Influence of support materials on the electroactive behavior, structure and gene expression of wild type and GSU1771-deficient mutant of Geobacter sulfurreducens biofilms.

Geobacter sulfurreducens DL1 is a metal-reducing dissimilatory bacterium frequently used to produce electricity in bioelectrochemical systems (BES). The biofilm formed on electrodes is one of the most important factors for efficient electron transfer; this is possible due to the production of type IV pili and c-type cytochromes that allow it to carry out extracellular electron transfer (EET) to final acceptors. In this study, we analyzed the biofilm formed on different support materials (glass, hematite (Fe2O3) on glass, fluorine-doped tin oxide (FTO) semiconductor glass, Fe2O3 on FTO, graphite, and stainless steel) by G. sulfurreducens DL1 (WT) and GSU1771-deficient strain mutant (Δgsu1771). GSU1771 is a transcriptional regulator that controls the expression of several genes involved in electron transfer. Different approaches and experimental tests were carried out with the biofilms grown on the different support materials including structure analysis by confocal laser scanning microscopy (CLSM), characterization of electrochemical activity, and quantification of relative gene expression by RT-qPCR. The gene expression of selected genes involved in EET was analyzed, observing an overexpression of pgcA, omcS, omcM, and omcF from Δgsu1771 biofilms compared to those from WT, also the overexpression of the epsH gene, which is involved in exopolysaccharide synthesis. Although we observed that for the Δgsu1771 mutant strain, the associated redox processes are similar to the WT strain, and more current is produced, we think that this could be associated with a higher relative expression of certain genes involved in EET and in the production of exopolysaccharides despite the chemical environment where the biofilm develops. This study supports that G. sulfurreducens is capable of adapting to the electrochemical environment where it grows.

Characterization of a catalase-peroxidase variant (L333V-KatG) identified in an INH-resistant Mycobacterium tuberculosis clinical isolate.

Mycobacterium tuberculosis catalase-peroxidase (Mt-KatG) is a bifunctional heme-dependent enzyme that has been shown to activate isoniazid (INH), the widely used antibiotic against tuberculosis (TB). The L333V-KatG variant has been associated with INH resistance in clinical M. tuberculosis isolates from Mexico. To understand better the mechanisms of INH activation, its catalytic properties (catalase, peroxidase, and IN-NAD formation) and crystal structure were compared with those of the wild-type enzyme (WT-KatG). The rate of IN-NAD formation mediated by WT-KatG was 23% greater than L333V-KatG when INH concentration is varied. In contrast to WT-KatG, the crystal structure of the L333V-KatG variant has a perhydroxy modification of the indole nitrogen of W107 from MYW adduct. L333V-KatG shows most of the active site residues in a similar position to WT-KatG; only R418 is in the R-conformation instead of the double R and Y conformation present in WT-KatG. L333V-KatG shows a small displacement respect to WT-KatG in the helix from R385 to L404 towards the mutation site, an increase in length of the coordination bond between H270 and heme Fe, and a longer H-bond between proximal D381 and W321, compared to WT-KatG; these small displacements could explain the altered redox potential of the heme, and result in a less active and stable enzyme.

Global transcriptional analysis of Geobacter sulfurreducens gsu1771 mutant biofilm grown on two different support structures.

Electroactive biofilms formation by the metal-reducing bacterium Geobacter sulfurreducens is a step crucial for bioelectricity generation and bioremediation. The transcriptional regulator GSU1771 controls the expression of essential genes involved in electron transfer and biofilm formation in G. sulfurreducens, with GSU1771-deficient producing thicker and more electroactive biofilms. Here, RNA-seq analyses were conducted to compare the global gene expression patterns of wild-type and Δgsu1771 mutant biofilms grown on non-conductive (glass) and conductive (graphite electrode) materials. The Δgsu1771 biofilm grown on the glass surface exhibited 467 differentially expressed (DE) genes (167 upregulated and 300 downregulated) versus the wild-type biofilm. In contrast, the Δgsu1771 biofilm grown on the graphite electrode exhibited 119 DE genes (79 upregulated and 40 downregulated) versus the wild-type biofilm. Among these DE genes, 67 were also differentially expressed in the Δgsu1771 biofilm grown on glass (56 with the same regulation and 11 exhibiting counter-regulation). Among the upregulated genes in the Δgsu1771 biofilms, we identified potential target genes involved in exopolysaccharide synthesis (gsu1961-63, gsu1959, gsu1972-73, gsu1976-77). RT-qPCR analyses were then conducted to confirm the differential expression of a selection of genes of interest. DNA-protein binding assays demonstrated the direct binding of the GSU1771 regulator to the promoter region of pgcA, pulF, relA, and gsu3356. Furthermore, heme-staining and western blotting revealed an increase in c-type cytochromes including OmcS and OmcZ in Δgsu1771 biofilms. Collectively, our findings demonstrated that GSU1771 is a global regulator that controls extracellular electron transfer and exopolysaccharide synthesis in G. sulfurreducens, which is crucial for electroconductive biofilm development.

GSU1771 regulates extracellular electron transfer and electroactive biofilm formation in Geobacter sulfurreducens: Genetic and electrochemical characterization.

Type IV pili and the >100c-type cytochromes in Geobacter sulfurreducens are essential for extracellular electron transfer (EET) towards metal oxides and electrodes. A previous report about a mutation in the gsu1771 gene indicated an enhanced reduction of insoluble Fe(III) oxides coupled with increased pilA expression. Herein, a marker-free gsu1771-deficient mutant was constructed and characterized to assess the role of this regulator in EET and the formation of electroactive biofilms. Deleting this gene delayed microbial growth in the acetate/fumarate media (electron donor and acceptor, respectively). However, this mutant reduced soluble and insoluble Fe(III) oxides more efficiently. Heme staining, western blot, and RT-qPCR analyses demonstrated that GSU1771 regulates the transcription of several genes (including pilA) and many c-type cytochromes involved in EET, suggesting the broad regulatory role of this protein. DNA-protein binding assays indicated that GSU1771 directly regulates the transcription of pilA, omcE, omcS, and omcZ. Additionally, gsu1771-deficient mutant biofilms are thicker than wild-type strains. Electrochemical studies revealed that the current produced by this biofilm was markedly higher than the wild-type strains (approximately 100-fold). Thus, demonstrating the role of GSU1771 in the EET pathway and establishing a methodology to develop highly electroactive G. sulfurreducens mutants.