Analysis of Genes Involved in Metal Resistance and Cytochrome C Maturation in Shewanella Oneidensis MR-1

Metals play crucial roles in many cellular processes where they form active centers of metabolic enzymes or participate in electron transfer reactions during respiration. At high concentrations, metals can be toxic and result in the formation of reactive oxygen species and protein denaturation. Bacteria have evolved homeostasis systems to maintain intracellular concentrations of various metals and avoid their toxic effects. The aim of this project is to identify and characterize metal homeostasis systems in the metal reducer Shewanella oneidensis MR-1. This bacterium can use metals and radionuclides as electron acceptors during anaerobic respiration and is therefore a good candidate for bioremediation of metal-contaminated environments. Furthermore, this bacterium is able to maintain low internal levels of heavy metals through the use of multiple efflux pumps such as the P-type ATPase - CopA, and the HME RND efflux pump – CzcCBA. This study aims to understand the role of these efflux pumps and their regulators in metal resistance. Shewanella oneidensis also expresses a large number of c-type cytochromes, many of which function as terminal reductases. All of these proteins contain the typical heme-binding motif CXXCH and require the Ccm proteins for maturation. SirA, the terminal sulfite reductase, also possesses an atypical heme binding site CX15CH which requires a specialized system for heme attachment. S. oneidensis MR-1 encodes two cytochrome c synthetases (CcmF and SirE) and two apocytochrome c chaperones (CcmI and SirG). In this study we show that both apocytochrome c chaperones, CcmI and SirG, are required for the maturation of SirA and they each interact with the terminal sulfite reductase independently of each other, even in the absence of other components of the cytochrome c maturation system

Identification of a new pebp2 alpha A2 isoform from zebrafish runx2 capable of inducing osteocalcin gene expression in vitro

Introduction: RUNX2 (also known as CBFA1/Osf2/AML3/PEBP2 alpha A) is a transcription factor essential for bone formation in mammals, as well as for osteoblast and chondrocyte differentiation, through regulation of expression of several bone- and cartilage-related genes. Since its discovery, Runx2 has been the subject of intense studies, mainly focused in unveiling regulatory targets of this transcription factor in high vertebrates. However, no single study has been published addressing the role of Runx2 in bone metabolism of low vertebrates. While analyzing the zebrafish (Danio rerio) runx2 gene, we identified the presence of two orthologs of RUNX2, which we named runx2a and runx2b and cloned a pebp2 alpha A-like transcript of the runx2b gene, which we named pebp2 alpha A2. Materials and Methods: Zebrafish runx2b gene and cDNA were isolated by RT-PCR and sequence data mining. The 3D structure of runx2b runt domain was modeled using mouse Runx1 runt as template. The regulatory effect of pebp2 alpha A2 on osteocalcin expression was analyzed by transient co-transfection experiments using a luciferase reporter gene. Phylogenetic analysis of available Runx sequences was performed with TREE-PUZZLE 5.2. and MrBayes. Results and Conclusions: We showed that the runx2b gene structure is highly conserved between mammals and fish. Zebrafish runx2b has two promoter regions separated by a large intron. Sequence analysis suggested that the runx2b gene encodes three distinct isoforms, by a combination of alternative splicing and differential promoter activation, as described for the human gene. We have cloned a pebp2 alpha A-like transcript of the runx2b gene, which we named pebp2 alpha A2, and showed its high degree of sequence similarity with the mammalian pebp2 alpha A. The cloned zebrafish osteocalcin promoter was found to contain three putative runx2-binding elements, and one of them, located at -221 from the ATG, was capable of mediating pebp2 alpha A2 transactivation. In addition, cross-species transactivation was also confirmed because the mouse Cbfa1 was able to induce the zebrafish osteocalcin promoter, whereas the zebrafish pebp2 alpha A2 activated the murine osteocalcin promoter. These results are consistent with the high degree of evolutionary conservation of these proteins. The 3D structure of the runx2b runt domain was modeled based on the runt domain of mouse Runx1. Results show a high degree of similarity in the 3D configuration of the DNA binding regions from both domains, with significant differences only observed in non-DNA binding regions or in DNA-binding regions known to accommodate considerable structure flexibility. Phylogenetic analysis was used to clarify the relationship between the isoforms of each of the two zebrafish Runx2 orthologs and other Runx proteins. Both zebrafish runx2 genes clustered with other Runx2 sequences. The duplication event seemed, however, to be so old that, whereas Runx2b clearly clusters with the other fish sequences, it is unclear whether Runx2a clusters with Runx2 from higher vertebrates or from other fish.info:eu-repo/semantics/publishedVersio