Effects of increased and deregulated expression of cell division genes on the morphology and on antibiotic production of streptomycetes

This paper describes the effects of increased expression of the cell division genes ftsZ, ftsQ, and ssgA on the development of both solid- and liquid-grown mycelium of Streptomyces coelicolor and Streptomyces lividans. Over-expression of ftsZ in S. coelicolor M145 inhibited aerial mycelium formation and blocked sporulation. Such deficient sporulation was also observed for the ftsZ mutant. Over-expression of ftsZ also inhibited morphological differentiation in S. lividans 1326, although aerial mycelium formation was less reduced. Furthermore, antibiotic production was increased in both strains, and in particular the otherwise dormant actinorhodin biosynthesis cluster of S. lividans was activated in liquid- and solid-grown cultures. No significant alterations were observed when the gene dosage of ftsQ was increased. Analysis by transmission electron microscopy of an S. coelicolor strain over-expressing ssgA showed that septum formation had strongly increased in comparison to wild-type S. coelicolor, showing that SsgA clearly influences Streptomyces cell division. The morphology of the hyphae was affected such that irregular septa were produced with a significantly wider diameter, thereby forming spore-like compartments. This suggests that ssgA can induce a process similar to submerged sporulation in Streptomyces strains that otherwise fail to do so. A working model is proposed for the regulation of septum formation and of submerged sporulation.     

Genetic control of fungal differentiation: The three sporulation pathways of Neurospora crassa

Sporulation in the mold Neurospora crussa can proceed along three very different pathways, leading to the production of three types of spores. Two asexual sporulation pathways that lead to the formation of macroconidia and microconidia involve budding from hyphae by two different mechanisms. A much more complex sexual reproductive pathway involves the formation of a fruiting body called a perithecium, in which meiosis takes place and ascospores are formed in sac-like cells called asci. Numerous mutations exist that affect these developmental pathways, and genes have been isolated that are expressed preferentially during sporulation. The Neurospora sporulation pathways offer a simple system with which to study mechanisms and regulation of development that are usually obscured by complex cell-cell interactions involved in animal and plant development.     

Endospore formation in Hanseniaspora pseudoguilliermondii: a key characteristics of the species

In the course of a study on yeast diversity in Japan and Thailand, we isolated two yeast strains with bipolar budding patterns. Physiological and phylogenetic analysis suggested that these two strains were identical to Hanseniaspora pseudoguilliermondii. However, these strains produced hat-shaped ascospores and endospores, the latter of which was an unknown characteristic of the species. Endospores were produced on yeast extract–malt extract (YM) plates, though ascospores were produced on cornmeal agar of H. pseudoguilliermondii cultures. Endospores were formed in a twin-cell structure composed of a mother cell and a daughter cell, which did not separate after budding. Unlike the cell wall of the endospores, that of ascospore was stained with a chitin-specific stain. This was a feature distinguishing endospores and ascospores. Cell morphology of H. pseudoguilliermondii was compared with other species of the genus by observing their type strains. Other Hanseniaspora species did not show endospore formation under the same condition in which H. pseudoguilliermondii did. Therefore, the formation of endospores was considered to be a species-delimiting character of H. pseudoguilliermondii.     

蛹虫草子囊孢子萌发及其后代群体培养性状观察

从野生和人工栽培的蛹虫草子实体共分离了6个子囊孢子群体,对子囊孢子形态、萌发过程、培养性状及子实体产生等进行了观察。结果表明,蛹虫草子囊孢子为线形、多细胞,169.78~364.57×1.72~2.04μm;每个子囊孢子有56~114个细胞,每个细胞大小为1.77~4.53×1.72~2.04μm。子囊孢子弹射后13h开始萌发,30h后开始形成分生孢子。多数子囊孢子的每个细胞都能萌发,但少数子囊孢子的部分细胞不萌发。子囊孢子群体的培养性状表现多样性,以I型所占比列较大。野生群体的菌落颜色以杏橙色和淡柠檬黄色为主,而人工栽培的群体以橙铬色所占比例最大。不同个体的菌落生长速率有差异。野生群体产生扇形突变的比例高于人工栽培群体。在人工栽培的CM群体中出现了气生菌丝较多、白色、生长缓慢的个体。菌落为I型,橙铬色或杏橙色,生长速率正常,无突变的单子囊孢子菌株产生子实体的可能性较大,子实体产量较高,但大部分单子囊孢子菌株不能产生发育良好的子实体。     

The fine structure of ascospore shape and development inCeratocystis fimbriata

Ascospore development inCeratocystis fimbriata Ell. & Halst. commenced in an eight-nucleate ascus. A single vesicle formed along the periphery of the ascus from fragments of ascospore delimiting membranes, surrounded all eight nuclei and eventually invaginated, first forming pouches with open ends, then finally enclosing each of the eight nuclei in a separate sac, thus delimiting ascospores. Pairing of the ascospores followed and brim formation occurred at the contact area between two ascospores. Osmiophilic bodies contributed to the formation of brim-like appendages by fusing to the ascospore walls. Additional brims were observed at opposite ends of the ascospores giving them a double-brimmed appearance.Abbreviations AV ascus vesicle - DM delimiting membrane - EV electron translucent bodies - G granules - M mitochondria - N nucleus - OB osmiophilic bodies - PMV plasmamembrane vesicles - PW primary wall - SW secondary wall     

bgs2+, a sporulation-specific glucan synthase homologue is required for proper ascospore wall maturation in fission yeast

The formation of the ascospore cell wall of Schizosaccharomyces pombe requires the co-ordinated activity of enzymes involved in the biosynthesis of its components, such as glucans. We have cloned the bgs2+ gene. bgs2+ belongs to the glucan synthase family of S. pombe and is homologous to the Saccharomyces cerevisiae FKS1 and FKS2 genes. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth, but homozygous bgs2Delta diploids do show a sporulation defect. Although meiosis takes place normally, ascospores are unable to mature, and their wall differs from that of wild-type ascospores. Moreover, bgs2Delta zygotes were not able to release ascospores spontaneously, and the ascospores were unable to germinate. We show that expression of bgs2+ is restricted to sporulation and that a bgs2-green fluorescent protein (GFP) fusion protein localizes to the ascospore envelope. The glucan synthase activity in sporulating diploids bearing a bgs2 deletion was diminished in comparison with that of the wild-type diploids, a fact that underscores the importance of the bgs2+ gene and glucan synthesis for the proper formation and maturation of the ascospore wall.     

Manipulation of Major Membrane Lipid Synthesis and Its Effects on Sporulation in Saccharomyces cerevisiae

During the sporulation process of Saccharomyces cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether certain species of lipids are required for spore formation in yeast. In this study, we analyzed the requirement of the synthesis of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and ergosterol for spore formation using strains in which the synthesis of these lipids can be controlled. When synthesis of PE and PC was repressed, sporulation efficiency decreased. This suggests that synthesis of these phospholipids is vital to proper sporulation. In addition, sporulation was also impaired in cells with a lowered sterol content, raising the possibility that sterol content is also important for spore formation.     

Sporulation in saprotrophic and clinical strains of <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> (Bain. &; Sart.) Thom &; Church under various environmental conditions

Molecular genetic characteristics and capacity for sporulation under different levels of temperature and humidity were compared for three saprotrophic and four clinical strains of A. sydowii. Analysis of the ITS and D1/D2 loci of these A. sydowii strains revealed two clades, each including both the clinical and saprotrophic strains. The differences in sporulation in the saprotrophic and clinical strains of the potentially pathogenic microscopic fungus A. sydowii under different environmental conditions were demonstrated. In the clinical A. sydowii strains, the level of spore formation was generally higher, especially at humidity levels of 0.90 and 0.95 a_w and 20–25°C. The level of spore formation for the clinical strains inoculated into sterile soil was several times higher than for the saprotrophic ones. On the contrary, nonsterile soils (sod-podzolic and urban soils) exhibited a fungistatic effect against A. sydowii populations.     

A Demonstration of the Role of het Genes in Heterokaryon Formation in Neurospora under Simulated Field Conditions

An experimental system was developed for assessing the role of het genes in heterokaryon formation in Neurospora in nature. Burned sugar cane segments planted in soil were infected using a mixture of mutant ascospores of two genotypes. Neurospora ramified in the cane and erupted as distinct pustules of conidia. When ascospores carried identical het alleles, the (macro) conidial pustules which formed were heterokaryotic. On the other hand, when ascospores carried dissimilar het alleles, the pustules were homokaryotic. These results showed that stable heterokaryons between compatible strains can form in nature. When two strains are growing together on a natural substrate, heterozygosity at het loci serves to maintain their individuality.     

A study of copulation,sporulation and meiotic segregation in Candida lipolytica

Summary We have attempted to optimize conjugation and sporulation in Candida lipolytica, by studying the conditions of culture and growth.Copulation between compatible strains is a rare event, particularly in the case of auxotrophic mutants. However, diploids can be selected for on minimal medium provided parents are suitable auxotrophs. These diploids can multiply vegetatively for many generations. They can also be induced to sporulate at a very high frequency.Free ascospores were isolated by means of paraffin oil and segregations of markers could be studied. At first quite irregular, these segregations improved following a number of brother-sister matings. At the same time, the mean number of spores per ascus as well as spore germinability were considerably increased.     

Initiation of solvent production,clostridial stage and endospore formation in Clostridium acetobutylicum P262

Summary A minimal medium was used to investigate the triggers regulating the initiation of solvent production and differentiation in Clostridium acetobutylicum P262. The accumulation of acid end-products caused the inhibition of cell division and the initiation of solvent production and cell differentiation. Initiation only occurred with a narrow pH range. Glucose or ammonium limited cultures failed to achieve the necessary threshold of acid end-products and solvent production and differentiation were not initiated. The addition of acid end-products or ammonium to cultures containing suboptimal levels of glucose or nitrogen respectively, enhanced solvent production. Resuspension of cells in media containing the threshold level of acid end-products and residual glucose induced endospore formation. Glucose or ammonium limitation did not induce sporulation and there was a requirement for glucose and ammonium during solventogenesis and endospore formation. Initiation of solvent production and clostridial stage formation were essential for sporulation. The induction of endospore formation in C. acetobutylicum P262 differs from that in the aerobic endospore forming bacteria where sporulation is initiated by nutrient starvation.     

冬虫夏草不同部位来源的菌株及多次传代固体发酵特性

从西藏新鲜冬虫夏草不同部位分离菌株,并进行固体培养,比较菌丝生长、分生孢子产生情况及不同传代次数菌株的固体培养特性,以获得产生分生孢子数量最多的菌株用于蝙蝠蛾幼虫的侵染。结果表明：单子囊孢子、双子囊孢子和多子囊孢子菌株在斜面上出草能力较好,固体发酵时菌丝生长旺盛,分生孢子产生较多,显著高于组织分离菌株;组织分离菌株中,子座来源的菌株在谷壳和泥炭土培养基中,其产孢能力高于虫体来源的菌株;多次传代（5次传代）菌株在固体斜面上有较明显的退化现象,但在液体及固体发酵方面有着较好的生长以及分生孢子产量。     

Carbohydrate Metabolism During Ascospore Development in Yeast

Carbohydrate metabolism, under sporulation conditions, was compared in sporulating and non-sporulating diploids of Saccharomyces cerevisiae. Total carbohydrate was fractionated into trehalose, glycogen, mannan, and an alkali-insoluble fraction composed of glucan and insoluble glycogen. The behavior of three fractions was essentially the same in both sporulating and non-sporulating strains; trehalose, mannan, and the insoluble fraction were all synthesized to about the same extent regardless of a strain's ability to undergo meiosis or sporulation. In contrast, aspects of soluble glycogen metabolism depended on sporulation. Although glycogen synthesis took place in both sporulating and non-sporulating strains, only sporulating strains exhibited a period of glycogen degradation, which coincided with the final maturation of ascospores. We also determined the carbohydrate composition of spores isolated from mature asci. Spores contained all components present in vegetative cells, but in different proportions. In cells, the most abundant carbohydrate was mannan, followed by glycogen, then trehalose, and finally the alkali-insoluble fraction; in spores, trehalose was most abundant, followed by the alkali-insoluble fraction, glycogen, and mannan in that order.     

Development of breeding stocks of the yeast Saccharomycopsis lipolytica by methods of moderate inbreeding

Sporulation parameters of genetically labelled strains, derived from a wild strain of the alkane-utilizing yeast Saccharomycopsis lipolytica were improved by a breeding program using brother-sister crosses. Sporulation frequency, the number of four-spored asci and viability of ascospores could be significantly enhanced. To date a number of genetically well-defined strains is available that have good sporulation parameters and show a 1:1 segregation pattern of markers suitable for genetic analysis.     

iTRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.     

Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation‐specific genes

Three classes of low‐G+C Gram‐positive bacteria (Firmicutes), Bacilli, Clostridia and Negativicutes, include numerous members that are capable of producing heat‐resistant endospores. Spore‐forming firmicutes include many environmentally important organisms, such as insect pathogens and cellulose‐degrading industrial strains, as well as human pathogens responsible for such diseases as anthrax, botulism, gas gangrene and tetanus. In the best‐studied model organism Bacillus subtilis, sporulation involves over 500 genes, many of which are conserved among other bacilli and clostridia. This work aimed to define the genomic requirements for sporulation through an analysis of the presence of sporulation genes in various firmicutes, including those with smaller genomes than B. subtilis. Cultivable spore‐formers were found to have genomes larger than 2300 kb and encompass over 2150 protein‐coding genes of which 60 are orthologues of genes that are apparently essential for sporulation in B. subtilis. Clostridial spore‐formers lack, among others, spoIIB, sda, spoVID and safA genes and have non‐orthologous displacements of spoIIQ and spoIVFA, suggesting substantial differences between bacilli and clostridia in the engulfment and spore coat formation steps. Many B. subtilis sporulation genes, particularly those encoding small acid‐soluble spore proteins and spore coat proteins, were found only in the family Bacillaceae, or even in a subset of Bacillus spp. Phylogenetic profiles of sporulation genes, compiled in this work, confirm the presence of a common sporulation gene core, but also illuminate the diversity of the sporulation processes within various lineages. These profiles should help further experimental studies of uncharacterized widespread sporulation genes, which would ultimately allow delineation of the minimal set(s) of sporulation‐specific genes in Bacilli and Clostridia.     

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae.

Summary Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34° C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34° C.     

Dependence of inessential late gene expression on early meiotic events in Saccharomyces cerevisiae

Summary SPR3 is one of at least nine genes which are expressed in sporulating Saccharomyces cerevisiae cells at the time of meiosis I. We show below that strains homozygous for null alleles of SPR3 are capable of normal meiosis and the production of viable ascospores. We have also monitored SPR3 expression in a series of strains that are defective in meiotic development, using an SPR3: lacZ fusion carried on a single copy plasmid. -Galactosidase activity occurred at wild-type levels in diploid strains homozygous for mutations in spo13, rad50, rad57 and cdc9, but was greatly reduced in strains carrying cdc8 or spo7 defects. We conclude that SPR3 expression is a valid monitor of early meiotic development, even though the gene is inessential for the sporulation process.     

Timing of mitochondrial DNA synthesis during meiosis in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) synthesis was examined during meiosis in Saccharomyces cerevisiae using an aneuploid strain disomic (n + 1) for chromosome III. The aneuploid has the advantage over true diploid strains in that it completes early meiotic events, including premeiotic chromosome replication, but does not form mature ascospores. Thus, differential extraction problems, resulting from the simultaneous presence of both unsporulated cells and spores in the population, are eliminated. The kinetic of mtDNA synthesis was monitored by determining the actual mtDNA content of cells following analytical CsCl centrifugation of cell extracts. MtDNA synthesis started soon after the cells were placed in sporulation medium and continued at an approximately constant rate until 24 h, resulting in slightly more than a doubling of the mitochondrial DNA content per cell. [¹⁴C]uracil was incorporated into mtDNA during the entire developmental period. Extensive preferential labeling of mtDNA occurred between 24 and 50 h, when no net DNA synthesis was observed.