Deficiency of essential GTP‐binding protein ObgE in Escherichia coli inhibits chromosome partition

Abstract

GTP‐binding proteins are involved in cell proliferation, development, signal transduction, protein elongation, etc. and construct the GTPase superfamily, whose structures and sequence motifs (G‐1 to G‐5) are highly conserved from prokaryote to eukaryote. Obg of Bacillus subtilis and Obg homologues of other bacteria belong to the GTPase superfamily and have been suggested as being essential for cell growth, development and monitoring of intracellular levels of GTP. We identified the Obg homologue in Escherichia coli, a protein previously known as YhbZ, which we have renamed ObgE. Double cross‐over experiments showed that the obgE gene is essential for growth in E. coli. From characterization of the obgE temperature‐sensitive mutant, we found that DNA replication was not inhibited, that the nucleoids did not partition and instead remained in the middle of cell, and that the cells elongated. Overproduction of ObgE also resulted in aberrant chromosome segregation. These data suggested that ObgE is involved directly or indirectly in E. coli chromosome partitioning. Characterization studies showed that ObgE is abundant in normal cells, partially associated with the membrane and does not associate with ribosomes such as in Obg of B. subtilis. We purified ObgE protein from a cell extract of E. coli, and the purified ObgE had GTPase activity and DNA‐binding ability.

Chromosome segregation control by Escherichia coli ObgE GTPase

“…Confirming the results of Kobayashi et al (2001) with the temperature-sensitive allele of obgE, large DAPI-staining nucleoids were seen after only 1 h of ObgE depletion, when levels of ObgE were still detectable at about 15% of that prior to the downshift (Fig. 1B).…”

“…It has been previously reported that ObgE interacts with ribosomal proteins, and depletion of ObgE effects pre-16S-rRNA processing and ribosomal protein levels (Kobayashi et al, 2001). Both E. coli and C. crescentus have been reported to copurify with the ribosome, although this interaction has been reported to be weak (for Caulobacter) (Lin et al, 2004) or variable (for E. coli ) (Kobayashi et al, 2001;Sato et al, 2005).…”

“…Both E. coli and C. crescentus have been reported to copurify with the ribosome, although this interaction has been reported to be weak (for Caulobacter) (Lin et al, 2004) or variable (for E. coli ) (Kobayashi et al, 2001;Sato et al, 2005). It has been suggested that ObgE is required for ribosome biogenesis and that observed cell cycle effects may be an indirect consequence of loss of translation capacity (Sato et al, 2005).…”

“…The Obg/CgtA protein is an essential GTPase conserved throughout the eubacteria and implicated, in E. coli, for control of chromosome segregation (Kobayashi et al, 2001). We discovered a mutation in obgE that conferred extreme sensitivity to replication inhibitors and has characteristic phenotypes of a cell cycle checkpoint control defect (Foti et al, 2005).…”

“…Previously, the essential role of E. coli ObgE and Caulobacter CgtA had been examined using temperature-sensitive alleles (Kobayashi et al, 2001;Datta et al, 2004), which could have complex effects on the function of the protein. Our results are consistent with a role for ObgE in chromosome partitioning, with defects in chromosome segregation observed early after shut-off of the ara promoter even before depletion of ObgE protein.…”