Ganguli, M. | Search bioRxiv

bioRxiv2018

Higher order assembling of the mycobacterial essential polar growth factor DivIVA/Wag31

How proteins localize at the pole remains an enigma. DivIVA/Wag31, which is an essential pole organizing protein in mycobacteria, can assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. A single-site phosphorylation in Wag31 is linked to the regulation of peptidoglycan biosynthesis for optimal mycobacterial growth. The structural organization of polar scaffold formed by coiled-coil rich Wag31, which is a target for anti-tubercular agent amino-pyrimidine sulfonamide, remains to be determined. Here, we report biophysical characterizations of a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of mycobacterial Wag31 using circular dichroism, small angle solution X-ray scattering and atomic force microscopy. While data obtained from both variants of Wag31 in solution states suggested formation of alpha-helical, large, elongated particles, their structural organizations were different. Atomic force microscopic images of Wag31 indicate polymer formation, with occasional curving, sharp bending and branching. Observed structural features in this first view of the polymeric forms of Wag31 suggest a basis for higher order network scaffold formation for polar protein localization.

biophysics↗

bioRxiv2017

Redox-Dependent Condensation Of Mycobacterial Genome By WhiB4

Oxidative stress response in bacteria is generally mediated through coordination between the regulators of oxidant-remediation systems (e.g. OxyR, SoxR) and nucleoid condensation (e.g. Dps, Fis). However, these genetic factors are either absent or rendered nonfunctional in the human pathogen Mycobacterium tuberculosis (Mtb). Therefore, how Mtb organizes genome architecture and regulates gene expression to counterbalance oxidative imbalance during infection is not known. Here, we report that an intracellular redox-sensor, WhiB4, dynamically links genome condensation and oxidative stress response in Mtb. Disruption of WhiB4 affects the expression of genes involved in maintaining redox homeostasis, central carbon metabolism (CCM), respiration, cell wall biogenesis, DNA repair and protein quality control under oxidative stress. Notably, disulfide-linked oligomerization of WhiB4 in response to oxidative stress activates the proteins ability to condense DNA in vitro and in vivo. Further, overexpression of WhiB4 led to hypercondensation of nucleoids, redox imbalance and increased susceptibility to oxidative stress, whereas WhiB4 disruption reversed this effect. In accordance with the findings in vitro, ChIP-Seq data demonstrated non-specific binding of WhiB4 to GC-rich regions of the Mtb genome. Lastly, data indicate that WhiB4 deletion affected the expression of only a fraction of genes preferentially bound by the protein, suggesting its indirect effect on gene expression. We propose that WhiB4 is a novel redox-dependent nucleoid condensing protein that structurally couples Mtbs response to oxidative stress with genome organization and transcription.\n\nSignificance StatementMycobacterium tuberculosis (Mtb) needs to adapt in response to oxidative stress encountered inside human phagocytes. In other bacteria, condensation state of nucleoids modulates gene expression to coordinate oxidative stress response. However, this relation remains elusive in Mtb. We performed molecular dissection of a mechanism controlled by an intracellular redox sensor, WhiB4, in organizing both chromosomal structure and selective expression of adaptive traits to counter oxidative stress in Mtb. Using high-resolution sequencing, transcriptomics, imaging, and redox biosensor, we describe how WhiB4 modulates nucleoid condensation, global gene expression, and redox-homeostasis. WhiB4 over-expression hypercondensed nucleoids and perturbed redox homeostasis whereas WhiB4 disruption had an opposite effect. Our study discovered an empirical role for WhiB4 in integrating redox signals with nucleoid condensation in Mtb.

microbiology↗