Cloning of Iron Transport Related Gene TBSR1 and Construction an Expressed Strain in Shewanella oneidensis MR-1

Abstract

Iron is one of the essential trace elements for living organisms, widely involved in key life processes such as redox reactions, electron transfer, and metabolic regulation. Despite the abundance of iron in nature, it is mainly present in insoluble forms, making it difficult for organisms to directly obtain usable iron ions. Therefore, bacteria need to maintain intracellular iron homeostasis through efficient iron uptake and regulatory systems to meet the demands of optimal growth and physiological activities. The dual nature of iron - being essential yet potentially toxic - poses a significant challenge to bacterial survival. Iron deficiency can inhibit the activity of key enzymes, while iron excess can generate hydroxyl radicals through the Fenton reaction, causing oxidative damage. Gram-negative bacteria achieve dynamic iron homeostasis through iron carrier systems, TonB-dependent receptors, and the Fur protein regulatory network. Recent studies have shown that the iron homeostasis mechanisms of environmental bacteria, such as Shewanella, exhibit high diversity, and the interaction between their regulatory pathways and metabolic networks is becoming a research hotspot. Shewanella, as a representative strain of Gram-negative γ-proteobacteria, has become an ideal model for understanding bacterial iron metabolism due to its diverse respiratory pathways and high iron protein requirements. This experiment focuses on the iron carrier receptor gene TBSR1 of Shewanella, hypothesizing its involvement in the recognition and transport of iron carriers. To explore the function of TBSR1, this study constructed a TBSR1 overexpression strain using molecular cloning techniques: specific primers were designed to amplify the TBSR1 gene, which was then cloned into the expression vector pBbB5k using homologous recombination to construct the recombinant plasmid pBbB5k-TBSR1. Subsequently, the recombinant plasmid was introduced into the Escherichia coli WM3064 conjugation strain by electroporation and then transferred into Shewanella through conjugation. Finally, the successful acquisition of the TBSR1 overexpression strain was verified through colony PCR, plasmid digestion, and qPCR. The experimental results showed that the mRNA expression level of TBSR1 in the mutant strain was significantly upregulated compared to the wild type, confirming the effectiveness of the gene overexpression system. This study lays the foundation for subsequent analysis of the molecular mechanism and physiological function of TBSR1 in iron uptake.