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

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 (ihfA and ihfB) are found in a single copy. However, in some Deltaproteobacteria, like Geobacter sulfurreducens, 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 ihfA-1, ihfA-2, ihfB-1, and ihfB-2 from G. sulfurreducens. Unlike the ΔihfA-2 or ΔihfB-1 strains, single gene deletion in ihfA-1 or ihfB-2, provokes an impairment in fumarate and Fe(III) citrate reduction. Accordingly, sqRT-PCR experiments showed that ihfA-1 and ihfB-2 were expressed at higher levels than ihfA-2 and ihfB-1. In addition, RNA-Seq analysis of the ΔihfA-1 and ΔihfB-2 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 pilA-repressor, GSU1771, and downregulation of the triheme-cytochrome (pgcA) and the aconitate hydratase (acnA) genes by RT-qPCR. EMSA experiments also demonstrated the direct binding of IHF to the upstream promoter regions of GSU1771, pgcA and acnA. PilA changes in ΔihfA-1 and ΔihfB-2 strains were also verified by immunoblotting. Additionally, heme-staining of subcellular fractions in ΔihfA-1 and ΔihfB-2 strains revealed a remarkable deficit of c-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 ihfA-1 and ihfB-2. Also, a role of IHF controlling expression of multiple genes (other than RpoN-dependent) affects G. sulfurreducens physiology and extracellular electron transfer.

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On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes

Biochemical Society Transactions ◽

10.1042/bst20120099 ◽

2012 ◽

Vol 40 (6) ◽

pp. 1295-1301 ◽

Cited By ~ 18

Author(s):

Leonor Morgado ◽

Ana P. Fernandes ◽

Joana M. Dantas ◽

Marta A. Silva ◽

Carlos A. Salgueiro

Keyword(s):

Electron Transfer ◽

Functional Characterization ◽

Cost Effective ◽

Genetically Engineered ◽

Geobacter Sulfurreducens ◽

Extracellular Electron Transfer ◽

The Road ◽

On The Road ◽

Haem Proteins

Extracellular electron transfer is one of the physiological hallmarks of Geobacter sulfurreducens, allowing these bacteria to reduce toxic and/or radioactive metals and grow on electrode surfaces. Aiming to functionally optimize the respiratory electron-transfer chains, such properties can be explored through genetically engineered strains. Geobacter species comprise a large number of different multihaem c-type cytochromes involved in the extracellular electron-transfer pathways. The functional characterization of multihaem proteins is particularly complex because of the coexistence of several microstates in solution, connecting the fully reduced and oxidized states. NMR spectroscopy has been used to monitor the stepwise oxidation of each individual haem and thus to obtain information on each microstate. For the structural study of these proteins, a cost-effective isotopic labelling of the protein polypeptide chains was combined with the comparative analysis of 1H-13C HSQC (heteronuclear single-quantum correlation) NMR spectra obtained for labelled and unlabelled samples. These new methodological approaches allowed us to study G. sulfurreducens haem proteins functionally and structurally, revealing functional mechanisms and key residues involved in their electron-transfer capabilities. Such advances can now be applied to the design of engineered haem proteins to improve the bioremediation and electricity-harvesting skills of G. sulfurreducens.

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Determining the DNA sequence elements required for binding integration host factor to two different target sites.

Journal of Bacteriology ◽

10.1128/jb.176.10.2999-3006.1994 ◽

1994 ◽

Vol 176 (10) ◽

pp. 2999-3006 ◽

Cited By ~ 44

Author(s):

L M Hales ◽

R I Gumport ◽

J F Gardner

Keyword(s):

Dna Sequence ◽

Integration Host Factor ◽

Host Factor ◽

Target Sites ◽

Sequence Elements

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Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens

Applied and Environmental Microbiology ◽

10.1128/aem.01967-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 7178-7186 ◽

Cited By ~ 19

Author(s):

Chi Ho Chan ◽

Caleb E. Levar ◽

Lori Zacharoff ◽

Jonathan P. Badalamenti ◽

Daniel R. Bond

Keyword(s):

Gene Clusters ◽

Genetic System ◽

Geobacter Sulfurreducens ◽

Expression Vectors ◽

Extracellular Electron Transfer ◽

Gene Encoding ◽

Biofilm Growth ◽

Content Type ◽

Biofilm Development ◽

Citrate Reduction

ABSTRACTMetal reduction by members of theGeobacteraceaeis encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent ofGeobacterbiofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacteriumGeobacter sulfurreducens. A motile strain ofG. sulfurreducenswas constructed by precise removal of a transposon interrupting thefgrMflagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochromeimcH. We also show that RK2-based plasmids were maintained inG. sulfurreducensfor over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutiveGeobacterpromoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcHmutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerousG. sulfurreducensstrain constructs simultaneously and shows the sensitivity ofimcHexpression by the vanillate system. These tools will enable more sophisticated genetic studies inG. sulfurreducenswithout polar insertion effects or need for multiple antibiotics.

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