Timothy M. Vogel

Transformation of halogenated aliphatic compounds

This article summarizes and systematizes the current understanding of abiotic and biotic chemistry of halogenated aliphatic compounds. Knowledge of abiotic transformations can provide a conceptual framework for understanding biologically mediated transformations. Most abiotic transformations are slow, but they can still be significant within the time scales commonly associated with ground water movement. In contrast, biotic transformations typically proceed much faster, provided that there are sufficient substrate and nutrients and a microbial population that can mediate such transformation. Recent studies, which describe transformations of halogenated aliphatic compounds in microbial and mammalian systems, are also discussed. These studies reveal broad patterns of transformation in biological systems in general. 114 references, 8 figures, 12 tables.

Extraction of DNA from soil

There is an increased interest in the extraction of nucleic acids from various environmental samples, since molecular techniques allow less biased access to a greater portion of uncultivable microorganisms. Two strategies have been developed to improve DNA recovery in terms of yield, purity and unbiased representation of the microbial diversity. The first approach consists of the direct extraction of nucleic acids from soil through in situ cell lysis followed by DNA purification. The alternative approach is based on the separation of bacteria from the soil particles followed by cell lysis and then DNA purification. Several published methods describe the recovery of highly purified nucleic acids that are well-suited for molecular purposes even though a new challenge concerns the recovery of large bacterial DNAs essential for functional investigation of gene clusters and biosynthetic pathways. This review presents an overview of the available methods to achieve this challenging objective.

In Situ Transfer of Antibiotic Resistance Genes from Transgenic (Transplastomic) Tobacco Plants to Bacteria

ABSTRACT Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plants transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.

Modeling transport and biodegradation of benzene and toluene in sandy aquifer material: Comparisons With experimental measurements

A one-dimensional numerical model is developed for simulating the biodegradation and transport of benzene and toluene in the subsurface environment. Modeled processes include mass exchange between the constituent phases (solid, liquid, gas, and biomass), advective and dispersive transport, and biotransformation, as well as microbial biomass production. Two substrates, two electron acceptors, one trace nutrient, and two microbial populations are modeled. Resulting governing equations include five nonlinear partial differential equations describing component transport in the bulk pore fluids, five nonlinear algebraic equations governing interphase mass exchange, and two ordinary differential equations governing microbial growth. These equations are solved through application of a Galerkin finite element method and a set iterative solution scheme. The utility and validity of the modeling approach is explored through comparisons with laboratory column experiments. Model parameters were estimated independently through laboratory batch experiments, aquifer slurry studies, or from the literature. Simulations are found to provide reasonable agreement with measurements of benzene and toluene biodegradation in saturated continuous-flow columns packed with aquifer material. Sensitivity analyses and comparisons with column data suggest that model predictions are highly dependent upon the microbial parameters, particularly the initial active biomass concentration, the maximum specific substrate utilization rate, and the half-saturation coefficient. The importance of the accurate estimation of these microbial parameters is emphasized.

Accessing the soil metagenome for studies of microbial diversity.

ABSTRACT Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome.

Kinetics of aerobic biodegradation of benzene and toluene in sandy aquifer material

Monods equation adequately described aerobic biodegradation rates of benzene and toluene by the microbial population of a sandy aquifer when these compounds were initially present at concentrations lower than 100 mg/l each. Concentrations higher than 100 mg/l were inhibitory, and no benzene or toluene degradation was observed when these compounds were initially present at 250 mg/l each. The Monod coefficients were calculated as k = 8.3 g-benzene/g-cells/day and Ks = 12.2 mg/l for benzene, and k = 9.9 g-toluene/g-cells/day and Ks = 17.4 mg/l for toluene. Specific first-order coefficients would be 0.68 l/mg.day for benzene and 0.57 l.mg.day for toluene.

Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium.

Lindane-degrading activity under aerobic conditions has been observed in two bacterial strains: UT26, phenotypically identified as Sphingomonas paucimobilis, and a new single unidentified isolate named RP5557T. The rrs (16S rDNA) sequences for both strains and the phenotypic characteristics for the unidentified isolate RP5557T were determined. RP5557T does not have high identity (less than 90% in all cases) with any sequence in the GenBank or RDP databases. A phylogenetic analysis based on rrs sequences indicated that RP5557T belongs to the gamma-Proteobacteria in a coherent phylum that includes the genera Xanthomonas and Xylella (100% bootstrap), whereas UT26 is clearly separate from the Xanthomonas cluster. Based on the phylogenetic analyses and on the phenotypic characteristics, a new genus, Rhodanobacter, containing a single species, Rhodanobacter lindaniclasticus, is proposed for strain RP5557T (= LMG 18385T), which becomes the type strain.

Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones.

ABSTRACT The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.

Structure, fluctuation and magnitude of a natural grassland soil metagenome.

The soil ecosystem is critical for human health, affecting aspects of the environment from key agricultural and edaphic parameters to critical influence on climate change. Soil has more unknown biodiversity than any other ecosystem. We have applied diverse DNA extraction methods coupled with high throughput pyrosequencing to explore 4.88 × 109 bp of metagenomic sequence data from the longest continually studied soil environment (Park Grass experiment at Rothamsted Research in the UK). Results emphasize important DNA extraction biases and unexpectedly low seasonal and vertical soil metagenomic functional class variations. Clustering-based subsystems and carbohydrate metabolism had the largest quantity of annotated reads assigned although <50% of reads were assigned at an E value cutoff of 10−5. In addition, with the more detailed subsystems, cAMP signaling in bacteria (3.24±0.27% of the annotated reads) and the Ton and Tol transport systems (1.69±0.11%) were relatively highly represented. The most highly represented genome from the database was that for a Bradyrhizobium species. The metagenomic variance created by integrating natural and methodological fluctuations represents a global picture of the Rothamsted soil metagenome that can be used for specific questions and future inter-environmental metagenomic comparisons. However, only 1% of annotated sequences correspond to already sequenced genomes at 96% similarity and E values of <10−5, thus, considerable genomic reconstructions efforts still have to be performed.

Antibiotic-resistant soil bacteria in transgenic plant fields

Understanding the prevalence and polymorphism of antibiotic resistance genes in soil bacteria and their potential to be transferred horizontally is required to evaluate the likelihood and ecological (and possibly clinical) consequences of the transfer of these genes from transgenic plants to soil bacteria. In this study, we combined culture-dependent and -independent approaches to study the prevalence and diversity of bla genes in soil bacteria and the potential impact that a 10-successive-year culture of the transgenic Bt176 corn, which has a blaTEM marker gene, could have had on the soil bacterial community. The bla gene encoding resistance to ampicillin belongs to the beta-lactam antibiotic family, which is widely used in medicine but is readily compromised by bacterial antibiotic resistance. Our results indicate that soil bacteria are naturally resistant to a broad spectrum of beta-lactam antibiotics, including the third cephalosporin generation, which has a slightly stronger discriminating effect on soil isolates than other cephalosporins. These high resistance levels for a wide range of antibiotics are partly due to the polymorphism of bla genes, which occur frequently among soil bacteria. The blaTEM116 gene of the transgenic corn Bt176 investigated here is among those frequently found, thus reducing any risk of introducing a new bacterial resistance trait from the transgenic material. In addition, no significant differences were observed in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn fields, although the bacterial populations were different.