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Abstract
Vibrio vulnificus, a gram-negative halophilic marine bacterium and opportunistic pathogen, must withstand various environmental changes, especially the simultaneous change of temperature and salinity (SCTS) from 25°C/2.5% to 37°C/0.9% upon entering the human body. Previous studies have suggested that temperature and salinity may affect the production of metalloprotease VvpE via the LuxS-mediated autoinducer-2 quorum sensing system (AI-2-QSS). However, this hypothesis remains to be verified through coherent experiments. In this study, SCTS stimulated V. vulnificus growth with no increase in total growth levels. The SCTS-mediated prolongation of the stationary growth phase resulted in a significant increase in growth phase-dependent luxS and vvpE transcriptions; however, SCTS did not affect luxS or vvpE transcription levels during the exponential growth phase. SCTS also advanced extracellular VvpE production, which was consistent with vvpE transcription and V. vulnificus growth. SCTS-mediated modulation of vvpE expression was slightly attenuated but still observed in the background of a luxS mutation which seriously repressed vvpE expression. These results indicate that SCTS stimulates luxS and vvpE expression by stimulating V. vulnificus growth; however, the LuxS-mediated AI-2-QSS plays only a minor role, if any, in the SCTS-mediated modulation of vvpE expression.
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Figure 1.
Effect of the simultaneous change of temperature and salinity from 25°C/2.5% to 37°C/0.9% on luxS transcription. After preconditioning by culturing at 25°C/2.5% overnight, the V. vulnificus RC138 strain with the PluxS::lacZ transcriptional fusion was transferred into fresh broths and cultured with vigorous shaking at 25°C/2.5% or 37°C/0.9%. Bacterial growth (A) was expressed as the optical density of culture aliquots at a wavelength of 600 nm. Accumulated β-galactosidase activity in culture aliquots was expressed as the Miller unit, and plotted against culture time (B) and bacterial growth (C). Means and standard deviations were from triplicate measurements.
Figure 2.
Effect of a luxS mutation on the regulation of vvpE transcription by the simultaneous change of temperature and salinity from 25°C/2.5% to 37°C/0.9%. After preconditioning by culturing at 25°C/2.5% overnight, the two V. vulnificus strains with PvvpE::lacZ transcriptional fusion, CMM2106 with wild-type luxS (A to C) and CMM2207 with mutated luxS (D to F) strains, were transferred to fresh Heart Infusion broths and cultured with vigorous shaking at 25 °C/2.5% or 37°C/0.9%. Bacterial growth (A and D) was expressed as the optical density of culture aliquots at a wavelength of 600 nm (OD600). Accumulated β-galactosidase activity in culture aliquots was expressed as the Miller unit, and plotted against culture time (B and E) and bacterial growth (C and F). Means and standard deviations were from triplicate measurements.
Figure 3.
Effect of a luxS mutation on the regulation of extracellular VvpE production by the simultaneous change of temperature and salinity from 25°C/2.5% to 37°C/0.9%. After preconditioning by culturing at 25°C/2.5% overnight, the V. vulnificus M06-24/O, CMM2201 (with the mutated luxS gene) and CMM2211 (with the in trans complemented luxS gene) strains were transferred to fresh Heart Infusion broths and cultured with vigorous shaking at 25°C/2.5% or 37°C/0.9%. (A) Bacterial growth was expressed as the optical density of culture aliquots at a wavelength of 600 nm (OD600). (B) Culture supernatants were obtained by centrifugation of culture aliquots to measure extracellular VvpE production by Western blotting. A representative one of the twice repeated experiments is shown. Arrows indicate the two forms of VvpE.
Table 1.
Bacterial strains, plasmids and primers used in this study
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