Presentation Information
[P01-131]Analysis of lipopolysaccharide of the toluene-degrading bacterium Acinetobacter sp. Tol 5
○Haru Chikusa1, Shogo Yoshimoto1, Shori Inoue1, Katsutoshi Hori1 (1. Dept. Biomolecular Engineering, Grad. Sch. Engineering, Nagoya University (Japan))
Keywords:
Lipopolysaccharide,Cell surface,Gram-negative bacteria,Toluene
[Purpose]
Acinetobacter sp. Tol 5 is a toluene-degrading bacterium that exhibits exceptionally high adhesiveness and the ability to utilize a wide range of hydrophobic compounds, making it a promising platform for gas-phase bioproduction using immobilized cells. Despite its biotechnological potential, the mechanism underlying its interaction with and uptake of hydrophobic compounds remains poorly understood. Because lipopolysaccharide (LPS) is a major structural component of the outer membrane that affects membrane integrity, permeability, and cell surface interactions, it is a plausible determinant of these unique phenotypes. In this study, we aimed to identify genes involved in LPS biosynthesis in Tol 5 and to evaluate how LPS structure influences its cell surface properties and responses to hydrophobic compounds.
[Methods]
Putative genes involved in LPS biosynthesis in Tol 5 were identified by BLAST analysis based on known LPS biosynthesis genes in A. baumannii. Deletion mutants targeting these putative LPS biosynthesis genes were then constructed using cytidine base editing. The resulting mutants were subjected to LPS extraction and analyzed to evaluate the effects of LPS truncation on cell surface properties, including growth on substrates, tolerance to organic solvents, and uptake capacity for hydrophobic compounds.
[Results]
LPS extraction analysis showed that Tol 5 possesses rough-type LPS lacking an O-antigen, and that the constructed mutants produced shorter LPS than the wild type. Functional characterization further revealed that the constructed mutants exhibited reduced growth on hydrophobic small molecules. In contrast, no marked change was observed in organic solvent tolerance, whereas the uptake capacity for hydrophobic small molecules was enhanced relative to the wild type. These results indicate that alteration of LPS structure affects the interaction of Tol 5 with hydrophobic compounds and significantly changes its cell surface-associated phenotypes.
[Consideration]
Although LPS truncation did not alter organic solvent tolerance, it enhanced the uptake of hydrophobic small molecules while reducing growth on these compounds. This result suggests that the increased permeability or accessibility of hydrophobic compounds in the LPS mutants promoted their excessive intracellular accumulation, thereby increasing intracellular toxicity and impairing growth.
[Conclusion]
We successfully identified genes involved in LPS biosynthesis in Tol 5. In addition, it was revealed that Tol 5 possesses rough-type LPS lacking an O-antigen. Our results further suggest that LPS structure contributes to the regulation of hydrophobic compound uptake and cell surface properties in Tol 5.
[Reference]
VanOtterloo LM, et al., mBio, 15(3):e0301323
Acinetobacter sp. Tol 5 is a toluene-degrading bacterium that exhibits exceptionally high adhesiveness and the ability to utilize a wide range of hydrophobic compounds, making it a promising platform for gas-phase bioproduction using immobilized cells. Despite its biotechnological potential, the mechanism underlying its interaction with and uptake of hydrophobic compounds remains poorly understood. Because lipopolysaccharide (LPS) is a major structural component of the outer membrane that affects membrane integrity, permeability, and cell surface interactions, it is a plausible determinant of these unique phenotypes. In this study, we aimed to identify genes involved in LPS biosynthesis in Tol 5 and to evaluate how LPS structure influences its cell surface properties and responses to hydrophobic compounds.
[Methods]
Putative genes involved in LPS biosynthesis in Tol 5 were identified by BLAST analysis based on known LPS biosynthesis genes in A. baumannii. Deletion mutants targeting these putative LPS biosynthesis genes were then constructed using cytidine base editing. The resulting mutants were subjected to LPS extraction and analyzed to evaluate the effects of LPS truncation on cell surface properties, including growth on substrates, tolerance to organic solvents, and uptake capacity for hydrophobic compounds.
[Results]
LPS extraction analysis showed that Tol 5 possesses rough-type LPS lacking an O-antigen, and that the constructed mutants produced shorter LPS than the wild type. Functional characterization further revealed that the constructed mutants exhibited reduced growth on hydrophobic small molecules. In contrast, no marked change was observed in organic solvent tolerance, whereas the uptake capacity for hydrophobic small molecules was enhanced relative to the wild type. These results indicate that alteration of LPS structure affects the interaction of Tol 5 with hydrophobic compounds and significantly changes its cell surface-associated phenotypes.
[Consideration]
Although LPS truncation did not alter organic solvent tolerance, it enhanced the uptake of hydrophobic small molecules while reducing growth on these compounds. This result suggests that the increased permeability or accessibility of hydrophobic compounds in the LPS mutants promoted their excessive intracellular accumulation, thereby increasing intracellular toxicity and impairing growth.
[Conclusion]
We successfully identified genes involved in LPS biosynthesis in Tol 5. In addition, it was revealed that Tol 5 possesses rough-type LPS lacking an O-antigen. Our results further suggest that LPS structure contributes to the regulation of hydrophobic compound uptake and cell surface properties in Tol 5.
[Reference]
VanOtterloo LM, et al., mBio, 15(3):e0301323
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