AUTHOR=Andrade Angel , Hernández-Eligio Alberto , Tirado Ana Lilia , Vega-Alvarado Leticia , Olvera Maricela , Morett Enrique , Juárez Katy 

TITLE=Specialization of the Reiterated Copies of the Heterodimeric Integration Host Factor Genes in Geobacter sulfurreducens

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.626443

DOI=10.3389/fmicb.2021.626443

ISSN=1664-302X

ABSTRACT=<p>Integration host factor (IHF) is a widely distributed small heterodimeric protein member of the bacterial Nucleoid-Associated Proteins (NAPs), implicated in multiple DNA regulatory processes. IHF recognizes a specific DNA sequence and induces a large bend of the nucleic acid. IHF function has been mainly linked with the regulation of RpoN-dependent promoters, where IHF commonly recognizes a DNA sequence between the enhancer-binding region and the promoter, facilitating a close contact between the upstream bound activator and the promoter bound, RNA polymerase. In most proteobacteria, the genes encoding IHF subunits (<italic>ihfA</italic> and <italic>ihfB</italic>) are found in a single copy. However, in some Deltaproteobacteria, like <italic>Geobacter sulfurreducens</italic>, those genes are duplicated. To date, the functionality of IHF reiterated encoding genes is unknown. In this work, we achieved the functional characterization of the <italic>ihfA-1, ihfA-2, ihfB-1</italic>, and <italic>ihfB-2</italic> from <italic>G</italic>. <italic>sulfurreducens.</italic> Unlike the Δ<italic>ihfA-2</italic> or Δ<italic>ihfB-1</italic> strains, single gene deletion in <italic>ihfA-1</italic> or <italic>ihfB-2</italic>, provokes an impairment in fumarate and Fe(III) citrate reduction. Accordingly, sqRT-PCR experiments showed that <italic>ihfA-1</italic> and <italic>ihfB-2</italic> were expressed at higher levels than <italic>ihfA-2</italic> and <italic>ihfB-1</italic>. In addition, RNA-Seq analysis of the Δ<italic>ihfA-1</italic> and Δ<italic>ihfB-2</italic> strains revealed a total of 89 and 122 differentially expressed genes, respectively. Furthermore, transcriptional changes in 25 genes were shared in both mutant strains. Among these genes, we confirmed the upregulation of the <italic>pilA</italic>-repressor, GSU1771, and downregulation of the triheme-cytochrome (<italic>pgcA</italic>) and the aconitate hydratase (<italic>acnA</italic>) genes by RT-qPCR. EMSA experiments also demonstrated the direct binding of IHF to the upstream promoter regions of GSU1771, <italic>pgcA</italic> and <italic>acnA</italic>. PilA changes in Δ<italic>ihfA-1</italic> and Δ<italic>ihfB-2</italic> strains were also verified by immunoblotting. Additionally, heme-staining of subcellular fractions in Δ<italic>ihfA-1</italic> and Δ<italic>ihfB-2</italic> strains revealed a remarkable deficit of <italic>c</italic>-type cytochromes. Overall, our data indicate that at least during fumarate and Fe(III) citrate reduction, the functional IHF regulator is likely assembled by the products of <italic>ihfA-1</italic> and <italic>ihfB-2</italic>. Also, a role of IHF controlling expression of multiple genes (other than RpoN-dependent) affects <italic>G. sulfurreducens</italic> physiology and extracellular electron transfer.</p>