The role of two major nucleoid associated proteins in Streptomyces, HupA and HupS, in stress survival and gene expression regulation

Abstract

Streptomyces are sporulating soil bacteria with enormous potential for secondary metabolites biosynthesis. Regulatory networks governing Streptomyces coelicolor differentiation and secondary metabolites production are complex and composed of numerous regulatory proteins ranging from specific transcriptional regulators to sigma factors. Nucleoid associated proteins (NAPs) are also believed to contribute to regulation of gene expression. Upon DNA binding these proteins impact DNA accessibility. Among NAPs HU proteins are the most widespread and abundant. Unlike other bacteria, the Streptomyces genome encodes two HU homologs: HupA and HupS, differing in structure and expression profile. In this study, we explore whether HupA and HupS affect S. coelicolor growth under optimal and stressful conditions and how they control global gene expression. By testing both single and double mutants we address the question of both HU homologs complementarity. The lack of both hup genes led to growth and sporulation inhibition, as well as increased spore fragility. Our data indicate a synergy between the functions of HupA and HupS during S. coelicolor growth. We also demonstrate, that both HU homologs can be considered global transcription regulators influencing expression of numerous genes encoding proteins linked to chromosome topology, secondary metabolites production and transcription. We identify the independent HupA and HupS regulons as well as genes under the control of both HupA and HupS proteins. Our data indicate some extent of redundancy as well as independent function of both homologs.

Importance Streptomyces belong to the bacterial family widely used in the production of antibiotics as well as research for new bioactive substances with antimicrobial properties. Gene expression in Streptomyces, and consequently the production of secondary metabolites, is controlled by a vast and complex network of transcriptional regulators. Our data indicate that two proteins, HupA and HupS, involved in the maintenance of chromosome structure, also participate in this regulatory network. Their presence appears to important for S. coelicolor’s adaptation for survival in unfavorable conditions such as high temperature. The lack of one or both HU proteins affects the expression of many genes, indicating that they act as global transcriptional regulators.

Introduction

Nucleoid associated proteins (NAPs) are bacterial proteins that perform a role similar to eukaryotic histones. By coating, bridging and bending DNA molecules, these proteins organize and compact DNA. They also share other properties with histones, such as small size, a high content of basic amino acids, and a lack of (or very low) DNA sequence specificity. What is more, similarly to histones, by modifying the accessibility of DNA to transcriptional machineries, they play a role in the regulation of gene expression (1, 2).

In many bacterial cells, the most abundant NAP is a small, positively charged protein HU (Heat Unstable) (3). In Escherichia coli, HU abundance, estimated to be between 30, 000 and 55, 000 HU molecules per cell, reaches its peak during exponential growth (4). Like other NAPs, this protein exhibits little sequence specificity, but shows increased affinity to supercoiled, single stranded or distorted DNA (5–7). Interestingly, the impact of HU binding on DNA structure in vitro depends on protein/DNA molar ratio and osmolarity. At law salt concentration, HU promotes DNA compaction, while at high salt concentration, its binding leads to the formation of “rigid filaments” (8–10). In vivo HU homologs affect gene expression and change the distribution of RNA polymerase by altering DNA topology within promoter regions or promoting long-range DNA contacts (11, 12). Recent studies have revealed that HU homologs influence transcription of genes connected to stress response or virulence in many bacterial species including: E. coli (13, 14) Salmonella enterica (15), Vibrio parahaemolyticus (16), Francisella tularensis (17) and Helicobacter pylori (18).

Most of the Proteobacteria and Bacteroidetes, like E. coli, possess two HU homologs that form homo- or heterodimers, while other phyla have only one HU homolog which forms a homodimer (19). In E. coli two HU homologs, HUα and HUβ, share 69% amino acid identity (20), but differ in affinity for different DNA structures, binding modes and their level during culture growth (5, 21, 22). Notably, actinobacterial HU homologs form a distinct group and are characterized by the presence of a long, positively charged C-terminal domain (23). In Mycobacteria, the presence of C-terminal domain is necessary for DNA binding (24, 25). Interestingly, a few actinobacterial orders, namely Streptomycetales, Propionibacteriales, Kineosporiales and Micrococcales, possess two HU homologs differing in structure: one with an extended C-terminal domain and one similar to E. coli HU.

Streptomyces are soil-dwelling bacteria known for antibiotic production and a complex life cycle that includes sporulation. Streptomyces coelicolor long, linear chromosome (8.6 Mbp) contains more than 20 biosynthetic gene clusters involved in production of secondary metabolites (26). Most of them remain inactive during growth, while a few are activated before the start of sporulation (27). The structure of the Streptomyces chromosome also changes during growth, from uncondensed in vegetative hyphae to tightly packaged in unigenomic spores or late stationary phase (28, 29). These changes of Streptomyces chromosome organisation seem to be related, among other factors, to changes in the levels of two HU homologs. According to proteomic and transcriptomic data, HupA is the most abundant NAP during vegetative growth (30, 31) and binds preferentially at the central region of chromosome (32). HupS levels increase during sporulation and the protein shows enhanced binding in terminal regions of chromosome (29). In contrast to E. coli HU homologs, HupA and HupS share little sequence homology. HupA is more similar to the canonical E. coli HU, but has only 38% identity with N-terminal domain of HupS. The long positively charged C-terminal domain of HupS contains multiple lysine repeats, similar to those found in other Actinobacterial proteins such as topoisomerase I (TopA) (30, 33–35). Interestingly, the lack of only one HU homolog moderately affects Streptomyces growth. Deletion of hupA in both S. coelicolor and S. lividans reduced their growth rate (32, 36), while hupS deletion in S. coelicolor and S. venezuelae resulted in diminished compaction of chromosome in spores, which were more sensitive to high temperatures than wild type spores (29, 33).

Given the minimal phenotype of either hupA or hupS deletion mutants, we expected that the functions of HupA and HupS may be partially complementary. The cooperation between HupA and HupS has not yet been described. Thus, in this work we sought to examine the consequences of hupA and hupS deletion for S. coelicolor growth under optimal and stressful conditions. Given that NAPs binding impacts transcription, the phenotype of hupS and hupA mutant strains could be at least partially attributed to their influence on transcription. Therefore, we also set out to establish HupA and HupS regulons in S. coelicolor. We show that both HU homologs regulate genes involved in secondary metabolism and stress response. Comparison of HupA and HupS regulons allowed us to determine the extent of HupA and HupS cooperation. Taken together, our results suggest that, apart from their structural roles, HupA and HupS binding has as impact on global gene expression, facilitating survival under various environmental conditions.

Materials and Methods

Growth conditions and genetic modifications of bacterial strains

The E. coli and S. coelicolor strains used are listed in Supplementary Table S1. The culture conditions, antibiotic concentrations, and transformation and conjugation methods followed the general procedures for E. coli (37) and Streptomyces (38). For plate cultures of S. coelicolor strains, minimal medium supplemented with 1% mannitol (MM) or soy flour medium (SFM) was used. For the growth rate evaluation, S. coelicolor cultures in YEME/TSB were inoculated with spores to final 0.01 U/ml (1 U of spores increases medium absorbance by 1) and cultured in microplates (250 μl per well), for 72h at 30°C using a Bioscreen C (Automated Growth Curves Analysis System, Growth Curves USA), with five experimental replicates for each strain. S. coelicolor growth curves were analyzed using the log-logistic model, for each strain half-time was determined.

In order to construct strain lacking hupA and hupS genes we plasmid pKF289 (33) was introduced into ASMK011 strain (ΔhupA::scar). After conjugation colonies resistant to hygromycin were obtained yielding strain ASMK019 (ΔhupA::scar ΔhupS::higro), which was verified using PCR. In order to create a complementation strain, hupA gene with its native promoter was amplified using hupA_pSET_FW and hupA_pSET_RV primers, and then ligated into a pSET152 vector. Obtained was plasmid was introduced into ASMK019 strain. After conjugation colonies resistant to hygromycin and apramycin were obtained yielding strain ASMK019.2 (ΔhupA::scar ΔhupS::higro pSET152hupA). DNA manipulations were carried out by standard protocols (37). The genetic modifications of the obtained strains were verified by PCR and sequencing. The oligonucleotides used for PCR are listed in Supplementary Table S2.

Stress sensitivity analyses

First, spore concentrations were measured using a Thoma Cell Counting Chamber and Leica DM6 B fluorescence microscope equipped with a 40x objective. Spores were subjected to either increased temperature (60°C for 15-45 min) or detergent (2.5-10% SDS for 1h in room temperature). Next serial dilutions of spores were plated on SFM medium. To test UV sensitivity, spores were first plated and then exposed to UV light for 15-45s. For oxidative stress analysis serial dilutions of spores were plated on the SFM medium containing increasing concentration of H₂O₂ (0 – 1 mM) and incubated in 30°C for 5 days. After 5 days of incubation at 30°C the number of growing colonies was counted to determine the percentage of plated spores that survived the stress.

Microscopy analysis

For microscopy analysis S. coelicolor spores were cultured for 44 hours on microscopy coverslips inserted at a 45° angle in a MM solid medium containing 1% mannitol and then mycelia were fixed with a 2.8% paraformaldehyde/0.00875% glutaraldehyde mixture for 10 min at room temperature. After fixation samples were digested with lysozyme (2 mg/ml in 20 mM Tris–HCl supplemented with 10 mM EDTA and 0.9% glucose) for 2 min, washed with PBS, blocked with 2% BSA in a PBS buffer for 10 min and incubated with 0.1–1 μg/ml DAPI (4’, 6-diamidyno-2-fenyloindol, Molecular Probes) and WGA-Texas Red (Wheat Germ Agglutinin-Texas Red) for 60 min. Fluorescence microscopy was performed using a Leica DM6 B fluorescence microscope equipped with a 100x oil immersion objective. Sporulating hyphae were analyzed using custom protocols involving Fiji (39) and R software (40), code is available at https://github.com/astrzalka/sporecounter.

RNA isolation and RNA-seq bioinformatic analysis

For RNA-seq, total RNA was isolated from 30 ml YEME/TSB cultures. Cultures were inoculated with S. coelicolor spores (amount of spores was normalized by OD measurement of preliminary cultures), and cultivated in flasks with spring coils for 24-36h in 30°C. For osmotic stress experiment growth medium was supplemented with NaCl to final concentration 0.5M. Mycelia were collected at two time points (exponential and early stationary growth, determined individually for each strain based on the growth curve) by collecting 2 ml from the culture and centrifugation. Cell pellets were frozen and stored at -70°C for subsequent RNA isolation. RNA was isolated using the RNeasy Mini Kit (Qiagen) following manufacturer’s instructions, DNA digestions was performed using on column digestion with DNase-I (Qiagen) and TURBO DNase I (Ambion). RNA quality and concentration was measured using Nanodrop (Thermo Fisher Scientific) and Qubit (Thermo Fisher Scientific). The absence of DNA in the sample was confirmed using PCR.

Library preparation and RNA-sequencing was performed by Genewiz (Germany). Trimmomatic software (version 0.39) (41) was used to remove adapter sequences from sequenced reads. Obtained reads were mapped to S. coelicolor genome (NC_003888.3) using Bowtie2 software (version 2.3.5.1) (42, 43) and processed with samtools (version 1.10) (44). On average 4 x 10⁶ reads mapped successfully to S. coelicolor genome. Differential analysis was performed using R packages Rsubread (version 2.10) and edgeR (version 3.38) (45, 46) following a protocol described in (47). Gene count matrix was normalized using egdeR and quasi-likelihood negative binomial was fitted to the data. Differential expression was tested using glmTtreat function with 1.5 fold change threshold. Genes were considered to be differentially expressed if false discovery rate (FDR) was below 0.05 threshold and Log₂FC value was above 1.5. For data visualization ggplot2 (version 3.3.6) (48), ggVennDiagram (version 1.2.0) (49) and tidyHeatmap (version 1.6.0) (50) R packages were used. Cluster analysis was performed using clust programme (version 1.10.8) (51). RNA isolation and Reverse-Transcription and Quantitative PCR (RT-qPCR)

RNA for RT-qPCRwas isolated from 5 mL YEME/TSB liquid medium S. coelicolor cultures cultivated for 24-36 h. Mycelia were collected by centrifugation, frozen and stored at -70°C for subsequent RNA isolation. RNA was isolated using the RNeasy Mini Kit (Qiagen) following manufacturer’s instructions, digested with TURBO DNase I (Invitrogen) and checked for chromosomal DNA contamination using PCR. A total of 500 ng of RNA was used for cDNA synthesis using the Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific) in a final volume of 20 μl. Obtained cDNA was diluted to 100 μl and directly used for quantitative PCRs performed with PowerUp SYBR Green Master Mix (Applied Biosystems). The relative level of transcript of interest was quantified using the comparative ΔΔCt method using the hrdB transcript as the endogenous control (StepOne Plus real-time PCR system, Applied Biosystems).

Results and Discussion

Deletion of hupA and hupS has a synergistic effect on Streptomyces growth and development

Previous reports concerning the role of HU homologs in Streptomyces showed only a moderate phenotype of deletion strains; specifically: the ΔhupA mutant exhibited slower growth (32, 36), while the ΔhupS mutant displayed decreased nucleoid compaction and increased spore sensitivity to thermal stress (29, 33). Given the high level of both proteins in the cell, we wondered if the functions of HU homologs could be redundant in Streptomyces and whether HupA or HupS would compensate for the loss of the other homolog. To test this hypothesis, we constructed a double deletion ΔhupAΔhupS S. coelicolor strain and compared its growth under various conditions to that of the ΔhupA and ΔhupS strains.

The growth of ΔhupAΔhupS strain was more inhibited than that of either of single mutant (Fig. 1 A). Both the ΔhupAΔhupS and ΔhupA strains showed a significant delay in the initiation of growth in liquid medium (half time 28.7 and 27.3 h, based on a log-logistic growth model, respectively) compared to wildtype and ΔhupS strains (17.5 and 16.3 hours, respectively). However, after the complementation of the double deletion strain with hupA gene, an improvement in growth was observed (Fig. S1 A). A similar growth delay was also observed for ΔhupAΔhupS strain during culture on solid medium (Fig. 1 B). Moreover, ΔhupAΔhupS colonies remained white, as this strain did not produce the characteristic for S. coelicolor spores grey-brown pigment (Fig. 1 B). Microscopic observations confirmed, however, that the double deletion strain was able to sporulate (Fig. S1 B), but only after a prolonged incubation (∼7 days) compared to 3 days for the wild type and ΔhupS strains, and 4 days for the ΔhupA strain. The analysis of sporogenic hyphae in the mutant strains also showed that the ΔhupAΔhupS strain had chromosome segregation defects, with 10.4% of spores lacking DNA; in comparison, the wild type strain had only 1.5% anucleate spores. Segregation defects were detectable also in single deletion strains: 3.9% spores lacked DNA in the ΔhupA strain and 4.4% in the ΔhupS strain (Fig. 1 C, D).

Figure 1 Deletion hupA or/and hupS inhibits growth and increases stress sensitivity of S. coelicolor spores

A. Growth curves of wild type (purple), ΔhupA (red), ΔhupS (green) and ΔhupAΔhupS (blue) strains in liquid ‘79’ medium. Bold lines represent log-logistic model while striped lines show half time calculated by the model.

B. Colonies of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains on solid SFM medium after incubation in 30°C for 72 and 96 hours. Grey appearance of colonies indicates sporulation.

C. Microscopic images of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS hyphae stained with DAPI (blue, DNA) and WGA-Texas Red (red, cell wall). White arrows indicate positions of anucleate prepsore compartments. Scale bar 2 μm.

D. Percentage of anucleate spores in sporulating hyphae of wild type (purple), ΔhupA (red), ΔhupS (green) and ΔhupAΔhupS (blue) strains. Grey lines represent 95% confidence interval.

E. Growth of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains on solid SFM medium after exposure of spores to high temperature (60°C) or detergent (5% SDS) for 30 minutes before plating.

F. Percentage of germinating spores of wild type (purple), ΔhupA (red), ΔhupS (green) and ΔhupAΔhupS (blue) strains after exposure to high temperature (60°C), detergent (5% SDS) or UV light.

Given the lack of spore pigmentation and chromosome segregation defects in the ΔhupAΔhupS strain, we expected that its spores would be less viable than those of ΔhupA or ΔhupS spores. Indeed, spores from the ΔhupAΔhupS strain were significantly less resistant to all tested stress factors: high temperature, presence of SDS as well as exposure to UV light or hydrogen dioxide. Colony forming unit (CFU) calculations showed that less than 0.00001% of ΔhupAΔhupS spores survived a 45 minutes incubation in 60°C compared to survival of 40.5% of wild type spores and 1.3% and 0.07% of ΔhupA and ΔhupS spores, respectively. Treatment with SDS affected solely the spores of ΔhupAΔhupS strain (less than 1% spores germinating), while for wild type and single deletion strains, treatment with SDS increased the germination rate from 60% to around 100% (Fig. 1 E, F). UV light exposure for 60 seconds resulted in the survival of less than 0.1% of Δ hupS and ΔhupAΔhupS spores, whereas spores of ΔhupA and wild type strains were less affected and 0.6 and 2% spores still formed colonies, respectively (Fig. 1 F). The presence of H₂O₂ in both liquid and solid medium strongly inhibited the growth of ΔhupS and ΔhupAΔhupS strains, while ΔhupA strain tolerated higher concentrations of H₂O_2. The H₂O₂ concentration of 1.75 mM led to growth inhibition on ΔhupA strain but not wild type strain (Fig. S2). Thus, factors causing DNA damage such as UV light or hydrogen dioxide, were found to be particularly harmful to spores of strains lacking HupS.

In summary, the deletion of both genes encoding HU homologs, hupA and hupS, in S. coelicolor resulted in a more severe phenotype than that of the single deletion mutants: more pronounced growth retardation, chromosome segregation defects and changes in colony pigmentation. Severity of ΔhupAΔhupS strain phenotype compared to single deletion mutants suggested a synergy between HupA and HupS. The absence of both HU homologs decreased also spores resistance to stress conditions. The elevated sensitivity to some of the stress factors, such as UV light or reactive oxygen species, could be explained either by lack of the physical protection of DNA or by transcriptional changes of genes involved in stress response.

HupA and HupS regulons partially overlap

Given that diminished stress response may result from the impact of HU homologs on gene expression, we set out to test whether the elimination of hupA or hupS would lead to transcriptional changes in S. coelicolor. Since the lack of HupA and HupS in S. coelicolor resulted in somewhat different phenotypes, we expected that their regulatory networks may not entirely overlap. To establish the HupA and HupS regulons and compare them to transcriptional changes in the double mutant strain, we performed an RNA-seq experiment for the ΔhupA, ΔhupS and ΔhupAΔhupS strains, in comparison to wild type control, all grown in liquid YEME/TSB medium. For each strain two timepoints were chosen based on the growth curves (Fig. S3): the middle of exponential growth (20 h for wild type strain, ΔhupA and ΔhupS, 24h for ΔhupAΔhupS strain) and the early stationary phase of growth (26 h for wild type strain, ΔhupA and ΔhupS, 30h for ΔhupAΔhupS strain). Differential expression analysis of all S. coelicolor genes using edgeR package determined which genes were affected by either hupA or hupS deletions when compared to wild type strain at the two tested time points (Table S3).

The number of genes whose transcription changed in DhupA was similar during exponential growth and early stationary phase (272 and 299 genes, respectively), while hupS deletion affected expression of more genes during exponential growth than in the stationary phase (431 and 140, respectively) (Fig. 2 A, B). Gene expression was most altered in the double deletion mutant, with 451 genes changed during exponential growth and 343 genes during the stationary phase (Fig. 2 A, B). Interestingly, in ΔhupAΔhupS strain 213 genes were affected at both analysed time points (while in the DhupA and ΔhupS it was only 43 and 64 genes, respectively) (Fig. S4). This may suggest that double deletion strain did not undergo a distinct transition between growth phases or that it was still at an earlier stage of growth than either ΔhupA or ΔhupS strains.

Figure 2 Global changes of gene expression in ΔhupA, ΔhupS and ΔhupAΔhupS strains

A. Volcano plots showing altered gene expression in ΔhupA, ΔhupS and ΔhupAΔhupS strains at exponential and stationary growth phase compared to the wild type strain. For each strain significantly changes genes (FDR ≤ 0.05, |Log₂FC| > 1.5) are shown in red. Chosen differentially expressed genes are labelled.

B. Venn diagrams showing significantly changed genes unique and common for analyzed strains; ΔhupA, ΔhupS and ΔhupAΔhupS during exponential and stationary growth.

C. Scatterplots showing correlation between ΔhupA, ΔhupS and ΔhupAΔhupS transcriptomes from exponential and stationary growth compared to wild type strain. Pearson correlation coefficient is shown on each plot. Genes found to be significant in both comparisons (FDR ≤ 0.05, |Log₂FC| > 1.5) are shown in red, while genes significant in only one of the comparisons are marked as blue or green, grey dots represent genes not affected by hupA and/or hupS deletions.

Most often, the deletion of hupA and/or hupS resulted in transcription upregulation (88% and 55% of genes in the ΔhupA mutant, 80% and 71% in the ΔhupS and 83% and 76% in the ΔhupAΔhupS strains during exponential and stationary growth, respectively) (Fig. 2 A). To confirm the obtained results, ten representative genes from the putative hupAS regulon were chosen for replication analysis and their expression pattern was confirmed by an RT-PCR experiment (Pearson correlation coefficients 0.57 and 0.53 between Log₂FC values for exponential and stationary growth, respectively) (Fig S5C).

To further compare the transcriptional changes between hupA and/or hupS deletion strains, we calculated the Pearson correlation coefficient of obtained Log₂FC values from comparisons of mutant strains to the wild type strain (Fig. 2 B). We found a moderate correlation between the hupA and hupS strains at both stages of growth (Pearson coefficient = 0.56 and 0.58 at exponential and stationary growth, respectively). Interestingly, the transcriptional changes detected in the double deletion mutant ΔhupAΔhupS were similar to those in the single deletion strains only during exponential growth (Pearson coefficient: 0.63 and 0.71 when compared to hupA and hupS strains, respectively), while during the stationary phase this strain was visibly distinct (Pearson coefficient: 0.15 and 019 when compared to hupA and hupS strains, respectively). The high similarity between RNA-seq results obtained for all deletion mutants at an earlier stage of growth and the remarkable difference of the ΔhupAΔhupS strain from the other two strains in stationary phase could also be seen on the Principal Component Analysis (PCA) plot (Fig. S4, S5A).

Based on the pattern of regulation by HupA/HupS, differentially expressed genes could be divided into four major categories. The first group (HupA|S regulon) contained genes for which the presence of one HU homolog was sufficient to maintain the expression pattern. These genes were thus differentially expressed only in the ΔhupAΔhupS strain – 148 and 255 genes during exponential and stationary growth, respectively. The second group (HupA&S regulon) required both HupA and HupS to maintain wild type level of expression. Therefore, this group was comprised of genes whose expression changed in all tested strains, 149 and 11 genes during exponential and stationary growth, respectively. The last two groups (HupA regulon and HupS regulon) contained genes whose expression changed in the ΔhupS strain, but not in the ΔhupA strain or in the ΔhupA strain, but not in the ΔhupS strain. Surprisngly, the HupS regulon was larger than the HupA regulon during exponential growth (257 and 98 genes, respectively) while HupA regulon was larger during stationary phase (Fig. 2C).

The larger number of genes under the control of HupA in the stationary phase and under control of HupS during exponential growth is somewhat contradictory to expectations based on the fact that HupA is the most abundant NAP during vegetative growth, while HupS levels increase during sporulation. The moderate overlap between HupA and HupS regulons and the existence of HupA|S and HupA&S regulons, indicates some extent of cooperation between these two HU homologs. This cooperation may explain their synergistic impact on phenotype, although the details of such cooperation remain to be elucidated. On the other hand, the separate HupA and HupS regulons may correspond to the different binding pattern of both proteins. While HupA was shown to predominantly bind within the central region of the S. coelicolor chromosome, HupS in S. venezuealae preferentially bound within the arms regions (29, 32). Finally, it is worth highlighting that both HU homologs seemingly most often played the role of transcriptional repressors. A similar function has been noted for Lsr2 in S. venezeulae, where removal of Lsr2 activated a number of secondary metabolite clusters (52). In S. coelicolor, a novel NAP called Gbn was also found to have a supressive effect on gene expression (53). The comparable roles of HupA and HupS positions them among other proteins crucial for controlling Streptomyces metabolism.

Deletion of hupA and hupS genes alters the expression of genes involved in chromosome structure and topology maintenance

The fragility of ΔhupAΔhupS strain spores, and to a lesser extent of the ΔhupA and ΔhupS strains, could be attributed either to lack of DNA protection by HU homologs or to transcriptional changes of genes vital for S. coelicolor spore maturation. Diminished resistance to stress conditions such as UV light, heat or free radicals has often been described for HU mutant strains of various bacterial species (33, 54–57). Therefore we investigated how the transcriptional activity of genes important for DNA structure and topology or spore maturation was changed in hupA and/or hupS deletion mutants (Fig. 3A).

Figure 3 Deletion of hupA and/or hupS changes expression of genes connected to DNA topology maintenance and encoding global regulators

A. Heatmap showing normalized expression (CPM) of genes connected with DNA topology and spore development in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth.

B. Heatmap showing normalized expression (CPM) of SigB regulon genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth.

C. Selected genes from differentiation and stress response network that are upregulated (blue) or downregulated (red) in ΔhupAΔhupS strain during stationary growth.

A, B Genes marked with diamond were found to be significant in comparison to the wild type strain (FDR ≤ 0.05).

Since HupA is one of the most abundant NAPs in S. coelicolor during vegetative growth (30, 35), its deletion should be compensated by an upregulation of gene(s) encoding other NAP(s). Studies of S. lividans suggested that increased expression of hupS could partially suppress the effects of hupA deletion (36). However, we have not found any evidence of hupS upregulation in the ΔhupA strain or hupA upregulation in the ΔhupS strain. Instead, in both ΔhupS and ΔhupA mutants, we observed a significant increase in dpsA expression (sco0596), which encodes a NAP involved mainly in DNA protection in stressful conditions (58) and sIHF (sco1480), a NAP responsible for chromosome condensation and segregation (59, 60). Expression of dpsA and sIHF increased during exponential growth in all mutant strains as compared to the wild type (Log₂FC for dpsA in ΔhupA: 3.45, in ΔhupS: 3.40 and in ΔhupAΔhupS: 3.41; for sIHF in ΔhupA: 1.55, in ΔhupS: 1.24, in ΔhupAΔhupS: 1.70), while in stationary phase expression of those genes increased only in the ΔhupAΔhupS strain (Log₂FC for dpsA: 2.61; for sIHF: 1.56) (Fig. 3 A, C). Deletion of sIHF was earlier shown to result in a phenotype similar to that of hupA and/or hupS mutants, namely reduced viability of spores and inhibition of sporulation (61). This suggests an existence of a functional overlap between HupA, HupS and sIHF in S. coelicolor. Interestingly, transcription of gene encoding another NAP named Gbn (sco1839) also increased in the ΔhupAΔhupS strain (Log₂FC ΔhupAΔhupS: 1.75 and 1.31 during exponential and stationary growth, respectively) (Fig. 3 A). Deletion of gbn had a different effect then hupAhupS deletion and led to accelerated development, while overexpression of gbn delayed sporulation (53). Finally, unlike the above described NAP genes, the expression of lsrL (sco4076) decreased in the ΔhupAΔhupS strain (Log₂FC ΔhupAΔhupS exponential: -1.24, stationary: -1.48). LsrL is a homolog of Lsr2, but little is known about its function in Streptomyces. Interestingly, the expression of lsr2 remained unchanged in all tested strains.

HupA protein is also crucial for DNA supercoiling homeostasis in S. coelicolor and cooperates with topoisomerase I (TopA) in maintaining chromosome topology (32). Here, we did not detect any changes in expression of either topA (sco3543) or parE/C (sco5822, sco5836) genes encoding topoisomerase IV, but in strains with hupA deletion (ΔhupA and ΔhupAΔhupS), we observed an upregulation of the gyrA/gyrB operon (sco3873-sco3874) encoding gyrase, placing gyrAB in the HupA regulon (Fig. 3 A). This result corroborates an earlier observation that the DhupA mutant was more sensitive to gyrase inhibition by novobiocin than the wild type strain (32). Thus, the increased gyrase activity could be necessary to compensate for the lack of HupA.

Diminished UV and oxidative stress resistance of hupA and hupS spores could be linked to the role of HU in homologous replication or RecA-dependant DNA repair (56, 62). However, recA expression in S. coelicolor hupA and/or hupS mutants was not affected. Nevertheless, we found that some spore associated genes which were repressed in the ΔhupAΔhupS strain during stationary growth, e.g., sapA (sco0409) and rdlB (sco2719) encoding spore coat proteins important for spore hydrophobicity (63–65) (Fig. 3 A, C). These changes could account for increased spores sensitivity to stress factors. Interestingly, even though mutant strain colonies were paler than the wild type, the genes responsible for spore pigmentation (e.g. whiE) were not affected.

Summarizing, genes encoding numerous proteins involved in DNA structure maintenance and protection, such as DpsA, sIHF and gyrase, were affected by either hupA or hupS deletion. This indicates at least partially independent functions of HupA and HupS in maintaining chromosome structure. Remarkably, in the exponential phase, the elimination of either HupA or HupS led to upregulation of expression, thus placing those genes in the HupA&S regulon. This suggests that in this phase of growth, in the absence of HupA and HupS, other NAPs may compensate for their loss and maintain chromosome organisation. However, in the stationary phase, the elimination of both HU homologs was required to activate other NAP encoding genes. This observation may be explained by modified chromosome structure during stationary phase (66). This may also explain the remarkably increased sensitivity of ΔhupAΔhupS spores.

HupA and HupS are a part of the Streptomyces regulatory network

The observed HupA- and HupS-dependent changes in global transcriptional activity might be explained by a direct impact of these NAPs on particular gene expression or by an indirect effect mediated by modified levels of regulators and/or sigma factors. To explore the latter possibility, we utilized the RNA-seq dataset to identify transcription regulators whose expression was altered in hupA/hupS deletion strains and whose regulatory networks have already been established. We found that genes encoding SigB (sco0600), ArgR (sco1576) and OsdR (sco0204) fulfilled these criteria. Next, we analysed how HupA- and/or HupS-dependent modification of these genes impacted their regulatory networks. We also investigated whether HupA and HupS are involved in the regulatory network controlling S. coelicolor life cycle.

SigB (sco0600) is a sigma factor, which acts as a major osmotic stress regulator and, through SigM (sco7314) and SigL (sco7278), regulates Streptomyces differentiation and stress response (67) (Fig. 3 B). The expression of sigB increased in ΔhupA and ΔhupS strains during exponential growth and in the ΔhupAΔhupS strain during exponential and stationary growth (Fig. S5 B, C), placing sigB in the HupA&S regulon during exponential growth and in the HupA|S regulon during stationary growth. In Streptomyces, SigB activity is controlled by its anti-sigma factor RsbA (sco0599) and two anti-anti sigma factors: RsbB (sco0598) and RsbV (sco7325). Only rsbV expression was elevated in ΔhupA and/or ΔhupS strains. Out of 92 genes reported to belong to the SigB regulon about one-fourth were upregulated in the ΔhupAΔhupS strain at both time points (25 genes, hypergeometric test p-value: 6.65*10^-12) (Fig. 3 B). Remarkably, during exponential growth, most of these genes were also upregulated in either ΔhupA or ΔhupS deletion strains, while during stationary growth their expression was unchanged, reflecting the pattern of sigB expression levels. Genes from the SigB regulon that were upregulated by hupA and hupS deletions included dpsA, sigL, sigM, ectABCD, rsbV and sco7590 (catalase) (Fig. 3 B, C). Thus, SigB network can serve as an example of HupA and HupS indirect influence, where changes of expression of a single sigma factor propagated through an entire regulatory network. However, a large fraction of genes from the SigB regulon were not found to be upregulated in neither hupA nor hupS deletion strains. This could be explained by either the low sensitivity of RNA-seq method to small changes in gene expression or by the influence of other factors independent of hupA and/or hupS deletion.

Another global regulator that was affected by the double deletion of hupA and hupS genes was ArgR. The expression of argR (sco1576) increased during stationary growth in ΔhupAΔhupS strain (Log₂FC ΔhupAΔhupS: 2.69), placing it in the hupA|S regulon. According to published data, ArgR controls the expression of around 1500 genes and usually acts as a repressor (68), but for our analysis, we only considered 90 genes whose expression was altered by argR deletion at all tested time points. Surprisingly, we found that the upregulation of argR in the ΔhupAΔhupS strain was accompanied by upregulation of ∼ 30 genes belonging to ArgR cluster. These changes were observed during exponential growth of all analyzed mutant strains and in the ΔhupAΔhupS strain during stationary growth (hypergeometric test p-value: 1.15*10^-16). (Fig S6 A). In E. coli, HU proteins act as co-repressors with the GalR protein and hupAB deletion leads to the destabilization of repression loops and expression of the gal operon (69, 70). A similar mechanism could perhaps explain the observed upregulation of the arg operon despite the increased expression of the argR repressor gene found in the ΔhupAΔhupS strain.

In ΔhupA, ΔhupS and ΔhupAΔhupS mutants, we found a significant increase in the expression of genes encoding two-component system OsdKR (sco0203-sco0204) which thus fall into the HupA&S regulon (exponential growth, Log₂FC ΔhupA: 1.44 and 2.65, ΔhupS: 1.28 and 2.07, ΔhupAΔhupS: 1.41 and 2.50). OsdR plays an important role in the control of stress and development related genes, and is an orthologue of Mycobacterium tuberculosis DevR protein (71, 72). The core regulon of OsdR/K system lies between genes sco0167 and sco0219. These genes were shown to be activated by OsdR and involved in stress response, spore maturation and nitrogen metabolism in S. coelicolor (71). In all tested hupA and/or hupS strains, the expression of the “core” OsdR/K genes increased during exponential growth, but genes belonging to the OsdR regulon located in other parts of S. coelicolor chromosome were not affected (Fig. S6B). Interestingly, 17 genes belonging to the OsdR regulon were earlier shown to be influenced by TopA depletion (35), suggesting that this cluster could be controlled by DNA supercoiling. The increased expression of genes belonging to the osdR regulon during early stationary growth in all tested strains is in agreement with previously published transcriptomic data (31, 71).

Lastly, we observed an induction of rfsA gene (sco4677) but only in the ΔhupAΔhupS strain (stationary growth, Log₂FC: 2.27). This gene encodes an anti-sigma factor interacting with SigF. rfsA null mutants are characterized by faster development (73, 74). Additionally, RfsA is able to negatively regulate BldG (sco3579) by phosphorylation, linking it to the Streptomyces sporulation regulatory network (75). Interaction of RfsA with two anti-anti-sigma factors was described (74), but only one of them (sco0781) was induced in both strains lacking hupS. Expression of sigF (sco4035) decreased in the ΔhupAΔhupS strain during stationary growth. Though this result was not highly significant (FDR = 0.064), it could explain the spore fragility of ΔhupAΔhupS since sigF null mutants in Streptomyces are characterized by lessened resistance to detergent treatment (76, 77). Moreover, another anti-sigma factor, sco7328, was upregulated in the ΔhupAΔhupS strain. This protein similarly to RfsA phosporylates BldG, but also inhibits activity of SigM, SigG (sco7341) and SigK (78) (Fig. 3 C). The expression pattern of rsfA, sigF and sco7328 places them in the HupA|S regulon.

To sum up, the analysis of SigB regulon represents an example of a predictable secondary impact of hupA and hupS deletion resulting from sigB upregulation. In contrast, the ArgR regulon indicates the involvement of HU homologs in more complex regulatory circuits, while OsdR regulon expression could be influenced by structural changes of S. coelicolor chromosome caused by either hupA or hupS deletion. On the other hand, RsfA upregulation could partially explain the slower growth of the ΔhupAΔhupS strain.

Deletion of hupA and/or hupS affects production of secondary metabolites

Given that the chromosome of S. coelicolor, similarly to other Streptomyces species, encodes numerous biosynthetic gene clusters, we set out to examine if the deletion of hupA and/or hupS in S. coelicolor, like lsr2 deletion in S. venezuelae, could activate those clusters. We found that out of 22 secondary metabolic clusters present in S. coelicolor (Bentley, 2002) 4 exhibited changes of gene expression in hupA and/or hupS deletion strains. The most striking example was the red cluster (sco5877-sco5898) encoding the red oligopyrrole prodiginine antibiotic - undecyloprodigiosin (79). Undecyloprodigiosins were suggested to have antimalarial and anticancer properties (80), and in S. coelicolor they were implicated in controlled cell death during development (81). Expression of the red cluster is under the positive control of pathway specific regulators RedD and RedZ and is upregulated during stationary phase, especially during growth in liquid media (31, 82, 83). The expression of almost entire red cluster was elevated in the ΔhupS strain (but not in the DhupA or DhupADhupS strains, placing it in the HupS regulon) at both tested time-points as compared to the wild type (Log₂FC range for the red cluster, ΔhupS stationary growth: 1.95 – 4.23), except for the transcription regulator redZ (sco5881). The fact that the double deletion of hupA and hupS did not lead to activation of the red cluster suggests that HupA presence is required for its expression. Indeed, redZ was downregulated in the ΔhupA strain during stationary growth (Fig. 4 A). Additionally, two-component systems: ecrA1/A2 (sco2517-sco2518) and ecrE1/E2 (sco6421-sco6422) which are involved in transcriptional control of the red cluster (84, 85) were also upregulated during stationary growth in ΔhupS strain. Notably, the overexpression of red cluster was earlier observed in the strain with deletion of sIHF (60, 61). Overproduction of RED antibiotic was confirmed by plate cultures showing an abundance of red pigment produced by the ΔhupS strain (Fig. 4 B). Interestingly during growth on solid medium, ΔhupA and ΔhupAΔhupS strains did not produce the characteristic for S. coelicolor blue pigment (actinorhodin) (Fig. 4 B), which could be explained either by the growth delay or transcriptional influence of hupA deletion on the actinorhodin cluster transcription. However our RNA-seq data did not show any significant changes in the act cluster genes expression (Fig. S7). To sum up, HupA is required for the activation of gene encoding activator RedZ while HupS inhibits red cluster expression possibly by downregulation of the two component system genes.

Figure 4 Deletion of hupA and/or hupS changes expression of genes associated with production of secondary metabolites

A. Heatmap showing normalized expression (CPM) of the red cluster genes in wild type, Δ hupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth.

B. Production of undecyloprodigiosin (red metabolite) during plate culture of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains incubated of solid SFM medium for 72 hours in 30°C.

C. Heatmap showing normalized expression (CPM) of the carotenoid gene cluster in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth.

D. Heatmap showing normalized expression (CPM) of ectoine gene cluster in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth.

E. Heatmap showing normalized expression (CPM) of desferroxamine cluster genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth.

A, C, D, E Genes marked with diamond were found to be significant in comparison to the wild type strain (FDR ≤ 0.05). Genes encoding established transcription regulators are marked in grey.

The other biosynthetic gene cluster affected by the elimination of HupS was the carotenoid cluster (sco0185-sco0194). Expression of the crt cluster was shown to be highest during exponential growth (31). The function of carotenoids in Streptomyces has not been fully established yet, but it is suggested that they are involved in protection from photo-oxidative damage (86). Expression of the crt cluster was upregulated strongly in the ΔhupS strain during stationary growth (Log₂FC range for crt cluster = 2.09 - 4.54) and to a lesser degree in the ΔhupA (Log₂FC = -0.05 - 2.72) and ΔhupAΔhupS (Log₂FC = 0.42 - 2.24) strains (Fig. 4 C), placing it in the HupA&S regulon. Interestingly, genes belonging to the crt cluster are located between genes belonging to the osdR regulon, which expression was also elevated in either ΔhupA or ΔhupS mutants during exponential growth (Fig S6). Expression of the crt cluster is light induced and controlled by litQR (sco0193-sco0194) and litSAB genes, with litS being essential for crt expression (sco0195-sco0197) (Takano, 2005). Notably, the expression of litSAB genes in ΔhupA and/or ΔhupS strains was not significantly different from the wild type strain with the exception of litA, which was upregulated in ΔhupAΔhupS strain during stationary growth (Fig. 4 C).

Elimination of HupA and/or HupS activated the ectoine cluster (sco1864-sco1887). Ectoine production in Streptomyces has been linked with survival in high salt or temperature conditions (87). Earlier transcriptional studies showed that the ect cluster expression is highest during exponential growth and decreases at later stages of growth on both solid and liquid media (31, 82, 88). Deletion of hupA and/or hupS genes led to an overexpression of ectABCD genes at both analysed time points (Log₂FC range for ect cluster exponential growth, ΔhupA: 2.54 - 2.94; ΔhupS: 2.68 - 4.00; ΔhupAΔhupS: 1.37 – 2.15) thus placing this cluster in the HupA&S regulon (Fig. 4 D). The ectoine cluster was shown to be negatively controlled by the GlnR transcription factor (sco4159) (89) and expression of glnR decreased in ΔhupA and ΔhupS strains during stationary growth. Additionaly ect cluster expression is dependent upon SigB (67, 90), and expression of sigB was elevated in all three hupA and/or hupS deletion strains (Fig. 4 D). Thus, ectoine cluster activation may be explained by increased levels od SigB and lowered levels of GlnR in the absence of HU homologs.

Contrary to the previously described clusters, the desferroxamine cluster (sco2782-sco2785) was downregulated in the absence of HupA or HupS. The des cluster encodes genes necessary for the production of desfeeroxamine, a nonpeptide hydroxamate siderophore (91). Desferrioxamine are produced by many Streptomyces species in iron deficiency conditions and at an early stage of growth on solid media (88, 92). Expression of desABCD genes decreased in ΔhupA and ΔhupS strains during stationary growth (Log₂FC range ΔhupA: -2.24 to -3.08; ΔhupS: -2.65 to -3.30), but not in the double deletion ΔhupAΔhupS strain (Fig. 4E). desABCD expression is governed by the iron repressor dmdR1 (sco4394) (93) but dmdR1 transcription was not affected by hupA and/or hupS deletion (Fig. 4 E). Thus, the mechanism of des cluster regulation by HupA and HupS is unclear.

To sum up, we found that expression of four BGC was modified in the absence of HupA and/or HupS. Expression of secondary clusters responsible for the production of RED and carotenoid compounds were activated by hupS deletion, however the presence of HupA was required for this activation. That suggests an interplay between both HU homologs in the transcriptional regulation of these clusters. In contrast, ectoine cluster was upregulated in the absence of at least one HU homolog. For three out of four clusters, the changes of expression could be at least partially explained by modified levels of the pathway specific (RedZ) or global (SigB) regulators.

Effective response to osmotic stress depends upon the presence of HupA

Several sigma factors whose expression levels were affected by hupA and/or hupS deletion participate in S. coelicolor’s response to osmotic stress (i.e. SigB). Similarly, ectoine, whose biosynthesis gene cluster expression was elevated in hupA and/or hupS deletion mutants, is important for Streptomyces survival in high salt environments (87). These observations prompted us to determine the effect of hupA and/or hupS deletion on S. coelicolor survival and gene expression in osmotic stress. To this end, we cultured ΔhupA, ΔhupS and ΔhupAΔhupS strains in liquid YEME/TSB medium supplemented with 0.5 M NaCl, to assess their growth rate, and next, we to determine the transcription profile using RNA-seq (Table S3).

Addition of 0.5 M NaCl slowed down growth of all tested strains, but the inhibition of growth was most pronounced for ΔhupA and ΔhupAΔhupS strains (Fig. S3). NaCl supplementation altered expression of a substantial number of genes in the wild type strain, with more genes affected during the stationary than the exponential phase (142 and of 338 genes, respectively). Deletion of hupA or hupS also resulted in a significant change in transcryptomic activity compared to these strains cultured in normal medium (659 and 455 genes, respectively during stationary growth). However, the double deletion mutant was largely unaffected by osmotic stress, with only 95 changed genes during stationary growth in the NaCl-supplemented medium (Fig. 5 A). Next, we calculated the correlations between individual strains’ responses to the osmotic stress. The ΔhupS strain transcriptome was the most similar to the wild type strain (Pearson coefficient = 0.71), while ΔhupA and ΔhupAΔhupS strains were significantly different from the wild type strain (Pearson coefficient 0.25 and 0.31, respectively). Both strains lacking the hupA gene were remarkably similar to each other with a 0.79 correlation coefficient and 432 shared genes (Fig 5B, C, S8).

Figure 5 hupA deletion changes S. coelicolor response to growth in osmotic stress

A. Volcano plots showing changes in gene expression in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains from exponential and stationary phase of growth in NaCl supplemented medium (0.5 M NaCl) compared to the same strain grown in standard YEME/TSB medium. For each strain significantly changes genes (FDR ≤ 0.05, |Log₂FC| > 1.5) are shown in red. Chosen differentially expressed genes are labeled.

B. Scatterplots showing correlation between wild type and ΔhupA, ΔhupS and ΔhupAΔhupS Log₂FC values in medium supplemented with salt. Log₂FC values were calculated from comparison with wild type strain grown in standard medium. Pearson correlation coefficient is shown on each plot. Genes found to be significant in both comparisons (FDR ≤ 0.05, |Log₂FC| > 1.5) are shown in red, while genes significant in only one of the comparisons are marked as blue or green, grey dots represent genes not affected by hupA and/or hupS deletions.

C. Correlation between ΔhupA and ΔhupAΔhupS transcriptomes during stationary growth in NaCl supplemented medium. Pearson correlation coefficient is shown on the plot. Genes found to be significant in both comparisons (FDR ≤ 0.05, |Log₂FC| > 1.5) are shown in red, while genes significant in only one of the comparisons are marked as blue or green, grey dots represent genes not affected by hupA and double hupAhupS deletions.

D. Regulatory networks network involving selected upregulated (blue) or downregulated (red) genes in ΔhupAΔhupS strain during stationary growth in NaCl supplemented medium (0.5 M NaCl).

These observations suggest that the presence of HupA is necessary for S. coelicolor response to osmotic stress. This situation could partly explained by the fact that several genes overexpressed in the wild type strain in NaCl supplemented medium, like ectABCD or sigB, are a part of the HupA&S regulon (Fig. 5 A). Moreover, among the genes most affected by hupA deletion, we found sigE (sco3356); a sigma factor related to osmotic stress (94, 95). SigE controls the expression of genes mainly involved in maintaining cell wall and membrane integrity (95). HupA could be one of SigE binding partners (96). Lack of SigE could explain some of the transcriptomic changes observed in both ΔhupA strains. Indeed, analysis of the SigE regulon revealed the presence of 42 genes that were strongly repressed in either ΔhupA or ΔhupAΔhupS strains but induced in the wild type and ΔhupS strains during osmotic stress (hypergeomertic test p-value: 2.49 * 10^-14) (Fig. S8 D). On the other hand, in vitro studies concerning the HupA protein showed that its binding to DNA is affected by NaCl concentration (10). Perhaps altered distribution of HupA on the S. coelicolor chromosome is one of the sources of observed transcriptional alterations.

Gene clusters within HupAS regulons

Lastly, to improve gene classification into the four major regulons (HupA, HupS, HupA&S and HupA|S) by taking into account the influence of salt, we performed a cluster analysis of the entire dataset obtained for the four strains tested at two timepoints under two growth conditions. Using the clust programme we found 15 clusters of genes (with sizes ranging between 11 and 577) (Fig. S10), containing a total of 1601 S. coelicolor genes. Among the obtained clusters, three contained genes whose expression seemed to be affected by the lack of either of the HU homologs (clusters 9, 10 and 13) and thus belonging to the HupA&S regulon. Two of those clusters, 9 (39 genes) and 10 (142 genes), included many genes identified in the earlier differential expression analysis as controlled by both HupA and HupS, such as: dpsA, sIHF, sigM, sigL, rsbV, catB, sigK in cluster 10, and: sigB, ectABCD in cluster 9. The expression of genes from cluster 10 seemed to be largely unaffected by growth in osmotic stress, while genes belonging to cluster 9 were upregulated during stationary growth in osmotic stress conditions in all tested strains (Fig. 6). Interestingly, cluster 13 contained only genes located between 150 kb and 213 kb on the S. coelicolor genome and belonging to the OsdR regulon described earlier. Possibly, the control of cluster 13 expression involves changes in chromosome organization and/or supercoiling, explaining its sensitivity to both hupA and/or hupS deletion and TopA depletion (35) (Fig. 6).

Figure 6 Clusters of genes within hupAS regulons

A. Cluster identification - results of clust analysis of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS transcriptomes from exponential (red) and stationary (blue) phase of growth in normal and NaCl supplemented medium. Plot shows six chosen clusters out of 15 obtained.

B. t-distributed stochastic neighbour embedding (t-SNE) analysis of normalized wild type ΔhupA, ΔhupS and ΔhupAΔhupS transcriptomes from exponential and stationary phase of growth in normal and NaCl supplemented medium. Clusters: HupA&S regulon (cluster 9 – dark purple, cluster 10 – purple, cluster 13 - orange), HupS regulon (cluster 11 – pink), HupA regulon clusters affected by growth in NaCl supplemented medium (clusters 6, 7, 8 – red, clusters 1, 2, 14 – yellow). Positions of genes associated with stress response, secondary metabolites production, spore development and NAPs are shown on the plot.

Cluster 11 (33 genes) contained genes whose expression changed only in ΔhupS or ΔhupAΔhupS strains, thus constituting the HupS regulon. The expression of those genes was not influenced by growth in osmotic stress conditions and was also similar at both tested growth stages. Seemingly, those genes were controlled solely by HupS. This group included cvn12 conservon (sco2879-sco2884), anti-anti sigma factor sco0781, putative LysR regulator (sco2734), putative stress response protein (sco5806), and operon sco2521-sco2526. SCO2525 is a putative methyltransferase necessary for normal growth of S. coelicolor (97) (Fig. 6). Genes affected only by hupA deletion (HupA regulon) belonged to six clusters (Clusters 1, 2, 6, 7, 8, 14), and their expression changed only during osmotic stress. Cluster 7 contained, among others, genes belonging to the red cluster (Fig. S9), sigE, and lsrL. The expression of those genes increased during stationary growth in osmotic stress in the wild type and ΔhupS strain, but remained low in ΔhupA and ΔhupAΔhupS strains (Fig. 6, S10). This analysis confirmed that genes belonging HupA and HupA&S regulons are involved in osmotic stress response, while HupS regulon genes are not sensitive to increased salt concentration.

Conclusions

In summary, the presence of both HupA and HupS is necessary for proper growth and development of S. coelicolor. The absence of both HupA and HupS results in severe growth inhibition and impaired stress survival, significantly more pronounced than that of either single deletions strains, indicating a synergy between the actions of the two HU homologs. The increased sensitivity of spores to DNA damaging factors may be explained by diminished protection of DNA.

RNA-seq analysis showed that by binding to DNA, HupA and HupS act as global transcription factors, altering the expression of multiple genes, mostly upregulating them. Genes upregulated in the absence of at least one of HU homologs were involved in DNA protection (NAPs, gyrase), transcription regulation (e.g. sigma factors) or stress survival (e.g. osmotic stress). HupS was involved in controlling the expression of secondary metabolites clusters (e.g. the red cluster), while HupA’s control of gene expression, separate from HupS, was mostly evident during growth in osmotic stress (Fig. 7). The identification of HupA&S and HupA|S regulons suggests a cooperation between the two HU homologs in Streptomyces.

Figure 7 HupA and HupS regulons

Data Availability

The raw RNA-Seq data, as well as the processed data generated in this study, have been deposited in the ArrayExpress database (EMBL-EBI) under accession code E-MTAB-13846.

Funding

This work was funded by the Polish National Science Centre: SONATINA grant 2018/28/C/NZ1/00241.

Supplementary Data

Table S1 Streptomyces coelicolor strains used in the study

Table S2 Oligonucleotides used in the study

Fig. S1. Growth and sporulation of ΔhupAΔhupS strain.

A. Growth curves of wild type (purple), ΔhupAΔhupS (blue) and ΔhupAΔhupS::hupA (orange) strains cultured in ‘79’ medium. Solid lines show the log-logistic model while vertical lines represent the half time calculated by the model.

B. Microscopic images of spores of wild type S. coelicolor and ΔhupAΔhupS strains from 7-day old colonies grown on SFM solid medium. Scale 5 μm.

Fig. S2 Impact of hupA and/or hupS deletion on S. coelicolor growth in the presence of H₂O₂

A. Growth curves of wild type (purple), ΔhupA (red), ΔhupS (green) and ΔhupAΔhupS (blue) strains cultured in ‘79’ medium supplemented with increasing concentrations of H₂O₂. Solid lines show the growth curve smoothed by the loess algorithm.

B. Growth of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains on solid SFM medium containing increasing concentrations of H₂O₂. Pictures were taken after 5 days of incubation in 30°C.

Fig. S3 Impact of osmotic stress on the growth of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains

Growth curves of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains in standard (red) and NaCl supplemented (blue, 0.5 M NaCl) YEME/TSB medium. Vertical lines show growth half-time calculated by log-logistic model.

Fig. S4 Comparison of transcriptomes of ΔhupA, ΔhupS, ΔhupAΔhupS strains during different phases of growth

A. Scatterplots showing correlation between ΔhupA, ΔhupS and ΔhupAΔhupS transcriptomes from exponential phase compared to stationary phase. Pearson correlation coefficient is shown on each plot. Genes found to be significant in both comparisons (FDR ≤ 0.05, |Log₂FC| > 1.5) are shown in red, while genes significant in only one of the comparisons are marked as blue or green, grey dots represent genes not affected by hupA and/or hupS deletions.

B. Venn diagrams showing significantly changed genes common for analyzed strains ΔhupA, ΔhupS and ΔhupAΔhupS during exponential and stationary growth.

Fig. S5 RNA-sea and RT-PCR analysis of gene expression

A. Principal component analysis (PCA) of the normalized RNA-seq CPM (Counts Per Million) data of S. coelicolor strains in response to hupA (red), hupS (green) or hupAhupS (blue) deletion during exponential (circles) or stationary growth (triangles).

B. Correlation between Log₂FC values obtained from RNA-seq and RT-PCR for genes: dpsA, sigB, gyrB, fabH3, ecrA1, sIHF, lsr, sco0781, gvpA1 from ΔhupA (red), ΔhupS (green) and ΔhupAΔhupS (blue) strains when compared to the wild type strain.

C. Table showing Log₂FC values of several genes in strains ΔhupA, ΔhupS and ΔhupAΔhupS when compared to wild type strain during exponential and stationary growth, NA value indicates that FDR value was above the 0.05 threshold.

Fig. S6 Genes clusters regulated by HupA and HupS

A. Heatmap showing normalized expression (CPM) of ArgR regulon genes for ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth.

B. Heatmap showing normalized expression (CPM) of OsdR regulon genes for ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth.

A, B Genes marked with diamond were found to be significant in comparison to the wild type strain (FDR ≤ 0.05).

Fig. S7

Heatmap showing normalized expression (CPM) of actinorhodin cluster genes for ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth. Genes marked with diamond are those whose expression was found to be significantly altered in comparison to the wild type strain (FDR ≤ 0.05).

Fig. S8 Changes of gene expression induced by osmotic stress conditions

A. Volcano plots showing modified gene expression in ΔhupA, ΔhupS and ΔhupAΔhupS during exponential and stationary growth in medium supplemented with 0.5 M NaCl compared to the wild type strain. For each strain significantly changes genes (FDR ≤ 0.05, |Log₂FC| > 1.5) are shown in red. Chosen differentially expressed genes are labelled.

B. Venn diagrams showing significantly changed genes common for strains ΔhupA, ΔhupS and ΔhupAΔhupS during exponential or stationary growth in medium supplemented with 0.5 M NaCl.

C. Heatmap showing normalized expression (CPM) of 150 genes with the lowest FDR value calculated for ΔhupAΔhupS strain and wild type comparison in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth in NaCl supplemented medium (0.5 M NaCl).

D. Heatmap showing normalized expression (CPM) of SigE regulon genes for ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth in NaCl supplemented medium (0.5 M NaCl).

C, D Genes marked with diamond were found to be significant in comparison to the wild type strain (FDR ≤ 0.05, |Log₂FC| > 1.5).

Fig. S9 Modified expression of the genes within biostynthetic gene clusters, ΔhupA, ΔhupS and ΔhupAΔhupS strains during growth at osmotic stress as compared to the wild type strain

A. Heatmap showing normalized expression (CPM) of the red cluster genes in wild type, Δ hupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth in NaCl supplemented medium (0.5 M NaCl).

B. Heatmap showing normalized expression (CPM) of the carotenoid cluster genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth in NaCl supplemented medium (0.5 M NaCl)

C. Heatmap showing normalized expression (CPM) of ectoine cluster genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth in NaCl supplemented medium (0.5 M NaCl)

D. Heatmap showing normalized expression (CPM) of desferroxamine cluster genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during exponential and stationary growth in NaCl supplemented medium (0.5 M NaCl)

A, B, C, D Genes marked with diamond were found to be significant in comparison to the wild type strain (FDR ≤ 0.05).

Fig. S10 All clusters of genes identified based on transcriptional changes in ΔhupA, ΔhupS and ΔhupAΔhupS

Results of clust analysis of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS transcriptomes from exponential (red) and stationary (blue) growth in normal and NaCl supplemented medium.

Acknowledgements

We thank Klas Flärdh for the ΔhupS strain (K304). We thank Govind Chandra for help in bioinformatic analysis. We thank Mark Buttner for helpful collaboration.