New loci required for Streptomyces coelicolor morphological and physiological differentiation.

Streptomyces coelicolor colonies differentiate both morphologically, producing aerial spore chains, and physiologically, producing antibiotics as secondary metabolites. Single mutations, which block both aspects of differentiation, define bld (bald colony) genes. To identify new bld genes, mutagenized colonies were screened for blocks in the earliest stage of sporulation, the formation of aerial mycelia, and blocks in antibiotic synthesis. The mutations in 12 mutants were mapped; in each strain, the pleiotropic phenotype was due to a single mutation. Seven of the strains contained mutations in known bld loci, bldA and bldB. Three strains contained mutations in a new locus, bldG, and two contained mutations in another new locus, bldH. Like the previously defined bldA mutants, the bldG and bldH mutants were developmentally blocked on glucose. On a variety of carbon sources whose utilization was subject to glucose repression, the developmental blocks were partially relieved for bldG (and bldA) mutants and fully relieved for bldH mutants. These results are compatible with an hypothesis which suggests that there are two alternative controls on S. coelicolor differentiation, one of which is glucose repressible.     

The bld mutants of Streptomyces coelicolor are defective in the regulation of carbon utilization, morphogenesis and cell–cell signalling

Mutants of Streptomyces coelicolor blocked at the earliest visible stage of morphological differentiation are called bld mutants. These mutants fail to form aerial hyphae on rich medium and most are defective in antibiotic production. One striking feature of these mutants is that, with the exception of bldB , their morphological defect is carbon-source dependent. In our investigation of catabolite control in Streptomyces , we identified mutants that were resistant to glucose repression and were also bld . The existence of these new bld mutants led us to examine the catabolite control phenotype of the previously described bld mutants which were not known to contain defects in carbon regulation. We report here that all of the characterized bld mutants of S. coelicolor are defective in the regulation of galP1 , and that at least one of the bld mutants, bldB , is globally deregulated for carbon utilization. Complementation of the morphological defect of bldA and bldB mutants with a cloned copy of the wild-type bld gene simultaneously restored normal regulation of galP1 , indicating that both aspects of the mutant phenotype are caused by the same lesion. We suggest a new interpretation for the role of the bld genes in development in Streptomyces. We suggest that the primary defect in bld mutants is in the regulation of carbon utilization, not specifically in the activation of genes whose products regulate the development pathway as previously suggested. We speculate that the inability of bld mutants to initiate morphogenesis is a secondary consequence of their inability to sense and/or signal starvation.     

Loss of Spatial Control of the Mitotic Spindle Apparatus in a Chlamydomonas Reinhardtii Mutant Strain Lacking Basal Bodies

The bld2-1 mutation in the green alga Chlamydomonas reinhardtii is the only known mutation that results in the loss of centrioles/basal bodies and the loss of coordination between spindle position and cleavage furrow position during cell division. Based on several different assays, bld2-1 cells lack basal bodies in >99% of cells. The stereotypical cytoskeletal morphology and precise positioning of the cleavage furrow observed in wild-type cells is disrupted in bld2-1 cells. The positions of the mitotic spindle and of the cleavage furrow are not correlated with respect to each other or with a specific cellular landmark during cell division in bld2-1 cells. Actin has a variable distribution during mitosis in bld2-1 cells, but this aberrant distribution is not correlated with the spindle positioning defect. In both wild-type and bld2-1 cells, the position of the cleavage furrow is coincident with a specialized set of microtubules found in green algae known as the rootlet microtubules. We propose that the rootlet microtubules perform the functions of astral microtubules and that functional centrioles are necessary for the organization of the cytoskeletal superstructure critical for correct spindle and cleavage furrow placement in Chlamydomonas.     

Tn4563 transposition in Streptomyces coelicolor and its application to isolation of new morphological mutants.

The Tn3-like transposon Tn4556 (and its derivatives Tn4560 and Tn4563) has been used for insertion mapping of genetic loci cloned on plasmids, but it has been difficult to obtain chromosomal insertions, largely because of the lack of a strong selection against transposon donor molecules. In this communication, we report two efficient selection techniques for transposition and their use in the isolation of chromosomal insertion mutations. A number of independent Streptomyces coelicolor morphological mutants (bld and whi) were obtained. Two of the bld mutations were mapped to locations on the chromosome by SCP1-mediated conjugation; at least one mutation, bld-5m1, appears to define a novel locus involved in control of S. coelicolor morphogenesis and antibiotic production.     

Extragenic bypass suppressors of mutations in the essential gene BLD2 promote assembly of basal bodies with abnormal microtubules in Chlamydomonas reinhardtii

bld2-1 mutant Chlamydomonas reinhardtii strains assemble basal bodies with singlet microtubules; bld2-1 cells display flagellar assembly defects as well as positioning defects of the mitotic spindle and cleavage furrow. To further understand the role of the BLD2 gene, we have isolated three new bld2 alleles and three partially dominant extragenic suppressors, rgn1-1, rgn1-2, and rgn1-3. bld2 rgn1-1 strains have phenotypes intermediate between those of bld2 and wild-type strains with respect to flagellar number, microtubule rootlet organization, cleavage furrow positioning, and basal body structural phenotypes. Instead of the triplet microtubules of wild-type cells, bld2 rgn1-1 basal bodies have mixtures of no, singlet, doublet, and triplet microtubules. The bld2-4 allele was made by insertional mutagenesis and identified in a noncomplementation screen in a diploid strain. The bld2-4 allele has a lethal phenotype based on mitotic segregation in diploid strains and in haploid strains generated by meiotic recombination. The lethal phenotype in haploid strains is suppressed by rgn1-1; these suppressed strains have similar phenotypes to other bld2 rgn1-1 double mutants. It is likely that BLD2 is an essential gene that is needed for basal body assembly and function.     

An oligopeptide permease responsible for the import of an extracellular signal governing aerial mycelium formation in Streptomyces coelicolor

Morphological differentiation in the filamentous bacterium Streptomyces coelicolor is believed to involve a mechanism of extracellular signalling that culminates with the formation of an aerial mycelium. We have identified a gene cluster designated bldK in which insertional and deletion mutations cause a block in aerial mycelium formation. Extracellular complementation experiments indicate that bldK defines a step in a cascade of extracellular signals; colonies of a bldK -mutant strain extracellularly complement bld261 -mutant colonies, and are themselves extracellularly complemented by bldA -and bldH -mutant colonies. The bldK locus, which is located at 5 o'clock on the genetic map and within Ase  I fragment 'N' on the physical map, consists of five adjacent open reading frames. These genes specify homologues of the subunits of the oligopeptide-permease family of ATP-binding cassette (ABC) membrane-spanning transporters. Because bldK mutations confer resistance to the toxic tripeptide bialaphos, it is inferred that BldK is an oligopeptide importer. We propose that the BldK transporter is responsible for the import of an extracellular signalling molecule produced under the control of the wild-type product of the bld261 gene. The BldK-imported signal, in turn, causes the production of a second extracellular signal molecule that depends on the products of bldA and bldH for its action.     

圈卷产色链霉菌分化及其特性的研究

链霉菌分化的分子生物学研究是一个饶有兴趣并富有挑战性的世界前沿研究课题.在原核生物的分化研究中,主要以枯草杆菌(Bacillus subtilis)作为模式系统,但枯草杆菌的生命周期尤其是分化过程远比链霉菌简单,不象链霉菌有基质菌丝、气生菌丝、菌丝螺旋和孢子分隔那样的发育分化过程[1].在真核生物中也有分化研究的报道,如构巢曲霉、酵母等.由于真核生物的基因结构比原核生物复杂得多,弄清分化中基因调控的关系就更加困难.因此,选用链霉菌作分化研究的材料有其独一无二的优越性.国际上有关链霉菌的分子生物学研究,近年来主要以链霉菌的抗生素生物合成基因和链霉菌的分化基因两个大的方面作为研究的热点和主攻方向,并展开了一些开拓性的研究.     

Katanin Localization Requires Triplet Microtubules in Chlamydomonas reinhardtii

Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.     

Cloning and expression of the plasmid-encoded benzene dioxygenase genes from Pseudomonas putida ML2

Three different bld mutants from S. griseus ATCC 10137 were isolated by nitrosoguanidine mutagenesis. They simultaneously lost the capability of antibiotic production and the formation of pigments. The three bld mutants were differently affected by different carbon sources. Two of these mutants showed a high efficiency of transformation with several plasmid vectors, in contrast to the low efficiency of transformation showed by the wild type. We showed that S. griseus ATCC 10137 and the three bld mutants possess an enzymatic activity that protects their DNAs against the digestion by SacI. Antibiotic and pigment production, and low transformability with plasmid DNA were together restored in spontaneous spo+ revertants.     

Initiation of aerial mycelium formation in Streptomyces

In the past two years, the isolation of extracellular factors involved in the initiation of aerial mycelium formation, the identification of metabolic defects in certain developmental mutants, and the characterisation of three further bld genes and several gamma-butyrolactone receptor genes have led to new ideas about the mechanisms that initiate aerial mycelium formation in Streptomyces. The emerging picture suggests the integration of numerous signals from both inside and outside the cell.     

Properties of a Class of Genes Required for Ray Morphogenesis in Caenorhabditis Elegans

We have identified eight mutations that define at least five terminal differentiation genes (ram genes) whose products are required during the extension of the male-specific ray sensilla in Caenorhabditis elegans. ram gene mutations result in morphological abnormalities in the sensory rays but do not appear to interfere with ray functions. A similar ray morphology phenotype was observed in males harboring mutations in three previously defined genes, dpy-11, dpy-18 and sqt-1, that also affect body shape. One of these genes, sqt-1, is known to encode a collagen. Mutations in different ram genes failed to complement, from which we infer that their gene products functionally interact. For one ram gene, failure to complement was shown to result from haploinsufficiency. Intergenic noncomplementation did not extend to the body morphology genes. The temperature-sensitive periods of both ram and body morphology mutations corresponded to the period of development in which ray extension occurs. We propose that ram gene products act together in a critical interaction between the rays and the cuticle required for wild-type ray morphology.     

Interaction of hairless, delta, enhancer of split and notch genes of Drosophila melanogaster as expressed in adult morphology

The interaction of three neurogenic loci viz. Delta, Enhancer of split and Notch, and a related gene, Hairless, of Drosophila melanogaster was investigated at the adult morphology level by measuring the effects of the mutations of the three other genes on the expression of the recessive lethal antimorphic Abruptex mutations of the Notch locus. The Abruptex mutations were also coupled in cis or trans with facet-glossy or split mutations of the Notch locus. In some of the experiments, the genotype of the fly was homozygous for either facet-glossy or split mutation or their wild type alleles but heterozygous for the Abruptex. Facet-glossy is located in a large intron of the locus, whereas split is located in the same exon as Abruptex. In all compounds studied, Delta suppressed the expression of Abruptex while Hairless and Enhancer of split enhanced it. The interactions of the four genes studied were allele specific, suggesting an interaction at the protein level. The comparison of the results presented in this study on the interaction of the neurogenic genes with other results on the same subject suggests that the interactions are similar in embryonic and imaginal development.     

SapB and the chaplins: connections between morphogenetic proteins in Streptomyces coelicolor

Morphogenesis in the streptomycetes features the differentiation of substrate-associated vegetative hyphae into upwardly growing aerial filaments. This transition requires the activity of bld genes and the secretion of biosurfactants that reduce the surface tension at the colony-air interface enabling the emergence of nascent aerial hyphae. Streptomyces coelicolor produces two classes of surface-active molecules, SapB and the chaplins. While both molecules are important for aerial development, nothing is known about the functional redundancy or interaction of these surfactants apart from the observation that aerial hyphae formation can proceed via one of two pathways: a SapB-dependent pathway when cells are grown on rich medium and a SapB-independent pathway on poorly utilized carbon sources such as mannitol. We used mutant analysis to show that while the chaplins are important, but not required, for development on rich medium, they are essential for differentiation on MS (soy flour mannitol) medium, and the corresponding developmental defects could be suppressed by the presence of SapB. Furthermore, the chaplins are produced by conditional bld mutants during aerial hyphae formation when grown on the permissive medium, MS, suggesting that the previously uncharacterized SapB-independent pathway is chaplin dependent. In contrast, a bld mutant blocked in aerial morphogenesis on all media makes neither SapB nor chaplins. Finally, we show that a constructed null mutant that lacks all chaplin and SapB biosynthetic genes fails to differentiate in any growth condition. We propose that the biosurfactant activities of both SapB and the chaplins are essential for normal aerial hyphae formation on rich medium, while chaplin biosynthesis and secretion alone drives aerial morphogenesis on MS medium.     

The Identification of Transposon-Tagged Mutations in Essential Genes That Affect Cell Morphology in Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae reproduces by budding, and many genes are required for proper bud development. Mutations in some of these genes cause cells to die with an unusual terminal morphology--elongated or otherwise aberrantly shaped buds. To gain insight into bud development, we set out to identify novel genes that encode proteins required for proper bud morphogenesis. Previous studies screened collections of conditional mutations to identify genes required for essential functions, including bud formation. However, genes that are not susceptible to the generation of mutations that cause a conditional phenotype will not be identified in such screens. To identify a more comprehensive collection of mutants, we used transposon mutagenesis to generate a large collection of lethal disruption mutations. This collection was used to identify 209 mutants with disruptions that cause an aberrant terminal bud morphology. The disruption mutations in 33 of these mutants identify three previously uncharacterized genes as essential, and the mutant phenotypes suggest roles for their products in bud morphogenesis.     

Regulation of Streptomyces development: reach for the sky!

Streptomyces coelicolor is characterized by a complex life cycle and serves as a model system for bacterial development. After a feeding substrate mycelium has been formed, this filamentous bacterium differentiates by forming aerial hyphae that septate into spores. The bld cascade regulates initiation of aerial growth, whereas the whi genes control spore formation. Recent findings indicate the existence of another regulatory pathway that operates after aerial hyphae have started to grow into the air, which we call the sky pathway. This pathway controls the expression of the chaplin and rodlin genes. These genes encode proteins that assemble into a rodlet layer that provides surface hydrophobicity to aerial hyphae and spores.     

Characterization of X-Linked Recessive Lethal Mutations Affecting Embryonic Morphogenesis in Drosophila Melanogaster

This study attempted to assay the zygotic contribution of X chromosome genes to the genetic control of embryonic morphogenesis in Drosophila melanogaster. A systematic screen for X-linked genes which affect the morphology of the embryo was undertaken, employing the phenotype of whole mount embryos as the major screening criterion. Of 800 EMS-induced lethal mutations analyzed, only 14% were embryonic lethal, and of these only a minority affected embryonic morphogenesis. By recombination and complementation analyses, the mutations that affected embryonic morphogenesis were sequestered into 26 complementation groups. Fourteen of the loci correspond to genes previously identified in a large-scale screen in which fixed cuticles were examined, and 12 new loci have been identified. Most of the mutations which disrupt embryonic morphology had specific and uniform mutant phenotypes. Mutations were recovered which disrupt major morphogenetic events such as gastrulation, germ band retraction and head involution. No mutations were found which arrest the embryos prior to blastoderm formation. However, a novel class was found, one comprised of mutations which interfere with the development of internal structures but not cuticular structures. Nevertheless, saturation of the X chromosome for genes important for embryonic morphogenesis is probably incomplete.     

Site-specific mutagenesis in the TR-DNA region of octopine-type Ti plasmids

Site-specific insertion and deletion mutations affecting all six of the eukaryotic-like genes in the TR-DNA region of the octopine-type Ti plasmids pTil5955 or pTiA6 have been generated. None of the mutations affected virulence or tumor morphology on sunflower. Mutations in the coding regions of two of the genes resulted in tumors without any detectable mannopine, mannopinic acid or agropine, and mutations in either the coding region or in the 3′ untranslated region of a third gene eliminated biosynthesis of agropine, but not mannopine or mannopinic acid. Detection of two previously unobserved silver nitrate-positive substance in tumors incited by one of the mutant strains, together with data on the presence of opines in tumors incited by coinoculation with mixtures of different mutant strains, allowed us to propose the functional order of all three genes involved in the biosynthesis of mannopine, mannopinic acid and agropine. TR-DNA was absent in tumors incited by anAgrobacterium tumefaciens strain harboring a Ti plasmid in which the right border of the TR-DNA region was deleted.