Bacterial Membrane Vesicles for In Vitro Catalysis.

The use of biological systems in manufacturing and medical applications has seen a dramatic rise in recent years as scientists and engineers have gained a greater understanding of both the strengths and limitations of biological systems. Biomanufacturing, or the use of biology for the production of biomolecules, chemical precursors, and others, is one particular area on the rise as enzymatic systems have been shown to be highly advantageous in limiting the need for harsh chemical processes and the formation of toxic products. Unfortunately, biological production of some products can be limited due to their toxic nature or reduced reaction efficiency due to competing metabolic pathways. In nature, microbes often secrete enzymes directly into the environment or encapsulate them within membrane vesicles to allow catalysis to occur outside the cell for the purpose of environmental conditioning, nutrient acquisition, or community interactions. Of particular interest to biotechnology applications, researchers have shown that membrane vesicle encapsulation often confers improved stability, solvent tolerance, and other benefits that are highly conducive to industrial manufacturing practices. While still an emerging field, this review will provide an introduction to biocatalysis and bacterial membrane vesicles, highlight the use of vesicles in catalytic processes in nature, describe successes of engineering vesicle/enzyme systems for biocatalysis, and end with a perspective on future directions, using selected examples to illustrate these systems' potential as an enabling tool for biotechnology and biomanufacturing.

Isolation and Characterization of Membrane Vesicles from Lactobacillus Species.

Throughout their life cycle, bacteria shed portions of their outermost membrane comprised of proteins, lipids, and a diversity of other biomolecules. These biological nanoparticles have been shown to have a range of highly diverse biological activities, including pathogenesis, community regulation, and cellular defense (among others). In recent publications, we have isolated and characterized membrane vesicles (MVs) from several species of Lactobacilli, microbes classified as commensals within the human gut microbiome ( Dean et al., 2019 and 2020). With increasing scientific understanding of host-microbe interactions, the gut-brain axis, and tailored probiotics for therapeutic or performance increasing applications, the protocols described herein will be useful to researchers developing new strategies for gut community engineering or the targeted delivery of bio-active molecules. Graphic abstract: Figure 1. Atomic force microscopic image of Lactobacillus casei ATCC 393 bacteria margins (white arrows) and membrane vesicles (black arrows).

Woodland strawberry axillary bud fate is dictated by a crosstalk of environmental and endogenous factors.

Plant architecture is defined by fates and positions of meristematic tissues and has direct consequences on yield potential and environmental adaptation of the plant. In strawberries (Fragaria vesca L. and F. × ananassa Duch.), shoot apical meristems can remain vegetative or differentiate into a terminal inflorescence meristem. Strawberry axillary buds (AXBs) are located in leaf axils and can either remain dormant or follow one of the two possible developmental fates. AXBs can either develop into stolons needed for clonal reproduction or into branch crowns (BCs) that can bear their own terminal inflorescences under favorable conditions. Although AXB fate has direct consequences on yield potential and vegetative propagation of strawberries, the regulation of AXB fate has so far remained obscure. We subjected a number of woodland strawberry (F. vesca L.) natural accessions and transgenic genotypes to different environmental conditions and growth regulator treatments to demonstrate that strawberry AXB fate is regulated either by environmental or endogenous factors, depending on the AXB position on the plant. We confirm that the F. vesca GIBBERELLIN20-oxidase4 (FvGA20ox4) gene is indispensable for stolon development and under tight environmental regulation. Moreover, our data show that apical dominance inhibits the outgrowth of the youngest AXB as BCs, although the effect of apical dominance can be overrun by the activity of FvGA20ox4. Finally, we demonstrate that the FvGA20ox4 is photoperiodically regulated via FvSOC1 (F. vesca SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) at 18°C, but at higher temperature of 22°C an unidentified FvSOC1-independent pathway promotes stolon development.

Harnessing the potential of Lactobacillus species for therapeutic delivery at the lumenal-mucosal interface.

Lactobacillus species have been studied for over 30 years in their role as commensal organisms in the human gut. Recently there has been a surge of interest in their abilities to natively and recombinantly stimulate immune activities, and studies have identified strains and novel molecules that convey particular advantages for applications as both immune adjuvants and immunomodulators. In this review, we discuss the recent advances in Lactobacillus-related activity at the gut/microbiota interface, the efforts to probe the boundaries of the direct and indirect therapeutic potential of these bacteria, and highlight the continued interest in harnessing the native capacity for the production of biogenic compounds shown to influence nervous system activity. Taken together, these aspects underscore Lactobacillus species as versatile therapeutic delivery vehicles capable of effector production at the lumenal-mucosal interface, and further establish a foundation of efficacy upon which future engineered strains can expand.

Allelic Variation of MYB10 Is the Major Force Controlling Natural Variation in Skin and Flesh Color in Strawberry (Fragaria spp.) Fruit.

The fruits of diploid and octoploid strawberry (Fragaria spp) show substantial natural variation in color due to distinct anthocyanin accumulation and distribution patterns. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 is the main activator in strawberry fruit. Here, we show that mutations in MYB10 cause most of the variation in anthocyanin accumulation and distribution observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F ×ananassa). Using a mapping-by-sequencing approach, we identified a gypsy-transposon in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional loss-of-function mutations in MYB10 were identified among geographically diverse white-fruited F. vesca ecotypes. Genetic and transcriptomic analyses of octoploid Fragaria spp revealed that FaMYB10-2, one of three MYB10 homoeologs identified, regulates anthocyanin biosynthesis in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression. Our findings suggest that cis-regulatory elements in FaEnSpm-2 are responsible for enhanced MYB10-2 expression and anthocyanin biosynthesis in strawberry fruit flesh.

Overexpression of Arabidopsis microRNA167 induces salicylic acid-dependent defense against Pseudomonas syringae through the regulation of its targets ARF6 and ARF8.

microRNAs are powerful regulators of growth, development, and stress responses in plants. The Arabidopsis thaliana microRNA miR167 was previously found to regulate diverse processes including flower development, root development, and response to osmotic stress by controlling the patterns of expression of its target genes AUXIN RESPONSE FACTOR 6 (ARF6), ARF8, and IAA-Ala RESISTANT 3. Here, we report that miR167 also modulates defense against pathogens through ARF6 and ARF8. miR167 is differentially expressed in response to the bacterial pathogen Pseudomonas syringae, and overexpression of miR167 confers very high levels of resistance. This resistance appears to be due to suppression of auxin responses and is partially dependent upon salicylic acid signaling, and also depends upon altered stomatal behavior in these plants. Closure of stomata upon the detection of P. syringae is an important aspect of the basal defense response, as it prevents bacterial cells from entering the leaf interior and causing infection. Plants overexpressing miR167 constitutively maintain small stomatal apertures, resulting in very high resistance when the pathogen is inoculated onto the leaf surface. Additionally, the systemic acquired resistance (SAR) response is severely compromised in plants overexpressing miR167, in agreement with previous work showing that the activation of SAR requires intact auxin signaling responses. This work highlights a new role for miR167, and also emphasizes the importance of hormonal balance in short- and long-term defense and of stomata as an initial barrier to pathogen entry.

Corrigendum: Arabidopsis G-Protein ß Subunit AGB1 Interacts With BES1 to Regulate Brassinosteroid Signaling and Cell Elongation.

[This corrects the article DOI: 10.3389/fpls.2017.02225.].

Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery.

Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii. We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii, which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics.

An Arabidopsis DISEASE RELATED NONSPECIFIC LIPID TRANSFER PROTEIN 1 is required for resistance against various phytopathogens and tolerance to salt stress.

Synchronous and timely regulation of multiple genes results in an effective defense response that decides the fate of the host when challenged with pathogens or unexpected changes in environmental conditions. One such gene, which is downregulated in response to multiple bacterial pathogens, is a putative nonspecific lipid transfer protein (nsLTP) of unknown function that we have named DISEASE RELATED NONSPECIFIC LIPID TRANSFER PROTEIN 1 (DRN1). We show that upon pathogen challenge, DRN1 is strongly downregulated, while a putative DRN1-targeting novel microRNA (miRNA) named DRN1 Regulating miRNA (DmiR) is reciprocally upregulated. Furthermore, we provide evidence that DRN1 is required for defense against bacterial and fungal pathogens as well as for normal seedling growth under salinity stress. Although nsLTP family members from different plant species are known to be a significant source of food allergens and are often associated with antimicrobial properties, our knowledge on the biological functions and regulation of this gene family is limited. Our current work not only sheds light on the mechanism of regulation but also helps in the functional characterization of DRN1, a putative nsLTP family member of hitherto unknown function.

Bacterial Membrane Vesicles as Mediators of Microbe - Microbe and Microbe - Host Community Interactions.

Bacterial membrane vesicles are proteoliposomal nanoparticles produced by both Gram-negative and Gram-positive bacteria. As they originate from the outer surface of the bacteria, their composition and content is generally similar to the parent bacterium's membrane and cytoplasm. However, there is ample evidence that preferential packaging of proteins, metabolites, and toxins into vesicles does occur. Incorporation into vesicles imparts a number of benefits to the cargo, including protection from degradation by other bacteria, the host organism, or environmental factors, maintenance of a favorable microenvironment for enzymatic activity, and increased potential for long-distance movement. This enables vesicles to serve specialized functions tailored to changing or challenging environments, particularly in regard to microbial community interactions including quorum sensing, biofilm formation, antibiotic resistance, antimicrobial peptide expression and deployment, and nutrient acquisition. Additionally, based on their contents, vesicles play crucial roles in host-microbe interactions as carriers of virulence factors and other modulators of host cell function. Here, we discuss recent advances in our understanding of how vesicles function as signals both within microbial communities and between pathogenic or commensal microbes and their mammalian hosts. We also highlight a few areas that are currently ripe for additional research, including the mechanisms of selective cargo packaging into membrane vesicles and of cargo processing once it enters mammalian host cells, the function of vesicles in transfer of nucleic acids among bacteria, and the possibility of engineering commensal bacteria to deliver cargo of interest to mammalian hosts in a controlled manner.

The Arabidopsis SENESCENCE-ASSOCIATED GENE 13 Regulates Dark-Induced Senescence and Plays Contrasting Roles in Defense Against Bacterial and Fungal Pathogens.

SENESCENCE-ASSOCIATED GENE 13 (SAG13) of Arabidopsis is a widely conserved gene of unknown function that has been extensively used as a marker of plant senescence. SAG13 induction occurs during plant cell death processes, including senescence and hypersensitive response, a type of programmed cell death that occurs in response to pathogens. This implies that SAG13 expression is regulated through at least two different signaling pathways affecting these two different processes. Our work highlights a contrasting role for SAG13 in regulating resistance against disease-causing biotrophic bacterial and necrotrophic fungal pathogens with contrasting infection strategies. We provide further evidence that SAG13 is not only induced during oxidative stress but also plays a role in protecting the plant against other stresses. SAG13 is also required for normal seed germination, seedling growth, and anthocyanin accumulation. The work presented here provides evidence for the role of SAG13 in regulating multiple plant processes including senescence, defense, seed germination, and abiotic stress responses. SAG13 is a valuable molecular marker for these processes and is conserved in multiple plant species, and this knowledge has important implications for crop improvement.

Broad range shuttle vector construction and promoter evaluation for the use of Lactobacillus plantarum WCFS1 as a microbial engineering platform.

As the field of synthetic biology grows, efforts to deploy complex genetic circuits in nonlaboratory strains of bacteria will continue to be a focus of research laboratories. Members of the Lactobacillus genus are good targets for synthetic biology research as several species are already used in many foods and as probiotics. Additionally, Lactobacilli offer a relatively safe vehicle for microbiological treatment of various health issues considering these commensals are often minor constituents of the gut microbial community and maintain allochthonous behavior. In order to generate a foundation for engineering, we developed a shuttle vector for subcloning in Escherichia coli and used it to characterize the transcriptional and translational activities of a number of promoters native to Lactobacillus plantarum WCFS1. Additionally, we demonstrated the use of this vector system in multiple Lactobacillus species, and provided examples of non-native promoter recognition by both L. plantarum and E. coli strains that might allow a shortcut assessment of circuit outputs. A variety of promoter activities were observed covering a range of protein expression levels peaking at various times throughout growth, and subsequent directed mutations were demonstrated and suggested to further increase the degree of output tuning. We believe these data show the potential for L. plantarum WCFS1 to be used as a nontraditional synthetic biology chassis and provide evidence that our system can be transitioned to other probiotic Lactobacillus species as well.

An eFP browser for visualizing strawberry fruit and flower transcriptomes.

Wild strawberry Fragaria vesca is emerging as an important model system for the cultivated strawberry due to its diploid genome and availability of extensive transcriptome data and a range of molecular genetic tools. Being able to better utilize these tools, especially the transcriptome data, will greatly facilitate research progress in strawberry and other Rosaceae fruit crops. The electronic fluorescent pictograph (eFP) software is a useful and popular tool to display transcriptome data visually, and is widely used in other model organisms including Arabidopsis and mouse. Here we applied eFP to display wild strawberry RNA sequencing (RNA-seq) data from 42 different tissues and stages, including various flower and fruit developmental stages. In addition, we generated eight additional RNA-seq data sets to represent tissues from ripening-stage receptacle fruit from yellow-colored and red-colored wild strawberry varieties. Differential gene expression analysis between these eight data sets provides additional information for understanding fruit-quality traits. Together, this work greatly facilitates the utility of the extensive transcriptome data for investigating strawberry flower and fruit development as well as fruit-quality traits.

JMJ27, an Arabidopsis H3K9 histone demethylase, modulates defense against Pseudomonas syringae and flowering time.

Histone methylation is known to dynamically regulate diverse developmental and physiological processes. Histone methyl marks are written by methyltransferases and erased by demethylases, and result in modification of chromatin structure to repress or activate transcription. However, little is known about how histone methylation may regulate defense mechanisms and flowering time in plants. Here we report characterization of JmjC DOMAIN-CONTAINING PROTEIN 27 (JMJ27), an Arabidopsis JHDM2 (JmjC domain-containing histone demethylase 2) family protein, which modulates defense against pathogens and flowering time. JMJ27 is a nuclear protein containing a zinc-finger motif and a catalytic JmjC domain with conserved Fe(II) and α-ketoglutarate binding sites, and displays H3K9me1/2 demethylase activity both in vitro and in vivo. JMJ27 is induced in response to virulent Pseudomonas syringae pathogens and is required for resistance against these pathogens. JMJ27 is a negative modulator of WRKY25 (a repressor of defense) and a positive modulator of several pathogenesis-related (PR) proteins. Additionally, loss of JMJ27 function leads to early flowering. JMJ27 negatively modulates the major flowering regulator CONSTANS (CO) and positively modulates FLOWERING LOCUS C (FLC). Taken together, our results indicate that JMJ27 functions as a histone demethylase to modulate both physiological (defense) and developmental (flowering time) processes in Arabidopsis.

Arabidopsis G-Protein ß Subunit AGB1 Interacts with BES1 to Regulate Brassinosteroid Signaling and Cell Elongation.

In Arabidopsis, brassinosteroids (BR) are major growth-promoting hormones, which integrate with the heterotrimeric guanine nucleotide-binding protein (G-protein) signals and cooperatively modulate cell division and elongation. However, the mechanisms of interaction between BR and G-protein are not well understood. Here, we show that the G-protein ß subunit AGB1 directly interacts with the BR transcription factor BES1 in vitro and in vivo. An AGB1-null mutant, agb1-2, displays BR hyposensitivity and brassinazole (BRZ, BR biosynthesis inhibitor) hypersensitivity, which suggests that AGB1 positively mediates the BR signaling pathway. Moreover, we demonstrate that AGB1 synergistically regulates expression of BES1 target genes, including the BR biosynthesis genes CPD and DWF4 and the SAUR family genes required for promoting cell elongation. Further, Western blot analysis of BES1 phosphorylation states indicates that the interaction between AGB1 and BES1 alters the phosphorylation status of BES1 and increases the ratio of dephosphorylated to phosphorylated BES1, which leads to accumulation of dephosphorylated BES1 in the nucleus. Finally, AGB1 promotes BES1 binding to BR target genes and stimulates the transcriptional activity of BES1. Taken together, our results demonstrate that AGB1 positively regulates cell elongation by affecting the phosphorylation status and transcriptional activity of BES1.

Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry.

Fragaria vesca is a species of diploid strawberry being developed as a model for the octoploid garden strawberry. This work sequenced and compared the genomes of three F. vesca accessions: 'Hawaii 4', 'Rügen', and 'Yellow Wonder'. Genome-scale analyses of shared and distinct SNPs among these three accessions have revealed that 'Rügen' and 'Yellow Wonder' are more similar to each other than they are to 'Hawaii 4'. Though all three accessions are inbred seven generations, each accession still possesses extensive heterozygosity, highlighting the inherent differences between individual plants even of the same accession. The identification of the impact of each SNP as well as the large number of Indel markers provides a foundation for locating candidate mutations underlying phenotypic variations among these F. vesca accessions and for mapping new mutations generated through forward genetics screens. Through systematic analysis of SNP variants affecting genes in anthocyanin biosynthesis and regulation, a candidate SNP in FveMYB10 was identified and then functionally confirmed to be responsible for the yellow color fruits made by many F. vesca accessions. As a whole, this study provides further resources for F. vesca and establishes a foundation for linking traits of economic importance to specific genes and variants.

Grassland root communities: species distributions and how they are linked to aboveground abundance.

There is little comprehensive information on the distribution of root systems among coexisting species, despite the expected importance of those distributions in determining the composition and diversity of plant communities. This gap in knowledge is particularly acute for grasslands, which possess large numbers of species with morphologically indistinguishable roots. In this study we adapted a molecular method, fluorescent fragment length polymorphism, to identify root fragments and determine species root distributions in two grasslands in Yellowstone National Park (YNP). Aboveground biomass was measured, and soil cores (2 cm in diameter) were collected to depths of 40 cm and 90 cm in an upland, dry grassland and a mesic, slope-bottom grassland, respectively, at peak foliar expansion. Cores were subdivided, and species that occurred in each 10-cm interval were identified. The results indicated that the average number of species in 10-cm intervals (31 cm3) throughout the sampled soil profile was 3.9 and 2.8 species at a dry grassland and a mesic grassland, respectively. By contrast, there was an average of 6.7 and 14.1 species per 0.5 m2, determined by the presence of shoot material, at dry and mesic sites, respectively. There was no relationship between soil depth and number of species per 10-cm interval in either grassland, despite the exponential decline of root biomass with soil depth at both sites. There also was no relationship between root frequency (i.e., the percentage of samples in which a species occurred) and soil depth for the vast majority of species at both sites. The preponderance of species were distributed throughout the soil profile at both sites. Assembly analyses indicated that species root occurrences were randomly assorted in all soil intervals at both sites, with the exception that Festuca idahoensis segregated from Artemisia tridentata and Pseudoroegnaria spicata in 10-20 cm soil at the dry grassland. Root frequency throughout the entire sampled soil profile was positively associated with shoot biomass among species. Together these results indicated the importance of large, well-proliferated root systems in establishing aboveground dominance. The findings suggest that spatial belowground segregation of species probably plays a minor role in fostering resource partitioning and species coexistence in these YNP grasslands.