Proteomic profiling of Escherichia coli cytoplasmic proteins under sublethal boric acid stress

Boron is an essential micronutrient for plants and certain bacteria, where it plays critical roles in cellular processes at low concentrations. However, elevated levels of boron-containing compounds, such as boric acid, exhibit antimicrobial toxicity. Although the physiological effects of boric acid on bacteria have been partially characterized, its proteome-wide impacts remain poorly elucidated. This study employs a 2D-PAGE-based proteomic approach to investigate how sublethal boric acid stress alters the cytoplasmic proteome of Escherichia coli BW25113.

E. coli BW25113 cultures were grown to mid-log phase in tryptic soy broth (TSB) and exposed to 70 mM boric acid (a sublethal concentration) or left untreated as a control. Cytoplasmic protein extracts were subjected to 2D-PAGE analysis to identify differentially expressed proteins. Selected protein spots were excised, identified via MALDI-TOF mass spectrometry, and validated by RT-PCR to assess corresponding mRNA expression levels.

Proteomic analysis revealed 12 differentially regulated cytoplasmic proteins under boric acid stress. Upregulated proteins included SodA, KduD, KduI, DeoB, Icd, AceE, RpsM, TdcE, Tuf1, LexA, and LamB, while GatY was downregulated. Functional annotation linked these proteins to oxidative stress defense (SodA), carbohydrate metabolism (KduD, KduI, DeoB), energy production (Icd, AceE), translation (RpsM, Tuf1), and membrane integrity (LamB). RT-PCR validation confirmed transcriptional upregulation of sodA, kduD, and kduI, corroborating proteomic findings. These results suggest that boric acid disrupts metabolic homeostasis, induces oxidative stress, and modulates structural and translational processes in E. coli.

This study provides the first proteomic evidence of E. coli's cytoplasmic response to boric acid stress, highlighting its multifaceted effects on metabolic, oxidative, and translational pathways. The upregulation of KduI and KduD, enzymes involved in carbohydrate utilization, points to potential adaptive mechanisms for boron detoxification. Further investigation into these targets could elucidate molecular strategies for bacterial boron tolerance and inform the development of boron-based antimicrobials.