Yun Luo

Document Type

Thesis - University Access Only

Award Date

2006

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Bacteria play an important role in ecosystem functioning, especially in soil where they are believed to catalyze a range of degradative processes. Yet knowledge of the in situ physiology of bacteria in soil is sorely lacking. Members of Bacillus cereus group are gram-positive spore-forming rods, commonly isolated from soils. Yet the ecology and physiology of the B. cereus group and other Bacillus in soil are still poorly understood. Although some earlier literature argued that Bacillus spores can germinate and become active vegetative cells when readily decomposable organic matter was available, the current paradigm is that B. cereus sensu lato can only germinate and grow in animal or insect hosts, and survive in soil as spores. In order to study the growth of B. cereus A TCC 14579 under soil nutrient conditions, an in terra approach is described in this study, using filter-sterilized soil extracted soluble organic matter (SESOM) and artificial soil microcosms (ASM) saturated with SESOM. B. cereus displayed a life cycle in soil, able to germinate, grow, and subsequently sporulate in both the liquid SESOM and in ASM inserted into wells in agar media. Vegetative cells grew as filaments in liquid SESOM without separating, reminiscent of Arthromitus found in insect guts. Then filaments and cells coalesced to form clumps, which were three-dimensional multicellular structures, encasing the ensuing spores in an extracellular matrix. Two genes, purA and galE were identified to be required for the formation of multicellular structures. Analysis of the extracellular matrix showed that it contained DNA, protein. The DNA content of the matrix matched random sequences of the genome. Four predominant proteins, an OppA homologue, ATP synthase beta chain, enolase, and glutamine synthetase (GlnA), were identified in the matrix, indicating that they did not originate from lysed cells. The formation of multicellular structure and extracellular matrix is similar to the concept of a biofilm, which is the predominant growth mode of bacteria in nature. These biofilms formed in the absence of an interface, indicating that B. cereus biofilms in soil develop in response to the chemical environment rather than the presence of a surface. The proteomic analysis of exponential growing populations showed that cells grown in SESOM differentially expressed about 400 proteins, of which 45 proteins were identified by MALDI-TOF mass spectrometry. These identified proteins covered a range of cellular metabolism functions, suggesting a comprehensive shift in gene regulation and expression. In ASM, B. cereus and some soil-isolated Bacillus were able to translocate from the point of inoculation through soil microcosms as bundles of parallel chains, supporting the notion of translocation through the artificial soil matrix by extension of multicellular filaments through growth and cell division. All these results indicate that B. cereus is a saprophytic bacterium that is able to grow in soil and translocate through soil by employing a multicellular mode of growth. Furthermore B. cereus adapts to grow on soil organic matter, and forms biofilms in the spaces between soil particles.

Library of Congress Subject Headings

Bacillus cereus

Soil microbiology

Biofilms

Format

application/pdf

Number of Pages

171

Publisher

South Dakota State University

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