Document Type

Dissertation - University Access Only

Award Date

2011

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

Abstract

Growing interest in the field of microbial ecology is expanding our knowledge of microbial diversity. However, gathering of information is constrained by the techniques used to generate the data. A variety of techniques and tools have been developed to analyze microbial communities, but limitations exist for each method. A comprehensive examination of different techniques can help identify their respective strengths and weaknesses and determine which is most effective for particular studies. This dissertation focused on how the application of analytical techniques affects the representation of bacterial communities. Antibiotic growth promotants (AGP) have long been used to promote growth in farming animals, but the increasing problem of antibiotic resistance has prompted the development of alternative strategies. While the mechanisms behind growth promotants are still not well understood, much evidence suggests that effects of antibiotics on intestinal bacteria are a main component. Comparative analysis of 16S rRNA sequence data from the ilea of 12 chlortetracycline (CTC) treated and 12 control weanling pigs was performed using DOTUR, SONS, LIB SHUFF, and JMPIN. The identified bacterial community changes between treated and untreated pigs supports that bacterial community composition may play a role in the growth promotant effect, and presents some possible avenues for development of non-antibiotic growth promotion strategies. V Denaturing gradient gel electrophoresis (DGGE) is a popular tool for the analysis of microbial communities. The technique hinges on use of a GC-clamp attached to one of the primers to resolve the amplified communities into distinct banding profiles. Following the observation of differing amplification efficiencies between primer batches, the DGGE fingerprinting technique was interrogated to determine if different batches of equivalent GC-clamp primers could affect community profiles. Five sets of GC-clamp primers targeting the 16S rRNA V3-5 region were used to compare DGGE profiles generated from two soil bacterial communities and three bacterial pure cultures. The nucleotide sequences of the primers were also determined and an in-si/ico examination of their possible effects on melting temperature was performed. The DGGE profiles varied between primer batches of the same sequence as much or more than alternate primers for both the soil and pure culture DNA. Profiles generated from replicates of the same primer batch were confirmed to be more similar than those from different primer batches. Sequences of the GC-clamp portion of the primer deviated from the design and a greater error rate occurred with a poor Ge-clamp design. The use of a single batch of Ge-clamp primers and careful Ge-clamp design is recommended to decrease the effects of primer variation occurring within a project. Limitations from culture-based methods to represent bacterial communities have lead to a reliance on culture-independent methods to analyze microbial communities, usually based on ribosomal gene sequences. While DNA is popular target for sequence analysis it does not reflect cellular activity, leading to the use of rRNA for studies determining cellular activity. Stable isotope probing (SIP) techniques allow the determination of active microbial communities using both DNA and RNA, but have previously been limited to the study of particular groups of microorganisms. The introduction of H2 18O as a SIP substrate has provided a potential universal label for the study of entire microbial communities. Changes in soil bacterial community representation from different types of nucleic acids were determined through comparisons of total and H2 18O SIP-labeled DNA and RNA. The V3-5 16S rRNA nucleic acid pools were characterized through pyrosequencing and DGGE. Analysis revealed that total and SIP-labeled libraries of the same form of nucleic acid retained similar structures while DNA and RNA bacterial communities had substantial differences. The intrinsic bias of each form of sema:ntide was reflected in the recovered representations of bacterial community diversity and structure. Since the number of ribosomes present in bacteria has the potential for greater variation than the l 6S rRNA gene copy number, DNA semantides present less potential bias for bacterial community studies. The diversity of the SIP-labeled bacterial communities also suggested that H2 18O may prove an effective universal label for active bacterial communities. The historic limitations of culture-dependent methods have led to the current paradigm stating that only a tiny fraction of bacteria are culturable. The number and Vil types of bacteria identified using culture-dependent methods has been previously limited by small sample sizes and frequent poor choices in growth conditions, providing a limited representation of culturable diversity. Culture-dependent methods utilizing eight different types of growth media and culture-independent techniques utilizing DNA and RNA extractions were used to recover large sequence libraries for comparison using pyrosequencing and DGGE community fingerprinting. While the majority of operational taxonomic units (OTUs) in the culture-independent library were not found in the culturedependent library, the culture-dependent library was also dominated by OTUs not shared with the culture-independent library. Culture-based methods revealed OTUs that were not recovered from direct soil nucleic acid extractions. This suggests that a polyphasic approach is able to recover greater bacterial diversity and highlights the need to reevaluate how we perceive recoverable bacterial diversity. While the disparity of finding bacterial taxa unique to culture-independent methods can be explained by the known difficulty in culturing certain bacteria, the recovery of taxa unique to culture-dependent methods is more complicated. Effectiveness in lysis procedures during soil extractions is essential to bacterial representation in the recovered nucleic acids. Certain bacteria are naturally resistant to lysis, while others have the ability to form protective structures to help them survive in the harsh conditions of the soil environment. Endospores represent one of the most resistant structures formed by bacteria, and they appear to have a ubiquitous presence in soil. A comparison of vegetative cell and endospore recovery of Bacillus cereus and B. subtilis from soil was performed to determine if inadequate lysis of endospores could lead to their underrepresentation in culture-independent nucleic acid extractions. Realtime PCR was utilized to determine the percentage recoveries for both vegetative cells and endospores. The results suggested that endospores were recovered significantly less than vegetative cells and the type of soil affected the efficiency of recovery. However, the real-time PCR data indicated that a greater number of genomes were recovered from soil than had been inoculated according to plate counts. This suggested that extracellular DNA, particularly in B. cereus, may have influenced the results. Further investigation of the presence of extracellular DNA in Bacillus endospores and vegetative cells will be necessary to clearly evaluate this data.

Library of Congress Subject Headings

Microbial ecology
Bacteria -- Ecology
Soil microbiology

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