VTechWorks Repository :: Browsing by Author "Yang, Zhaomin"

Browsing by Author "Yang, Zhaomin"

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Adherence and Biofilm Formation of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus in household plumbing
Mullis, Summer (Virginia Tech, 2012-09-05)
Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and found in drinking water distribution systems and household plumbing. They are opportunistic pathogens of humans, causing lung disease. Their ability to adhere and form biofilm is attributed to a waxy, lipid-rich, cell envelope. This highly hydrophobic envelope also contributes to the characteristic antibiotic-, chlorine-, and disinfectant- resistance of NTM. NTM in household plumbing reside primarily in biofilms and the ability to form biofilm has been linked to virulence. Shedding of cells from biofilm and the subsequent aerosolization of microorganisms through showerheads presents a significant public health risk, particularly to those individuals with associated risk factors. Three species of NTM, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus, were examined for adherence and biofilm formation on surfaces common to household plumbing systems, including glass, copper, stainless steel, polyvinyl chloride, and galvanized steel. All experiments were conducted with sterile, Blacksburg tap water in a CDC Biofilm Reactor. Highest adherence was observed by M. avium on galvanized steel surfaces, reaching 15,100 CFU/cm2 surface at 6 hours incubation at room temperature. After 3 weeks incubation at room temperature, biofilm formation of M. avium was also highest on galvanized steel surfaces, reaching 14,000,000 CFU/cm2 surface. Lowest adherence was observed by M. abscessus on polyvinyl chloride (PVC) surfaces, reaching 40 CFU/cm2. Lowest biofilm formation was observed by M. intracellulare on glass surfaces, reaching 5,900 CFU/cm2. Surfaces, such as galvanized (zinc), on which high adherence and biofilm formation was observed, should be avoided in household plumbing systems of NTM patients and individuals at risk for developing NTM disease. Additionally, surfaces such as copper, harbor fewer NTM and may provide a safer alternative for household plumbing of NTM patients.
Analysis of Myxococcus xanthus Vegetative Biofilms With Microtiter Plates
Dye, Keane J.; Yang, Zhaomin (Frontiers, 2022-04-29)
The bacterium Myxococcus xanthus forms both developmental and vegetative types of biofilms. While the former has been studied on both agar plates and submerged surfaces, the latter has been investigated predominantly on agar surfaces as swarming colonies. Here we describe the development of a microplate-based assay for the submerged biofilms of M. xanthus under vegetative conditions. We examined the impacts of inoculation, aeration, and temperature to optimize the conditions for the assay. Aeration was observed to be critical for the effective development of submerged biofilms by M. xanthus, an obligate aerobic bacterium. In addition, temperature plays an important role in the development of M. xanthus submerged biofilms. It is well established that the formation of submerged biofilms by many bacteria requires both exopolysaccharide (EPS) and the type IV pilus (T4P). EPS constitutes part of the biofilm matrix that maintains and organizes bacterial biofilms while the T4P facilitates surface attachment as adhesins. For validation, we used our biofilm assay to examine a multitude of M. xanthus strains with various EPS and T4P phenotypes. The results indicate that the levels of EPS, but not of piliation, positively correlate with submerged biofilm formation in M. xanthus.
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.
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.
Comparative characterization of Arabidopsis Subfamily III beta-galactosidases
Gantulga, Dashzeveg (Virginia Tech, 2008-12-05)
The Arabidopsis genome encodes 17 putative beta-galactosidases belonging to Glycosyl Hydrolase (GH) family 35, which have been classified into seven subfamilies based on sequence homology. The largest of these, Subfamily III, consists of six genes, Gal-1 (At3g13750), Gal-2 (At3g52840), Gal-3 (At4g36360), Gal-4 (At5g56870), Gal-5 (At1g45130), and Gal-12 (At4g26140) that share 60-81% sequence identity at the amino acid level. All six proteins have a signal peptide that may target them to the cell exterior. We report purification and biochemical characterization of all six members of Subfamily III, each expressed as a recombinant protein in Pichia pastoris and one also in native form, purified from Arabidopsis leaves, with a special emphasis on substrate specificities. Organ specific expression of the six Gal genes was examined by analysis of the microarray databases and by semi-quantitative RT-PCR. The relative abundance and size of the Gal-1, Gal-2, Gal-5, and Gal-12 proteins was studied by immunoblotting using isoform-specific anti-peptide antibodies. The protein expression patterns of the Gal genes were generally consistent with microarray and RT-PCR data, though some discrepancies were observed suggesting distinct mechanisms of regulation for transcription and translation. Localization of total beta-galactosidase activity was visualized using the substrate, 5-bromo-4-chloro-3-indolyl-beta-D-galatopyranoside (X-Gal), to stain whole plants. Subcellular localization of the four isoforms examined by immuno-dotblotting and western blotting showed that Gal-1, Gal-2, Gal-5 and Gal-12 are present in apoplastic and cell wall bound protein extracts. Immuno-EM analysis of Gal-1 and Gal-12 showed that these proteins are localized in the cell walls of vascular and epidermal tissues in mature root. Taken together, the biochemical properties, expression patterns, and subcellular localization of these isozymes indicate that the Subfamily III beta-galactosidases all have potential functions in restructuring the cell wall during plant growth and development.
Computationally and Experimentally Exploring the Type IV Pilus Assembly ATPase for Antivirulence Drug Discovery
Ramos, Jazel Mae Silvela (Virginia Tech, 2023-08-10)
Disease caused by antibiotic resistant (ABR) bacteria has become a widespread global public health issue as humanity's existing collection of effective antibiotics dwindles. ABR bacteria are responsible for approximately 5 million deaths worldwide annually, which is predicted to reach 10 million yearly by 2050. Antivirulence therapeutics have been explored in recent times as another approach to tackling the global ABR pandemic by disrupting the function of virulence factors that promote disease development. The bacterial type IV pilus (T4P) is a prevalent virulence factor in many ABR pathogens, contributing to bacterial pathogenesis by facilitating cell motility, surface adhesion, and biofilm formation. Critically, the T4P facilitates early stages of disease, providing a means to invade and colonize a host. T4P assembly is driven by the PilB/PilF motor ATPase that localizes to the cytoplasmic face of the inner membrane to drive pilus biogenesis by ATP hydrolysis. The thesis work here explores computational and experimental methods for the discovery of antivirulence therapeutics targeting the T4P assembly ATPase PilB. A computational model of Chloracidobacterium thermophilum PilB was generated by homology modeling and molecular docking was performed to analyze the binding characteristics of six anti-PilB inhibitory compounds identified in previous studies. Computational docking aligns with the existing body of work and reveals important protein-ligand interactions and characteristics, particularly involving the ATP binding domain of PilB. This work supports the use of PilB in structure-based virtual screening to identify novel compounds targeting PilB. Additionally, through heterologous expression and chromatography methods, the ATPase core of Neisseria gonorrhoeae PilF was successfully expressed and purified as an active ATPase. This work optimized conditions for its ATPase activity in vitro. Additionally, this thesis documents the experimental attempt to express and purify Clostridioides difficile PilB as an active ATPase. Two of the seven C. difficile PilB variant proteins expressed led to soluble protein while one construct remains to be explored. The results of these studies provide insight for future methodology design for antivirulence therapeutic research targeting the T4P assembly ATPase using both in silico and in vitro methods.
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
Construction and Use of Transposon MycoTetOP(2) for Isolation of Conditional Mycobacteria Mutants
Riggs-Shute, Sarah D.; Falkinham, Joseph O. III; Yang, Zhaomin (2020-01-21)
Mycobacteria are unique in many aspects of their biology. The development of genetic tools to identify genes critical for their growth by forward genetic analysis holds great promises to advance our understanding of their cellular, physiological and biochemical processes. Here we report the development of a novel transposon, MycoTetOP(2), to aid the identification of such genes by direct transposon mutagenesis. This mariner-based transposon contains nested anhydrotetracycline (ATc)-inducible promoters to drive transcription outward from both of its ends. In addition, it includes the Escherichia coli R6K gamma origin to facilitate the identification of insertion sites. MycoTetOP(2) was placed in a shuttle plasmid with a temperature-sensitive DNA replication origin in mycobacteria. This allows propagation of mycobacteria harboring the plasmid at a permissive temperature. The resulting population of cells can then be subjected to a temperature shift to select for transposon mutants. This transposon and its delivery system, once constructed, were tested in the fast-growing model Mycobacterium smegmatis and 13 mutants with ATc-dependent growth were isolated. The identification of the insertion sites in these mutants led to nine unique genetic loci with genes critical for essential processes in both M. smegmatis and Mycobacterium tuberculosis. These results demonstrate that MycoTetOP(2) and its delivery vector provide valuable tools for the studies of mycobacteria by forward genetics.
A CRISPR with Roles in Myxococcus xanthus Development and Exopolysaccharide Production
Wallace, Regina A.; Black, Wesley P.; Yang, Xianshuang; Yang, Zhaomin (American Society for Microbiology, 2014-09-08)
The Gram-negative soil bacterium Myxococcus xanthus utilizes its social (S) gliding motility to move on surfaces during its vegetative and developmental cycles. It is known that S motility requires the type IV pilus (T4P) and the exopolysaccharide (EPS) to function. The T4P is the S motility motor, and it powers cell movement by retraction. As the key regulator of the S motor, EPS is proposed to be the anchor and trigger for T4P retraction. The production of EPS is regulated in turn by the T4P in M. xanthus, and T4P(-) mutants are S- and EPS-. In this study, a Delta pilA strain (T4P(-) and EPS-) was mutagenized by a transposon and screened for EPS- mutants. A pilA suppressor isolated as such harbored an insertion in the 3rd clustered regularly interspaced short palindromic repeat (CRISPR3) in M. xanthus. Evidence indicates that this transposon insertion, designated CRISPR3(star), is a gain-of-function (GOF) mutation. Moreover, CRISPR3(star) eliminated developmental aggregation in both the wild-type and the pilA mutant backgrounds. Upstream of CRISPR3 are genes encoding the repeat-associated mysterious proteins (RAMPs). These RAMP genes are indispensable for CRISPR3(star) to affect development and EPS in M. xanthus. Analysis by reverse transcription (RT)-PCR suggested that CRISPR3(star) led to an increase in the processing of the RNA transcribed from CRISPR3. We propose that certain CRISPR3 transcripts, once expressed and processed, target genes critical for M. xanthus fruiting body development and EPS production in a RAMP-dependent manner.
CRISPR3 Regulates Exopolysaccharide Production in Myxococcus xanthus
Wallace, Regina A. (Virginia Tech, 2013-10-10)
Myxococcus xanthus, a model organism for studying development and Type IV pili (T4P), harbors three Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) on its chromosome. CRISPR systems, which function as an adaptive immune system in prokaryotes, are classified into three types based on CRISPR-associated genes. Evidence suggests that these three types mediate immunity slightly differently. M. xanthus CRISPR1 and CRISPR2 are Type I systems while CRISPR3 is a Type III-B system. In a genetic screen, a mariner transposon insertion in the 13th spacer of CRISPR3 (3SP13) was found to restore exopolysaccharide (EPS) production to a pilA mutant. Since the deletion of CRISPR3 failed to suppress a pilA mutation and expression of CRISPR3 from a heterologous promoter led to pilA suppression, it was concluded that the 3SP13 transposon insertion is a gain-of-function mutation. Deletion of the adjacent Repeat Associated Mysterious Proteins (RAMP) genes indicated that they are essential for the 3SP13 transposon insertion to suppress pilA, providing evidence that Type III-B systems may be involved in the regulation of chromosomal genes. We suggest that one of the spacers, once expressed and processed, may inhibit the expression of a negative regulator of EPS production in M. xanthus.
Crosstalk Signaling Between Circadian Clock Components and Iron Metabolism
Schiffhauer, Samuel Peter (Virginia Tech, 2017-04-25)
Circadian rhythms are daily molecular oscillations within cells ranging from prokaryotes to humans. This rhythm is self sustaining, and receives external cues in order to synchronize an organism's behavior and physiology with the environment. Many metabolites utilized in metabolic processes seem to follow a pattern of circadian oscillation. Iron, an essential component in cellular processes such as respiration and DNA synthesis, is obtained almost exclusively through diet, yet little is known about how the clock governs iron metabolism. The regulation of iron within the cell is very tightly controlled, as iron is highly reactive in the generation of oxidative stress and the excretion of excess iron is very limited. There are limited findings indicating that there are molecular ties between the circadian clock and the regulation of iron metabolism. The first half of my dissertation focuses on the role of the circadian clock in modulating expression of iron metabolic components. We found that key components of iron import, in TFRC, and export, in SLC40A1, show altered expression in response to changes in the expression of clock transcription components. Furthermore, in circadian synchronized HepG2 hepatocytes TFRC and SLC40A1 showed rhythms in their mRNA expression, although expression of these genes was highly altered in conditions of high iron availability. We also examined IREB2, which expresses a master regulator of iron concentration in IRP2. IRP2 showed rhythms in phase with circadian component PER2, and IRP2's rhythmicity was lost under iron overload conditions. We observed that the ability of these three critical iron metabolic components to respond to sudden increases in available iron was mitigated in cells with clock impairment. Whole cistrome and transcriptome analysis was used to determine that rhythmicity in TFRC and SLC40A1 are not equal in their recruitment of circadian protein binding or in the stage of transcription in which circadian rhythms are generated. The cumulative effect of all of this regulation is that rhythmic variation in intracellular hepatic ferrous iron is clock controlled. The second half of my dissertation focuses on understanding how iron uptake influences clock resetting. Initially, iron was added to the cells in the form of ferrous sulfate, or chelated out of the cells using 2-2'-dipyridyl and clock gene expression was monitored. Altered rhythmicity of these components was seen at both the mRNA and protein level in cells with disrupted iron homeostasis. Then, we measured changes in period, phase, and amplitude of these rhythms, ultimately using a luciferase reporter cell line to demonstrate that even slight changes in cellular iron produce an effect on rhythmic period. We find that the circadian clock and iron metabolism pathway are intimately related, and that the intracellular iron concentration plays a role in circadian clock behavior. Overall, our research illustrates the importance of the circadian clock in liver metabolism and physiology. Improper iron metabolism due to genetic or dietary shortcomings is common in humans, and our work builds on the importance of chronotherapy in treatment of these conditions. Conversely, our research into the effect intracellular iron has on the clock contributes to the growing body of research into how circadian clocks, especially the peripheral clock of the liver, receive input from a range of metabolites in conjunction with signals from the master oscillator of the suprachiasmatic nucleus.
Cyclic-di-GMP and ADP bind to separate domains of PilB as mutual allosteric effectors
Dye, Keane J.; Yang, Zhaomin (2020-01-10)
PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N- and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.
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.
Determination of the Binding Site and the Key Amino Acids on Maize β-Glucosidase Isozyme Glu1 Involved in Binding to β-Glucosidase Aggregating Factor (BGAF)
Yu, Hyun Young (Virginia Tech, 2009-04-10)
β-Glucosidase zymograms of certain maize genotypes (nulls) do not show any activity bands after electrophoresis. We have shown that a chimeric lectin called β-glucosidase aggregating factor (BGAF) is responsible for the absence of β-glucosidase activity bands on zymograms. BGAF specifically binds to maize β-glucosidase isozymes Glu1 and Glu2 and forms large, insoluble complexes. Furthermore, we have previously shown that the N-terminal (Glu⁵⁰-Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions contain residues that make up the BGAF binding site on maize Glu1. However, sequence comparison between sorghum β-glucosidases (dhurrinases, Dhr1 and Dhr2), to which BGAF does not bind, and maize β-glucosidases, and an examination of the 3-D structure of Glu1 suggested that the BGAF binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²-Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹-Thr²⁹, together with C-terminal region Phe⁴⁶⁶-Ala⁵¹², affects the size of Glu1-BGAF complexes. To determine the importance of each region for binding, we determined the dissociation constants (K_d) of chimeric β-glucosidase-BGAF interactions. The results demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²-Thr⁸² on Glu1 for BGAF binding, we constructed chimeric Dhr2 (C-11, Dhr2 whose Val⁷²-Glu⁸² region was replaced with the Ile⁷²-Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²-Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding. We also constructed mutant β-glucosidases to identify and define the contribution of individual amino acids in the above three regions to BGAF binding. In the N-terminal region (Ile⁷²-Thr⁸²), critical region for BGAF binding, Glu1 mutants K81E and T82Y failed to bind BGAF in the gel-shift assay and their frontal affinity chromatography (FAC) profiles were essentially similar to that of sorghum β-glucosidase (dhurrinase 2, Dhr2), a non-binder, indicating that these two amino acids within Ile⁷²-Thr⁸² region are essential for BGAF binding. In the extreme N-terminal (Ser¹-Thr²⁹) and C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions, N481E [substitution of asparagine-481 with glutamic acid (as in Dhr)] showed lower affinity for BGAF, whereas none of the single amino acid substitutions in the Ser¹-Thr²⁹ region showed any effect on BGAF binding indicating that these regions play a minor role. To further confirm the importance of lysine-81 and threonine-82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four unique amino acids (isoleucine-72, asparagine-75, lysine-81, and threonine-82) of Glu1 in the peptide span Ile⁷²-Thr⁸² are required to impart BGAF binding ability to Dhr2. The sequence comparison among plant β-glucosidases supports the hypothesis that BGAF binding is specific to maize β-glucosidases because only maize β-glucosidases have threonine at position 82.
Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds
Dye, Keane J.; Vogelaar, Nancy J.; O'Hara, Megan; Sobrado, Pablo; Santos, Webster; Carlier, Paul R.; Yang, Zhaomin (American Society for Microbiology, 2022-12)
Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson's disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics.IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.
Effects of Protein Domains on Localization of Penicillin-Binding Proteins 2a and 2b in Bacillus Subtilis
Xue, Yong (Virginia Tech, 2008-09-24)
Peptidoglycan not only protects bacterial cells against intracellular pressure but also provides the cells with a defined morphology. Penicillin-binding proteins (PBPs) catalyze the polymerization of the peptidoglycan in Bacillus subtilis. PBP2a and PBP2b are class B PBPs which have been known to have transpeptidase activities and they localize at different positions on the cell membrane. PBP2a spreads around the cylindrical wall as well as some at the septum, and PPB2b localizes exclusively to the septum and some at the cell poles. Both PBP2a and PBP2b are composed of four domains: S, N, P, and C domains from the N- to C- terminus. A FLAG epitope was tagged to the C-terminal ends of PBP2a and PBP2b. Cells with FLAG tagged PBP2a or PBP2b grow as well as wild type strain. Expression of PBP2a-FLAG and PBP2b-FLAG can be detected by western blotting using anti FLAG antibody. The expression of wild type PBP2a/PBP2b in these strains was tightly controlled by a xylose promoter. The FLAG fusion didn't influence the normal membrane localizations of PBP2a or PBP2b. PBP2a/2b mutant strains with the S and/or N domains switched between PBP2a and PBP2b were constructed. All these domain-switch proteins were tagged with a FLAG at the C-terminus. The expression of these recombinant proteins can be detected by western blotting. None of these domain-switch proteins was able to complement the wild type PBP2a and PBP2b and cells with only these recombinant proteins but no wild type proteins were non-viable. Cellular localization of these domain switch proteins were visualized using immunofluorescence microscopy. Proteins containing the PBP2a S domain had the same localization patterns as wild type PBP2a. Proteins that have the PBP2b S domain localized specifically at the septum and cell poles, which is similar to the wild type PBP2b. These results indicate that the S domain is the determinant to direct PBP2a and PBP2b to their cellular destinations.
Evolutionary Genomics of Dominant Bacterial and Archaeal Lineages in the Ocean
Martinez Gutierrez, Carolina Alejandra (Virginia Tech, 2023-01-20)
The ocean plays essential roles in Earth's biochemistry. Most of the nutrient transformations that fuel trophic webs in the ocean are mediated by microorganisms. The extent of phylogenetic and metabolic diversity of key culture and uncultured marine microbial clades started to be revealed due to progress in sequencing technologies, however we still lack a comprehensive understanding of the evolutionary processes that led to the microbial diversity we see in the ocean today. In this dissertation, I apply phylogenomic and comparative genomic methods to explore the evolutionary genomics of bacterial and archaeal clades that are relevant due to their abundance and biogeochemical activities in the ocean. In Chapter 1, I review relevant literature regarding the evolutionary genomics of marine bacteria and archaea, with emphasis on the origins of marine microbial diversity and the evolution of genome architecture. In Chapter 2, I use a comparative framework to get insights into the evolutionary forces driving genome streamlining in the Ca. Marinimicrobia, a clade widely distributed in the ocean. This project shows that differences in the environmental conditions found along the water column led to contrasting mechanisms of evolution and ultimately genome architectures. In Chapter 3, I assess the phylogenetic signal and congruence of marker genes commonly used for phylogenetic studies of bacteria and archaea and propose a pipeline and a set of genes that provide a robust phylogenetic signal for the reconstruction of multi-domain phylogenies. In Chapter 4, I apply a phylogeny-based statistical approach to evaluate how tightly genome size in bacteria and archaea is linked to evolutionary ii history, including marine clades. I present evidence suggesting that phylogenetic history and environmental complexity are strong drivers of genome size in prokaryotes. Lastly, in Chapter 5, I estimate the emergence time of marine bacterial and archaeal clades in the context of the Prokaryotic Tree of Life and demonstrate that the diversification of these groups is linked to the three main oxygenation periods occurring throughout Earth's history. I also identify the metabolic novelties that likely led to the colonization of marine realms. Here I present methodological frameworks in the fields of comparative genomics and phylogenomics to study the evolution of marine microbial diversity and show evidence suggesting that the main evolutionary processes leading to the extant diversity seen in the ocean today are intimately linked to geological and biological innovations occurring throughout Earth's history.
Functional Studies of Penicillin-binding Protein 1 in Bacillus subtilis
Liu, Lin (Virginia Tech, 2007-04-30)
The penicillin-binding proteins (PBPs) synthesize and modify peptidoglycan (PG), the main structural element of the bacterial cell wall. PBPs and PG synthesis are highly conserved in all bacteria and both have been important targets for antibiotic and antibacterial development. In the Gram positive bacterium Bacillus subtilis, PBP1 is composed of the four domains S, N, P, and C in order from the N- to C-terminus. It plays important roles in vegetative PG synthesis. Compared to the wild type B. subtilis, the PBP1 null mutant has decreased growth rate, cell diameter, and PG crosslinking; the cell population has more long cells; and the colonies have raised and smooth edges. In this work, we constructed six mutant forms of PBP1 that were tagged with a C-terminal FLAG epitope, to complement the wild type gene. We examined the colony and cell morphologies, and PBP1 localization in the mutant strains. The removal of the cytoplasmic region of the PBP1 S domain and the replacement of PBP1 S domain by PBP4 S domain did not change the colony morphologies, and each of these two mutations had minor effects on growth rate, cell diameter, PG crosslinking and generation of long cells in the cell population. The single point mutation in the active site of the N or P domain presumably removed the enzymatic activity, and each mutation caused slower growth rate, decreased cell diameter and PG crosslinking. The point mutation in the P domain had a minor effect on the colony morphology and formation of long cells; while the mutation in the N domain altered the colony morphology, and resulted in high percentage of long cells that is comparable to the PBP1 null mutant. The C domain of PBP1 has no apparent enzymatic activity, but the loss of it altered the colony morphology, and caused slower growth rate, decreased cell diameter, and PG crosslinking. In the wild type B. subtilis, PBP1 localizes to the septum. This septum localization specificity was lost in strains expressing PBP1 without the C domain, with PBP4 S domain, or with a point mutation in the active site of the N domain. PBP1 with a point mutation in the active site of the P domain, or without the cytoplasmic region of the S domain, had decreased septum localization specificity. These findings were used to develop a model of how PBP1 domain functioning in B. subtilis.
Genetic Analysis of the Quorum Sensing Regulator EsaR
Koziski, Jessica Marie (Virginia Tech, 2008-07-24)
Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt disease in maize plants. The bacteria are injected into the plant by corn flea beetles during feeding. They colonize the xylem and overproduce a capsular exopolysaccharide (EPS) at high cell densities. The production of EPS is regulated by an EsaI/EsaR quorum sensing mechanism, homologous to the LuxI/R system. Although activation of the EPS encoding genes by EsaR occurs after it complexes to the AHL (3-oxo-C6-HSL), unlike the LuxI/R system, this activation occurs by a different mechanism. At low cell densities, dimerized EsaR acts as a repressor. At a high cell population, derepression of the EPS genes occurs via an unknown mechanism once the AHL complexes to EsaR. Hence, a random mutagenesis genetic approach to isolate EsaR* variants that are immune to the effects of AHL has been utilized. Error-prone PCR and site-directed mutagenesis were used to generate desired mutants, which were subsequently screened for their ability to repress transcription in the presence of AHL. Several individual amino acids playing a critical role in the AHL-insensitive phenotype have been identified and mapped onto a homology model of EsaR. A separate study attempted to localize the dimerization region and analyze the stability of the N-terminal domain of EsaR. Truncations of EsaR at amino acids 169 and 178, without and with the extended linker region respectively, were generated using PCR. Dimerization assays similar to those by Choi and Greenberg in 1991 were performed but proved to be unsuccessful. However, the N-terminal domain is stable as determined by western blotting, which may facilitate its future structural analysis. Together, these efforts have contributed to the molecular understanding of AHL-dependent derepression of EsaR.

Browsing by Author "Yang, Zhaomin"

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