FIG 2.

Construction of the intracellular alkane detection system. (A) Schematic diagram of the construction process. alkMa and almA are alkane hydroxylases responsible for C₂₈ oxidation in Acinetobacter venetianus RAG-1; alkRa is a putative transcriptional activator that is essential for the expression of alkMa; altL is the potential alkane transporter; gfp represents the green fluorescent protein gene, which replaced the alkMa gene in ΔalmA/alkMa::gfp-pKRG and ΔaltL/almA/alkMa::gfp-pKRG as a reporter. almA was also deleted in ΔalmA/alkMa::gfp-pKRG and ΔaltL/almA/alkMa::gfp-pKRG to eliminate the process of C₂₈ degradation in RAG-1. The expression of gfp can be activated in the presence of alkRa and C₂₈; hence, the expression of GFP can be used as a reporter to reflect the presence of intracellular alkanes in ΔalmA/alkMa::gfp-pKRG. (B) Transcriptional levels of genes alkRa and alkMa in strains WT, ΔalkRa, and ΔalkRa/PalkRa with C₂₈ as the sole carbon source. The expression of alkRa and alkMa in the WT strain was used as a control. (C) GFP fluorescence in strain ΔalmA/alkMa::gfp-pKRG in the absence or presence of C₂₈. GFP fluorescence was calculated as relative fluorescence units (F/OD), which represent fluorescence values per OD₆₀₀. SA represents BSM medium supplemented with 0.5% sodium acetate, and SA+C28 represents BSM medium supplemented with 0.5% sodium acetate and 0.1% C₂₈. Values are shown as means ± SD (n = 3) and were analyzed by independent-sample t test (**, P < 0.01; ***, P < 0.001).