Identification, characterization and evolutionary history of the Escherichia coli glycogen operon

Representing one of the major storage carbohydrate in many bacteria, glycogen is a branched homopolysaccharide of ¿-1,4-linked glucose subunits with ¿-1,6-linked glucose at the branching points that accumulates under conditions of limiting growth when an excess of carbon is available and other nutrients are deficient. The exact role of this polyglucan in bacteria is not as clear-cut as in animal and yeast cells, but some studies have linked glycogen to extended bacterial survival, symbiotic performance, colonization and virulence. It is widely accepted that genes involved in Escherichia coli and Salmonella enterica glycogen metabolism are clustered in two tandemly arranged operons: glgBX (encompassing the genes coding for glycogen branching (GlgB) and debranching (GlgX) enzymes), and glgCAP (encoding the GlgC and GlgA anabolic enzymes, as well as the catabolic glycogen phosphorylase (GlgP)). However, the data regarding regulatory aspects of the expression of glycogen genes in E. coli are contradictory, thus questioning the presence of two operons. To get inshight into the trascriptional organization of glycogen genes, in the first chapter of this work I characterized glg genes transcription using RT (reverse transcriptase)-PCR approach. This analysisW revealed that E. coli cells possess transcripts comprising the five glgBXCAP genes. glg::lacZY expression analyses in cells lacking the region immediately upstream of the glgB gene revealed an almost total abolishment of glgB, glgX and glgC expression and a reduced expression of glgA and glgP. Similar type of analyses showed that glgA and glgP expression was almost totally abolished in cells lacking glgA upstream sequences, including glgC, glgB and the asd-glgB intergenic region upstream of glgB. All the data indicate that the five glgBXCAP genes are transcribed in a single transcriptional unit under the control of promoter sequences upstream of glgB and that an alternative suboperonic promoter driving glgA and glgP expression is located within glgC. Using computer searches for putative bacterial promoters and 5¿ RACE (rapid amplification of cDNA ends) techniques I identified the -35 and -10 sequences of both promoters as well as the trasnscription start sites. Finally, I measured glg::lacZY expression on cells lacking the relA or phoP regulatory genes. These analyses indicated that both glgBXCAP operon and the suboperonic promoter form part of RelA and PhoP-PhoQ regulons.

Bibliographic Details

Main Author:	Almagro, Goizeder
Other Authors:	Pozueta Romero, Javier
Format:	tesis doctoral biblioteca
Published:	Universidad Pública de Navarra 2014-07
Online Access:	http://hdl.handle.net/10261/142593 http://dx.doi.org/10.13039/501100007273
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