Progress 10/01/00 to 09/30/05
Outputs
This project encompassed two lines of investigation. One line focused on the elucidation of mechanisms by which microbes degrade pollutants in soil. From these studies, we identified new isolates of bacteria able to degrade pollutants when these compounds are sorbed to soil constituents. This is significant because most pollutants sorb strongly to soil constituents and most bacteria are unable to degrade the sorbed compounds. We are presently in the process of identifying the characteristics of these organisms that enable them to degrade sorbed compounds. Our other work has focused on determining how Nitrosomonas europaea, a soil bacterium that carries out a central step in the nitrogen cycle, adapts to stresses it encounters in soil. The project is in part technology development, and we are exploring new ways to employ proteomics to study responses of N. europaea. A "proteome" is the complete set of proteins that an organism can produce. Because proteins mediate
activities of an organism, changes in proteome composition could gives us new insights into how bacteria interact with the soil environment. We have also identified molecules (acyl-homoserine lactones) produced by N. europaea that may affect its activity in soil.
Impacts
These studies have contributed methods that may be applied for environmental analysis, organisms that may be useful for bioremediation, and basic knowledge that furthers our understanding of microbial processes in the environment.
Publications
- Hickey, W.J. 2004. Microbiology and Biochemistry of Xenobiotic Compound Degradation. p. 437-467. In D.M. Sylvia et al. (eds.) Soil Microbiology: Environmental and Agricultural Perspectives, 2nd Ed. Oxford University Press, New York, NY.
- Jencova, V., H. Strnad, P. Ulbrich, W.J. Hickey and V. Paces. 2004. Chlorocatechol catabolic enzymes from Achromobacter xylosoxidans A8 pA81 DNA. Int. J. Biodeg. Biodet. 54:175-181.
- Vacca, D.J., W.F. Bleam and W.J. Hickey 2005. Isolation of soil bacteria adapted to degrade humic acid-sorbed phenanthrene. Appl. Environ. Microbiol. 71:000-000 (In press; submitted 9/04, accepted 12/04)
- Burton, E.O., H.W. Read, M.C. Pellitteri and W.J. Hickey. 200_.Identification of acyl-homoserine lactone signal molecules produced by Nitrosomonas europaea strain Schmidt. Appl. Environ. Microbiol. (Submitted 10/04)
- Hickey, W.J. 200_. Nanopods: A novel cellular appendage produced by Burkholderia sp. strain Cs1-4. (In Review)
- Pellitterri, M.C. and W.J. Hickey. 2005. Proteomic analysis of ammonium starvation in Nitrosomonas europaea. Amer. Soc. Microbiol. Atlanta, GA; 5-10 June 2005.
- Starkenburg, S., L. Sayavedra-Soto, P. Chain, M.L. Land, P. Bottomley, F. Larimer, M. Klotz, D. Arp, W. J. Hickey. 2005 The genome sequence of the chemolithotrophic nitrite-oxidizing bacterium Nitrobacter winogradskyi Nb-255. Amer. Soc. Microbiol. Atlanta, GA; 5-10 June 2005.
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Progress 01/01/03 to 12/31/03
Outputs
This project encompassed three lines of investigation. One of these focused on the elucidation of mechanisms by which microbes degraded xenobiotic compounds. From our studies, we discovered a new type of transporter system bacteria may utilize to access xenobiotics. We also identified a unique cluster of biodegradation genes carried on a mobile element. In a second line of investigation, we developed molecular methods for tracking bacterial and fungal pathogens in soil. The third study group focused in the investigation of bacteria adapted to interact with humic acids (HA) in a manner that allows these organisms to access and degrade polynuclear aromatic hydrocarbons (PAH) sorbed by HA. A wide variety of bacteria were isolated that degraded non-sorbed phenanthrene (NSP), but only three strains of Burkholderia sp. and one strain of Delftia acidovorans were isolated that degraded humic acid-sorbed phenanthene (HASP); this phenotype was termed competence. Competence was
not a graded characteristic; bacteria were either able to degrade HASP or they were not. The characteristics of competent cells that supported this phenotype, and the nature of the interaction occurring between humics and bacterial cells, are unknown. However, competence determinants were physically associated with the cells; diffusible agents such as biosurfactants did not have a significant role. As yet, known physical differences between competent and closely related non-competent strains are phospholipid fatty acid content and extracelluar matrix production by the former. Further studies are needed to elucidate the biochemical and biophysical basis of this novel microbial activity.
Impacts
These studies have contributed methods that may be applied for environmental analysis, organisms that may be useful for bioremediation, and basic knowledge that furthers our understanding of microbial processes in the environment.
Publications
- Yuroff, A.S., Sabat, G. and Hickey, W.J. 2003. Transporter-mediated uptake of 2-chloro- and 2-hydroxy-benzoate by Pseudomonas huttiensis strain D1. Appl. Environ. Microbiol. 69:7401-7408.
- Hickey, W.J., Vacca, D.J., Pellitterri, M. and Bleam, W.F. 2003. Isolation and analysis of bacteria adapted to degrade humic-sorbed polynuclear aromatic hydrocarbons (PAH). ASA-CSA-SSSA, Denver, CO; 2-6 Nov 2003.
- Padilla-Crespo, E., Vacca, D. and Hickey, W.J. 2003. Diversity of Burkholderia polycyclic aromatic hydrocarbons (PAH) degradation genes. Abstracts of NIEHS Superfund Sci. Mtg, Los Angeles, CA; 13-15 October 2003.
- Rose, P., Harkin, J.M. and Hickey, W.J. 2003. Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction. J. Microbiol. Meth. 52:29-38.
- Reeleder, R.D., Campell, B.B., Tomlinson, L.D., Miller, J. and Hickey, W.J. 2003. The extraction of fungal DNA from multiple large soil samples. Can. J Plant Pathol. 25:182-191.
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Progress 01/01/02 to 12/31/02
Outputs
Work during the past year focused on determination of the abilities of isolates to degrade humic acid (HA)-sorbed PAH, and examination of factors that may affect the abilities of the organisms to degrade HA-sorbed PAH. Degradation of HA-sorbed PAH was examined by comparing mineralization of [14C]phenanthrene by cultures growing in 2 percent HA solutions that effected in the absence of HA (buffer solutions). The HA and buffer solutions each contained a total of 7.5 ug phenanthrene in 500 uL. In the HA solution, the amount of mineralization effected by twelve organisms isolated by the standard approach (PAH added as crystals to a buffer solution) was only 2.3 percent of that achieved in the buffer solution, which averaged ca. 4.1 ug in a one week incubation. In contrast, for the four cultures of Burkholderia isolated by using the HA systems, mineralization in the HA and buffer solutions was not significantly different and amounted to ca. 4.5 ug. Based on equilibrium
dialysis measurements, the amount of phenanthrene desorbed in one week from the HA was estimated to be a maximum of ca. 1.9 ug. Thus, the organisms must have accessed and degraded phenanthrene sorbed into the HA. This fact, along with the comparatively poor mineralization activity of the standard isolates in the HA solutions, led us to conclude that the phenanthrene-degraders isolated by using the HA solutions had adaptations that allowed them to more effectively degrade sorbed PAH. Investigations into the nature of these adaptations included tests for biosurfactant production, and for cell surface alterations that may enhance mass transfer rates. Results of the former indicate that biosurfactant production did not facilitate PAH degradation in the HA solutions. Cell surface analyses are currently ongoing. Results so far indicate that overall the isolates from the HA approach have more hydrophilic surfaces than those from the standard approach, which may facilitate the interaction of
the organisms with the HA. We are currently examining differences in HA-binding by the cells, variations in lipopolysaccaride content and the occurrence of membrane transporters.
Impacts
PAH are widespread pollutants sorbed by soil organic matter for which bioremediation may be a cost effective solution. These results of our studies will improve our basic understanding of the mechanisms underlying PAH biodegradation and also yield organisms that may be more effective in removing PAH from soil, and thus, be candidates for use as agents to accelerate bioremediation.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Our efforts during the past year have focused on the isolation of PAH-degrading bacteria, analysis of the phylogenetic diversity of these isolates, and examination of the organisms' sorptive characteristics. [14-C]Phenanthrene equilibrium dialysis studies showed 2% humic acid (HA) solutions increased the apparent solubility of phenanthrene by 25-fold. HA solution containing sorbed phenanthrene was used as an enrichment system to isolate bacteria that might be adapted to better interact with soil organic material than isolates obtained with synthetic sorbents or without a sorbent. Seven pure cultures were acquired from these experiments, and based on 16S rRNA gene analysis all were identified as Sphingomonas. Repetitive element PCR (BOXA1R primer) was used to acquire genomic fingerprints of these isolates. All HA isolates showed identical patterns that were identical to fingerprints of two Sphingomonas strains that we previously isolated by the standard approach
(adding PAH as crystals, no sorbent), suggesting the same strain (designated 12H6) was obtained by both approaches. The genomic fingerprints of another Sphingomonas isolated by the standard approach (strain Sg5) differed from that of strain 12H6 by two bands. Additional HA enrichments have been established with other contaminated soils to determine the extent to which Sphingomonas may be commonly selected for by using this approach. Also, mineralization experiments were initiated, but not yet completed, to determine whether the HA-isolated sphingomonads are better able to degrade HA-sorbed phenanthrene than are isolates obtained via the standard approach. Previous investigators have suggested a feature of soil bacteria with enhanced abilities to degrade sorbed chemicals is a hydrophobic cell surface. However, in our meaurement of surface hydrophobicity (sorption to hydrophobic microtiter plates) the HA isolates showed lower sorption strength than those acquired from the standard
approach. Little is known about the genes encoding PAH degradation in sphingpomonds. Plasmids of ca. 194 kb were detected in strains Sg5 and 12H6 by using pulsed-field gel electrophoresis. A positive control used in these experiments was the PAH-degrader Sphingomonas strain F199, which contains a fully sequenced 120 kb plasmid (pF199). By using PCR, we amplified an oxygenase gene from pF199 that has been proposed to initiate phenanthrene degradation this strain. When this gene was used to probe the pulsed field gels, there was hybridization to pF199 but not to the plasmids in strains Sg5 or 12H6.
Impacts
PAH are widespread pollutants sorbed by soil organic matter for which bioremediation may be a cost effective solution. These experiments may help us identify organisms that are most useful for these applications.
Publications
- Vacca, D., E. Kim, W.F. Bleam and W.J. Hickey. 2001. Diversity of polynuclear aromatic hydrocarbon-degrading bacteria isolated from differing enrichment methods. In Agronomy abstracts (CD-ROM). ASA, Madison, WI.
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