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‘Exploring’ the regulation of Streptomyces growth and development
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ScienceDirect ‘Exploring’ the regulation of Streptomyces growth and development Stephanie E Jones1,2 and Marie A Elliot1,2 The Streptomyces life cycle encompasses three wellestablished developmental stages: vegetative hyphae, aerial hyphae and spores. Many regulators governing the transitions between these life cycle stages have been identified, and recent work is shedding light on their specific functions. A new discovery has shown Streptomyces can deviate from this classic life cycle through a process termed ‘exploration’, where cells rapidly traverse solid surfaces. Exploration does not require any of the traditional developmental regulators, and therefore provides an exciting new context in which to uncover novel developmental pathways. Here, we summarize our understanding of how Streptomyces exploration is controlled, and we speculate on how insight into classical regulation and stress response systems can inform future research into the regulation of exploratory growth. Addresses 1 M.G. DeGroote Institute for Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1 2 Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1 Corresponding author: Elliot, Marie A ([email protected])
Current Opinion in Microbiology 2018, 42:25–30 This review comes from a themed issue on Cell regulation Edited by Jan-Willem Veening and Rita Tamayo
https://doi.org/10.1016/j.mib.2017.09.009 1369-5274/ã 2017 Elsevier Ltd. All rights reserved.
Introduction to Streptomyces development and exploration Within the soil, microbes are highly abundant, and they grow using remarkably diverse strategies. Amongst these soil-dwelling microbes, Streptomyces are a ubiquitous group of Gram-positive bacteria that are best known for their ability to produce an extraordinary range of antibiotics and other medicinally/agriculturally useful compounds [1]. Streptomyces also have a well-studied, complex life cycle that classically encompasses three morphologically differentiated growth stages [2] (Figure 1). The Streptomyces life cycle initiates with germ tubes emerging from a previously dormant spore. These tubes www.sciencedirect.com
extend by polar growth at the hyphal tips, with additional branches emerging from the lateral walls at random intervals. Continuous cycles of polar growth and branching ultimately result in the formation of a dense network of filaments termed the vegetative mycelium. This branching mycelium anchors the Streptomyces colony to its growth substrate, similar to the way a plant root system anchors a plant in the soil. Polar growth is directed by the ‘polarisome’, a multiprotein complex involved in recruiting the peptidoglycan biosynthetic machinery to the hyphal tips [3–7]. Chromosomes replicate during vegetative growth, and the resulting multinucleate hyphae are delimited by occasional crosswalls [8,9]. In response to nutrient depletion, Streptomyces commence their second differentiated growth stage. Here, nonbranching aerial hyphae coated in a hydrophobic protein layer (consisting of chaplin proteins and the surfactant SapB [10–12]) rise into the air, away from the vegetative mycelium (Figure 1). The transition from branching vegetative growth to hydrophobic, nonbranching aerial growth requires regulators termed the bld genes — so named for the ‘bald’ colony phenotype of bld mutants, where they lack the characteristic fuzzy aerial hyphae that cover wild type colonies. Once aerial growth ceases, the long, multigenomic filaments undergo synchronous septation. This generates chains of single genome prespore compartments, which then mature to form dormant, pigmented spores. The transition from aerial hyphal growth to sporulation requires regulators termed ‘whi’ (for ‘white’) genes, as whi mutants lack the pigment associated with mature, wild type spores. Recent work has shown that Streptomyces can escape this classically defined life cycle using a new mode of growth termed ‘exploration’ [13,14] (Figure 1). This exploration phenomenon was discovered during the co-culture of Streptomyces venezuelae with yeast — a situation that mimics the polymicrobial communities in the soil. Exploration occurs in response to fungi and other metabolic cues, and involves the rapid spread of Streptomyces colonies over solid surfaces including agar, plastics and rocks. During exploratory growth, cells grow as nonbranching vegetative hyphae, and proceed at a rate more than 10 that of vegetative growth. In recent years, there have been important advances in understanding the roles of the bld and whi genes in regulating the transitions between the different canonical life cycle stages [15,16,17,18,19,20]. Exploration Current Opinion in Microbiology 2018, 42:25–30
26 Cell regulation
Figure 1
WhiB WhiA
WhiG
WhiI BIdM
WhiE
BIdD-(c-di-GMP)
other BId regulators
BIdN
spore chains
aerial hyphae
BIdM BIdM
BIdO vegetative mycelium
classic Streptomyces life cycle
germination spore unknown regulators trimethylamine CydCD required
Streptomyces exploration
? ? Streptomyces explorer cells
Streptomyces aerial hyphae and spores yeast abundant amino acids glucose consumption
medium alkalinization
induction of exploration
Current Opinion in Microbiology
The classic Streptomyces life cycle and Streptomyces exploration. Left: Germ tubes emerge from a single dormant spore, and Streptomyces can enter either the classic life cycle (top) or initiate exploratory growth (bottom). Top: Germ tubes extend by polar growth at the hyphal tips, and additional branches emerge from the lateral walls, forming a dense network of branching hyphae termed the vegetative mycelium. Within the vegetative mycelium, BldD bound to c-di-GMP represses other bld genes, including genes encoding BldM and the sigma factor BldN, and various whi genes, including those encoding WhiB, WhiA, WhiG and WhiI. Within the vegetative mycelium, a BldM homodimer represses the sporulation regulator WhiB. When BldD-mediated repression is relieved, other Bld regulators activate genes required for Streptomyces to raise aerial hyphae. Subsequently, aerial hyphae coated in a hydrophobic sheath rise into the air. Within the aerial hyphae, the recently discovered regulator BldO initially represses the expression of whiB. When this repression is relieved, WhiB together with WhiA, and WhiI together with BldM, activate other genes necessary for sporulation. Aerial hyphae differentiate into long chains of dormant exospores, and spores can be dispersed to begin the life cycle anew. Bottom: After germination, Streptomyces can diverge from their classic life cycle. Combinations of low glucose and concentrated amino acid sources trigger exploration. When Streptomyces are grown beside fungi, Streptomyces first grows on top of the yeast, raising aerial hyphae and forming spores. Once the yeast has consumed the local glucose supply, Streptomyces exploration begins. Explorer cells grow as nonbranching vegetative hyphae, in contrast to the branching vegetative mycelium seen in the classic Streptomyces life cycle. Explorer cells release the volatile compound trimethylamine (TMA — indicated in purple), which raises the pH of the surrounding medium from 7.0 to 9.5. Explorer cells require genes encoding the cytochrome bd oxidase complex, presumably to withstand the highly alkaline environment. TMA promotes continued exploration of the producer colony, and can stimulate exploration by nearby Streptomyces colonies. Known Bld and Whi regulators are not required for exploration. Other regulators involved in activating/repressing genes required for exploration are currently unknown.
does not directly require these regulators, and we are in the early stages of understanding the cues and genes involved in the control of exploration. In this review, we discuss our current understanding of factors governing exploration, highlight some of the recent advances in understanding the roles of regulators involved in the canonical Streptomyces life cycle, and explore connections between these two life cycle trajectories. Current Opinion in Microbiology 2018, 42:25–30
Models matter: S. coelicolor versus S. venezuelae The foundation for understanding Streptomyces development was laid by studies conducted in Streptomyces coelicolor, a species that fully differentiates on solid medium but not in liquid culture. In recent years, there has been a shift to studying development in S. venezuelae, which like S. coelicolor undergoes its full life cycle during growth on www.sciencedirect.com
Control of exploration in Streptomyces Jones and Elliot 27
solid medium, but also sporulates to completion in liquid culture. Notably, S. venezuelae is capable of exploratory growth, while S. coelicolor does not explore, at least under conditions tested to date [13]. This could explain in part, why Streptomyces exploration had not been previously reported, despite decades of developmental investigation. This raises the obvious question of whether exploration is in any way tied to the ability to undergo submerged sporulation. This does not seem to be the case, as Streptomyces griseus — another streptomycete that can sporulate in liquid culture — is not exploration competent under conditions that promote S. venezuelae exploration [13]. In all, 10% of streptomycetes tested seem to be able to explore. Whether the others are unable to do so, or whether they explore in response to different signals remains to be determined.
Understanding Streptomyces exploration Exploration does not require classical developmental regulators
Explorer cells appear to be a unique Streptomyces cell type, in that they share properties of both vegetative hyphae and aerial hyphae [13]. Like vegetative hyphae, they are hydrophilic, and like aerial hyphae, they do not branch. Mutant strains lacking the Bld or Whi regulators can explore in the same way as wild type strains, indicating the transition from vegetative growth to aerial hyphal growth is not required for exploration. The one exception to this is the bldN mutant strain, which explores more slowly than wild type. BldN is a sigma factor that activates the expression of both the developmental regulatory gene bldM, and the chp genes [21], which encode the chaplin proteins that assemble into the hydrophobic sheath covering aerial hyphae and spores [10,11]. Given the hydrophilic nature of explorer cells, it seems unlikely that the hydrophobic chaplin proteins play a key role in exploration, so this implies that either BldM or another BldM/ BldN regulon member may facilitate efficient exploration. BldM is an ‘orphaned’ response regulator (it lacks a cognate kinase, and does not require phosphorylation for activity), and is unusual in that it acts both as a homodimer, and as a heterodimer in association with WhiI (another orphaned response regulator) [17]. Notably, neither bldM nor whiI mutants exhibit altered exploration [13], suggesting that BldN may have additional regulon members that help to promote exploration. New bld regulators continue to be discovered. These include the recently characterized BldO, which controls the expression of the sporulation regulator whiB [19,22]. Whether other — as yet untested — canonical developmental regulators impact exploration remains to be seen. BldD is a master repressor of bld and whi gene expression [23,24], but unlike most other bld mutants (apart from bldO), bldD mutants are not blocked at vegetative growth, and instead transition directly from germination to www.sciencedirect.com
sporulation and are seen to hypersporulate [16]. It was therefore particularly surprising that the bldD mutant strain appeared to explore in a manner indistinguishable from wild type, presumably as nonbranching vegetative hyphae, although this remains to be confirmed [13]. This suggests that exploration may not require typical vegetative growth, and/or may be a process that is entirely independent of any BldD-governed pathway. It is tempting to speculate that exploration may have evolved as a bet-hedging strategy to help ensure survival in the event of mutations in the classic developmental regulators. Mutations in any bld or whi gene would block the sporulation/dispersal capabilities of Streptomyces, with exploration then providing cells with an alternative means of escaping to a more favourable growth environment. Nutritional cues governing exploration and development
Exploration by S. venezuelae relies on distinct nutritional factors [13]. It requires both a rich peptide source, and a low glucose environment (Figure 1). In the S. venezuelaeyeast association on glucose-containing medium, the yeast titrates glucose from the medium, and this relieves repression of exploration by S. venezuelae. Accordingly, cells grown without yeast in a glucose-free environment can initiate exploration on their own. The transition from vegetative to aerial growth in the classical life cycle is also thought to have a nutritional component, with it being widely assumed that nutrient depletion promotes the initiation of aerial hyphae formation. While the specific cues that are sensed have yet to be identified, the accumulation of different developmental regulators is affected by glucose levels [25], and the developmental defects associated with a number of bld mutations can be suppressed by growing these strains on alternative carbon sources [12,26]. The glucose-repressible nature of exploration suggests a role for carbon catabolite repression. In Streptomyces, this is thought to be mediated in part by the glucose kinase enzyme [27,28]. A glucose kinase-independent pathway has also been characterized in S. coelicolor, in which pyruvate phosphate dikinase (PPDK) figures prominently [25]. Whether the activity of either of these enzymes is required for exploration remains to be tested, but it is worth noting that RNA-sequencing experiments comparing transcript levels in exploring versus stationary cultures revealed significant upregulation of the PPDK-encoding gene [13]. pH profoundly influences exploration and development
Genetic screens for strains deficient in exploration, coupled with whole-genome sequencing, identified genes encoding the cytochrome bd oxidase complex as being essential for exploration [14]. This complex catalyzes electron transfer in the electron transport chain without simultaneous pumping of protons across the membrane [29], and functions as part of the alkaline stress response in other bacteria [30]. Explorer cells raise the pH of solid medium from 7.0 to 9.5 — which is considered highly Current Opinion in Microbiology 2018, 42:25–30
28 Cell regulation
alkaline for Streptomyces. It has been hypothesized that the cytochrome bd oxidase complex is essential in allowing explorers to tolerate alkaline environments. Consistent with this proposal, transcription profiling experiments revealed the five gene clusters most highly upregulated in explorers were associated with alkaline stress responses in other bacteria. How the cytochrome complexes and alkaline stress responsive genes are controlled in Streptomyces is currently unknown. Additional genetic screens are required to identify the regulators responsible for activating these adaptive gene clusters. Explorer cells raise the pH of the medium by releasing the volatile organic compound (VOC) trimethylamine (TMA) [13] (Figure 1). Remarkably, this small, nitrogenous compound can induce exploration in physically separated Streptomyces while also killing other soil microbes, serving as both an exploration-inducing airborne signal and a competitive weapon. TMA-mediated induction of exploration appears to result from a rise in pH, rather than a TMA-specific signalling cascade, as other alkaline VOCs can also induce exploration. The genes directing the production of TMA in Streptomyces remain to be determined. It will be interesting to see whether these TMA-associated genes are integrated in dedicated regulatory networks, or whether TMA is naturally produced as a by-product of the metabolism of nitrogen-rich peptide sources. While aerial hyphae formation and sporulation are not known to require TMA or a high alkaline environment, aerial hyphae development is pH sensitive and can be inhibited by acidic conditions. Indeed, mutations in primary metabolic genes (e.g. citA and cya) and some bld genes, result in increased secretion of organic acids and a concomitant inability to sporulate [31]. This raises the tantalizing possibility that TMA emission by exploring cultures could rescue the developmental defects of some sporulation-deficient mutants. Exploration as a form of Streptomyces motility
Explorer cells can rapidly traverse solid surfaces, and this spreading is phenotypically similar to sliding motility — a growth-dependent mode of microbial movement facilitated by a secreted surfactant [32]. Streptomyces produce two known surfactants: SapB and the chaplin proteins. Given that the production of both of these surfactants requires the activity of the bld genes, and these genes are largely dispensable for exploration [13], it seems unlikely that either SapB or the chaplins are the exploration/sliding motility-promoting surfactants. Many bacteria move by sliding motility [33], including Pseudomonas [34], Serratia [35], Legionella [36], Bacillus [37], Salmonella [38], Sinorhizobium [39] and Mycobacterium [40]. Interestingly, the regulators and surfactants permitting sliding seem to be genus-specific. For example, in Current Opinion in Microbiology 2018, 42:25–30
Bacillus subtilis, the master sporulation regulator SpoOA activates the transcription of genes encoding products necessary for the secretion of surfactin molecules [41]. In contrast, the opportunistic human pathogen Pseudomonas aeruginosa uses the two-component system GacA/GacS to control the production of exopolysaccharides and rhamnolipid surfactants [34]. Factors that repress sliding are, however, much more broadly conserved. In particular, levels of the second messenger cyclic di-GMP (c-diGMP), are inversely correlated with motility in many organisms, with low levels associated with motility, and high levels associated with biofilm formation [34,42,43]. c-di-GMP modulates the activity of BldD — a central regulator in classic Streptomyces development [16]. BldD dimerization is coordinated by a c-di-GMP tetramer, and this allows BldD to bind and repress its DNA targets. When c-di-GMP levels fall, BldD dimers dissociate, relieving repression of its target genes and triggering development into aerial hyphae and spores. While BldD is not required for exploratory growth, the role of c-di-GMP in exploration has not yet been tested, and will be an important avenue for future investigation.
Conclusions Exploration is a novel mode of Streptomyces growth, triggered by combinations of interkingdom interactions, nutritional/metabolic cues, and airborne signals. Exploration does not require many of the well-characterized Bld or Whi regulators, indicating that exploratory growth is regulated either by undiscovered Bld/Whi proteins, or by as yet unknown regulatory pathways. Advances in our understanding of classic Streptomyces development have largely been achieved through the application of next generation sequencing technologies, and we expect similar techniques combined with genetic screens will allow us to delineate the regulatory pathways underlying exploration.
Conflicts of interest None.
Acknowledgements S.E.J. is supported by a Vanier Scholarship. This work has been supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants and Discovery Accelerator Supplements programs.
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mediates effective transcription factor dimerization to control Streptomyces development. Cell 2014, 158:1136-1147. Reveals how the activity of BldD, which sits at the top of the Streptomyces developmental regulatory cascade, is controlled. This study reports the first tetrameric form of c-di-GMP, and shows that this unusual second messenger configuration coordinates BldD dimerization, with dimers being the active, transcriptionally repressive form of this regulator. Notably, this work also reveals that bldD mutants are in fact hypersporulators, and are not blocked at vegetative growth. 17. Al-Bassam MM, Bibb MJ, Bush MJ, Chandra G, Buttner MJ: Response regulator heterodimer formation controls a key stage in Streptomyces development. PLoS Genet 2014, 10: e1004554. Shows the response regulator BldM targets two sets of genes: one set as a homodimer during early stages of development, and another set as a heterodimer with the response regulator WhiI during later stages of development. This paper demonstrates the potential for response regulator heterodimer formation, and provides an example of two regulatory pathways converging to provide precise, timely regulation over the Streptomyces life cycle. 18. Bush MJ, Chandra G, Bibb MJ, Findlay KC, Buttner MJ: Genome wide chromatin immunoprecipitation sequencing analysis shows that WhiB is a transcription factor that co-controls its regulon with WhiA to initiate developmental cell division in Streptomyces. mBio 2016, 7:1-13. Reveals that mutants of the sporulation regulators WhiB and WhiA have identical phenotypes, and that the two proteins depend on each other for targetting a common set of genes. This paper provides another example of partnering regulators, and shows that the actinobacterial-specific, ironsulfur-containing WhiB-like proteins are transcription factors. 19. Bush MJ, Chandra G, Findlay KC, Buttner MJ: Multi-layered inhibition of Streptomyces development: BldO is a dedicated repressor of whiB. Mol Microbiol 2017, 104:700-711. Characterizes the new regulator BldO, and demonstrates that this regulator represses a single gene, whiB. BldO, like BldD, keeps Streptomyces cells in the vegetative growth stage, and prevents premature entry into sporulation. This paper provides an example of the multi-layered regulation governing the first transition stage in Streptomyces development. 20. Schlimpert S, Wasserstrom S, Chandra G, Bibb MJ, Findlay KC, Fla¨rdh K, Buttner MJ: Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation. Proc Natl Acad Sci U S A 2017 http://dx.doi.org/10.1073/pnas.1704612114. Multigenomic Streptomyces hyphae septate into chains of unigenomic spores, and the bacterial tubulin homolog FtsZ is essental for septum synthesis. This paper shows that two Streptomyces dynamin-like proteins, controlled by the WhiH regulator, act in conjunction with the cell division machinery and function to stabilize FtsZ rings. These results reveal a new function for the poorly understood bacterial dynamins. 21. Bibb MJ, Domonkos A, Chandra G, Buttner MJ: Expression of the chaplin and rodlin hydrophobic sheath proteins in Streptomyces venezuelae is controlled by s(BldN) and a cognate anti-sigma factor, RsbN. Mol Microbiol 2012, 84:10331049. 22. Fla¨rdh K, McCormick JR: The Streptomyces O-B one connection: a force within layered repression of a key developmental decision. Mol Microbiol 2017, 4:1-8. 23. Hengst CD den, Tran NT, Bibb MJ, Chandra G, Leskiw BK, Buttner MJ: Genes essential for morphological development and antibiotic production in Streptomyces coelicolor are targets of BldD during vegetative growth. Mol Microbiol 2010, 78:361-379. 24. Elliot MA, Bibb MJ, Buttner MJ, Leskiw BK: BldD is a direct regulator of key developmental genes in Streptomyces coelicolor A3(2). Mol Microbiol 2001, 40:257-269. 25. Gubbens J, Janus M, Florea BI, Overkleeft HS, Van Wezel GP: Identification of glucose kinase-dependent and -independent pathways for carbon control of primary metabolism, development and antibiotic production in Streptomyces coelicolor by quantitative proteomics. Mol Microbiol 2012, 86:1490-1507. 26. Pope MK, Green BD, Westpheling J: The bld mutants of Streptomyces coelicolor are defective in the regulation of carbon utilization, morphogenesis and cell–cell signalling. Mol Microbiol 1996, 19:747-756. Current Opinion in Microbiology 2018, 42:25–30
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ScienceDirect ‘Exploring’ the regulation of Streptomyces growth and development Stephanie E Jones1,2 and Marie A Elliot1,2 The Streptomyces life cycle encompasses three wellestablished developmental stages: vegetative hyphae, aerial hyphae and spores. Many regulators governing the transitions between these life cycle stages have been identified, and recent work is shedding light on their specific functions. A new discovery has shown Streptomyces can deviate from this classic life cycle through a process termed ‘exploration’, where cells rapidly traverse solid surfaces. Exploration does not require any of the traditional developmental regulators, and therefore provides an exciting new context in which to uncover novel developmental pathways. Here, we summarize our understanding of how Streptomyces exploration is controlled, and we speculate on how insight into classical regulation and stress response systems can inform future research into the regulation of exploratory growth. Addresses 1 M.G. DeGroote Institute for Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1 2 Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1 Corresponding author: Elliot, Marie A ([email protected])
Current Opinion in Microbiology 2018, 42:25–30 This review comes from a themed issue on Cell regulation Edited by Jan-Willem Veening and Rita Tamayo
https://doi.org/10.1016/j.mib.2017.09.009 1369-5274/ã 2017 Elsevier Ltd. All rights reserved.
Introduction to Streptomyces development and exploration Within the soil, microbes are highly abundant, and they grow using remarkably diverse strategies. Amongst these soil-dwelling microbes, Streptomyces are a ubiquitous group of Gram-positive bacteria that are best known for their ability to produce an extraordinary range of antibiotics and other medicinally/agriculturally useful compounds [1]. Streptomyces also have a well-studied, complex life cycle that classically encompasses three morphologically differentiated growth stages [2] (Figure 1). The Streptomyces life cycle initiates with germ tubes emerging from a previously dormant spore. These tubes www.sciencedirect.com
extend by polar growth at the hyphal tips, with additional branches emerging from the lateral walls at random intervals. Continuous cycles of polar growth and branching ultimately result in the formation of a dense network of filaments termed the vegetative mycelium. This branching mycelium anchors the Streptomyces colony to its growth substrate, similar to the way a plant root system anchors a plant in the soil. Polar growth is directed by the ‘polarisome’, a multiprotein complex involved in recruiting the peptidoglycan biosynthetic machinery to the hyphal tips [3–7]. Chromosomes replicate during vegetative growth, and the resulting multinucleate hyphae are delimited by occasional crosswalls [8,9]. In response to nutrient depletion, Streptomyces commence their second differentiated growth stage. Here, nonbranching aerial hyphae coated in a hydrophobic protein layer (consisting of chaplin proteins and the surfactant SapB [10–12]) rise into the air, away from the vegetative mycelium (Figure 1). The transition from branching vegetative growth to hydrophobic, nonbranching aerial growth requires regulators termed the bld genes — so named for the ‘bald’ colony phenotype of bld mutants, where they lack the characteristic fuzzy aerial hyphae that cover wild type colonies. Once aerial growth ceases, the long, multigenomic filaments undergo synchronous septation. This generates chains of single genome prespore compartments, which then mature to form dormant, pigmented spores. The transition from aerial hyphal growth to sporulation requires regulators termed ‘whi’ (for ‘white’) genes, as whi mutants lack the pigment associated with mature, wild type spores. Recent work has shown that Streptomyces can escape this classically defined life cycle using a new mode of growth termed ‘exploration’ [13,14] (Figure 1). This exploration phenomenon was discovered during the co-culture of Streptomyces venezuelae with yeast — a situation that mimics the polymicrobial communities in the soil. Exploration occurs in response to fungi and other metabolic cues, and involves the rapid spread of Streptomyces colonies over solid surfaces including agar, plastics and rocks. During exploratory growth, cells grow as nonbranching vegetative hyphae, and proceed at a rate more than 10 that of vegetative growth. In recent years, there have been important advances in understanding the roles of the bld and whi genes in regulating the transitions between the different canonical life cycle stages [15,16,17,18,19,20]. Exploration Current Opinion in Microbiology 2018, 42:25–30
26 Cell regulation
Figure 1
WhiB WhiA
WhiG
WhiI BIdM
WhiE
BIdD-(c-di-GMP)
other BId regulators
BIdN
spore chains
aerial hyphae
BIdM BIdM
BIdO vegetative mycelium
classic Streptomyces life cycle
germination spore unknown regulators trimethylamine CydCD required
Streptomyces exploration
? ? Streptomyces explorer cells
Streptomyces aerial hyphae and spores yeast abundant amino acids glucose consumption
medium alkalinization
induction of exploration
Current Opinion in Microbiology
The classic Streptomyces life cycle and Streptomyces exploration. Left: Germ tubes emerge from a single dormant spore, and Streptomyces can enter either the classic life cycle (top) or initiate exploratory growth (bottom). Top: Germ tubes extend by polar growth at the hyphal tips, and additional branches emerge from the lateral walls, forming a dense network of branching hyphae termed the vegetative mycelium. Within the vegetative mycelium, BldD bound to c-di-GMP represses other bld genes, including genes encoding BldM and the sigma factor BldN, and various whi genes, including those encoding WhiB, WhiA, WhiG and WhiI. Within the vegetative mycelium, a BldM homodimer represses the sporulation regulator WhiB. When BldD-mediated repression is relieved, other Bld regulators activate genes required for Streptomyces to raise aerial hyphae. Subsequently, aerial hyphae coated in a hydrophobic sheath rise into the air. Within the aerial hyphae, the recently discovered regulator BldO initially represses the expression of whiB. When this repression is relieved, WhiB together with WhiA, and WhiI together with BldM, activate other genes necessary for sporulation. Aerial hyphae differentiate into long chains of dormant exospores, and spores can be dispersed to begin the life cycle anew. Bottom: After germination, Streptomyces can diverge from their classic life cycle. Combinations of low glucose and concentrated amino acid sources trigger exploration. When Streptomyces are grown beside fungi, Streptomyces first grows on top of the yeast, raising aerial hyphae and forming spores. Once the yeast has consumed the local glucose supply, Streptomyces exploration begins. Explorer cells grow as nonbranching vegetative hyphae, in contrast to the branching vegetative mycelium seen in the classic Streptomyces life cycle. Explorer cells release the volatile compound trimethylamine (TMA — indicated in purple), which raises the pH of the surrounding medium from 7.0 to 9.5. Explorer cells require genes encoding the cytochrome bd oxidase complex, presumably to withstand the highly alkaline environment. TMA promotes continued exploration of the producer colony, and can stimulate exploration by nearby Streptomyces colonies. Known Bld and Whi regulators are not required for exploration. Other regulators involved in activating/repressing genes required for exploration are currently unknown.
does not directly require these regulators, and we are in the early stages of understanding the cues and genes involved in the control of exploration. In this review, we discuss our current understanding of factors governing exploration, highlight some of the recent advances in understanding the roles of regulators involved in the canonical Streptomyces life cycle, and explore connections between these two life cycle trajectories. Current Opinion in Microbiology 2018, 42:25–30
Models matter: S. coelicolor versus S. venezuelae The foundation for understanding Streptomyces development was laid by studies conducted in Streptomyces coelicolor, a species that fully differentiates on solid medium but not in liquid culture. In recent years, there has been a shift to studying development in S. venezuelae, which like S. coelicolor undergoes its full life cycle during growth on www.sciencedirect.com
Control of exploration in Streptomyces Jones and Elliot 27
solid medium, but also sporulates to completion in liquid culture. Notably, S. venezuelae is capable of exploratory growth, while S. coelicolor does not explore, at least under conditions tested to date [13]. This could explain in part, why Streptomyces exploration had not been previously reported, despite decades of developmental investigation. This raises the obvious question of whether exploration is in any way tied to the ability to undergo submerged sporulation. This does not seem to be the case, as Streptomyces griseus — another streptomycete that can sporulate in liquid culture — is not exploration competent under conditions that promote S. venezuelae exploration [13]. In all, 10% of streptomycetes tested seem to be able to explore. Whether the others are unable to do so, or whether they explore in response to different signals remains to be determined.
Understanding Streptomyces exploration Exploration does not require classical developmental regulators
Explorer cells appear to be a unique Streptomyces cell type, in that they share properties of both vegetative hyphae and aerial hyphae [13]. Like vegetative hyphae, they are hydrophilic, and like aerial hyphae, they do not branch. Mutant strains lacking the Bld or Whi regulators can explore in the same way as wild type strains, indicating the transition from vegetative growth to aerial hyphal growth is not required for exploration. The one exception to this is the bldN mutant strain, which explores more slowly than wild type. BldN is a sigma factor that activates the expression of both the developmental regulatory gene bldM, and the chp genes [21], which encode the chaplin proteins that assemble into the hydrophobic sheath covering aerial hyphae and spores [10,11]. Given the hydrophilic nature of explorer cells, it seems unlikely that the hydrophobic chaplin proteins play a key role in exploration, so this implies that either BldM or another BldM/ BldN regulon member may facilitate efficient exploration. BldM is an ‘orphaned’ response regulator (it lacks a cognate kinase, and does not require phosphorylation for activity), and is unusual in that it acts both as a homodimer, and as a heterodimer in association with WhiI (another orphaned response regulator) [17]. Notably, neither bldM nor whiI mutants exhibit altered exploration [13], suggesting that BldN may have additional regulon members that help to promote exploration. New bld regulators continue to be discovered. These include the recently characterized BldO, which controls the expression of the sporulation regulator whiB [19,22]. Whether other — as yet untested — canonical developmental regulators impact exploration remains to be seen. BldD is a master repressor of bld and whi gene expression [23,24], but unlike most other bld mutants (apart from bldO), bldD mutants are not blocked at vegetative growth, and instead transition directly from germination to www.sciencedirect.com
sporulation and are seen to hypersporulate [16]. It was therefore particularly surprising that the bldD mutant strain appeared to explore in a manner indistinguishable from wild type, presumably as nonbranching vegetative hyphae, although this remains to be confirmed [13]. This suggests that exploration may not require typical vegetative growth, and/or may be a process that is entirely independent of any BldD-governed pathway. It is tempting to speculate that exploration may have evolved as a bet-hedging strategy to help ensure survival in the event of mutations in the classic developmental regulators. Mutations in any bld or whi gene would block the sporulation/dispersal capabilities of Streptomyces, with exploration then providing cells with an alternative means of escaping to a more favourable growth environment. Nutritional cues governing exploration and development
Exploration by S. venezuelae relies on distinct nutritional factors [13]. It requires both a rich peptide source, and a low glucose environment (Figure 1). In the S. venezuelaeyeast association on glucose-containing medium, the yeast titrates glucose from the medium, and this relieves repression of exploration by S. venezuelae. Accordingly, cells grown without yeast in a glucose-free environment can initiate exploration on their own. The transition from vegetative to aerial growth in the classical life cycle is also thought to have a nutritional component, with it being widely assumed that nutrient depletion promotes the initiation of aerial hyphae formation. While the specific cues that are sensed have yet to be identified, the accumulation of different developmental regulators is affected by glucose levels [25], and the developmental defects associated with a number of bld mutations can be suppressed by growing these strains on alternative carbon sources [12,26]. The glucose-repressible nature of exploration suggests a role for carbon catabolite repression. In Streptomyces, this is thought to be mediated in part by the glucose kinase enzyme [27,28]. A glucose kinase-independent pathway has also been characterized in S. coelicolor, in which pyruvate phosphate dikinase (PPDK) figures prominently [25]. Whether the activity of either of these enzymes is required for exploration remains to be tested, but it is worth noting that RNA-sequencing experiments comparing transcript levels in exploring versus stationary cultures revealed significant upregulation of the PPDK-encoding gene [13]. pH profoundly influences exploration and development
Genetic screens for strains deficient in exploration, coupled with whole-genome sequencing, identified genes encoding the cytochrome bd oxidase complex as being essential for exploration [14]. This complex catalyzes electron transfer in the electron transport chain without simultaneous pumping of protons across the membrane [29], and functions as part of the alkaline stress response in other bacteria [30]. Explorer cells raise the pH of solid medium from 7.0 to 9.5 — which is considered highly Current Opinion in Microbiology 2018, 42:25–30
28 Cell regulation
alkaline for Streptomyces. It has been hypothesized that the cytochrome bd oxidase complex is essential in allowing explorers to tolerate alkaline environments. Consistent with this proposal, transcription profiling experiments revealed the five gene clusters most highly upregulated in explorers were associated with alkaline stress responses in other bacteria. How the cytochrome complexes and alkaline stress responsive genes are controlled in Streptomyces is currently unknown. Additional genetic screens are required to identify the regulators responsible for activating these adaptive gene clusters. Explorer cells raise the pH of the medium by releasing the volatile organic compound (VOC) trimethylamine (TMA) [13] (Figure 1). Remarkably, this small, nitrogenous compound can induce exploration in physically separated Streptomyces while also killing other soil microbes, serving as both an exploration-inducing airborne signal and a competitive weapon. TMA-mediated induction of exploration appears to result from a rise in pH, rather than a TMA-specific signalling cascade, as other alkaline VOCs can also induce exploration. The genes directing the production of TMA in Streptomyces remain to be determined. It will be interesting to see whether these TMA-associated genes are integrated in dedicated regulatory networks, or whether TMA is naturally produced as a by-product of the metabolism of nitrogen-rich peptide sources. While aerial hyphae formation and sporulation are not known to require TMA or a high alkaline environment, aerial hyphae development is pH sensitive and can be inhibited by acidic conditions. Indeed, mutations in primary metabolic genes (e.g. citA and cya) and some bld genes, result in increased secretion of organic acids and a concomitant inability to sporulate [31]. This raises the tantalizing possibility that TMA emission by exploring cultures could rescue the developmental defects of some sporulation-deficient mutants. Exploration as a form of Streptomyces motility
Explorer cells can rapidly traverse solid surfaces, and this spreading is phenotypically similar to sliding motility — a growth-dependent mode of microbial movement facilitated by a secreted surfactant [32]. Streptomyces produce two known surfactants: SapB and the chaplin proteins. Given that the production of both of these surfactants requires the activity of the bld genes, and these genes are largely dispensable for exploration [13], it seems unlikely that either SapB or the chaplins are the exploration/sliding motility-promoting surfactants. Many bacteria move by sliding motility [33], including Pseudomonas [34], Serratia [35], Legionella [36], Bacillus [37], Salmonella [38], Sinorhizobium [39] and Mycobacterium [40]. Interestingly, the regulators and surfactants permitting sliding seem to be genus-specific. For example, in Current Opinion in Microbiology 2018, 42:25–30
Bacillus subtilis, the master sporulation regulator SpoOA activates the transcription of genes encoding products necessary for the secretion of surfactin molecules [41]. In contrast, the opportunistic human pathogen Pseudomonas aeruginosa uses the two-component system GacA/GacS to control the production of exopolysaccharides and rhamnolipid surfactants [34]. Factors that repress sliding are, however, much more broadly conserved. In particular, levels of the second messenger cyclic di-GMP (c-diGMP), are inversely correlated with motility in many organisms, with low levels associated with motility, and high levels associated with biofilm formation [34,42,43]. c-di-GMP modulates the activity of BldD — a central regulator in classic Streptomyces development [16]. BldD dimerization is coordinated by a c-di-GMP tetramer, and this allows BldD to bind and repress its DNA targets. When c-di-GMP levels fall, BldD dimers dissociate, relieving repression of its target genes and triggering development into aerial hyphae and spores. While BldD is not required for exploratory growth, the role of c-di-GMP in exploration has not yet been tested, and will be an important avenue for future investigation.
Conclusions Exploration is a novel mode of Streptomyces growth, triggered by combinations of interkingdom interactions, nutritional/metabolic cues, and airborne signals. Exploration does not require many of the well-characterized Bld or Whi regulators, indicating that exploratory growth is regulated either by undiscovered Bld/Whi proteins, or by as yet unknown regulatory pathways. Advances in our understanding of classic Streptomyces development have largely been achieved through the application of next generation sequencing technologies, and we expect similar techniques combined with genetic screens will allow us to delineate the regulatory pathways underlying exploration.
Conflicts of interest None.
Acknowledgements S.E.J. is supported by a Vanier Scholarship. This work has been supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants and Discovery Accelerator Supplements programs.
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