Deborah T. Newby

Comparison of 2,4-Dichlorophenoxyacetic Acid Degradation and Plasmid Transfer in Soil Resulting from Bioaugmentation with Two Different pJP4 Donors

ABSTRACT A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection,Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. colistrain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of theBurkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.

Estimates of biogenic methane production rates in deep marine sediments at Hydrate Ridge, Cascadia Margin

ABSTRACT Methane hydrate found in marine sediments is thought to contain gigaton quantities of methane and is considered an important potential fuel source and climate-forcing agent. Much of the methane in hydrates is biogenic, so models that predict the presence and distribution of hydrates require accurate rates of in situ methanogenesis. We estimated the in situ methanogenesis rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor, Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. Quantitative PCR (QPCR) directed at the methyl coenzyme M reductase subunit A gene (mcrA) indicated that 75% of the HR sediments analyzed contained <1,000 methanogens/g. The highest numbers of methanogens were found mostly from sediments <10 m below seafloor. By considering methanogenesis rates for starved methanogens (adjusted to account for in situ temperatures) and the numbers of methanogens at selected depths, we derived an upper estimate of <4.25 fmol methane produced/g sediment/day for the samples with fewer methanogens than the QPCR method could detect. The actual rates could vary depending on the real number of methanogens and various seafloor parameters that influence microbial activity. However, our calculated rate is lower than rates previously reported for such sediments and close to the rate derived using geochemical modeling of the sediments. These data will help to improve models that predict microbial gas generation in marine sediments and determine the potential influence of this source of methane on the global carbon cycle.

Detection and Characterization of Plasmid pJP4 Transfer to Indigenous Soil Bacteria

ABSTRACT Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism,Escherichia coli ATCC 15224, by plate mating withRalstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 107 and 108 transconjugants g of dry soil−1 for samples supplemented with 500 and 1,000 μg of 2,4-D g of dry soil−1, respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genusBurkholderia or the genus Pseudomonas. No mercury-resistant, 2,4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.

The role of selective pressure and selfish DNA in horizontal gene transfer and soil microbial community adaptation

Abstract Recent advances in genome sequencing and horizontal gene transfer in soil have led to new insights on soil microbial community adaptation. In this review, we document and evaluate the role of selective pressure and selfish DNA in propagating horizontal gene transfer in soil through the use of a model system involving the organic pesticide 2,4-dichlorophenoxyacetic acid and the metal cadmium. This review provides a theoretical framework for microbial adaptation, wherein it is the selfish nature of DNA that provides the initial stimulus for adaptation rather than the host cells themselves. Subsequent to selfish DNA transfer, if useful to host cells, the transferred DNA may become integrated into the host chromosome. Following these events, ultimately the growth of more fit individuals within the newly created ecological niche allows for adaptation of the soil microbial community.

Soil: A Public Health Threat or Savior?

Soil is the most complicated biomaterial on the planet due to complex soil architecture and billions of soil microbes with extreme biotic diversity. Soil is potentially a source of human pathogens, which can be defined as geo-indigenous, geo-transportable, or geo-treatable. Such pathogens cumulatively can and do result in multiple human fatalities annually. A striking example is Helminths, with current infections worldwide estimated to be around two billion. However, soil can also be a source of antibiotics and other natural products that enhance human health. Soilborne antibiotics are used to treat human infections, but can also result in antibiotic-resistant bacteria. Natural products isolated from soil resulted in 60% of new cancer drugs between the period 1983–1994. Soils are also crucial to human health through their impact on human nutrition. Finally, from a global perspective, soils are vital to the future well-being of nations through their impact on climate change and global warming. A critical review of soil with respect to public health leads to the conclusion that overall soil is a public health savior. The value of soil using a systems approach is estimated to be

Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture: Bioaugmentation effects on soil microorganisms

20 trillion, and is by far the most valuable ecosystem in the world.

Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1000 μg 3-chlorobenzoate g-1 dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraderswere enumerated on minimal salts media containing 3-chlorobenzoate. Culturableheterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1000 μg 3-chlorobenzoate g-1 dry soil inoculated microcosms, degradersincreased from the initial inoculum and decreased following degradation of 3-CB.Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 × 107 colony-forming-units g-1 dry soil in the 500 μg 3-chlorobenzoate g-1 dry soil microcosms, but none were detected in the 1000 μg 3-chlorobenzoate g-1 dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species.In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1000 μg 3-chlorobenzoate g-1 dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.

Dispersal of plasmid pJP4 in unsaturated and saturated 2,4-dichlorophenoxyacetic acid contaminated soil

The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.

Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100‐bp insertion in helix 6

Little is known regarding plasmid fate within contaminated soils. Column studies were used to evaluate dissemination of plasmid pJP4 under unsaturated or saturated flow conditions in a 2,4-dichlorophenoxyacetic acid (2,4-D) contaminated soil. Columns were destructively sampled following 1 week of percolation to assess the vertical distribution of donors, transconjugants, and 2,4-D concentrations within the soil. In unsaturated soil, pJP4 was detected in both culturable donor and transconjugant cells within soil 10.5 cm from the inoculated end of the column. In saturated soil, no transconjugants were detected; however, donors were found throughout the entire length of the column (30.5 cm). These results suggest that donor transport, particularly in conjunction with plasmid transfer to indigenous recipients, allows for significant dispersal of introduced genes through contaminated soil.

Nucleic Acid-Based Methods of Analysis

Two different versions of the 16S rRNA gene, one of which contained an unusual 100-bp insertion in helix 6, were detected in isolate UFO1 acquired from the Oak Ridge Integrated Field-Research Challenge (ORIFRC) site in Tennessee. rRNA was extracted from UFO1 and analyzed by reverse transcriptase-quantitative PCR with insert- and non-insert-specific primers; only the noninsert 16S rRNA gene sequence was detected. Similarly, PCR-based screening of a cDNA library (190 clones) constructed from reverse-transcribed rRNA from UFO1 did not detect any clones containing the 100-bp insert. Examination of cDNA with primers specific to the insert-bearing 16S rRNA gene, but downstream of the insert, suggests that the insert was excised from rRNA. Inspection of other 16S rRNA genes in the GenBank database revealed that a homologous insert sequence, also found in helix 6, has been reported in other environmental clones, including those acquired from ORIFRC enrichments. These findings demonstrate the existence of widely divergent copies of the 16S rRNA gene within the same organism, which may confound 16S rRNA gene-based methods of estimating microbial diversity in environmental samples.