Whole-genome characterization and comparative genomics of a novel freshwater cyanobacteria species: Pseudanabaena punensis.

Cyanobacteria are emerging as a potential source of novel, beneficial bioactive compounds. However, some cyanobacteria species can harm water quality and public health through the production of toxins. Therefore, surveying the occurrence and generating genomic resources of cyanobacteria producing harmful compounds could help develop the control methods necessary to manage their growth and limit the release contaminants into the water bodies. Here, we describe a novel strain, Pseudanabaena punensis isolated from the open ends of pipelines supplying freshwater. This isolate was characterized morphologically, biochemically and by whole-genome sequence analysis. We also provide genomic information for P. punensis to help understand and highlight the features unique to this isolate. Morphological and genetic (analysis using 16S rRNA and rbcL genes) data were used to assign this novel strain to phylogenetic and taxonomic groups. The isolate was identified as a filamentous and non-heterocystous cyanobacteria. Based on morphological and 16S rRNA phylogeny, this isolate shares characteristics with the Pseudanabaenaceae family, but remains distinct from well-characterized species suggesting its polyphyletic assemblage. The whole-genome sequence analysis suggests greater genomic and phenotypic plasticity. Genome-wide sequence and comparative genomic analyses, comparing against several closely related species, revealed diverse and important genes associated with synthesizing bioactive compounds, multi-drug resistance pathway, heavy metal resistance, and virulence factors. This isolate also produces several important fatty acids with potential industrial applications. The observations described in this study emphasize both industrial applications and risks associated with the freshwater contamination, and therefore genomic resources provided in this study offer an opportunity for further investigations.

Metabolic engineering of phosphite metabolism in Synechococcus elongatus PCC 7942 as an effective measure to control biological contaminants in outdoor raceway ponds.

BACKGROUND: The use of cyanobacteria and microalgae as cell factories to produce biofuels and added-value bioproducts has received great attention during the last two decades. Important investments have been made by public and private sectors to develop this field. However, it has been a challenge to develop a viable and cost-effective platform for cultivation of cyanobacteria and microalgae under outdoor conditions. Dealing with contamination caused by bacteria, weedy algae/cyanobacteria and other organisms is a major constraint to establish effective cultivation processes. RESULTS: Here, we describe the implementation in the cyanobacterium Synechococcus elongatus PCC 7942 of a phosphorus selective nutrition system to control biological contamination during cultivation. The system is based on metabolic engineering of S. elongatus to metabolize phosphite, a phosphorus source not normally metabolized by most organisms, by expressing a bacterial phosphite oxidoreductase (PtxD). Engineered S. elongatus strains expressing PtxD grow at a similar rate on media supplemented with phosphite as the non-transformed control supplemented with phosphate. We show that when grown in media containing phosphite as the sole phosphorus source in glass flasks, the engineered strain was able to grow and outcompete biological contaminants even when the system was intentionally inoculated with natural competitors isolated from an irrigation canal. The PtxD/phosphite system was successfully used for outdoor cultivation of engineered S. elongatus in 100-L cylindrical reactors and 1000-L raceway ponds, under non-axenic conditions and without the need of sterilizing containers and media. Finally, we also show that the PtxD/phosphite system can be used as selectable marker for S. elongatus PCC 7942 transgenic strains selection, eliminating the need of antibiotic resistance genes. CONCLUSIONS: Our results suggest that the PtxD/phosphite system is a stable and sufficiently robust strategy to control biological contaminants without the need of sterilization or other complex aseptic procedures. Our data show that the PtxD/phosphite system can be used as selectable marker and allows production of the cyanobacterium S. elongatus PCC 7942 in non-axenic outdoor reactors at lower cost, which in principle should be applicable to other cyanobacteria and microalgae engineered to metabolize phosphite.

Selective fertilization with phosphite allows unhindered growth of cotton plants expressing the ptxD gene while suppressing weeds.

Weeds, which have been the bane of agriculture since the beginning of civilization, are managed manually, mechanically, and, more recently, by chemicals. However, chemical control options are rapidly shrinking due to the recent rise in the number of herbicide-resistant weeds in crop fields, with few alternatives on the horizon. Therefore, there is an urgent need for alternative weed suppression systems to sustain crop productivity while reducing our dependence on herbicides and tillage. Such a development will also allay some of the negative perceptions associated with the use of herbicide-resistance genes and heavy dependence on herbicides. Transgenic plants expressing the bacterial phosphite dehydrogenase (ptxD) gene gain an ability to convert phosphite (Phi) into orthophosphate [Pi, the metabolizable form of phosphorus (P)]. Such plants allow for a selective fertilization scheme, based on Phi as the sole source of P for the crop, while offering an effective alternative for suppressing weed growth. Here, we show that, when P is supplied in the form of Phi, ptxD-expressing cotton (Gossypium hirsutum L.) plants outcompete, in both artificial substrates and natural soils from agricultural fields, three different monocot and dicot weed species intentionally introduced in the experiments, as well as weeds naturally present in the tested soils. Importantly, the ptxD/Phi system proved highly efficacious in inhibiting the growth of glyphosate-resistant Palmer amaranth. With over 250 weed species resistant to currently available herbicides, ptxD-transgenic plants fertilized with Phi could provide an effective alternative to suppressing the growth of these weeds while providing adequate nutrition to the crop.

ptxD gene in combination with phosphite serves as a highly effective selection system to generate transgenic cotton (Gossypium hirsutum L.).

KEY MESSAGE: This report demonstrates the usefulness of ptxD/phosphite as a selection system that not only provides a highly efficient and simple means to generate transgenic cotton plants, but also helps address many of the concerns related to the use of antibiotic and herbicide resistance genes in the production of transgenic crops. Two of the most popular dominant selectable marker systems for plant transformation are based on either antibiotic or herbicide resistance genes. Due to concerns regarding their safety and in order to stack multiple traits in a single plant, there is a need for alternative selectable marker genes. The ptxD gene, derived from Pseudomonas stutzeri WM88, that confers to cells the ability to convert phosphite (Phi) into orthophosphate (Pi) offers an alternative selectable marker gene as demonstrated for tobacco and maize. Here, we show that the ptxD gene in combination with a protocol based on selection medium containing Phi, as the sole source of phosphorus (P), can serve as an effective and efficient system to select for transformed cells and generate transgenic cotton plants. Fluorescence microscopy examination of the cultures under selection and molecular analyses on the regenerated plants demonstrate the efficacy of the system in recovering cotton transformants following Agrobacterium-mediated transformation. Under the ptxD/Phi selection, an average of 3.43 transgenic events per 100 infected explants were recovered as opposed to only 0.41% recovery when bar/phosphinothricin (PPT) selection was used. The event recovery rates for nptII/kanamycin and hpt/hygromycin systems were 2.88 and 2.47%, respectively. Molecular analysis on regenerated events showed a selection efficiency of ~ 97% under the ptxD/Phi system. Thus, ptxD/Phi has proven to be a very efficient, positive selection system for the generation of transgenic cotton plants with equal or higher transformation efficiencies compared to the commonly used, negative selection systems.

Biotechnology of nutrient uptake and assimilation in plants.

Plants require a complex balance of mineral nutrients to reproduce successfully. Because the availability of many of these nutrients in the soil is compromised by several factors, such as soil pH, cation presence, and microbial activity, crop plants depend directly on nutrients applied as fertilizers to achieve high yields. However, the excessive use of fertilizers is a major environmental concern due to nutrient leaching that causes water eutrophication and promotes toxic algae blooms. This situation generates the urgent need for crop plants with increased nutrient use efficiency and better-designed fertilization schemes. The plant biology revolution triggered by the development of efficient gene transfer systems for plant cells together with the more recent development of next-generation DNA and RNA sequencing and other omics platforms have advanced considerably our understanding on the molecular basis of plant nutrition and how plants respond to nutritional stress. To date, genes encoding sensors, transcription factors, transporters, and metabolic enzymes have been identified as potential candidates to improve nutrient use efficiency. In addition, the study of other genetic resources, such as bacteria and fungi, allows the identification of alternative mechanisms of nutrient assimilation, which are potentially applicable in plants. Although significant progress in this respect has been achieved by conventional breeding, in this review we focus on the biotechnological approaches reported to date aimed at boosting the use of the three most limiting nutrients in the majority of arable lands: nitrogen, phosphorus, and iron.

A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism.

Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large-scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods.