Aerial Mycelium Formation Research Articles

A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2).

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sigmaH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sigmaH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2-egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sigmaH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2-egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sigmaH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.

Modulation of actinorhodin biosynthesis in Streptomyces lividans by glucose repression of afsR2 gene transcription.

While the biosynthetic gene cluster encoding the pigmented antibiotic actinorhodin (ACT) is present in the two closely related bacterial species, Streptomyces lividans and Streptomyces coelicolor, it normally is expressed only in S. coelicolor-generating the deep-blue colonies responsible for the S. coelicolor name. However, multiple copies of the two regulatory genes, afsR and afsR2, activate ACT production in S. lividans, indicating that this streptomycete encodes a functional ACT biosynthetic pathway. Here we report that the occurrence of ACT biosynthesis in S. lividans is determined conditionally by the carbon source used for culture. We found that the growth of S. lividans on solid media containing glucose prevents ACT production in this species by repressing the synthesis of afsR2 mRNA; a shift to glycerol as the sole carbon source dramatically relieved this repression, leading to extensive ACT synthesis and obliterating this phenotypic distinction between S. lividans and S. coelicolor. Transcription from the afsR2 promoter during growth in glycerol was dependent on afsR gene function and was developmentally regulated, occurring specifically at the time of aerial mycelium formation and coinciding temporally with the onset of ACT production. In liquid media, where morphological differentiation does not occur, ACT production in the absence of glucose increased as S. lividans cells entered stationary phase, but unlike ACT biosynthesis on solid media, occurred by a mechanism that did not require either afsR or afsR2. Our results identify parallel medium-dependent pathways that regulate ACT biosynthesis in S. lividans and further demonstrate that the production of this antibiotic in S. lividans grown on agar can be modulated by carbon source through the regulation of afsR2 mRNA synthesis.

A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens.

A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.

Intracellular protease activity during the growth of Streptomyces coelicolor

Multiple intracellular proteases were produced by Streptomyces coelicolor throughout growth as surface cultures. Zymography revealed two constitutive, gelatinolytic proteases of approximate molecular masses 32.5 and 36.5 kDa. In addition, transient expression of a large (183.5 kDa) protease preceded aerial mycelium formation and following this, during sporulation, an additional protease of mass 27.5 kDa was produced.

Characterization of mutants defective in melanogenesis and a gene for tyrosinase of Streptomyces griseus.

Nitrosoguanidine-induced melanin-negative mutants of Streptomyces griseus were classified according to their ability to produce streptomycin and A-factor, and to form aerial mycelium. A large proportion of the mutants showed deficiency in either antibiotic production and morphological development or both, suggesting close regulatory correlation between melanogenesis and morphological and physiological differentiation. The tyrosinase-encoding mel operon of S. griseus was cloned and examined for its role in melanogenesis of this organism. As in other Streptomyces homologues, the operon consisted of two open reading frames, melC1 encoding the putative cofactor and melC2 encoding the tyrosinase. Regardless of the distinct sequence similarity, introduction of the operon on plasmids failed to confer melanin production in the melanin-negative mutants, and the disruption of melC2 barely affected the melanin productivity, which indicated the presence of another enzyme involved in the melanogenesis in S. griseus. On the other hand, mel on a high-copy-number plasmid caused precocious aerial mycelium formation in Streptomyces lividans TK21 suggesting a stimulatory role of tyrosinase in morphological development.

An oligoribonuclease gene in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) serves as a microbial hormone that switches on many genes required for streptomycin production and morphological development. An open reading frame (Orf1) showing high sequence similarity to oligoribonucleases of various origins is present just downstream of adpA, one of the A-factor-dependent genes. Orf1 was named OrnA (oligoribonuclease A) because it showed 3'-to-5' exo-oligoribonuclease activity, releasing [(32)P]CMP from ApCpC[(32)P]pC used as a substrate. Reverse transcription-PCR and S1 nuclease mapping analyses revealed that ornA was transcribed from two promoters; one was a developmentally regulated, A-factor-dependent promoter in front of adpA, and the other was a constitutive promoter in front of the ornA coding sequence. Transcription of ornA was thus additively enhanced at the initiation stage for secondary metabolism and aerial mycelium formation. ornA-disrupted strains grew slowly and scarcely formed aerial mycelium. ornA homologues were distributed in a wide variety of Streptomyces species, including S. coelicolor A3(2), as determined by Southern hybridization analysis. Disruption of the ornA homologue in S. coelicolor A3(2) also caused phenotypes similar to those of the S. griseus DeltaornA strains. The OrnA oligoribonucleases in Streptomyces species are therefore not essential but play an important role in vegetative growth and in the initiation of differentiation.

An A-factor-dependent extracytoplasmic function sigma factor (sigma(AdsA)) that is essential for morphological development in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function sigma factor belonging to a subgroup of the primary sigma(70) family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named sigma(AdsA). Transcription of adsA depended on A-factor and AdpA, since adsA was transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that sigma(AdsA) is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomyces species examined by Southern hybridization suggests a common role in morphogenesis in this genus.

Sigma(BldN), an extracytoplasmic function RNA polymerase sigma factor required for aerial mycelium formation in Streptomyces coelicolor A3(2).

Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N'-nitro-N-nitrosoguanidine)-induced whi strains (N. J. Ryding et al., J. Bacteriol. 181:5419-5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed that whiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the "bald" phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficient bld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC, bldF, bldK, or bldJ or on bldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended on bldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for sigma(BldN) holoenzyme in vitro.

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating. Mutants defective in the formation of aerial mycelium and spores (bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores (whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating. Mutants defective in the formation of aerial mycelium and spores ( bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores ( whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.

A developmentally regulated catalase required for proper differentiation and osmoprotection of Streptomyces coelicolor.

Streptomyces coelicolor produces at least three catalases, the expression of which varies under different conditions. We characterized a gene (catB) for developmentally controlled catalase of 779 amino acids (83408 Da), homologous to KatE of Escherichia coli and Bacillus subtilis. Expression of the catB gene increased at the stationary phase in liquid culture and after the onset of differentiation on solid culture. It was also increased by osmotic treatments. Transcription was initiated from a promoter (catBp), whose sequence (ATGCCTCG-N13-GGGTAC) resembled promoters recognized by sigmaB of B. subtilis. CatB protein underwent proteolytic cleavage of its N-terminal 95 amino acids and was secreted to the medium when cells sporulated. Disruption of the catB gene caused impairment in the formation of aerial mycelium and reduction in the synthesis of undecylprodigiosin. On the contrary, hyperproduction of actinorhodin was observed in accordance with the increase in actII-ORF4 transcription. In addition, catB mutant became hypersensitive to osmotic stresses. These results suggest that regulated synthesis of CatB protein is necessary to ensure proper differentiation as well as to protect S. coelicolor cells against osmotic stresses.

Carbon-source-dependent transcriptional control involved in the initiation of cellular differentiation in Streptomyces griseus.

Carbon source is one of the environmental factors that affects cellular differentiation of Streptomyces. We have identified the craA gene as a putative negative regulator involved in the carbon-source-dependent initiation of cellular differentiation in Streptomyces griseus. Carbon-source-dependent transcriptional repression of craA, which is caused by binding of a putative repressor protein to its promoter region, is proposed to result in the initiation of aerial mycelium formation. The presence of a craA homologue in the chromosome of Streptomyces coelicolor A3(2) implicates the existence of a similar regulatory mechanism in this organism. The repression of craA-promoter activity in glucose media could be alleviated not only by replacing glucose with maltose but also by supplying copper, which suggests that the stimulatory effect of copper on cellular differentiation in Streptomyces is excerted via abolishment of glucose-repression of craA.

Mated: a putative peptide transporter of Schizophyllum commune expressed in dikaryons.

A gene that exhibits sexually induced transcript accumulation, mtd1, was identified physically linked to a locus that is also under control of the mating-type genes. Genomic and cDNA sequence analyses were performed to determine a large open reading frame which starts with the amino-acid sequence M-A-T-E-D. The ORF showed similarities to a novel family of peptide transporters with at least 12 putative transmembrane structures. A gene disruption resulted in a growth-phenotype with slow aerial mycelium formation that could be complemented with peptone or uracil added to the growth medium.

An AfsK/AfsR system involved in the response of aerial mycelium formation to glucose in Streptomyces griseus.

In Streptomyces coelicolor A3(2), a protein serine/threonine kinase (AfsK) and its target protein (AfsR) control secondary metabolism. AfsK and AfsR homologues (AfsK-g and AfsR-g) from Streptomyces griseus showed high end-to-end similarity in amino acid sequence with the respective S. coelicolor A3(2) proteins, as determined by cloning and nucleotide sequencing. AfsK-g and a fusion protein between AfsK-g and thioredoxin (TRX-AfsK-g) produced in high yield as inclusion bodies in Escherichia coli were solubilized with urea, purified by column chromatography and then refolded to an active form by dialysis to gradually remove the urea. AfsR-g was also fused to glutathione S-transferase (GST-AfsR-g); the fusion product in the soluble fraction in E. coli was purified. Incubation of AfsK-g or TRX-AfsK-g in the presence of [gamma-32P]ATP yielded autophosphorylated products containing phosphoserine and phosphothreonine residues. In addition, TRX-AfsK-g phosphorylated serine and threonine residues of GST-AfsR-g in the presence of [gamma-32P]ATP. Disruption of chromosomal afsK-g had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The afsK-g disruptants did not form aerial mycelium or spores on media containing glucose at concentrations higher than 1%, but did form spores on mannitol- and glycerol-containing media; this suggests that afsK-g is essential for morphogenesis in the presence of glucose. Introduction of afsK-g restored aerial mycelium formation in the disruptants. The phenotype of afsR-g disruptants was similar to that of afsK-g disruptants; introduction of afsR-g restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in S. griseus is conditionally needed for morphological differentiation, whereas in S. coelicolor A3(2) it is conditionally involved in secondary metabolism.

Involvement of amfC in physiological and morphological development in Streptomyces coelicolor A3(2).

amfC plays a regulatory role in aerial mycelium formation in Streptomyces griseus and is distributed widely among Streptomyces species. Disruption of the chromosomal amfC gene in Streptomyces coelicolor A3(2) severely reduced formation of aerial hyphae, indicating that amfC is important in morphological development. In addition, the disruption caused S. coelicolor A3(2) M130 to produce much less actinorhodin, and to produce undecylprodigiosin at a later stage of growth, indicating that amfC also regulates secondary metabolism. S1 nuclease mapping showed that transcription of actII-ORF4, the pathway-specific transcriptional activator in the act gene cluster, was greatly reduced in the amfC disruptants. The defect in secondary metabolite formation was suppressed or overcome by a mutation in sre-1. Consequently, an amfC-disrupted strain derived from S. coelicolor A3(2) M145, an actinorhodin-overproducing strain due to the sre-1 mutation, still produced a large amount of actinorhodin. Extra copies of amfC in strains M130 and M145 did not change spore-chain morphology or secondary metabolite formation. However, the spores in these strains remained white even after prolonged incubation. Since only spore pigmentation was affected, all known whi genes, except whiE, responsible for the polyketide spore pigment formation, were assumed to function normally. S1 nuclease mapping showed that transcription of whiEP1, one of the promoters in the whiE locus, was reduced in S. coelicolor A3(2) containing extra copies of amfC. Introducing amfC into several other Streptomyces species, such as Streptomyces lividans, Streptomyces lavendulae and Streptomyces lipmanii, also abolished spore pigment formation. An increase in the amount of AmfC appeared to disturb the maturation of spores.

A putative regulatory element for carbon-source-dependent differentiation in Streptomyces griseus.

To identify negative regulatory genes for cellular differentiation in Streptomyces griseus, DNA fragments repressing the normal developmental processes were cloned on a high-copy-number plasmid. One of these DNA fragments markedly repressed aerial mycelium and spore formation on solid media containing glucose or galactose, but not on media containing maltose or mannitol. The fragment contained three complete ORFs; precise subcloning revealed that a 249 bp fragment located in the promoter region between ORF1 and ORF3 was sufficient for repression. Quantification of the promoter activities by using a thermostable malate dehydrogenase gene as a reporter showed that the promoter for ORF3 (P(ORF3)) maintained high activity in mycelia grown in the presence of glucose but lost activity rapidly in maltose medium. P(ORF3) activity increased markedly when the promoter sequence was introduced on a high-copy-number plasmid. The results suggested that carbon-source-dependent deactivation of P(ORF3) mediated by a transcriptional repressor may initiate differentiation in S. griseus.

Purification, Characterization, and Role of Nucleases and Serine Proteases in Streptomyces Differentiation: ANALOGIES WITH THE BIOCHEMICAL PROCESSES DESCRIBED IN LATE STEPS OF EUKARYOTIC APOPTOSIS

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.

Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid pIJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with antiphosphotyrosine antibody.

Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces.

The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP-dependent protease family Clp were cloned, mapped and characterized. S. coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes. Disruption of the clpP1 gene in S. lividans and S. coelicolor blocks differentiation at the substrate mycelium step. Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S. lividans, S. albus and S. coelicolor. Overproduction of ClpX accelerates actinorhodin production in S. coelicolor and activates its production in S. lividans.

Extracellular complementation and the identification of additional genes involved in aerial mycelium formation in Streptomyces coelicolor.

Morphogenesis in the bacterium Streptomyces coelicolor involves the formation of a lawn of hair-like aerial hyphae on the colony surface that stands up in the air and differentiates into chains of spores. bld mutants are defective in the formation of this aerial mycelium and grow as smooth, hairless colonies. When certain pairs of bld mutants are grown close to one another on rich sporulation medium, they exhibit extracellular complementation such that one mutant restores aerial mycelium formation to the other. The extracellular complementation relationships of most of the previously isolated bld mutants placed them in a hierarchy of extracellular complementation groups. We have screened for further bld mutants with precautions intended to maximize the discovery of additional genes. Most of the 50 newly isolated mutant strains occupy one of three of the previously described positions in the hierarchy, behaving like bldK, bldC, or bldD mutants. We show that the mutations in some of the strains that behave like bldK are bldK alleles but that others fall in a cluster at a position on the chromosome distinct from that of any known bld gene. We name this locus bldL. By introducing cloned genes into the strains that exhibit bldC or bldD-like extracellular complementation phenotypes, we show that most of these strains are likely to contain mutations in genes other than bldC or bldD. These results indicate that the genetic control of aerial mycelium formation is more complex than previously recognized and support the idea that a high proportion of bld genes are directly or indirectly involved in the production of substances that are exchanged between cells during morphological differentiation.