The rpoZ Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in Streptomyces kasugaensis

ABSTRACT The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (ω), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete ω of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.

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Identification of a Gene Negatively Affecting Antibiotic Production and Morphological Differentiation in Streptomyces coelicolor A3(2)

Journal of Bacteriology ◽

10.1128/jb.00933-06 ◽

2006 ◽

Vol 188 (24) ◽

pp. 8368-8375 ◽

Cited By ~ 36

Author(s):

Wencheng Li ◽

Xin Ying ◽

Yuzheng Guo ◽

Zhen Yu ◽

Xiufen Zhou ◽

...

Keyword(s):

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Regulatory Gene ◽

Morphological Differentiation ◽

Aerial Mycelium ◽

Protein Domain ◽

Chromosomal Dna ◽

Reading Frame ◽

Directed Mutation ◽

In The Wild

ABSTRACT SC7A1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolor A3(2). Its insertion into the chromosome of S. coelicolor strains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frame SCO5582 as nsdA, a gene n egatively affecting S treptomyces d ifferentiation. The disruption of chromosomal nsdA caused the overproduction of spores and of three of four known S. coelicolor antibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying that nsdA in the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster. nsdA expression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, a Streptomyces protein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in some Streptomyces genomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of the nsdA-like gene SCO4114 had no obvious phenotypic effects on S. coelicolor. The nsdA orthologue SAV2652 in S. avermitilis could complement the S. coelicolor nsdA-null mutant phenotype.

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Inactivation or amplification of the spa2 gene, encoding a potential stationary-phase regulator, affects differentiation in Streptomyces ambofaciens

Canadian Journal of Microbiology ◽

10.1139/m97-160 ◽

1997 ◽

Vol 43 (12) ◽

pp. 1118-1125 ◽

Cited By ~ 1

Author(s):

Martine Aubert ◽

Elisabeth Weber ◽

Brigitte Gintz ◽

Bernard Decaris ◽

Keith F. Chater

Keyword(s):

Stationary Phase ◽

Antibiotic Production ◽

Morphological Differentiation ◽

Aerial Mycelium ◽

Detectable Effect ◽

Multicopy Plasmid ◽

Gene Encoding ◽

Wild Type Phenotype ◽

Streptomyces Ambofaciens ◽

Mycelial Development

The deduced product of the spa2 gene of Streptomyces ambofaciens is a homologue of RspA, involved in stationary-phase σs factor regulation in Escherichia coli. This suggests that Spa2 could play a part in stationary-phase-associated differentiation in S. ambofaciens. The disruption of spa2 led to reductions in aerial mycelial development and associated spore pigmentation. The mutant phenotype reverted to the wild-type phenotype when the disruption construct spontaneously excised. The spa2 disruption had no detectable effect on growth rates in different media or antibiotic production and resistance. When spa2 was placed on a multicopy plasmid, a severe defect in formation and pigmentation of aerial mycelium resulted. These results strongly suggest that Spa2 is involved in a complex manner in the morphological differentiation process.Key words: Streptomyces, differentiation, stationary-phase regulator.

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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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The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

Applied and Environmental Microbiology ◽

10.1128/aem.00465-11 ◽

2011 ◽

Vol 77 (21) ◽

pp. 7586-7594 ◽

Cited By ~ 27

Author(s):

Fernando Santos-Beneit ◽

Mónica Barriuso-Iglesias ◽

Lorena T. Fernández-Martínez ◽

Miriam Martínez-Castro ◽

Alberto Sola-Landa ◽

...

Keyword(s):

Rna Polymerase ◽

Response Regulator ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Morphological Differentiation ◽

Luciferase Reporter ◽

Antibiotic Biosynthesis ◽

Binding Assays ◽

Content Type ◽

Phosphate Depletion

ABSTRACTThe RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ββ′ core of this enzyme in bacteria. We have characterized therpoZgene ofStreptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of therpoZgene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZstrain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZmutant with the wild-typerpoZallele restored both phenotype and antibiotic production. Interestingly, therpoZgene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed thatrpoZpromoter activity was increased in a ΔphoPbackground, it can be concluded thatrpoZis controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation inStreptomyces.

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The rpoZ Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in Streptomyces kasugaensis

Journal of Bacteriology ◽

10.1128/jb.185.1.386.2003 ◽

2003 ◽

Vol 185 (1) ◽

pp. 386-386 ◽

Cited By ~ 1

Author(s):

Ikuo Kojima ◽

Kano Kasuga ◽

Masayuki Kobayashi ◽

Akira Fukasawa ◽

Satoshi Mizuno ◽

...

Keyword(s):

Rna Polymerase ◽

Antibiotic Production ◽

Morphological Differentiation ◽

Gene Encoding

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Novel Genes That Influence Development in Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.186.11.3570-3577.2004 ◽

2004 ◽

Vol 186 (11) ◽

pp. 3570-3577 ◽

Cited By ~ 20

Author(s):

Amy M. Gehring ◽

Stephanie T. Wang ◽

Daniel B. Kearns ◽

Narie Yoo Storer ◽

Richard Losick

Keyword(s):

Soil Bacteria ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Morphological Differentiation ◽

Aerial Mycelium ◽

Colony Growth ◽

Methionine Synthase ◽

Regulatory Proteins ◽

Methionine Biosynthesis ◽

Acyl Coenzyme A

ABSTRACT Filamentous soil bacteria of the genus Streptomyces carry out complex developmental cycles that result in sporulation and production of numerous secondary metabolites with pharmaceutically important activities. To further characterize the molecular basis of these developmental events, we screened for mutants of Streptomyces coelicolor that exhibit aberrant morphological differentiation and/or secondary metabolite production. On the basis of this screening analysis and the subsequent complementation analysis of the mutants obtained we assigned developmental roles to a gene involved in methionine biosynthesis (metH) and two previously uncharacterized genes (SCO6938 and SCO2525) and we reidentified two previously described developmental genes (bldA and bldM). In contrast to most previously studied genes involved in development, the genes newly identified in the present study all appear to encode biosynthetic enzymes instead of regulatory proteins. The MetH methionine synthase appears to be required for conversion of aerial hyphae into chains of spores, SCO6938 is a probable acyl coenzyme A dehydrogenase that contributes to the proper timing of aerial mycelium formation and antibiotic production, and SCO2525 is a putative methyltransferase that influences various aspects of colony growth and development.

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RNA Polymerase Sigma Factor That Blocks Morphological Differentiation by Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.183.20.5991-5996.2001 ◽

2001 ◽

Vol 183 (20) ◽

pp. 5991-5996 ◽

Cited By ~ 35

Author(s):

Amy M. Gehring ◽

Narie J. Yoo ◽

Richard Losick

Keyword(s):

Rna Polymerase ◽

Sigma Factor ◽

Insertional Mutagenesis ◽

Early Stage ◽

Streptomyces Coelicolor ◽

Morphological Differentiation ◽

Aerial Mycelium ◽

Growth Defect ◽

Insertion Mutant

ABSTRACT The filamentous bacterium Streptomyces coelicolorundergoes a complicated process of morphological differentiation that begins with the formation of an aerial mycelium and culminates in sporulation. Genes required for the initiation of aerial mycelium formation have been termed bld (bald), describing the smooth, undifferentiated colonies of mutant strains. By using an insertional mutagenesis protocol that relies on in vitro transposition, we have isolated a bld mutant harboring an insertion in a previously uncharacterized gene, SCE59.12c, renamed here rsuA. The insertion mutant exhibited no measurable growth defect but failed to produce an aerial mycelium and showed a significant delay in the production of the polyketide antibiotic actinorhodin. The rsuA gene encodes an apparent anti-sigma factor and is located immediately downstream ofSCE59.13c, renamed here sigU, whose product is inferred to be a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors. The absence ofrsuA in a strain that contained sigUcaused a block in development, and the overexpression ofsigU in an otherwise wild-type strain caused a delay in aerial mycelium formation. However, a strain in which bothrsuA and sigU had been deleted was able to undergo morphological differentiation normally. We conclude that thersuA-encoded anti-sigma factor is responsible for antagonizing the function of the sigma factor encoded bysigU. We also conclude that thesigU-encoded sigma factor is not normally required for development but that its uncontrolled activity obstructs morphological differentiation at an early stage.

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A bipartite DNA-binding domain in yeast Reb1p.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1173 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1173-1182 ◽

Cited By ~ 33

Author(s):

B E Morrow ◽

Q Ju ◽

J R Warner

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Kluyveromyces Lactis ◽

Binding Domain ◽

Dna Binding Domain ◽

Coding Region ◽

Active Growth ◽

Yeast Saccharomyces Cerevisiae

The REB1 gene encodes a DNA-binding protein (Reb1p) that is essential for growth of the yeast Saccharomyces cerevisiae. Reb1p binds to sites within transcriptional control regions of genes transcribed by either RNA polymerase I or RNA polymerase II. The sequence of REB1 predicts a protein of 809 amino acids. To define the DNA-binding domain of Reb1p, a series of 5' and 3' deletions within the coding region was constructed in a bacterial expression vector. Analysis of the truncated Reb1p proteins revealed that nearly 400 amino acids of the C-terminal portion of the protein are required for maximal DNA-binding activity. To further define the important structural features of Reb1p, the REB1 homolog from a related yeast, Kluyveromyces lactis, was cloned by genetic complementation. The K. lactis REB1 gene supports active growth of an S. cerevisiae strain whose REB1 gene has been deleted. The Reb1p proteins of the two organisms generate almost identical footprints on DNA, yet the K. lactis REB1 gene encodes a polypeptide of only 595 amino acids. Comparison of the two Reb1p sequences revealed that within the region necessary for the binding of Reb1p to DNA were two long regions of nearly perfect identity, separated in the S. cerevisiae Reb1p by nearly 150 amino acids but in the K. lactis Reb1p by only 40 amino acids. The first includes a 105-amino-acid region related to the DNA-binding domain of the myb oncoprotein; the second bears a faint resemblance to myb. The hypothesis that the DNA-binding domain of Reb1p is formed from these two conserved regions was confirmed by deletion of as many as 90 amino acids between them, with little effect on the DNA-binding ability of the resultant protein. We suggest that the DNA-binding domain of Reb1p is made up of two myb-like regions that, unlike myb itself, are separated by as many as 150 amino acids. Since Reb1p protects only 15 to 20 nucleotides in a chemical or enzymatic footprint assay, the protein must fold such that the two components of the binding site are adjacent.

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