A systematic evaluation of the value and potential of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure has been undertaken. The reproducibility and robustness of the method has been assessed using environmental DNA samples isolated directly from PCB-polluted or pristine soil, and subsequent polymerase chain reaction (PCR) amplification of total community 16S rDNA. An initial investigation to assess the variability both within and between different polyacrylamide gel electrophoresis (PAGE) runs showed that almost identical community profiles were consistently produced from the same sample. Similarly, very little variability was observed as a result of variation between replicate restriction digestions, PCR amplifications or between replicate DNA isolations. Decreasing concentrations of template DNA produced a decline in both the complexity and the intensity of fragments present in the community profile, with no additional fragments detected in the higher dilutions that were not already present when more original template DNA was used. Reducing the number of cycles of PCR produced similar results. The greatest variation between profiles generated from the same DNA sample was produced using different Taq DNA polymerases, while lower levels of variability were found between PCR products that had been produced using different annealing temperatures. Incomplete digestion by the restriction enzyme may, as a result of the generation of partially digested fragments, lead to an overestimation of the overall diversity within a community. The results obtained indicate that, once standardized, T-RFLP analysis is a highly reproducible and robust technique that yields high-quality fingerprints consisting of fragments of precise sizes, which, in principle, could be phylogenetically assigned, once an appropriate database is constructed.

An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics

Phytoremediation is a promising technology for the cleanup of polluted environments. The technology has so far been used mainly to remove toxic heavy metals from contaminated soil, but there is a growing interest in broadening its applications to remove/degrade organic pollutants in the environment. Both plants and soil microorganisms have certain limitations with respect to their individual abilities to remove/breakdown organic compounds. A synergistic action by both rhizosphere microorganisms that leads to increased availability of hydrophobic compounds, and plants that leads to their removal and/or degradation, may overcome many of the limitations, and thus provide a useful basis for enhancing remediation of contaminated environments. The review of literature presented in this article provides an insight to the nature of plant-microbial interactions in the rhizosphere, with a focus on those processes that are relevant to the breakdown and/or removal of organic pollutants. Due consideration has been given to identify opportunities for utilising the plant-microbial synergy in the rhizosphere to enhance remediation of contaminated environments. The literature review has highlighted the existence of a synergistic interaction between plants and microbial communities in the rhizosphere. This interaction benefits both microorganisms through provision of nutrients by root exudates, and plants through enhanced nutrient uptake and reduced toxicity of soil contaminants. The ability of the plant-microbial interaction to tackle some of the most recalcitrant organic chemicals is of particular interest with regard to enhancing and extending the scope of remediation technologies. Plant-microbial interactions in the rhizosphere offer very useful means for remediating environments contaminated with recalcitrant organic compounds. A better knowledge of plant-microbial interactions will provide a basis for improving the efficacy of biological remediations. Further research is, however, needed to investigate different feedback mechanisms that select and regulate microbial activity in the rhizosphere.

Utilising the Synergy between Plants and Rhizosphere Microorganisms to Enhance Breakdown of Organic Pollutants in the Environment (15 pp)

Monitoring complex bacterial communities using culture-independent molecular techniques: application to soil environment.

http://www.people.vcu.edu/%7erbfranklin/pdf%20of%20publications%20(make%20links%20to%20us)/Franklin%20&%20Mills%20(FEMS%202003).pdf

Multi-scale variation in spatial heterogeneity for microbial community structure in an eastern Virginia agricultural field.

This review summarizes aspects of the current knowledge about the ecology of ammonia-oxidizing and denitrifying bacteria. The development of molecular techniques has contributed enormously to the rapid recent progress in the field. Different techniques for doing so are discussed. The characterization of ammonia-oxidizing and -denitrifying bacteria by sequencing the genes encoding 16S rRNA and functional proteins opened the possibility of constructing specific probes. It is now possible to monitor the occurrence of a particular species of these bacteria in any habitat and to get an estimate of the relative abundance of different types, even if they are not culturable as yet. These data indicate that the composition of nitrifying and denitrifying communities is complex and apparently subject to large fluctuations, both in time and in space. More attempts are needed to enrich and isolate those bacteria which dominate the processes, and to characterize them by a combination of physiological, biochemical and molecular techniques. While PCR and probing with nucleotides or antibodies are primarily used to study the structure of nitrifying and denitrifying communities, studies of their function in natural habitats, which require quantification at the transcriptional level, are currently not possible.

https://www.io-warnemuende.de/tl_files/bio/ag-umweltmikrobio/pdf/BotheWard_2000.pdf

Molecular analysis of ammonia oxidation and denitrification in natural environments

DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types

In Sweden, human cases of tularemia caused by Francisella tularensis holarctica are assumed to be transmitted by mosquitoes, but how mosquito vectors acquire and transmit the bacterium is not clear. To determine how transmission of this bacterium occurs, mosquito larvae were collected in an area where tularemia is endemic, brought to the laboratory, and reared to adults in their original pond water. Screening of adult mosquitoes by real-time PCR demonstrated F. tularensis lpnA sequences in 14 of the 48 mosquito pools tested; lpnA sequences were demonstrated in 6 of 9 identifi ed mosquito species. Further analysis confi rmed the presence of F. tularensis holarctica–specifi c 30-bp deletion region sequences (FtM19inDel) in water from breeding containers and in 3 mosquito species (Aedes sticticus, Ae. vexans, and Ae. punctor) known to take blood from humans. Our results suggest that the mosquitoes that transmit F. tularensis holarctica during tularemia outbreaks acquire the bacterium already as larvae.

/pdf/transstadial-transmission-of-francisella-tularensis-2qvcyhmwwl.pdf

Transstadial Transmission of Francisella tularensis holarctica in Mosquitoes, Sweden

Biomonitoring complex microbial communities using random amplified polymorphic DNA and principal component analysis

During the last two decades major changes of the benthic fauna have occurred in the northern Baltic Sea, the Gulf of Bothnia. The native amphipod, Monoporeia affinis, has shown a large-scale abundance decrease, while polychaetes, Marenzelleria spp. have invaded the system. Marenzelleria co-exist with the native fauna in the southern Baltic Sea, but in the north the pelagic production might be too low to allow co-existence. Thus, M. affinis might have been out-competed by Marenzelleria in the Gulf of Bothnia. This hypothesis was tested in a competition experiment with a high and a low fresh phytoplankton food supply. When exposed to high food supply both species showed stable or increased biomass over the four week test period. In low food supply, however, M. affinis was found to have a competitive advantage. The experimental data were also related to Baltic Sea monitoring data on primary production, sedimentation and invertebrate abundances. Data from the northern Baltic Sea show that the dominance in the benthic community by M. affinis was replaced by Marenzelleria around 2001. The amphipod decrease might be explained by a marked decrease in primary production during this period. Combining monitoring and experimental data suggests that the invasion of Marenzelleria did not cause the decrease of M. affinis in the northern Baltic Sea; it rather took advantage of the density gap that had occurred. A shift may thus have been established in the Bothnian Sea benthic community.

Ann-Christin Andersson

Papers

DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types

Transstadial Transmission of Francisella tularensis holarctica in Mosquitoes, Sweden

Biomonitoring complex microbial communities using random amplified polymorphic DNA and principal component analysis

Benthic competition and population dynamics of Monoporeia affinis and Marenzelleria sp in the northern Baltic Sea