Linkage of Marine Bacterial Polyunsaturated Fatty Acid and Long-Chain Hydrocarbon Biosynthesis

Various marine gamma-proteobacteria produce omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (20:5, EPA) and docosahexaenoic acid (22:6, DHA), which are incorporated into membrane phospholipids. Five genes, designated pfaABCDE, encode the polyketide/fatty acid synthase necessary for production of these long-chain fatty acids. In addition to de novo biosynthesis of EPA and DHA, the “Pfa synthase” is also involved with production of a long-chain polyunsaturated hydrocarbon product (31:9, PUHC) in conjunction with the oleABCD hydrocarbon biosynthesis pathway. In this work, we demonstrate that OleA mediates the linkage between these two pathways in vivo. Co-expression of pfaA-E along with oleA from Shewanella pealeana in Escherichia coli yielded the expected product, a 31:8 ketone along with a dramatic ∼10-fold reduction in EPA content. The decrease in EPA content was independent of 31:8 ketone production as co-expression of an OleA active site mutant also led to identical decreases in EPA content. We also demonstrate that a gene linked with either pfa and/or ole operons in diverse bacterial lineages, herein designated pfaT, plays a role in maintaining optimal production of Pfa synthase derived products in Photobacterium and Shewanella species

Molecular Mechanisms Driving Switch Behavior in Xylem Cell Differentiation.

Plant xylem cells conduct water and mineral nutrients. Although most plant cells are totipotent, xylem cells are unusual and undergo terminal differentiation. Many genes regulating this process are well characterized, including the Vascular-related NAC Domain 7 (VND7), MYB46, and MYB83 transcription factors, which are proposed to act in interconnected feedforward loops (FFLs). Less is known regarding the molecular mechanisms underlying the terminal transition to xylem cell differentiation. Here, we generate whole-root and single-cell data, which demonstrate that VND7 initiates sharp switching of root cells to xylem cell identity. Based on these data, we identified 4 candidate VND7 downstream target genes capable of generating this switch. Although MYB46 responds to VND7 induction, it is not among these targets. This system provides an important model to study the emergent properties that may give rise to totipotency relative to terminal differentiation and reveals xylem cell subtypes

Widespread Occurrence of Secondary Lipid Biosynthesis Potential in Microbial Lineages

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as “Pfa synthases”. In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of “secondary lipids” to describe these biosynthetic pathways and products, a proposition consistent with the “secondary metabolite” vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages

Bacterial and Archaeal Communities and Bottom Waters of the Abyssal Patterns and Future Monitoring Considerations

Bacteria and archaea are key contributors to deep-sea biogeochemical cycles and food webs. The disruptions these microbial communities may experience during and following polymetallic nodule mining in the Clarion-Clipperton Zone (CCZ) of the North Pacific Ocean could therefore have broad ecological effects. Our goals in this synthesis are to characterize the current understanding of biodiversity and biogeography of bacteria and archaea in the CCZ and to identify gaps in the baseline data and sampling approaches, prior to the onset of mining in the region. This is part of a large effort to compile biogeographic patterns in the CCZ, and to assess the representivity of no-mining Areas of Particular Environmental Interest, across a range of taxa. Here, we review published studies and an additional new dataset focused on 16S ribosomal RNA (rRNA) gene amplicon characterization of abyssal bacterial and archaeal communities, particularly focused on spatial patterns. Deep-sea habitats (nodules, sediments, and bottom seawater) each hosted significantly different microbial communities. An east-vs.-west CCZ regional distinction was present in nodule communities, although the magnitude was small and likely not detectable without a high-resolution analysis. Within habitats, spatial variability was driven by differences in relative abundances of taxa, rather than by abundant taxon turnover. Our results further support observations that nodules in the CCZ have distinct archaeal communities from those in more productive surrounding regions, with higher relative abundances of presumed chemolithoautotrophic Nitrosopumilaceae suggesting possible trophic effects of nodule removal. Collectively, these results indicate that bacteria and archaea in the CCZ display previously undetected, subtle, regional-scale biogeography. However, the currently available microbial community surveys are spatially limited and suffer from sampling and analytical differences that frequently confound inter-comparison; making definitive management decisions from such a limited dataset could be problematic. We suggest a number of future research priorities and sampling recommendations that may help to alleviate dataset incompatibilities and to address challenges posed by rapidly advancing DNA sequencing technology for monitoring bacterial and archaeal biodiversity in the CCZ. Most critically, we advocate for selection of a standardized 16S rRNA gene amplification approach for use in the anticipated large-scale, contractor driven biodiversity monitoring in the region

Engineered Root Bacteria Release Plant-Available Phosphate from Phytate.

Microorganisms that release plant-available phosphate from natural soil phosphate stores may serve as biological alternatives to costly and environmentally damaging phosphate fertilizers. To explore this possibility, we engineered a collection of root bacteria to release plant-available orthophosphate from phytate, an abundant phosphate source in many soils. We identified 82 phylogenetically diverse phytase genes, refactored their sequences for optimal expression in Proteobacteria, and then synthesized and engineered them into the genomes of three root-colonizing bacteria. Liquid culture assays revealed 41 engineered strains with high levels of phytate hydrolysis. Among these, we identified 12 strains across three bacterial hosts that confer a growth advantage on the model plant Arabidopsis thaliana when phytate is the sole phosphate source. These data demonstrate that DNA synthesis approaches can be used to generate plant-associated strains with novel phosphate-solubilizing capabilities.IMPORTANCE Phosphate fertilizers are essential for high-yield agriculture yet are costly and environmentally damaging. Microbes that release soluble phosphate from naturally occurring sources in the soil are appealing, as they may reduce the need for such fertilizers. In this study, we used synthetic biology approaches to create a collection of engineered root-associated microbes with the ability to release phosphate from phytate. We demonstrate that these strains improve plant growth under phosphorus-limited conditions. This represents a first step in the development of phosphate-mining bacteria for future use in crop systems

Engineered Root Bacteria Release Plant-Available Phosphate from Phytate.

Microorganisms that release plant-available phosphate from natural soil phosphate stores may serve as biological alternatives to costly and environmentally damaging phosphate fertilizers. To explore this possibility, we engineered a collection of root bacteria to release plant-available orthophosphate from phytate, an abundant phosphate source in many soils. We identified 82 phylogenetically diverse phytase genes, refactored their sequences for optimal expression in Proteobacteria, and then synthesized and engineered them into the genomes of three root-colonizing bacteria. Liquid culture assays revealed 41 engineered strains with high levels of phytate hydrolysis. Among these, we identified 12 strains across three bacterial hosts that confer a growth advantage on the model plant Arabidopsis thaliana when phytate is the sole phosphate source. These data demonstrate that DNA synthesis approaches can be used to generate plant-associated strains with novel phosphate-solubilizing capabilities.IMPORTANCE Phosphate fertilizers are essential for high-yield agriculture yet are costly and environmentally damaging. Microbes that release soluble phosphate from naturally occurring sources in the soil are appealing, as they may reduce the need for such fertilizers. In this study, we used synthetic biology approaches to create a collection of engineered root-associated microbes with the ability to release phosphate from phytate. We demonstrate that these strains improve plant growth under phosphorus-limited conditions. This represents a first step in the development of phosphate-mining bacteria for future use in crop systems

From the surface to the deep-sea: bacterial distributions across polymetallic nodule fields in the Clarion-Clipperton zone of the Pacific Ocean

Marine bacteria regulate fluxes of matter and energy essential for pelagic and benthic organisms and may also be involved in the formation and maintenance of commercially valuable abyssal polymetallic nodules. Future mining of these nodule fields is predicted to have substantial effects on biodiversity and physicochemical conditions in mined areas. Yet, the identity and distributions of bacterial populations in deep-sea sediments and associated polymetallic nodules has received relatively little attention. We examined bacterial communities using high-throughput sequencing of bacterial 16S rRNA gene fragments from samples collected in the water column, sediment, and polymetallic nodules in the Pacific Ocean (bottom depth ≥4,000 m) in the eastern Clarion-Clipperton Zone. Operational taxonomic units (OTUs; defined at 99% 16S rRNA gene identity) affiliated with JTB255 (Gammaproteobacteria) and Rhodospirillaceae (Alphaproteobacteria) had higher relative abundances in the nodule and sediment habitats compared to the water column. Rhodobiaceae family and Vibrio OTUs had higher relative abundance in nodule samples, but were less abundant in sediment and water column samples. Bacterial communities in sediments and associated with nodules were generally similar; however, 5,861 and 6,827 OTUs found in the water column were retrieved from sediment and nodule habitats, respectively. Cyanobacterial OTUs clustering among Prochlorococcus and Synechococcus were detected in both sediments and nodules, with greater representation among nodule samples. Such results suggest that vertical export of typically abundant photic-zone microbes may be an important process in delivery of water column microorganisms to abyssal habitats, potentially influencing the structure and function of communities in polymetallic nodule fields