R-3 Repository :: Browsing by Author "Bennett, George N."

Browsing by Author "Bennett, George N."

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A rapid, flexible method for incorporating controlled antibiotic release into porous polymethylmethacrylate space maintainers for craniofacial reconstruction
(Royal Society of Chemistry, 2016) Mountziaris, Paschalia M.; Shah, Sarita R.; Lam, Johnny; Bennett, George N.; Mikos, Antonios G.
Severe injuries in the craniofacial complex, resulting from trauma or pathology, present several challenges to functional and aesthetic reconstruction. The anatomy and position of the craniofacial region make it vulnerable to injury and subsequent local infection due to external bacteria as well as those from neighbouring structures like the sinuses, nasal passages, and mouth. Porous polymethylmethacrylate (PMMA) “space maintainers” have proven useful in staged craniofacial reconstruction by promoting healing of overlying soft tissue prior to reconstruction of craniofacial bones. We describe herein a method by which the porosity of a prefabricated porous PMMA space maintainer, generated by porogen leaching, can be loaded with a thermogelling copolymer-based drug delivery system. Porogen leaching, space maintainer prewetting, and thermogel loading all significantly affected the loading of a model antibiotic, colistin. Weeks-long release of antibiotic at clinically relevant levels was achieved with several formulations. In vitro assays confirmed that the released colistin maintained its antibiotic activity against several bacterial targets. Our results suggest that this method is a valuable tool in the development of novel therapeutic approaches for the treatment of severe complex, infected craniofacial injuries.
A review of parameters and heuristics for guiding metabolic pathfinding
(Springer International Publishing, 2017-09-15) Kim, Sarah M.; Peña, Matthew I.; Moll, Mark; Bennett, George N.; Kavraki, Lydia E.
Abstract Recent developments in metabolic engineering have led to the successful biosynthesis of valuable products, such as the precursor of the antimalarial compound, artemisinin, and opioid precursor, thebaine. Synthesizing these traditionally plant-derived compounds in genetically modified yeast cells introduces the possibility of significantly reducing the total time and resources required for their production, and in turn, allows these valuable compounds to become cheaper and more readily available. Most biosynthesis pathways used in metabolic engineering applications have been discovered manually, requiring a tedious search of existing literature and metabolic databases. However, the recent rapid development of available metabolic information has enabled the development of automated approaches for identifying novel pathways. Computer-assisted pathfinding has the potential to save biochemists time in the initial discovery steps of metabolic engineering. In this paper, we review the parameters and heuristics used to guide the search in recent pathfinding algorithms. These parameters and heuristics capture information on the metabolic network structure, compound structures, reaction features, and organism-specificity of pathways. No one metabolic pathfinding algorithm or search parameter stands out as the best to use broadly for solving the pathfinding problem, as each method and parameter has its own strengths and shortcomings. As assisted pathfinding approaches continue to become more sophisticated, the development of better methods for visualizing pathway results and integrating these results into existing metabolic engineering practices is also important for encouraging wider use of these pathfinding methods.
Aerobic succinate production in bacteria
(2007-07-17) San, Ka-Yiu; Bennett, George N.; Lin, Henry; Rice University; United States Patent and Trademark Office
Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.
Aerobic succinate production in bacteria
(2011-05-03) San, Ka-Yiu; Bennett, George N.; Lin, Henry; Rice University; United States Patent and Trademark Office
Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.
Aerobic succinate production in bacteria
(2007-08-28) San, Ka-Yiu; Bennett, George N.; Lin, Henry; Rice University; United States Patent and Trademark Office
Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.
Anticipation of Nitric Oxide Stress in the Human Commensal Fungus Candida albicans
(2013-07-24) Lynn, Jed; Bennett, George N.; Braam, Janet; Segatori, Laura; Stewart, Charles R.
Candida albicans is the most common human commensal fungus, able to colonize host niches such as skin, mouth and gastrointestinal tract. Colonization of diverse microenvironments requires the ability to evade or overcome innate host protection and adapt to rapid transitions between environments with different stresses and nutrient availability. Colonization of the gastrointestinal tract requires passage through the stomach containing toxic levels of nitric oxide, generated from acidification of nitrite in the low pH of the stomach. Although resistance of C. albicans to nitric oxide is mediated by the flavohemoglobin Yhb1, little is known about the physiologically relevant ligands that regulate YHB1 expression. Here I propose the hypothesis that nontoxic saliva chemicals induce YHB1 expression and promote resistance to nitric oxide generated in the stomach. Supporting this hypothesis is the observation that two ions actively concentrated in the saliva – nitrate and thiocyanate – induce YHB1 expression. Indeed, whole-genome transcriptional analysis of C. albicans treated with nitrate or thiocyanate produce gene expression profiles nearly identical to cells treated with nitrite or nitric oxide. Pretreatment of C. albicans with either of these two nontoxic compounds increases resistance of the yeast to nitric oxide. I propose that this is an evolved response in which C. albicans anticipates nitric oxide stress generated in the stomach. C. albicans thus upregulates nitric oxide stress response genes in response to saliva signals that precede nitric oxide formation further on in the gut. Only a few examples of anticipatory signaling have so far been identified and it is not known how common this type of regulation is among microbes. Expression of the YHB1 gene in response to nitric oxide is regulated by the transcription factor Cta4. I show that Cta4 binds to the YHB1 promoter in vivo as a homodimer and is necessary, but not sufficient, for nitric oxide, nitrate and thiocyanate induced expression of YHB1. Based on these data I propose a model in which Cta4 transcriptional activation is inhibited under non-inducing conditions by a negative regulator. Understanding the mechanism by which C. albicans senses and responds to nitric oxide, nitrate and thiocyanate remains a question for future research.
Biosynthesis of Medium-Chain ω-Hydroxy Fatty Acids by AlkBGT of Pseudomonas putida GPo1 With Native FadL in Engineered Escherichia coli
(Frontiers, 2019) He, Qiaofei; Bennett, George N.; San, Ka-Yiu; Wu, Hui
Hydroxy fatty acids (HFAs) are valuable compounds that are widely used in medical, cosmetic and food fields. Production of ω-HFAs via bioconversion by engineered Escherichia coli has received a lot of attention because this process is environmentally friendly. In this study, a whole-cell bio-catalysis strategy was established to synthesize medium-chain ω-HFAs based on the AlkBGT hydroxylation system from Pseudomonas putida GPo1. The effects of blocking the β-oxidation of fatty acids (FAs) and enhancing the transportation of FAs on ω-HFAs bio-production were also investigated. When fadE and fadD were deleted, the consumption of decanoic acid decreased, and the yield of ω-hydroxydecanoic acid was enhanced remarkably. Additionally, the co-expression of the FA transporter protein, FadL, played an important role in increasing the conversion rate of ω-hydroxydecanoic acid. As a result, the concentration and yield of ω-hydroxydecanoic acid in NH03(pBGT-fadL) increased to 309 mg/L and 0.86 mol/mol, respectively. This whole-cell bio-catalysis system was further applied to the biosynthesis of ω-hydroxyoctanoic acid and ω-hydroxydodecanoic acid using octanoic acid and dodecanoic acid as substrates, respectively. The concentrations of ω-hydroxyoctanoic acid and ω-hydroxydodecanoic acid reached 275.48 and 249.03 mg/L, with yields of 0.63 and 0.56 mol/mol, respectively. This study demonstrated that the overexpression of AlkBGT coupled with native FadL is an efficient strategy to synthesize medium-chain ω-HFAs from medium-chain FAs in fadE and fadD mutant E. coli strains.
Blocking sporulation by inhibiting SpoIIE
(2008-10-07) Bennett, George N.; Scotcher, Miles C.; Rice University; United States Patent and Trademark Office
We have shown that the control of solventogenesis and sporulation can be genetically uncoupled in C. acetobutylicum. In strain 824(pASspo), the absence of SpoIIE causes sporulation to be blocked at stage II. The cell remains in a vegetative state, and this allows solvent production to proceed for longer and for solvents to accumulate more rapidly and to a higher concentration. The characteristic drop in OD600 observed in wild type and control strains of C. acetobutylicum after 48-72 hours as the cells transition from the solventogenic phase to sporulation is notably absent in the fermentations of 824(pASspo). Mutant S (wild type background, spoIIE disrupted), Mutant BS (Mutant B background, spoIIE disrupted), Mutant HS (Mutant H background, spoIIE disrupted) and Mutant bukS (buk- background, spoIIE disrupted) were generated to create stable solvent producing bacteria with complete inactivation of the SpoIIE protein. Similarity between the SpoIIE protein of C. acetobutylicum, B. subtilis, and other Clostridial species indicates that the techniques used in C. acetobutylicum can be applied to other solvent producing Clostridia.
Blocking sporulation by inhibiting SpoIIE
(2011-12-27) Bennett, George N.; Scotcher, Miles C.; Rice University; United States Patent and Trademark Office
We have shown that the control of solventogenesis and sporulation can be genetically uncoupled in C. acetobutylicum. In strain 824(pASspo), the absence of SpoIIE causes sporulation to be blocked at stage II. The cell remains in a vegetative state, and this allows solvent production to proceed for longer and for solvents to accumulate more rapidly and to a higher concentration. The characteristic drop in OD600 observed in wild type and control strains of C. acetobutylicum after 48-72 hours as the cells transition from the solventogenic phase to sporulation is notably absent in the fermentations of 824(pASspo). Mutant S (wild type background, spoIIE disrupted), Mutant BS (Mutant B background, spoIIE disrupted), Mutant HS (Mutant H background, spoIIE disrupted) and Mutant bukS (buk- background, spoIIE disrupted) were generated to create stable solvent producing bacteria with complete inactivation of the SpoIIE protein. Similarity between the SpoIIE protein of C. acetobutylicum, B. subtilis, and other Clostridial species indicates that the techniques used in C. acetobutylicum can be applied to other solvent producing Clostridia.
Characterization of the acetone production pathway genes from Clostridium acetobutylicum ATCC 824
(1991) Petersen, Daniel James; Bennett, George N.
In Clostridium acetobutylicum the three enzymes thiolase, CoA-transferase, and acetoacetate decarboxylase serve to catalyze the formation of acetone from acetyl-CoA. This process is unique to butyric-acid clostridia and has long-term industrial significance. These three enzymes are known to he induced at the onset of solvent production. Both the acetoacetate decarboxylase and CoA-transferase activities are often spontaneously lost in degenerative mutants. To further investigate the regulatory mechanisms governing the expression of these enzymes, the genes encoding each of the three enzymes were cloned and expressed in Escherichia coli. The CoA-transferase is an $\alpha\sb2$,$\beta\sb2$ heterotetramer of 23 and 26 kDa subunits. Cloning revealed the enzyme is encoded by two genes (actA and actB). Genes for both subunits of the CoA-transferase were located in an operon arrangement. DNA sequence analysis suggests that these genes are co-transcribed as part of a larger operon from an upstream promoter. The adc gene, encoding the acetoacetate decarboxylase, was cloned and physically mapped downstream from the CoA-transferase genes. However, it is not part of the actAB operon, being oriented in the opposite direction. DNA sequencing analysis demonstrates transcription of the adc as a monocistronic operon. The 65 bp intergenic region between the C-termini of the adc and actB genes is characterized by a novel stem-loop structure which appears capable of rho-independent transcription termination function in both directions in C. acetobutylticum. The cloned adc gene was successfully reintroduced into a C. acetobutylicum adc$\sp-$ mutant, resulting in wild-type regulation and expression of the gene. The gene encoding the thiolase (thl) was also cloned, although it is not located near the other acetone production genes. The cloned thiolase is transcribed from its own promoter. No other thiolase genes were identified. A profound bias for adenine or thymidine in the third ("wobble") position of each codon was demonstrated in all four genes sequenced. Despite preferential usage of codons rarely used in E. coli, the transcription of the genes, assembly of subunits, and high levels of enzyme activity indicate the genes were well expressed in E. coli.
Cofactor engineering for advancing chemical biotechnology
(Elsevier, 2013) Wang, Yipeng; San, Ka-Yiu; Bennett, George N.
Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.
Effects of Antibiotic Physicochemical Properties on Their Release Kinetics from Biodegradable Polymer Microparticles
(Springer, 2014) Shah, Sarita R.; Henslee, Allan M.; Spicer, Patrick P.; Yokota, Shun; Petrichenko, Sophia; Allahabadi, Sachin; Bennett, George N.; Wong, Mark E.; Kasper, F. Kurtis; Mikos, Antonios G.
Purpose: This study investigated the effects of the physicochemical properties of antibiotics on the morphology, loading efficiency, size, release kinetics, and antibiotic efficacy of loaded poly(DL-lactic-co-glycolic acid) (PLGA) microparticles (MPs) at different loading percentages. Methods: Cefazolin, ciprofloxacin, clindamycin, colistin, doxycycline, and vancomycin were loaded at 10 and 20 wt% into PLGA MPs using a water-in-oil-in water double emulsion fabrication protocol. Microparticle morphology, size, loading efficiency, release kinetics, and antibiotic efficacy were assessed. Results: The results from this study demonstrate that the chemical nature of loaded antibiotics, especially charge and molecular weight, influence the incorporation into and release of antibiotics from PLGA MPs. Drugs with molecular weights less than 600 Da displayed biphasic release while those with molecular weights greater than 1,000 Da displayed triphasic release kinetics. Large molecular weight drugs also had a longer delay before release than smaller molecular weight drugs. The negatively charged antibiotic cefazolin had lower loading efficiency than positively charged antibiotics. Microparticle size appeared to be mainly controlled by fabrication parameters, and partition and solubility coefficients did not appear to have an obvious effect on loading efficiency or release. Released antibiotics maintained their efficacy against susceptible strains over the duration of release. Duration of release varied between 17 and 49 days based on the type of antibiotic loaded. Conclusions: The data from this study indicate that the chemical nature of antibiotics affects properties of antibiotic-loaded PLGA MPs and allows for general prediction of loading and release kinetics.
Engineered bacteria produce succinate from sucrose
(2015-02-24) San, Ka-Yiu; Bennett, George N.; Wang, Jian; Rice University; United States Patent and Trademark Office
Bacteria optimized to produce succinate and other feedstocks by growing on low cost carbon sources, such as sucrose.
Engineering Silver Nanoparticles: Towards a Tunable Antimicrobial
(2014-03-11) Puppala, Hema Lakshmi; Colvin, Vicki L.; Billups, W. Edward; Bennett, George N.
Overwhelming production of commercially available products containing silver nanoparticles (AgNPs) underscores the studies determining their fate in the environment. In order to regulate the use, assess the environmental impact and develop eco-responsible silver products, models that can predict AgNP toxicity based on physicochemical properties are vital. With that vision, this thesis developed well-characterized model libraries of uniform AgNPs stabilized with oleate in the range of 2-45 nm diameter with variable surface coating and investigated the dissolution properties that link AgNP structure to antimicrobial activity. High temperature organic synthesis allowed controlled growth of AgNPs (σ<15%) by an Ostwald ripening mechanism in the first few hours, and followed by size dependent growth rates yielding uniform nanocrystals. Characterization of these materials revealed a crystalline nature, bidentate binding mode of oleate and non-oxidized pristine silver surface. Phase transfer of these AgNPs from organics to water was facilitated by encapsulation and ligand exchange methods using amphiphilic polymers and methoxy poly (ethylene glycol) (mPEGSH) respectively. Among these surface coatings, steric stabilization by mPEGSH not only helped retain their optical properties but also reduced the dissolution (<1(w/w)%) of AgNPs. This enhanced the stability in various environmentally relevant high ionic strength media (such as Hoaglands, EPA hard water and OECD medium), thereby increasing the shelf life. In addition, size, surface coating, pH of the medium and grafting density of the polymer mediated the dissolution of AgNPs. For instance, the rate of dissolution was decreased by 40% when the polymer coating possessed a mushroom conformation and increased with reducing core size. Analogous to dissolution, physicochemical properties also influenced the antimicrobial activity which were studied by minimum inhibitory concentration (MIC) and bactericidal efficacy assays. For example, surface passivation with mPEGSH prevented the oxidation of active silver atoms on the surface, and resulted in reduced toxicity against E. coli. Moreover citrate stabilized AgNPs when surface modified with mPEGSH had reduced toxicity, which was correlated with residual Ag+ in AgNP solution. Therefore this study demonstrates that processes in the environment that increase stability of AgNPs could make them more persistent due to low dissolution. Furthermore, the size and surface chemistry effects of AgNPs studied here make the intrinsic antimicrobial property of silver tunable and hence more versatile. This work also served as a material support for research on investigating toxicity of AgNPs to C. elegans, Daphnia Magna, Populus and Arabidopsis. In the future, this data will be used to develop nanomaterial bioavailability & environmental exposure (nanoBEE) models that predict the environmental impact of AgNPs.
Engineering the Production of Itaconic Acid in Escherichia coli
(2011) Lin, Tao; Bennett, George N.
Itaconic acid (IA) has many versatile applications in science and medicine. Engineering Escherichia coli can provide a reliable route for IA production, where cis -aconitate decarboxylase (CAD) is one essential enzyme in the process. The synthetic cad gene has been cloned into various vectors to improve the expression rate and folding activities. The strain bearing plasmid pTrc99a- cad displays a significant amount of IA production, while the CAD protein expressed from the same plasmid displays the highest enzymatic activity in vitro. To improve the reliability of forming the cis -aconitate intermediate, the aconitase genes from Aspergillus terreus have been cloned into the pTrc99a vector. The expression of aconitate hydratase leads to a peak at 10.3 min in the HPLC spectra after 4 hours incubation. The peak will be analyzed to determine whether it is cis -aconitate. The obtained data will provide data on possible metabolic pathways of IA production in Aspergillus terreus.
Evaluation of antibiotic releasing porous polymethylmethacrylate space maintainers in an infected composite tissue defect model
(Elsevier, 2013-11) Spicer, Patrick P.; Shah, Sarita R.; Henslee, Allan M.; Watson, Brendan M.; Kinard, Lucas A.; Kretlow, James D.; Bevil, Kristin; Kattchee, Lauren; Bennett, George N.; Demian, Nagi M.; Mende, Katrin; Murray, Clinton K.; Jansen, John A.; Wong, Mark E.; Mikos, Antonios G.; Kasper, F.Kurtis
This study evaluated the in vitro and in vivo performance of antibiotic-releasing porous polymethylmethacrylate (PMMA)-based space maintainers comprising a gelatin hydrogel porogen and a poly(DL-lactic-co-glycolic acid) (PLGA) particulate carrier for antibiotic delivery. Colistin was released in vitro from either gelatin or PLGA microparticle loaded PMMA constructs, with gelatin-loaded constructs releasing colistin over approximately 7 days and PLGA microparticle-loaded constructs releasing colistin up to 8 weeks. Three formulations with either a burst release or extended release in different doses were tested in a rabbit mandibular defect inoculated with Acinetobacter baumannii (2 × 107 colony forming units/mL). In addition, one material control that released antibiotic but was not inoculated with A. baumannii was tested. A. baumannii was not detectable in any animal after 12 weeks by culture of the defect, saliva, or blood. Defects with high-dose, extended-release implants had greater soft tissue healing compared to defects with burst release implants, with 8 out of 10 animals showing healed mucosae compared to 2 out of 10 with healed mucosae, respectively. Extended release of locally delivered colistin via a PLGA microparticle carrier improved soft tissue healing over the implants compared to burst release of colistin from a gelatin carrier.
Evolutionary Relationships Between Low Potential Ferredoxin and Flavodoxin Electron Carriers
(Frontiers, 2019) Campbell, Ian J.; Bennett, George N.; Silberg, Jonathan J.
Proteins from the ferredoxin (Fd) and flavodoxin (Fld) families function as low potential electrical transfer hubs in cells, at times mediating electron transfer between overlapping sets of oxidoreductases. To better understand protein electron carrier (PEC) use across the domains of life, we evaluated the distribution of genes encoding [4Fe-4S] Fd, [2Fe-2S] Fd, and Fld electron carriers in over 7,000 organisms. Our analysis targeted genes encoding small PEC genes encoding proteins having ≤200 residues. We find that the average number of small PEC genes per Archaea (~13), Bacteria (~8), and Eukarya (~3) genome varies, with some organisms containing as many as 54 total PEC genes. Organisms fall into three groups, including those lacking genes encoding low potential PECs (3%), specialists with a single PEC gene type (20%), and generalists that utilize multiple PEC types (77%). Mapping PEC gene usage onto an evolutionary tree highlights the prevalence of [4Fe-4S] Fds in ancient organisms that are deeply rooted, the expansion of [2Fe-2S] Fds with the advent of photosynthesis and a concomitant decrease in [4Fe-4S] Fds, and the expansion of Flds in organisms that inhabit low-iron host environments. Surprisingly, [4Fe-4S] Fds present a similar abundance in aerobes as [2Fe-2S] Fds. This bioinformatic study highlights understudied PECs whose structure, stability, and partner specificity should be further characterized.
Flavodoxin protein electron carriers: bioinformatic analysis and interactions with sulfite reductases
(2020-08-14) Guseva, Anna; Silberg, Jonathan J.; Bennett, George N.; Ajo-Franklin, Caroline M.
Flavodoxins (Flds) are oxidoreductases that distribute electrons to different metabolic pathways through interactions with an array of partner proteins. The aim of my thesis is to understand Fld evolution, establish whether Flds are encoded within the same genomes as Fd-dependent sulfite reductases (SIRs), and demonstrate that a cellular assay can monitor Fld electron transfer (ET) to SIRs. Using bioinformatics, I identify numerous microbes whose genomes encode both Fld and SIR genes. Additionally, I show that Flds can support ET to SIR using a synthetic pathway where protein-mediated ET is monitored using the growth of an Escherichia coli auxotroph that depends upon Fld transferring electrons from a Fd:NADP+ reductase to SIR. My results represent the first evidence that Flds support ET to assimilatory SIRs. Additionally, they show how a synthetic ET pathway in cells can be leveraged to rapidly compare the ET efficiencies of different Flds.
Genetic Catabolic Probes to Assess the Natural Attenuation of 1,4-Dioxane
(2013-12-06) Li, Mengyan; Alvarez, Pedro J.; Li, Qilin; Bennett, George N.; Fiorenza, Stephanie
Remediation of aquifers contaminated with 1,4-dioxane (dioxane) is a difficult task because dioxane can be recalcitrant to biodegradation, is not easily removed by volatilization or adsorption, and is highly mobile in groundwater. Monitored natural attenuation (MNA), which relies primarily on biodegradation, is often the most cost-effective approach to manage large and dilute groundwater plumes of priority pollutants, such as those formed by dioxane. However, the burden of proof that MNA is an appropriate solution lies on the proponent, which requires demonstration of the presence and expression of relevant biodegradation capabilities. Therefore, an innovative micro-extraction of aqueous samples coupled with gas chromatography/mass spectrometry (GC/MS) was developed to monitor dioxane attenuation with low part-per-billion detection sensitivity. Soluble di-iron monooxygenases (SDIMOs), especially group-5 SDIMOs (e.g., tetrahydrofuran [THF]/dioxane monooxygenases), are of significant interest due to their potential role in the initializing the cleavage of cyclic ethers. In this study, seven gene clusters encoding SDIMOs were annotated in the genome of Pseudonocardia dioxanivorans CB1190, a well-characterized bacterial dioxane degrader. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) results revealed that only the dxmA gene encoding the large hydroxylase of the putative dioxane monooxygenase was significantly up-regulated when fed with dioxane and its structural analog, THF, compared to controls fed with acetate. This is in accordance with previous evidence implicating the key role of dioxane/THF in initiating the oxidation of cyclic ethers. Additional efforts to evaluate the presence of SDIMO genes in groundwater samples were undertaken using advanced molecular tools, such as functional gene array (i.e. GeoChip) and PCR-DGGE. Both assays demonstrated the widespread distribution of various dioxane-degrading SDIMO genes at a dioxane-impacted site in Alaska. Notably, a group-5 thmA-like gene was enriched in source-zone samples with higher dioxane concentrations, suggesting selective pressure by dioxane. Microcosm assays with 14C-labeled dioxane showed that the highest mineralization capacity corresponded to the source area, which was presumably more acclimated and contained a higher abundance of SDIMO genes. Thus, converging lines of evidence based on both pure bacterial cultures and complex environmental samples corroborate the usefulness of THF/dioxane monooxygenases as biomarkers of dioxane natural attenuation. A primer/probe set was then developed to target bacterial genes encoding the large hydroxylase subunits of THF/dioxane monooxygenases using Taqman (5’-nuclease) chemistry. The probe targets conserved regions surrounding the active site, thus enabling detection of multiple dioxane degraders. Real-time PCR using reference strain genomic DNA demonstrated the high selectivity (no false positives) and sensitivity of this probe. Microcosm tests prepared with groundwater samples from 16 monitoring wells at five different dioxane-impacted sites showed that enrichment of this catabolic gene (up to 114-fold) was significantly correlated to the amount of dioxane degraded. A significant correlation was also found between biodegradation rates and the abundance of thmA/dxmA genes, suggesting them as reliable indicators of dioxane biodegradation activity. Furthermore, pyrosequencing-based metagenomics and 16S rDNA profiling was used to understand how dioxane contamination incidents along with chlorinated solvents and other hydrocarbons have affected the indigenous microbial communities and the microbes that are critical to carbon cycling at the site in Alaska. Actinobacteria and Proteobacteria were the dominant bacterial phyla. However, shifting of the microbial communities structures among various sampling locations was significantly related to the types and presence of external carbon sources (e.g., synthetic chemicals and naturally released gases) at the site. This indicates that the indigenous microbes have adapted to the local environment probably due to long term of acclimation, and may be contributing to the presence of natural attenuation. This study comprehensively evaluated the essential roles of THF/dioxane monooxygenases in dioxane degradation in dioxane-degrading bacterial strains and environment samples. This is the first report to elicit the impact from dioxane and other co-contaminants on shaping functional and phylogenetic structures of microbial communities at a dioxane-impacted site. The development of the novel catabolic biomarker (thmA/dxmA) is of great research and engineering value to unequivocally characterize both dioxane biodegradation potential and activity for enhanced MNA forensics.
Genetic factors affecting the regulation of solventogenesis in Clostridium acetobutylicum ATCC824
(2005) Scotcher, Miles Christopher; Bennett, George N.
The obligate anaerobe Clostridium acetobutylicum produces the acids acetate and butyrate during exponential growth. At the transition to stationary growth, these acids are converted to the solvents acetone and butanol. The genes involved in these metabolic pathways have been identified and characterized, but little is known about the control of their expression. This thesis focuses on the identification and further investigation of genes involved in the control of solventogenesis. solR was previously identified through genetic studies as a controller of solventogenesis. We have shown that the original construct used to increase solR expression can complement the high solvent phenotype of the solR-deleted mutants B and H when the solR open reading frame is either intact or partially deleted, thus demonstrating that the solR open reading frame is not required to restore solvent levels. Using a chloramphenicol acetyl-transferase reporter system harboring fragments of the alcohol dehydrogenase ( adhE) promoter, we have shown that a region beyond the 0A box is needed for full induction of the promoter, and that the presence of sequences around a possible processing site designated P2 may have a negative role in adhE expression. We identified two transcription factors, sinR and abrB310, which may be involved in the control of solventogenesis. We showed that abrB310 is expressed strongly and transiently at the onset of solventogenesis, and antisense RNA targeted against abrB310 caused a delay and decrease in solvent production, and also in sporulation. We propose that abrB310 is a regulator of the transition between acidogenic and solventogenic growth. We identified a homologue to the Bacillus subtilis early sporulation phosphatase and membrane protein, SpoIIE. Using reporter vectors, we showed that spoIIE is expressed in wild type C. acetobutylicum cells during late solventogenesis, but not in strain SKO1, where the sporulation initiator spo0A is deleted. Antisense RNA targeted against spoIIE caused a prolongation of solventogenesis characterized by increased solvent production and defective sporulation.

Browsing by Author "Bennett, George N."

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