A response regulator‐like protein that functions at an intermediate stage of sporulation in <i>Streptomyces coelicolor</i> A3(2)

Aínsa, José A.; Parry, Huw D.; Chater, Keith F.

doi:10.1046/j.1365-2958.1999.01630.x

Cited by 71 publications

(100 citation statements)

References 44 publications

(78 reference statements)

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“…The high precision of the recombination occurring between 39-bp sequences was more extensively documented in experiments with another gene, whiI (28). Cosmid DNA samples from 50 transformants were all shown to contain the desired gene replacement both by restriction analysis and by PCR, using primers annealing 100 bp outside the 39-bp tail sequences.…”

Section: Resultsmentioning

confidence: 98%

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Gust

Challis

Fowler

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Streptomycetes are high G؉C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and -Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT RK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.FLP recombinase ͉ lambda Red recombinase ͉ oriT ͉ SCO5222 ͉ SCO6073

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Section: Resultsmentioning

confidence: 98%

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Gust

Challis

Fowler

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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1,315

1,554

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“…The complex early whi mutant phenotypes suggest that some or all of the six "early" whi genes encode regulatory elements. Indeed, whiG encodes a sigma factor ( WhiG [11,37]), whiH encodes a repressor-like protein (31), and whiI encodes a response regulator-like protein (1); the product of whiB has features that resemble those of some transcription factors (13,34). Mutations in some other more recently discovered genes also affect sporulation septation but in a more allele-specific manner (30).…”

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confidence: 99%

“…whiA and whiB are therefore believed to play a role in the cessation of aerial growth. Flärdh et al (15) and Aínsa et al (1) proposed that this is part of a hypothetical sequentially dependent series of developmental decisions. In this model, newly emerged aerial hyphae first switch to a sporulation-specific mode of elongation through the action of WhiG , eventually undergoing a WhiA-and WhiBdependent orderly cessation of elongation and DNA replication, presumably in response to some signal(s); it is suggested that growth cessation then sets off further signals, which activate the WhiH and WhiI proteins, and the activated forms of these proteins switch on sporulation septation.…”

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confidence: 99%

WhiA, a Protein of Unknown Function Conserved among Gram-Positive Bacteria, Is Essential for Sporulation in Streptomyces coelicolor A3(2)

et al. 2000

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The whiA sporulation gene of Streptomyces coelicolor A3(2), which plays a key role in switching aerial hyphae away from continued extension growth and toward sporulation septation, was cloned by complementation of whiA mutants. DNA sequencing of the wild-type allele and five whiA mutations verified that whiA is a gene encoding a protein with homologues in all gram-positive bacteria whose genome sequence is known, whether of high or low G؉C content. No function has been attributed to any of these WhiA-like proteins. In most cases, as in S. coelicolor, the whiA-like gene is downstream of other conserved genes in an operon-like cluster. Phenotypic analysis of a constructed disruption mutant confirmed that whiA is essential for sporulation. whiA is transcribed from at least two promoters, the most downstream of which is located within the preceding gene and is strongly up-regulated when colonies are undergoing sporulation. The up-regulation depends on a functional whiA gene, suggesting positive autoregulation, although it is not known whether this is direct or indirect. Unlike the promoters of some other sporulation-regulatory genes, the whiA promoter does not depend on the sporulation-specific factor encoded by whiG.Dispersal of the mycelial organism Streptomyces coelicolor A3(2) occurs by the formation of long chains of spores from aerial hyphae. A critical stage in this process is the subdivision of a multigenomic apical aerial hyphal compartment into many unigenomic prespore compartments by the synchronous formation of regularly spaced sporulation septa (26,38). At least six genetic loci (whiA, whiB, whiG, whiH, whiI, and whiJ) are needed for sporulation septation (8, 10), in addition to cell division genes that are also involved in vegetative growth (24,25). These six early whi loci appear to play no role in vegetative growth. Mutations in them have pleiotropic effects on the later stages of sporulation, including weak or undetectable transcription of the genes responsible for the production of grey spore pigment (20), hence the white colony phenotype after which they were named (18). Transcription of sigF, which encodes a late sporulation sigma factor, is also dependent on these early whi genes (21, 28). The complex early whi mutant phenotypes suggest that some or all of the six "early" whi genes encode regulatory elements. Indeed, whiG encodes a sigma factor ( WhiG [11,37]), whiH encodes a repressor-like protein (31), and whiI encodes a response regulator-like protein (1); the product of whiB has features that resemble those of some transcription factors (13,34). Mutations in some other more recently discovered genes also affect sporulation septation but in a more allele-specific manner (30).This paper is concerned with whiA. Like whiB mutants, whiA and whiA whiB double mutants have tightly coiled aerial hyphae that are markedly longer than normal spore chains, contain uncondensed DNA, and lack sporulation septa and readily detectable FtsZ (9, 15, 33). whiA and whiB are therefore believed to play a role in th...

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“…The expression of glgBII therefore depends on whiG, but not on whiH or I (both of which are themselves directly and completely whiG-dependent; Ryding et al, 1998;Aínsa et al, 2000) or whiA or B (which are whiG-independent; Aínsa et al, 1999). The abundance of aerial-mycelium-associated glycogen in whiG mutants may reflect the absence of WhiH from such mutants, if WhiH does indeed influence the quantity of glycogen in aerial hyphae (see above).…”

Section: Discussionmentioning

confidence: 98%

“…Mutants in the remaining well-studied early whi genes display at least some coiling of aerial hyphal tips (Chater et al, 1989;Flärdh et al, 1999). Among theses genes, whiA encodes a protein of unknown function with orthologues in several other Gram-positive bacteria (Aínsa et al, 2000); whiB belongs to a gene group whose products code for actinomycete-specific small cysteine-rich regulatory proteins of unknown structure (Davis & Chater, 1992;Soliveri et al, 2000); whiH encodes a member of the GntR family of transcription factors (Ryding et al, 1998); and whiI encodes an atypical member of the kind of response regulator usually associated with histidine protein kinases in bacterial two-component systems (Aínsa et al, 1999). Both whiH and whiI are direct targets for RNA polymerase holoenzyme containing s WhiG (Ryding et al, 1998;Aínsa et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

The interplay of glycogen metabolism and differentiation provides an insight into the developmental biology of Streptomyces coelicolor

Yeo

Chater

2005

Microbiology

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Mycelial colonies of the developmentally complex actinomycete Streptomyces coelicolor growing on solid medium contain glycogen in two distinct locations. Phase I deposits are found in a substrate mycelium region bordering the developing aerial mycelium. Their production involves GlgBI, one of two glycogen branching enzyme isoforms. Phase II deposits occur in the upper regions of aerial hyphae, in long tip cells that are dividing, or have just divided, into unigenomic prespore compartments. Their formation involves a second branching enzyme isoform, GlgBII. To find out if the gene for the second isoform, glgBII, is regulated by any of the well-studied whiA, B, G, H or I genes needed for sporulation septation, glgBI or glgBII was disrupted in a set of whi mutants, and the glycogen phenotypes examined by transmission electron microscopy. In the whiG mutants, deposits were found throughout the aerial mycelium and the adjacent region of the substrate mycelium, but the morphology of all the deposits, i.e. whether they were in the form of granules of branched glycogen or large blobs of unbranched glycan, depended solely on GlgBI. In contrast, the whiA, B, H and I mutations had no obvious effect on the pattern of glycogen deposition, or on the spatial specificity of the branching enzyme isoforms (though phase II glycogen deposits were reduced in size and abundance in the whiA and B mutants, and increased in the whiH mutant). These results indicate that glgBII is regulated (directly or indirectly) by whiG, and not by any of the other whi genes tested, and that the aerial hyphae of a whiG mutant are atypical in being physiologically similar to the substrate hyphae from which they emerge. A new role for aerial hyphae is proposed.

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A response regulator‐like protein that functions at an intermediate stage of sporulation in Streptomyces coelicolor A3(2)

Cited by 71 publications

References 44 publications

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

WhiA, a Protein of Unknown Function Conserved among Gram-Positive Bacteria, Is Essential for Sporulation in Streptomyces coelicolor A3(2)

The interplay of glycogen metabolism and differentiation provides an insight into the developmental biology of Streptomyces coelicolor

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