Structural and DNA binding properties of mycobacterial integration host factor mIHF

Abstract

In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered Nterminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the x-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (~300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA. Highlights-The structure of mIHF was solved by solution NMR spectroscopy-mIHF is an alpha-helical protein with two DNA-binding sites-mIHF introduces left-handed loops into DNA-DNA unwinds and re-compacts in a mIHF-level dependent manner

Nanoscale surface structures of DNA bound to Deinococcus radiodurans HU unveiled by atomic force microscopy

“…[10][11][12][13][14] In particular, AFM has been successfully used to investigate the interactions of bacterial nucleoid-associated proteins (NAPs), the various roles of which have been reviewed elsewhere, 15 with DNA, an essential step for chromosome packaging in bacteria. AFM has supplied a direct visualization of NAPs binding to plasmid [16][17][18][19][20][21] and linearized DNAs, [22][23][24][25] as well as the formation of higher order structure gradually induced by NAPs from the protein-free state of DNA. 26 Among bacterial species, NAPs from E. coli have been most extensively studied.…”

Mycobacterial IHF is a highly dynamic nucleoid-associated protein that assists HupB in organizing chromatin

“…To date, the studies on mIHF have been largely limited to in vitro analyses of mIHF-DNA interactions (Pedulla and Hatfull, 1998;Mishra et al, 2013;Sharadamma et al, 2015;Odermatt et al, 2020a).…”

“…These differences raised the question of whether mycobacterial IHF (mIHF) may have cellular functions analogous to those of E. coli IHF, such as contributing to the basic cellular processes (e.g., replication initiation; Ryan et al, 2002 ). Similar to E. coli IHF, mIHF was shown to be a DNA-bending protein that can also create rigid filaments and introduce left-hand loops, as assessed by Atomic Force Microscopy (AFM; Mishra et al, 2013 ; Odermatt et al, 2020a ). Meanwhile, RNA-seq experiments revealed that M. tuberculosis IHF (mtIHF) influences the expression levels of many genes, including those involved in virulence ( Odermatt et al, 2018 ).…”

“…To date, the studies on mIHF have been largely limited to in vitro analyses of mIHF-DNA interactions ( Pedulla and Hatfull, 1998 ; Mishra et al, 2013 ; Sharadamma et al, 2015 ; Odermatt et al, 2020a ). In the present study, we sought to explore the biological function of mIHF at the single-cell level and to understand its role in the organization of the mycobacterial chromosome alongside with HupB.…”

“…Characteristics of HupB and msIHF. (A) Biochemical characteristics of msIHF and HupB (Kumar et al, 2010;Bhowmick et al, 2014;Odermatt et al, 2018Odermatt et al, , 2020a. as does Possibly the dynamic, non-specific binding of msIHF could be required in certain conditions (e.g., under stress) to regulate gene expression and/or compact chromosomal DNA.…”