MicrobiologyBytes

What would happen if all the leaves fell off the trees and did not rot? We’d be buried under them and all plants would run out of nutrients and die, then we would starve. So the seemingly non-sexy buisness of rotting is rather important when it comes to element and nutrient cycles.

In ecological studies, leaf litter degradation is often estimated by measuring parameters such as soil respiration, litter mass loss or the activities of specific microbial enzymes in soil extracts. In microbiology, the degradation of plant-derived compounds such as lignocellulose has been studied using a few microbial model species and has recently led to the sequencing of the genomes of different saprotrophic fungal species which use different strategies to degrade plant material, thus revealing the full enzymatic machinery implicated in this process. Under natural conditions, litter degradation is generally carried out by consortia of species that either act simultaneously or replace one another on a common piece of plant debris in a sometimes predictable manner and not by a single microbial species. It can therefore be anticipated that the molecular machinery deployed to completely mineralize litter in the field is far more complex and diverse than the machinery observed in a single microbial genome. In addition, it is likely that the diversity of this machinery is partly controlled by litter chemistry and complexity and therefore by plant community composition.

By allowing access to the genome contents of the different microorganisms present in a common environment (metagenomics) or to the set of genes they express (metatranscriptomics), environmental genomics offers a novel opportunity to decipher at the molecular level, complex ecological processes such as plant organic matter degradation, thus bridging the gap between global field measurements and targeted genomic approaches.

Metatranscriptomics Reveals the Diversity of Genes Expressed by Eukaryotes in Forest Soils. (2012) PLoS ONE 7(1): e28967. doi:10.1371/journal.pone.0028967
Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides), sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin) and glycoside hydrolases represented 0.5% (beech soil)–0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases) were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a potentially significant contribution of non-fungal eukaryotes in the actual hydrolysis of soil organic matter.

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During the 1960s a ‘sexual revolution’ occurred in many Western human societies. In a similar fashion, mycologists now find themselves caught up in a fungal sexual revolution as previously accepted norms for fungal sex are being overturned. In particular need of reappraisal is the view that a significant number of fungal species are restricted solely to asexual reproduction. Fungi are unusual amongst eukaryotic organisms in that at least 20% of all species lack a known sexual state and instead appear to rely solely on asexual methods of reproduction. This is very surprising because a sexual phase in the life cycle, even if only intermittent, has several evolutionary advantages over purely asexual reproduction. In the specific case of fungi, sexual reproduction also confers the ability to produce dormant survival structures and a transient ‘capacitor’ diploid state that might allow selection of evolutionarily favourable sets of genes in a fitness ‘selection arena’.

However, recent experimental and genomic discoveries are now challenging the supposed asexual status of mitosporic fungi. Instead it is becoming apparent that many species have a previously unidentified ‘cryptic’ or ‘covert’ sexual state and are ‘holding back the truth’ about their sexuality. This review focuses on lessons being learnt from investigations involving Aspergillus and Penicillium species that are helping pave the way to fungal sexual realisation.

A fungal sexual revolution: Aspergillus and Penicillium show the way. Curr Opin Microbiol. Oct 25 2011
Fungi have some of the most diverse sex lives in nature, ranging from self-fertility to obligate outcrossing systems with several thousand different sexes, although at least 20% of fungal species have no known sexual stage. However, recent evidence suggests that many supposed ‘asexual’ species do indeed have the potential to undergo sexual reproduction. Using experimental and genomic findings from Aspergillus and Penicillium species as examples, it is argued that evidence such as the presence and expression of apparently functional sex-related genes, the distribution of mating-type genes, detection of recombination from population genetic analyses, and the discovery of extant sexual cycles reveal an on-going revolution in the understanding of fungal asexuality.

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The fungus Candida albicans is often a benign member of the mucosal flora; however, it commonly causes mucosal disease with substantial morbidity and in vulnerable patients it causes life-threatening bloodstream infections. A striking feature of its biology is its ability to grow in yeast, pseudohyphal and hyphal forms. The hyphal form has an important role in causing disease by invading epithelial cells and causing tissue damage. This review describes our current understanding of the network of signal transduction pathways that monitors environmental cues to activate a programme of hypha-specific gene transcription, and the molecular processes that drive the highly polarized growth of hyphae.

Growth of Candida albicans hyphae. (October 2011) Nature Reviews Microbiology 9: 737-748 doi:10.1038/nrmicro2636

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One of the great advances in medical technology has unwittingly spawned a serious threat to public health. Implanted medical devices, from cardiac stents to artificial hip joints, are commonly infected with biofilms, complex microbial communities that can prove remarkably resistant to host defenses and treatment. It appears, however, that biofilms, even those arising from the same microbe species, may harbor innate differences in their response to treatments like antifungal agents. Understanding how these microbe colonies might produce structures that appear similar but have very different physical properties and functions is a critical step in figuring out how to overcome antimicrobial resistance.

In humans, Candida albicans can cause problems like oral thrush and yeast infections. Far more serious is its increasing tendency to colonize catheters, heart valves, and other medical devices, where it serves as a seeding source for potentially deadly bloodstream infections. The finding that C. albicans can form two different types of biofilm is interesting not only because it is an example of how similar structures can have very different functions, but also because it provides information about how signaling pathways may evolve by modifying preexisting signaling modules for entirely new purposes. This phenomenon may be widespread, extending beyond slimy fungal biofilms to a variety of organisms. On a practical note, when researchers are designing new methods to discourage fungal biofilms it will be useful for them to keep in mind that there are two types of biofilms being formed by C. albicans.

A Tale of Two Biofilms. 2011 PLoS Biol 9(8): e1001119. doi:10.1371/journal.pbio.1001119

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At the edge of a fungal colony, leading hyphae grow into new territory in search of food. Behind the colony edge, the hyphae interconnect to form a three-dimensional network that is optimized to extract nutrients from the surrounding medium in order to fuel continued exploration. Colony growth can be fast (about 10–100 μm min⁻¹, depending on the organism, nutrient availability and temperature) and involves the continuous synthesis of all the cellular constituents that are necessary for rapid cell expansion. A major driving force for cell expansion is pressure.

Pressure is a thermodynamic state property that affects the life of all organisms. In cells that lack a cell wall, excessive pressure can result in cell lysis and death. In cells that do have a wall (most bacteria, algae, fungi and plants), an internal hydrostatic pressure (turgor) provides both mechanical support for free-standing structures and a force that drives cellular expansion, substrate penetration and other processes. Extreme examples from fungi are the projectile release of spores at >100,000 × g (g is the acceleration due to gravity at the Earth’s surface) in ascomycetes and zygomycetes.

This review describes the roles of turgor and pressure in fungal growth. How is turgor regulated? How does it affect tip growth? Do intra-hyphal pressure gradients play a part in fungal growth (such as in the transport of new materials to the growing tip)? These areas of active research are revealing the mechanisms of hyphal growth in filamentous fungi and are relevant to applied research on pathogenicity and the control of fungal diseases.

How does a hypha grow? The biophysics of pressurized growth in fungi. (2011) Nature Reviews Microbiology 9, 509-518 doi:10.1038/nrmicro2591
The mechanisms underlying the growth of fungal hyphae are rooted in the physical property of cell pressure. Internal hydrostatic pressure (turgor) is one of the major forces driving the localized expansion at the hyphal tip which causes the characteristic filamentous shape of the hypha. Calcium gradients regulate tip growth, and secretory vesicles that contribute to this process are actively transported to the growing tip by molecular motors that move along cytoskeletal structures. Turgor is controlled by an osmotic mitogen-activated protein kinase cascade that causes de novo synthesis of osmolytes and uptake of ions from the external medium. However, as discussed in this Review, turgor and pressure have additional roles in hyphal growth, such as causing the mass flow of cytoplasm from the basal mycelial network towards the expanding hyphal tips at the colony edge.

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The fungal kingdom is vast, spanning 1.5 to as many as 5 million species diverse as unicellular yeasts, filamentous fungi, mushrooms, lichens, and both plant and animal pathogens. The fungi are closely aligned with animals in one of the six to eight supergroups of eukaryotes, the opisthokonts. The animal and fungal kingdoms last shared a common ancestor 1 billion years ago, more recently than other groups of eukaryotes. As a consequence of their close evolutionary history and shared cellular machinery with metazoans, fungi are exceptional models for mammalian biology, but prove more difficult to treat in infected animals. The last common ancestor to the fungal/metazoan lineages is thought to have been unicellular, aquatic, and motile with a posterior flagellum, and certain extant species closely resemble this hypothesized ancestor. Species within the fungal kingdom were traditionally assigned to four phyla, including the basal fungi (Chytridiomycota, Zygomycota) and the more recently derived monophyletic lineage, the dikarya (Ascomycota, Basidiomycota). The fungal tree of life project has revealed that the basal lineages are polyphyletic, and thus there are as many as eight to ten fungal phyla. Fungi that infect vertebrates are found in all of the major lineages, and virulence arose multiple times independently. A sobering recent development involves the species Batrachochytrium dendrobatidis from the basal fungal phylum, the Chytridiomycota, which has emerged to cause global amphibian declines and extinctions. Genomics is revolutionizing our view of the fungal kingdom, and genome sequences for zygomycete pathogens (Rhizopus, Mucor), skin-associated fungi (dermatophytes, Malassezia), and the Candida pathogenic species clade promise to provide insights into the origins of virulence. Here we survey the diversity of fungal pathogens and illustrate key principles revealed by genomics involving sexual reproduction and sex determination, loss of conserved pathways in derived fungal lineages that are retained in basal fungi, and shared and divergent virulence strategies of successful human pathogens, including dimorphic and trimorphic transitions in form. The overarching conclusion is that fungal pathogens of animals have arisen repeatedly and independently throughout the fungal tree of life, and while they share general properties, there are also unique features to the virulence strategies of each successful microbial pathogen.

Microbial pathogens in the fungal kingdom. (2011) Fungal Biology Reviews 25(1): 48-60

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As the number of immunocompromised individuals grows, fungal pathogens are becoming ever more important. In this article in Microbiology Today (pdf) Ken Haynes discusses how functional genomics technologies are helping to combat these less than well known eukaryotic adversaries:

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Biologists debated for more than 200 years about which organisms should be counted as fungi. In less than 5 years, DNA sequencing provided a multitude of new characters for analysis and identified about 10 phyla as members of the monophyletic kingdom Fungi. Mycologists benefited from early developments applied directly to fungi. The “universal primers,” so popular in the early 1990s for the polymerase chain reaction (PCR), actually were designed for fungi. Use of the PCR was a monumental advance for those who studied minute, often unculturable, organisms.

Fungi interact with all major groups of organisms. By their descent from an ancestor shared with animals about a billion years ago plus or minus 500 million years, the fungi constitute a major eukaryotic lineage equal in numbers to animals and exceeding plants. But how many fungal species are there?

The Fungi: 1, 2, 3 … 5.1 million species? American Journal of Botany, March 2 2011 doi: 10.3732/ajb.1000298
Premise of the study: Fungi are major decomposers in certain ecosystems and essential associates of many organisms. They provide enzymes and drugs and serve as experimental organisms. In 1991, a landmark paper estimated that there are 1.5 million fungi on the Earth. Because only 70000 fungi had been described at that time, the estimate has been the impetus to search for previously unknown fungi. Fungal habitats include soil, water, and organisms that may harbor large numbers of understudied fungi, estimated to outnumber plants by at least 6 to 1. More recent estimates based on high-throughput sequencing methods suggest that as many as 5.1 million fungal species exist.
Methods: Technological advances make it possible to apply molecular methods to develop a stable classification and to discover and identify fungal taxa.
Key results: Molecular methods have dramatically increased our knowledge of Fungi in less than 20 years, revealing a monophyletic kingdom and increased diversity among early-diverging lineages. Mycologists are making significant advances in species discovery, but many fungi remain to be discovered.
Conclusions: Fungi are essential to the survival of many groups of organisms with which they form associations. They also attract attention as predators of invertebrate animals, pathogens of potatoes and rice and humans and bats, killers of frogs and crayfish, producers of secondary metabolites to lower cholesterol, and subjects of prize-winning research. Molecular tools in use and under development can be used to discover the world’s unknown fungi in less than 1000 years predicted at current new species acquisition rates.

Related:

• There’s an awful lot of zombie ant fungi in Brazil
• The Great Indoors
• New Strain of Virulent Airborne Fungus Looks Set to Spread

Four new fungi in the genus Ophiocordyceps have been identified. These fungi belong to a group of “zombifying” fungi that infect ants and then manipulate their behavior, eventually killing the ants after securing a prime location for spore dispersal.

Beyond this important milestone, the paper also draws attention to undiscovered, complex, biological interactions in threatened habitats. The four new species all come from the Atlantic Rainforest of Brazil which is the most heavily degraded biodiversity hotspot on the planet. Ninety-two percent of its original coverage is gone. The effect of biodiversity loss on community structure is well known. What researchers don’t know is how parasites, such as these zombie-inducing fungi, cope with fragmentation. The authors show that each of the four species is highly specialized on one ant species and has a suite of adaptations and spore types to ensure infection. The life-cycle of these fungi that infect, manipulate and kill ants before growing spore producing stalks from their heads is remarkably complicated. The present work establishes the identification tools to move forward and ask how forest fragmentation affects such disease dynamics.

Hidden Diversity Behind the Zombie-Ant Fungus Ophiocordyceps unilateralis: Four New Species Described from Carpenter Ants in Minas Gerais, Brazil. (2011) PLoS ONE 6(3): e17024. doi:10.1371/journal.pone.0017024
Background: Ophiocordyceps unilateralis (Clavicipitaceae: Hypocreales) is a fungal pathogen specific to ants of the tribe Camponotini (Formicinae: Formicidae) with a pantropical distribution. This so-called zombie or brain-manipulating fungus alters the behaviour of the ant host, causing it to die in an exposed position, typically clinging onto and biting into the adaxial surface of shrub leaves. We (HCE and DPH) are currently undertaking a worldwide survey to assess the taxonomy and ecology of this highly variable species.
Methods: We formally describe and name four new species belonging to the O. unilateralis species complex collected from remnant Atlantic rainforest in the south-eastern region (Zona da Mata) of the State of Minas Gerais, Brazil. Fully illustrated descriptions of both the asexual (anamorph) and sexual (teleomorph) stages are provided for each species. The new names are registered in Index Fungorum (registration.indexfungorum.org) and have received IF numbers. This paper is also a test case for the electronic publication of new names in mycology.
Conclusions: We are only just beginning to understand the taxonomy and ecology of the Ophiocordyceps unilateralis species complex associated with carpenter ants; macroscopically characterised by a single stalk arising from the dorsal neck region of the ant host on which the anamorph occupies the terminal region and the teleomorph occurs as lateral cushions or plates. Each of the four ant species collected – Camponotus rufipes, C. balzani, C. melanoticus and C. novogranadensis – is attacked by a distinct species of Ophiocordyceps readily separated using traditional micromorphology. The new taxa are named according to their ant host.