A Single Mechanosensitive Channel ProtectsFrancisella tularensissubsp.holarcticafrom Hypoosmotic Shock and Promotes Survival in the Aquatic Environment

ABSTRACTFrancisella tularensissubsp.holarcticais found in North America and much of Europe and causes the disease tularemia in humans and animals. An aquatic cycle has been described for this subspecies, which has caused waterborne outbreaks of tularemia in at least 10 countries. In this study, we sought to identify the mechanosensitive channel(s) required for the bacterium to survive the transition from mammalian hosts to freshwater, which is likely essential for the transmission of the bacterium between susceptible hosts. A single 165-amino-acid MscS-type mechanosensitive channel (FtMscS) was found to protectF. tularensissubsp.holarcticafrom hypoosmotic shock, despite lacking much of the cytoplasmic vestibule domain found in well-characterized MscS proteins from other organisms. The deletion of this channel did not affect virulence within the mammalian host; however,FtMscS was required to survive the transition from the host niche to freshwater. The deletion ofFtMscS did not alter the sensitivity ofF. tularensissubsp.holarcticato detergents, H2O2, or antibiotics, suggesting that the role ofFtMscS is specific to protection from hypoosmotic shock. The deletion ofFtMscS also led to a reduced average cell size without altering gross cell morphology. The mechanosensitive channel identified and characterized in this study likely contributes to the transmission of tularemia between hosts by allowing the bacterium to survive the transition from mammalian hosts to freshwater.IMPORTANCEThe contamination of freshwater byFrancisella tularensissubsp.holarcticahas resulted in a number of outbreaks of tularemia. Invariably, the contamination originates from the carcasses or excreta of infected animals and thus involves an abrupt osmotic downshock as the bacteria enter freshwater. HowF. tularensissurvives this drastic change in osmolarity has not been clear, but here we report that a single mechanosensitive channel protects the bacterium from osmotic downshock. This channel is functional despite lacking much of the cytoplasmic vestibule domain that is present in better-studied organisms such asEscherichia coli; this report builds on previous studies that have suggested that parts of this domain are dispensable for downshock protection. These findings extend our understanding of the aquatic cycle and ecological persistence ofF. tularensis, with further implications for mechanosensitive channel biology.

Study on Pure Aqueous Extraction Method of PHB/Ectoine Co-Products Based on Osmotic Downshock

The simultaneously co-production of Poly-β-hydroxybutyrate (PHB) and ectoine in a process (PHB/Ect co-production) and co-products extraction have great significant for reducing the manufacture cost and promoting industrialization of PHB and ectoine. The pure aqueous extraction method based on osmotic downshock was used for the extraction of PHB/Ect co-products byH. salina. The effects of osmotic pressure, extraction temperature and extraction time on the extraction rate of PHB were investigated. The ectoine was extracted and purified by the techniques of hollow fiber and ion exchange. The optimal conditions for PHB extraction were osmotic downshock in pure water, extraction temperature at 60 °C and extraction for 4 h. The extraction rate of PHB was 87.5%. The extraction rate of ectoine was 84.2%.

Study on Synthesis of PHB by Moderate Halophile and Aqueous Extraction of PHB

PHB (Poly-β-hydroxybutyrate) has been widely in the areas of industrial packaging, pharmacy and agriculture. Currently, its production cost, especially in extraction and purification are high. A moderate halophilic bacterium was used for PHB synthesis and an aqueous extraction method based on osmotic downshock was investigated in this paper to further improve the extraction efficiency. PHB synthesised by moderate halophile with certain NaCl concentration can be released with cell lysis by osmotic downshock. Meanwhile the high temperature, ultrasonic and SDS (Sodium dodecyl sulfate) can enhance the release. After 48 h of fermentation, the production of PHB by Halomonas salina was 6.5 g/L. The extraction rate of PHB was 24.6% with osmotic downshock and 35.4% with additional ultrasonic assistance at 65 °C. A further addition of 0.5% SDS made the extraction rate increase to 75.4%. Thereinto, the addition of 6% NaCl had significantly improved the aqueous extraction rate to 92.3%.

Genetic Complementation of the Obligate Marine Actinobacterium Salinispora tropica with the Large Mechanosensitive Channel GenemscLRescues Cells from Osmotic Downshock

ABSTRACTMarine actinomycetes in the genusSalinisporafail to grow when seawater is replaced with deionized (DI) water in complex growth media. While bioinformatic analyses have led to the identification of a number of candidate marine adaptation genes, there is currently no experimental evidence to support the genetic basis for the osmotic requirements associated with this taxon. One hypothesis is that the lineage-specific loss ofmscLis responsible for the failure of strains to grow in media prepared with DI water. ThemscLgene encodes a conserved transmembrane protein that reduces turgor pressure under conditions of acute osmotic downshock. In the present study, themscLgene from aMicromonosporastrain capable of growth on media prepared with DI water was transformed intoS. tropicastrain CNB-440. The single-copy, chromosomal genetic complementation yielded a recombinantSalinispora mscL+strain that demonstrated an increased capacity to survive osmotic downshock. The enhanced survival of theS. tropicatransformant provides experimental evidence that the loss ofmscLis associated with the failure ofSalinisporaspp. to grow in low-osmotic-strength media.