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).

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.

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.

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.

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.

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.