Radomir Schmidt

Whole-Genome Analysis of the Methyl tert-Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1.

Methylibium petroleiphilum PM1 is a methylotroph distinguished by its ability to completely metabolize the fuel oxygenate methyl tert-butyl ether (MTBE). Strain PM1 also degrades aromatic (benzene, toluene, and xylene) and straight-chain (C(5) to C(12)) hydrocarbons present in petroleum products. Whole-genome analysis of PM1 revealed an approximately 4-Mb circular chromosome and an approximately 600-kb megaplasmid, containing 3,831 and 646 genes, respectively. Aromatic hydrocarbon and alkane degradation, metal resistance, and methylotrophy are encoded on the chromosome. The megaplasmid contains an unusual t-RNA island, numerous insertion sequences, and large repeated elements, including a 40-kb region also present on the chromosome and a 29-kb tandem repeat encoding phosphonate transport and cobalamin biosynthesis. The megaplasmid also codes for alkane degradation and was shown to play an essential role in MTBE degradation through plasmid-curing experiments. Discrepancies between the insertion sequence element distribution patterns, the distributions of best BLASTP hits among major phylogenetic groups, and the G+C contents of the chromosome (69.2%) and plasmid (66%), together with comparative genome hybridization experiments, suggest that the plasmid was recently acquired and apparently carries the genetic information responsible for PM1s ability to degrade MTBE. Comparative genomic hybridization analysis with two PM1-like MTBE-degrading environmental isolates (approximately 99% identical 16S rRNA gene sequences) showed that the plasmid was highly conserved (ca. 99% identical), whereas the chromosomes were too diverse to conduct resequencing analysis. PM1s genome sequence provides a foundation for investigating MTBE biodegradation and exploring the genetic regulation of multiple biodegradation pathways in M. petroleiphilum and other MTBE-degrading beta-proteobacteria.

Comparative Transcriptome Analysis of Methylibium petroleiphilum PM1 Exposed to the Fuel Oxygenates Methyl tert-Butyl Ether and Ethanol

ABSTRACT High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.

Autophosphorylation and dephosphorylation by soluble forms of the nitrate-responsive sensors NarX and NarQ from Escherichia coli K-12.

NarX-NarL and NarQ-NarP are paralogous two-component regulatory systems that control Escherichia coli gene expression in response to the respiratory oxidants nitrate and nitrite. Nitrate stimulates the autophosphorylation rates of the NarX and NarQ sensors, which then phosphorylate the response regulators NarL and NarP to activate and repress target operon transcription. Here, we investigated both the autophosphorylation and dephosphorylation of soluble sensors in which the maltose binding protein (MBP) has replaced the amino-terminal transmembrane sensory domain. The apparent affinities (K(m)) for ADP were similar for both proteins, about 2 microM, whereas the affinity of MBP-NarQ for ATP was lower, about 23 microM. At a saturating concentration of ATP, the rate constant of MBP-NarX autophosphorylation (about 0.5 x 10(-4) s(-1)) was lower than that observed for MBP-NarQ (about 2.2 x 10(-4) s(-1)). At a saturating concentration of ADP, the rate constant of dephosphorylation was higher than that of autophosphorylation, about 0.03 s(-1) for MBP-NarX and about 0.01 s(-1) for MBP-NarQ. For other studied sensors, the published affinities for ADP range from about 16 microM (KinA) to about 40 microM (NtrB). This suggests that only a small proportion of NarX and NarQ remain phosphorylated in the absence of nitrate, resulting in efficient response regulator dephosphorylation by the remaining unphosphorylated sensors.

Involvement of a novel enzyme, MdpA, in methyl tert-butyl ether degradation in Methylibium petroleiphilum PM1.

ABSTRACT 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.

Selenium Biotransformations in an Engineered Aquatic Ecosystem for Bioremediation of Agricultural Wastewater via Brine Shrimp Production

An engineered aquatic ecosystem was specifically designed to bioremediate selenium (Se), occurring as oxidized inorganic selenate from hypersalinized agricultural drainage water while producing brine shrimp enriched in organic Se and omega-3 and omega-6 fatty acids for use in value added nutraceutical food supplements. Selenate was successfully bioremediated by microalgal metabolism into organic Se (seleno-amino acids) and partially removed via gaseous volatile Se formation. Furthermore, filter-feeding brine shrimp that accumulated this organic Se were removed by net harvest. Thriving in this engineered pond system, brine shrimp ( Artemia franciscana Kellogg) and brine fly (Ephydridae sp.) have major ecological relevance as important food sources for large populations of waterfowl, breeding, and migratory shore birds. This aquatic ecosystem was an ideal model for study because it mimics trophic interactions in a Se polluted wetland. Inorganic selenate in drainage water was metabolized differently in microalgae, bacteria, and diatoms where it was accumulated and reduced into various inorganic forms (selenite, selenide, or elemental Se) or partially incorporated into organic Se mainly as selenomethionine. Brine shrimp and brine fly larva then bioaccumulated Se from ingesting aquatic microorganisms and further metabolized Se predominately into organic Se forms. Importantly, adult brine flies, which hatched from aquatic larva, bioaccumulated the highest Se concentrations of all organisms tested.

Gene mdpC plays a regulatory role in the methyl-tert-butyl ether degradation pathway of Methylibium petroleiphilum strain PM1

Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC(-). We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA.

Microbial biosafety of pilot-scale bioreactor treating MTBE and TBA-contaminated drinking water supply

A pilot-scale sand-based fluidized bed bioreactor (FBBR) was utilized to treat both methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) from a contaminated aquifer. To evaluate the potential for re-use of the treated water, we tested for a panel of water quality indicator microorganisms and potential waterborne pathogens including total coliforms, Escherichia coli, Salmonella and Shigella spp., Campylobacter jejuni, Aeromonas hydrophila, Legionella pneumophila, Vibrio cholerae, Yersinia enterocolytica and Mycobacterium avium in both influent and treated waters from the bioreactor. Total bacteria decreased during FBBR treatment. E. coli, Salmonella and Shigella spp., C. jejuni, V. cholerae, Y. enterocolytica and M. avium were not detected in aquifer water or bioreactor treated water samples. For those pathogens detected, including total coliforms, L. pneumophila and A. hydrophila, numbers were usually lower in treated water than influent samples, suggesting removal during treatment. The detection of particular bacterial species reflected their presence or absence in the influent waters.

Long-term use of cover crops and no-till shift soil microbial community life strategies in agricultural soil

Reducing tillage and growing cover crops, widely recommended practices for boosting soil health, have major impacts on soil communities. Surprisingly little is known about their impacts on soil microbial functional diversity, and especially so in irrigated Mediterranean ecosystems. In long-term experimental plots at the West Side Research and Extension Center in California’s Central Valley, we characterized soil microbial communities in the presence or absence of physical disturbance due to tillage, in the presence or absence of cover crops, and at three depths: 0–5, 5–15 and 15–30 cm. This characterization included qPCR for bacterial and archaeal abundances, DNA sequencing of the 16S rRNA gene, and phylogenetic estimation of two ecologically important microbial traits (rRNA gene copy number and genome size). Total (bacterial + archaeal) diversity was higher in no-till than standard till; diversity increased with depth in no-till but decreased with depth in standard till. Total bacterial numbers were higher in cover cropped plots at all depths, while no-till treatments showed higher numbers in 0–5 cm but lower numbers at lower depths compared to standard tillage. Trait estimates suggested that different farming practices and depths favored distinctly different microbial life strategies. Tillage in the absence of cover crops shifted microbial communities towards fast growing competitors, while no-till shifted them toward slow growing stress tolerators. Across all treatment combinations, increasing depth resulted in a shift towards stress tolerators. Cover crops shifted the communities towards ruderals–organisms with wider metabolic capacities and moderate rates of growth. Overall, our results are consistent with decreasing nutrient availability with soil depth and under no-till treatments, bursts of nutrient availability and niche homogenization under standard tillage, and increases in C supply and variety provided by cover crops. Understanding how agricultural practices shift microbial abundance, diversity and life strategies, such as presented here, can assist with designing farming systems that can support high yields, while enhancing C sequestration and increasing resilience to climate change.