Molecular evolution of the fungi: relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes.

Establishing the phylogeny of fungi and protists often has proved difficult owing to the simple morphologies and convergent characters in these organisms. We used DNA sequences of nuclear small-subunit ribosomal RNA genes to determine phylogenetic relationships among three major classes of organisms considered to be fungi--Basidiomycetes, Ascomycetes and Chytridiomycetes--and to assess the taxonomic position of Neocallimastix, an economically important anaerobic rumen microorganism whose classification is controversial. The Basidiomycetes and Ascomycetes, two classes of nonflagellated fungi, are the most closely related taxa. Chytridiomycetes, though bearing flagella, group with these higher fungi rather than with the protists. Neocallimastix, a eukaryote lacking mitochondria and variously classified as a protist or as a fungus, shows closest molecular affinities with the Chytridiomycete fungi in the order Spizellomycetales.     

How modern systematics relates to the rumen fungi

The zoosporic fungi comprise a polyphyletic grouping of four classes, the Plasmodiophoromycetes, Oomycetes, Hyphochytriomycetes and Chytridiomycetes. Apart from their absorptive mode of nutrition and the presence of zoospores in some stage of their life cycle, there is little these classes have in common. The zoosporic species of rumen fungi are classified in the Class Chytridiomycetes which is a monophyletic group with extreme diversity in thallus morphology, reproduction and zoospore cytology. The rumen fungi have many characteristics in common with the Spizellomycetaceae but have been given their own family, the Neocallimasticaceae. There are arguments for reducing this family to synonymy with the Spizellomycetaceae, or elevating it to a new order, but before a rational decision can be made, more rumen fungi require detailed examination, especially their zoospore ultrastructure.     

Cytochromes of aquatic fungi

The cytochrome systems of two classes of aquatic fungi, the Oomycetes and Chytridiomycetes, were studied by means of reduced-minus-oxidized difference spectra at room and at low temperature. At room temperature, all of these fungi have a c-type cytochrome with an absorption maximum at 551 mmu and a b-type cytochrome at 564 mmu. The Oomycetes have a-type cytochromes at 605 mmu, and the Chytridiomycetes have a-type cytochromes at 606 mmu (Blastocladiales) or at 609 mmu (Monoblepharidales). Additional b-type cytochromes are found at 557 mmu in the Oomycetes and at approximately 560 mmu in the Chytridiomycetes. The data obtained from spectra at low temperature are consistent with these conclusions. Thus, the difference spectra reveal variation between the cytochrome systems of these two classes of aquatic fungi.     

In vitro degradation of wheat straw by anaerobic fungi from small ruminants

Anaerobic ruminal fungi may play an active role in fibre degradation as evidenced by the production of different fibrolytic enzymes in culture filtrate. In the present study, 16 anaerobic fungal strains were isolated from ruminal and faecal samples of sheep and goats. Based on their morphological characteristics they were identified as species of Anaeromyces, Orpinomyces, Piromyces and Neocallimastix. Isolated Neocallimastix sp. from goat rumen showed a maximum activity of CMCase (47.9 mIU ml(-1)) and filter paper cellulase (48.3 mIU ml(-1)), while Anaeromyces sp. from sheep rumen showed a maximum xylanolytic activity (48.3 mIU ml(-1)). The cellobiase activity for all the isolates ranged from 178.0-182.7 mIU ml(-1). Based on the enzymatic activities, isolated Anaeromyces sp. from sheep rumen and Neocallimastix sp. from goat rumen were selected for their potential of in vitro fibre degradation. The highest in vitro digestibility of NDF (23.2%) and DM (34.4%) was shown for Neocallimastix sp. from goat rumen, as compared to the digestibility of NDF and DM in the control group of 17.5 and 25.0%, respectively.     

Characterization of an anaerobic fungus from llama faeces.

An anaerobic fungus was isolated from llama faeces. Based on its morphological characteristics, polyflagellated zoospores, extensive rhizoid system and the formation of monocentric colonies, the fungus is assigned to the genus Neocallimastix. Neocallimastix sp. L2 is able to grow on several poly-, oligo- and monosaccharides. It differs from other Neocallimastix isolates in its inability to ferment inulin. Neocallimastix sp. L2 requires CO2 for growth. In the presence of 100% CO2 in the gas phase glucose is fermented to H2, CO2, formate, acetate, lactate, succinate and ethanol (33.8, 15.4, 74.3, 69.2, 26.7, 8.2, and 28.7 mmol per 100 mmol glucose, respectively). Reduced sulphur compounds can be used as sulphur source and ammonium or amino acids as nitrogen source. The temperature range for glucose fermentation is from 37 to 42 degrees C with an optimum of around 38 degrees C. The pH range for glucose fermentation is from pH 6 to pH 8 with a broad optimum between pH 6.5 and pH 7.5. The zoospores of Neocallimastix sp. L2 contain ribosomal 'globules' and hydrogenosomes. In the kinetosomes of the zoospores spurs, scoops and skirts are visible. In both the rhizoids and the sporangia 'crystal bodies' and hydrogenosomes are present. Mitochondria were not detected in either of these life stages.     

垃圾填埋场中厌氧真菌18SrDNA的PCR扩增及鉴定

采用机械破壁法直接从来自7个不同地区的垃圾填埋场滤液样本中提取真菌DNA,应用真菌通用引物NS1和NS8扩增18SrDNA（约1800bp),多聚酶链式反应（PCR）产物的琼脂糖凝胶电泳结果表明所有的样本均得到了扩增;以PCR产物作为模板,采用厌氧真菌Chytridiomycetes科的专用引物Chyt-719和Chyt-1553进行二次PCR扩增（约857bp),该阳性扩增产物克隆和测序结果首次表明在食草动物瘤胃中存在的厌氧真菌Chytridiomycetes也存在于垃圾填埋场中,且为Neocallimastix属。     

The effects of co-cultivation with the acetogen Acetitomaculum ruminis on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix sp. strain L2

Abstract The effects of co-cultivation with the hydrogen-utilizing acetogenic bacterium Acetitomaculum ruminis on the fermentative activities of the rumen fungi Neocallimastix patriciarum or Neocallimastix sp. L2 were investigated. In both co-cultures acetate production increased, making it the predominant fermentation product, as the accumulation of lactate, formate, ethanol, H₂ and (in the case of Neocallimastix sp. L2) succinate all decreased. The effects of co-cultivation with Methanobrevibacter smithii were more pronounced. Decreased activities of lactate dehydrogenase, alcohol dehydrogenase and (in the case of Neocallimastix sp. L2) fumarate reductase accompanied the shift in fermentation product formation. The rate of glucose utilization and the fungal biomass yield were also increased in the co-culture.     

Hyaloraphidium curvatum is not a green alga, but a lower fungus; Amoebidium parasiticum is not a fungus, but a member of the DRIPs

The unicellular heterotrophic protist Hyaloraphidium is classified with a family of green algae, the Ankistrodesmaceae. The only species that exists in pure culture and that is available for taxonomic studies is H. curvatum. Comparative 18S ribosomal RNA sequence analyses showed that H. curvatum belongs to the fungi rather than to the algae. Within the fungi, H. curvatum preferentially clustered with Chytridiomycetes. Unlike Chytridiomycetes, H. curvatum propagates by autosporulation, and the presence of flagella has never been reported. Transmission electron microscopy indicated that H. curvatum in some respects resembles Chytridiomycetes, but no elements of a flagellar apparatus were detected. The habitus of H. curvatum is unlike that of other fungi except the trichomycete Amoebidium parasiticum. The cell wall sugar composition of H. curvatum was unique, but to some extent resembled that of A. parasiticum. However, H. curvatum and A. parasiticum are not closely related to each other according to 18S rRNA sequence data. Moreover, A. parasiticum clustered with protistan animals, the Mesomycetozoa (DRIPs). Combined molecular, ultrastructural and chemical data do not allow assignment of H. curvatum to any recognized clade of fungi. This suggests that H. curvatum may represent an independent evolutionary lineage within the fungi.     

A rumen anaerobic fungus of the genus Neocallimastix: ultrastructure of the polyflagellate zoospore and young thallus

The ultrastructure of the zoosporic, rumen fungal anaerobe, Neocallimastix sp. R1, was determined and compared to that of the two known species of Neocallimastix. Zoospores of the new isolate were generally ovoid in shape, but without the waisted appearance of N. frontalis zoospores. They possessed similar organelles to the other two species, but with different localisation. The flagellar rootlet system was broadly similar to N. frontalis and N. patriciarum, however, a previously undescribed, large organelle was found to be associated near the kinetosomal apparatus in some Neocallimastix sp. R1 zoospores. Well developed flagella, complete with basal bodies, were observed in young thalli.     

A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.     

The fermentative characteristics of anaerobic rumen fungi

Substrate utilization and fermentation characteristics of rumen fungi of the genus Neocallimastix are described. Preliminary observations on the removal of monosaccharides from plant cell walls and the effect of fermentation products on growth of Neocallimastix sp. (isolate R1) are presented. The properties of rumen fungi are discussed in relation to their role in the rumen.     

A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: support for the hypothesis that hydrogenosomes are modified mitochondria

The hydrogenosomal malic enzyme (ME) was purified from the anaerobic fungus Neocallimastix frontalis . Using reverse genetics, the corresponding cDNA was isolated and characterized. The deduced amino acid sequence of the ME showed high similarity to ME from metazoa, plants and protists. Putative functional domains for malate and NAD⁺/NADP⁺ binding were identified. Phylogenetic analysis of the deduced amino acid sequence of the new ME suggests that it is homologous to reference bacterial and eukaryotic ME. Most interestingly, the cDNA codes for a protein which contains a 27-amino-acid N-terminus which is not present on the purified mature protein. This presequence shares features with known mitochondrial targeting signals, including an enrichment in Ala, Leu, Ser, and Arg, and the presence of an Arg at position –2 relative to amino acid 1 of the mature protein. This is the first report of a mitochondrial-like targeting signal on a hydrogenosomal enzyme from an anaerobic fungus and provides support for the hypothesis that hydrogenosomes in Neocallimastix frontalis might be modified mitochondria.     

Growth and survival of rumen fungi

The life cycle and growth kinetics of an anaerobic rumen fungus (Neocallimastix R1) in liquid and solid media are described, together with its response to light, temperature and oxygen. These results are discussed in relation to the survival of rumen fungi in saliva and faeces of sheep, and the possible routes for the transfer of anaerobic fungi between ruminants. The thallus and life cycle of Neocallimastix R1 are compared with those of aerobic chytrids.     

Comparative biochemistry of oxygen toxicity in lactic acid-forming aquatic fungi

Taxonomically diverse aquatic fungi ranging in oxidative capabilities from obligate aerobes to aerotolerant anaerobes were examined for growth under hyperbaric (0.9 atm) O₂, and for the ability to degrade H₂O₂ and O ₂ ^- . The results support the presumption that several Oomycetes and Chytridiomycetes are biochemically adapted to environments low in O₂. Results further indicate significant differences between Oomycetes and Chytridiomycetes in the enzymatic means of dealing with O ₂ ^- and H₂O₂, supporting the recent concept of a great evolutionary divergence between the groups. In general, facultative anaerobes and aerotolerant anaerobes were more severely inhibited by hyperbaric O₂, and they exhibited lower superoxide dismutase (SOD), catalase and peroxidase activities than did strongly-oxidative species. SOD activity, which was detected in all isolates, was insensitive to cyanide in Oomycetes but cyanide sensitive in the Blastocladiales (Chytridiomycetes). All strongly-oxidative Oomycetes exhibited readily-detectable catalase and peroxidase activities, while activities were very low or absent in strongly-fermentative species. As with the Oomycetes, peroxidase activities among the Blastocladiales were high in aerobes and low in strong fermenters. Surprisingly, however, none of the Blastocladiales, including strongly-oxidative species, exhibited substantial catalase activity. Catalase and SOD activities in faculatively anaerobic Oomycetes increased with increasing O₂ concentration; but even in hyperbaric (0.5 atm) O₂, activities for both enzymes in the aerotolerant anaerobe Aqualinderella fermentans were very low compared with activities in aerobes.Abbreviation SOD Superoxide dismutase     

Effect of coumarin on glucose uptake by anaerobic rumen fungi in the presence and absence of Methanobrevibacter smithii

The effect of coumarin (1,2 benzopyrone) on glucose utilisation by the anaerobic rumen fungi Neocallimastix frontalis and N. patriciarum has been compared with the effect of p-coumaric acid. Both compounds largely inhibited glucose utilisation by N. patriciarum strain Cx when present in the medium at a concentration of 2.5 mM, and had a similar effect on N. frontalis strain RE1 at 5 mM. Although in earlier studies co-culturing rumen fungi with Methanobrevibacter smithii enhanced resistance to ionophores, no comparable protective effect of M. smithii was found in the present study.     

Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles

Mitochondria are one of the hallmarks of eukaryotic cells, exporting ATP in exchange for cytosolic ADP using ADP/ATP carriers (AAC) located in the inner mitochondrial membrane. In contrast, several evolutionarily important anaerobic eukaryotes lack mitochondria but contain hydrogenosomes, peculiar organelles of controversial ancestry that also supply ATP but, like some fermentative bacteria, make molecular hydrogen in the process. We have now identified genes from two species of the hydrogenosome-containing fungus Neocallimastix that have three-fold sequence repeats and signature motifs that, along with phylogenetic analysis, identify them as AACs. When expressed in a mitochondrial AAC- deficient yeast strain, the hydrogenosomal protein was correctly targeted to the yeast mitochondria inner membrane and yielded mitochondria able to perform ADP/ATP exchange. Characteristic inhibitors of mitochondrial AACs blocked adenine nucleotide exchange by the Neocallimastix protein. Thus, our data demonstrate that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange. These experiments provide some of the strongest evidence yet that yeast mitochondria and Neocallimastix hydrogenosomes are but two manifestations of the same fundamental organelle.     

The effect of lasalocid on glucose uptake, hydrogen production and the solubilization of straw by the anaerobic rumen fungus Neocallimastix frontalis

Three isolates of Neocallimastix frontalis grown in pure culture with glucose as substrate differed in their response to the presence of lasalocid in the growth medium. For two strains (RE1 and RK21) hydrogen production and glucose uptake were reduced to 50% or less of the control values in the presence of lasalocid at 0–25 μg/ ml. A third strain (PNK2) retained significant fermentative ability in the presence of at least four times this amount of lasalocid. Lasalocid was rather less inhibitory to the solubilization of straw by these fungi, PNK2 again providing the most resistant strain. It is concluded that the use of lasalocid in ruminant feeds is likely to have a strain selective effect on the rumen population of Neocallimastix.     

On the induction of zoosporogenesis in the rumen phycomycetes Neocallimastix frontalis, Piromonas communis and Sphaeromonas communis.

The plant components inducing zoosporogenesis in the rumen phycomycetes Neocallimastix frontalis, Sphaeromonas communis and Piromonas communis were widely distributed in the plant kingdom with no apparent taxonomic relationship. In Lolium perenne L. (perennial rye-grass) and Hordeum distichon (barley), the components were principally present in the leaves and aerial tissues. Sufficient inducer was present in the normal diet of the host animal to trigger the differentiation and release of the zoospores from all the sporangia of each phycomycete species present in the rumen fluid tested. The inducers were unstable to oxygen, especially at elevated temperatures, and were destroyed by rumen micro-organisms. They may be similar compounds for each species.     

To See or Not to See: Do Front of Pack Nutrition Labels Affect Attention to Overall Nutrition Information?

Background

Front of pack (FOP) nutrition labels are concise labels located on the front of food packages that provide truncated nutrition information. These labels are rapidly gaining prominence worldwide, presumably because they attract attention and their simplified formats enable rapid comparisons of nutritional value.

Methods

Eye tracking was conducted as US consumers interacted with actual packages with and without FOP labels to (1) assess if the presence of an FOP label increases attention to nutrition information when viewers are not specifically tasked with nutrition-related goals; and (2) study the effect of FOP presence on consumer use of more comprehensive, traditional nutrition information presented in the Nutritional Facts Panel (NFP), a mandatory label for most packaged foods in the US.

Results

Our results indicate that colored FOP labels enhanced the probability that any nutrition information was attended, and resulted in faster detection and longer viewing of nutrition information. However, for cereal packages, these benefits were at the expense of attention to the more comprehensive NFP. Our results are consistent with a potential short cut effect of FOP labels, such that if an FOP was present, participants spent less time attending the more comprehensive NFP. For crackers, FOP labels increased time spent attending to nutrition information, but we found no evidence that their presence reduced the time spent on the nutrition information in the NFP.

Conclusions

The finding that FOP labels increased attention to overall nutrition information by people who did not have an explicit nutritional goal suggests that these labels may have an advantage in conveying nutrition information to a wide segment of the population. However, for some food types this benefit may come with a short-cut effect; that is, decreased attention to more comprehensive nutrition information. These results have implications for policy and warrant further research into the mechanisms by which FOP labels impact use of nutrition information by consumers for different foods.     

Nascent synthesis and secretion of cellobiase inNeocallimastix frontalis EB188

De novo synthesis and the secretion of cellobiase fromNeocallimastix frontalis EB188 were studied. Cellobiase was secreted rapidly in cellulose switch cultures. Chemical inhibition of protein synthesis and radiotracer studies suggested secretion was dependent on nascent protein synthesis. Inhibitors of protein glycosylation also inhibited secretion. An 85,000 dalton protein (and several others) represented the principal differences in de novo synthesis between cellulose- and glucose-switched cultures. Concentrations of the 85,000 daltons protein increased with culture incubation time and ultimately accounted for 7% of the total protein secreted. This protein was purified by gel electrophoresis separations and was determined to be a cellobiase. Secretion of this cellobiase was correlated with its radiolabeling. The possibility of cellobiase induction representing a specialized gene control system inNeocallimastix frontalis EB188 is discussed.