Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of secondary metabolite gene clusters. Bioinformatic analysis of the genome of this organism predicts the presence of 27 gene clusters for secondary metabolites. We have used three different strategies for the activation of some of these silent/cryptic gene clusters in S. albus J1074: two hybrid polyketide-non-ribosomal peptides (PK-NRP) (antimycin and 6-epi-alteramides), a type I PK (candicidin), a non-ribosomal peptides (NRP) (indigoidine) and glycosylated compounds (paulomycins). By insertion of a strong and constitutive promoter in front of selected genes of two clusters, production of the blue pigment indigoidine and of two novel members of the polycyclic tetramate macrolactam family (6-epi-alteramides A and B) was activated. Overexpression of positive regulatory genes from the same organism also activated the biosynthesis of 6-epi-alteramides and heterologous expression of the regulatory gene pimM of the pimaricin cluster activated the simultaneous production of candicidins and antimycins, suggesting some kind of cross-regulation between both clusters. A cluster for glycosylated compounds (paulomycins) was also identified by comparison of the high-performance liquid chromatography profiles of the wild-type strain with that of a mutant in which two key enzymes of the cluster were simultaneously deleted.

SummarySchizosaccharomyces pombe Rgf3p is a specific Rho1 GEF that regulates cell wall β-glucan biosynthesis through the GTPase Rho1p

Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. We have identified a new GEF, designated Rgf1p, which specifically regulates Rho1p during polarized growth. The phenotype of rgf1 null cells was very similar to that seen after depletion of Rho1p, 30% of cells being lysed. In addition, rgf1(+) deletion caused hypersensitivity to the antifungal drug Caspofungin and defects in the establishment of bipolar growth. rho1(+), but none of the other GTPases of the Rho-family, suppressed the rgf1Delta phenotypes. Moreover, deletion of rgf1(+) suppressed the severe growth defect in rga1(+) null mutants (a Rho1-GAP, negative regulator). Rgf1p and Rho1p coimmunoprecipitated and overexpression of rgf1(+) specifically increased the GTP-bound Rho1p; it caused changes in cell morphology, and a large increase in beta(1,3)-glucan synthase activity. These effects were similar to those elicited when the hyperactive rho1-G15V allele was expressed. A genetic relationship was observed between Rgf1p, Bgs4p (beta[1,3]-glucan synthase), and Pck1p (protein kinase C [PKC] homologue); Bgs4p and Pck1p suppressed the hypersensitivity to Caspofungin in rgf1Delta mutants. Rgf1p localized to the growing ends and the septum, where Rho1, Pck1p, and Bgs4p are known to function. Our results suggest that Rgf1p probably activates the Rho functions necessary for coordinating actin deposition with cell wall biosynthesis during bipolar growth, allowing the cells to remodel their wall without risk of rupture.

The Rho family of GTPases are highly conserved molecular switches that control some of the most fundamental processes of cell biology, including morphogenesis, vesicular transport, cell division and motility. Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to extracellular stimuli. In fission yeast, there are seven Dbl-related GEFs and they activate six Rho-type GTPases within a particular spatio-temporal context. The failure to do so might have consequences reflected in aberrant phenotypes and in some cases lead to cell death. In this review, we briefly summarize the role of Rho GTPases and Rho-GEFs in the establishment and maintenance of cell polarity and cell integrity in Schizosaccharomyces pombe.

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Schizosaccharomyces pombe Rho1p is essential, directly activates b-1,3-glucan synthase, and participates in the regulation of morphogenesis. In S. pombe, Rho1p is activated by at least three guanine nucleotide exchange factors (GEFs): Rgf1p, Rgf2p, and Rgf3p. In this study we show that Rgf2p is a Rho1p GEF required for sporulation. The rgf2 1 deletion did not affect forespore membrane formation and the nuclei were encapsulated properly. However, the mutant ascospores appeared dark and immature. The rgf2D zygotes were not able to release the ascospores spontaneously, and the germination efficiency was greatly reduced compared to wild-type (wt) spores. This phenotype resembles that of the mutants in bgs2 1 , which encodes a sporulation-specific glucan synthase subunit. In fact, glucan synthase activity was diminished in sporulating rgf2D diploids. Rgf2p also plays a role in b-glucan biosynthesis during vegetative growth. Overexpression of rgf2 1 specifically increased GTP-bound Rho1p, caused changes in cell morphology, and elicited an increase in b-1,3-glucan synthase activity. Moreover, the simultaneous disruption of rgf1 1 and rgf2 1 was lethal and both Rgf1p and Rgf2p were able to partially substitute for each other. Our results suggest that Rgf1p and Rgf2p are alternative GEFs with an essential overlapping function in Rho1p activation during vegetative growth.

Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.

Summary Fission yeast possesses a family of (1,3)‐α‐glucan synthase‐related genes; one of them, mok1+/ags1+, plays an essential function in morphogenesis during vegetative growth. Here we show that three mok1+ paralogues –mok12+, mok13+ and mok14+– are required for sporulation to succeed, acting at different stages of the spore wall maturation process. Mutation of mok12+ affected the efficiency of spore formation and spore viability. Deletion of mok13+ does not affect spore viability but the spores showed reduced resistance to stress conditions. mok14Δ mutant spores failed to accumulate the amylose‐like spore wall‐specific polymer. mok12+, mok13+ and mok14+ expression was restricted to sporulating cells and the proteins localized to the spore envelope but with different timing. mok11+ was also induced during the sporulation process although its deletion did not show apparently a sporulation defect. In vegetative cells, β‐glucans are more abundant than α‐glucans (55% versus 28%). In spores, the situation was the opposite, α‐glucans accounted for 46% while β‐glucans were approximately 38% of the total polysaccharides. We found at least two types of α‐glucan polymers, Mok12p and Mok13p, were involved in the synthesis of the greater part of α‐glucan in the spores envelope, a polymer that is mainly digested with α‐1,3 glucanase, while Mok14p, homologous to starch synthases, was required for the synthesis of the iodine‐reactive polymer that is made of α‐1,4 glucose residues.