A novel and efficient strategy for the biodegradation of di(2-ethylhexyl) phthalate by Fusarium culmorum.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is used worldwide and raises concerns because of its prevalence in the environment and potential toxicity. Herein, the capability of Fusarium culmorum to degrade a high concentration (3 g/L) of DEHP as the sole carbon and energy source in solid-state fermentation (SSF) was studied. Cultures grown on glucose were used as controls. The biodegradation of DEHP by F. culmorum reached 96.9% within 312 h. This fungus produced a 3-fold higher esterase activity in DEHP-supplemented cultures than in control cultures (1288.9 and 443.2 U/L, respectively). In DEHP-supplemented cultures, nine bands with esterase activity (24.6, 31.2, 34.2, 39.5, 42.8, 62.1, 74.5, 134.5, and 214.5 kDa) were observed by zymography, which were different from those in control cultures and from those previously reported for cultures grown in submerged fermentation. This is the first study to report the DEHP biodegradation pathway by a microorganism grown in SSF. The study findings uncovered a novel biodegradation strategy by which high concentrations of DEHP could be biodegraded using two alternative pathways simultaneously. F. culmorum has an outstanding capability to efficiently degrade DEHP by inducing esterase production, representing an ecologically promising alternative for the development of environmental biotechnologies, which might help mitigate the negative impacts of environmental contamination by this phthalate. KEY POINTS: • F. culmorum has potential to tolerate and remove di(2-ethylhexyl) phthalate (DEHP) • Solid-state fermentation is an efficient system for DEHP degradation by F. culmorum • High concentrations of DEHP induce high levels of esterase production by F. culmorum.

Improvement of Akanthomyces lecanii resistance to tebuconazole through UV-C radiation and selective pressure on microbial evolution and growth arenas.

Tebuconazole (TEB) is a fungicide widely used in agriculture; however, its constant application has increased the emergence of resistant plant pathogenic fungal strains and reduced the effectiveness of fungi as biological control agents; for instance, the entomopathogenic and hyperparasitic fungus Akanthomyces lecanii, suitable for simultaneous biological control of insect pest and plant pathogenic fungi, is highly sensitive to fungicides. We carried out the induction of resistance to TEB in two wild type strains of A. lecanii by UV radiation and selective pressure in increasing fungicide gradients using a modified Microbial Evolution and Growth Arena (MEGA), to produce A. lecanii strains that can be used as biological control agent in the presence of tebuconazole. Nine UV-induced and three naturally adapted A. lecanii strains were resistant to TEB at the agriculturally recommended dose, and three irradiated strains were resistant to TEB concentration ten times higher; moreover, growth, sporulation rates, production of hydrolytic enzymes, and virulence against the hemipteran Coccus viridis, a major pest of coffee crops, were not affected in the TEB-resistant strains. These A. lecanii TEB-resistant strains would have a greater opportunity to develop and to establish themselves in fields where the fungicide is present and can be used in a combined biological-chemical strategy to improve insect and plant pathogenic fungal control in agriculture. Also, the selective pressure through modified MEGA plate methodology can be used for the adaptation of entomopathogenic filamentous fungi to withstand other chemical or abiotic stresses that limits its effectiveness for pest control.

The plastomes of Hyalomonas oviformis and Hyalogonium fusiforme evolved dissimilar architectures after the loss of photosynthesis.

The loss of photosynthesis in land plants and algae is typically associated with parasitism but can also occur in free-living species, including chlamydomonadalean green algae. The plastid genomes (ptDNAs) of colorless chlamydomonadaleans are surprisingly diverse in architecture, including highly expanded forms (Polytoma uvella and Leontynka pallida) as well as outright genome loss (Polytomella species). Here, we explore the ptDNAs of Hyalomonas (Hm.) oviformis (SAG 62-27; formerly known as Polytoma oviforme) and Hyalogonium (Hg.) fusiforme (SAG 62-1c), each representing independent losses of photosynthesis within the Chlamydophyceae. The Hm. oviformis ptDNA is moderately sized (132 kb) with a reduced gene complement (but still encoding the ATPase subunits) and is in fact smaller than that of its photosynthetic relative Hyalomonas chlamydogama SAG 11-48b (198.3 kb). The Hg. fusiforme plastome, however, is the largest yet observed in nonphotosynthetic plants or algae (~463 kb) and has a coding repertoire that is almost identical to that of its photosynthetic relatives in the genus Chlorogonium. Furthermore, the ptDNA of Hg. fusiforme shows no clear evidence of pseudogenization, which is consistent with our analyses showing that Hg. fusiforme is the nonphotosynthetic lineage of most recent origin among known colorless Chlamydophyceae. Together, these new ptDNAs clearly show that, in contrast to parasitic algae, plastid genome compaction is not an obligatory route following the loss of photosynthesis in free-living algae, and that certain chlamydomonadalean algae have a remarkable propensity for genomic expansion, which can persist regardless of the trophic strategy.

An unexpected guest: a green microalga associated with the arsenic-tolerant shrub Acacia farnesiana.

The best-known plant endophytes include mainly fungi and bacteria, but there are also a few records of microalgae growing endophytically in vascular land plants, some of which belong to the genus Coccomyxa. In this study, we isolated a single-celled photosynthetic microorganism from the arsenic-tolerant shrub Acacia farnesiana, thus we hypothesized that it is an endophytic arsenic-tolerant microalga. The microorganism was identified as belonging to the genus Coccomyxa, and the observation of algal cells within the root tissues strongly suggests its endophytic nature. The alga's tolerance to arsenate (AsV) and its influence on the fitness of A. farnesiana in the presence of AsV were evaluated. Coccomyxa sp. can tolerate up to 2000 µM of AsV for periods shorter than 10 days, however, AsV-tolerance decreased significantly in longer exposure periods. The association with the microalga increased the pigment content in aboveground tissues of A. farnesiana seedlings exposed to AsV for 50 days, without changes in plant growth or arsenic accumulation. This work describes the association, probably endophytic, between an angiosperm and a microalga, confirming the ability of the genus Coccomyxa to form associations with land plants and broadening the known variety of plant endophytes.

Polyurethane foam as an inert support using concentrated media improves quality and spore production from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) (Bacillales:Bacillaceae) is a gram-positive bacterium that produces spores, several virulence factors and insecticidal toxins, making this microorganism the most used biopesticide worldwide. The use of inert supports such as polyurethane foam (PUF) in solid cultures has been a great alternative to produce various metabolites, including those produced by Bt. In this study we compared the yields, productivity and quality of the spores by two wild strains of Bt, (Y15 and EA3), grown in media with high substrate concentration in both culture systems: liquid and solid (PUF as solid inert support). Both strains showed 2.5- to 30-fold increases in spore production and productivity in solid culture, which showed an even greater increase when considering the spores retained in the PUF observed by scanning electron microscopy. Moreover, spore produced in solid culture showed up to sevenfold higher survival after a heat-shock treatment, relative to spores from liquid culture. The infectivity against larvae of Galleria mellonella (Lepidoptera:Pyralidae) improved also in spores from solid cultures. This comparison showed that the culture of Bt on solid support has clear advantages over liquid culture in terms of the production and quality of spores, and that those advantages can be attributed only to the culture system, as the same media composition was used in both systems.

Solid-state fermentation increases secretome complexity in Aspergillus brasiliensis.

Aspergillus is used for the industrial production of enzymes and organic acids, mainly by submerged fermentation (SmF). However, solid-state fermentation (SSF) offers several advantages over SmF. Although differences related to lower catabolite repression and substrate inhibition, as well as higher extracellular enzyme production in SSF compared to SmF have been shown, the mechanisms undelaying such differences are still unknown. To explain some differences among SSF and SmF, the secretome of Aspergillus brasiliensis obtained from cultures in a homogeneous physiological state with high glucose concentrations was analyzed. Of the regulated proteins produced by SmF, 74% were downregulated by increasing the glucose concentration, whereas all those produced by SSF were upregulated. The most abundant and upregulated protein found in SSF was the transaldolase, which could perform a moonlighting function in fungal adhesion to the solid support. This study evidenced that SSF: (i) improves the kinetic parameters in relation to SmF, (ii) prevents the catabolite repression, (iii) increases the branching level of hyphae and oxidative metabolism, as well as the concentration and diversity of secreted proteins, and (iv) favors the secretion of typically intracellular proteins that could be involved in fungal adhesion. All these differences can be related to the fact that molds are more specialized to growth in solid materials because they mimic their natural habitat.

Plastid Genomes from Diverse Glaucophyte Genera Reveal a Largely Conserved Gene Content and Limited Architectural Diversity.

Plastid genome (ptDNA) data of Glaucophyta have been limited for many years to the genus Cyanophora. Here, we sequenced the ptDNAs of Gloeochaete wittrockiana, Cyanoptyche gloeocystis, Glaucocystis incrassata, and Glaucocystis sp. BBH. The reported sequences are the first genome-scale plastid data available for these three poorly studied glaucophyte genera. Although the Glaucophyta plastids appear morphologically "ancestral," they actually bear derived genomes not radically different from those of red algae or viridiplants. The glaucophyte plastid coding capacity is highly conserved (112 genes shared) and the architecture of the plastid chromosomes is relatively simple. Phylogenomic analyses recovered Glaucophyta as the earliest diverging Archaeplastida lineage, but the position of viridiplants as the first branching group was not rejected by the approximately unbiased test. Pairwise distances estimated from 19 different plastid genes revealed that the highest sequence divergence between glaucophyte genera is frequently higher than distances between species of different classes within red algae or viridiplants. Gene synteny and sequence similarity in the ptDNAs of the two Glaucocystis species analyzed is conserved. However, the ptDNA of Gla. incrassata contains a 7.9-kb insertion not detected in Glaucocystis sp. BBH. The insertion contains ten open reading frames that include four coding regions similar to bacterial serine recombinases (two open reading frames), DNA primases, and peptidoglycan aminohydrolases. These three enzymes, often encoded in bacterial plasmids and bacteriophage genomes, are known to participate in the mobilization and replication of DNA mobile elements. It is therefore plausible that the insertion in Gla. incrassata ptDNA is derived from a DNA mobile element.

The oxygen concentration in cultures modulates protein expression and enzymatic antioxidant responses in Metarhizium lepidiotae conidia.

Conidia from Metarhizium spp. are used for integrated pest control; however, environmental factors diminish the effectivity of these programs. Several approaches tried to improve conidia resistance to overcome this limitation, although little is known about the mechanisms involved in this effect. Here we measured the activity of antioxidant enzymes and conidia virulence, comparing the proteomic profiles of Metarhiziumlepidiotae CP-OAX conidia produced under normal (21% O2) and high oxygen atmospheres (pulses with 30% O2). We detected a higher virulence against Tenebrio molitor larvae, in addition to an increase in ultraviolet light tolerance in conidia produced under 30% O2, which correlates with increased glutathione reductase activity. Two-dimensional gel electrophoresis (2D SDS-PAGE) of proteins extracted in conidia harvested from both experimental conditions revealed a group of proteins that was observed only in conidia from oxidant atmospheres. Some of those proteins were directly involved in oxidative stress responses, whereas others were involved in conidial virulence, thermo-tolerance, and the central metabolism. Thus, a high atmospheric oxygen concentration (30%) activates antioxidant defence and general stress response mechanisms involved in conidia resistance to adverse environmental factors, which can ultimately translate into higher effectivity for the use of entomopathogenic fungi conidia in pest control.

The plastid genomes of nonphotosynthetic algae are not so small after all.

The thing about plastid genomes in nonphotosynthetic plants and algae is that they are usually very small and highly compact. This is not surprising: a heterotrophic existence means that genes for photosynthesis can be easily discarded. But the loss of photosynthesis cannot explain why the plastomes of heterotrophs are so often depauperate in noncoding DNA. If plastid genomes from photosynthetic taxa can span the gamut of compactness, why can't those of nonphotosynthetic species? Well, recently we showed that they can. The free-living, heterotrophic green alga Polytoma uvella has a plastid genome boasting more than 165 kilobases of noncoding DNA, making it the most bloated plastome yet found in a heterotroph. In this addendum to the primary study, we elaborate on why the P. uvella plastome is so inflated, discussing the potential impact of a free-living vs. parasitic lifestyle on plastid genome expansion in nonphotosynthetic lineages.

Complete chloroplast genomes of the Chlamydomonas reinhardtii nonphotosynthetic mutants CC-1375, CC-373, CC-4199, CC-2359 and CC-1051.

The chloroplast genomes (cpDNA) of five Chlamydomonas reinhardtii nonphotosynthetic mutants were sequenced. The architecture, gene content, and synteny of the cpDNAs from the five mutants are identical to the C. reinhardtii 'wild-type' plastome. A small number of differences at sequence level between coding regions of the reference genome and the cpDNAs of the mutants were detected. The vast majority of the sequence differences were synonymous and likely due to nucleotide substitutions preceding the generation of the mutant strains, but not caused by the erosion of the cpDNA following the loss of photosynthesis.

The Plastid Genome of Polytoma uvella Is the Largest Known among Colorless Algae and Plants and Reflects Contrasting Evolutionary Paths to Nonphotosynthetic Lifestyles.

The loss of photosynthesis is frequently associated with parasitic or pathogenic lifestyles, but it also can occur in free-living, plastid-bearing lineages. A common consequence of becoming nonphotosynthetic is the reduction in size and gene content of the plastid genome. In exceptional circumstances, it can even result in the complete loss of the plastid DNA (ptDNA) and its associated gene expression system, as reported recently in several lineages, including the nonphotosynthetic green algal genus Polytomella Closely related to Polytomella is the polyphyletic genus Polytoma, the members of which lost photosynthesis independently of Polytomella Species from both genera are free-living organisms that contain nonphotosynthetic plastids, but unlike Polytomella, Polytoma members have retained a genome in their colorless plastid. Here, we present the plastid genome of Polytoma uvella: to our knowledge, the first report of ptDNA from a nonphotosynthetic chlamydomonadalean alga. The P. uvella ptDNA contains 25 protein-coding genes, most of which are related to gene expression and none are connected to photosynthesis. However, despite its reduced coding capacity, the P. uvella ptDNA is inflated with short repeats and is tens of kilobases larger than the ptDNAs of its closest known photosynthetic relatives, Chlamydomonas leiostraca and Chlamydomonas applanata In fact, at approximately 230 kb, the ptDNA of P. uvella represents the largest plastid genome currently reported from a nonphotosynthetic alga or plant. Overall, the P. uvella and Polytomella plastid genomes reveal two very different evolutionary paths following the loss of photosynthesis: expansion and complete deletion, respectively. We hypothesize that recombination-based DNA-repair mechanisms are at least partially responsible for the different evolutionary outcomes observed in such closely related nonphotosynthetic algae.

When the lights go out: the evolutionary fate of free-living colorless green algae.

The endosymbiotic origin of plastids was a launching point for eukaryotic evolution. The autotrophic abilities bestowed by plastids are responsible for much of the eukaryotic diversity we observe today. But despite its many advantages, photosynthesis has been lost numerous times and in disparate lineages throughout eukaryote evolution. For example, among green algae, several groups have lost photosynthesis independently and in response to different selective pressures; these include the parasitic/pathogenic trebouxiophyte genera Helicosporidium and Prototheca, and the free-living chlamydomonadalean genera Polytomella and Polytoma. Here, we examine the published data on colorless green algae and argue that investigations into the different evolutionary routes leading to their current nonphotosynthetic lifestyles provide exceptional opportunities to understand the ecological and genomic factors involved in the loss of photosynthesis.

Massive and widespread organelle genomic expansion in the green algal genus Dunaliella.

The mitochondrial genomes of chlamydomonadalean green algae are renowned for their highly reduced and conserved gene repertoires, which are almost fixed at 12 genes across the entire lineage. The sizes of these genomes, however, are much more variable, with some species having small, compact mitochondrial DNAs (mtDNAs) and others having expanded ones. Earlier work demonstrated that the halophilic genus Dunaliella contains extremely inflated organelle genomes, but to date the mtDNA of only one isolate has been explored. Here, by surveying mtDNA architecture across the Chlamydomonadales, we show that various Dunaliella species have undergone massive levels of mitochondrial genomic expansion, harboring the most inflated, intron-dense mtDNAs available from chlorophyte green algae. The same also appears to be true for their plastid genomes, which are potentially among the largest of all plastid-containing eukaryotes. Genetic divergence data are used to investigate the underlying causes of such extreme organelle genomic architectures, and ultimately reveal order-of-magnitude differences in mitochondrial versus plastid mutation rates within Dunaliella.

What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes.

Allotopic expression is potentially a gene therapy for mtDNA-related diseases. Some OXPHOS proteins like ATP6 (subunit a of complex V) and COX3 (subunit III of complex IV) that are typically mtDNA-encoded, are naturally nucleus-encoded in the alga Chlamydomonas reinhardtii. The mitochondrial proteins whose genes have been relocated to the nucleus exhibit long mitochondrial targeting sequences ranging from 100 to 140 residues and a diminished overall mean hydrophobicity when compared with their mtDNA-encoded counterparts. We explored the allotopic expression of the human gene products COX3 and ATP6 that were re-designed for mitochondrial import by emulating the structural properties of the corresponding algal proteins. In vivo and in vitro data in homoplasmic human mutant cells carrying either a T8993G mutation in the mitochondrial atp6 gene or a 15bp deletion in the mtDNA-encoded cox3 gene suggest that these human mitochondrial proteins re-designed for nuclear expression are targeted to the mitochondria, but fail to functionally integrate into their corresponding OXPHOS complexes.

Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp.

Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.

Reconstructing the mitochondrial protein import machinery of Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii several nucleus-encoded proteins that participate in the mitochondrial oxidative phosphorylation are targeted to the organelle by unusually long mitochondrial targeting sequences. Here, we explored the components of the mitochondrial import machinery of the green alga. We mined the algal genome, searching for yeast and plant homologs, and reconstructed the mitochondrial import machinery. All the main translocation components were identified in Chlamydomonas as well as in Arabidopsis thaliana and in the recently sequenced moss Physcomitrella patens. Some of these components appear to be duplicated, as is the case of Tim22. In contrast, several yeast components that have relatively large hydrophilic regions exposed to the cytosol or to the intermembrane space seem to be absent in land plants and green algae. If present at all, these components of plants and algae may differ significantly from their yeast counterparts. We propose that long mitochondrial targeting sequences in some Chlamydomonas mitochondrial protein precursors are involved in preventing the aggregation of the hydrophobic proteins they carry.