The evolution of an osmotically inducible dps in the genus Streptomyces

Abstract

Dps proteins are found almost ubiquitously in bacterial genomes and there is now an appreciation of their multifaceted roles in various stress responses. Previous studies have shown that this family of proteins assemble into dodecamers and their quaternary structure is entirely critical to their function. Moreover, the numbers of dps genes per bacterial genome is variable; even amongst closely related species - however, for many genera this enigma is yet to be satisfactorily explained. We reconstruct the most probable evolutionary history of Dps in Streptomyces genomes. Typically, these bacteria encode for more than one Dps protein. We offer the explanation that variation in the number of dps per genome among closely related Streptomyces can be explained by gene duplication or lateral acquisition, and the former preceded a subsequent shift in expression patterns for one of the resultant paralogs. We show that the genome of S. coelicolor encodes for three Dps proteins including a tailless Dps. Our in vivo observations show that the tailless protein, unlike the other two Dps in S. coelicolor, does not readily oligomerise. Phylogenetic and bioinformatic analyses combined with expression studies indicate that in several Streptomyces species at least one Dps is significantly over-expressed during osmotic shock, but the identity of the ortholog varies. In silico analysis of dps promoter regions coupled with gene expression studies of duplicated dps genes shows that paralogous gene pairs are expressed differentially and this correlates with the presence of a sigB promoter. Lastly, we identify a rare novel clade of Dps and show that a representative of these proteins in S. coelicolor possesses a dodecameric quaternary structure of high stability.

Figure 1. Amino acid alignment of S. coelicolor Dps proteins.

Amino acid alignment of the three S. coelicolor Dps proteins showing the position of the five characteristic Dps helices along with the position of the different length N- and C-terminal tails.

Figure 2. A: Immunoblot of a native PAGE gel.

Immunoblot showing the in vivo oligomeric state of DpsA_Sc, DpsB_Sc and DpsC_Sc overexpressed using a thiostrepton inducible promoter. Overexpression of DpsA (lane A), DpsB (lane B) and DpsC (lane C) from a thiostrepton inducible promoter. Black arrow indicates the non-dodecameric DpsB. B: Coomassie blue stained native PAGE gel assessing the stability of assembled Dps_Sc oligomers. Coomassie blue stained native PAGE gel showing the stability of assembled S. coelicolor Dps oligomers after incubation with 8 M urea. Lane A−  =  DpsA_Sc without 8 M urea, lane A+  =  DpsA_Sc + 8 M urea, Lane C−  =  DpsC_Sc without 8 M Urea, lane C+  =  DpsC_Sc + 8 M urea.

Figure 3. Majority rule consensus phylogenetic tree of Actinobacterial Dps orthologous proteins.

Maximum-likelihood reconstructed phylogenetic tree of Actinobacterial Dps proteins. Indicated are three orthologous protein clusters (shaded boxes). DpsA_Sc, DpsB_Sc and DpsC_Sc indicates the position of S. coelicolor Dps in the tree. Paralogous gene pairs of DpsB in Streptomyces are indicated using matched Roman numerals. σB indicates those proteins where a putative sigB-like promoter was identified. Bootstrap values >60% are indicated next to major nodes.

Figure 4. Normalized fold change in dps transcript abundance among five Streptomyces in response to osmotic stress.

q RT PCR monitoring of dps transcript abundance after 1 hour incubation with 250 mM KCl in S. co.  =  S. coelicolor; S. gha  =  S. ghanaensis; S. alb  =  S. albus; S. av  =  S. avermitilis; S. ven.  =  S. venezuelae. Relationships to S. coelicolor dps orthologs are indicated in parentheses. dps transcript abundance is normalized to principal sigma factor hrdB. Paralogous gene pairs are sequentially numbered. Presence of a sigB-like promoter motif upstream of the ORF is indicated with an asterisk. Error bars represent standard deviation of the mean normalized transcript abundance. A broken Y-axis has been used.

Figure 5. dps location maps among 17 Streptomycete chromosomes.

Approximate chromosome location of dps orthologs in 17 Streptomycete chromosomes. Species names are abbreviated to the left of the diagram: S. gha  =  S. ghanaensis; S. griseof  =  S. griseoflavus; S. prist  =  S. pristinaespiralis; S. co.  =  S. coelicolor; S. liv.  =  S. lividans; S. alb  =  S. albus; S. flavo  =  S. flavogriseus; S.gris  =  S. griseus; S. virido  =  S.viridochromogenes; S. scab  =  S. scabies; S. aver  =  S. avermitilis; S. clav  =  S. clavuligerus; S. gris. gris  =  S. griseus subsp. griseus; S. him  =  S. himastatinicus; S. svic  =  S. sviceus; S. griseoaur  =  S. griseoaurantiacus; S. venez  =  S. venezuelae. Chromosomes were orientated based on the centrally located initiator protein (black arrow). Light grey arrows represent orthologs of dpsA_Sc, clear arrows represent orthologs of dpsC_Sc, striped arrows represent orthologs of dpsB_Sc. Figures above arrows represent approximate chromosome location in Mb. Figures in parentheses  =  chromosome size. Asterisks indicate those genes where we identified sigB-like promoter motifs upstream of ORFs. Scale bar  =  1 Mb.

Figure 6. Schematic representation of the genomic neighbourhood of dpsB_Sc in Streptomyces.

Consensus genomic neighbourhood around dpsB_Sc orthologs (without a sigB promoter) in completely sequenced and assembled Streptomyces genomes. The nomenclature of genes follows that of S. coelicolor orthologs. Numbers in parenthesis represents (as a percentage) the number of times that gene occurs in that position among all the Streptomyces tested. Genes that do not match 100% are not shown. H/p  =  hypothetical protein.