An Alternative σ Factor, σ8, Controls Avermectin Production and Multiple Stress Responses in Streptomyces avermitilis

Alternative σ factors in bacteria redirect RNA polymerase to recognize alternative promoters, thereby facilitating coordinated gene expression necessary for adaptive responses. The gene sig8 (sav_741) in Streptomyces avermitilis encodes an alternative σ factor, σ8, highly homologous to σB in Streptomyces coelicolor. Studies reported here demonstrate that σ8 is an important regulator of both avermectin production and stress responses in S. avermitilis. σ8 inhibited avermectin production by indirectly repressing expression of cluster-situated activator gene aveR, and by directly initiating transcription of its downstream gene sav_742, which encodes a direct repressor of ave structural genes. σ8 had no effect on cell growth or morphological differentiation under normal growth conditions. Growth of a sig8-deletion mutant was less than that of wild-type strain on YMS plates following treatment with heat, H2O2, diamide, NaCl, or KCl. sig8 transcription was strongly induced by these environmental stresses, indicating response by σ8 itself. A series of σ8-dependent genes responsive to heat, oxidative and osmotic stress were identified by EMSAs, qRT-PCR and in vitro transcription experiments. These findings indicate that σ8 plays an important role in mediating protective responses to various stress conditions by activating transcription of its target genes. Six σ8-binding promoter sequences were determined and consensus binding sequence BGVNVH-N15-GSNNHH (B: C, T or G, V: A, C or G, S: C or G, H: A, C or T, N: any nucleotide) was identified, leading to prediction of the σ8 regulon. The list consists of 940 putative σ8 target genes, assignable to 17 functional groups, suggesting the wide range of cellular functions controlled by σ8 in S. avermitilis.


INTRODUCTION
Soil-inhabiting filamentous Streptomyces are characterized by the ability to produce valuable secondary metabolites having antimicrobial, anticancer, anthelmintic, immunosuppressive, or other biological activities (Challis and Hopwood, 2003). Biosynthesis of secondary metabolites is usually accompanied by initiation of morphological differentiation, and precisely controlled by complex regulatory networks involving cluster-situated regulators (CSRs) and higher-level global/pleiotropic regulators in response to various environmental and endogenous signals (van Wezel and McDowall, 2011;Liu et al., 2013;Niu et al., 2016;Urem et al., 2016). Elucidation of these regulatory networks is essential for strain improvement by metabolic engineering approaches.
Streptomyces avermitilis is an important industrial microorganism used for production of avermectins, effective anthelmintic agents widely applied in agricultural and medical fields (Burg et al., 1979;Egerton et al., 1979). aveR, located at the left edge of the ave gene cluster, encodes a LuxR-family cluster-situated activator (Kitani et al., 2009;Guo et al., 2010). The aveR promoter is recognized by a housekeeping σ factor, σ hrdB (Zhuo et al., 2010). We showed that two extracytoplasmic function (ECF) σ factors, σ 6 (SAV663) and σ 25 (SAV3351), inhibit avermectin production by indirectly affecting aveR transcription (Jiang et al., 2011;Luo et al., 2014). The other 57 σ factors in S. avermitilis remain to be characterized. Elucidation of their functions will help to clarify the regulatory networks involved in avermectin biosynthesis.
With very few exceptions, bacterial σ factors belong to the σ 70 family, which is divided into four groups (Groups 1-4) based on differential possession of four conserved domains (σ 1 , σ 2 , σ 3 , and σ 4 ), phylogenic relationships and physical functions (Osterberg et al., 2011). Group 1 housekeeping sigma factors possess all four domains and are necessary for growth. Group 2 sigma factors also contain four domains, but are dispensable for growth. Group 3 alternative σ factors lack a σ 1 domain and are involved mainly in stress response and differentiation processes. Group 4 ECF sigma factors contain only σ 2 and σ 4 domains and usually respond to extracytoplasmic stimuli. The first identified alternative σ factor is σ B in Bacillus subtilis (Haldenwang and Losick, 1980), which functions as a master regulator that controls >200 genes in response to a wide variety of stress/starvation stimuli including glucose, phosphate, or oxygen starvation, heat or cold shock, ethanol, acid, or osmotic stress, nitric oxide, and antibioticinduced cell wall damage (Lee et al., 2005;Hecker et al., 2007). sigB and its homologes are widespread among Grampositive bacteria, and have diverse functions (Hecker et al., 2007). SCO0600, the σ B homolog in Streptomyces coelicolor, responds to osmotic stress, but not to heat, ethanol, or H 2 O 2 stress (Cho et al., 2001;Lee et al., 2004aLee et al., , 2005. σ B is also involved in secondary metabolism and morphological differentiation in S. coelicolor. The sigB deletion mutant of this species displayed overproduction of actinorhodin (ACT), reduced production of undecylprodigosin (RED) and lack of aerial mycelium formation on R2YE or NA plates (Cho et al., 2001). In Streptomyces hygroscopicus 5008, sigB transcription was enhanced by heat stress or treatment with a reactive oxygen species (ROS) inhibitor, suggesting involvement of σ B in response to changes in temperature or ROS level (Wei et al., 2012).
We demonstrated recently that SAV742, a novel AraC-family transcriptional regulator in S. avermitilis, directly represses ave structural genes and controls cell growth and morphological differentiation . The gene adjacent to sav_742, sav_741 (sig8), encodes an alternative σ factor, σ 8 , homologous to σ B in S. coelicolor. We describe here characterization of σ 8 as an important regulator that controls avermectin biosynthesis and multiple stress responses in S. avermitilis. σ 8 represses avermectin biosynthesis in part through direct initiation of sav_742 transcription. Unlike its S. coelicolor homolog σ B , σ 8 responds to heat, osmotic and oxidative stress by directly regulating expression of its own gene and certain other stress protection genes, but is not involved in morphological differentiation or cell growth. Moreover, we predicted the σ 8 regulon based on the consensus σ 8 -binding promoter sequence.

Primers, Plasmids, Strains, and Growth Conditions
The strains and plasmids used or constructed in this study are listed in Table 1, and the primers in Supplementary Table S1. Culture conditions for Escherichia coli and S. avermitilis strains were as described previously (Liu W. et al., 2015). MM, R2YE (Kieser et al., 2000) and YMS (Ikeda et al., 1988) plates were used for phenotypic observation of S. avermitilis strains. Insoluble fermentation medium FM-I  was used for routine avermectin production. Soluble fermentation medium FM-II  was used to grow mycelia for biomass analysis, and for RNA isolation following stress treatment.

Gene Deletion, Complementation, and Overexpression
A sig8 in-frame gene deletion mutant was constructed using a homologous recombination strategy. Two homologous fragments flanking sig8 were amplified by PCR from wild-type (WT) genomic DNA. A 529-bp 5 -flanking region (positions −337 to +192 relative to the sig8 start codon) was amplified with primers SD2A/SD2B, and a 541-bp 3 -flanking region (positions +835 to +1375) was amplified with primers SD3A/SD3B. The two fragments were cut with HindIII/BamHI and BamHI/EcoRI, respectively, and then simultaneously cloned into HindIII/EcoRI-digested pKC1139 to generate sig8 deletion vector pKCDsig8. Non-methylated pKCDsig8 was transformed into WT protoplasts, and double-crossover recombinant strains were isolated as reported previously (Zhao et al., 2007). Resulting sig8 deletion mutants were verified by colony PCR using primers SD23A/SD23B (flanking the exchange regions) and SD39A/SD39B (located within the deletion region of sig8) (Supplementary Figure S1), followed by DNA sequencing. When primers SD23A/SD23B were used, a 1.2-kb band appeared, whereas a 1.9-kb band was detected in WT genomic DNA. When primers SD39A/SD39B were used, only WT DNA generated a 390-bp band. We thus obtained sig8 gene deletion mutant Dsig8, in which 840-bp sig8 ORF was mostly deleted (from positions +193 to +834 relative to the start codon) (Supplementary Figure S1). The deletion part of sig8 covered coding region for all three functional domains (σ 2 , σ 3 , and σ 4 ); Thus, the remaining fragment was unlikely to be functional.
To construct a sig8sav_742 double deletion mutant, the pKCDsig8 vector was transformed into D742 protoplasts . The expected mutant, Dsig8-742, was isolated by selection of the Dsig8 mutant.
For complementation of Dsig8, a 1618-bp PCR fragment containing the sig8 promoter and open reading frame (ORF) was amplified with primers SD1B/SD1D, and inserted into the integrative vector pSET152 to give sig8-complemented vector pSET152-sig8, which was then transformed into Dsig8 to obtain complemented strain Csig8.

Production and Analysis of Avermectins
Fermentation of S. avermitilis strains and HPLC (highpressure liquid chromatography) analysis of avermectin yield in fermentation broth were performed as described previously .

Quantitative Real-Time RT-PCR (qRT-PCR)
S. avermitilis mycelia grown in FM-I or FM-II were collected at various time points for RNA isolation. Triturated samples were suspended in TRIzol reagent (Tiangen, China) for RNA isolation. Genomic DNA contamination was removed by treatment of RNA samples with RNase-free DNase I (TaKaRa, China). 4 µg total RNA was used for cDNA synthesis. qRT-PCR was performed using primers listed in Supplementary Table S1 to analyze transcription levels of the tested genes as described previously (Luo et al., 2014), with expression level of housekeeping gene 16S rRNA as internal control. Each experiment was repeated three times.

Electrophoretic Mobility Shift Assays (EMSAs)
EMSA probes carrying respective tested promoter regions were obtained by PCR using primers listed in Supplementary Table S1, and labeled at their 3 ends with digoxigenin (DIG). Conditions for binding reaction and detection were as described previously . To confirm binding specificity between His 6 -σ 8 and DNA probes, a ∼300-fold excess of each specific or nonspecific (hrdB) unlabeled probe was added to the reaction mixture before incubation.

In Vitro Transcription Assay
DNA templates containing respective promoter and partial coding region were amplified by PCR using primers listed in Supplementary Table S1. Conditions for transcription assays and detection of transcripts were as described previously (Luo et al., 2014). Reaction mixtures contained 0.4 pmol DNA template and various amounts of renatured His 6 -σ 8 . Transcription was initiated by addition of 3.7 pmol E. coli core RNA polymerase (core-RNAP) (New England Biolabs, USA).

Rapid Amplification of cDNA Ends (RACE)
The transcriptional start site (TSS) of selected genes was identified using a 5 /3 RACE kit (Roche, 2nd Generation). S. avermitilis strains were cultured in FM-II at 28 • C for 2 days, and then subjected to various stress conditions. Mycelia were harvested after various durations of stress treatment and used for RNA isolation. 4 µg total RNA was reverse transcribed with 40 pmol gene-specific primer SP1. Purified cDNAs were added to oligo-dA tails at the 3 end by TdT (terminal deoxynucleotidyl transferase, TaKaRa) treatment at 37 • C for 30 min. The tailed cDNA was used as template for first-round PCR using second inner gene-specific primer SP2 and oligo-dT anchor primer. The resulting PCR product was diluted to appropriate concentration, and used as template for second-round PCR with nested primer SP3 and an anchor primer. The purified final PCR product was sequenced (Invitrogen Biotechnology Corporation, China). TSS was determined as the first nucleotide following the oligo-dT sequence.

σ 8 Regulates Avermectin Production by Repressing ave Gene Expression during the Late Fermentation Stage
The sig8 (sav_741) gene in the S. avermitilis chromosome contains 840 nucleotides (nt) and encodes a 279-amino-acid σ 70family alternative σ factor, σ 8 . The downstream convergently transcribed gene sav_742, located 377 nt from sig8 ( Figure 1A), encodes an AraC-family transcriptional regulator that was recently characterized as a global regulator of avermectin biosynthesis, cell growth, and morphological development . The upstream convergently transcribed gene sav_740, located 215 nt from sig8, encodes a hypothetical protein. BLAST analysis revealed that σ 8 displays high amino acid sequence identities with S. coelicolor σ B (77.22%) and S. hygroscopicus σ B (78.29%).
To investigate the role of σ 8 in avermectin biosynthesis, we constructed sig8 deletion mutant Dsig8, complemented strain Csig8 and overexpression strain Osig8, and compared their avermectin yields with that of WT ATCC31267 cultured in FM-I for 10 days. Relative to WT yield, that of Dsig8 was ∼96% higher, that of Osig8 was ∼50% lower, and that of Csig8 was similar ( Figure 1B). Yields of two vector control strains, WT/pSET152 and WT/pKC1139, were almost the same as that of WT ( Figure 1B). These findings indicate that σ 8 has a negative effect on avermectin production.
To assess the effect of σ 8 on S. avermitilis growth, we measured biomasses of WT, Dsig8 and Csig8 cultured in soluble FM-II. Biomass of Dsig8 and Csig8 was similar to that of WT ( Figure 1C), indicating that σ 8 has no effect on cell growth, and that avermectin overproduction in Dsig8 does not result from alteration of growth. Dsig8 and Osig8 grew normally on YMS, R2YE, and MM plates (Supplementary Figure S2), indicating that σ 8 also has no effect on morphological differentiation.
To investigate the relationship between σ 8 and avermectin production, we examined the sig8 transcription profile by qRT-PCR using RNAs isolated from WT in FM-I. sig8 transcript was detected throughout the fermentation process. Its level increased gradually from day 1, reached a maximum on day 6, and remained high thereafter, suggesting that σ 8 plays its regulatory role mainly during the late stage of fermentation ( Figure 1D).
The effect of σ 8 on expression of ave genes was assessed by qRT-PCR analysis of transcription levels of aveR (CSR gene) and aveA1 (structural gene encoding AVES1 polyketide synthase) in WT and Dsig8 grown in FM-I for 2 days (early exponential phase) or 6 days (stationary phase). Transcription levels of aveR and aveA1 in Dsig8 did not differ from those in WT on day 2, but were notably higher on day 6 ( Figure 1E), consistent with the observed avermectin overproduction in Dsig8. These findings indicate that σ 8 controls avermectin production at the transcription level by repressing ave genes, primarily in the late fermentation stage. σ 8 Directly Activates Expression of Its Own Gene and sav_742, Indirectly Regulates ave Genes σ factors function as initiators of transcription. The negative regulatory effects of σ 8 on aveR and aveA1 are therefore likely to be indirect. To test this idea, we performed EMSAs using refolded soluble recombinant His 6 -σ 8 and probes aveRp and aveA1p prepared by labeling promoter regions of aveR and aveA1. His 6 -σ 8 did not bind to the probes even at high concentration (1 µM) (Figure 2A), confirming that σ 8 indirectly represses ave genes.  (Dsig8), complemented strain (Csig8) and overexpression strain (Osig8) grown in FM-I for 10 days. WT/pKC1139 and WT/pSET152: vector control strains constructed by transformation of plasmids pKC1139 and pSET152 into WT, respectively. Error bar: SD from three independent experiments. NS, not significant; * * * , P < 0.001 for comparison of values for mutant versus WT strains (Student's t-test). (C) Growth curves of WT, Dsig8 and Csig8 in soluble FM-II. (D) Transcriptional profile of sig8 in WT grown in FM-I. Values were calculated by normalization against internal control gene 16S rRNA at specific time points. The relative value of sig8 at day 1 was assigned as 1. (E) qRT-PCR analysis of aveR and aveA1 from WT and Dsig8 grown in FM-I at days 2 and 6. Transcription level of each gene was expressed relative to WT value at day 2, defined as 1. NS, not significant; * * * , P < 0.001 (Student's t-test).
σ B homologes typically initiate their own transcription (Hecker et al., 2007). The finding that σ 8 indirectly represses ave genes suggested that σ 8 initiates transcription of direct repressor(s) of aveR or aveA1. We recently characterized PhoP (Yang et al., 2015) and AvaR2  as direct repressors of aveR. We also found that SAV742 directly represses transcription of several ave structural genes including aveA1, but not aveR . To test the hypothesis that σ 8 directly controls sig8 and regulatory genes phoP, avaR2 and sav_742, we performed EMSAs, qRT-PCR analysis and in vitro transcription assays.
EMSA results revealed that His 6 -σ 8 formed complexes with promoter regions of sig8 (probe sig8p) and sav_742 (probe sav_742p), but not with those of avaR2 (probe avaR2p) or phoR-phoP operon (probe phoRp) (Figure 2A). No shifted band was observed for negative probe control hrdB or protein control BSA. Binding specificity was confirmed by competitive assays using excess unlabeled specific probe (lanes S) and non-specific probe hrdB (lanes N). qRT-PCR analysis showed that transcription levels of sig8 and sav_742 on days 2 and 6 were lower in Dsig8 than in WT (Figure 2B), indicating that σ 8 positively regulates expression of its own gene and adjacent gene sav_742. The lower sav_742 transcription level and higher avermectin yield in Dsig8 are consistent with SAV742's function as a repressor of avermectin production .
Avermectin production in Dsig8 was very close to that in sig8sav_742 double deletion mutant Dsig8-742 (Supplementary Figure S3), but was higher than that in sav_742 deletion mutant D742 , suggesting that other σ 8 targets may also affect avermectin production in Dsig8.

σ 8 Responds to Various Environmental Stresses
Alternative σ factors are usually involved in modulation of stress responses (Osterberg et al., 2011). To determine which type(s) of stress σ 8 responds to, we performed stress tests on YMS plates. Relative to growth of WT, that of sig8 deletion mutant Dsig8 was more sensitive to NaCl, KCl, H 2 O 2 , diamide, and heat (42 • C) stresses, but similarly sensitive to tert-butyl hydroperoxide (TBHP) and sucrose stresses ( Figure 3A). These findings indicate that σ 8 is required for WT levels of resistance to multiple stresses, e.g., heat stress, salt stress, and oxidative stress (from H 2 O 2 or diamide).
The observations that sig8 mutant Dsig8 was more sensitive to various environmental stresses, and that σ 8 was autoregulated, suggested that sig8 itself may be induced by these environmental stresses in a σ 8 -dependent manner. We tested this possibility by qRT-PCR comparison of sig8 transcription levels in WT and Dsig8 under stress conditions. Cells were cultured in soluble FM-II for 2 days, and then treated with heat (42 • C), H 2 O 2 , diamide, or NaCl. RNA samples were isolated from cells before (0 min) and after treatment (10, 20, 30, 40, 60, 80, and 100 min). For WT, sig8 transcription level increased to a maximal value within 10-60 min for each stress type (∼13-fold for heat; ∼20fold for H 2 O 2 , ∼17-fold for diamide; ∼54-fold for NaCl). For Dsig8, maximal induction was sharply decreased under each stress type (∼6-fold for heat; ∼2-fold for H 2 O 2 , ∼8-fold for diamide; ∼17-fold for NaCl) and delayed for heat (from 10 to 30 min) and salt (from 60 to 80 min) ( Figure 3B). These findings indicate that σ 8 itself responds to various environmental stresses at the transcription level in either a σ 8 -dependent or σ 8independent manner. The rapid and robust induction of sig8 in WT may be due to σ 8 -dependent control, whereas the decreased and/or delayed induction in Dsig8 may be due to σ 8 -independent control.
Identification of σ 8 Target Genes Related to Heat Stress σ 8 was strongly induced in response to a variety of stresses, suggesting that it mediates defensive systems against stresses. To identify σ 8 target genes that respond to heat stress, we initially performed a series of EMSAs using His 6 -σ 8 and potential promoter probes of heat shock genes, including dnaK1p for dnaK1-grpE1-dnaJ1-hspR operon, dnaK2p for dnaK2-grpE2-dnaJ2 operon, groES1p for groES1-groEL1 operon, groEL2p, htpGp, hsp18_1p and hsp18_2p. His 6 -σ 8 bound specifically to dnaK1p, but not to other probes tested ( Figure 4A). In vitro transcription analysis confirmed that dnaK1 transcription was initiated by σ 8 (Figure 4B). dnaK1 transcription level in Dsig8 was downregulated at two time points (Figure 4C). These findings demonstrate that σ 8 is a direct activator of dnaK1. grpE1, dnaJ1, and hspR are cotranscribed with dnaK1, and therefore are also σ 8 targets. dnaK1, grpE1, and dnaJ1 encode molecular chaperones for interaction with denatured proteins and facilitate refolding to the native state following heat stress, and hspR encodes a putative transcriptional repressor of its operon. Transcription levels of dnaK1 in WT and Dsig8 under heat stress were recorded to determine whether dnaK1p for its operon was induced in a σ 8 -dependent manner. In WT, dnaK1 transcription level increased rapidly to ∼45-fold after 10 min exposure to 42 • C, and then gradually declined. In Dsig8, maximal induction was reduced to ∼35-fold and delayed after 30 min ( Figure 4D). These findings indicate that σ 8 facilitates rapid adaption to heat stress mainly by direct regulation of the dnaK1-grpE1-dnaJ1-hspR operon. The slower, lower-degree induction of dnaK1p in Dsig8 presumably depends on other factors not addressed here.
For analysis of oxidative stress responses, WT and Dsig8 were treated with 1 mM H 2 O 2 or diamide for various durations. In WT, catR and oxyR were induced to maximal transcription level (∼20-and ∼23-fold, respectively) by H 2 O 2 within 40 min ( Figure 5D). In Dsig8, H 2 O 2 treatment caused only a slight increase of transcription level (∼4-fold induction) of these two  2 mM), and incubated at 28 • C for 3 days. For heat stress assay, spore suspensions were treated at 42 • C for 5 min and then spotted onto YMS plates. Each stress assay was repeated three times on YMS plates with consistent results. (B) Induction of sig8 transcription in WT and Dsig8 by stresses. RNAs used for qRT-PCR analysis were prepared from cells grown in FM-II for 2 days followed by treatment with 42 • C, 1 mM H 2 O 2 , 1 mM diamide, or 250 mM NaCl for the indicated times. Relative values are shown as fold change relative to sig8 transcription level at the first time point (0 min) in WT, which was assigned as 1.
Prediction of the σ 8 Regulon σ 8 in S. avermitilis evidently plays a pleiotropic role in control of avermectin production and in protection against a variety of stresses. To clarify broader roles of σ 8 in this species, more σ 8 target genes need to be identified. The recognition and binding sites of σ 70 -family factors are the -35 and -10 hexamers of its target promoters. We conducted 5 -RACE assays to determine promoter structures of several σ 8 targets and identified a consensus σ 8 -binding sequence. TSSs of sig8, dnaK1, oxyR, trxA3, sig22, and opuBC1 were determined by 5 -RACE analysis of WT gene transcripts under stress conditions (Supplementary Figure S4), leading to the putative -35 and -10 promoter sequences shown in Figure 7. Analysis of these promoter sequences using the PREDetector web-based program 1 revealed a consensus sequence BGVNVH-N 15 -GSNNHH (Figure 7), which resembles that of σ B -specific promoters of S. coelicolor (GNNTN-N 14−16 -GGGTAY) (Y: C or T) (Lee et al., 2004b).
RNA polymerase is not sensitive to variations in spacer length between the -35 and -10 regions, and the consensus σ Bbinding sequence is GNNTN-N 14−16 -GGGTAY. We therefore employed the sequence pattern BGVNVH-N 14−16 -GSNNHH to scan the S. avermitilis genome using the PREDetector program to predict candidate members of the σ 8 regulon, and selected putative promoters that were located within 300 nt upstream of the translational start codons. Using a cut-off score of ≥6.5, we identified 940 putative σ 8 target genes, of which 453 have unknown function or are unclassified (Supplementary Table  S2). The remaining 487 genes were assigned to 17 functional groups. Among these 487 genes, 179 are associated with regulatory functions, according to the KEGG pathways database for S. avermitilis 2 . These findings suggest the extent biological significance of σ 8 in S. avermitilis.

DISCUSSION
Important roles of alternative σ factors in Streptomyces species in regulation of secondary metabolism are suggested by several previous studies. In S. coelicolor, σ B (Cho et al., 2001;Lee et al., 2004b) and σ K (Mao et al., 2009) are involved in regulation of ACT and RED biosynthesis, although the regulatory mechanisms are unclear. In Streptomyces chattanoogensis L10, alternative σ factor WhiG ch promotes natamycin biosynthesis by directly 2 www.genome.jp/kegg-bin/show_organism?org=sma FIGURE 7 | Analysis of consensus σ 8 -binding promoter sequence using the PREDetector program. Putative -35 and -10 regions are underlined. Shading: translational start codons. Boldface: TSSs. In the sequence logo of σ 8 -binding consensus, appearance frequency of each base is proportional to the height of the corresponding letter. activating two structural genes, scnC and scnD (Liu S. P. et al., 2015). In the present study, we characterized alternative σ factor σ 8 in S. avermitilis, and demonstrated that it indirectly represses avermectin production through its effects on expression of CSR gene aveR and structural gene aveA1. σ 8 is the first among the 10 alternative σ factors in S. avermitilis to be characterized. Our findings augment the limited knowledge of regulation of secondary metabolism by alternative σ factors in Streptomyces.
AveR is an essential activator for transcription of the ave cluster . We showed that σ 8 initiates transcription of SAV742, which directly represses expression of ave structural genes aveA1, aveD, aveF, and aveA4 , but not that of aveR. Thus, σ 8 controls avermectin production through at least two pathways: (i) directly initiating transcription of sav_742; (ii) indirectly repressing expression of aveR. The observation that σ 8 has an indirect effect on aveR expression suggests that it regulates aveR through a "cascade" mechanism. However, our search for aveR upstream regulator(s) that mediate the repression of σ 8 on aveR showed that the two known aveR direct repressor genes, phoP (Yang et al., 2015) and avaR2 , are not targets of σ 8 . Future studies will identify such direct regulator(s) of aveR and clarify the mechanisms underlying σ 8 function in avermectin production.
Alternative σ factors help to control morphological differentiation and stress responses, in addition to secondary metabolism. In S. coelicolor, σ B is required for aerial mycelium formation and osmoprotection (Cho et al., 2001); σ B -dependent σ L and σ M are involved in sporulation; σ H is activated under heat stress and osmotic stress, and plays a crucial role in morphological development (Sevcikova et al., 2001); σ I responds to osmotic stress, but has no effect on differentiation (Homerova et al., 2012); σ F and σ WhiG affect spore formation (Chater et al., 1989;Potuckova et al., 1995;Lee et al., 2005). In S. hygroscopicus, sigB is induced by heat stress and ROS inhibitor (Wei et al., 2012). The present study showed that σ 8 is involved in protection against heat, osmotic and oxidative stresses, but has no effect on morphological differentiation. The differing functions of σ B homologes in various Streptomyces species presumably reflect differences in regulatory mechanisms for σ B activity/expression, and in σ B -mediated regulatory pathways for adaptation to various environmental stresses.
In many Gram-positive bacteria, activity of σ B homologs is modulated by their cognate anti-σ factors (Hecker et al., 2007), which inhibit transcription activity of σ B by binding to it. Under stress conditions, σ B is released free of its anti-σ factor, and subsequently initiates transcription of its target genes related to stress protection. The anti-σ factor gene is typically located adjacent to the sigB locus. In S. coelicolor, the anti-σ B factor gene rsbA (sco0599) is upstream of sigB (sco0600) (Lee et al., 2004a). In S. avermitilis, the rsbA-homolog gene prsR (sav_7158) is not located near sig8 (sav_741), and sig8-adjacent genes have no similarity to rsbA, suggesting that the regulatory mechanism of σ 8 activity is different from that of σ B . Further studies to identify regulators associated with σ 8 , based on co-immunoprecipitation assays within S. avermitilis cells, are in progress.
σ 8 responds rapidly to heat stress by directly activating transcription of the dnaK1-grpE1-dnaJ1-hspR operon. Heat shock proteins include not only chaperones for refolding denatured proteins, but also proteases for degrading more denatured proteins. S. avermitilis has three putative heat shock protease genes lonA, htpX1, and htpX2, and whether they are under direct control of σ 8 needs to be investigated. Under H 2 O 2 stress, σ 8 directly activates transcription of H 2 O 2 -sensing regulator genes catR and oxyR, but not of kat or ahpCD, suggesting that CatR and OxyR mediate H 2 O 2 induction of peroxide-scavenging enzymes. We showed that OxyR in S. avermitilis directly activates expression of antioxidant enzyme genes ahpCD, katA1, katA2, and katA3 in response to H 2 O 2 stress . The targets of CatR remain to be characterized. σ 8 thus exerts its protective effect against H 2 O 2 damage in S. avermitilis mainly through a cascade regulatory mechanism involving control of OxyR. σ R homologes and their predicted targets, e.g., trx and msh genes are well-conserved in Streptomyces (Kim et al., 2012). Two trx genes, three msh genes and sig22 (sigR homolog) in S. avermitilis are directly controlled by σ 8 in response to diamide, indicating that σ 8 facilitates a rapid response to thiol-oxidative stress through both direct and cascade regulatory mechanisms. σ 8 targets involved in osmoprotection, identified in this study, are ect genes, opuB genes, osaAB and katB. The specific substrate of OpuB transporter in S. avermitilis remains unknown. Our findings are consistent with the observations that ectoine is osmoprotectant, and osaAB and katB are required for σ Bdependent osmoprotection in S. coelicolor (Cho et al., 2000(Cho et al., , 2001Bursy et al., 2008;Fernández Martínez et al., 2009), indicating a conserved role of ectoine, osaAB and katB in osmoadaptation in Streptomyces.
Our present findings, taken together, demonstrate clearly that σ 8 is a pleiotropic regulator of avermectin production and responses to heat, osmotic and oxidative stresses in S. avermitilis. A proposed model of the σ 8 -mediated regulatory network is presented in Figure 8. σ 8 is a good example of a regulator that links stress responses to antibiotic production. Regulatory pathways of specific stress responses are potential targets for genetic manipulation to increase antibiotic yields. For example, disruption of osaB led to a 37% increase in avermectin yield (Godinez et al., 2015), and deletion of sig8 increased avermectin yield ∼96%. Continued elucidation of such regulatory mechanisms will contribute to improvements in antibiotic production.
Using the identified σ 8 consensus binding sequence, we predicted 940 putative σ 8 target genes. It is unlikely that σ 8 binds to all the predicted targets, more knowledge of the promoter sequence recognized by σ 8 is necessary for precise prediction of σ 8 regulon. Studies using high-throughput technologies (e.g., ChIP-seq) are in progress to experimentally verify additional σ 8 targets in S. avermitilis and thereby elucidate the wide range of cellular functions controlled by this important σ factor.

AUTHOR CONTRIBUTIONS
YW and DS: designed the research, DS: performed experiments, QW, ZC, and JL: contributed study materials, YW and DS: wrote the manuscript.

FUNDING
This study was supported by National Natural Science Foundation of China (grant no. 31170045).