Polyphasic Taxonomy Of Actinomycetes From The Dead Seaand Multiomics Study On The Halophilic Mechanism Of Halopolyspora AlbaAFM 10251

It is well known that there are special microorganisms in the extreme environments.The physiological structure and metabolic pathway of the special microorganisms are obviously different from the ordinary environmental microorganisms.It is of great significance to explore the special mechanisms for theoretical research and applied research.Halophilic microorganism is an important branch of the extremophiles that can survive in high salt environment,and their halophilic mechanisms have been a hot spot for microbiologists.Previous research by our group showed that the Dead Sea soil contains abundant halophilic and salt-tolerant actinomycetes.The deep exploration of these resources and the halophilic molecular mechanism of halophilic actinomycetes have important guiding effects for the subsequent development and utilization of such special microorganisms.In this paper,four strains of actinomycetes isolated from the Dead Sea soil were identified using polyphasic taxonomy technique and the genetic information of a new genus and species,Haloactinomyces alba AFM 10251,was analyzed by genome sequencing.Transcriptome and proteome analysis of the gene and protein expression of strain H.alba AFM 10251 under low salt(10%),medium salt(15%)and high salt(20%)conditions were conducted.The obtained results were as following:The ployphasic taxonomy results of phenotypic,chemical and genetic characteristics showed that strain AFM 10258~T was suggested as a new genus,named Haloactinomyces,and the species name was named Haloactinomyces alba;strain AFM 10111~T was suggested as a new species of the genus Amycolatopsis,named Amycolatopsis aureus;strain AFM 10238~T was suggested as a new species of the genus Saccharopolyspora,named Saccharopolyspora griseoalba;strain AFM 10040~T was suggested as a new species of the genus Haloactinomyces,named Haloactinomyces halophilus.The genome of the strain H.alba AFM 10251 contains one chromosome and two plasmids,and 4967 coding genes and 65 RNA coding genes are predicted.Through functional annotation,a total of 3883 functional annotations were obtained,accounting for 78.17%of the total predicted genes,meanwhile 404 unknown functional genes were obtained,accounting for 8.13%of the total predicted genes.The KEGG metabolic pathway analysis revealed the presence of proline,glutamic acid,glycine betaine,tetrahydropyrimidine and trehalose metabolic pathways in the H.alba AFM 10251 genome,and a small number genes related to K~+transport.There also exist some genes related to Na~+transport,such as the primary sodium pump,secondary sodium pump and other Na~+transporters.There were 2753 differentially expressed genes of strain H.alba AFM 10251 under three different salt concentrations,and 768 genes were significant differentially expressed genes.Their functions were mainly related to amino acid transport and metabolism,energy production,lipid transport and metabolism,transcription,protein synthesis and modification,ion transport.It is speculated that the strain may change the physiological state of the strain to adapt to different salt environments.Proteomics research successfully identified 3,255 proteins,of which 66.9%are acidic proteins,and the presence of a large number of acidic proteins may be more conducive to the survival of the strain under high salt conditions.The number of differential translated proteins in medium salt and high salt relative to low salt conditions were 275 and 854,respectively,indicating that the response proteins were increasing with the increase of salt concentration.It can be seen that there are also significant differences in the protein levels of the strain response to different salt stress.All genes related to the synthetic pathways of betaine,tetrahydropyrimidine,proline,trehalose and glutamic acid have high expression levels at both transcriptional and protein levels,and the genes involved in synthesis of the compatible solutes showed obvious difference.There is a significant difference in expression at salt concentration.The strain up-regulated the gene expression of proline and trehalose synthesis pathway at medium salt and high salt concentration,and down-regulated the gene expression related to betaine and tetrahydropyrimidine synthesis pathway.It was speculated that the strain may increase intracellular osmotic pressure mainly by increasing proline and trehalose synthesis in medium salt and high salt environments,while betaine and tetrahydropyrimidine were more important in strains dealing with low salt stress.The change in the gene expression related to the compatible solutes synthesis indicated that the osmotic pressure regulation of the strain may be altered by a plurality kinds of compatible solutes,and the intracellular osmotic pressure could be adjusted by changing the proportion of compatible solutes.Under high salt conditions,the expression of K~+transporter,KdpD and TrkA,and secondary sodium pumping protein,NhaA,were up-regulated at the transcriptional level and protein level;under medium salt conditions,the expression of the primary sodium pump constituent proteins NuoB and NuoD were up-regulated in transcription level and protein level.It was predicted that the strain may excrete excess Na~+by enhancing the expression of the primary sodium pump under the medium salt environment.Under high salt environments,it may increase the intracellular osmotic pressure by enhancing the importing of K~+,and meanwhile,enhancing the secondary sodium pump function to efflux excess Na~+.The strains adjusted the genes expression levels related to the five compatible solute synthesis pathways under different salt concentrations,and adjusted the genes expression levels related to K~+accumulation and Na~+efflux.It was speculated that the regulation of cell osmotic pressure was achieved by compatible solute synthesis,K~+accumulation and Na~+efflux.And the strain could adjust osmotic pressure by different regulatory pathways under different salt conditions.Thus the osmotic pressure regulation mechanism of the strain H.alba AFM10251 is complicated.In addition,we found that the proteomic analysis results are not completely consistent with the results of the transcriptome analysis,indicating that there were more complex post-transcriptional regulatory mechanisms.The study also found 54 proteins with unknown function were highly expressed or significantly differentially expressed under three different salt concentrations,which were obviously involved in the response to salt stress.The analysis showed that the homologues of these proteins are all halophilic or salt-tolerant bacteria strains,and their functions may also likely to be related to the regulation of osmotic pressure,which deserve high attention.