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Identification and characterization of a developmentally regulated protein, EshA, required for sporogenic hyphal branches in Streptomyces griseus.

ABSTRACT: To identify sporulation-specific proteins that might serve as targets of developmental regulatory factors in Streptomyces, we examined total proteins of Streptomyces griseus by two-dimensional gel electrophoresis. Among five proteins that were present at high levels during sporulation but absent from vegetative cells, two of the proteins, P3 and P4, were absent from developmental mutants that undergo aberrant morphogenesis. The deduced amino acid sequence of the gene that encodes P3 (EshA) showed extensive similarity to proteins from mycobacteria and a cyanobacterium, Synechococcus, that are abundant during nutritional stress but whose functions are unknown. Uniquely among these proteins, EshA contains a cyclic nucleotide-binding domain, suggesting that the activity of EshA may be modulated by a cyclic nucleotide. The eshA gene was strongly expressed from a single transcription start site only during sporulation, and accumulation of the eshA transcript depended on a developmental gene, bldA. During submerged sporulation, a null mutant strain that produced no EshA could not extend sporogenic hyphae from new branch points but instead accelerated septation and spore maturation at the preexisting vegetative filaments. These results indicated that EshA is required for the growth of sporogenic hyphae and localization of septation and spore maturation but not for spore viability.

SUBMITTER: Kwak J

PROVIDER: S-EPMC95199 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature

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Identification and characterization of a developmentally regulated protein, EshA, required for sporogenic hyphal branches in Streptomyces griseus.

Kwak J J McCue L A LA Trczianka K K Kendrick K E KE

Journal of bacteriology 20010501 10

To identify sporulation-specific proteins that might serve as targets of developmental regulatory factors in Streptomyces, we examined total proteins of Streptomyces griseus by two-dimensional gel electrophoresis. Among five proteins that were present at high levels during sporulation but absent from vegetative cells, two of the proteins, P3 and P4, were absent from developmental mutants that undergo aberrant morphogenesis. The deduced amino acid sequence of the gene that encodes P3 (EshA) showe ...[more]

PMID: 11325927

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Project description:BackgroundThe sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation.ResultsWe report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity.ConclusionAerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously unknown genes with important roles in sporulation. The transcriptomic data reported here should also serve as a basis for identification of further developmentally important genes in future functional studies.

Project description:BackgroundIn recent years, the sea anemone Nematostella vectensis has emerged as a critical model organism for comparative genomics and developmental biology. Although Nematostella is a member of the anthozoan cnidarians (known for producing an abundance of diverse fluorescent proteins (FPs)), endogenous patterns of Nematostella fluorescence have not been described and putative FPs encoded by the genome have not been characterized.Methodology/principal findingsWe described the spatiotemporal expression of endogenous red fluorescence during Nematostella development. Spatially, there are two patterns of red fluorescence, both restricted to the oral endoderm in developing polyps. One pattern is found in long fluorescent domains associated with the eight mesenteries and the other is found in short fluorescent domains situated between tentacles. Temporally, the long domains appear simultaneously at the 12-tentacle stage. In contrast, the short domains arise progressively between the 12- and 16-tentacle stage. To determine the source of the red fluorescence, we used bioinformatic approaches to identify all possible putative Nematostella FPs and a Drosophila S2 cell culture assay to validate NvFP-7R, a novel red fluorescent protein. We report that both the mRNA expression pattern and spectral signature of purified NvFP-7R closely match that of the endogenous red fluorescence. Strikingly, the red fluorescent pattern of NvFP-7R exhibits asymmetric expression along the directive axis, indicating that the nvfp-7r locus senses the positional information of the body plan. At the tissue level, NvFP-7R exhibits an unexpected subcellular localization and a complex complementary expression pattern in apposed epithelia sheets comprising each endodermal mesentery.Conclusions/significanceThese experiments not only identify NvFP-7R as a novel red fluorescent protein that could be employed as a research tool; they also uncover an unexpected spatio-temporal complexity of gene expression in an adult cnidarian. Perhaps most importantly, our results define Nematostella as a new model organism for understanding the biological function of fluorescent proteins in vivo.

Dataset Information

Identification and characterization of a developmentally regulated protein, EshA, required for sporogenic hyphal branches in Streptomyces griseus.

Publications

Identification and characterization of a developmentally regulated protein, EshA, required for sporogenic hyphal branches in Streptomyces griseus.

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