Antimicrobial And Defensive Effects Of Extracellular Protease From Haloarchaea And Identification Of A Haloarchaeal New Species

Haloarchaea is a type of aerobic or facultative anaerobic Archaea,which thrive in the salt lake,saline-alkali land,solar salt,subterranean salt mine and other hypersaline environments.They are not only being taken as a good model organism for investigating the molecular mechanism of adaptation extremely hypersaline environment,but also have a broad application prospectives in the remediation of pollution in salted environment and the production of biodegradable plastics.In this study,gene knockout and complementation strategy,culture-dependent and polyphasic taxonomy methods were used to dissect the multi-functions of extracellular protease from haloarchaea in antimicrobial and defensive effects against other haloarchaeal cells,the strategies of haloarchaea cells in response to low osmotic impact,and a new species isolated from hypersaline environment.The results indicated that:(1)hlyR4 was the sole gene encoding an extracellular serine protease in Hfx.mediterranei ATCC 33500 that exhibited antagonistic activity against some haloarchaeal strains.Deletion of the hlyR4 completely eliminated the inhibition activity of Hfx.mediterranei to other haloarchaea,while complementation of the hlyR4 restored its inhibition activity.Furthermore,Hfx.mediterranei strains containing the hlyR4 showed a resistance to halocins and halolysins in milieu to some extent.This function of the hlyR4 has been verified in Haloarcula hispanica.When hlyR4 was introduced into strain Haloarcula hispanica DF60,the strain acquired the ability of resisting the attacks from halocins and halolysins.In addition,hlyR4 was probabaly expressed constitutively that was different from other extracellular protease genes.(2)The content of the compatible solute betaine in the hypotonic condition resistant strain was much higher than that in the hypotonic condition sensitive strain(about 2.5 times),while the intracellular potassium concentration was opposite.After washing with distilled water for several times,the hypotonic condition sensitive cells were completely lysed,while the cells of the hypotonic condition resistant cells were barely or rarely lysed,and cells were still alive.The cell wall thickness of the hypotonic condition sensitive cells was very thin,only about 10-20 nm,while the cell wall thickness of the hypotonic condition resistant strain was significantly thicker than that of the lysis hypotonic condition sensitive cells reached about 100-140 nm,about 10-15 times that of the hypotonic condition sensitive cells.(3)A pleomorphic halophilic archaeon named strain ZC67~T,was isolated from the Yuanyongjing Salt Mine,Yunnan,China.Based on similarity search and phylogenetic analysis of the 16S rRNA gene sequence,the closest relative species of strain ZC67~T was Halorubrum saccharovorum JCM 8865~T,and the sequence similarity of 16S rRNA gene was 99.0%.The average nucleotide identity(ANI)and the average amino-acid identity(AAI)indexes bewtween ZC67~T and its closest relatives were 88.9%and 89.3%,respectively,which were far below 95%,the criterion for delineation of the new haloarchaeal species.In silico DNA relatedness between strain ZC67~T and its closest relatives was 37.8%,which is also less than the70%.The major polar lipids profile of strain ZC67~T was the same as that of the type sepcies of the genus Halorubrum.The DNA G+C content of ZC67~T was 66.3 mol%(genome calculation).In addition,in combination with other characteristics,such as phenotypic and physiological and biochemical differences,strain ZC67~T was proposed to represent a new species of Halorubrum.The strain ZC67~T was named Halorubrum amylolyticum ZC67~T due to its hydrolysis of starch.The type strain of this new species was ZC67~T(=CGMCC 1.15718~T=JCM 31850~T)?This study provides a new perspective for better understanding the complex interactions of microorganisms in extremely hypersaline environments,and criticalclues for learning how halophilic archaea,which mainly live in a hyperosmotic environment,cope with the low osmotic impact.In addition,this study will also accumulate valuable haloarchaeal genetics and species resources for exploring the adaptation mechanism of extremophiles to the environment.