Mycobacterium tuberculosis and M. bovis BCG Moreau Fumarate Reductase Operons Produce Different Polypeptides That May Be Related to Non-canonical Functions

Abstract

Tuberculosis is a world widespread disease, caused by Mycobacterium tuberculosis (M.tb). Although considered an obligate aerobe, this organism can resist life-limiting conditions such as microaerophily mainly due to its set of enzymes responsible for energy production and coenzyme restoration under these conditions. One of these enzymes is fumarate reductase, an heterotetrameric complex composed of a catalytic (FrdA), an iron-sulfur cluster (FrdB) and two transmembrane (FrdC and FrdD) subunits involved in anaerobic respiration and important for the maintenance of membrane potential. In this work, aiming to further characterize this enzyme function in mycobacteria, we analyzed the expression of FrdB-containing proteins in M.tb and Mycobacterium bovis Bacillus Calmette–Guérin (BCG) Moreau, the Brazilian vaccine strain against tuberculosis. We identified three isoforms in both mycobacteria, two of them corresponding to the predicted encoded polypeptides of M.tb (27 kDa) and BCG Moreau (40 kDa) frd sequences, as due to an insertion on the latter’s operon a fused FrdBC protein is expected. The third 52 kDa band can be explained by a transcriptional slippage event, typically occurring when mutation arises in a repetitive region within a coding sequence, thought to reduce its impact allowing the production of both native and variant forms. Comparative modeling of the M.tb and BCG Moreau predicted protein complexes allowed the detection of subtle overall differences, showing a high degree of structure and maybe functional resemblance among them. Axenic growth and macrophage infection assays show that the frd locus is important for proper bacterial development in both scenarios, and that both M.tb’s and BCG Moreau’s alleles can partially revert the hampered phenotype of the knockout strain. Altogether, our results show that the frdABCD operon of Mycobacteria may have evolved to possess other yet non-described functions, such as those necessary during aerobic logarithmic growth and early stage steps of infection.

Comparative Genomic Analysis of Metal-Tolerant Bacteria Reveals Significant Differences in Metal Adaptation Strategies

“…In S. marcescens X1 genome, a number of genes encoding the homologs of proteins responsible for Fe uptake pathways were discovered, including the ferric hydroxamate uptake (Fhu) system (FhuB, -C and -D) necessary for the transport of ferrichrome, and other Fe 3+ -compounds from the periplasm (45), elemental Fe(II/III) (Efe) complex (EfeO and EfeU) required for the acquisition and utilization of iron ( 46), the AfuABC system responsible for the uptake of Fe(III) (17), the ferrous iron transport (Feo) system (FeoA, -B and -C) involved in the transport of Fe(II) (47), and the Salmonella iron transporter (Sit) (SitA, -B, -C and -D) which plays an important role in Fe acquisition under iron-deficient conditions (48). Many iron-sulfur proteins, i.e., iron-sulfur cluster assembly protein (IscA)/iron-sulfur cluster assembly regulator (IscR), A-type iron-sulfur carrier protein (ErpA), fumarate reductase iron-sulfur subunit (FrdB), and formate dehydrogenase iron-sulfur subunit (FdoH), have been reported to play important roles in Fe homeostasis and many cellular processes (49)(50)(51)(52). Therefore, the determination of MTC and effect of iron on S. marcescens X1 show that this isolate exhibited high tolerance to iron (MTC of 1000 mg/L) and its growth was not obviously affected by middle concentration of iron (500 mg/L of FeSO 4 ).…”

M. bovis BCG Moreau N-Terminal Loss Leads to a Less Stable Dodecin With Lower Flavin Binding Capacity

“…But even with the long, continuous usage of this strain for vaccination of Brazilian children, little is known about BCG Moreau physiology, when compared to BCG Pasteur, one of the most studied BCG strains, M. bovis or M.tb. In the last decade, our group worked on its characterization, on the genomic (Gomes et al, 2011), proteomic (Berredo-Pinho et al, 2011Pagani et al, 2019) and functional context (Galvão et al, 2014;Monteiro-Maia et al, 2018;Schwarz et al, 2020;Schwarz et al, 2021). Comparison of the BCG Moreau and M.tb genomes confirms a single nucleotide polymorphism (SNP) within the start codon of the dodecin coding gene, already annotated, but not functionally described, for BCG Pasteur (BCG_1562c), leading to a predicted N-terminal shorter homolog in BCG.…”

Use of transcriptional slippage for diverse gene expression

“…It is mainly related to the intrinsic property of the RNA polymerase (RNAP), which under certain conditions (long A/T homopolymeric sequences) may introduce random and two-directional InDel errors, contributing to an increase in mRNA polymorphism of a given gene (Larsen et al, 2000;Tamas et al, 2008;Wagner et al, 1990;Wons et al, 2015), with changes in the primary reading frame (InDel frameshift). This leads to production of altered forms of proteins (Schwarz et al, 2021;Wons et al, 2015;Rockah-Shmuel et al, 2013). Such a mechanism turns out to be beneficial in case of genes with a single InDel mutation, as it can "repair" the mRNA sequence (including reinstatement of the correct reading frame) while keeping the gene's DNA sequence mutated (Gordon et al, 2013;Koscielniak et al, 2018;Rockah-Shmuel et al, 2013;Ta-mas et al, 2008;Wernegreen et al, 2010;Wons et al, 2015).…”