Browsing by Author "Mukhopadhyay, Biswarup"

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    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.
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    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.
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    Comparative and Functional Genomic Studies of Histophilus somni (Haemophilus somnus)
    Siddaramappa, Shivakumara Swamy (Virginia Tech, 2007-04-09)
    Histophilus somni is a commensal of the mucosal surfaces of respiratory and reproductive tracts of cattle and sheep. However, as an opportunistic pathogen, H. somni can cause diseases such as pneumonia, myocarditis, abortion, arthritis, and meningo-encephalitis. Previously, several virulence factors/mechanisms had been identified in H. somni of which the phase-variable lipooligosaccharide, induction of host cell apoptosis, intraphagocytic survival, and immunoglobulin Fc binding proteins were well characterized. To further understand the biological properties of H. somni, the genomes of pneumonia strain 2336 and preputial strain 129Pt have been sequenced. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, putative genes that encode proteases, restriction-modification enzymes, hemagglutinins, glycosyltransferases, kinases, helicases, and adhesins have been identified in H. somni. Most of the H. somni strain-specific genes were found to be associated with prophage-like sequences, plasmids, and/or transposons. Therefore, it is likely that these mobile genetic elements played a significant role in creating genomic diversity and phenotypic variability among strains of H. somni. Functional characterization of H. somni luxS in the genomic context revealed that the gene encodes S-ribosylhomocysteinase that can complement biosynthesis of AI-2 quorum sensing signal molecules in Escherichia coli DH5alpha. It was also found that several pathogenic isolates of H. somni form a prominent biofilm and that luxS as well as phosphorylcholine expression can influence biofilm formation by H. somni. In conclusion, comparative analyses of the genomes and functional characterization of putative genes have shed new light on the versatility and evolution of H. somni.
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    Comparative Genomics and Proteomic Analysis of Assimilatory Sulfate Reduction Pathways in Anaerobic Methanotrophic Archaea
    Yu, Hang; Susanti, Dwi; McGlynn, Shawn E.; Skennerton, Connor T.; Chourey, Karuna; Iyer, Ramsunder; Scheller, Silvan; Tavormina, Patricia L.; Hettich, Robert L.; Mukhopadhyay, Biswarup; Orphan, Victoria J. (Frontiers, 2018-12-03)
    Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F-420-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments.
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    Complete Genome Sequence of Bordetella pertussis Pelita III, the Production Strain for an Indonesian Whole-Cell Pertussis Vaccine
    Efendi, Yusuf Sofyan; Susanti, Dwi; Tritama, Erman; Pasier, Michelle; Putri, Gilang Nadia Niwan; Raharso, Sugeng; Iskandar; Aditiawati, Pingkan; Giri-Rachman, Ernawati; Mukhopadhyay, Biswarup; Purwantini, Endang (2017-04)
    PT Bio Farma, the sole World Health Organization-approved Indonesian vaccine producer, manufactures a whole-cell whooping cough vaccine (wP) that, as part of a pentavalent diphtheria-tetanus-pertussis/hepatitis B/Haemophilus influenzae b (DTP/HB/Hib) vaccine, is used in Indonesia and many other countries. We report here the whole-genome sequence for Bordetella pertussis Pelita III, PT Bio Farma's wP production strain.
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    The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota
    Anderson, Iain J.; Dharmarajan, Lakshmi; Rodriguez, Jason; Hooper, Sean; Porat, Iris; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Sun, Hui; Land, Miriam; Lapidus, Alla; Lucas, Susan; Barry, Kerrie W.; Huber, Harald; Zhulin, Igor B.; Whitman, William B.; Mukhopadhyay, Biswarup; Woese, Carl; Bristow, James; Kyrpides, Nikos C. (2009-04-02)
    Background Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. Results The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced - Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. Conclusion The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.
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    The Design of Antimicrobial Detachable Thin Films for the Study of Hepatic Infections
    Cassin, Margaret Emily (Virginia Tech, 2015-10-27)
    Microbial infections are a global problem. Due to the over and misuse of antibiotics, drug-resistant pathogens are becoming more common. It is imperative to explore broad spectrum antimicrobial approaches. In this work, we modified collagen/hyaluronic acid polyelectrolyte multilayers (PEMs) with the natural antimicrobial peptide, LL-37 to study hepatic infections. LL-37 was physisorbed and covalently linked to the surface of the PEMs. Escherichia coli DH10B were cultured in the presence of LL-37modified PEMs in bacterial adhesion and contact killing models. Physisorbed LL-37 PEMs prevented bacterial adhesion and could also kill pathogens in the surrounding environment due to the release of LL-37 from the film. Immobilized LL-37 PEMs resulted in less bacterial adhesion on the surface due to the presence of the peptide. Films were then placed in contact with primary rat hepatocytes as well as in hepatocyte/bacteria co-cultures. LL-37 input concentrations up to of 16μM did not exhibit cytotoxic effects on hepatocytes. The LL-37 modified PEMs exhibited a hepatoprotective effect on albumin and urea secretion functions in co-cultures. The hepatoprotective effects were dependent on the ratio of hepatocytes and bacteria as well as the concentration of LL-37. These findings are encouraging and demonstrate that LL-37 modified PEMs can be used to investigate hepatic infections caused by bacteria.
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    Development of an Antibiotic Resistance Free Bivalent Vaccine Against Swine Brucellosis and Swine Influenza
    Rajasekaran, Parthiban (Virginia Tech, 2009-12-09)
    Livestock across the world contract several infectious diseases of both bacterial and viral origin. Swine brucellosis caused by Brucella suis and swine influenza caused by Influenza A virus affect both domestic and feral swine populations. Both the diseases have zoonotic potential to cause disease in humans with serious complications apart from inflicting huge economic losses. Infected feral swine can also act as a source of spread and outbreak where the disease is not endemic. At present, there is no vaccine available for swine brucellosis. The currently used swine influenza vaccine may not be effective against influenza strains like the recent H1N1 strain that caused a pandemic. To develop an effective bivalent vaccine for swine against these two diseases, a leucine auxotroph of the USDA approved vaccine B. abortus strain RB51 was constructed along with leuB gene complementing plasmid pNS4 to over-express antigens from Brucella and influenza. This antibiotic resistance free system over-expressed Brucella derived antigens SOD, L7/L12 and WboA in three different constructs. Against a virulent challenge of B. suis, the candidate vaccine strain over-expressing both SOD and WboA protected mice more significantly than the control group and was also found to be better protective than other candidate vaccine strains over-expressing either SOD and L7/L12 together or SOD alone. Immunoassays (ELISA) suggested that the protection afforded is Th1 type mediated immune response, as cytokine IFN-γ and IgG2a antibody sub-isotype was observed in the splenocyte culture supernatant and serum samples respectively. The strain RB51leuB platform was not expressing influenza derived antigens Hemagglutinin (HA) and Nucleoprotein (NP) when screened for expression by immunoblot. Influenza antigens, HA, NP and ectodomain of matrix protein M2e, were not found to be expressing even after optimizing their codon usage to suit Brucella tRNA preference. However, RT-PCR showed that the influenza genes mRNA were produced. In conclusion, this dissertation describes the construction of an environmentally safe antigen over-expression platform and successful employment of the system as a candidate vaccine in protecting mice against B. suis challenge. This new platform is a potential candidate for developing vaccines against other infectious diseases of livestock. This document also discusses alternate strategies for expressing influenza antigens in a Brucella platform.
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    Differential expression profiling of proteomes of pathogenic and commensal strains of Staphylococcus aureus using SILAC
    Manickam, Manisha (Virginia Tech, 2011-11-29)
    Staphylococcus aureus (S. aureus) is the etiological agent of food-borne diseases, skin infections in humans and mastitis in bovines. S. aureus is also known to exist as a commensal on skin, nose and other mucosal surfaces of the host. This symbiotic association is a result of immune dampening or tolerance induced in the host by this pathogen. We proposed the variation in protein expression by commensal and pathogenic strain as an important factor behind the difference in pathogenicity. The identification of differentially expressed proteins was carried out using a quantitative mass spectrometry (MS)-based proteomic approach, known as stable isotope labeling of amino acids in cell culture (SILAC). Four commensal and pathogenic strains each were grown in the SILAC minimal media (RPMI 1640), containing light (12C) and heavy (13C) form of lysine, respectively, until early stationary growth phase. Various protein fractions, including cell wall, membrane and secreted, were extracted from the bacterial cultures and mixed in a 1:1 ratio. The relative abundance of proteins present in light and heavy labeled samples was determined using MS analysis. From a total of 151 differentially expressed proteins, 58 were found to be upregulated in the pathogenic strains. These proteins are involved in a variety of cellular functions, including immune modulation, iron-binding, cellular transport, redox reactions, and metabolic enzymes. The differentially expressed proteins can serve as putative candidates to improve current approach towards development of a vaccine against S. aureus.
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    The Effect of a Trace Element Supplement on the Biomethane Potential of Food Waste Anaerobic Digestion
    Graff, Kelly Mackenzie (Virginia Tech, 2022-06-15)
    Food waste is a desirable feedstock for anaerobic digestion because it is high in moisture and is an easily degradable material. However, mono-digestion of food waste often fails due to the accumulation of volatile fatty acids. Supplementing trace elements is one strategy to combat this issue. This study examined the effect of supplementing trace elements (iron, nickel, selenium, molybdenum, magnesium, zinc, calcium, copper, manganese, cobalt) on the methane yield and organic waste destruction of anaerobically digested food waste. Methane yield of food waste with and without the inorganic salt trace element was determined by the gas density-based biomethane potential method at mesophilic (37°C) conditions over 30 days. The three treatments were inoculum only, food waste and inoculum, and food waste and inoculum with an added trace element solution. There was no significant difference between treatments in terms of waste stabilization (percent volatile solids, total solids, and total chemical oxygen demand reduction) between treatments. The average cumulative biogas produced was 41% higher, and the average total cumulative methane produced was 23% higher in the treatment with the trace element supplement. Mean methane yield was not different (p > 0.05) between treatments over the 30 days, and there was no difference (p > 0.05) in biomethane potential between treatments. In addition, greenhouse gas reduction potential was estimated from food waste streams in Montgomery, VA using anaerobic digestion. The purpose of this work was to (1) estimate the total mass of food waste produced in Montgomery, VA in a year, (2) use the results from the biomethane potential analyses to inform the sizing of a theoretical community digester in Montgomery, VA, and (3) estimate the greenhouse gas reduction potential of anaerobically digesting the food waste instead of sending it to landfill. Greenhouse gas reduction was calculated using the Climate Action Reserve Organic Waste Digestion Project Protocol guidelines. The greenhouse gas reduction potential was estimated as 6,532 tonnes of carbon dioxide equivalent per year (tCO2e/year), with approximately 693 m3 methane produced per day. In one year, the digester would generate an estimated 7370 kWh of energy which has the potential to power 149 homes for a year in Montgomery, VA. In addition, 4130 tonnes/year of composted digestate would be available as fertilizer for surrounding farms.
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    Effect of methanogenic substrates on coenzyme F420-dependent N5,N10-methylene-H4MPT dehydrogenase, N5,N10-methenyl-H4MPT cyclohydrolase and F420-reducing hydrogenase activities in Methanosarcina barkeri
    Mukhopadhyay, Biswarup; Purwantini, Endang; Daniels, Lacy (1993)
    We measured F420-dependent N5,N10-methylenetetrahydro-methanopterin dehydrogenase, N5, N10-methenyltetrahydro-methanopterin cyclohydrolase, and F420-reducing hydrogenase levels in Methanosarcina barkeri grown on various substrates. Variation in dehydrogenase levels during growth on a specific substrate was usually <3-fold, and much less for cyclohydrolase. H2−CO2-, methanol-, and H2−CO2+ methanol-grown cells had roughly equivalent levels of dehydrogenase and cyclohydrolase. In acetate-grown cells cyclohydrolase level was lowered 2 to 3-fold and dehydrogenase 10 to 80-fold; this was not due to repression by acetate, since, if cultures growing on acetate were supplemented with methanol or H2−CO2, dehydrogenase levels increased 14 to 19-fold, and cyclohydrolase levels by 3 to 4-fold. Compared to H2−CO2- or methanol-grown cells, acetate-or H2−CO2 + methanol-grown cells had lower levels of and less growth phase-dependent variation in hydrogenase activity. Our data are consistent with the following hypotheses: 1. M. barkeri oxidizes methanol via a portion of the CO2-reduction pathway operated in the reverse direction. 2. When steps from CO2 to CH3-S-CoM in the CO2-reduction pathway (in either direction) are not used for methanogenesis, hydrogenase activity is lowered.
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    Effect of Nanoscale Surface Structures on Microbe-Surface Interactions
    Ye, Zhou (Virginia Tech, 2017-04-24)
    Bacteria in nature predominantly grow as biofilms on living and non-living surfaces. The development of biofilms on non-living surfaces is significantly affected by the surface micro/nano topography. The main goal of this dissertation is to study the interaction between microorganisms and nanopatterned surfaces. In order to engineer the surface with well-defined and repeatable nanoscale structures, a new, versatile and scalable nanofabrication method, termed Spun-Wrapped Aligned Nanofiber lithography (SWAN lithography) was developed. This technique enables high throughput fabrication of micro/nano-scale structures on planar and highly non-planar 3D objects with lateral feature size ranging from sub-50 nm to a few microns, which is difficult to achieve by any other method at present. This nanolithography technique was then utilized to fabricate nanostructured electrode surfaces to investigate the role of surface nanostructure size (i.e. 115 nm and 300 nm high) in current production of microbial fuel cells (MFCs). Through comparing the S. oneidensis attachment density and current density (normalized by surface area), we demonstrated the effect of the surface feature size which is independent of the effect on the surface area. In order to better understand the mechanism of microorganism adhesion on nanostructured surfaces, we developed a biophysical model that calculates the total energy of adhered cells as a function of nanostructure size and spacing. Using this model, we predict the attachment density trend for Candida albicans on nanofiber-textured surfaces. The model can be applied at the population level to design surface nanostructures that reduce cell attachment on medical catheters. The biophysical model was also utilized to study the motion of a single Candida albicans yeast cell and to identify the optimal attachment location on nanofiber coated surfaces, thus leading to a better understanding of the cell-substrate interaction upon attachment.
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    Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein
    Phan, Hong-Duc; Norris, Andrew S.; Du, Chen; Stachowski, Kye; Khairunisa, Bela H.; Sidharthan, Vaishnavi; Mukhopadhyay, Biswarup; Foster, Mark P.; Wysocki, Vicki H.; Gopalan, Venkat (Oxford University Press, 2022-08-12)
    RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5 ' leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-angstrom resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae-RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein-protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.
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    Enzymology and Physiology of a New Type of Phosphoenolpyruvate Carboxylase and the Development of a Pyruvate Carboxylase Expression System
    Kraszewski, Jessica (Virginia Tech, 2007-01-29)
    Our laboratory is interested in studying the junction of glycolysis and the tricarboxylic acid (TCA) cycle, specifically the enzymes phosphoenolpyruvate carboxykinase, pyruvate carboxylase and phosphoenolpyruvate carboxylase. All produce oxaloacetate (OAA) for the cell. OAA production is critical for cell carbon synthesis in the methanogenic archaea. Therefore OAA-generating enzymes are essential for the survival of methanogens. In part of this study we investigated archaeal-type phosphoenolpyruvate carboxylase (PpcA), a new type of phosphoenolpyruvate carboxylase, which is widespread in the archaea and is found in three bacterial species. The form of phosphoenolpyruvate carboxylase (Ppc) that is prevalent in bacteria and plants is not found in the archaea. Due to complications expressing PpcA in the soluble form and difficulty purifying this enzyme from methanogens, an in-depth investigation of this enzyme's biochemical properties has yet to occur. In this study we demonstrate the successful expression of a PpcA homolog in the soluble fraction of Escherichia coli. We purified the recombinant protein to homogeneity. This development provides the means to study the enzyme's biochemical properties and manipulate the primary sequence in order to identify residues critical to the enzyme's function. We also show that this PpcA homolog does have the postulated activity and investigate its biochemical properties. The data show that PpcA has unique properties in regard to the enzyme's substrate and its regulation by metabolites. Our data also reveal that PpcA is a membrane associated protein, unlike Ppc, which is a soluble protein. We also show that pyruvate carboxylase (Pyc) can be expressed recombinantly in Pseudomonas aeruginosa at levels sufficient for structure-function studies. This is a major step forward in the expression in Pyc because it cannot be expressed at high levels in Escherichia coli. These are important developments in studying the enzymes that connect glycolysis and the TCA cycle.
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    Establishing a physical and chemical framework for Amorphous Calcium Carbonate (ACC) biomineralization
    Mergelsberg, Sebastian Tobias (Virginia Tech, 2018-07-05)
    Recent advances in high-resolution analytical methods have brought about a paradigm shift in our understanding of how crystalline materials are formed. The scientific community now recognizes that many earth materials form by multiple pathways that involve metastable intermediates. Biogenic calcium carbonate minerals are now recognized to develop by aggregating molecules or clusters to form amorphous phases that later transform to one or more crystalline polymorphs. Amorphous calcium carbonate (ACC) is now recognized as a precursor to CaCO₃ biominerals in a wide variety of natural environments. Recent studies suggest an ACC pathway may imprint a different set of dependencies from those established for classical growth processes. Previous ACC studies provided important insights, but a quantitative understanding of controls on ACC composition when formed at near-physiological conditions is not established. The Mg content of ACC and calcite is of particular interest as a minor element that is frequently found in final crystalline products in calcified skeletons. This three-part dissertation investigated biological and well-characterized synthetic ACC using high-energy x-ray methods, Raman spectroscopy, and mechanical tests. The findings establish chemical and physical properties of ACC in the exoskeleton of crustaceans and show Mg and P levels are tuned in the mineral component to optimize exoskeleton function that could be sensitive to ecological or environmental conditions. Calcite and chitin crystallinity exhibit a similar body-part-specific pattern that correlates directly with the mechanical strength of the exoskeleton. Insights from this study suggest precise biological control of ACC chemistry in the to regulate exoskeleton properties. Laboratory measurements using quantitative methods and compositions that approximate the physiological conditions of crustaceans, demonstrate at least two types of ACC are formed by controlling Mg concentration and alkalinity. We also find temporal changes in the short-range ordering of ACC after precipitation that is dependent upon carbonate content. The findings from this study provide a quantitative basis for deciphering relationships between ACC structures, solution chemistry, and the final transformation products under biologically relevant conditions.
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    Evolving understanding of rumen methanogen ecophysiology
    Khairunisa, Bela Haifa; Heryakusuma, Christian; Ike, Kelechi; Mukhopadhyay, Biswarup; Susanti, Dwi (Frontiers, 2023-11-06)
    Production of methane by methanogenic archaea, or methanogens, in the rumen of ruminants is a thermodynamic necessity for microbial conversion of feed to volatile fatty acids, which are essential nutrients for the animals. On the other hand, methane is a greenhouse gas and its production causes energy loss for the animal. Accordingly, there are ongoing efforts toward developing effective strategies for mitigating methane emissions from ruminant livestock that require a detailed understanding of the diversity and ecophysiology of rumen methanogens. Rumen methanogens evolved from free-living autotrophic ancestors through genome streamlining involving gene loss and acquisition. The process yielded an oligotrophic lifestyle, and metabolically efficient and ecologically adapted descendants. This specialization poses serious challenges to the efforts of obtaining axenic cultures of rumen methanogens, and consequently, the information on their physiological properties remains in most part inferred from those of their non-rumen representatives. This review presents the current knowledge of rumen methanogens and their metabolic contributions to enteric methane production. It also identifies the respective critical gaps that need to be filled for aiding the efforts to mitigate methane emission from livestock operations and at the same time increasing the productivity in this critical agriculture sector.
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    Genetic resources for advanced biofuel production described with the Gene Ontology
    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, João C.; Mukhopadhyay, Biswarup; Tyler, Brett M. (Frontiers, 2014-10-10)
    Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary. The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology (http://www.mengo.biochem.vt.edu) project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. Here we review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.
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    A Genetic System for Methanocaldococcus jannaschii: An Evolutionary Deeply Rooted Hyperthermophilic Methanarchaeon
    Susanti, Dwi; Frazier, Mary C.; Mukhopadhyay, Biswarup (Frontiers, 2019-07-03)
    Phylogenetically deeply rooted methanogens belonging to the genus of Methanocaldococcus living in deep-sea hydrothermal vents derive energy exclusively from hydrogenotrophic methanogenesis, one of the oldest respiratory metabolisms on Earth. These hyperthermophilic, autotrophic archaea synthesize their biomolecules from inorganic substrates and perform high temperature biocatalysis producing methane, a valuable fuel and potent greenhouse gas. The information processing and stress response systems of archaea are highly homologous to those of the eukaryotes. For this broad relevance, Methanocaldococcus jannaschii, the first hyperthermophilic chemolithotrophic organism that was isolated from a deep-sea hydrothermal vent, was also the first archaeon and third organism for which the whole genome sequence was determined. The research that followed uncovered numerous novel information in multiple fields, including those described above. M. jannaschii was found to carry ancient redox control systems, precursors of dissimilatory sulfate reduction enzymes, and a eukaryotic-like protein translocation system. It provided a platform for structural genomics and tools for incorporating unnatural amino acids into proteins. However, the assignments of in vivo relevance to these findings or interrogations of unknown aspects of M. jannaschii through genetic manipulations remained out of reach, as the organism was genetically intractable. This report presents tools and methods that remove this block. It is now possible to knockout or modify a gene in M. jannaschii and genetically fuse a gene with an affinity tag sequence, thereby allowing facile isolation of a protein with M. jannaschii-specific attributes. These tools have helped to genetically validate the role of a novel coenzyme F420-dependent sulfite reductase in conferring resistance to sulfite in M. jannaschii and to demonstrate that the organism possesses a deazaflavin-dependent system for neutralizing oxygen.
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    An Intertwined Evolutionary History of Methanogenic Archaea and Sulfate Reduction
    Susanti, Dwi; Mukhopadhyay, Biswarup (Public Library of Science, 2012-09-21)
    Hydrogenotrophic methanogenesis and dissimilatory sulfate reduction, two of the oldest energy conserving respiratory systems on Earth, apparently could not have evolved in the same host, as sulfite, an intermediate of sulfate reduction, inhibits methanogenesis. However, certain methanogenic archaea metabolize sulfite employing a deazaflavin cofactor (F420)-dependent sulfite reductase (Fsr) where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F420H2 dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively. From genome analysis we found that Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP), both being abundant in methanogens. Dsr-LPs fell into two groups defined by following sequence features: Group I (simplest), carrying a coupled siroheme-[Fe4-S4] cluster and sulfite-binding Arg/Lys residues; Group III (most complex), with group I features, a Dsr-type peripheral [Fe4-S4] cluster and an additional [Fe4-S4] cluster. Group II Dsr-LPs with group I features and a Dsr-type peripheral [Fe4-S4] cluster were proposed as evolutionary intermediates. Group III is the precursor of Fsr-C. The freestanding Fsr-N homologs serve as F420H2 dehydrogenase unit of a putative novel glutamate synthase, previously described membrane-bound electron transport system in methanogens and of assimilatory type sulfite reductases in certain haloarchaea. Among archaea, only methanogens carried Dsr-LPs. They also possessed homologs of sulfate activation and reduction enzymes. This suggested a shared evolutionary history for methanogenesis and sulfate reduction, and Dsr-LPs could have been the source of the oldest (3.47-Gyr ago) biologically produced sulfide deposit.
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    Investigating the Distribution and Biosynthesis of Modified F430 Cofactors in Methanogenic and Methanotrophic Archaea
    Boswinkle, Kaleb Storm (Virginia Tech, 2022-07-05)
    Methanogenesis is the biological production of methane and is utilized by methanogenic archaea (methanogens) to generate energy. This process is responsible for 70% of total atmospheric methane, a potent greenhouse gas and an important energy source (natural gas). In the future, reversing methanogenesis in an engineered methanogenic strain could be realized to efficiently convert natural gas into liquid fuels. Methyl coenzyme M reductase (Mcr) catalyzes the final reaction of methanogenesis in methanogens and the first reaction in the anaerobic oxidation of methane (AOM) carried out by the anaerobic methanotrophs (ANME). Cofactor F430, a unique nickel-containing tetrapyrrole, serves as the prosthetic group and catalytic component of Mcr. Recently, multiple F430 variants have been discovered in several methanogenic species, including Methanococcus maripaludis, Methanosarcina acetivorans, and Methanocaldococcus jannaschii. A novel variant reported here has an exact mass of 1008.3478, a similar absorption spectrum as unmodified F430, and associates with purified Mcr from M. acetivorans. Based on the exact mass, this molecule is likely modified with a mercaptopropamide moiety. In some conditions, this modified F430 comprises 30-50% of the total F430 pool. We also report upon our work to identify the sulfur insertion enzyme required to produce methylthio-F430 that functions with Mcr in ANME-1. We hypothesized that the insertion of the methylthio moiety is likely catalyzed by a methylthiotransferase (MTTase) homolog present in ANME. However, purified ANME MTTase does not appear to catalyze this reaction, and instead catalyzes the methylthiolation of N6-threonylcarbamoyladenosine (t6A) in tRNA.