VTechWorks Repository :: Browsing by Author "Popham, David L."

Browsing by Author "Popham, David L."

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Amino Acid Residues in LuxR Critical for its Mechanism of Transcriptional Activation during Quorum Sensing
Trott, Amy Elizabeth (Virginia Tech, 2000-07-17)
Vibrio fischeri, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in V. fischeri, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or lux operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the lux operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An in vivo luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the lux operon in recombinant Escherichia coli. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the lux box) or to establish associations with RNA polymerase (RNAP) at the lux operon promoter necessary for transcriptional initiation. A third in vivo assay was used to test the ability of the altered forms of LuxR to bind to the lux box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the lux box in the DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the lux box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the lux box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the lux operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the lux operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.
Analysis of Peptidoglycan Structural Changes and Cortex Lytic Enzymes during Germination of Bacillus anthracis Spores
Dowd, Melissa Margaret (Virginia Tech, 2005-09-05)
Sporulation is a process of differentiation that allows capable cells to go into a dormant and resistant stage of life. To become active again, the spores must germinate into vegetative cells. One key process in spore germination is hydrolysis of the cortex peptidoglycan. This process has been studied in a variety of sporulating species; however, it has not been studied in Bacillus anthracis. A clear understanding of cortex degradation may provide information that will allow for better cleanup of spore contaminated sites. The structure of cortex peptidoglycan of Bacillus anthracis was characterized. The peptidoglycan of the dormant spores was extracted, digested with Mutanolysin, and analyzed using HPLC to determine the structure. The analyses revealed that the cortex peptidoglycan of B. anthracis was very similar to other Bacillus sp.. Spores were stimulated to germinate and cortex peptidoglycan was extracted and analyzed at various times. Bacillus anthracis appeared to hydrolyze its cortex more rapidly than other Bacillus species. While the spores of three species release the spore solute dipicolinic acid and resume metabolism at similar rates, the B. anthracis spores released 75% their cortex material within 10 minutes while the other species released only 20% in the same time frame. This suggests that the B. anthracis spore coats are more permeable to cortex fragments than those of the other species, or that B. anthracis rapidly cleaves the cortex into smaller fragments. Novel cortex fragments analyzed during B. anthracis germination were produced by a glucosaminidase; however, additional studies need to be performed for confirmation.
Analysis of the Regulons Controlled by Transcriptional Regulators LuxR and LitR in Vibrio fischeri
Qin, Nan (Virginia Tech, 2008-07-22)
Quorum sensing is a bacterial signaling system that controls gene expression in a population density-dependent manner. In Gram-negative proteobacteria, the cell density control of luminescence was first observed in the symbiotic marine bacterium Vibrio fischeri and this system is one of the best studied quorum sensing systems. Two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). A lacZ reporter was used to show that the promoters for qsrP, acfA, and ribB were directly activated via LuxR-3-oxo-C6-HSL in recombinant Escherichia coli. The sites of transcription initiation were established via primer extension analysis. Based on the position of the lux box-binding site near position â 40, all three promoters appear to have a class II-type promoter structure. Real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. In order to fully characterize the LuxR regulon in V. fischeri ES114, microarray analysis was performed in the Greenberg lab (University of Washington) and 18 LuxR-3-oxo-C6-HSL regulated promoters were found including 2 genes (qsrP and acfA) identified previously in MJ-100 in addition to the well-studied lux operon. In collaboration with them, full-length purified LuxR protein was used to show direct interaction between the LuxR protein and 7 genes/operons newly identified out of 13 genes/operons examined. The binding affinity between LuxR proteins and those genes was also measured. Based on the sequence of the lux boxes of the known genes regulated by LuxR and LitR, a position specific weight matrix (PSWM) was created and used to search through the intergenic regions of the V. fischeri ES114 genome. Some potential LuxR and LitR-regulated genes with high score were tested experimently to confirm direct activation. For the LuxR regulon, these possible LuxR-regulated promoters were cloned into a lacZ reporter and tested for their LuxR dependence. Beyond the genes found in microarray, the promoter of the intergenic region VFA0658-0659 was found to be activated by LuxR and 3-oxo-C6-HSL. For the LitR regulon, two LitR-regulated genes found in the microarray were also identified using PSWM and confirmed by real-time PCR to be dependent on LitR for expression. EMSA experiments showed that LitR can specifically bind to the litR boxes of LitR-regulated genes, litR and VF0170 which confirmed that the regulation is direct.
Analysis of the Roles of the cwlD Operon Products during Sporulation in Bacillus subtilis
Gilmore, 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.
Bacterial developmental checkpoint that directly monitors cell surface morphogenesis
Delerue, Thomas; Anantharaman, Vivek; Gilmore, Michael C.; Popham, David L.; Cava, Felipe; Aravind, L.; Ramamurthi, Kumaran S. (Cell Press, 2022-02-07)
Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous “coat” and an inner peptidoglycan “cortex,” separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.
Biochemical and genetic characterization of mercaptopyruvate sulfurtransferase and paralogous putative sulfurtransferases of Escherichia coli
Jutabha, 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 Nucleotide Sugar Monomers for Exopolysaccharide Production in Myxococcus Xanthus
Cadieux, Christena Linn (Virginia Tech, 2007-09-07)
Myxococcus xanthus displays social (S) motility, a form of surface motility that is key to the multicellular behaviors of this organism. S motility requires two cellular structures: type IV pili (TFP) and exopolysaccharides (EPS). Previous studies have shown that M. xanthus does not use glucose or any other sugar as a primary carbon source. However, eight monosaccharides, namely glucose, mannose, arabinose, galactose, xylose, rhamnose, N-acetyl-glucosamine, and N-acetyl-mannosamine, are found in M. xanthus EPS. In this study, pathways that M. xanthus could use to produce the activated sugar monomers to form EPS are proposed based on genomic data. Of the eight sugars, pathways for seven were disrupted by mutation and their effects on the EPS-dependent behaviors were analyzed. The results indicate that disruption of the two pathways leading to the production of activated rhamnose (GDP- and TDP-rhamnose) affected fruiting body formation (GDP form only) and dye binding ability (both forms) but not S motility. Disruptions of the xylose, mannose, and glucose pathways caused M. xanthus to lose S motility, fruiting body formation, and dye binding abilities. An interruption in the pathway for galactose production created a mutant with properties similar to a lipopolysaccharide (LPS) deficient strain. This discovery led us to study the phenotypes of all mutant strains for LPS production. The results suggest that all mutants may synthesize defective LPS configurations. Disruption of the UDP-N-acetyl-mannosamine pathway resulted in a wild type phenotype. In addition, it was discovered that interruption of the pathway for N-acetyl-glucosamine production was possible only by supplementing this amino-sugar in the growth medium. In an attempt to determine if other mutants could be recovered by sugar supplementation, it was discovered that the Δpgi mutant can be rescued by glucose supplementation. The Dif chemotaxis-like pathway is known to regulate EPS production in M. xanthus. DifA is the upstream sensor of the pathway. Previous studies had created a NarX-DifA chimeric protein, NafA, that enables the activation of the Dif pathway by nitrate, the signal for NarX. In this study, we constructed a Δpgi difA double mutant containing NafA. This strain was then subjected to various incubations with glucose and/or nitrate to determine whether the point of EPS regulation by the Dif pathway is down- or up-stream of the step catalyzed by Pgi (phosphoglucose isomerase). Preliminary results from this study are inconclusive.
Cd44-Hyaluronic Acid Interactions in Il-2 Induced Vascular Leak Syndrome
Mustafa, Amjad (Virginia Tech, 2001-06-15)
Immunotherapy with IL-2 is accompanied by severe toxicity leading to development of vascular leak syndrome (VLS). Previous studies from our laboratory demonstrated that CD44 knockout mice exhibit marked decrease in IL-2 induced VLS, thereby suggesting a role for CD44 in VLS. In the current study we tested whether use of mAbs against CD44 or hyaluronic acid (HA), the ligand for CD44, can abrogate IL-2 induced VLS. Administration of IL-2 (75,000 U/mouse, three times a day for 4 days) into C57BL/6 mice triggered significant VLS in the lungs and liver. Interestingly, HA caused a marked increase in IL-2-induced VLS in the lungs and liver. In contrast, use of anti-CD44 mAbs reduced IL-2-induced VLS in the lungs and liver. The change in VLS seen following HA or anti-CD44 mAbs treatment was not due to any defect in lymphocyte migration or homing to various organs because histopathological studies in these mice demonstrated significant and often greater perivascular infiltration of lymphocytes when compared to mice treated with IL-2 alone. However, HA treatment exhibited a marked increase in IL-2-induced lymphokine-activated killer (LAK) cell activity while anti-CD44 mAbs treatment led to a significant decrease in IL-2-induced LAK cell activity. These studies demonstrate that HA or anti CD44 mAbs may serve as a useful tool to selectively alter the LAK activity as well as to prevent the toxicity induced by IL-2. Altering CD44-HA interactions in vivo may offer a novel therapeutic approach to prevent endothelial cell injury by cytotoxic lymphocytes in a variety of clinical diseases.
Characterization of Bacillus Spore Membrane Proteomes and Investigation of Their Roles in the Spore Germination Process
Chen, Yan (Virginia Tech, 2014-09-23)
Components of the bacterial spore germination apparatus are crucial for survival and for initiation of infection by some pathogens. While some components of the germination apparatus are well conserved in spore-forming species, such as the spoVA operon, each species may possess a different and possibly unique germinant recognition mechanism. The significance of several individual proteins in the germination process has been characterized. However, the mechanisms of how these proteins perform their functions and the network connecting these proteins in the complete germination process are still a mystery. In this study, we characterized a Bacillus subtilis superdormant spore population and investigated the abundance of 11 germination-related proteins. The relative quantities of these proteins in dormant, germinating and superdormant spores suggested that variation in the levels of proteins, other than germinant receptor proteins may result in superdormancy. Specifically, variation in the abundance of the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Spore membrane proteomes of Bacillus anthracis and B. subtilis were characterized to generate a candidate protein list that can be further investigated. Proteins that were not previously known to be spore-associated were identified, and many of these proteins shared great similarity in both Bacillus species. A significant number of these proteins are implicated in functions that play major roles in spore formation and germination, such as amino acid or inorganic ion transport and protein fate determination. By analyzing the in vivo and in vitro activity of HtrC, we proved that the protease is responsible for YpeB proteolytic processing at specific sites during germination. However, without HtrC present in the spore, other proteases appear to degrade YpeB at a reduced rate. The activity of purified HtrC in vitro was stimulated by a relatively high concentration of Mn²⁺ or Ca²⁺ ions, but the mechanism behind the stimulation is not clear. We also demonstrated that YpeB and SleB, in the absence of their partner protein, were degraded by unknown proteases other than HtrC during spore formation. Identification and characterization of these unknown proteases would be a future direction for revealing the roles of proteases in spore germination.
Characterization of proteins involved in Bacillus subtilis spore formation and germination
Barat, Bidisha (Virginia Tech, 2020-05-22)
Members of the Bacillus genus, when faced with unfavorable environmental conditions such as depletion of nutrients, undergo an asymmetric division process ultimately leading to the formation of an endospore. In some instances, the spore serves as the infectious agent of an associated disease; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of unfavorable environmental conditions including traditional decontamination techniques. Spore resistance is due to the formation of specialized structures that contribute to spore dormancy through several mechanisms, including maintenance of the dehydrated state of the spore core. Spore germination is a rapid process resulting in the irrevocable transformation of the non-metabolizing dehydrated spore into a vegetative outgrowing bacterium. The exact mechanism by which individual proteins function in the germination pathway remains unknown. In this study, we have focused on the roles of putative ion transporters and other germination-active proteins in affecting spore formation and germination. Metal ions can activate enzymes during the sporulation process and/or be factors in spore resistance properties. In B. subtilis, six proteins within the spore membrane proteome (ChaA, YcnL,YflS, YloB, YugS, ZnuA) are similar to components of known cation transport systems. These proteins may play roles in the accumulation of ions during sporulation and/or the release of ions during germination. Multiple mutants altered in the putative ion transporter genes were generated, and the effects of these mutations were analyzed. All strains containing a yloB deletion showed a decrease in heat resistant cfu/ml, and >40% of the spores appeared phase dark during microscopy, indicating the formation of unstable spores. Studies were conducted to quantify the amounts of individual ions in phase-bright spores using atomic emission spectroscopy and to analyze the rate at which ions are released from germinating spores. The transport of Ca2+ from mother cell to forespore during sporulation seems to be affected in the yloB deletion mutant. This Ca2+ deficit apparently renders the spores unstable, heat sensitive, and partially germination defective, suggesting that a high-affinity transporter for Ca2+ is nonfunctional. To better understand the underlying mechanisms of germination, a high-throughput genetic screening method called transposon sequencing was used. This analysis identified genes that had not been previously implicated in germination. To investigate their functions, a number of functional assays of all the Ger mutant strains were performed that indicated a delay in stage I of germination. The mutant strains showed significant reduction in germination efficiency with L-valine: about 50% of the population failed to initiate germination suggesting a defect in the GerA-mediated response. The expression of gerA was studied using a lacZ transcriptional fusion followed by quantitative western blot analyses to determine abundance of GerA in mutant strains. The mutants were classified based upon normal or decreased gerA transcription and normal or reduced GerA protein. Further work involves understanding the functions of the identified genes and their correlation to the GerA receptor. Insight into ion transporters of spore-forming bacteria and understanding the germination apparatus may lead to promising new applications, detection methods, or therapeutics for spores, and may allow the development of better spore decontamination procedures.
Characterization of PspE, a Secreted Sulfurtransferase of Escherichia coli
Cheng, Hui (Virginia Tech, 2003-05-01)
PspE, encoded by the last gene of the phage shock protein operon, is one of the nine proteins of Escherichia coli that contain a rhodanese homology domain. PspE is synthesized as a precursor with a 19-amino acid signal sequence and secreted to the periplasm. Mature PspE is the smallest rhodanese of E. coli (85 amino acids) and catalyzes the transfer of sulfur from thiosulfate to cyanide forming thiocyanate and sulfite. Cation exchange chromatography of a freeze-thaw extract of a PspE-overexpressing strain yielded two major peaks of active, homogeneous PspE. The two peaks contained two forms of PspE (PspE1 and PspE2) of distinct size and/or charge that were distinguished by native polyacrylamide gel electrophoresis and gel chromatography. PspE2 was converted to the more compact PspE1 by treatment with thiosulfate, which suggested that PspE1 is the persulfide form. One equivalent of cyanizable sulfur was associated with PspE1, with much less present in PspE2. Consistent with the conclusion that the single active site cysteine of PspE1 contains a persulfide sulfur was the observation that this form was much more tolerant to chemical inactivation by thiol-specific modifying reagent DTNB (5,5′-dithiobis(2-nitrobenzoic acid)). Rhodanese activity was subject to inhibition by anions (sulfite, sulfate, chloride, phosphate and arsenate), suggesting PspE has a cationic site for substrate binding. Kinetic analysis revealed that PspE employs a double-displacement mechanism, as is the case for other rhodaneses. The Kms for SSO32- and CN- were 3.0 and 43 mM, respectively. PspE exhibited a kcat of 72 s-1. To aid in understanding the physiological role of PspE, a strain with a pspE gene disruption was constructed. Comparison of rhodanese activity in extracts of wild-type and mutant strains revealed that PspE is a major contributor of rhodanese activity in E. coli. The pspE mutant displayed no obvious growth defect or auxotrophies, and was capable of molybdopterin biosynthesis, indicating that pspE is not essential for production of sulfur-containing amino acid or cofactors. Growth of wild-type and mutant strains deficient in pspE and other sulfurtransferase paralogs in medium with cyanide or cadmium was compared. The results indicated that neither PspE nor other E. coli rhodanese paralogs play roles in cyanide or cadmium detoxification. The physiological role of PspE remains to be determined.
Characterization of symbiotically important processes in Sinorhizobium meliloti
Zatakia, Hardik M. (Virginia Tech, 2015-09-15)
Bacteria perform biological nitrogen fixation (BNF) which leads to conversion of N2 to ammonia. One of the best studied models of BNF is the symbiotic association of Sinorhizobium meliloti - Medicago sativa (alfalfa). Since alfalfa is a major source of animal feed and the fourth largest crop grown in the USA, enhanced understanding of this symbiosis can have implications for increasing crop yields, reducing environmental contamination and food costs. Studies discussed here focus on two symbiotically important bacterial traits, type IVb pili and chemotaxis. Chapter 2 characterizes S. meliloti type IVb pili encoded by flp-1 and establishes their role in nodulation. Bundle-forming pili were visualized in wild-type cells, while cells lacking pilA1, the pilin-encoding gene, showed an absence of pili. Competitive nodulation assays with alfalfa concluded that cells lacking pili had a significant nodulation defect. Regulation of pilA1 expression via a quorum sensing regulator, ExpR, was confirmed. Chapter 3 describes the role of the flp-2 cluster in establishing symbiosis. PilA2 is a pilin subunit encoded from flp-2. The pilA2 deletion strain was defective in nodulation by 31% as compared to the wild type. A non-significant change in nodulation was seen in pilA1pilA2 strain. Thus, both flp-1 and flp-2 have a significant role in establishing symbiosis. Chapter 4 focuses on the deviations of S. meliloti chemotaxis from the enterobacterial paradigm. Transcriptional fusions showed that S. meliloti chemoreceptors (MCPs) are class III genes and regulated by FlbT. Quantitative immunoblots determined the cellular amounts of chemoreceptors. Chemoreceptors were grouped in three classes; high, low, and extremely-low abundance, similar to the high and low abundance chemoreceptors of Escherichia coli. Importantly, the MCP:CheA ratio in an S. meliloti cell was observed to be 37:1, similar to that in Bacillus subtilis of 24:1, but quite different from that in E. coli of 3.4:1. In conclusion, our data indicates that soil bacteria may have optimized their chemotaxis system based on their milieu, which is different from enteric bacteria. These studies have enhanced our understanding of two symbiotically important processes in S. meliloti, and pave the way for future manipulations of the system to increase symbiosis and reduce our dependence on synthetic fertilizers.
Characterization of the Bacillus anthracis SleL Protein and its Role in Spore Germination
Lambert, Emily Anne (Virginia Tech, 2010-03-24)
Bacillus anthracis is a spore-forming bacterium that is included on the list of select agents compiled by the Centers for Disease Control. When a B. anthracis spore germinates, a protective layer of peptidoglycan known as the cortex must be depolymerized by germination-specific lytic enzymes (GSLEs) before the bacterium can become a metabolically active vegetative cell. By exploiting cortex lytic enzymes it may be possible to control germination. This could be beneficial in elucidating ways to enhance current decontamination methods. In this work we created in-frame deletion mutants to study not only the role of one GSLE, SleL, but by creating multi-deletion mutants, we were able to analyze how the protein cooperates with other lytic enzymes to efficiently hydrolyze the cortical PG. We determined that SleL plays an auxiliary role in complete peptidoglycan hydrolysis, secondary to cortex lytic enzymes CwlJ1, CwlJ2, and SleB. The loss of sleL results in a delay in the loss of optical density during germination. However, spores are capable of completing germination as long as CwlJ1 or SleB remains active. HPLC analysis of muropeptides collected from B. anthracis sleL strains indicates that SleL is an N-acetylglucosamidase that acts on cortical PG to produce small muropeptides which are quickly released from the germinating spore. By analyzing the in vitro and in vivo activities of SleL we confirmed the enzymatic activity of the protein, characterized its substrates, and studied the roles of its putative LysM domains in substrate binding and spore-protein association. We were able to show that purified SleL is capable of depolymerizing partially digested spore PG resulting in the production of N-acetylglucosaminidase products that are readily released as small muropeptides. In vitro, loss of the LysM domain(s) decreases hydrolysis effectiveness. The reduction in hydrolysis is likely due to LysM domains being involved in substrate recognition and PG binding. When the SleL derivatives are expressed in vivo those proteins lacking one or both LysM domains do not associate with the spore, suggesting that LysM is involved in directing protein localization.
Characterization of the Sinorhizobum Meliloti Chotaxis System
Castaneda Saldana, Rafael (Virginia Tech, 2019-12-19)
Increasing awareness to global climate change has drastically focused attention on finding solutions to reduce environmental impacts while still providing sufficient food for the increasing world population. Beneficial Nitrogen Fixing (BNF) microbes provide a possible solution by delivering biological nitrogen to plants resulting in reduced environmental impacts due to fertilizer runoff and eutrophication. One well studied model is that of Sinorhizobium meliloti and its legume host Medicago sativa (alfalfa), the fourth largest USA crop used for animal feed. Advancing research for this symbiosis model can provide solutions to enhance yield while minimizing environmental impacts. Chapter 2 focuses on the deviation of the S. meliloti chemotaxis system from the enteric paradigm. Quantitative immunoblots determined the cellular amounts of chemotaxis proteins. Overall, chemotaxis protein levels were approximately 10-fold lower in S. meliloti compared to B. subtilis and E. coli. Focusing on cellular stoichiometric ratios, S. meliloti generally exhibits drastically higher values for CheB, CheR, and CheY to the histidine kinase CheA monomer compared to E. coli and B. subtilis. Chapter 3 characterizes the role of McpX to quaternary ammonium compound (QAC) sensing. QACs are exuded by germinating alfalfa seeds. In vitro binding assays were performed to determine ligand binding characteristics. S. meliloti chemotaxis behavior to QACs was analyzed in in vivo capillary assays under real-time imaging. These studies strengthen our knowledge of the chemotaxis system in the symbiosis model of S. meliloti and alfalfa. The data can further be used to create a mathematical model of the dynamics of bacteria-host interaction. The results can be used to optimize chemotaxis to host plants to improve crop yield and protect watersheds.
Characterization of the thioredoxin system in Methanosarcina mazei
Loganathan, Usha R. (Virginia Tech, 2014-12-18)
Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation.
Characterization of two Bacillus subtilis penicillin-binding protein-coding genes, ykuA (pbpH) and yrrR (pbpI)
Wei, Yuping (Virginia Tech, 2002-08-06)
Penicillin-binding proteins (PBPs) are required in the synthesis of the cell wall of bacteria. In Bacillus subtilis, PBPs play important roles in the life cycle, including both vegetative growth and sporulation, and contribute to the formation of the different structures of vegetative cell wall and spore cortex. The B. subtilis genome sequencing project revealed there were two uncharacterized genes, ykuA and yrrR, with extensive sequence similarity to class B PBPs. These two genes are renamed and referred to henceforth as pbpH and pbpI, respectively. A sequence alignment of the predicted product of pbpH against the microbial protein database demonstrated that the most similar protein in B. subtilis is PBP2A and in E. coli is PBP2. This suggested that PbpH belongs to a group of the genes required for maintaining the rod shape of the cell. Study of a pbpH-lacZ fusion showed that pbpH was expressed weakly during vegetative growth and the expression reached the highest level at the transition from exponential phase to stationary phase. The combination of a pbpA deletion and the pbpH deletion was lethal and double mutant strains lacking pbpH and pbpC or pbpI (also named yrrR) were viable. The viable mutants were indistinguishable from the wild-type except that the vegetative PG of the pbpC pbpH strain had a slightly slightly lower amount of disaccharide tetrapeptide with 1 amidation and higher amount of disaccharide tripeptide tetrapeptide with 2 amidations when compared to others strains. This suggests that PbpC (PBP3) is involved in vegetative PG synthesis but only affects the PG structure with a very low efficiency. A pbpA pbpH double mutant containing a xylose-regulated pbpH gene inserted into the chromosome at the amyE locus was constructed. Depletion of PbpH resulted in an arrest in cell growth and a dramatic morphological change in both vegetative cells and outgrowing spores. Vegetative cells lacking pbpA and pbpH expression swelled and cell elongation was arrested, leading to the formation of pleiomorphic spherical cells and eventual lysis. In these cells, cell septations were randomly localized, cell walls and septa were thicker than those seen in wild type cells, and the average cell width and volume were larger than those of cells expressing pbpA or pbpH. The vegetative PG had an increased abundance of one unidentified muropeptide. Spores produced by the pbpA pbpH double mutant were able to initiate germination but the transition of the oval-shaped spores to rod-shape cells was blocked. The outgrowing cells were spherical, gradually enlarged, and eventually lysed. Outgrowth of these spores in the presence of xylose led to the formation of helical cells. Thus, PbpH is apparently required for maintenance of cell shape, specifically for cell elongation. PbpH and PBP2a play a redundant role homologous to that of PBP2 in E. coli. A sequence alignment of the predicted product of pbpI against the microbial protein database demonstrated that the most similar protein in B. subtilis is SpoVD and in E. coli is PBP3. This suggested that PbpI belongs to the group of the genes required for synthesis of the spore or septum PG. PbpI was identified using radio-labeled penicillin and found to run underneath PBP4 on SDS-PAGE. PbpI is therefore renamed PBP4b. Study of a pbpI-lacZ fusion showed that pbpI was expressed predominantly during early sporulation. A putative sigma F recognition site is present in the region upstream of pbpI and studies using mutant strains lacking sporulation-specific sigma factors demonstrated that the expression of pbpI is mainly dependent on sigma factor F. A pbpI single mutant, a pbpI pbpG double mutant, and a pbpI pbpF double mutant were indistinguishable from the wild-type. The sporulation defect of a pbpI pbpF pbpG triple mutant was indistinguishable from that of a pbpF pbpG double mutant. Structure parameters of the forespore PG in a pbpI spoVD strain are similar to that of a spoVD strain. These results indicate that PBP4b plays a unknown redundant role.
Characterization of two novel proteins containing the rhodanese homology domain: YgaP and YbbB of Escherichia coli
Ahmed, 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.
Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination
Diaz, Oscar R.; Sayer, Cameron V.; Popham, David L.; Shen, Aimee (American Society for Microbiology, 2018-06-27)
Clostridium difficile, also known as Clostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea. C. difficile infections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-l-lactam. C. difficile cortex degradation also depends on the Peptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-8-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of AgerS spores to those of spores lacking either CwID or PdaA, both of which mediate cortex modification in Bacillus subtilis, we determined that C. difficile GerS, CwID, and PdaA are all required to generate muramic-3-lactam. Both GerS and CwID were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwID directly interact and that CwID modulates GerS incorporation into mature spores. Since AgerS, AcwID, and Delta pdaA spores exhibited equivalent germination defects, our results indicate that C. difficile spore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination in C. difficile relative to B. subtilis and other spore-forming organisms. IMPORTANCE The Gram-positive, spore-forming bacterium Clostridium difficile is a leading cause of antibiotic-associated diarrhea. Because C. difficile is an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of Delta gerS spores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwID and PdaA. These analyses revealed that GerS, CwID, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in Delta gerS, Delta cwID, and Delta pdaA mutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwID are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight into C. difficile spore germination.
Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria
Riggs, Sarah Danielle (Virginia Tech, 2011-04-18)
The continuing emergence of multi-drug resistant Mycobacterium tuberculosis is threatening the ability to treat tuberculosis (TB) worldwide. The development of new anti-TB drugs requires new approaches and new drug targets. In this study, a mariner-based transposon, TnQuoVadis, was constructed to identify essential genes as potential drug targets. This transposon has an outward-facing anhydrotetracycline (ATc)-inducible promoter at each end. A mutant with TnQuoVadis inserted upstream of an essential gene may display normal growth in the presence of ATc, but exhibit no growth or severely diminished growth in the absence of ATc. TnQuoVadis was placed onto a vector with a temperature sensitive replication origin for more efficient mutagenesis of mycobacteria. In a preliminary genetic screen using the model organism Mycobacterium smegmatis, 13 mutants with ATc-dependent growth were identified. Identification of the insertion sites by cloning and sequencing indicated that there were nine genetic loci containing transposon insertions upstream of essential gene candidates in M. smegmatis. Further analysis of these genes indicated that many were previously known essential in both M. smegmatis and M. tuberculosis. These results demonstrate that TnQuoVadis and its delivery system can be utilized for the identification of essential genes in mycobacteria
Demonstration of Interactions Among Dif Proteins and the Identification of Kapb as a Regulator of Exopolysaccharide in Myxococcus Xanthus
Li, Zhuo (Virginia Tech, 2007-05-01)
Myxococcus xanthus Dif proteins are chemotaxis homologues that regulate exopolysaccharide (EPS) biogenesis. Previous genetic studies suggested that Dif protein might interact with one another as do the chemotaxis proteins in enterics. The interactions among Dif proteins were since investigated with the yeast two-hybrid (Y2H) system. The results indicate that DifC interacts with both DifA and DifE. Using a modified Y2H system, DifC was shown to be able to bring DifA and DifE into a protein complex. Further Y2H experiments demonstrated that the different conserved domains of DifE likely function as their counterparts of CheA-type kinases because the putative P2 domain of DifE interacts with DifD, P5 with DifC and the dimerization domain P3 with itself. Similarly, DifA can interact with itself through its C-terminal region. In addition, DifG was found to interact with the CheY homologue DifD. These findings support the notion that Dif proteins constitute a unique chemotaxis-like signal transduction pathway in M. xanthus. In addition, KapB, a TPR (Tetratricopeptide repeats) protein, was identified as an interacting partner of DifE byY2H library screening. Further analysis demonstrated that the N-terminal half of KapB interacted with the putative P2 domain of DifE. KapB had been previously reported to interact with several Serine/Threonine (Ser/Thr) kinase pathways including the Pkn4-Pfk pathway. This pathway is implicated in glycogen metabolism in M. xanthus by a previous report. In this study, kapB as well as pfkn deletion mutants were found to overproduce EPS. It was also found that the Dif pathway is involved in glycogen metabolism because the glycogen level is altered in dif mutants. These results indicate EPS biogenesis and glycogen metabolism may be coordinately regulated. This coordination of the Dif-regulated EPS production and the Pkn4-regulated glycogen metabolism appears to involve KapB. This is the first example of a TPR protein mediating the interplays of a histidine kinase pathway and a Ser/Thr kinase pathway.

Browsing by Author "Popham, David L."

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