Date of Award

January 2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Ann M. Flower

Abstract

In Escherichia coli, ribosomes are the basic protein-synthesizing cellular machines. Hence, regulation of ribosome synthesis and assembly as well as regulation of protein translation are very important for bacterial cells. However, the cellular mechanisms of both these processes are not yet completely understood and thus both are active fields of research. It has been suggested that the BipA protein is involved in the regulation of both these processes. The research described here focused on understanding the cellular function(s) of BipA in regulating protein synthesis and ribosome assembly.

BipA is a member of the elongation factor family of translational GTPases and shares protein domain homology to EF-G, EF-Tu and LepA. BipA is not essential for growth but has been proposed to be involved in the regulation of a variety of cellular processes which include protein synthesis, motility, capsule formation, antibiotic resistance, symbiosis, low temperature growth, and pathogenicity. These findings led us to the hypothesis that BipA is involved in regulating expression of target genes, presumably at the translational level. However, BipA also associates with the ribosome and the GTPase activity of BipA is induced in the presence of fully formed ribosomes and high levels of GTP. Additionally the cold-sensitive phenotype of a bipA mutant is similar to mutants of ribosome assembly factors. These characteristics of BipA supported an alternate hypothesis that BipA is involved in regulating ribosome assembly and/or biogenesis.

To test our first hypothesis we investigated the role of BipA as a regulator of translation by monitoring the effect of bipA deletion on the expression of different genes of the RcsBCD

pathway. The RcsBCD pathway regulates the expression of genes involved in the synthesis of flagella and capsule along with other genes and is regulated via either the RcsA-RcsB complex or via RcsB. Our results suggest that BipA regulates the expression of multiple genes of the Rcs pathway possibly by affecting RcsB expression.

To test our second hypothesis we determined the role of BipA in ribosome assembly, for which we compared the phenotypes of a bipA mutant to the phenotypes of a known ribosome assembly factor, DeaD. We also analyzed ribosome profiles and rRNA processing in strains lacking bipA and compared that to an isogenic wild-type. Our results suggest that BipA is involved in ribosome assembly, particularly in the biogenesis of the 50S ribosomal subunit.

Our results provide support for both of the starting hypotheses: expression studies indicate that deletion of bipA alters expression of multiple genes, and ribosomal profiling demonstrates dependence on BipA for proper assembly. It is possible that BipA facilitates these processes independent of one another, or that interference of one function indirectly leads to disruption of the other. While our results do not define the direct function of BipA, they expand our existing understanding of the protein and highlight the extent of cellular processes affected by BipA.

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