A Novel Gene, Le-Dd 10, Is Involved in Fruiting Body Formation of Lentinula Edodes

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened by using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd 1~21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.

Social selection within aggregative multicellular development drives morphological evolution

Aggregative multicellular development is a social process involving complex forms of cooperation among unicellular organisms. In some aggregative systems, development culminates in the construction of spore-packed fruiting bodies and often unfolds within genetically and behaviourally diverse conspecific cellular environments. Here, we use the bacterium Myxococcus xanthus to test whether the character of the cellular environment during aggregative development shapes its morphological evolution. We manipulated the cellular composition of Myxococcus development in an experiment in which evolving populations initiated from a single ancestor repeatedly co-developed with one of several non-evolving partners—a cooperator, three cheaters and three antagonists. Fruiting body morphology was found to diversify not only as a function of partner genotype but more broadly as a function of partner social character, with antagonistic partners selecting for greater fruiting body formation than cheaters or the cooperator. Yet even small degrees of genetic divergence between distinct cheater partners sufficed to drive treatment-level morphological divergence. Co-developmental partners also determined the magnitude and dynamics of stochastic morphological diversification and subsequent convergence. In summary, we find that even just a few genetic differences affecting developmental and social features can greatly impact morphological evolution of multicellular bodies and experimentally demonstrate that microbial warfare can promote cooperation.

Imaging Promoter Assay of Adenylyl Cyclase A Gene in Dictyostelium discoideum during Fruiting Body Formation by Dual-Color Bioluminescence Microscopy

Cyclic adenosine monophosphate (cAMP), which is derived from adenosine triphosphate through adenylyl cyclase A (acaA), acts as an intracellular secondary messenger and an extracellular chemotactic substance in important biological processes. In the social amoebae Dictyostelium discoideum, cAMP mediates cell aggregation, development, and differentiation to spore and stalk cells during fruiting body formation. The acaA gene is transcribed under the control of three different alternative promoters. This study aimed to develop a promoter assay for acaA in D. discoideum using bioluminescence microscopy. Here, we inserted green- and red-emitting luciferase genes into downstream of promoter regions 1 and 3, respectively. Promoter activities were visualized by bioluminescence microscopy. We confirmed the differential expression of acaA under the control of promoters 1 and 3 at the different stages of D. discoideum development. We also demonstrated the application of dual-color bioluminescence imaging in the development of an imaging promoter assay.

Mg, K-containing microparticle: A possible active principle of a culture extract produced by a microbial consortium

A synthetic microbial consortium called Effective Microorganisms (EM) consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects of EM∙XGOLD, a health drink produced by EM, on life cycle of Dictyostelium discoideum were described previously. Here, we report our attempt to identify the active principle, termed EMF, that brought about the observed effects. Throughout the purification processes, the presence of the active principle was monitored by promoted fruiting body formation. By liquid-liquid separation the activity was recovered in aqueous phase, which, after concentration, was further subjected to reverse-phase column chromatography. No activity was detected in any eluant, while almost all the activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures to the residual fraction did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, the residue revealed spherical particles of μm size range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence, on the surface of the particles, of magnesium and, to a certain extent, of potassium. In separate experiments, acid treatment and alkali neutralization of EM∙XGOLD completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticles to be an active principle of EM culture extract. How these particles exert their effect is currently under intensive investigation.

The Predatory Myxobacterium Citreicoccus inhibens gen. nov. sp. nov. Showed Antifungal Activity and Bacteriolytic Property against Phytopathogens

The application and promotion of biological control agents are limited because of poor efficacy and unstable performance in the field. Screening microorganisms with high antagonistic activity, effective adaptability, and high field-survival should be prospected. Myxobacteria are soil predatory bacteria with wide adaptability, which are considered as good antagonists. Here, we report a myxobacterium strain M34 isolated from subtropical forest soil in South China using the Escherichia coli baiting method. Based on the morphological observation, physiological test, biochemical characteristics, 16S rRNA gene sequence, and genomic data, strain M34 was identified as a novel genus and novel species, representing a new clade of Myxococcaceae, for which the name Citreicoccus inhibens gen. nov. sp. nov. is proposed (type strain M34T = GDMCC 1.2275T = KCTC 82453T). The typical features of M34, including fruiting body formation and extracellular fibrillar interconnection, indicated by microscopic observations, contributed to cell adaption in different environments. Furthermore, the strain showed antifungal activity against phytopathogenic fungi and predatory activity to both Gram-negative and Gram-positive phytopathogenic bacteria. The bioprotective mechanisms are attributed to the presence of pyrrolnitrin and derivative with antifungal activity and the extracellular proteins with lytic activity against pathogenic bacteria. Due to its multiple beneficial traits, strain M34 has the potential to be developed into a versatile biocontrol agent for the management of both fungal and bacterial phytopathogens.

Widespread density dependence of bacterial growth under acid stress

Benefits of cooperation intrinsically depend on density because biological interaction requires organismal proximity. Microbial cooperative traits are common, yet systematic tests for a shared cooperative phenotype across diverse species are rare, as are direct tests for the Allee effect - positive density dependence of fitness. Here we test for positive density dependence of growth under acid stress in five phylogenetically widespread bacterial species - three Gram-negative and two Gram-positive - and find the Allee effect in all five. However, social protection from acid stress appears to have evolved by different mechanisms across species. In Myxococcus xanthus, the acid-stress Allee effect is found to be mediated by pH-regulated secretion of a diffusible molecule present in supernatants of high-density cultures. In contrast, growth from low density under acid stress by the other species was not enhanced by high-density supernatant. Additionally, density dependence of Myxococcus fruiting-body formation during starvation is found to increase with acid stress, suggesting that abiotic stresses other than starvation shape the evolution of aggregative development. Our findings suggest that high cell density may protect against acid stress in most bacterial species and in Myxococcus may promote predation on microbes that acidify their local environment by secretion of metabolic byproducts.

Genomes of novel Myxococcota reveal severely curtailed machineries for predation and cellular differentiation

Cultured Myxococcota are predominantly aerobic soil inhabitants, characterized by their highly coordinated predation and cellular differentiation capacities. Little is currently known regarding yet-uncultured Myxococcota from anaerobic, non-soil habitats. We analyzed genomes representing one novel order (o__JAFGXQ01) and one novel family (f__JAFGIB01) in the Myxococcota from an anoxic freshwater spring (Zodletone spring) in Oklahoma, USA. Compared to their soil counterparts, anaerobic Myxococcota possess smaller genomes, and a smaller number of genes encoding biosynthetic gene clusters (BGCs), peptidases, one- and two-component signal transduction systems, and transcriptional regulators. Detailed analysis of thirteen distinct pathways/processes crucial to predation and cellular differentiation revealed severely curtailed machineries, with the notable absence of homologs for key transcription factors (e.g. FruA and MrpC), outer membrane exchange receptor (TraA), and the majority of sporulation-specific and A-motility-specific genes. Further, machine-learning approaches based on a set of 634 genes informative of social lifestyle predicted a non-social behavior for Zodletone Myxococcota. Metabolically, Zodletone Myxococcota genomes lacked aerobic respiratory capacities, but encoded genes suggestive of fermentation, dissimilatory nitrite reduction, and dissimilatory sulfate-reduction (in f_JAFGIB01) for energy acquisition. We propose that predation and cellular differentiation represent a niche adaptation strategy that evolved circa 500 Mya in response to the rise of soil as a distinct habitat on earth. Importance The Myxococcota is a phylogenetically coherent bacterial lineage that exhibits unique social traits. Cultured Myxococcota are predominantly aerobic soil-dwelling microorganisms that are capable of predation and fruiting body formation. However, multiple yet-uncultured lineages within the Myxococcota have been encountered in a wide range of non-soil, predominantly anaerobic habitats; and the metabolic capabilities, physiological preferences, and capacity of social behavior of such lineages remain unclear. Here, we analyzed genomes recovered from a metagenomic analysis of an anoxic freshwater spring in Oklahoma, USA that represent novel, yet-uncultured, orders and families in the Myxococcota. The genomes appear to lack the characteristic hallmarks for social behavior encountered in Myxococcota genomes, and displayed a significantly smaller genome size and a smaller number of genes encoding biosynthetic gene clusters, peptidases, signal transduction systems, and transcriptional regulators. Such perceived lack of social capacity was confirmed through detailed comparative genomic analysis of thirteen pathways associated with Myxococcota social behavior, as well as the implementation of machine learning approaches to predict social behavior based on genome composition. Metabolically, these novel Myxococcota are predicted to be strict anaerobes, utilizing fermentation, nitrate reduction, and dissimilarity sulfate reduction for energy acquisition. Our results highlight the broad patterns of metabolic diversity within the yet-uncultured Myxococcota and suggest that the evolution of predation and fruiting body formation in the Myxococcota has occurred in response to soil formation as a distinct habitat on earth.

Functional characterization of the developmental genes asm2, asm3, and spt3 required for fruiting body formation in the filamentous ascomycete Sordaria macrospora

The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

COMBINATION OF SAWDUST, “FILTER CAKE” AND CALCIUM CARBONATE AS GROWTH MEDIUM FOR THE PRODUCTION OF WHITE OYSTER MUSHROOM (PLEUROTUS OSTREATUS)

This study was aimed to investigate the effect of combining sawdust (SD), filter cake (FC) and calcium carbonate as growth medium for the production of white oyster mushroom. Isolate F2 oyster mushroom was cultured on malt extract agar (MEA) and used in the experiment. The culture medium consisted of two treatments: first, A treatment: combined medium (A0=100% SD; A1=100% FC; A2=70% FC and 30% SD; A3=50% FC and 50% SD; A4=30% FC and 70% SD) and second, K treatment: addition of calcium carbonate (K1=2%; K2=3%; K3=4%; K4=5% weight of medium). A Randomized design was used to analyze certain parameters, such as mycelial growth, presence of fruiting body, the number of fruiting bodies and the fresh weight of fruiting body at harvest. The results showed that the highest mycelial growth and fruiting body formation occurred on A0 treatment. However, a high number of fruiting bodies and a high fresh weight of fruiting body at harvest were obtained on A4 treatment. Interaction between the combined medium (A) and addition of calcium carbonate (K) showed that the highest mycelial growth occurred on A0K2 treatment 30 days after incubation. The composition of A4 treatment (30% FC and 70% SD) was found to be the optimal medium for the production of fruiting body. This finding shows that FC with additional nutrition could as a substitute of SD medium for cultivating white oyster mushroom.