Positive Regulation of Spoilage Potential and Biofilm Formation in Shewanella baltica OS155 via Quorum Sensing System Composed of DKP and Orphan LuxRs

Abstract

The spoilage potential and biofilm formation of Shewanella baltica are reported to be regulated by Quorum sensing (QS) system from the phenotype point of view, but the specific mechanism is not fully understood. In the present study, the QS autoinducers were detected by UHPLC-MS/MS, cell density-dependent luxR-type genes were obtained through autoregulation experiments among a series of candidates in S. baltica OS155 (The SSO of large yellow croaker). The direct interaction between cyclo-(L-Pro-L-Phe) (PP) and LuxR01 as well as LuxR02 proteins was revealed via in vitro binding assay. Deletion of luxR-type genes (luxR01 and luxR02) impaired spoilage potential and biofilm formation of S. baltica OS155 in various degrees. Transcriptional analysis and qRT-PCR validation showed that spoilage and biofilm-related genes torS, speF, and pomA were down-regulated in luxR01 and luxR02 deletion strains. In addition, exogenous PP promoted spoilage potential and biofilm formation, which could be attenuated by luxR01 or luxR02 deletion. Our results revealed an explicit QS system employing PP as autoinducer and two orphan LuxRs as receptors which positively regulated spoilage capacity and biofilm formation via transcriptional regulation of corresponding genes in S. baltica OS155, which provides potential specific targets for seafood preservation involving QS system.

Keywords: biofilm formation; diketopiperazines; orphan LuxR-type proteins; quorum sensing; spoilage.

FIGURE 1

Identification of cell density-dependent LuxR-type proteins in Shewanella baltica OS155. Effects of post exponential supernatant extract on the transcription of provisional luxR-type genes, including luxR01, luxR02, luxR03, luxR04, luxR05 and luxR06, were quantified by qRT-PCR in S. baltica OS155. Data was presented as the mean ± standard deviation (n = 3, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).

FIGURE 2

The direct interaction between cyclo-(L-Pro-L-Phe) (PP) molecule and LuxR01 as well as LuxR02 proteins. PP molecule captured by purified LuxR01 (A) and LuxR02 (B) in the mixture of autoinducer candidates was detected using UHPLC-MS/MS as compared with the pET-15b vector control (C). PP: cyclo-(L-Pro-L-Phe).

FIGURE 3

Spoilage potential of gene deletion mutants. The TMA and putrescine production of wild-type and each deletion mutant (SB7301, SB7302, SB7303, SB7304, SB7305, and SB7306) strain were measured at the addition of 10 mM (A1) and 100 mM (A2) TMAO, 0.5% (B1), and 2% (B2) L-Ornithine monohydrochloride and L-Arginine monohydrochloride, respectively. Data was presented as the mean ± standard deviation (n = 3, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 4

Biofilm formation activity of gene deletion mutants. (A) Biofilm formation of wild-type and each deletion mutant (SB7301, SB7302, SB7303, SB7304, SB7305, and SB7306) strains was quantified by microtiter plate assay. (B) Biofilm formation of wild-type, SB7301 and SB7302 mutant strains was analyzed by CLSM imaging in blend mode view (left panel) and section mode view (right panel). The scale bars represent 50 μm. (C) The mean maximum thickness of biofilms obtained from CLSM imaging. Data was presented as the mean ± standard deviation (n = 3, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).

FIGURE 5

Cyclo-(L-Pro-L-Phe) promotes biofilm formation in a LuxR-type protein-dependent manner. (A) Biofilm formation of wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was quantified by microtiter plate assay. (B) Biofilm formation of wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was analyzed by CLSM imaging in blend mode view (left panel) and section mode view (right panel). The scale bars represent 50 μm. (C) The mean maximum thickness of biofilm formed in wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was calculated by IMARIS software. Data was presented as the mean ± standard deviation (n = 3, ^∗p < 0.05, ^∗∗∗p < 0.001).

FIGURE 6

Cyclo-(L-Pro-L-Phe) promotes spoilage activity in a LuxR-type protein-dependent manner. The TMA (A) and putrescine (B) production of wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment were measured. Data was presented as the mean ± standard deviation (n = 3, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 7

Transcription verification in gene deletion mutants. The expression level of torS, speF, and pomA genes in wild-type strain, SB7301 and SB7302 mutants were quantified by qRT-PCR. Data was presented as the mean ± standard deviation (n = 3, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

FIGURE 8

Scheme representing the QS system in regulating spoilage potential and biofilm formation in S. baltica OS155. At high cell density, QS signal PP reaches a threshold level and is detected by specific receptor LuxR01 and/or LuxR02 protein, which acts as transcription regulator to alter the expression of torS, speF, and pomA. Among them, TorS functions as a signal-transduction cascade mediator and transphosphorylates TorR upon the presence of TMAO, which finally activate torA expression to reduce TMAO into TMA. speF gene is responsible for the production of putrescine from ornithine. pomA gene promotes the biofilm formation by regulating swimming motility.