Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis.

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.

Relationships between sediment microbial communities and pollutants in two California salt marshes.

Salt marshes are important ecosystems whose plant and microbial communities can alter terrestrially derived pollutants prior to coastal water discharge. However, knowledge regarding relationships between anthropogenic pollutant levels and salt marsh microbial communities is limited, and salt marshes on the West Coast of the United States are rarely examined. In this study, we investigated the relationships between microbial community composition and 24 pollutants (20 metals and 4 organics) in two California salt marshes. Multivariate ordination techniques were used to assess how bacterial community composition, as determined by terminal restriction fragment length polymorphism and phospholipid fatty acid analyses, was related to pollution. Sea urchin embryo toxicity measurements and plant tissue metabolite profiles were considered two other biometrics of pollution. Spatial effects were strongly manifested across marshes and across channel elevations within marshes. Utilizing partial canonical correspondence analysis, an ordination technique new to microbial ecology, we found that several metals were strongly associated with microbial community composition after accounting for spatial effects. The major patterns in plant metabolite profiles were consistent with patterns across microbial community profiles, but sea urchin embryo assays, which are commonly used to evaluate ecological toxicity, had no identifiable relationships with pollution. Whereas salt marshes are generally dynamic and complex habitats, microbial communities in these marshes appear to be relatively sensitive indicators of toxic pollutants.

Comparison of free-living and particle-associated bacterial communities in a coastal lagoon.

We analyzed, by terminal restriction fragment length polymorphisms (T-RFLPs) of PCR-amplified 16S rDNA, microbial diversity in water collected during the dry and wet seasons in a human-impacted coastal lagoon. Water samples were fractionated by prefiltration to differentiate particle-associated and free-living microbes. From a sample collected during the dry season, prefiltration removed 23 to 44% of bacteria, as assessed by direct counts and MPN, and 99% of phytoplankton, as assessed by chlorophyll a. Restriction with RsaI yielded fewer peaks than restriction with HhaI. Diversity indices calculated from T-RFLPs were higher in the lagoon than adjoining coastal waters and higher in the particle-associated than the free-living fraction. In the dry season, peaks found only in bulk and particle-associated T-RFLPs were consistent with plastid and cyanobacterial ribotypes. These peaks matched those observed in the sequence of a clone generated from the bulk fraction with plastid and cyanobacterial specific primers. This clone appeared related to plastids found in the diatom genus Skeletonema. Principal component analysis of T-RFLPs suggested that the difference between the free-living and particle-associated fractions in the dry season was less than temporal variability in this lagoon and that these fractions varied significantly only in the wet season. This fractionation of microbial populations into particle-associated and free-living guilds during the wet season, when water residence time in the lagoon is relatively low, suggests an external source of particle-associated bacteria such as erosion of upland soils by runoff.

Comparison of subsurface and surface soil bacterial communities in California grassland as assessed by terminal restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes.

The integrated biomass beneath the surface horizon in unsaturated soils is large and potentially important in nutrient and carbon cycling. Compared to surface soils, the ecology of these subsurface soils is weakly understood, particularly in terms of the composition of bacterial communities. We compared soil bacterial communities along two vertical transects by terminal restriction fragment length polymorphisms (TRFLPs) of PCR-amplified 16S rRNA genes to determine how surface and deep bacterial communities differ. DNA yield from soils collected from two Mediterranean grassland transects decreased exponentially from the surface to 4 m deep. Richness, as assessed by the number of peaks obtained after restriction with HhaI, MspI, RsaI, or HaeIII, and diversity, as assessed by the Shannon diversity indices, were lowest in the deepest sample. Lower diversity at depth is consistent with species-energy theory, which would predict relatively low diversity in the low organic matter horizons. Principal components analysis suggested that, in terms of HhaI and HaeIII generated TRFLPs, bacterial communities differed between depths. The most abundant amplicons cloned from the deepest sample contained sequences with restriction sites consistent with the largest peaks observed in TRFLPs generated from deep samples. These more abundant operational taxonomic units (OTUs) appeared related to Pseudomonas and Variovorax. Several OTUs were more related to each other than any previously described ribotypes. These OTUs showed similarity to bacteria from the divisions Actinobacteria and Firmicutes.

Identification and analysis of PCB dechlorinating anaerobic enrichments by amplification: accuracy of community structure based on restriction analysis and partial sequencing of 16S rRNA genes.

The composition of polychlorinated biphenyl (PCB) dechlorinating mixed communities was analysed by restriction fragment length polymorphism of PCR amplified rDNAs (ARDRA) and partial sequencing of 16S rRNA genes amplified from PCB degrading enrichments. Restriction analysis confirms that the 16S rRNA genes amplified from PCB dechlorinating communities vary depending on the PCB congener dechlorinated. Comparison of 16S rRNA sequences to published ribosomal databases indicates that the two most abundant Operational Taxonomic Units (OTUs) appear to be species of the genus Clostridium. The amount that the amplification procedure contributed to this result was determined by varying the amplification procedure and by creating an artificial template mixture. Varying the amount of template by sixfold in the amplification did not affect the distribution of OTUs but the number of OTUs observed decreased with decreasing template concentration. Comparison of products amplified from mixtures of 16S rDNA clones indicates that the more abundant Clostridium OTU did not amplify more efficiently than those of less abundant OTUs. Hybridization to a probe designed to detect the most abundant OTUs indicates that two other OTUs are closely related to this Clostridium species.

Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand.

Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils. Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by Pseudomonas aeruginosa, can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown. In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane. Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the gfp reporter gene either to the promoter region of pra, which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator rhlR. We assessed green fluorescent protein (GFP) production conferred by these gene fusions in P. aeruginosa PG201. GFP was produced in sand culture, indicating that the rhlR and pra genes are both transcribed in unsaturated porous media. Confocal laser scanning microscopy of liquid drops revealed that gfp expression was localized at the hexadecane-water interface. Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to P. aeruginosa biodegrading hexadecane in sand. Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production. Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms. Our findings suggest that P. aeruginosa likely produces surface-active compounds in sand culture. However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells. In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.