Quantitative Detection of Perchlorate-Reducing Bacteria by Real-Time PCR Targeting the Perchlorate Reductase Gene

A quantitative real-time PCR assay targeting the pcrA gene, encoding the catalytic subunit of perchlorate reductase, detected pcrA genes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. Partial pcrA sequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors.     

Involvement of a Novel Enzyme, MdpA, in Methyl tert-Butyl Ether Degradation in Methylibium petroleiphilum PM1

Methylibium petroleiphilum PM1 is a well-characterized environmental strain capable of complete metabolism of the fuel oxygenate methyl tert-butyl ether (MTBE). Using a molecular genetic system which we established to study MTBE metabolism by PM1, we demonstrated that the enzyme MdpA is involved in MTBE removal, based on insertional inactivation and complementation studies. MdpA is constitutively expressed at low levels but is strongly induced by MTBE. MdpA is also involved in the regulation of tert-butyl alcohol (TBA) removal under certain conditions but is not directly responsible for TBA degradation. Phylogenetic comparison of MdpA to related enzymes indicates close homology to the short-chain hydrolyzing alkane hydroxylases (AH1), a group that appears to be a distinct subfamily of the AHs. The unique, substrate-size-determining residue Thr⁵⁹ distinguishes MdpA from the AH1 subfamily as well as from AlkB enzymes linked to MTBE degradation in Mycobacterium austroafricanum.     

Reduction of Perchlorate and Nitrate by Microbial Communities in Vadose Soil

Perchlorate contamination is a concern because of the increasing frequency of its detection in soils and groundwater and its presumed inhibitory effect on human thyroid hormone production. Although significant perchlorate contamination occurs in the vadose (unsaturated) zone, little is known about perchlorate biodegradation potential by indigenous microorganisms in these soils. We measured the effects of electron donor (acetate and hydrogen) and nitrate addition on perchlorate reduction rates and microbial community composition in microcosm incubations of vadose soil. Acetate and hydrogen addition enhanced perchlorate reduction, and a longer lag period was observed for hydrogen (41 days) than for acetate (14 days). Initially, nitrate suppressed perchlorate reduction, but once perchlorate started to be degraded, the process was stimulated by nitrate. Changes in the bacterial community composition were observed in microcosms enriched with perchlorate and either acetate or hydrogen. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes recovered from these microcosms indicated that formerly reported perchlorate-reducing bacteria were present in the soil and that microbial community compositions were different between acetate- and hydrogen-amended microcosms. These results indicate that there is potential for perchlorate bioremediation by native microbial communities in vadose soil.     

Naturally Occurring Bacteria Similar to the Methyl tert-Butyl Ether (MTBE)-Degrading Strain PM1 Are Present in MTBE-Contaminated Groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10³ to 10⁴ cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 μg of MTBE/liter, densities of native PM1 increased to approximately 10⁵ cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.     

Survival and growth of Salmonella Enteritidis PT 30 in almond orchard soils

Aims:  To evaluate factors potentially contributing to the long-term persistence of Salmonella enterica serovar Enteritidis phage type (PT) 30 in an almond orchard. Methods and Results:  Surface and subsurface soil temperatures, and air temperatures in a radiation shelter, were recorded during a 12-month period, and were used to identify relevant storage temperatures (20 or 35°C) for microcosms of two different soil types (clay and sandy loams) with moisture levels near saturation or near field capacity. Salmonella Enteritidis PT 30 was inoculated into the microcosms at 6 log CFU g⁻¹ dry weight. Between 14 and 180 days of incubation, counts of S. Enteritidis PT 30 decreased rapidly at 35°C and were significantly different (P < 0·05) from counts at 20°C, regardless of the soil type or moisture level. Salmonella was detected by enrichment of 10-g samples from all microcosms after 180 days of incubation at 20°C, but from none of the microcosms held at 35°C. To measure the potential for the growth of S. Enteritidis PT 30 in clay loam soil, an aqueous extract of almond hulls (containing 1·6% mono and disaccharides) or equivalent volume of water was added 7 days after inoculation. Significant (P < 0·05) growth of S. Enteritidis PT 30 was observed within 8 or 24 h of adding hull extract, but not water, to soil. Conclusions:  Opportunities may exist for S. Enteritidis PT 30 to survive for an extended time in almond orchard soils and to grow in these soils where hull nutrients are released. Significance and Impact of the Study:  Temperature has a significant impact on the long-term survival of S. Enteritidis PT 30 in soil, and nutrients leached from almond hulls may result in Salmonella growth. These factors should be considered in the design of Good Agricultural Practices for almonds.     

Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR

The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (C_T) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with Escherichia coli cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE.     

Naturally occurring bacteria similar to the methyl tert-butyl ether (MTBE)-degrading strain PM1 are present in MTBE-contaminated groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10(3) to 10(4) cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 microg of MTBE/liter, densities of native PM1 increased to approximately 10(5) cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.     

Estimation of methanotroph abundance in a freshwater lake sediment

The numbers of methane-oxidizing bacteria (methanotrophs) in the sediments of Lake Washington were estimated using three culture-independent methods. Quantitative slot-blot hybridizations were performed with type I and type II methanotroph-specific probes. These data were compared to data from quantitative hybridizations using a pmoA-specific probe and a eubacterial probe. From the combined hybridization data, the methanotroph population in Lake Washington was estimated to be 3.6 x 10(8)-7.4 x 10(8) cells/g dry weight. Methanotroph community structure and number were also investigated using polar lipid fatty acid (PLFA) analysis. Analysis of biomarker PLFAs characteristic of both type I (16:1 omega 8) and type II (18:1 omega 8) methanotrophs was used to estimate the abundance of these bacteria in Lake Washington sediments. From the PLFA data, the methanotroph population in Lake Washington was estimated to be 7.1 x 10(8)-9.4 x 10(9) cells/g dry weight. As a third method of quantitation, we calculated the methanotroph population using the total methane oxidation rate for whole cells in Lake Washington sediment to be 1.3 x 10(8)-1.2 x 10(9) cells/g dry weight. The three independent estimates of the number of methanotrophs in Lake Washington sediment agree within a two- to fourfold range. These data suggest that the three techniques used in this study detect the functionally significant population of methanotrophs in Lake Washington. Furthermore, these techniques will be useful for obtaining estimates of methanotroph abundance in additional environments.     

Measuring growth of a phenanthrene-degrading bacterial inoculum in soil with a quantitative competitive polymerase chain reaction method

We measured growth of a phenanthrene-degrading bacterium, Arthrobacter, strain RP17, in Forbes soil, amended with 500 μg g(-1) phenanthrene using a quantitative competitive polymerase chain reaction method. The inoculum, which was not indigenous to Forbes soil, grew from 5.55x10(5) colony forming units (cfu) g(-1) to 1.97x10(7) cfu g(-1) within 100 h after the cells were added to the soil. Maximum population density was reached before the highest degradation rate was observed 150 h after the cells were added to soil. Population density remained stable even after 56% of the phenanthrene had mineralized. This study is one of the few documented examples of growth by a non-indigenous bacterium in a non-sterile soil amended with a pollutant.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]