Polydiglycosylphosphate Transferase PdtA (SCO2578) of Streptomyces coelicolor A3(2) Is Crucial for Proper Sporulation and Apical Tip Extension under Stress Conditions

ABSTRACTAlthough anionic glycopolymers are crucial components of the Gram-positive cell envelope, the relevance of anionic glycopolymers for vegetative growth and morphological differentiation ofStreptomyces coelicolorA3(2) is unknown. Here, we show that the LytR-CpsA-Psr (LCP) protein PdtA (SCO2578), a TagV-like glycopolymer transferase, has a dual function in theS. coelicolorA3(2) life cycle. Despite the presence of 10 additional LCP homologs, PdtA is crucial for proper sporulation. The integrity of the spore envelope was severely affected in apdtAdeletion mutant, resulting in 34% nonviable spores.pdtAdeletion caused a significant reduction in the polydiglycosylphosphate content of the spore envelope. Beyond that, apical tip extension and normal branching of vegetative mycelium were severely impaired on high-salt medium. This growth defect coincided with the mislocalization of peptidoglycan synthesis. Thus, PdtA itself or the polydiglycosylphosphate attached to the peptidoglycan by the glycopolymer transferase PdtA also has a crucial function in apical tip extension of vegetative hyphae under stress conditions.IMPORTANCEAnionic glycopolymers are underappreciated components of the Gram-positive cell envelope. They provide rigidity to the cell wall and position extracellular enzymes involved in peptidoglycan remodeling. AlthoughStreptomyces coelicolorA3(2), the model organism for bacterial antibiotic production, is known to produce two distinct cell wall-linked glycopolymers, teichulosonic acid and polydiglycosylphosphate, the role of these glycopolymers in theS. coelicolorA3(2) life cycle has not been addressed so far. This study reveals a crucial function of the anionic glycopolymer polydiglycosylphosphate for the growth and morphological differentiation ofS. coelicolorA3(2). Polydiglycosylphosphate is attached to the spore wall by the LytR-CpsA-Psr protein PdtA (SCO2578), a component of theStreptomycesspore wall-synthesizing complex (SSSC), to ensure the integrity of the spore envelope. Surprisingly, PdtA also has a crucial role in vegetative growth under stress conditions and is required for proper peptidoglycan incorporation during apical tip extension.

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Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis

Journal of Bacteriology ◽

10.1128/jb.00840-16 ◽

2017 ◽

Vol 199 (12) ◽

Cited By ~ 15

Author(s):

Yoshihiro Mouri ◽

Kenji Konishi ◽

Azusa Fujita ◽

Takeaki Tezuka ◽

Yasuo Ohnishi

Keyword(s):

Vegetative Growth ◽

Streptomyces Coelicolor ◽

Morphological Differentiation ◽

Transcriptional Regulator ◽

Saccharopolyspora Erythraea ◽

Transcriptional Analysis ◽

Morphological Development ◽

Sequencing Analysis ◽

Content Type ◽

Biological Studies

ABSTRACT The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces. AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangium-forming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD. 3′,5′-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5′-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3′ as the consensus AmBldD-binding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation. IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis. The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).

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Cell Wall Hydrolases Affect Germination, Vegetative Growth, and Sporulation in Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.00767-09 ◽

2009 ◽

Vol 191 (21) ◽

pp. 6501-6512 ◽

Cited By ~ 52

Author(s):

Henry J. Haiser ◽

Mary R. Yousef ◽

Marie A. Elliot

Keyword(s):

Cell Wall ◽

Life Cycle ◽

Vegetative Growth ◽

Streptomyces Coelicolor ◽

Hydrolytic Enzymes ◽

In Silico Analysis ◽

Cell Types ◽

Complex Life Cycle ◽

Cell Wall Remodeling ◽

Cell Growth And Differentiation

ABSTRACT Peptidoglycan is a major cell wall constituent of gram-positive bacteria. It is a dynamic macromolecule that is actively remodeled to enable cell growth and differentiation through a tightly choreographed interplay of hydrolytic and biosynthetic enzyme activities. The filamentous bacterium Streptomyces coelicolor has a complex life cycle that likely requires considerable cell wall remodeling to enable both extension of vegetative hyphae and formation of differentiated cell types. In silico analysis of the S. coelicolor genome enabled identification of 56 candidate cell wall hydrolase genes. We found that seven of these genes shared a highly conserved 5′ untranslated region and were expressed during both vegetative growth and sporulation; four of these genes were selected for more extensive biochemical and biological characterization. The proteins encoded by these genes, termed RpfA, SwlA, SwlB, and SwlC, were confirmed to be hydrolytic enzymes, as they could efficiently cleave S. coelicolor cell walls. Phenotypic analyses revealed that these enzymes are important throughout development; deletion of each hydrolase gene resulted in a mutant strain that was heat sensitive, defective in spore formation, and either altered in vegetative growth or delayed in spore germination. Our results indicate that these enzymes play key roles at multiple stages in the growth and development of S. coelicolor, highlighting both the lack of redundancy in hydrolase activity and the importance of cell wall remodeling in the S. coelicolor life cycle.

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In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00523-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4470-4480 ◽

Cited By ~ 29

Author(s):

Min Jung Kwun ◽

Gabriela Novotna ◽

Andrew R. Hesketh ◽

Lionel Hill ◽

Hee-Jeon Hong

Keyword(s):

Cell Wall ◽

Transcriptional Activation ◽

Streptomyces Coelicolor ◽

Constitutive Expression ◽

Transcriptional Response ◽

Vancomycin Resistance ◽

Content Type ◽

Expression Of Genes ◽

Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

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Cell-Biological Studies of Osmotic Shock Response in Streptomyces spp

Journal of Bacteriology ◽

10.1128/jb.00465-16 ◽

2016 ◽

Vol 199 (1) ◽

Cited By ~ 11

Author(s):

Katsuya Fuchino ◽

Klas Flärdh ◽

Paul Dyson ◽

Nora Ausmees

Keyword(s):

Cell Wall ◽

Cell Polarity ◽

Tip Growth ◽

Biological Study ◽

Polar Growth ◽

Hyphal Branching ◽

Content Type ◽

Biological Studies ◽

Wide Range ◽

Tip Extension

ABSTRACT Most bacteria are likely to face osmotic challenges, but there is yet much to learn about how such environmental changes affect the architecture of bacterial cells. Here, we report a cell-biological study in model organisms of the genus Streptomyces, which are actinobacteria that grow in a highly polarized fashion to form branching hyphae. The characteristic apical growth of Streptomyces hyphae is orchestrated by protein assemblies, called polarisomes, which contain coiled-coil proteins DivIVA and Scy, and recruit cell wall synthesis complexes and the stress-bearing cytoskeleton of FilP to the tip regions of the hyphae. We monitored cell growth and cell-architectural changes by time-lapse microscopy in osmotic upshift experiments. Hyperosmotic shock caused arrest of growth, loss of turgor, and hypercondensation of chromosomes. The recovery period was protracted, presumably due to the dehydrated state of the cytoplasm, before hyphae could restore their turgor and start to grow again. In most hyphae, this regrowth did not take place at the original hyphal tips. Instead, cell polarity was reprogrammed, and polarisomes were redistributed to new sites, leading to the emergence of multiple lateral branches from which growth occurred. Factors known to regulate the branching pattern of Streptomyces hyphae, such as the serine/threonine kinase AfsK and Scy, were not involved in reprogramming of cell polarity, indicating that different mechanisms may act under different environmental conditions to control hyphal branching. Our observations of hyphal morphology during the stress response indicate that turgor and sufficient hydration of cytoplasm are required for Streptomyces tip growth. IMPORTANCE Polar growth is an intricate manner of growth for accomplishing a complicated morphology, employed by a wide range of organisms across the kingdoms of life. The tip extension of Streptomyces hyphae is one of the most pronounced examples of polar growth among bacteria. The expansion of the cell wall by tip extension is thought to be facilitated by the turgor pressure, but it was unknown how external osmotic change influences Streptomyces tip growth. We report here that severe hyperosmotic stress causes cessation of growth, followed by reprogramming of cell polarity and rearrangement of growth zones to promote lateral hyphal branching. This phenomenon may represent a strategy of hyphal organisms to avoid osmotic stress encountered by the growing hyphal tip.

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The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

Infection and Immunity ◽

10.1128/iai.00005-19 ◽

2019 ◽

Vol 87 (8) ◽

Cited By ~ 6

Author(s):

Elodie Cuenot ◽

Transito Garcia-Garcia ◽

Thibaut Douche ◽

Olivier Gorgette ◽

Pascal Courtin ◽

...

Keyword(s):

Cell Wall ◽

Type Strain ◽

Wild Type Strain ◽

Cell Envelope ◽

Wild Type ◽

Hamster Model ◽

Content Type ◽

Physiological Processes ◽

Signal Transduction Networks ◽

Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

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High-Resolution Analysis of the Peptidoglycan Composition inStreptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.00290-18 ◽

2018 ◽

Vol 200 (20) ◽

Cited By ~ 10

Author(s):

Lizah T. van der Aart ◽

Gerwin K. Spijksma ◽

Amy Harms ◽

Waldemar Vollmer ◽

Thomas Hankemeier ◽

...

Keyword(s):

Cell Wall ◽

Mycelial Growth ◽

Antibiotic Production ◽

Hydrolytic Enzymes ◽

Morphological Differentiation ◽

Model Organisms ◽

Major Constituent ◽

Single Spore ◽

Content Type ◽

Liquid Chromatography Tandem Mass

ABSTRACTThe bacterial cell wall maintains cell shape and protects against bursting by turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously modified to enable cell growth and differentiation through the concerted activity of biosynthetic and hydrolytic enzymes. Streptomycetes are Gram-positive bacteria with a complex multicellular life style alternating between mycelial growth and the formation of reproductive spores. This involves cell wall remodeling at apical sites of the hyphae during cell elongation and autolytic degradation of the vegetative mycelium during the onset of development and antibiotic production. Here, we show that there are distinct differences in the cross-linking and maturation of the PGs between exponentially growing vegetative hyphae and the aerial hyphae that undergo sporulation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified over 80 different muropeptides, revealing that major PG hydrolysis takes place over the course of mycelial growth. Half of the dimers lacked one of the disaccharide units in transition-phase cells, most likely due to autolytic activity. The deacetylation of MurNAc to MurN was particularly pronounced in spores and strongly reduced in sporulation mutants with a deletion ofbldDorwhiG, suggesting that MurN is developmentally regulated. Altogether, our work highlights the dynamic and growth phase-dependent changes in the composition of the PG inStreptomyces.IMPORTANCEStreptomycetes are bacteria with a complex lifestyle and are model organisms for bacterial multicellularity. From a single spore, a large multigenomic multicellular mycelium is formed, which differentiates to form spores. Programmed cell death is an important event during the onset of morphological differentiation. In this work, we provide new insights into the changes in the peptidoglycan composition and over time, highlighting changes over the course of development and between growing mycelia and spores. This revealed dynamic changes in the peptidoglycan when the mycelia aged, with extensive peptidoglycan hydrolysis and, in particular, an increase in the proportion of 3-3 cross-links. Additionally, we identified a muropeptide that accumulates predominantly in the spores and may provide clues toward spore development.

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MreB Forms Subdiffraction Nanofilaments during Active Growth in Bacillus subtilis

mBio ◽

10.1128/mbio.01879-18 ◽

2019 ◽

Vol 10 (1) ◽

pp. e01879-18 ◽

Cited By ~ 9

Author(s):

Cyrille Billaudeau ◽

Zhizhong Yao ◽

Charlène Cornilleau ◽

Rut Carballido-López ◽

Arnaud Chastanet

Keyword(s):

Cell Wall ◽

Dynamic Properties ◽

Cell Envelope ◽

Side Wall ◽

Active Growth ◽

Content Type ◽

Gram Positive Bacteria ◽

Entire Cell ◽

The Subject ◽

True Structure

ABSTRACT The actin-like MreB protein is a key player of the machinery controlling the elongation and maintenance of the cell shape of most rod-shaped bacteria. This protein is known to be highly dynamic, moving along the short axis of cells, presumably reflecting the movement of cell wall synthetic machineries during the enzymatic assembly of the peptidoglycan mesh. The ability of MreB proteins to form polymers is not debated, but their structure, length, and conditions of establishment have remained unclear and the subject of conflicting reports. Here we analyze various strains of Bacillus subtilis, the model for Gram-positive bacteria, and we show that MreB forms subdiffraction-limited, less than 200 nm-long nanofilaments on average during active growth, while micron-long filaments are a consequence of artificial overaccumulation of the protein. Our results also show the absence of impact of the size of the filaments on their speed, orientation, and other dynamic properties conferring a large tolerance to B. subtilis toward the levels and consequently the lengths of MreB polymers. Our data indicate that the density of mobile filaments remains constant in various strains regardless of their MreB levels, suggesting that another factor determines this constant. IMPORTANCE The construction of the bacterial cell envelope is a fundamental topic, as it confers its integrity to bacteria and is consequently the target of numerous antibiotics. MreB is an essential protein suspected to regulate the cell wall synthetic machineries. Despite two decades of study, its localization remains the subject of controversies, its description ranging from helical filaments spanning the entire cell to small discrete entities. The true structure of these filaments is important because it impacts the model describing how the machineries building the cell wall are associated, how they are coordinated at the scale of the entire cell, and how MreB mediates this regulation. Our results shed light on this debate, revealing the size of native filaments in B. subtilis during growth. They argue against models where MreB filament size directly affects the speed of synthesis of the cell wall and where MreB would coordinate distant machineries along the side wall.

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Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.00302-15 ◽

2015 ◽

Vol 197 (17) ◽

pp. 2747-2753 ◽

Cited By ~ 21

Author(s):

Corey L. Compton ◽

Michael S. Fernandopulle ◽

Rohith T. Nagari ◽

Jason K. Sello

Keyword(s):

Oxidative Stress ◽

Posttranslational Modification ◽

Streptomyces Coelicolor ◽

Morphological Differentiation ◽

Life Cycles ◽

Content Type ◽

Genes Encoding ◽

Large Genus ◽

Functional Equivalent ◽

First Time

ABSTRACTPupylation is a posttranslational modification peculiar to actinobacteria wherein proteins are covalently modified with a small protein called the prokaryotic ubiquitin-like protein (Pup). Like ubiquitination in eukaryotes, this phenomenon has been associated with proteasome-mediated protein degradation in mycobacteria. Here, we report studies of pupylation in a streptomycete that is phylogentically related to mycobacteria. We constructed mutants ofStreptomyces coelicolorlacking PafA (Pup ligase), the proteasome, and the Pup-proteasome system. We found that these mutants share a high susceptibility to oxidative stress compared to that of the wild-type strain. Remarkably, we found that thepafAnull mutant has a sporulation defect not seen in strains lacking the Pup-proteasome system. In proteomics experiments facilitated by an affinity-tagged variant of Pup, we identified 110 pupylated proteins inS. coelicolorstrains having and lacking genes encoding the 20S proteasome. Our findings shed new light on this unusual posttranslational modification and its role inStreptomycesphysiology.IMPORTANCEThe presence of 20S proteasomes reminiscent of those in eukaryotes and a functional equivalent of ubiquitin, known as the prokaryotic ubiquitin-like protein (Pup), in actinobacteria have motivated reevaluations of protein homeostasis in prokaryotes. Though the Pup-proteasome system has been studied extensively in mycobacteria, it is much less understood in streptomycetes, members of a large genus of actinobacteria known for highly choreographed life cycles in which phases of morphological differentiation, sporulation, and secondary metabolism are often regulated by protein metabolism. Here, we define constituents of the pupylome inStreptomyces coelicolorfor the first time and present new evidence that links pupylation and the oxidative stress response in this bacterium. Surprisingly, we found that the Pup ligase has a Pup-independent role in sporulation.

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The Level of AdpA Directly Affects Expression of Developmental Genes in Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.05734-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6358-6365 ◽

Cited By ~ 29

Author(s):

Marcin Wolański ◽

Rafał Donczew ◽

Agnieszka Kois-Ostrowska ◽

Paweł Masiewicz ◽

Dagmara Jakimowicz ◽

...

Keyword(s):

Early Stage ◽

Response Regulator ◽

Streptomyces Coelicolor ◽

Streptomyces Griseus ◽

Morphological Differentiation ◽

Aerial Mycelium ◽

Content Type ◽

Protein Functions

AdpA is a key regulator of morphological differentiation inStreptomyces. In contrast toStreptomyces griseus, relatively little is known about AdpA protein functions inStreptomyces coelicolor. Here, we report for the first time the translation accumulation profile of theS. coelicoloradpA(adpASc) gene; the level ofS. coelicolorAdpA (AdpASc) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpAScspecifically binds theadpAScpromoter regionin vitroandin vivo, suggesting that its expression is autoregulated; surprisingly, in contrast toS. griseus, the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpAScon the expression of several genes whose products play key roles in the differentiation ofS. coelicolor: STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpAScprotein on the expression of the analyzed genes presumably results mainly from different affinities of AdpAScprotein to individual promoters.

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The Leeuwenhoek Lecture, 1987 - Towards an understanding of gene switching in Streptomyces , the basis of sporulation and antibiotic production

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1988.0067 ◽

1988 ◽

Vol 235 (1279) ◽

pp. 121-138 ◽

Cited By ~ 58

Keyword(s):

Life Cycle ◽

Secondary Metabolites ◽

Vegetative Growth ◽

Antibiotic Production ◽

Morphological Differentiation ◽

Aerial Mycelium ◽

Secondary Phase ◽

Antibiotic Biosynthesis ◽

Regulatory Cascade ◽

Pharmacologically Active

Streptomycetes are soil bacteria that differ from the genetically well-known Escherichia coli in two striking characteristics. (1) Instead of consisting of an alternation of growth and fission of morphologically simple, undifferentiated rods, the streptomycete life cycle involves the formation of a system of elongated, branching hyphae which, after a period of vegetative growth, respond to specific signals by producing specialized spore-bearing structures. (2) The streptomycetes produce an unrivalled range of chemically diverse ‘secondary metabolites’, which we recognize as antibiotics, herbicides and pharmacologically active molecules, and which presumably play an important role in the streptomycete life cycle in nature. This ‘physiological’ differentiation is often temporally associated with the morphological differentiation of sporulation and there are common elements in the regulation of the two sets of processes. In the model system provided by Streptomyces coelicolor A3(2), the isolation of several whole clusters of linked antibiotic biosynthetic pathway genes, and some key regulatory genes involved in sporulation, has made it possible to study the basis for the switching on and off of particular sets of genes during morphological and ‘physiological’ differentiation. Genetic analysis clearly reveals a regulatory cascade operating at several levels in a ‘physiological’ branch of the differentiation control system. At the lowest level, within individual clusters of antibiotic biosynthesis genes are genes with a role as activators of the structural genes for the pathway enzymes, and also resistance genes. It is attractive to speculate that the latter play a dual role: protecting the organism from self-destruction by its own potentially lethal product, and forming an essential component of a regulatory circuit that activates the biosynthetic genes, thus ensuring that resistance is established before any antibiotic is made. A next higher level of regulation is revealed by the isolation of mutations in a gene ( afsB ) required for expression (probably at the level of transcription) of all five known secondary metabolic pathways in the organism. At a higher level still, the bldA gene, whose product seems to be a tRNA essential to translate the rare (in high [G + C] Streptomyces DNA) TTA leucine codon, controls or influences the whole gamut of morphological and ‘physiological’ differentiation, because bldA mutants fail to produce either secondary metabolites or aerial mycelium and spores, while growing normally in the vegetative phase. Thus a decision to switch from vegetative growth to the secondary phase of colonial development may be taken at the level of translation. In the ‘morphological’ branch of the proposed regulatory cascade, a key gene is whiG whose product, essential for the earliest known step in the metamorphosis of aerial hyphae into spore chains, appears to be an RNA polymerase sigma factor which is not needed for transcription of vegetative genes, but seems to control, at the level of transcription, the decision to sporulate.

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