Biological Trace Information Extracted from Bioaerosols Using NGS Analysis

Bioaerosols are atmospheric particles with a biological trace, such as viruses, bacteria, fungi, and plant material such as pollen and plant debris. In this study, we analyzed the biological information in bioaerosols using next generation sequencing of the trace DNA. The samples were collected using an Andersen air sampler and separated into two groups according to particulate matter (PM) size: small (PM2.5) and large (PM10). Amplification and sequencing of the bacterial 16S rDNA gene, prokaryotic internal transcribed spacer 1 (ITS1) region and DNA sequence of a plant chloroplast gene (rbcL) were carried out using several sets of specific primers targeting animal and plant sequences. Lots of bacterial information was detected from the bioaerosols. The most abundant bacteria in several samples were of the Actinobacteria (class), Alphaproteobacteria, Bacilli, and Clostridia. For the animal detection using internal transcribed spacer 1, only uncultured fungi were detected in more than half of the hits, with a high number of Cladosporium sp. in the samples. For the plant identification, the ITS1 information only matched fungal species. However, targeting of the rbcL region revealed diverse plant information, such as Medicago papillosa. In conclusion, traces of bacteria, fungi, and plants could be detected in the bioaerosols, but not of animals using our primers.

The Assembly of Super-Complexes in the Plant Chloroplast

Increasing evidence has revealed that the enzymes of several biological pathways assemble into larger supramolecular structures called super-complexes. Indeed, those such as association of the mitochondrial respiratory chain complexes play an essential role in respiratory activity and promote metabolic fitness. Dynamically assembled super-complexes are able to alternate between participating in large complexes and existing in a free state. However, the functional significance of the super-complexes is not entirely clear. It has been proposed that the organization of respiratory enzymes into super-complexes could reduce oxidative damage and increase metabolism efficiency. There are several protein complexes that have been revealed in the plant chloroplast, yet little research has been focused on the formation of super-complexes in this organelle. The photosystem I and light-harvesting complex I super-complex’s structure suggests that energy absorbed by light-harvesting complex I could be efficiently transferred to the PSI core by avoiding concentration quenching. Here, we will discuss in detail core complexes of photosystem I and II, the chloroplast ATPase the chloroplast electron transport chain, the Calvin–Benson cycle and a plastid localized purinosome. In addition, we will also describe the methods to identify these complexes.

Plug and play: Is “directed endosymbiosis” of chloroplasts possible?

The origin of mammalian mitochondria and plant chloroplasts is thought to be endosymbiosis. Millennia ago, a bacterium related to typhus-causing bacteria may have been consumed by a proto-eukaryote and over time evolved into an organelle inside eukaryotic cells, known as a mitochondrion. The plant chloroplast is believed to have evolved in a similar fashion from cyanobacteria. This project attempted to use “directed endosymbiosis” (my term) to investigate if chloroplasts can be taken up by a land animal and continue to function. It has been shown previously that mouse fibroblasts could incorporate isolated chloroplasts when co-cultured. Photosynthetic bacteria containing chloroplasts have been successfully injected into zebrafish embryos, mammalian cells, and ischemic rodent hearts. The photosynthetic alga Chlamydomonas reinhardtii (C. reinhardtii) has also been injected into zebrafish embryos. However, to the best of my knowledge, injection of isolated chloroplasts into a land animal embryo has not been attempted before.In four pilot experiments, solutions of chloroplasts in PBS were microinjected into Drosophila melanogaster (D. melanogaster) embryos to determine if the embryos would tolerate the foreign protein. Interestingly, results indicated that a portion of the D. melanogaster embryos appeared to tolerate the injections and survive to adulthood. To determine if chloroplasts had indeed been transferred, larvae were placed under fluorescent microscopy. Chlorophyll (serving as the reporter) was found to be present in several larvae and to decline in amount over time. To investigate if the chloroplasts still functioned, a radiotracer food intake assay was performed. It was hypothesized that if the chloroplasts were generating ATP (and possibly glucose), the larvae might need less food. Results indicated a decrease in intake, however this might have occurred for other reasons.

Evidence from simulation studies for selective constraints on the codon usage of the Angiosperm psbA gene

The codon usage of the Angiosperm psbA gene is atypical for flowering plant chloroplast genes but similar to the codon usage observed in highly expressed plastid genes from some other Plantae, particularly Chlorobionta, lineages. The pattern of codon bias in these genes is suggestive of selection for a set of translationally optimal codons but the degree of bias towards these optimal codons is much weaker in the flowering plant psbA gene than in high expression plastid genes from lineages such as certain green algal groups. Two scenarios have been proposed to explain these observations. One is that the flowering plant psbA gene is currently under weak selective constraints for translation efficiency, the other is that there are no current selective constraints and we are observing the remnants of an ancestral codon adaptation that is decaying under mutational pressure. We test these two models using simulations studies that incorporate the context-dependent mutational properties of plant chloroplast DNA. We first reconstruct ancestral sequences and then simulate their evolution in the absence of selection on codon usage by using mutation dynamics estimated from intergenic regions. The results show that psbA has a significantly higher level of codon adaptation than expected while other chloroplast genes are within the range predicted by the simulations. These results suggest that there have been selective constraints on the codon usage of the flowering plant psbA gene during Angiosperm evolution.

Localization of the putative recombinase Pf-int to the apicoplast of Plasmodium falciparum

Diseases caused by apicomplexan parasites, such as malaria and toxoplasmosis cause ∼200 million (worldwide) and 1 million (Europe) infections, respectively, every year. Apicomplexa possess a non-photosynthetic organelle homologous to the plant chloroplast, the so-called apicoplast, that is essential for their growth and survival. This study focused on the Int recombinase, the first protein discovered in Plasmodium spp. with the features of a site-specific recombinase, and which has an apicoplast targeting leader sequence at its amino-terminus. Int is conserved amongst several apicomplexan parasites. In the human toxoplasmosis parasite, Toxoplasma, Int localizes to the apicoplast and Pf-Int, the P. falciparum member, belongs to the group of non-mutable essential genes in P. falciparum. A conserved protein that has been shown to be essential at least in one species and that localizes to an essential organelle may become a novel drug target. Therefore, the aim of this study was to confirm the sub-cellular localization of Int in the human malaria parasite P. falciparum. Using western blot analysis and immunofluorescence microscopy of P. falciparum asexual blood stages, we observed that Int partially co-localized with the apicoplast (to discrete foci adjacent to the nucleus).

Engineered Accumulation of Bicarbonate in Plant Chloroplasts: Known Knowns and Known Unknowns

Heterologous synthesis of a biophysical CO2-concentrating mechanism (CCM) in plant chloroplasts offers significant potential to improve the photosynthetic efficiency of C3 plants and could translate into substantial increases in crop yield. In organisms utilizing a biophysical CCM, this mechanism efficiently surrounds a high turnover rate Rubisco with elevated CO2 concentrations to maximize carboxylation rates. A critical feature of both native biophysical CCMs and one engineered into a C3 plant chloroplast is functional bicarbonate (HCO3−) transporters and vectorial CO2-to-HCO3− converters. Engineering strategies aim to locate these transporters and conversion systems to the C3 chloroplast, enabling elevation of HCO3− concentrations within the chloroplast stroma. Several CCM components have been identified in proteobacteria, cyanobacteria, and microalgae as likely candidates for this approach, yet their successful functional expression in C3 plant chloroplasts remains elusive. Here, we discuss the challenges in expressing and regulating functional HCO3− transporter, and CO2-to-HCO3− converter candidates in chloroplast membranes as an essential step in engineering a biophysical CCM within plant chloroplasts. We highlight the broad technical and physiological concerns which must be considered in proposed engineering strategies, and present our current status of both knowledge and knowledge-gaps which will affect successful engineering outcomes.

Genome-wide identification and characterization of DNA/RNA differences associated with Fusarium graminearum infection in wheat

RNA editing (DNA/RNA differences) as a post-transcriptional modification approach to enrich genetic information, plays the crucial role in regulating diverse biological processes in eukaryotes. Although it has been extensively studied in plant chloroplast and mitochondria genome, RNA editing in plant nuclear genome, especially those associated with Fusarium head blight (FHB), is not well studied at present. Here, we investigated the DNA/RNA differences associated with FHB through a novel method by comparing the RNA-seq data from Fusarium-infected and control samples from 4 wheat genotypes. A total of 187 DNA/RNA differences were identified in 36 wheat genes, representing the first landscape of the FHB-responsive RNA editome in wheat. Furthermore, all of these 36 edited genes were located in the FHB related co-expression gene modules, which may involve in regulating FHB response. Finally, the effects of DNA/RNA differences were systematically investigated to show that they could cause the change of RNA structure and protein structure in edited genes. In particular, the G to C editing (chr3A_487854715) in TraesCS3A02G263900, which is the orthology of OsRACK1, resulted that it was targeted by tae-miR9664-3p to control its expression in different genotype through different editing efficiency, suggesting RNA editing could mediate miRNA to participate in the regulation network of FHB tolerance. This study reported the first wheat DNA/RNA differences associated with FHB, which not only contribute to better understand the molecular basis underlying FHB tolerance, but also shed light on improving FHB tolerance through epigenetic method in wheat and beyond.

Arabidopsis Seedling Lethal 1 Interacting With Plastid-Encoded RNA Polymerase Complex Proteins Is Essential for Chloroplast Development

Mitochondrial transcription termination factors (mTERFs) are highly conserved proteins in metazoans. Plants have many more mTERF proteins than animals. The functions and the underlying mechanisms of plants’ mTERFs remain largely unknown. In plants, mTERF family proteins are present in both mitochondria and plastids and are involved in gene expression in these organelles through different mechanisms. In this study, we screened Arabidopsis mutants with pigment-defective phenotypes and isolated a T-DNA insertion mutant exhibiting seedling-lethal and albino phenotypes [seedling lethal 1 (sl1)]. The SL1 gene encodes an mTERF protein localized in the chloroplast stroma. The sl1 mutant showed severe defects in chloroplast development, photosystem assembly, and the accumulation of photosynthetic proteins. Furthermore, the transcript levels of some plastid-encoded proteins were significantly reduced in the mutant, suggesting that SL1/mTERF3 may function in the chloroplast gene expression. Indeed, SL1/mTERF3 interacted with PAP12/PTAC7, PAP5/PTAC12, and PAP7/PTAC14 in the subgroup of DNA/RNA metabolism in the plastid-encoded RNA polymerase (PEP) complex. Taken together, the characterization of the plant chloroplast mTERF protein, SL1/mTERF3, that associated with PEP complex proteins provided new insights into RNA transcription in the chloroplast.

Characterization of variability of the intergenic spacers cpDNA trnH–psbA, trnY–trnT AND rpoB–trnC in representatives of Pisum L. (Tribe Fabeae)

Background. Plant chloroplast genome have conservative structure, but its nucleotide sequence is polymorphous due to which cpDNA fragments are often used in taxonomic and phylogenetic studies. Despite the widespread distribution and use of Fabeae species, mainly peas (Pisum), data on the intraspecific diversity of cpDNA fragments are almost absent. The aim of the work was to analyze the intraspecific variability of three cpDNA spacers in Pisum. Materials and methods. As a result of the work, intergenic spacers trnYtrnT, trnHpsbA and rpoBtrnC in 38 accessions of the Pisum and related Fabeae species were sequenced. Despite the fact that the selected chloroplast fragments are generally considered to be sufficiently variable in plants and are often used for phylogenetic studies, Pisum accessions have been found to have no intraspecific differences in two of the three spacers sequences analyzed. Results and conclusion. A total 97 SNPs were detected in Pisum accessions, seven of them distinguished P. sativum from P. fulvum. The most variable of the analyzed fragments was the intergenic spacer rpoBtrnC. Based on rpoBtrnC sequence 17 haplotypes in P. sativum and four haplotypes in P. fulvum were revealed. The cpDNA sequencing data were used for a phylogenetic analysis. On the obtained tree Vavilovia formosa accession formed a separate branch from pea accessions. All Pisum accessions fall in one cluster, split into distinct P. sativum and P. fulvum subclusters (BI = 99%).