Erik Vijgenboom and Bart Keijser

1. INTRODUCTION

Redox active metal ions such as copper are potentially dangerous to a cell because of their habit of engaging in all kinds of reaction generating, among others, reactive oxygen species. However, these metal ions play a crucial role as a cofactor in a variety of enzymes and electron-transport proteins. Recently, it has become evident that copper ions play an intriguing role in the morphogenesis of Streptomyces. This Gram-positive bacterium undergoes a complicated cycle of morphological differentiation to ensure its propagation. Here, we review what is currently known about Streptomyces morphogenesis and the link with copper, and what developments are to be expected in the near future.

2. THE LIFE CYCLE OF STREPTOMYCES

From a bacterial point of view, the filamentous Gram-positive soil bacteria Streptomyces have a very complex lifestyle (Fig. 1A). Their vegetative mycelium consists of a network of branched hyphae. Perhaps, as a consequence of their hyphal growth habit, they rely on the formation of exospores for propagation. In addition they produce a wide range of secondary metabolites (antibiotics, fungicides). Because of the fascinating and complex multicellular processes of development Streptomyces are the system of choice for studies of prokaryotic morphological and physiological development, but they are also an attractive industrial organism.

The complete life cycle of Streptomyces consists of two phases: the vegetative growth and the reproductive growth. Vegetative growth is characterized by the production of branched hyphae with irregular crosswalls dividing the hyphae in compartments. Growth mainly occurs through extension of cell walls at the hyphal tips. At variable intervals, septation occurs, resulting in multigenome compartments because partitioning of sibling DNA molecules does not take place. In the final stage of vegetative growth, when entering the stationary phase, many streptomycetes produce a variety of secondary metabolites, which include 60% or more of the known antibiotics. Because of nutrient limitation, stress response, and cellular signaling, streptomycetes initiate the reproductive growth stage by the formation of aerial mycelium. These hyphae grow perpendicular onto the vegetative mycelium and erect themselves from the plane of the agar. The vegetative mycelium supplies the nutrients needed for aerial growth, so the aerial mycelium is both physically and physiologically dependent on the vegetative mycelium. The early stages of aerial hyphae formation show straight

From: Handbook of Copper Pharmacology and Toxicology Edited by: E. J. Massaro © Humana Press Inc., Totowa, NJ

Free spore

Free spore

Fig. 1. (A) The life cycle of streptomycetes. The first step in the development of a new colony is the germination of a spore. Two germ tubes are formed that develop into a branched vegetative mycelium. One of the factors involved in the germination is cAMP (1). The reproductive part of the life cycle is initiated by the erection of aerial hyphae. At this point, the vegetative mycelium becomes a substrate mycelium that feeds the aerial mycelium. Developmental mutants affected in this process are designated bid (bald) mutants because they lack the fluffy white aerial mycelium. After coiling and regular septation, spores are developed at the tips of the aerial hyphae. Mutants blocked in this stage of differentiation are called whi (white) mutants because they do not develop the gray color of the pigment associated with the spores. (B) Photograph of vegetative mycelium (straight hyphae in the center), aerial hyphae (coiled hyphae at the top), and spore chains (bottom) taken with a phase-contrast microscope.

Fig. 1. (A) The life cycle of streptomycetes. The first step in the development of a new colony is the germination of a spore. Two germ tubes are formed that develop into a branched vegetative mycelium. One of the factors involved in the germination is cAMP (1). The reproductive part of the life cycle is initiated by the erection of aerial hyphae. At this point, the vegetative mycelium becomes a substrate mycelium that feeds the aerial mycelium. Developmental mutants affected in this process are designated bid (bald) mutants because they lack the fluffy white aerial mycelium. After coiling and regular septation, spores are developed at the tips of the aerial hyphae. Mutants blocked in this stage of differentiation are called whi (white) mutants because they do not develop the gray color of the pigment associated with the spores. (B) Photograph of vegetative mycelium (straight hyphae in the center), aerial hyphae (coiled hyphae at the top), and spore chains (bottom) taken with a phase-contrast microscope.

hyphae that eventually will start to coil. In the final stage of the reproductive growth, regular-sized compartments are formed at the tips of the aerial hyphae that develop into spores (Fig. 1B). During spore formation, partitioning of the genomes does occur, resulting in one genome per compartment. For most Streptomyces strains, the reproductive cycle is only switched on when grown on solid media. A few Streptomyces strains are capable of spore production in liquid broth, so-called submerged sporulation (2-5), but this will be not discussed here.

The various stages of differentiation during growth on solid media can easily be followed because the formation of aerial hyphae gives colonies a white and fluffy appearance, whereas upon spore formation, colonies turn gray because of the production of a gray spore pigment. The onset of the physiological differentiation, production of secondary metabolites, is intimately associated with the start of morphological development, suggesting that these two processes share common regulatory elements. As for the morphogenesis, the physiological differentiation can be monitored easily because many of the secondary metabolites are pigmented.

Insight in the underlying processes that initiate developmental pathways has been gained by studying mutants that are disturbed in their ability to differentiate. Screening programs using NTG -, UV -, or transposon mutagenesis rendered two classes of developmental mutants: those no longer able to form aerial hyphae, the bid (bald) mutants, and those disturbed in their capacity to form mature gray spores, the whi (white) mutants.

Analogies between the developmental process in Streptomyces and other spore-producing microorganisms such as Bacillus subtilis have been drawn (6-8). These ideas suggest that although the morphogenesis in these bacteria is rather different, endospore versus exospore, unicellular versus hyphal growth, the basic layout of developmental routes can be similar or at least contain similar components.

2.1. bld Mutants

The majority of developmental mutants have been generated in Streptomyces coelicolor A(3)2; therefore, this strain will be discussed in more detail. Strains lacking the ability to produce aerial hyphae are classified as bld (bald) mutants. A dozen or more bld mutants have been described in literature, but only a few have been studied at the molecular level. Many of these mutants are not only impaired in the earliest stages of morphogenesis but are also affected in their secondary metabolism and catabolite repression (9). Changes in secondary metabolism in S. coelicolor are obvious because two of the secondary metabolites, the blue-pigmented polyketide antibiotic actinorhodin (Act) and the red-pigmented tripyrolle undesylprodigiosin (Red), are colored. Most bld mutants turned out to be conditionally affected in their ability to initiate aerial mycelium growth. On media with glucose as the carbon source, they display the characteristic bald appearance, whereas on mannitol containing media, these mutants do produce aerial hyphae and spores (10). In some of the mutants, growth on a poor carbon source, such as mannitol, also results in a partial restoration of antibiotic production. One explanation for the medium-dependent phenotype of the bld mutants is the existence of at least two developmental pathways in this phase of the differentiation: one only active on minimal medium containing mannitol and the other on complex-rich media.

An interesting observation was made when bld mutants were grown on solid medium, one next to the other, without actual contact between the hyphae. Of each pair of bld mutants, one was observed to stimulate the development of aerial hyphae formation of the other in the border region between the two strains. This stimulation was always a one-way communication: one strain acted as the donor and the other as the recipient that was rescued for aerial hyphae formation. Analysis of pairs of all bld mutants showed that most of them could be arranged in a hierarchical order (11-13). Each mutant was able to stimulate the development of all mutants lower in this cascade. An explanation of this cascade is the involvement of at least five extracellular signals, where the presence of each signal causes the production or export of the next. So far, only one substance (signal 1) that would qualify as a signaling compound has been partially purified. This signal is a 665-Dalton peptide supposed to contain at least serine and glycine residues, and proposed to be associated with the function of the bldK gene cluster (14). The bldK locus consists of five genes with similarity to the oligopeptide-permease family of ATP-binding cassette (ABC) membrane-spanning transporters. The bldK mutation does not only result in a block in differentiation but also confers resistance to the toxic tripeptide bialaphos, confirming that the bldK gene product is an oligopeptide importer (12).

One would expect to find more elements of the proposed bld-signaling cascade by analyzing the gene products of the bld genes. However, this approach has not been successful until now. The best characterized mutant, bldA, does not encode a protein but an extremely rare leucyl tRNA molecule translating the UUA codon. These codons are very infrequent in genes of Streptomyces because the high GC contents (70%) of the genome and a strong bias for a G or C at the third position of codons (15). The involvement of this tRNA in the regulation of the onset of aerial mycelium formation could be through regulatory proteins of which the mRNAs contain one or more UUA codons. However, this appears not to be the case. Only for the regulatory proteins of the synthesis clusters of act and red has a clear regulatory role of bldA been found (16-18). Therefore, the molecular basis of the bald pheno-type of the bldA mutant is still not clear.

The bldB and bldD mutants are putative transcriptional regulators with unidentified targets (19,20). One mutant, bldF, has an extremely high production of the red pigment and it is not clear whether the phenotype is the result of this overproduction or an additional block of the developmental pathway (21). One of the most recently characterized bld mutants is bldN (22). BldN is an extracytoplasmic function RNA polymerase sigma factor but does not fit cleanly in the proposed "bld-cascade." A target for BldN is one of the two promoters of bldM that encodes a response regulator and belongs to the last step of the bld-cascade. An explanation for the fact that not all bld genes fit in the cascade could be an indirect role in the production of extracellular signaling molecules.

In a different approach to get information on the morphological differentiation, extracellular proteins produced during development have been studied. One of them, SapB, has a molecular weight of 2 kDa, consists of 18 amino acids, is most likely modified on the sixth amino acid residue, and is believed to be produced nonribosomal (9). Purified SapB turned out to be able to restore aerial mycelium formation in a bld mutant. This implied that SapB is an important factor in morphogenesis, although the aerial hyphae erected by the bld mutants upon the addition of SapB did not coil, septate, and form chains of spores as the wild-type aerial hyphae do (23). In this context, the sole function of SapB seems to be the facilitation of the erection of vegetative hyphae and so mimicking the early stages of the developmental controlled formation of aerial hyphae. Therefore, SapB does not appear to have a regulatory function concerned with the onset of differentiation. On minimal medium supplied with mannitol, bld mutants do form aerial hyphae and spores, but do not produce detectable amounts of SapB (24). This supports the hypothesis that at least two pathways of aerial mycelium formation do exist and that SapB is not required per se for aerial hyphae formation. This kind of morphogenic protein is not unique to S. coelicolor. Streptofactin, a similar protein isolated from S. tendae, can restore aerial hyphae formation to S. tendae bld mutants as well as S. coelicolor bld mutants (25). Also, SC3, a hydrophobin isolated from the fungus Schizophyllum commune, does restore erection of vegetative hyphae to some extent (26). What do these proteins have in common? They all are surface-active peptides that lower the surface tension and have the tendency to concentrate at hydrophobic-hydrophilic interfaces. The model for the action of these hydrophobinlike peptides discerns two actions: (1) reduction of surface tension to allow the hydrophilic vegetative hyphae to break through the liquid surface and (2) providing the aerial hyphae with a hydrophobic coat. The function of SapB in formation of aerial hyphae seems to be restricted to the first action, the reduction of the surface tension. Although the surface of S. coelicolor aerial hyphae is hydrophobic, no SapB could be detected (26).

2.2. whi Mutants

Mutants that fail to produce mature gray pigmented spores are called whi (white) mutants. In total, eight loci have been identified in which mutations result in the whi phenotype. The whiE mutants are all affected in the locus that contains the genes for the production of the spore pigment that has resemblance to the polyketides. These mutants are, from a morphological point of view, not defective. The functions of most of the gene products of the other whi genes are not elucidated. Six loci harbor the genes involved in early sporulation events. Mutations in these genes result in a diminished formation of sporulation septa. The whiG gene codes for a o-factor and whiG mutants are arrested in the earliest stages of aerial hyphae development and have straight aerial hyphae. The WhiG protein is expressed throughout growth, indicating that its activity must be repressed during vegetative growth. This is most likely done by an anti-o factor, which is in agreement with the characteristics of the o-factor family, of which WhiG is a member (27). Once the aerial hyphae start to coil, the whiB and whiA gene products come into play. Disruption mutants of these genes have extremely long aerial hyphae, suggesting that the WhiB and WhiA proteins somehow have control over the final length of the aerial hyphae (28,29). The function of both proteins is unknown. WhiB belongs to a group of small proteins with many charged residues and four cysteines in a particular order. WhiB-like proteins have only been detected in actinomycetes (30).

One of several genes involved in the late stages of aerial hyphae development, spore formation and maturation, is whiH. Mutations in this gene, either point mutations or disruptions, result in strains that have a pale gray color as the result of low whiE expression. Further characteristics of this mutant are condensation and aberrant partitioning of the nucleoids (29). Mutations in the whiD locus result in strains with defective spores. The size of the spores is irregular and inspection by scanning electron microscope indicates also possible lysis of spores (31).

Under the assumption that the available genetic map of eight whi loci was not complete, a renewed effort was made to identify new whi mutants. This approach has resulted in five sporulation loci that are distinct from the known ones (32). Another approach using insertional mutagenesis has come up with several genes involved in morphogenesis, among which there is one new whi mutant (33). Studies not directly aimed at the identification of developmental mutants have shown that not all developmental genes show up in mutant screens using mutagens such as UV and NTG. The gene ssgA, encoding for a small protein with unknown function, was identified in S. griseus as the suppressor of submerged sporulation (34). In S. coelicolor, an ortholog of the ssgA gene was isolated that upon overexpression stimulates the septum formation in liquid-grown cultures. Disruption of the ssgA gene resulted in a mutant defective in spore formation but capable of aerial hyphae production. Therefore, ssgA classifies as a whi gene and has a crucial function in septum formation (35).

2.3. Other Factors Affecting Development

Evidence for the involvement of y-butyrolactones in the regulation of the onset of secondary metabolism and the onset of differentiation is accumulating. Similar small diffusible signaling molecules operate in various other bacteria as regulators of a wide variety of processes (36). A-factor (2-isocapryloyl-3^-hydroxymethyl-y-butyrolactone) was the first of these bacterial hormones to be identified in S. griseus (37) and shown to act in nanomolar concentration as a "all-or-none" switch for both morphological development and streptomycin production. Later, several butyrolactone autoregulators were purified form S. coelicolor (38,39). The butyrolactone receptors are supposed DNA-binding proteins that take care of the regulation of genes needed for secondary metabolism and morphogenesis (40-42). Another diffusible signal molecule suggested to serve in the regulation of antibiotic synthesis and morphological development is cAMP. An adenylate cyclase disruption mutant is conditionally impaired in aerial hyphae production and displays a severe delay in germ-tube formation (1,43).

Many other genes have been reported that, upon mutation, disturb morphological development and/or secondary metabolism. The AfsK/AfsR couple consists of a serine/threonine kinase and its target protein. These components of a signal transduction pathway are present in S. griseus and S. coelicolor but do not seem to control the same developmental steps. In S. coelicolor, the AfsK/R system is needed for secondary metabolism, whereas the same system is needed for morphological development in S. griseus (44). Mutations in the abs loci uncouple antibiotic biosynthesis from morphological development. None of the four antibiotics/pigments of S. coelicolor are produced in an absA mutant, but sporulation was unaffected (45). Another group of these mutants, defined as the absB locus, were identified as RNase III mutants (46).

Last but not least, the RNA polymerase o factors should be mentioned. Streptomycetes produce a range of o factors that are implicated in the transcriptional regulation of a variety of genes, including some involved in differentiation (47-49). The best known examples are WhiG (see Section 2.2.) and oF that are required for normal spore maturation (27).

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