibition by CO of hydrogen evolution by these altered MoFe proteins is likely from a lowering of the rate of both electron and proton transfer to the H+- reduction site(s). Some altered MoFe proteins but not wild type MoFe protein can produce C2H6 from C2H2. This observation suggested a lower apparent binding affinity for C2H2 and a slower proton transfer to C2H2 reduction with these altered MoFe proteins, which allow the intermediate to stay at the site longer and be further reduced by two electrons and two protons to give C2H6. These changes in the biochemical properties of these altered MoFe proteins indicate that the Alpha Gln-191 residue is intimately involved in substrate binding and reduction including proton delivery to substrate.
- Azotobacter vinelandii Nitrogenase: Multiple Substrate-Reduction Sites and Effects of pH on Substrate Reduction and CO InhibitionLi, Hong (Virginia Tech, 2002-04-30)Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein's interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997). Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion. Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may "fine-tune" the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates.
- Azotobacter vinelandii nitrogenase: role of the MoFe protein α-subunit histidine-195 residue in catalysisKim, ChulHwan (Virginia Tech, 1994-06-05)Site-directed mutagenesis and gene replacement procedures were used to isolate mutant strains of Azotobacter vinelandii that produce altered MoFe proteins where the α-subunit residue-195 position, normally occupied by a histidine residue, was individually substituted by a variety of other amino acids. Structural studies have revealed that this histidine residue is associated with the FeMo-cofactor binding domain and probably provides an NH→S hydrogen bond to a central bridging sulfide located within FeMo-cofactor. The present study investigates the role of the α-histidine-195 residue in nitrogenase catalysis by examining the altered MoFe proteins. Comparisons of the catalytic and spectroscopic properties of altered MoFe proteins produced by the Azotobacter vinelandii mutant strains suggest that the α-histidine-195 residue has a structural role which serves to keep the FeMo-cofactor attached to the MoFe protein and to correctly position the FeMo-cofactor within the polypeptide matrix such that N₂ binding is accommodated. Substitution of the α-His-195 residue by a glutamine residue results in an altered MoFe protein that binds but does not reduce N₂, the physiological substrate. Stopped-flow spectroscopic analyses indicate that the α-195gln MoFe protein is unable to reduce N₂ even though the altered MoFe protein can reach the redox state necessary for N₂ reduction. Although, N₂ is not a substrate for the altered MoFe protein, it is an inhibitor of both acetylene and proton reduction, both of which are otherwise effectively reduced by the altered MoFe protein. This result provides evidence that N₂ inhibits proton and acetylene reduction by simple occupancy of the active site. The α-195gln MoFe protein catalyzes HD formation in the presence of N₂ and D₂. Moreover, N₂ binding at the active site of the altered MoFe protein is inhibited by the addition of D₂. These observations indicate that binding of nitrogen to the enzyme is necessary but its reduction is not required for the formation of HD. N₂ uncouples MgATP from proton reduction catalyzed by the α-195gln MoFe protein, but does so without lowering the overall rate of MgA TP hydrolysis. Thus, the quasi-unidirectional flow of electrons from the Fe protein to the MoFe protein that occurs during nitrogenase turnover is controlled, in part, by the substrate serving as an effective electron sink. N₂-induced uncoupling of ATP hydrolysis from substrate reduction by the α-195gln MoFe protein is reversed by the addition of H₂ (D₂) in the assay atmosphere. This observation can successfully be explained if it-is assumed that the altered MoFe protein has a much greater binding affinity for H₂ (D₂) than for N₂. Substitution of the α-histidie-195 residue by glutamine also imparts hypersensitivity of acetylene reduction and N2 binding to inhibition by CO, indicating that the imidazole group of the α-histidine- 195 residue might protect an Fe contained within FeMo-cofactor from attack by CO.
- Biochemical and genetic characterization of mercaptopyruvate sulfurtransferase and paralogous putative sulfurtransferases of Escherichia coliJutabha, Promjit (Virginia Tech, 2001-06-11)Sulfurtransferases, including mercaptopyruvate sulfurtransferase and rhodanese, are widely distributed in living organisms. Mercaptopyruvate sulfurtransferase and rhodanese catalyze the transfer of sulfur from mercaptopyruvate and thiosulfate, respectively, to sulfur acceptors such as thiols or cyanide. There is evidence to suggest that rhodanese can mobilize sulfur from thiosulfate for in vitro formation of iron-sulfur clusters. Additionally, primary sequence analysis reveals that MoeB from some organisms, as well as ThiI of Escherichia coli, contain a C-terminal sulfurtransferase domain. MoeB is required for molybdopterin biosynthesis, whereas ThiI is necessary for biosynthesis of thiamin and 4-thiouridine in transfer ribonucleic acid. These observations led to the hypothesis that sulfurtransferases might be involved in sulfur transfer for biosynthesis of some sulfur-containing cofactors (e.g., biotin, lipoic acid, thiamin and molybdopterin). Results of a BLAST search revealed that E. coli has at least eight potential sulfurtransferases, besides ThiI. Previously, a glpE-encoded rhodanese of E. coli was characterized in our laboratory. In this dissertation, a mercaptopyruvate sulfurtransferase and corresponding gene (sseA) of E. coli were identified. In addition, the possibility that mercaptopyruvate sulfurtransferase could participate or work in concert with a cysteine desulfurase, IscS, in the biosynthesis of cofactors was examined. Cloning of the sseA gene and biochemical characterization of the corresponding protein were used to show that SseA is a mercaptopyruvate sulfurtransferase of E. coli. A strain with a chromosomal insertion mutation in sseA was constructed in order to characterize the physiological function of mercaptopyruvate sulfurtransferase. However, the lack of SseA did not result in a discernable phenotypic change. Redundancy of sulfurtransferases in E. coli may prevent the appearance of a phenotypic change due to the loss of a single sulfurtransferase. Subsequently, other paralogous genes for putative sulfurtransferases, including ynjE and yceA, were cloned. Strains with individual deletions of the chromosomal ynjE and yceA genes were also constructed. Finally, strains with multiple deficiency in potential sulfurtransferase genes, including sseA, ynjE and glpE, as well as iscS, were created. However, no phenotype associated with combinations of sseA, glpE and/or ynjE deficiency was identified. Therefore, the physiological functions of mercaptopyruvate sulfurtransferase and related sulfurtransferases remain unknown.
- Biosynthesis of Iron-Sulfur ClustersYuvaniyama, Pramvadee (Virginia Tech, 1999-11-17)It is not known whether biosynthesis of [Fe-S] clusters occurs through a spontaneous self-assembly process or an enzymatic process. However, in the Azotobacter vinelandii nitrogenase system, it has been proposed that NifS and NifU are involved in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfur for the in vitro catalytic formation of [Fe-S] clusters. The activity of the NifU protein has not yet been established, but NifU could have functions complementary to NifS by mobilizing iron or serving as an intermediate site necessary for nitrogenase-specific [Fe-S] cluster assembly. A second iron-binding site within NifU was predicted to serve these functions because two identical [2Fe-2S] clusters that had previously been identified within the homodimeric NifU are tightly bound, and the NifU primary sequence is rich in cysteine residues. In this dissertation, I examined the possibility that NifU might mobilize iron or serve as an intermediate site for [Fe-S] cluster assembly, as well as the possibility that NifU could work in concert with NifS. Primary sequence comparisons, amino acid substitution experiments, and biophysical characterization of recombinantly-produced NifU fragments were used to show that NifU has a modular structure. One module is contained in approximately the C-terminal half of NifU and provides the binding site for the [2Fe-2S] cluster previously identified (the permanent [2Fe-2S] cluster). Cysteine residues Cys¹³⁷, Cys¹³⁹, Cys¹⁷⁵, and Cys¹⁷⁵ serve as ligands to the [2Fe-2S] cluster. Another module (referred to as NifU-1) is contained in approximately the N-terminal third of NifU and provides a second iron-binding site (rubredoxin-like Fe(III)-binding site). Cysteine residues Cys35, Cys⁶², Cys¹⁰⁶>, and a putative non-cysteine ligand of unknown origin provide coordination to the iron at this site. The significance of these iron-binding sites was also accessed by showing that cysteine residues involved in providing the rubredoxin-like Fe(III)-binding site and those that provide the [2Fe-2S] cluster binding site are all required for the full physiological function of NifU. The two other cysteine residues contained within NifU, Cys²⁷² and Cys²⁷⁵, are neither necessary for binding iron at either site nor are they required for the full physiological function of NifU. These results provide the basis for a model where iron bound at the rubredoxin-like sites within NifU-1 (one iron per monomer) is proposed to be destined for [Fe-S] cluster formation. It was possible to find in vitro evidence supporting this idea. First, it was demonstrated that NifU and NifS are able to form a transient complex. Second, in the presence of NifS as well as L-cysteine and a reducing agent, the Fe(III) contained at the rubredoxin-like sites within the NifU-1 or NifU homodimer can rearrange to form a transient [2Fe-2S] cluster between the two subunits. Finally, a mutant form of NifU-1 was isolated that appears to be trapped in the [2Fe-2S] cluster-containing form, and this [2Fe-2S] cluster (the transient [2Fe-2S] cluster) can be released from the polypeptide matrix upon reduction with dithionite. Previous work has shown that the permanent [2Fe-2S] clusters of as-isolated NifU are in the oxidized form but can be reduced chemically. The transient [2Fe-2S] cluster formed between rubredoxin-like sites, in contrast, is reductively labile. If the transient cluster serves as an intermediate [Fe-S] cluster to be destined for [Fe-S] cluster assembly, I propose that the permanent [2Fe-2S] clusters could have redox roles participating in either one or all of the following events. The permanent [2Fe-2S] clusters could have a redox function in the acquisition of iron for initial binding at the mononuclear sites. They could also provide reducing equivalents for releasing the transient [2Fe-2S] cluster. In addition, upon releasing the transient [2Fe-2S] cluster, the permanent [2Fe-2S] clusters could provide the appropriate oxidation state of the irons to be destined to nitrogenase metallocluster core formation. Finally, because proteins homologous to NifU and NifS are widely distributed in nature, it is suggested that the mechanism for NifU and NifS in the formation of nitrogenase-specific [Fe-S] clusters could represent a general mechanism for [Fe-S] cluster synthesis in other systems.
- Biosynthesis of the Nitrogenase FeMo-cofactor from Azotobacter vinelandii: Involvement of the NifEN complex, NifX and the Fe proteinGoodwin, Paul Joshua (Virginia Tech, 1999-10-25)The iron-molybdenum cofactor (FeMo-cofactor) of nitrogenase is the subject of one the most intensive biochemical/genetic detective cases of modern science. At the active site of nitrogenase, the FeMo-cofactor not only represents the heart of biological nitrogen fixation, but its synthesis also serves as a model for complex metallocluster biosynthesis. Research in the Dean Lab is focused on furthering the understanding of Fe-S cluster biosynthesis in the nitrogenase enzyme system. Throughout the years, scientists from a broad range of disciplines have focused their intellectual might on deciphering not only the chemistry of the FeMo-cofactor, but also the biosynthesis of this unique metallocluster. Recent advances in the study of FeMo-cofactor biosynthesis have produced considerable insight regarding the complex series of biological reactions necessary for the synthesis of this metallocluster. The work contained within this dissertation represents my efforts to further the understanding of FeMo-cofactor biosynthesis. The concept of a molecular scaffold in FeMo-cofactor biosynthesis is generally accepted in the field of nitrogenase. Previous work has implicated the products of nifE and nifN as providing the assembly site for FeMo-cofactor synthesis. Researchers were able to purify this molecular scaffold, commonly referred to as the NifEN complex, however, detailed characterization was precluded by the inability to obtain sufficient quantities of NifEN. In an effort to fully characterize the NifEN complex, we initiated a gene fusion approach for the high level production NifEN. In addition to gene fusion, a poly-histidine tag was incorporated into NifEN, allowing purification through the application of immobilized metal-affinity chromatography (IMAC). NifEN obtained in this way was characterized using a variety of biophysical techniques and found to contain two [4Fe-4S] clusters in each NifEN tetramer. These clusters were also shown to be completely ligated by cysteine residues. With the information obtained from this study, it is concluded that the [4Fe-4S] clusters of the NifEN complex are likely to play either a structural or a redox role rather than being transferred and becoming incorporated into the FeMo-cofactor. In addition to the biophysical characterization of the NifEN complex, a separate study was started to characterize the apo-MoFe protein. In this study we used IMAC to purify a poly-histidine-tagged apo-MoFe protein produced by a nifB-deletion mutant of A. vinelandii. Using the poly-histidine fusion approach, apo-MoFe protein was obtained in sufficient quantities for detailed catalytic, kinetic and spectroscopic analyses. This multidisciplinary approach confirmed that apo-MoFe protein contained intact P clusters and P cluster environments, as well as the ability to interact with the Fe protein. It was also shown for the first time that this tetrameric form of purified apo-MoFe protein could be activated by the addition of preformed FeMo-cofactor. The NifEN complex was further characterized to investigate the presence of bound FeMo-cofactor intermediates. NifEN purified by IMAC is produced in the absence of the nitrogenase structural genes (nifHDK). In this genetic background, it is believed that the FeMo-cofactor biosynthetic machinery will become obstructed with unprocessed FeMo-cofactor intermediates, such as the Fe-S precursors of FeMo-cofactor, NifB-cofactor. Previous work indicated that NifEN can exist in either a charged or discharged form, based on the presence or absence of the FeMo-cofactor precursor, NifB-cofactor. EPR and VTMCD spectroscopies showed the presence of a new paramagnetic signal associated with NifEN that is believed to be in the charged or precursor bound state. This represents the first spectroscopic evidence for a precursor to the FeMo-cofactor. Furthermore, an interaction of NifEN and NifX was examined by size exclusion chromatography. From this study, NifX exhibited the capacity to bind a chromophore, presumably an FeMo-cofactor precursor, from the NifEN complex. NifX was also capable of binding to isolated FeMo-cofactor and the FeMo-cofactor precursor, NifB-cofactor. Finally, preliminary investigations involving interaction between the Fe protein and NifEN were initiated. Recent findings indicate that NifEN and the Fe protein have the capacity to interact specifically with one another. The interaction of NifEN and Fe protein appears to be dependent on the association of FeMo-cofactor precursor with NifEN. The NifEN complex also has the capacity to accept electrons from the Fe protein in a MgATP dependent manner. The ability of NifEN to accept electrons from the Fe protein may be involved in the role of Fe protein in FeMo-cofactor biosynthesis.
- Characterization and Molecular Analysis of Fragilysin: The Bacteroides fragilis ToxinObiso, Richard J. Jr. (Virginia Tech, 1997-05-06)Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250
- Characterization and site-directed mutagenesis of NifU from Azotobacter vinelandiiJack, Richard F. (Virginia Tech, 1995-10-05)In order to elucidate the function of the nifU gene product in nitrogenase maturation in Azotobacter vinelandii. the gene product has been hyperexpressed in Escherichia coli and characterized by various biophysical techniques. Following the initial characterization, site-directed mutagenesis of conserved cysteinyl residues was performed in order to gain further insight into the structure/function relationship of NifU. Both the Fe protein and the MoFe protein of nitrogenase require processing by additional nif genes including nifM (Fe protein), and nifE, N, B, H, V, and Q (MoFe protein). Two additional genes, nifU and nifS, are required for the maturation of both nitrogenase component proteins. It has been proposed that they may somehow be involved in metallocluster biosynthesis (Jacobson et al., 1989b). Our laboratory has determined that the nifS gene product (Nifs) is a pyridoxal-phosphate containing enzyme capable of catalyzing the desulfurization of L-cysteine and can provide the inorganic sulfide necessary for in vitro metallocluster biosynthesis of the Fe protein (Zheng, et al., 1993: Zheng, et al., 1994).
- Characterization of AgaR and YihW, Members of the DeoR Family of Transcriptional Regulators, and GlpE, a Rhodanese Belonging to the GlpR Regulon, Also a Member of the DeoR FamilyRay, William Keith (Virginia Tech, 1999-08-02)AgaR, a protein in Escherichia coli thought to control the metabolism of N-acetylgalactosamine, is a member of the DeoR family of transcriptional regulators. Three transcriptional promoters within a cluster of genes containing the gene for AgaR were identified, specific for agaR, agaZ and agaS, and the transcription start sites mapped. Transcription from these promoters was specifically induced by N-acetylgalactosamine or galactosamine, though K-12 strains lacked the ability to utilize these as sole sources of carbon. The activity of these promoters was constitutively elevated in a strain in which agaR had been disrupted confirming that the promoters are subject to negative regulation by AgaR. AgaR-His6, purified using immobilized metal affinity chromatography, was used for DNase I footprint analysis of the promoter regions. Four operator sites bound by AgaR were identified. A putative consensus binding sequence for AgaR was proposed based on these four sites. In vivo and in vitro analysis of the agaZ promoter indicated that this promoter was activated by the cAMP-cAMP receptor protein (CRP). Expression from the aga promoters was less sensitive to catabolite repression in revertants capable of N-acetylgalactosamine utilization, suggesting that these revertants have mutation(s) that result in an elevated level of inducer for AgaR. A cluster of genes at minute 87.7 of the E. coli genome contains a gene that encodes another member of the DeoR family of transcriptional regulators. This protein, YihW, is more similar to GlpR, transcriptional regulator of sn-glycerol 3-phosphate metabolism in E. coli, than other members of the DeoR family. Despite the high degree of similarity, YihW lacked the ability to repress PglpK, a promoter known to be controlled by GlpR. A variant of YihW containing substitutions in the putative recognition helix to more closely match the recognition helix of GlpR was also unable to repress PglpK. Transcriptional promoters identified in this cluster of genes were negatively regulated by YihW. Regulation of genes involved in the metabolism of sn-glycerol 3-phosphate in E. coli by GlpR has been well characterized. However, the function of a protein (GlpE) encoded by a gene cotranscribed with that for GlpR was unknown prior to this work. GlpE was identified as a single-domain, 12-kDa rhodanese (thiosulfate:cyanide sulfurtransferase). The enzyme was purified to near homogeneity and characterized. As shown for other characterized rhodaneses, kinetic analysis revealed that catalysis occurs via an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. Km (SSO₃²⁻) and Km (CN⁻) were determined to be 78 mM and 17 mM, respectively. The native molecular mass of GlpE was 22.5 kDa indicating that GlpE functions as a dimer. GlpE exhibited a kcat of 230 s-1. Thioredoxin, a small multifunctional dithiol protein, served as sulfur-acceptor substrate for GlpE with an apparent Km of 34 mM when thiosulfate was near its Km, suggesting thioredoxin may be a physiological substrate.
- Characterization of an altered MoFe protein from a nifV- strain from Azotobacter vinelandiiComaratta, Leonard M. (Virginia Tech, 1998-12-03)The site of substrate binding and reduction for the nitrogenase complex is located on the iron molybdenum cofactor (FeMo-co) which is contained within the a-subunit of the molybdenum iron protein. FeMo co consists of a metal sulfur core composed of an FeS cluster bridged by three inorganic sulfides to a MoFeS cluster. An organic acid, homocitrate, is coordinated to the Mo atom through its 2-carboxy and 2-hydroxy groups. Homocitrate is formed by the condensation of acetyl-CoA and a-ketoglutarate, which is catalyzed by a homocitrate synthase encoded by nifV. By deleting the nifV gene from Azotobacter vinelandii we were able to study the role of homocitrate in nitrogenase catalysis. A poly-histidine tail was incorporated into the C-termini of the a-subunit permitting isolation of the homocitrateless MoFe protein by using metal affinity chromatography. We have found that the addition of a poly-histidine tag does not alter the catalytic behavior of the native enzyme. In NifV- strains of Klebsiella pneumoniae, citrate has been found to replace homocitrate as the organic constituent of FeMo-co. We have found no evidence this is so in A. vinelandii. Gas chromatography mass spectrophotometry studies indicate little or no organic acids are associated with FeMo-co. We examined the catalytic properties of the NifV- MoFe protein In the mutant, H2 evolution is inhibited by the addition of CO, unlike in the wild type. We have found that the NifV- MoFe protein from A. vinelandii is able to catalyze the reduction of acetylene to both ethylene and ethane.
- The characterization of Clostridium beijerinckii NRRL B592 cells transformed with plasmids containing the butanol-production genes under the control of constitutive promotersTollin, Craig Jeffrey (Virginia Tech, 2012-09-18)Clostridium beijerinckii is a spore-forming, obligate anaerobe that is capable of producing butanol, acetone and isopropanol. These industrial chemicals are traditionally known as solvents. The regulation of solventogenic fermentation is linked to the onset of sporulation, so that by the time the organism begins to produce solvents, it is also entering into spore formation and metabolic slowdown. The goal of this research project was to study the effect of placing the solvent-production genes from C. beijerinckii under the control of constitutive promoters from other genes, in an attempt to allow an earlier start of butanol production during the growth phase than is the case with the wild-type cells. The aldehyde dehydrogenase from C. beijerinckii NRRL B593 (ald) and alcohol dehydrogenase from C. beijerinckii NRRL B592 (adhA) were placed under the control of the promoter from the acid-producing operon (the BCS operon) in one vector, and under the control of the promoter from the ferredoxin gene in another. In both cases, aldehyde dehydrogenase activity was produced earlier in the growth phase in transformed cells, but alcohol dehydrogenase activity was not. The adhA gene from C. beijerinckii NRRL B592 was paired with the adhB gene from the same organism in a third vector, both under the control of the promoter from the BCS operon. In cells transformed with this vector, alcohol dehydrogenase activity was observed earlier in the growth phase than it was in wild-type NRRL B592 cells.
- Characterization of the Components of Carbon Catabolite Repression in Clostridium perfringensHorton, William Henry Clay (Virginia Tech, 2004-12-08)Clostridium perfringens is a versatile pathogen capable of causing a wide array of diseases, ranging from clostridial food poisoning to tissue infections such as gas gangrene. An important factor in virulence as well as in the distribution of C. perfringens is its ability to form an endospore. The symptoms of C. perfringens food poisoning are directly correlated to the release of an enterotoxin at the end of the sporulation process. The sporulation process in C. perfringens is subject to carbon catabolite repression (CCR) by sugars, especially glucose. CCR is a regulatory pathway that alters transcription based on carbon source availability. In Gram-positive bacteria, the HPr kinase/phosphatase is responsible for this nutritional sensing by phosphorylating or dephosphorylating the serine-46 residue of HPr. HPr-Ser-P then forms a complex with the transcriptional regulator CcpA to regulate transcription. We were able to show here that purified recombinant C. perfringens HPr kinase/phosphatase was able to phosphorylate the serine-46 residue of HPr. When the codon for this serine residue is mutated through PCR mutagenesis to encode alanine, phosphorylation could not take place. We have also shown that in gel retardation assays, CcpA and HPr-Ser-P were able to bind to two DNA fragments containing putative C. perfringens CRE-sites, sequences where CcpA binds to regulate transcription. The genome sequence of a food poisoning strain of C. perfringens was searched for potential CRE-sites using degenerate sequences designed to match those CRE-sites CcpA was shown to bind. DNA fragments containing these newly identified CRE-sites were then used in gel retardation assays to determine whether CcpA binds to these CRE-sites, making them candidates for CCR regulation. These results, combined with comparisons of metabolic characteristics of a ccpA- strain versus wild-type C. perfringens, provide evidence that CcpA participates in the regulation of carbon catabolite repression in the pathogenic bacterium C. perfringens
- Characterization of the structure and function of a Bacteroides thetaiotaomicron 16S rRNA promoterThorson, Mary Leah (Virginia Tech, 2003-06-06)The bacteroides group is a subdivision in the Cytophaga-Flavobacterium-Bacteroides phylum. This group is as phylogenetically distinct from other Gram-negative enterics, including Escherichia coli, as they are from Gram-positive organisms. Furthermore, there is no cross expression between genes of E. coli and Bacteroides species. It is thought that this difference in gene expression lies in part at the level of transcription initiation and is due to the sequences within the promoter region itself. A putative consensus sequence for Bacteroides promoters has been published by C. Jeff Smith’s research group based on alignments of the sequences upstream of certain regulated genes. However, this consensus has not been found within all putative Bacteroides promoters. In this study, the promoter structure and function of a strong housekeeping B. thetaiotaomicron 16S rRNA promoter was examined and compared to an E. coli 16S rRNA promoter. Our hypothesis is that there are significant differences between the promoters of these two organisms. Analysis of B. thetaiotaomicron sequence upstream of the 16S rRNA gene has revealed the same overall structure known for E. coli 16S rRNA promoters in that there are two putative promoters separated by approximately 150 bp. However, the B. thetaiotaomicron 16S rRNA promoter contains the proposed Bacteroides —7 and —33 consensus sequences instead of the well known E. coli —10 and —35 consensus sequences. The biological activity of the B. thetaiotaomicron 16S rRNA full-length promoter was confirmed using a Bacteroides lux reporter system. A newly designed Bacteroides lux reporter was used to analyze specific regions of the B. thetaiotaomicron 16S rRNA promoter. In addition, by pairing the B. thetaiotaomicron 16S rRNA promoter with an E. coli ribosomal binding site, and vice-versa, the improved lux reporter was used to further confirm that the difference in gene expression between the two species lies at the level of transcription in E. coli. In Bacteroides, however, transcription and translation may work together to create a barrier to efficient gene expression of foreign genes.
- Characterization of two novel proteins containing the rhodanese homology domain: YgaP and YbbB of Escherichia coliAhmed, Farzana (Virginia Tech, 2003-07-07)Rhodanese homology domains are ubiquitous structural modules found in eubacteria, eukaryotes and archaea. The rhodanese homology domain may comprise the entire structure of a protein. Alternatively it is found as tandemly repeated modules in which the C-terminal domain displays the properly structured active site. Finally it is found as a member of many multidomain proteins. Although some members of this family of proteins show sulfurtransferase activity in vitro, their specific physiological functions remain largely undefined. Fusion of a rhodanese domain to different protein domains of known or unknown functions provides important clues to the diverse roles for these proteins. Nine proteins containing the rhodanese homology domain are predicted in Escherichia coli. In this work, two of these proteins: YgaP and YbbB were characterized using bioinformatics, biochemical and genetic approaches. YgaP is a single domain rhodanese that is predicted to contain an amino-terminal rhodanese domain (118 amino acids) and a hydrophobic carboxy-terminal domain (56 amino acids). The ygaP gene was cloned into a vector that directed overexpression of a membrane-associated rhodanese activity. The cellular location of YgaP was determined by using sucrose density layer ultracentrifugation. YgaP and rhodanese activity co-sedimented with the cytoplasmic membrane marker D-lactate dehydrogenase, and was not present in the outer membrane fractions, indicating YgaP is a cytoplasmic membrane protein. A polyhistidine-tagged variant of YgaP was subsequently solubilized from the membrane by detergent extraction and purified by metal chelate chromatography. Similar to the other characterized rhodaneses, purified YgaP-His6 as well as the membrane-associated native form of the protein displayed a double displacement (ping-pong) mechanism. YgaP is unique in that it is the first membrane-associated rhodanese to be described. To understand the physiological role of YgaP, a strain with ygaP gene disruption was constructed. No obvious phenotype resulted from deletion of ygaP. The ybbB gene of E. coli has an interesting genome organization in several Gram-negative bacteria including Pseudomonas aeruginosa and Azotobacter vinelandii where it is predicted to be in the same operon with selD, encoding selenophosphate synthetase. Thus the role of YbbB in selenium metabolism was investigated. A strain with ybbB gene deletion was constructed and tested for its ability to incorporate 75Se into tRNA and protein. It was shown that the disruption of ybbB prevented specific incorporation of selenium into tRNA but not into proteins in vivo. The modified nucleoside missing in tRNAs of the DybbB strain was identified as 5-methylaminomethyl-2-selenouridine (mnm5se2U), which has previously been shown to be present in the wobble position of the anticodon of E. coli tRNAsLys, Glu and Gln. Data from HPLC analysis showed that the deletion of ybbB did not affect the production of 5-methylaminomethyl-2-thiouridine (mnm5s2U), the precursor to mnm5se2U, suggesting that YbbB is not required for sulfur transfer but is rather involved in selenation of tRNAs. YbbB was subsequently expressed with a C-terminal histidine tag and purified for initial characterization. Purified YbbB-His6 migrated as a 43 kDa monomer under denaturing conditions and displayed spectral properties that suggested its interaction with tRNA. Finally, it was shown that Cys97, which aligns with the active site cysteine of rhodanese and is conserved in all known YbbB homologs, is required for YbbB activity. However, Cys96, which is not conserved, is not required for activity.
- The Cloning of a Putative Regulatory Gene and the sol Region from Clostridium beijerinckiiHong, Rui (Virginia Tech, 1999-08-09)The solvent-producing clostridia are well known for their ability to produce acetone, butanol and isopropanol in industrial fermentation. Production of these compounds occurs in cells that have completed a metabolic switch under specific growth conditions. Knowledge of the regulation of the metabolic switch will make the industrial process more reliable. From an isopropanol-producing strain Clostridium beijerinckii NRRL B593, a gene which encodes a putative NtrC-like regulatory protein was cloned and sequenced. The gene codes for a polypeptide of 632 amino acids and has been designated the stc gene. Expression of the stc gene was confirmed by RT-PCR. The co-presence of the stc gene with the adh gene which encodes a primary/secondary alcohol dehydrogenase in isopropanol-producing clostridia suggests that the stc gene may be functionally related to isopropanol production. From C. beijerinckii NRRL B592, a region which encompassed the solvent-production genes ald (aldehyde dehydrogenase), ctfA and ctfB (acetoacetate: butyrate/acetate CoA-transferase) and part of adc (acetoacetate decarboxylase) was cloned and sequenced. The organization of these genes was similar to that in C. beijerinckii NRRL B593. Northern analysis indicated that these four genes were co-transcribed on the same messenger RNA in C. beijerinckii NRRL B593. Therefore, in C. beijerinckii, the sol operon consists of the ald -ctfA-ctfB-adc genes, which differs from the sol operon in Clostridium acetobutylicum.
- CoA-transferase and 3-hydroxybutyryl-CoA dehydrogenase: acetoacetyl-CoA-reacting enzymes from Clostridium beijerinckii NRRL B593Colby, Gary D. (Virginia Tech, 1993-07-05)In acetone/butanol-producing clostridia, the metabolic intermediate acetoacetyl-CoA can be directed toward butyrate or butanol formation by the reaction catalyzed by 3-hydroxybutyryl-CoA dehydrogenase, or toward acetone formation by the reaction catalyzed by acetoacetate:acetate/butyrate CoA-transferase. 3-Hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 or 1.1.1.157) has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beijerinckii strain NRRL B593. The identities of 34 of the 35 N-terminal amino acid residues have been determined. The enzyme exhibited a native Mr of 213,000 and a subunit Mr of 30,800. It is specific for the (S)-enantiomer of 3-hydroxybutyryl-CoA. Michaelis constants for NADH and acetoacetyl-CoA were 8.6 and 14 µM, respectively. The maximum velocity of the enzyme was 540 µmol/(min mg) for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as cosubstrate; however, NAD(H) appeared to be the physiological substrate. In the presence of 9.5 µM NADH, the enzyme was inhibited by acetoacetyl-CoA at concentrations as low as 20 µM, but the inhibition was relieved as the concentration of NADH was increased, suggesting a possible mechanism for modulating the energy efficiency during growth. Acetoacetate:acetate/butyrate CoA-transferase (EC 2.8.3.9) has been purified 308-fold to apparent homogeneity from the same organism. The enzyme exhibited a native Mr of 89,100. The subunits of the enzyme were separated by preparative SDS-PAGE, and exhibited M, values of 28,400 and 25,200. The identities of the 34 N-terminal amino acids of the large subunit and 38 of the 39 N-terminal amino acids of the small subunit were determined. The N-terminal region of the two subunits showed significant similarity with several other CoA transferase enzymes. Michaelis constants for butyrate and acetoacetyl-CoA were 11.7 mM and 107 µM, respectively, while those for acetate and acetoacetyl-CoA were 424 mM and 118 µM, respectively. The value of kcat/Km was approximately 100 times higher with butyrate than with acetate. Implications of the properties of these two enzymes for the acetone-butanol fermentation are discussed, and a model for the induction of the enzymes responsible for solvent production is suggested.
- Development of a reporter system for the study of gene expression for solvent production in Clostridium beijerinckii NRRL B592 and Clostridium acetobutylicum ATCC 824Li, Guang-Shan (Virginia Tech, 1998-09-23)To study the regulation of gene expression, a good reporter system is very useful. The lack of a good reporter system for the solvent-producing clostridia hindered the progress of research in this area. The objective of this study was to develop a reporter system to facilitate the elucidation of the control mechanism for the expression of solvent-producing genes. A potential reporter gene was found in Clostridium beijerinckii NRRL B593, which contains an adh gene encoding a primary-secondary alcohol dehydrogenase and this adh gene is not present in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NRRL B592. The adh gene was cloned into the E. coli -Clostridium shuttle vectors to generate plasmids. An electro-transformation procedure was developed for C. beijerinckii NRRL B592. Shuttle plasmids were transformed into C. beijerinckii NRRL B592 or C. acetobutylicum ATCC 824. The copy number of the plasmids in C. beijerinckii was 4. Isopropanol production suggested that the adh gene was expressed in transformants of C. acetobutylicum ATCC 824 and C. beijerinckii NRRL B592. Northern analysis indicated that the expression of the adh gene was regulated at the transcriptional level in the transformants of C. beijerinckii. The transcriptional start site for the adh gene was identified by the primer extension method. A promoter-probing vector was constructed and tested with the promoter from the ferredoxin(fer) gene. The expression of the adh gene under the control of the fer promoter was at a low and similar level during acidogenesis and solventogenesis. The expression pattern of the adh gene under the control of the promoter of the adh gene differed from that under the control of the promoter of the fer gene.
- The Effect of Nitrates, pH, and Dissolved Inorganic Carbon Concentrations on the Extracellular Polysaccharide of Three Strains of Cyanobacteria Belonging to the Family NostocaceaeHorn, Kevin J. (Virginia Tech, 2008-05-30)Three strains of cyanobacteria (Anabaena PCC7120, A. variabilis and Nostoc commune), all belonging to the family Nostocaceae, were found to be capable of modulating the production and chemical composition of extracellular polysaccharides (EPS) in response to carbon and nitrogen availability as well as pH. While the carbohydrate compositions of the glycans produced by the different organisms were indicative of their recent evolutionary divergence, there were measurable differences that were dependent upon growth conditions. The EPS resulting from biofilm growth conditions was reduced in glucuronic acid levels in both Anabaena variabilis ATCC 29413 and Anabaena PCC 7120. Under planktonic conditions, the glycan from A. variabilis contained glucuronic acid when grown in nitrate-free BG-11₀ medium whereas A. PCC 7120 produced similar levels in standard BG-11 medium. This suggests that phylogeneticallyrelated cyanobacteria respond very differently to changes in their local environment. The pH of BG-11 cultures increased to 9-10 for all three strains of cyanobacteria. The increase resulted in an increase in the amount of dissolved inorganic carbon available in the medium, creating an imbalance in the carbon-nitrogen ratio, with the complete consumption of 17.65 mmol L⁻¹ nitrates raising the pH to near 10 in BG-11 medium. While increased carbon availability has been shown to induce capsulated morphologies in strains of cyanobacteria, only Nostoc commune DRH-1 exhibited this behavior, and only when grown in BG-11 medium. Carbon and nitrogen availability as well as pH modulate the monosaccharide composition of the glycan generated by cyanobacteria investigated. The different characteristics of the glycans produced can affect the survivability of the organisms and the community structure of cyanobacterial biofilms and microbial mats found in nature. As cyanobacteria are ubiquitous organism both now and in the past, they play a pivotal role in the biological and geological processes of the Earth, controlling the availability and cycling of carbon and nitrogen both actively and passively.
- Enzymology of butanol formation in Clostridium BeijerinckiiYan, Run-Tao (Virginia Tech, 1991)The present study encompasses an investigation of the expression of solvent forming enzymes and purification and characterization of butanol-forming enzymes. More sensitive and accurate procedures for the determination of acids and solvents in cultures have been developed, which led to the recognition of the onset of solvent production at the mid-exponential phase, about two h earlier than previously reported. Activities of solvent-forming enzymes started to increase about one h before the onset of measurable solvent production and the activities of solvent-forming enzymes did not increase simultaneously. CoA-acylating aldehyde dehydrogenase (ALDH) was purified to near homogeneity. The ALDH showed a native M.. of 100,000, and a subunit Mr of 55,000. ALDH could use either NAD(H) or NADP(H) as the coenzyme. ALDH was oxygenlabile. The O₂-inactivated enzyme could be reactivated by incubating the enzyme with CoA. Both NADH- and NADPH-dependent alcohol dehydrogenase activities were present in crude extracts. The ratio of NADPH-dependent activity to NADH-dependent activity (the PID ratio) varied in crude extracts. The PID ratio was affected by O~ ionic strength, pH, growth stage of cell, Fe in culture medium and temperature. Two ADHs have been identified in crude extracts. The NADPH-dependent ADH (P-ADH) could be separated from the NADH/NADPH-dependent ADH (D/P-ADH). The D/P-ADH has been extensively purified. The D/P-ADH showed a native Mr of 70,000 and subunits with Mr of 45,300 and 40,000. The D/P-ADH activity could be inactivated by a,a' -dipyridyl and restored by Fe2+.