The SCO2102 Protein Harbouring a DnaA II Protein-Interaction Domain Is Essential for the SCO2103 Methylenetetrahydrofolate Reductase Positioning at Streptomyces Sporulating Hyphae, Enhancing DNA Replication during Sporulation

Abstract

Streptomyces DNA replication starts with the DnaA binding to the origin of replication. Differently to most bacteria, cytokinesis only occurs during sporulation. Cytokinesis is modulated by the divisome, an orderly succession of proteins initiated by FtsZ. Here, we characterised SCO2102, a protein harbouring a DnaA II protein-protein interaction domain highly conserved in Streptomyces. The ΔSCO2102 knockout shows highly delayed sporulation. SCO2102-mCherry frequently co-localises with FtsZ-eGFP during sporulation and greatly reduces FtsZ-eGFP Z-ladder formation, suggesting a role of SCO2102 in sporulation. SCO2102 localises up-stream of SCO2103, a methylenetetrahydrofolate reductase involved in methionine and dTMP synthesis. SCO2102/SCO2103 expression is highly regulated, involving two promoters and a conditional transcription terminator. The ΔSCO2103 knockout shows reduced DNA synthesis and a non-sporulating phenotype. SCO2102-mCherry co-localises with SCO2103-eGFP during sporulation, and SCO2102 is essential for the SCO2103 positioning at sporulating hyphae, since SCO2103-eGFP fluorescent spots are absent in the ΔSCO2102 knockout. We propose a model in which SCO2102 positions SCO2103 in sporulating hyphae, facilitating nucleotide biosynthesis for chromosomal replication. To the best of our knowledge, SCO2102 is the first protein harbouring a DnaA II domain specifically found during sporulation, whereas SCO2103 is the first methylenetetrahydrofolate reductase found to be essential for Streptomyces sporulation.

Keywords: DnaA; SCO2102 DnaA homologue; SCO2103 MTHFR; Streptomyces; cell division; differentiation; sporulation.

Figure 1

SCO2102/2103 genetic region, gene homologies and regulatory elements. (a) SCO2102/03 chromosomal region and homologies. (b) Alignment of DnaA proteins from different model bacteria (adapted from Zawilak-Pawlik et al. [7]). Amino acid numbering in the scheme correspond to E. coli DnaA [6]. The S. coelicolor DnaA (SCO3879) region homologue to the SCO2102 DnaA domain is highlighted in red. (c) Terminator/antiterminator conformations of the conditional terminator identified in the SCO2103 ORF (predicted using the online algorithm developed by Millman et al. [18]).

Figure 2

SCO2102/2103 gene expression in the S. coelicolor wild-type strain. SCO2102 and SCO2103 transcript abundance in GYM solid cultures. Asterisks indicate significant differences compared to the 16 h sample. Transcript abundance of the SCO4758 housekeeping gene [20] was used as control. Three biological replicates were processed.

Figure 3

ΔSCO2102/SCO2103 sporulation on solid cultures. (a–d) Wild-type in different culture media. (e–h) ΔSCO2103 bald phenotype in different culture media. (i–l) ΔSCO2103 bald phenotype in methionine-amended media. (m) Absence of growth of ΔSCO2103 in minimal medium without methionine. (n) ΔSCO2102 sporulation growing on SFM solid cultures. Macroscopic view of the Petri plates (grey colour indicates sporulation), laser-scanning confocal fluorescence images of hyphae stained with SYTO9 and PI and phase-contrast mode images are shown. Representative images from at least three biological replicates are shown. Scale bars indicate 5 µm.

Figure 4

SCO2102/2103 transcript abundances in solid GYM 48 h cultures. (a) SCO2102/2103 transcript abundances in the wild-type and the ΔSCO2102/SCO2103 knockout mutants. (b) SCO2102 transcript abundances in the ΔSCO2102 knockout complemented with the SCO2102 gene controlled by P1 and P2 with and without the conditional transcriptional terminator. Asterisks indicate significant differences compared to the wild-type strain. Three biological replicates were processed.

Figure 5

Sporulation timing in GYM solid cultures of the ΔSCO2102/SCO2103 mutants and their complemented strains. (a–f) Control cultures of the wild-type strain with and without pNG3. (g–n) ΔSCO2103 sporulation with and without different combinations of P1, P2 and the SCO2102-03 genes cloned into pNG3. (o–t) ΔSCO2102 sporulation with and without different combinations of P1, P2 and SCO2102 cloned into pNG3. Normal sporulation (72 h culture) is labelled in green. The constructions used to complement the ΔSCO2102/SCO2103 mutants are outlined and numbered as 1–4. Delayed sporulation is labelled in red. Macroscopic view of the Petri plates (grey colour indicates sporulation), and laser-scanning confocal fluorescence images of hyphae stained with SYTO9 and PI are shown. Representative images from at least three biological replicates are shown. Scale bars indicate 8 µm.

Figure 6

Antibiotic production in ΔSCO2102, ΔSCO2103 and their complemented strains. Antibiotic production was measured in three biological replicates from liquid sucrose-free R5A medium at 168 h once the maximum production was reached. (a,b) Actinorhodin. (c,d) Undecylprodigiosin. The constructions used to complement the ΔSCO2102/SCO2103 mutants are numbered as in Figure 5. Significant differences compared to the wild-type strain are labelled by asterisks.

Figure 7

FtsZ-eGFP and SCO2102-mCherry dynamics in sporulating SFM solid cultures. (a–n) FtsZ-eGFP (green) and SCO2102-mCherry (red) co-expression and cellular localisation in SFM solid cultures. Arrows indicate co-localising FtsZ-eGFP and SCO2102-mCherry spots. Arrow heads indicate FtsZ-eGFP or SCO2102-mCherry spots that do not co-localise. (o–r) FtsZ-eGFP (green) expression in the S. coelicolor wild-type strain. Z-ladders are observed during sporulation (72-h culture). Fluorescence and phase-contrast microscope images are shown. Scale bars indicate 5 µm.

Figure 8

SCO2102-mCherry and SCO2103-eGFP dynamics in sporulating SFM solid cultures. (a–f) SCO2103-eGFP (green) and SCO2102-mCherry (red) cellular localisation in the S. coelicolor wild-type strain. All SCO2102-mCherry spots co-localise with SCO2103-eGFP spots (arrows), but there are SCO2103-eGFP spots that do not co-localise with SCO2102-mCherry spots. (g–l) SCO2103-eGFP expressed in the wild-type and the ΔSCO2102 knockout strains. Fluorescence and phase-contrast microscope images are shown. Scale bars 5 µm.

Figure 9

Chromosomal DNA at the wild-type sporulation time points in GYM cultures of the wild-type strain and the ΔSCO2102/2103 knockout mutants. Significant difference compared to the wild-type strain is labelled by an asterisk.

Figure 10

Model outlining the dynamics of FtsZ (green), SCO2102 (red) and SCO2103 (orange) in sporulating hyphae. SCO2102 and FtsZ co-localise in the wild-type sporulating hyphae. SCO2102 is essential for positioning SCO2103 at sporulating hyphae, increasing MTHFR activity and nucleotide synthesis (dTMP), enhancing chromosomal DNA replication accompanying sporulation. SCO2103 is not positioned at sporulating hyphae in the ΔSCO2102 mutant, reducing dTMP accessibility and delaying sporulation. The absence of SCO2103 MTHFR activity in the ΔSCO2103 mutant would block the DNA synthesis enhanced by SCO2103, generating a non-sporulating phenotype.