Susan M. Barns

Empirical and Theoretical Bacterial Diversity in Four Arizona Soils

ABSTRACT Understanding patterns of biodiversity in microbial communities is severely constrained by the difficulty of adequately sampling these complex systems. We illustrate the problem with empirical data from small surveys (200-member 16S rRNA gene clone libraries) of four bacterial soil communities from two locations in Arizona. Among the four surveys, nearly 500 species-level groups (Dunbar et al., Appl. Environ. Microbiol.65:662-1669, 1999) and 21 bacterial divisions were documented, including four new candidate divisions provisionally designated SC1, SC2, SC3, and SC4. We devised a simple approach to constructing theoretical null models of bacterial species abundance. These null models provide, for the first time, detailed descriptions of soil bacterial community structure that can be used to guide experimental design. Models based on a lognormal distribution were consistent with the observed sizes of the four communities and the richness of the clone surveys. Predictions from the models showed that the species richness of small surveys from complex communities is reproducible, whereas the species composition is not. By using the models, we can now estimate the required survey scale to document specified fractions of community diversity. For example, documentation of half the species in each model community would require surveys of 16,284 to 44,000 individuals. However, quantitative comparisons of half the species in two communities would require surveys at least 10-fold larger for each community.

Comparison of Soil Bacterial Communities in Rhizospheres of Three Plant Species and the Interspaces in an Arid Grassland

ABSTRACT Soil bacteria are important contributors to primary productivity and nutrient cycling in arid land ecosystems, and their populations may be greatly affected by changes in environmental conditions. In parallel studies, the composition of the total bacterial community and of members of the Acidobacterium division were assessed in arid grassland soils using terminal restriction fragment length polymorphism (TRF, also known as T-RFLP) analysis of 16S rRNA genes amplified from soil DNA. Bacterial communities associated with the rhizospheres of the native bunchgrasses Stipa hymenoides and Hilaria jamesii, the invading annual grass Bromus tectorum, and the interspaces colonized by cyanobacterial soil crusts were compared at three depths. When used in a replicated field-scale study, TRF analysis was useful for identifying broad-scale, consistent differences in the bacterial communities in different soil locations, over the natural microscale heterogeneity of the soil. The compositions of the total bacterial community and Acidobacterium division in the soil crust interspaces were significantly different from those of the plant rhizospheres. Major differences were also observed in the rhizospheres of the three plant species and were most apparent with analysis of the Acidobacterium division. The total bacterial community and the Acidobacterium division bacteria were affected by soil depth in both the interspaces and plant rhizospheres. This study provides a baseline for monitoring bacterial community structure and dynamics with changes in plant cover and environmental conditions in the arid grasslands.

Acidobacteria Phylum Sequences in Uranium-Contaminated Subsurface Sediments Greatly Expand the Known Diversity within the Phylum

ABSTRACT The abundance and composition of bacteria of the phylum Acidobacteria were surveyed in subsurface sediments from uranium-contaminated sites using amplification of 16S rRNA genes followed by clone/sequence analysis. Analysis of sequences from this study and public databases produced a revised and greatly expanded phylogeny of the Acidobacteria phylum consisting of 26 subgroups.

Detection of Diverse New Francisella-Like Bacteria in Environmental Samples

ABSTRACT Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.

Changes in nitrogen-fixing and ammonia-oxidizing bacterial communities in soil of a mixed conifer forest after wildfire.

ABSTRACT This study was undertaken to examine the effects of forest fire on two important groups of N-cycling bacteria in soil, the nitrogen-fixing and ammonia-oxidizing bacteria. Sequence and terminal restriction fragment length polymorphism (T-RFLP) analysis of nifH and amoA PCR amplicons was performed on DNA samples from unburned, moderately burned, and severely burned soils of a mixed conifer forest. PCR results indicated that the soil biomass and proportion of nitrogen-fixing and ammonia-oxidizing species was less in soil from the fire-impacted sites than from the unburned sites. The number of dominant nifH sequence types was greater in fire-impacted soils, and nifH sequences that were most closely related to those from the spore-forming taxa Clostridium and Paenibacillus were more abundant in the burned soils. In T-RFLP patterns of the ammonia-oxidizing community, terminal restriction fragments (TRFs) representing amoA cluster 1, 2, or 4 Nitrosospira spp. were dominant (80 to 90%) in unburned soils, while TRFs representing amoA cluster 3A Nitrosospira spp. dominated (65 to 95%) in fire-impacted soils. The dominance of amoA cluster 3A Nitrosospira spp. sequence types was positively correlated with soil pH (5.6 to 7.5) and NH3-N levels (0.002 to 0.976 ppm), both of which were higher in burned soils. The decreased microbial biomass and shift in nitrogen-fixing and ammonia-oxidizing communities were still evident in fire-impacted soils collected 14 months after the fire.

Comparative diversity and composition of cyanobacteria in three predominant soil crusts of the Colorado Plateau

Terminal restriction fragment length polymorphism (TRF or T-RFLP) analysis and 16S rDNA sequence analysis from clone libraries were used to examine cyanobacterial diversity in three types of predominant soil crusts in an arid grassland. Total DNA was extracted from cyanobacteria-, lichen-, or moss-dominated crusts that represent different successional stages in crust development, and which contribute different amounts of carbon and nitrogen into the ecosystem. Cyanobacterial 16S rRNA genes were amplified by PCR using cyanobacteria-specific 16S rDNA primers. Both TRF and clone sequence analyses indicated that the cyanobacterial crust type is dominated by strains of Microcoleus vaginatus, but also contains other cyanobacterial genera. In the moss crust, M. vaginatus-related sequences were also the most abundant types, together with sequences from moss chloroplasts. In contrast, sequences obtained from the lichen crust were surprisingly diverse, representing numerous genera, but including only two from M. vaginatus relatives. By obtaining clone sequence information, we were able to infer the composition of many peaks observed in TRF profiles, and all peaks predicted for clone sequences were observed in TRF analysis. This study provides the first TRF analysis of biological soil crusts and the first DNA-based comparison of cyanobacterial diversity between lichen-, cyano- and moss-dominated crusts. Results indicate that for this phylogenetic group, TRF analysis, in conjunction with limited sequence analysis, can provide accurate information about the composition and relative abundance of cyanobacterial types in soil crust communities.

Phylogeny, ribosomal RNA gene typing and relative abundance of new Pseudomonas species (sensu stricto) isolated from two pinyon-juniper woodland soils of the arid southwest U.S.

Rhizosphere-inhabiting Pseudomonas species interact with plant roots and may be important for plant performance under stressful environmental conditions. A comparison was conducted of culturable Pseudomonas isolates associated with pinyon rhizosphere and between-tree interspace areas in a hot, dry, volcanic cinder field and an adjacent sandy loam soil, in order to identify Pseudomonas species which may be involved in pinyon pine survival under stressful conditions. From a collection of 800 isolates, eleven isolates exhibiting different colony morphology were selected for 16S ribosomal RNA gene sequencing. Phylogenetic analysis of rDNA sequences from the eleven field isolates, forty-six described Pseudomonas species, and thirty-four previously characterized environmental isolates indicated that the isolates from the cinders and sandy loam soil clustered into three groups. The field isolates were distinct from any of the named species or other environmental isolates. Oligonucleotide primer pairs that differentiated three field isolate groups were designed from the 16S rDNA sequences, and eight hundred Pseudomonas field isolates cultured from pinyon rhizospheres and interspaces in the cinders and sandy loam soils were typed into the three groups using PCR assays. The composition of Pseudomonas populations in four environments was significantly different. The relative abundance of the three rDNA-based groups appeared to be affected by both the soil type and the pinyon rhizosphere.