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Helicobacter pylori single-stranded DNA binding protein - functional characterization and modulation of H-pylori DnaB helicase activity
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 2, 519-531 p.Article in journal (Refereed) Published
Abstract [en]

Helicobacter pylori, an important bacterial pathogen, causes gastric ulcer and gastric adenocarcinoma in humans. The fundamentals of basic biology such as DNA replication are poorly understood in this pathogen. In the present study, we report the cloning and functional characterization of the single-stranded DNA (ssDNA) binding protein from H. pylori. The N-terminal DNA binding domain shows significant homology with E. coli single-stranded DNA binding protein (SSB), whereas the C-terminal domain shows less homology. The overall DNA-binding activity and tetramerization properties, however, remain unaffected. In in vitro experiments with purified proteins, H. pylori (Hp) SSB bound specifically to ssDNA and modulated the enzymatic ATPase and helicase activity of HpDnaB helicase. HpSSB and HpDnaB proteins were co-localized in sharp, distinct foci in exponentially growing H. pylori cells, whereas both were spread over large areas in its dormant coccoid form, suggesting the absence of active replication forks in the latter. These results confirm the multiple roles of SSB during DNA replication and provide evidence for altered replicative metabolism in the spiral and coccoid forms that may be central to the bacterial physiology and pathogenesis.

Place, publisher, year, edition, pages
2009. Vol. 276, no 2, 519-531 p.
Keyword [en]
DNA replication, helicase, Helicobacter pylori, replication foci, single-stranded DNA binding protein
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-129274DOI: 10.1111/j.1742-4658.2008.06799.xISI: 000261978000018OAI: oai:DiVA.org:uu-129274DiVA: diva2:342758
Available from: 2010-08-11 Created: 2010-08-10 Last updated: 2011-11-03Bibliographically approved
In thesis
1. Dynamic Organization of Molecular Machines in Bacteria
Open this publication in new window or tab >>Dynamic Organization of Molecular Machines in Bacteria
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism.

In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phase

Bacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase.

In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 48 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 847
molecular machines, bacteria, cell cycle, ribosome, replisome, divisome, spore, E. coli, H. pylori, Mycobacteria
National Category
Research subject
urn:nbn:se:uu:diva-157691 (URN)978-91-554-8140-7 (ISBN)
Public defence
2011-10-14, Room B41, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 13:15 (English)
Available from: 2011-09-23 Created: 2011-08-22 Last updated: 2013-03-14Bibliographically approved

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