Denaturing Gradient Gel Electrophoresis

Soil microbial forensics can be defined as the study of how microorganisms can be applied to forensic investigations. The field of soil microbial forensics is of increasing interest and applies techniques commonly used in diverse disciplines in order to identify microbes and determine their abundances, complexities, and interactions with soil and surrounding objects. Emerging new techniques are also providing insights into the complexity of microbes in soil. Soil may harbor unique microbes that may reflect specific physical and chemical characteristics indicating site specificity. While applications of some of these techniques in the field of soil microbial forensics are still in early stages, we are still gaining insight into how microorganisms may be more robustly used in forensic investigations.

Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious “house” flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.

About 145,000 metric tons of smear-ripened cheeses are made in Europe, mainly in France, Belgium, Austria, and Germany. Although relatively small in quantity compared with total European cheese production of almost 9 million metric tons, this amount is important because of the large number of varieties of smear cheeses made, each with its own distinctive flavor, e.g., Port Salut, Pont-l’Évêque, Munster, Vacherin Mont-d'Or, and Taleggio. Smear cheeses are characterized by the development of microbial consortia on the cheese surface early in ripening, composed of yeast and bacteria, which give some of the cheeses (the so-called red smear cheeses) their characteristic red or orange glistening color. This is a direct result of ripening the cheeses at relatively high temperatures (∼14°C) and high relative humidities (>95%), conditions which promote the growth of microorganisms present on the cheese surface. The red or orange color which develops on the surface of many smear cheeses during ripening is due to the production of pigments by the yeast and bacteria growing on the surface. The biochemical activity of the yeast and bacteria are mainly responsible for flavor development in these cheeses. Like every cheese made, smear cheeses also contain high numbers of starter and nonstarter lactic acid bacteria (LAB), but these are found throughout the cheese and are not considered further here.

This chapter talks about the development of culture-independent, molecular methods that have revolutionized the field and the understanding of molecular ecology. Through the use of these techniques, it is now apparent that the earlier culture-based studies were not a representative reflection of the dominant microorganisms in many psychrophilic habitats. Cyanobacteria present in Dry Valleys mineral soils are considered to be the major primary producers and contribute significantly to microbial diversity. Lithic communities are classified by the specific environmental niche they reside in, and hypoliths, chasmoliths, and cryptoendoliths are further discussed in this chapter. The majority of bacteria isolated from permafrost are aerobic and include a number of coryneforms, endospore formers, sulfate reducers, nitrifying and denitrifying bacteria, and cellulose degraders. The microbial mat bacterial diversity of 10 Dry Valleys lakes was assessed by culturing techniques (heterotrophic growth conditions and fatty acid analysis). Microbial mats from Markham and Ward Hunt Ice Shelves showed species homogeneity in the vertical profile, which has not been seen previously in Antarctic mats, possibly due to differences in mat thickness. The stratified Antarctic mats from the McMurdo Ice Shelf were up to 8 cm thick in places, while the Arctic mats in this study were approximately 2 cm. Using metagenomic methods researchers can assess the diversity of culturable and uncultured organisms, including rare taxa.

Polar environments can be among the simplest environments on Earth, making them excellent models to understand ecosystem processes and the responses of microorganisms to environmental perturbations. This vulnerability and the rate of the ongoing change make polar environments a priority in climate change and bioremediation studies. This chapter covers studies based on large-scale 16S rRNA gene libraries, environmental microarrays, large-insert clone libraries, and shotgun genome sequencing that are applying metagenomic techniques to characterize and understand polar ecosystems. A recent study at sites ranging from the Falkland Islands all the way to the base of the Antarctic Peninsula used 454 GS FLX Titanium sequencing of the 16S rRNA gene to compare the bacterial community structure and diversity in warmed versus control plots. This study revealed that soil warming induced significant shifts in the major soil bacterial groups like Acidobacteria and α-Proteobacteria, which led to changes in soil respiration. A recent shotgun metagenomic study in the Canadian High Arctic sequenced a permafrost sample and its overlying active-layer soil and focused particularly on genes that might be important for greenhouse gas emissions following permafrost thaw. The only metagenomic study of polar freshwater ecosystems published to date looked at the viral communities of an Antarctic lake. This study revealed that a large proportion of the viral sequences retrieved from the lake were from eukaryotic viruses and not from bacteriophages. Emerging technologies could also be interesting to apply to polar environments. For instance, bioremediation studies could benefit from metabolomics, proteomics, and newly developed reactome arrays.

The human microbiome includes bacteria, viruses, and small eukaryotes, such as fungi, and this chapter focuses on the bacterial members of the microbiome. The Human Microbiome Project (HMP) aims at developing tools and resources for characterization of the human microbiota and to relate this microbiota to human health and disease. The goals of the jumpstart phase have been to sequence 900 reference genomes to provide a catalog of genomes for metagenomic studies, to sample at least 300 healthy adults between 18 and 40 years of age at five body sites, and to develop sequencing and analysis protocols for the samples derived from human subjects. The second phase of the HMP includes human microbiome studies that target particular disease states. In a recent study, four phyla comprised 92.3% of bacterial DNA sequences analyzed from multiple human sources, including hair, oral cavity, skin, genitourinary, and gastrointestinal tract. A study by Pei et al. showed that the distal esophageal microbiomes of four adults had compositions similar to that of the oropharynx, with the exception that no spirochetes were found in the esophagus. The chapter concludes by highlighting that pathogen discovery efforts will be enhanced by new metagenomics strategies, and these studies may uncover single etiologic agents of infections as well as relative shifts in groups of bacterial pathogens that may contribute to human disease.

This chapter focuses on the microbial ecology of the gut, with emphasis on information gleaned from recent molecular studies. Most attention has been devoted to bacterial components of the gut microbiota and, thus, they are the focus of this chapter. The first metagenomic study of the human gut resulted in 78 million base pairs of DNA sequences from two American individuals. This study cataloged the combined gene complement of the microbiome, including functional genes. A section discusses some dramatic differences that have been observed in the gut microbiota of infants that are fed formula compared to infants that are breast-fed. An interesting example of the importance of host physiology in shaping the composition of the microbiota was shown in reciprocal transplantations of gut microbiota between mice and zebrafish. The chapter primarily discusses Crohn's disease (CD) because of the large number of recent reports that have focused on the correlation of the gut microbiota to this particular disease. Therefore, the increased production of butyrate resulted in greater host responses to colonization. Although this model gave some new insights into the complex ecology of the gut microbiota, it is yet unclear whether the interactions observed between Eubacterium rectale and Bacteroides thetaiotaomicron are representative of common interactions between Bacteroidetes and Firmicutes. The study of genetically matched twins and defined model systems are examples of approaches that have promise to help define diagnostic targets and disease biomarkers.

This chapter focuses on RNA-based stable isotope probing (RNA-SIP) that was developed to take advantage of the features of RNA that make it an excellent biomarker for linking environmental processes—rapid turnover rates independent of cellular replication, coupled to in-depth phylogenetic information within the molecule itself. The primary aim of an RNA extraction protocol for RNA-SIP is to generate over 1µg of quantifiable RNA. Nucleic acids appear in almost all gradient fractions as revealed by high-sensitivity methods for detecting them, such as reverse transcription PCR (RT-PCR) or PCR. The chapter also talks about the RNA-SIP- directed investigation that ultimately led to the isolation of a novel Thauera strain responsible for the observed phenol degradation and to confirmation that it could be used to revive sludge that had lost phenol-degrading activity. These findings changed one's understanding of the microbes responsible for phenol degradation in aerated sludge, revealed the pitfalls of both conventional culturing and basic molecular approaches, and highlighted the importance of methods linking function with phylogeny. Manefield used RNA-SIP to compare the communities dominating the assimilation of carbon from phenol in near-identical wastewater treatment bioreactors that were operated in the same manner but differed in wastewater treatment performance. This study revealed that Acidovorax species were responsible for phenol degradation and the poor performance of one reactor was associated with two different Acidovorax populations, while the strong performance of the other was associated with a single dominant Acidovorax lineage.

This chapter reviews the use of 18O-water in stable isotope probing (SIP) studies. Research groups made important contributions to understanding of adenosine triphosphate (ATP) production in mitochondria and bacteria. Richards and Boyer, employing 18O-water, showed that oxygen atoms from water can be transferred to DNA inside E. coli cells. Subsequently, it was shown that 18O-water may also be used to label DNA formed in soil. Water is a small molecule and therefore can rapidly diffuse throughout the soil environment, so that the label is relatively homogenously distributed throughout the sample and all soil organisms are exposed to similar concentrations of label. However, it is unlikely that the label will be completely homogenously distributed in soil. SIP with 18O-water may also be used to study the impact of environmental conditions such as temperature or pH on microbial population dynamics in soil. Finally, SIP with 18O-water is suitable for studies on the impact of complex nutrient sources on microbial population dynamics in soil. For instance, it is well known that plant litter quality, often gauged by measuring the lignin to nitrogen ratio, affects decomposition rates and therefore multiple nutrient cycles in soil.

This chapter summarizes the applications of stable isotope probing (SIP) technology in plant-soil systems and presents an overview of the progress achieved in the understanding of the plant-soil microbe interactions and their role in ecosystem functioning. The applications of phospholipid fatty acids-based SIP (PLFA-SIP) and then the applications of DNA- and RNA-based SIP in upland soils and flooded rice field soils, respectively, are described. Several studies have exploited PLFA-SIP technology to determine the plant-microbe interactions driven by rhizosphere carbon flow. In these studies, the living plants, either in the field or laboratory, are exposed to 13C-labeled CO2, and the microbial PLFAs are collected from rhizosphere soil. After pulse-labeling of rice plants with 13CO2 in a microcosm, soil samples were divided into rhizosphere and bulk soil, and the bulk soil samples were further partitioned vertically into upper layer and lower layer and horizontally into five layers with an increasing distance from roots. A study performed on grassland soil and on peatland soil, targeted mainly the root symbiont's arbuscular mycorrhizal (AM) fungi and the bacteria possibly associated with them. RNA-SIP revealed that AM fungi were labeled with 13C immediately after plant assimilation, suggesting that AM fungi preferentially used assimilates provided by plants rather than previously fixed carbon. Combining SIP with techniques such as metatranscriptomics, pyrosequencing, and community systems biology, promises a better and deeper understanding of plant-microbe interactions.