AUTHOR=Shoyama Fernanda Miyagaki , Janetanakit Taveesak , Bannantine John P. , Barletta Raul G. , Sreevatsan Srinand 

TITLE=Elucidating the Regulon of a Fur-like Protein in Mycobacterium avium subsp. paratuberculosis (MAP)

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.00598

DOI=10.3389/fmicb.2020.00598

ISSN=1664-302X

ABSTRACT=<p>Intracellular iron concentration is tightly regulated to maintain cell viability. Iron plays important roles in electron transport, nucleic acid synthesis, and oxidative stress. A <italic>Mycobacterium avium</italic> subsp. <italic>paratuberculosis</italic> (<italic>MAP</italic>)-specific genomic island carries a putative metal transport operon that includes <italic>MAP3773c</italic>, which encodes a Fur-like protein. Although well characterized as a global regulator of iron homeostasis in multiple bacteria, the function of Fur (ferric uptake regulator) in <italic>MAP</italic> is unknown as this organism also carries IdeR (iron dependent regulator), a native iron regulatory protein specific to mycobacteria. Computational analysis using PRODORIC identified 23 different pathways involved in respiration, metabolism, and virulence that were likely regulated by <italic>MAP3773c</italic>. Thus, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was performed to confirm the putative regulon of <italic>MAP3773c</italic> (Fur-like protein) in <italic>MAP</italic>. ChIP-Seq revealed enriched binding to 58 regions by Fur under iron-replete and -deplete conditions, located mostly within open reading frames (ORFs). Three ChIP peaks were identified in genes that are directly related to iron regulation: <italic>MAP3638c</italic> (hemophore-like protein), <italic>MAP3736c</italic> (Fur box), and <italic>MAP3776c</italic> (ABC transporter). Fur box consensus sequence was identified, and binding specificity and dependence on Mn<sup>2+</sup> availability was confirmed by a chemiluminescent electrophoresis mobility shift assay (EMSA). The results confirmed that <italic>MAP3773c</italic> is a Fur ortholog that recognizes a 19 bp DNA sequence motif (Fur box) and it is involved in metal homeostasis. This work provides a regulatory network of <italic>MAP</italic> Fur binding sites during iron-replete and -deplete conditions, highlighting unique properties of Fur regulon in <italic>MAP.</italic></p>