Role of RpoN from Labrenzia aggregata LZB033 (Rhodobacteraceae) in flagellar formation, motility, biofilm formation and environmental adaptation

Labrenzia aggregata LZB033 ( Rhodobacteraceae ), which produces dimethylsulfoniopropionate (DMSP) and reduces nitrate to nitrogen, was isolated from seawater of the East China Sea. Its genome encodes a large number of transcriptional regulators, which may be important for its adaptation to diverse marine environments. The alternative σ 54 factor (RpoN) is a central regulator of many bacteria, regulating the transcription of multiple genes and controlling important cellular functions. However, the exact role of RpoN in Labrenzia is unknown. In this study, an in-frame rpoN deletion mutant was constructed in LZB033, and the function of RpoN was determined. To systematically identify RpoN-controlled genes, we performed a detailed analysis of gene expression differences between the wild-type strain and theΔ rpoN mutant using RNA sequencing. The expression of 175 genes was shown to be controlled by RpoN. Subsequent phenotypic assays showed that the Δ rpoN mutant was attenuated in flagellar biosynthesis and swimming motility, utilized up to thirteen carbon substrates differently, lacked the ability to assimilate malic acid, and displayed markedly decreased biofilm formation. In addition, stress response assays showed that the Δ rpoN mutant was impaired in the ability to survive under different challenge conditions, including osmotic stress, oxidative stress, temperature changes and acid stress. Moreover, both the DMSP synthesis and catabolism rates of LZB033 decreased after rpoN was knocked out. Our work provides essential insight into the regulatory function of RpoN, revealing that RpoN is a key determinant for LZB033 flagellar formation, motility, biofilm formation and environmental fitness, as well as DMSP production and degradation. Importance This study established an in-frame gene deletion method in the Alphaproteobacteria Labrenzia aggregata LZB033 and generated an rpoN gene mutant. Comparing the transcriptomes and phenotypic characteristics between the mutant and wild-type strains confirmed the role of RpoN in L. aggregata LZB033 flagellar formation, motility, biofilm formation and carbon usage. Most importantly, RpoN is a key factor for survival under different environmental challenge conditions. Furthermore, the ability of synthesize and metabolize dimethylsulfoniopropionate (DMSP) was related to RpoN. These features revealed RpoN to be an important regulator of stress resistance and survival for L. aggregata LZB033 in marine environments.