Affiliation:
1. Laboratory of Molecular Biology of Microorganisms, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
2. Department of Molecular Microbiology, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland
Abstract
ABSTRACT
The bacterial chromosome undergoes dynamic changes in response to ongoing cellular processes and adaptation to environmental conditions. Among the many proteins involved in maintaining this dynamism, the most abundant is the nucleoid-associated protein (NAP) HU. In mycobacteria, the HU homolog, HupB, possesses an additional C-terminal domain that resembles that of eukaryotic histones H1/H5. Recently, we demonstrated that the highly abundant HupB protein occupies the entirety of the
Mycobacterium smegmatis
chromosome and that the HupB-binding sites exhibit a bias from the origin (
oriC
) to the terminus (
ter
). In this study, we used HupB fused with enhanced green fluorescent protein (EGFP) to perform the first analysis of chromosome dynamics and to track the
oriC
and replication machinery directly on the chromosome during the mycobacterial cell cycle. We show that the chromosome is located in an off-center position that reflects the unequal division and growth of mycobacterial cells. Moreover, unlike the situation in
E. coli
, the sister
oriC
regions of
M. smegmatis
move asymmetrically along the mycobacterial nucleoid. Interestingly, in this slow-growing organism, the initiation of the next round of replication precedes the physical separation of sister chromosomes. Finally, we show that HupB is involved in the precise timing of replication initiation.
IMPORTANCE
Although our view of mycobacterial nucleoid organization has evolved considerably over time, we still know little about the dynamics of the mycobacterial nucleoid during the cell cycle. HupB is a highly abundant mycobacterial nucleoid-associated protein (NAP) with an indispensable histone-like tail. It was previously suggested as a potential target for antibiotic therapy against tuberculosis. Here, we fused HupB with enhanced green fluorescent protein (EGFP) to study the dynamics of the mycobacterial chromosome in real time and to monitor the replication process directly on the chromosome. Our results reveal that, unlike the situation in
Escherichia coli
, the nucleoid of an apically growing mycobacterium is positioned asymmetrically within the cell throughout the cell cycle. We show that HupB is involved in controlling the timing of replication initiation. Since tuberculosis remains a serious health problem, studies concerning mycobacterial cell biology are of great importance.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
16 articles.
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