Title

Enhancer Binding Proteins Initiate and Propagate Multicellular Development in Myxococcus xanthus

Date of Award

12-2011

Degree Type

Dissertation/Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

Anthony Garza

Keywords

Biofilms, Enhancer binding proteins, Sigma 54 promoters, Two-component systems

Subject Categories

Biology

Abstract

Myxococcus xanthus responds to starvation by engaging a programmed series of morphological changes that yield multicellular fruiting bodies filled with dormant spores. Previous work suggests that M. xanthus uses a series of transcriptional activators called enhancer binding proteins (EBPs) to coordinate expression of many developmental genes. This work focuses on six EBPs (Nla4, Nla18, Nla6, Nla28, ActB and MXAN4899) that regulate early development. It is during this critical stage when information from both external and internal environments must be integrated and cells must decide to continue to grow and divide, or to arrest growth and commit to development. We find that the early developmental EBPs function sequentially during development. DNA microarray analysis and quantitative PCR revealed a transcriptional hierarchy among the early-acting EBP genes. EBPs work in conjunction with σ54 promoters, and bioinformatics revealed that five of the EBP gene operons have putative σ54 promoters, which suggested that the EBPs might form a cascade involving direct transcriptional regulation. Subsequent electrophoretic mobility shift assays (EMSAs) performed with the purified DNA binding domains of the six EBPs and fragments of the EBP gene promoters, indicated that an EBP functioning at one developmental stage directly binds to the promoter region of the EBP gene that functions at the next developmental stage. In addition to activation of downstream EBP genes within the cascade, it is probable that each EBP in the cascade activates many genes that are important for development. Using a combination of DNA microarray analysis and bioinformatics, we identified hundreds of operons that are activated in the early stages of development that are potential targets of the EBPs in the cascade. To further investigate the role of EBP-mediated transcription during development, we focused on Nla6, and identified developmental target operons for this EBP. Alignment of the promoter regions positive for Nla6 binding revealed a 10 bp tandem repeat as a putative Nla6 binding site. Using both in vitro and in vivo methods, we find that the 10bp consensus is important for Nla6 binding to the target promoters, and for expression of target operons. Using the confirmed consensus and the sequence of the M. xanthus genome, we identified 42 putative developmental promoter targets for Nla6. EMSAs were used to confirm that Nla6 binds to six of the putative target promoters. Additionally, we find that mutations in four of the targets tested caused developmental phenotypes similar to that of an nla6 mutant strain. The abundance of putative EBP targets and their functional importance to development, suggest that the cascade is part of a complex regulatory network that is essential to the initiation and propagation of multicellular development.

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