Dissertations / Theses on the topic 'Microbial biotechnology ; Soil biochemistry'

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

This study demonstrated a technique for fabricating simple, low-cost Paper Microbial fuel cells (PMFC’s) in the model of a previous study to, for the first time, produce voltage from wastewater effluent. The PMFC’s were created by stacking and gluing the main components of an MFC together: reservoir layer; anode; cation exchange membrane (CEM); air cathode. A wax printer was used to create the hydrophobic borders of the PMFC’s on filter paper, and graphite paint was applied to the paper to create the anode. The CEM’s considered were filter paper, wax, and Nafion, with Nafion being the most efficient. Finally, the air cathode was made using carbon veil, and leads (or resistors) were placed in both anode and cathode layers for voltage measurement. Confirming previous studies’ results, the PMFC’s had a rapid startup time and sustained voltage for at least 10 minutes. The study also found that: Nafion was the best CEM; painting one side of the anode had the highest voltage; higher surface area increased voltage; increased time from sampling decreased voltage. Thus, this study proved that the small, low-cost PMFC devices described in previous studies can produce a voltage using primary effluent, and showed that the surface area of the PMFC could be optimized to increase voltage.

This study was done to determine the effectiveness of a commercially available bioaugmentation product, BiOWiSHTM-Aqua FOG, for remediating petroleum-contaminated sandy soil. Biodegradation enhancement by BiOWiSHTM-Aqua FOG was evaluated in laboratory microcosms by directly measuring total petroleum hydrocarbon (TPH) and indirectly using respirometry. Attempts were made to enrich hydrocarbon-degrading bacteria in BiOWiSHTM-Aqua FOG, and the resulting enrichment cultures were screened using respirometry as well. Potential hydrocarbon-degrading bacteria in BiOWiSHTM-Aqua FOG were isolated. Experiments were performed at bench-scale using microcosm bottles containing sand contaminated with either motor oil or No. 2 diesel fuel. The microcosms were incubated at 25oC under aerobic conditions. TPH measurements of soil in the microcosms at 0, 25 and 56 days indicated that the addition of 500-ppm BiOWiSHTM-Aqua FOG improved biodegradation of the motor oil-contaminated soil by 45%. However, BiOWiSHTM-Aqua FOG did not have a measurable effect on biodegradation in the diesel-contaminated soil. In the respirometry experiments, BiOWiSHTM-Aqua FOG and two hydrocarbon-enriched BiOWiSHTM-Aqua FOG cultures were evaluated indirectly by the measurement of microbial carbon dioxide production and oxygen uptake using a MicroOxymaxTM respirometer. The respirometry experiments showed that in the six-day period following motor oil-contamination of soil, the addition of BiOWiSHTM-Aqua FOG substantially improves biodegradation rates. The added organisms in the product out-performed the indigenous organisms in the 5-6 days following contamination of the soil. The CO2 production observed in the BiOWiSHTM microcosms contaminated with motor oil was much greater than CO2 production without motor oil, which confirms that the observed metabolism can be attributed to motor oil biodegradation rather than metabolism of other organic material in the soil. Enriched consortia consistently generated far less CO2 than microcosms with the 500 ppm BiOWiSHTM-Aqua FOG. Stoichiometric calculations suggested that BiOWiSHTM-Aqua FOG removed approximately 1400 ppm TPH (14%) from the soil in 6.5 days, while an enrichment culture of BiOWiSHTM-Aqua FOG only reduced TPH levels by 459 ppm (5%). This result suggests that increased biodegradation rate in bioaugmented soil is aided by biodiversity in the augmenting inoculum. A potential hydrocarbon-degrading candidate organism was isolated from the product and cultured on Bushnell-Haas agar and plate-count agar (PCA). While at least two distinct colony types were successfully grown on media with motor oil, these same colonies appeared on Bushnell-Haas agar with no apparent carbon source, and survived repeated transfers onto this same medium. Therefore, their status as hydrocarbon-degraders is inconclusive. More thorough enrichment work could be pursued, especially using soil samples collected from petroleum-contaminated sites.

Of the organic matter in soils typically < 1% by weight is dissolved in the soil solution (dissolved organic matter; DOM). DOM is a continuum of molecules of various sizes and chemical structures which has largely been operationally defined as the fraction of total organic carbon in an aqueous solution that passes through a 0.45 µm filter. Although only representing a relatively small proportion, it represents the most mobile part of soil organic carbon and is probably enriched with highly labile compounds. DOM acts as a source of nutrients for both soil and aquatic micro-organisms, influences the fate and transport of organic and inorganic contaminants, presents a potential water treatment problem and may indicate the mobilisation rate of key terrestrial carbon stores. The objective of this research was to ascertain some of the biologically relevant characteristics of soil DOM and specifically to determine: (1) the influence of method and time of extraction of DOM from the soil on its biochemical composition and concentration; (2) the dynamics of DOM biodegradation; and, (3) the effects of repeated applications of trace amounts of DOM on the rate of soil carbon mineralization. To examine the influence of method and time of extraction on the composition and concentration of DOM, soil solution was collected from a raised peat bog in Central Scotland using water extraction, field suction lysimetry, and centrifugation techniques on a bimonthly basis over the period of a year (Aug 2003 – Jun 2004). Samples were analysed for dissolved organic carbon (DOC), dissolved organic nitrogen (DON), protein, carbohydrate and amino acid content. For all of the sampled months except June the biochemical composition of DOC varied with extraction method, suggesting the biological, chemical and/or physical influences on DOC production and loss are different within the differently sized soil pores. Water-extractable DOC generally contained the greatest proportion of carbohydrate, protein and/or amino acid of the three extraction methods. Time of extraction had a significant effect on the composition of water- and suction-extracted DOC: the total % carbohydrate + protein + amino acid C was significantly higher in Oct than Dec, Feb and Jun for water-extracted DOC and significantly greater in Dec than Aug, Apr and Jun for suction-extracted DOC. There was no significant change in the total % carbohydrate + protein + amino acid C of centrifuge-extracted DOC during the sampled year. Time of extraction also had a significant effect on the % protein + amino acid N in water- and centrifuge-extracted DON: Oct levels were significantly higher than Feb for water-extracted DON and significantly higher in Aug and Apr for centrifuge-extracted DON. Concentrations of total DOC and total DON were also found to be dependent on time of extraction. DOC concentrations showed a similar pattern of variation over the year for all methods of extraction, with concentrations relatively constant for most of the year, rising in April to reach a peak in Jun. DON concentrations in water- and centrifuge-extracted DON peaked later, in Aug. There were no significant seasonal changes in the concentration of suction-extracted DON. A lack of correlation between DOC and DON concentrations suggested that DOC and DON production and/or loss are under different controls. Laboratory-based incubation experiments were carried out to examine the dynamics of DOC biodegradation. Over a 70 day incubation period at 20oC, the DOM from two types of peat (raised and blanket) and four samples of a mineral soil (calcaric gleysol), each previously exposed to a different management strategy, were found to be comprised of a rapidly degradable pools (half-life: 3 – 8 days) and a more stable pool (half-life: 0.4 to 6 years). For all soil types/treatments, excepting raised peat, the total net loss of DOC from the culture medium was greater than could be accounted for by the process of mineralization alone. A comparison between net loss of DOC and loss of DOC to CO2 and microbial biomass determined by direct microscopy suggested that at least some of the differences between DOC mineralised and net DOC loss were due to microbial assimilation and release. Changes in the microbial biomass during the decomposition process showed proliferation followed by decline over 15 days. The protein and carbohydrate fractions showed a complex pattern of both degradation and production throughout the incubation. The effects of repeated applications of trace amounts of litter-derived DOC on the rate of carbon mineralization over a 35 day period were investigated in a laboratory based incubation experiment. The addition of trace amounts of litter-derived DOC every 7 and 10.5 days appeared to ‘trigger’ microbial activity causing an increase in CO2 mineralisation such that extra C mineralised exceeded DOC additions by more than 2 fold. Acceleration in the rate of extra C mineralised 7 days after the second addition suggested that either the microbial production of enzymes responsible for biodegradation and/or an increase in microbial biomass, are only initiated once a critical concentration of a specific substrate or substrates has been achieved. The addition of ‘DOC + nutrients’ every 3.5 days had no effect on the total rate of mineralization. To date DOC has tended to be operationally defined according to its chemical and physical properties. An understanding of the composition, production and loss of DOC from a biological perspective is essential if we are to be able to predict the effects of environmental change on the rate of mineralization of soil organic matter. This research has shown that the pools of DOC extracted, using three different methods commonly used in current research, are biochemically distinct and respond differently to the seasons. This suggests some degree of compartmentalisation of biological processes within the soil matrix. The observed similarities between the characteristics of the decomposition dynamics of both peatland and agricultural DOC suggests that either there is little difference in substrate quality between the two systems or that the microbial community have adapted in each case to maximise their utilisation of the available substrate. The dependency of the concentration and biochemical composition of DOC on the seasons requires further work to ascertain which biotic and/or abiotic factors are exerting control. Published research has focussed on factors such as temperature, wet/dry cycles, and freeze/thawing. The effect of the frequency of doses of trace amounts of DOC on increasing the rate of soil organic C mineralization, evident from this research, suggests that the interval between periods of rainfall may be relevant. It also emphasises how it can be useful to use knowledge of a biological process as the starting point in determining which factors may be exerting control on DOC production and loss.

Protein-protein interactions (PPIs) are essential features of cellular processes including DNA replication, transcription, translation, recombination, and repair. In my study, the protein interactions of bacterial DNA topoisomerase I, an essential enzyme, were investigated. The topoisomerase I in bacteria relaxes excess negative supercoiling on DNA and maintains genomic stability. Investigating the PPI network of DNA topoisomerase I can further our understanding of the various functional roles of this enzyme. My study is focused on topoisomerase I of Escherichia coli and Mycobacterium smegmatis. Firstly, we have explored the biochemical mechanisms for an interaction between RNA Polymerase, and topoisomerase I in E. coli. Molecular docking and molecular dynamic simulations have predicted that the interactions are mediated through electrostatic, and hydrogen bonding. The predicted Lysine residues (K627, K664) of topoisomerase I that are involved in the electrostatic interactions were mutated to Alanine, and its effect on the binding efficiency with RNA polymerase was reported. In a separate study, PPI partners of topoisomerase I in mycobacteria were identified. Knowledge gained from the study can provide valuable insights into the physiological functions of a validated drug target, DNA topoisomerase I, in pathogenic mycobacteria. Co-immunoprecipitation and pull-down assays were coupled to mass spectrometry for identification of the protein partners of mycobacterial topoisomerase I. The study has identified RNA polymerase, and putative helicases (DEAD/DEAH BOX helicases) as potential protein partners of mycobacterial topoisomerase I. My results indicated that the tail region of the CTD-topoisomerase I was required for direct physical interaction with the RNAP beta’ subunit. My studies have also verified the physiological relevance of the topoisomerase I - RNA polymerase interactions for survival under antibiotic, and oxidative stress. Lastly, I report a direct physical interaction between E. coli topoisomerase I and RecA by pull-down assays. Previous studies have shown that RecA, a DNA repair protein, can stimulate the relaxation activity of E. coli topoisomerase I. Our new results showed that the stimulatory effect can be attributed to the physical interaction of topoisomerase I with RecA.

Solos apresentam hidrocarbonetos gasosos em quantidades variáveis e acredita-se que as formações de reservatórios de óleo podem ser detectáveis indiretamente utilizando-se bactérias no solo capazes de degradá-los. O presente estudo teve como objetivo caracterizar comunidades microbianas envolvidas com o metabolismo desses hidrocarbonetos. As amostras de solo Np (área não petrolífera) e Solo P (área petrolífera) foram analisadas através da construção de bibliotecas do gene RNAr 16S de bactérias e arquéias e de genes catabólicos que codificam enzimas monooxigenases solúveis (SDIMO). As comunidades apresentaram estrutura diferente em relação aos grupos de bactérias e arqueias e análise dos genes catabólicos indicou maior riqueza e diversidade no solo P. A maior parte do clones se mostrou filogeneticamente mais próxima de sequências de enzimas de bactérias não cultivadas proveniente de amostras ambientais. Análises de cromatografia gasosa realizadas logo após a coleta detectaram maiores níveis de metano no solo P e maiores níveis de etano e propano no solo Np. A técnica de PCR quantitativo (Real Time PCR) mostrou um número maior de cópias do rRNA 16S no solo Np, mas não foi eficiente em quantificar os genes degradadores de gases leves presentes no solo.<br>Gaseous hydrocarbons occur in sub-surface soil in highly variable amounts and oil reservoirs formations are supposed to be indirectly detectable through soil microbial populations capable of consuming it. The goal of the present work was to characterize microbial communities involved in short-chain alkane metabolism in soils in and off sedimentary basin areas (named P and Np soil, respectively). Three clone libraries were constructed for each sample, one 16S rRNA gene library for each of the Domains Bacteria and Archaea, and one for the catabolic gene coding for the soluble di-iron monooxygenase (SDIMO) enzyme. Bacterial and archaeal communities structures were different between the samples. Analysis of the catabolic genes presented higher values of richness and diversity in soil P. The sequences from soil samples were more closely related to each other than to reference sequences. Short-chain hydrocarbon measures performed just after samples were collected showed higher levels of methane and lower levels of ethane and propane in soil P in comparison to soil Np. A real-time PCR method was not successful in yielding the catabolic gene quantification suggesting that such genes occur in very low abundance in the soil samples under study.

The genetic information found in each species of organism is unique, and can be used as a tool to differentiate at the molecular level. This has caused rapid genotyping methods to become the cornerstone of a new area of research dependent on reading the genome as a form of identification. One of these specific identification methods, known as pyroprinting, relies on the small variation of DNA sequences within the same species to develop a unique, reproducible fingerprint. By simultaneously pyrosequencing multiple polymorphic loci within the ribosomal operons known as the intergenic transcribed spacers, a reproducible output is obtained, known as a pyroprint, which can be used like a fingerprint to identify that organism. This section of the genome not only differs between species but also between isolated bacteria within that species, allowing for the differentiation of species subtypes, referred to as strains. While this is a viable method for generating reproducible fingerprints from individual strains it may be possible to obtain identical fingerprints from non-identical organisms. The following report uses direct sequence comparison and in silico pyrosequencing of E. coli isolates housed in the Center for Applications in Biotechnology at California Polytechnic State University, San Luis Obispo that have matching pyroprints to show that it is possible to receive near identical pyroprints from non-identical sequences of intergenic transcribed spacers. Although the exact likelihood and cause of this false positive result remains undetermined due to limitations in the sequencing method, its existence questions the accuracy of using pyroprints of the ITS regions as a method of strain classification.

Matthew R. Borglin This thesis demonstrates efficacy of Octenyl Succinic Anhydride (OSA), as a biofilm sanitizer. Biofilms allow bacteria to adhere to solid surfaces with the use of excreted polymeric compounds. For example, surfaces found in food production or processing facilities such as the interior of a raw milk holding tank, are some of the most susceptible to biofilm contamination. When present, biofilms can cause a variety of negative effects, which include; reduction of product shelf life, corrosion, and outbreaks of foodborne illnesses. The close association of biofilms with the majority of foodborne illness cases led the US Environmental Protection Agency (EPA) to create a new category of sanitizer specifically designed for treatment of mature biofilms. The efficacy of sanitizers in this new regulatory category is determined by the EPA protocols MB-19 and MB-20. The EPA’s protocols outline methods for cultivating, treating, and measuring effects on Pseudomonas aeruginosa biofilms in a continuous flow stir bar bioreactor. Biofilm modification by OSA was verified by the presence of octenyl esters on OSA treated biofilms with single point Raman spectrophotometry. OSA modified biofilm’s antimicrobial properties were first investigated with crystal violet staining in 96-well microtiter plates with inconclusive results. However, effective antimicrobial properties where apparent when using the CDC Biofilm Reactor. OSA treatments consistently returned a 6-log CFU/coupon reduction in biomass compared to controls. Inhibition of planktonic and/or biofilm regrowth was demonstrated using the 96-well plate methodology. This thesis demonstrated the effectiveness of OSA chemical esterification reaction as a biofilm treatment. In doing so, this work suggests a new approach for biofilm remediation by chemically modifying the structural components of biofilm.

Breast cancer mortality is mainly due to distant recurrence of the disease arising from dormant tumor cells established by cancer therapies. Patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy. Although immunotherapy of breast cancer by means of anti-HER2 antibodies prolongs survival of breast cancer patients, disease recurrence remains a major challenge. On the other hand administration of human vaccines against infectious disease in a preventive setting or during latency/dormancy has been successful in offering a cure. Here, we sought to use adoptive immunotherapy (AIT) at the time of tumor dormancy in order to prevent progression of breast cancer. We used a low dose immunogenic chemotherapy by means of 5-FU, Adriamycin, and Cyclophosphamide (FAC) in order to stabilize tumor progression prior to AIT using autologous tumor-reactive lymphocytes. Low dose FAC established local tumor dormancy, inhibited distant tumor dormancy occurring long before distant metastasis, and induced predominate a Ki67- quiescent type of tumor dormancy, which is less susceptible to tumor immunoediting. Dormant tumor cells expressed the cell survival pathways, including the endothelin receptor/ligand (ETRA, ETRB and ET-1) and PD-L1, thereby protecting them from elimination by AIT. In addition, tumor-reactive CD8+ T cells also produced ET-1 as a survival ligand for ETRA positive tumor cells. A combination of AIT with the blockade of tumor cell survival pathways resulted in a significant improvement of AIT against tumor dormancy. We also showed that the inhibition Bcl-xL downstream of the tumor cell survival pathways is specifically effective against dormant tumor cells, suggesting a combination of AIT with small molecules inhibitors of Bcl-xL. Altogether, we showed that distant tumor dormancy is established long before distant recurrence of breast cancer, and that the expression of several tumor cell survival pathways in dormant cells protects them from immunotherapy. Our results suggest that immunotherapeutic targeting of tumor dormancy combined with the blockade of a common downstream cell survival pathway could prevent tumor progression and recurrence of the disease.

Microalgae can be grown on municipal wastewater media to both treat the wastewater and produce feedstock for algae biofuel production. However the reliability of treatment must be demonstrated, as well as high areal algae productivity on recycled wastewater media and efficient sedimentation harvesting. This processes was studied at pilot scale in the present research. A pilot facility was operated with nine CO2-supplemented raceway ponds, each with a 33-m2 surface area and a 0.3-m depth, continuously from March 6, 2013 through September 24, 2014. The ponds were operated as three sets of triplicates with two sets continuously fed primary-clarified municipal wastewater at either a 2-day or 3-day hydraulic residence time (HRT), and one set fed the clarified effluent of the 3-day pond set. This second pond-in-series was operated with a 3-day HRT. Areal biomass productivity is reported as gross and net, the former based only on biomass in the pond effluents and the latter subtracting the volatile suspended solids in the influent from those in the effluent. An estimate was also made of autotrophic biomass productivity, as differentiated from heterotrophic growth. Over a year, net productivity averaged 83 metric tons per hectare per year (MT/ha-yr) for the 2-day HRT ponds, 52 MT/ha-yr for the 3-day HRT ponds, and 44 MT/ha-yr for the 3-day HRT ponds receiving clarified effluent of the first set of 3-day HRT ponds (i.e., recycled water). The lower net productivity of the pond receiving water recycling was attributed to two factors. First, the relatively high influent suspended solids concentrations were subtracted from the effluent suspended solids concentrations before net productivity was calculated. Second, the recycled water contained less soluble organic matter than the primary-clarified wastewater leading to less heterotrophic biomass production. The accumulation of inhibitory allelochemicals is a possible third cause of lower productivity , but no specific information was collected on allelopathy. Algae were harvested from pond effluent by sedimentation, with harvest efficiency most affected by the extent of natural bioflocculation occurring in the ponds. Some forms of bioflocculation are thought to be mediated by bacteria, which often make-up a substantial fraction of the settled flocs. Pond samples settled in 1-L Imhoff cones averaged/L total suspended solids after 24 hours of settling; but all ponds fell short of meeting an averaged/L total suspended solids after a 2 hour interval which would be ideally achieved for wastewater effluent. No relationship was seen between settling performance and the bacterial content of flocs. Soluble carbonaceous biochemical oxygen demand (scBOD5) removal by the raceway ponds was sufficient to meet wastewater treatment requirements year around. Influent scBOD5 concentrations averaged 83 mg/L, and the effluent averaged 5.1 mg/L and 4.2 mg/L for the 2-day and 3-day HRT pond sets, respectively. The variable with the greatest influence on productivity in all pond sets, and settling performance in the recycled water pond set, was season (i.e., co-correlated variables of solar insolation and pond temperature). Neither productivity nor settling appeared to be related to prominent algae genera or prevalence of grazers. The high net productivity achieved with a growth medium of primary clarifier effluent and the generally high settleability of algal-bacterial flocs indicate a good potential for algae wastewater treatment and biofuel production. However, the settling of algae grown on recycled water needs improvement to achieve the full potential of wastewater-grown algae biofuel production.

碩士<br>逢甲大學<br>環境工程與科學學系<br>107<br>Soil microorganisms are important decomposers in nature that participate in the biochemical reaction of the soil and promote the cycling of various elements. Numerous studies have shown that soil microorganisms closely related to soil quality. In Taiwan with the rapid industrial development, results the soil pollution on farmland. Heavy metals in the soil have been studied to prove pressure on soil microorganisms. In this study used next-generation sequencing techniques to analyze soil microbial composition and diversity that before and after improvement of pollution in farmland contaminated with heavy metals. In this way, the effects of heavy metal pollution on soil microbial community structure are known. 　　Results showed that the dominant species in heavy metal contaminated environments are often resistant to heavy metals. After the improvement of heavy metal contaminated, there are bacteria with heavy metal resistance and plant growth related species such as Rhizobium. Community diversity and species richness before contamination improvement are better than improved. The proportion of bacterial phylum and dominant species has increased or decreased.

Biological interactions occur on multiple length scales, ranging from molecular to population wide interactions. This work describes the study of two specific areas of biological interactions in microbial systems: intracellular protein-protein interactions and cell-to-cell interactions. The implementation of optical and atomic force microscopy and the methodologies developed during this study proved to be invaluable tools for investigating these systems. Identifying and characterizing protein interactions are fundamental steps toward understanding complex cellular networks. We have developed a unique methodology which combines an imaging-based protein interaction assay with a fluorescence recovery after photobleaching technique (FRAP). Protein interactions are readily detected by co-localization of two proteins of interest fused to green fluorescent protein (GFP) and DivIVA, a cell division protein from Bacillus subtilis. We demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions over four orders of magnitude. FRAP data was analyzed using a combination of various image processing techniques and analytical models. This combined approach made it possible to estimate cell morphology parameters such as length, diameter, the effective laser probe volume, as well as to the mobile protein concentration in vivo, the number of bound molecules at the cellular poles, and the biophysical parameter koff. Cells not only utilize molecular interactions in the intracellular environment, but also express proteins, polysaccharides and other complex molecules to mediate interactions with the surrounding extracellular environment. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Further analyses suggest that the extracellular matrix differs between the cheA1 and the cheY1 deletion mutants, despite similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition.

A thermophilic fungus was isolated from composted horse manure. The organism was as a Chaetomium sp. by sequencing the highly conserved ITS region of the fungus and comparing to known regions in a genomic database and was referred to as TM-417. TM-417 was found to have an optimal growth temperature of 45 oC and an optimal pH of 7.0. An extracellular DNase and RNase was found to be produced by the isolate and were purified 145.58-fold and 127.6-fold respectively using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4-15% gradient polyacrylamide gels. Both DNase and RNase were dependent on metal co-factors for activity. The metal ion Mg2+ was the preferred ion for the DNase, whereas for the RNase, Zn2+ and Mn2+ yielded an increase in enzyme activity over that with Mg2+. The purified DNase demonstrated maximum activity at pH 6.0 with no activity at pH 2.0 or 10.0. The RNase exhibited two peaks of maximum activity, on at pH 3.0 and the other at pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65oC. The optimal temperature for the RNase was 70oC. The molecular of the DNase and RNase were determined to be 56 kDa and 69kDa respectively using a Sephadex G-75 column. A standard curve was generated using several standard proteins of known molecular weight.