Mechanism Of Nucleoid Associated Protein Lsr2 Regulating The Expression Of Cryptic Gene Clusters In Streptomyces Roseosporus

Background:Streptomyces are a class of gram-positive bacteria with high GC contents.They can produce abundant secondary metabolites with various structures.These metabolites have a wide range of biological activities and applications in clinic,agriculture and biotechnology.Genomic sequencing have revealed that Streptomyces have greater potentials to produce new metabolites.Compared with biosynthetic gene clusters of known natural products,genomes of Streptomyces contain numerous cryptic biosynthetic gene clusters which are not expressed under laboratory conditions.Streptomyces roseosporus is known for its ability to produce daptomycin which has unique mechanism of action.Streptomyces roseosporus contains 26 biosynthetic gene clusters.There are numerous compounds to explore in addition to several products that have been identified.Nucleoid associated proteins are abundant proteins that influence the organization of bacterial chromosomes.In Streptomyces,nucleoid associated proteins include Hup A,Hup S,s IHF,Lsr2,Bld D and Crp.Nucleoid associated proteins usually bind to different regions of genomes with low sequence specificity and influence global chromosome organization by wrapping,bending or bridging DNA.And they also influence transcriptional patterns in many cases.Lsr2 is a nucleoid associated protein in actinomycetes and is the functional homologous protein of H-NS.Both of them are dissimilar in sequence and structure,but Lsr2 can complete the phenotype of E.coli hns mutant.In Mycobacterium tuberculosis,Lsr2 is able to interact with DNA minor groove and target AT-rich sequences.Lsr2 blocks RNA polymerase and causes transcription repression by bending or bridging DNA in the promoter regions.There are two Lsr2 homologous proteins in all Streptomyces,but its role and mechanism in Streptomyces roseosporus are not clear.It will provide approaches to active cryptic biosynthetic gene clusters and obtain new compounds by studying the mechanism that how Lsr2 influences the transcriptional patterns in Streptomyces roseosropus.Objective:This paper is aimed at investigating the effect of Lsr2 on the chromosome,especially on the biosynthetic gene clusters and the regulatory mechanism in Streptomyces roseosporus.Finally provide a new strategy for activating cryptic biosynthetic gene clusters and discovering new compounds.Method:1.Identify homologous proteins in Streptomyces through performing BLAST with Lsr2 protein sequence in Mycobacterium tuberculosis Lsr2.Then construct the phylogenetic tree of identified homologous proteins in Streptomyces.2.Detect the expression levels of lsr2 and lsr L over time in Streptomyces roseosporus by RT-q PCR.Then construct the deletion strains Δlsr2,Δlsr L and Δlsr2/L by CRISPR Cas9 technology and the phenotypes were detected.Determine which gene has biological function according to previous results and make verification by constructing complement strain.3.In order to study the effect and regulatory mechanism of Lsr2 in Streptomyces roseosporus,detect the transcriptome of the wild type and Δlsr2 fermented in YMS medium through RNA-seq.Then analyze the changes in transcription levels by bioinformatics.4.To determine if Δlsr2 did produce the compound mureidomycin,extract and concentrate the fermentation broth of Δlsr2 and the positive control strain Sros-h A and detect mureidomycin by HPLC and LC-MS.5.To explore the regulatory mechanism of mureidomycin biosynthetic gene cluster by Lsr2,construct deletion strain and overexpression strain of gene mur A to detect mureidomycin.Then employ EMSA to detect the binding capacity of Lsr2 with mur Ap and Mur A with promoter regions in mur gene cluster.6.To study the functional domains of Lsr2,determine the key amino acids at N/Cterminals of Lsr2 in Streptomyces roseosporus by sequence alignment and construct point mutation vector for protein expression and complementation.Then test the functional domains by in vitro biochemical experiments and biological activity assay.7.To investigate whether homologous Lsr2 proteins have the similar abilities to inhibit biosynthetic gene clusters in other Streptomyces,construct lsr2 deletion strains of Streptomyces venezuela,Streptomyces lividans and Streptomyces albus.Detect secondary metabolites by biological activity assay.Result:1.Lsr2 homologous of six Streptomyces species were identified.Compared with Lsr2 from Mycobacterium tuberculosis,one homolog sharing greater sequence similarity was SSGG＿02869(Lsr2),and the other one was SSGG＿03199(Lsr L)in Streptomyces roseosporus.2.RT-q PCR result showed that the expression level of lsr2 was higher than lsr L at all time points,and the transcription of lsr L maintained at a very low level.Compared with the wild type,the loss of lsr2 caused changes on sporulation and secondary metabolism and deletion of lsr L had no effect.The lsr2 complement strain recovered to the wild type.3.The RNA-seq data showed that about 14% genes were differentially expressed after deleting lsr2.Transcription levels of 11 biosynthetic gene clusters changed and the expression of nine gene clusters was inhibited by Lsr2.4.The fermentation products of Δlsr2 and Sros-h A were extracted and concentrated to detect mureidomycin by HPLC and LC-MS.It was confirmed that they both did produce mureidomycin.5.Mur A was a Lux R-tpye transcriptional regulator.Biological activity assay and EMSA indicated that Mur A was a positive regulator of mur gene cluster and it binded to the promoter regions in the gene cluster.Lsr2 was able to bind to mur Ap and inhibited the biosynthesis of mureidomycin.6.The Lsr2 Npm/Cpm protein expression vectors were constructed and Lsr2Npm/Cpm were expressed and purified.EMSA result revealed the binding capacity of Cterminal domain.The point mutation strains lsr2 CM Npm/Cpm were constructed and neither of them could recover to the wild type.7.The lsr2 deletion strains SVEN Δlsr2,SLIV Δlsr2 and SA Δlsr2 were constructed.It was found that lsr2 deletion had effect on the metabolism of Streptomyces lividans and Streptomyces albus.SLIV Δlsr2 produced a red pigment and SA Δlsr2 produce a compound with anti Cryptococcus neoformans activity.No metabolic change was detected in SVEN Δlsr2.Conclusion:There are two Lsr2 homologs in Streptomyces species.The two genes SSGG＿02869and SSGG＿03199 in Streptomyces roseosporus encode Lsr2 and Lsr L respectively.Lsr2 has no significant effect on the growth of Streptomyces roseosporus.Lsr2 acts as a negative regulator that regulates the expression of cryptic gene clusters.Lsr2 inhibits expressions of multiple secondary metabolic gene clusters.Lsr2 inhibits the biosynthesis of mureidomycin by binding the promoter region of mur A,which is the pathway specific regulatory gene of mur gene cluster.Explore the effect of Lsr2 on secondary metabolism in other Streptomyces.This study lays a foundation of expounding the regulatory mechanism of Lsr2 and activating cryptic biosynthetic gene clusters.