Tag: large square pots

Parathion also contains a thioester linkage that is analogous to the chemical structure of several chemical warfare agents, including sarin. Consequently, parathion is an excellent model for studying enhanced biodegrada–tion of environmental contaminants. Mineralization of parathion requires three unique catabolic properties: hydrolysis of parathion, mineralization of p-nitrophenol, and mineralization of diethyl thiophos–phate . The objective of this research was to develop a coculture capable of hydrolyzing parathion and degrading its metabolite p-nitrophenol; evaluate the kinetics of the reaction; and test the suitability of the coculture for use in a flow-through biofilm reactor for parathion bio–degradation.Escherichia coli strain SD2 was constructed by introducing plasmid pWM513, harboring the genes for parathion hydrolysis and ampicillin resistance, and plasmid pMAG1, carrying the green fluorescent pro–tein gene and tetracycline resistance, into Escherichia coli DH10B. The plasmids were inserted by electropo–ration and strain SD2 was selected using media containing both ampicillin and tetracycline. Strain SD2 was used together with Pseudomonas putida KT2440 carrying plasmid pPNP, harboring the genes for p–nitrophenol degradation and also tetracycline resistance. Strain KT2440 is naturally resistant to ampicillin, so the two strains could be cultured in media containing both antibiotics, and consequently maintained the genes required to degrade parathion. The ability of the coculture to degrade 0.5 mM parathion was evaluated during growth of the strains in a minimal medium containing glucose as the carbon source. P-nitrophenol accumulated in the medium during the growth of strain SD2 alone,drainage gutter resulting from parathion hydrolysis .

In contrast, the accumulation of p-nitrophenol was only transient in the coculture, as a result of the bio–degradation activity of strain KT2440. Kinetic analysis indicated that 2 mM p–nitrophenol was fully inhibitory to the growth of the coculture; consequently, this concentration represented the upper limit for parathion bio–degradation . The coculture was used to cultivate a biofilm in a par–allel plate flow cell for imaging by confocal microscopy. After 72 hours of growth in continuous mode, the biofilm was stained with a red fluores–cent nucleic acid dye and imaged using a confocal microscope. The dye caused the Pseudomonas strain to appear red, while the Escherichia coli strain appeared green and yellow as a result of the colocalization of green color fluorescing from its green fluorescent protein. The images indicated that the biofilm was dominated by Pseudomonas, although E. coli was stably maintained. The results suggest that the two strains could be used as part of a flow-through biofilm reactor for detoxification of parathion. Several industries in the United States and Puerto Rico are dedicated to the manufacture of filters of different materials, including nitrocellulose . These filters are very important in ensuring the purity of ingredients in food and pharmaceutical industries. However, the nitro–cellulose used for manufacturing these filters is considered a hazardous waste for its ignitability , high flammability and oxidizing properties. The Department of Defense also has sites contaminated with NC since it is a major component in explosives. Nitrocellulose exhibits good chemical stability, which for years made incineration the preferred method for dis–posing of the NC wastes. Alkaline hydrolysis appears to be a rapid process for nitrocellulose degradation, but research is limited. This investigation involves the eluci–dation of the mechanism of alkaline hydrolysis and its combination with biodegradation.Table 1 shows the research methods and tools employed in this study. Every step in Table 1 seems to play an important role for either hydrolysis or bio–degradation itself. Interpretation of the species left after degradation time facilitates analysis. Comparison between liquid and agar media is important to really determine if physical attachment is another factor for better fungus growth. Figure 1 shows a possible precipitation of products after acidification on the 9 mL NaOH treatment.

Figure 2 shows fungus growth from 10 mcL of spores suspen–sion placed on a petri dish containing NC as sole carbon source. HPLC assays have been made on a growth curve in a culture tube . A total of 0.1 g of NC hydrolyzed in 6 mL of SSC buffer was used. The strongest peaks are shown. The sample represents one-third of the total area. A total of 20 mcL were injected in a 50:50 acetonitrile:water mixture. Assays are being done using wavelengths of 210 nm and 214 nm, due to the abundance of probably dicarboxilic acids in the hydrolyzed NC.Polycyclic aromatic hydrocarbons are a con–cern in the environment because they are toxic and carcinogenic. Polycyclic aromatics are more recalci–trant in the soil than other hydrocarbons because they are hydrophobic and tend to migrate into the soil . Most PAHs occur as a result of fossil fuel combustion; thus, high concentra–tions of PAHs are found at the sites of active and inactive oil refineries . This study focuses on oil and petroleum contami–nated soil samples taken from Alameda Naval Air Station at Alameda Point, Calif. Pacific Coast Oil Works refinery used the site between 1879 and 1903. After the refinery closed in the 1930s, the U.S. Army and then the U.S. Navy owned the property. In 1991, jet fuel spilled from the jet engine test facility on the site. Heavy rains resulted in jet fuel in the overflow of storm drains. Damage to the storm drains during the Loma Prieta earthquake in 1989 may have caused ground water contamination. Recent studies showed total petroleum hydrocarbon amounts rang–ing from 100 to 10,000 mg/kg soil. Due to the long term and high concentration of oil in the soil it is likely that there are microorganisms capable of degrading the oil products. This study will examine the use of Inipol EAP 22, the same fertilizer used in the Exxon Valdez oil spill clean-up, to obtain optimal growth conditions of the naturally occurring bacteria using CO2 output to monitor the degradation of PAHs and hydrocarbons. Inipol EAP 22 is particularly attractive for this site since it is oleophilic and should make the strongly sorbed PAH components more bio–available, thereby stimulating bio–degradation.More than 8,000 Department of Defense sites need clean-up efforts .

TNT was the most commonly occurring compound within the contaminated samples from these sites. Fortunately, TNT is biodegradable, making in situ bio–remediation a cost–effective and rapid alternative for site cleanup. The toxicity of nitroarenes and their metabolites have been studied in a variety of biological systems but we have been unable to find any studies related to chemotaxis of nitroaromatic compounds in the scientific literature. Chemotaxis allows bacteria to respond to chemical gradients, seeking higher levels of potential nutrients and lower levels of inhibitors. Organisms that have developed mechanisms with which to beneficially orient themselves with respect to these gradients of different types may have a competitive advantage over other organisms. Also, the value of motility as a survival factor for bacteria in environments where nutrients or harmful agents are discontinuously dis–tributed, e.g., contaminated sites, seems obvious. The present study will target how bacteria can use nitroaromatic compounds as a source of nutrients and as a dispersal mechanism in soil,large square pots especially as it may apply to in situ bio–remediation.Chemotaxis provides a means for bacteria to respond to environmental gradients of potential nutrients and toxins, resulting in direct motility towards or away from these substances . The P. fluorecens bacterial strain used responded impressively according to our predictions . This is the first time that bacteria have been demonstrated to be attracted to explosive compounds. The strong attraction of soil bacteria to DNT was also verified using soil perfusion column leachate from field samples taken at Ft. Ord, Calif. In these leachate tests, the chemotactic indices observed for DNT are some of the highest rates ever observed for any type of chemotaxis, including the ones observed for P. fluorecens for concentrations of 2,4 and 2,6-dinitrotoluene that ranged from 0.12 M to 1.20×10-3 M. The results of this work have important implications for the ecology of TNT-degrading bacteria; it also may suggest ways that micro–environments containing explosives might be controlled to increase bio–degradation rates in situ.More than 8,000 Department of Defense sites need clean-up efforts . In a compilation of soil samples collected from 44 Army ammunition plants, arsenals and depots, 28% contained detectable levels of explosives. TNT was the most commonly occurring compound within the contaminated samples, and was seen in 66% of those samples. Facilities that may be contaminated with explosives include manufacturing plants, ordnance works, Army ammunition plants, ammunition depots, Army and Naval proving grounds, burning grounds, artillery impact ranges, explosives disposal sites, bombing ranges, firing ranges, and ordnance test and evaluation facilities.

Due to its toxicity and recalcitrance, the U.S. Environmental Protection Agency has listed TNT as a priority pollutant . Bio-remediation of organic contaminated soils has proven to be one of the fastest and cheapest remediation technologies available. TNT and its daughter products are highly recalcitrant, especially in highly weathered soils, i.e., soils that have been exposed to the environment for a number of years under sub–optimal microbial activity conditions. The present study examines weathered and unweathered soils and looks at the ability of a number of bio–stimulants to increase total microbial respiration.Two-gallon buckets were converted to soil bioreactors . These reactors use the same CO2 trapping principle as the bio–meter flasks, but allow for a much greater soil sample. The KOH trap was a funnel plugged at the bottom with a rubber stopper, which was held in place with epoxy. These traps rested on top of the soil, and could hold up to 80 ml of solution. Larger ascarite-top filters were made using 50 ml Corning vials. A hole was drilled into the bottom of each vial, through which a piece of plastic tubing would fit. This tube was connected to a stopcock, which let the filtered air into the bucket during the times when the KOH was changed. The attached diagram shows the bio–meter bucket design. The soil in each bucket was mixed well prior to the respiration measurements. After the bucket was sealed, the KOH was added to its trap, and the buckets sat for four days. In this four-day period, the KOH was changed and titrated as necessary, with intervals from an hour to a day, depending on the rate of respiration in the soil samples. These intervals change because a faster respiration rate can create enough CO2 to saturate the KOH solution. Therefore, the KOH monitoring a more active soil sample must be titrated much more often than in a slowly respiring sample. For each bucket, three experiments were run: an unamended control, where the soil was taken directly from the buckets in which it was collected, and lasting 3.57 days, an inorganic nutrient amendment, where 1000 ml of MSM was added to each bucket, and lasting 2.93 days, and an organic nutrient amendment, where an aqueous molasses solution was added to each bucket, and lasting 4.80 days. Respiration was measured daily and samples for HPLC analyses of contaminants were taken initially and at the end of the treatment.The respiration analysis indicates that unweathered soil responds faster and in greater magnitudes to nutrient amendments . The results suggest that the weathered soils may have stressed populations and cannot react as quickly to the amendments, especially because they actually reduce their respiration rate in response to MSM addition. The molasses-amended samples all respired at a greater rate than with the other amendments. This result implies that molasses may be used as an effective nutrient source to increase bacterial activity. Three of the four molasses-amended samples are among the lowest four in concentrations of the suspected contaminant located by the HPLC. The HPLC results also suggest a correlation between bacterial activity and bio–degradation. Glycerol trinitrate , also known as nitroglyc–erin, is extensively utilized for the production of explosives and pharmaceuticals. GTN is a hazardous waste product of increasing environmental concern. Current disposal techniques, such as open-air burning and incineration, are expensive and can produce hazardous waste byproducts. Bio–remediation systems could remediate explosive contaminants at approximately a tenfold lower treatment cost and with increased public acceptance. An ecological investigation of GTN-contaminated sites at the Naval Surface Warfare Station in Maryland resulted in the isolation of a Bacillus thuringiensis/cereus strain able to degrade GTN. HPLC and TLC analysis by other researchers of GTN metabolism in cell-free systems suggested that there was a sequential denitration to dinitrate isomers, mononitrate isomers and ultimately to glycerol.

Together, this evidence suggests the presence of a larger cis-regulatory module that may include up to five other transcription factors besides WUS. Slightly further downstream, another conspicuous feature of the 3’ enhancer is the presence of two large and perfectly conserved sequence blocks, spanning 42 and 32 bp, respectively . Both the size and the degree of sequence conservation in these two regions were exceptional in that they exceeded those found in the coding region of AtCLV3. The two regions also overlapped with the DNA family transposable element At2TE50665, suggesting that these may represent coding sequences of the transposon, rather than AtCLV3 regulators. When examined with the 5bp sliding window, the two conserved blocks were found to be surrounded on both sides by a strikingly periodic arrangement of three cis-motifs, spaced 11bp apart, strongly reminiscent of the pattern associated with the WUS-binding region. Superficially, the region around the two conserved blocks resembled an inverted repeat , though the underlying sequence in each half showed little or no sequence similarity. The repeated occurrence of this pattern suggests that the cis-motifs are organized around a higher-ordered protein structure, each of which may bridge up to 5-7 unique transcription factors. Such clustersof cis-motifs can be described as cis-regulatory modules, which in recognition of their similar structure, are provisionally identified here as CRM 1, 2, and 3 . Of the three modules, evidence from the WUS binding sites and a promoter deletion analysis, suggests that only CRM1 has a direct role in AtCLV3 activation. Although CRM2 and CRM3 might belong to a transposable element,drainage gutter the overall sequence of At2TE50665 is poorly conserved. It is also an old transposon, which likely shared a common ancestor with all five orthologs more than 20mya.

Suspecting functional diversification, the two large sequence blocks were subjected to additional BLAST searches in order to similar motifs in the A. thaliana genome. The genes located next to the motifs were identified, and there expression patterns of a select subset of identified genes were compared using microarray data using the eFP browser. In all, 32 genes were identified, most of which contained only one of the two conserved blocks.Unexpectedly, this analysis revealed that many genes had similar expression patterns in the lateral root cap, the columella, and root procambium tissue . This root expression pattern was also found to be shared between At2G27240 and AtCLV3, though the root expression of CLV3 was much reduced compared to its levels in the SAM. Further alignment of the oligomers returned by the blast searches identified three potential cismotifs, each of which was found to correlate with these expression patterns. Motifs were clearly related to root expression patterns, whereas the third motif was less tissue specific, but associated with several disparate structures in the shoot. However, the predicted transcription factor binding sites found within CRM2 and CRM3 did not strongly support any of these putative functions. The AAATCTAT motif overlapped with a predicted cytokinin response element , and an AGAMOUS binding site. Cytokinin responses occur in both root and shoot tissues, though AGAMOUS expression is clearly confined to the flowers, indicating a shoot-related function. The root-related function of the TGGCGATTTCG motif was at least partially supported by a predicted “right part of the root hair cis-element” motif, but the associated transcription factor is unknown. Finally, the ATTATCCTTAAT motif overlapped with two predicted targets, one for AtHB-2/HAT4, which has root and shoot expression patterns, and the other was a computer identified “sucrose response element” originally identified from lateral buds.

To test the function of the identified regulatory regions, a series of deletions were performed in the pCLV3m:H2B-YFP reporter construct . This construct containes three previously described mutated WUS binding sites, which enhanced expression of the reporter by 120% . Five large deletions, each about 500bp long were initially performed in the regulatory regions, three of which occurred in the upstream 1.5kb region and two in the downstream 1.2kb . Based on initial findings, two of the initial deletions were subdivided into smaller segments, identified here as 3.1, 5.1, and 5.2 . Most deletions produced a binary on-off response in the SAM, though a faint signal remained in the flower meristems of deletions 4 and 5, which occurred in 25% of all independent alleles. Only deletion 5.1 produced an intermediate fluorescent signal, though the pattern indicates reflection off the surface of the SAM, rather than actual fluorescence. Overall the deletions revealed that the AtCLV3 promoter is located in a small region between -154 and +25 bp TSS, while the 3’ enhancer required sequences between +584 and +1389 bp TSS . These regions closely correspond to the previously identified conserved footprints, and contained 8 out of 10 footprints in the 5’ promoter, all four footprints in the 3’UTR, and 14 out of 18 in the 3’enhancer region.Previous deletions of AtCLV3 found the 5’ promoter region was less than 812 bp long, and detected a large 3’ enhancer approximately 950bp long. These findings are broadly consistent with the results of the present study, where deletions 1, 2, 3, and 3.1 further narrowed down the 5’ promoter to just 154bp, and deletions 4, 5, 5.1 and 5.2 support the existence of a regulatory region just 805bp long in the 3’ enhancer. However, the two studies disagree in the functional annotation of these regulatory regions, as the present analysis found only positive regulatory regions , while both positive and negative regulatory functions were found in a previous deletion analysis. Disregarding the 5’ end of deletion V1 which had no significant footprint in the present analysis, a comparison of the two studies revealed that the previously identified negative regulatory region, corresponds to a 334 bp sequence containing CRM2 and CRM3.

Meanwhile, the positive regulatory region corresponded to a 290 bp sequence containing CRM1. The source of the discrepancy is obscure, but might potentially be related to the different techniques used to observe CLV3 expression. The previous deletion analysis relied on a GUS reporter system that used whole plant extracts, which might have missed ectopic expression patterns, while the present study did not attempt to observe any other tissue outside of the SAM. Thus it would be of interest to examine similar deletion constructs in a future study, to see if ectopic expression patterns actually occur following the loss of negative regulatory region. The slight increase in the reporter expression in the mutant control compared to a wild-type pCLV3:H2B-YFP reporter is consistent with the repressive function of WUS transcription factors, but suggests that WUS alone is insufficient to repress CLV3 expression. The failure to detect strong reporter activity in deletions 5 and 5.1 might also indicate an interaction between their regulatory modules, as the presence of CRM1 alone could not activate the reporter in the absence of CRM2 and CRM3. The reverse is also true, as CRM2/3 were not able to activate the reporter in the absence of CRM1 . It is not clear how the modules might interact with each other, as CRM1 is separated from CRM2/3 by 280-335bp, indicating that they are located on non-adjacent nucleosomes. There are however, four conserved regions located between CRM1 and CRM2 , which if recognized by additional DNA binding proteins, might help bridge the nucleosome gap. The AtCLV3 expression pattern occurs in an inverted cone-shaped domain in the apex of the SAM,large square pots and displays layer-specific patterns. The L1 is often strongest, while the signal intensity fades with tissue depth. In tangential sections, L2 cells often have noticeably weaker expression levels . However, the L2 “gap” often disappears in perfect longitudinal sections, suggesting that AtCLV3 is actually expressed in two closely spaced domains: a broad L1 sheet in the CZ, and a smaller, but roughly spherical domain directly underneath. Ideally, it would be possible to predict these two patterns using the conserved cis-motifs identified in this study, but unfortunately the function of individual regulatory regions often cannot be completely determined with the available data. Deletion 3.1 for example , suggests that Motif #2 and the redundantly predicted MYB site have no apparent function in the 5’ promoter, yet this contrasts with their unique and strongly conserved footprints. There is also predicted cytokinin-response element located at -102bp, but it is poorly conserved among the four orthologous sequences. A predicted AGL15 binding site might produce the L1 pattern by partially suppressing L2-L4 expression, as AGL15 is known to interact with transcriptional repressors such as TOPLESS and SAP18. However, an examination microarray data with the eFP browser suggests that AGL15 is only transcribed in a small subset of root tissues, where it is unlikely to affect the SAM.

However, considering that the AGAMOUS-LIKE gene family contains more than 100 members , it is possible that one or more of them might function redundantly to suppress AtCLV3 in a subset of SAM cells. If these four cis-elements are removed from consideration, the remaining portion of the AtCLV3 5’ promoter is surprisingly small, and is potentially less than 66bp long. Within this small region are three predicted cis-motifs, in addition to the previously noted initiator for a TATA-less promoter, and several conserved regions around the transcriptional start site. Two of the cismotifs, GT-1 and AGAMOUS, partially overlap with each other and are clear transcriptional activators. The presence of the AG site might also explain the weak expression pattern found in the flower meristems of deletions 4 and 5. The role of GT-1 is harder to explain though, as it is homogenously expressed in most plant tissues, and presumably would lead to widespread ectopic expression of AtCLV3. However, the absence of such ectopic expression patterns might be explained in terms of a nearby auxin response element, which is recognized by the AUXIN RESPONSE FACTOR1. Based on the pDR5rev:3xVENUS-N7 reporter, no auxin responses occured in the SAM itself, but they can be readily detected in the apices of lateral anlagen. Although it is not immediately clear how this might relate to AtCLV3 expression patterns in the CZ, a review of the ARF gene family finds that it includes 5 activators and 17 repressors. The cone-shaped expression domain of AtCLV3 is thus most consistent with repressive auxin responses in the peripheral zone, and might even suggest that the “cone” shape is actually pyramidal based on the auxin response foci observed with the pDR5rev:3xVENUS-N7 reporter. This model is also consistent with the peripheral expression of ARF3 and ARF9, both of which have been demonstrated to be transcriptional repressors. However, this interpretation is at odds with the transcriptional activator ARF5/MONOPTEROS, which is also largely expressed in the peripheral zone, and trace amounts extend into the CZ. As AtCLV3 itself is known to be up regulated by auxin responses at least within the narrow confines of the CZ, the potential functional significance of the GT-1 site might be questioned by an alternative regulatory hypothesis. It is equally probable for example, that AtCLV3 is activated in the CZ primarily by ARF5 or other ARF paralogs, and repressed though an unrelated molecule produced in the peripheral zone. So long as this occurs at levels below the detection threshold of the pDR5rev:3xVENUS-N7 reporter, this model would be indistinguishable from the GT-1 activation/peripheral auxin repression model. Currently, the only evidence that might be able to discriminate between these two hypothesis is rather indirect, and relies on the enhancement of cytokinin responses through the alcohol inducible RNAi system to silence ARR7/15 expression . Interestingly, the CLV3 expression level was reduced in this system, which is consistent with auxin-based activation. In contrast with the promoter, the 3’ enhancer region of AtCLV3 is quite large, with conserved regions spanning a minimum of 460 bp. This region quite likely contains three cis-regulatory modules, each of which may contain 5-7 unique cis-motifs, and together they might support upwards of 20 different transcription factors from multiple gene families. Clearly, regulation of AtCLV3 from the 3’ enhancer is likely to be complicated. One hint about how this might occur lies in the regular spacing of cis-motifs 11-15 bp apart, which is suggestive of helical phasing. The motifs themselves show little or no sequence similarity between modules, implying that transcription factors themselves are interchangeable, while their spacing pattern is governed by a higher-order protein complex that bridges all 5-7 cis-motifs simultaneously.