Fluid flow structures gut microbiota biofilm communities by distributing public goods

Bacterial gut commensals experience a biologically and physically complex mucosal environment. While many chemical factors mediate the composition and structure of these microbial communities, less is known about the role of mechanics. Here, we demonstrate that fluid flow impacts the spatial organization and composition of gut biofilm communities by shaping how different species interact metabolically. We first demonstrate that a model community composed of Bacteroides thetaiotaomicron (Bt) and Bacteroides fragilis (Bf), two representative human commensals, can form robust biofilms in flow. We identified dextran as a polysaccharide readily metabolized by Bt but not Bf, but whose fermentation generates a public good enabling Bf growth. By combining simulations with experiments, we demonstrate that in flow, Bt biofilms share dextran metabolic by-products, promoting Bf biofilm formation. By transporting this public good, flow structures the spatial organization of the community, positioning the Bf population downstream from Bt. We show that sufficiently strong flows abolish Bf biofilm formation by limiting the effective public good concentration at the surface. Physical factors such as flow may therefore contribute to the composition of intestinal microbial communities, potentially impacting host health.ISSN:0027-8424ISSN:1091-649

ACA pumps maintain leaf excitability during herbivore onslaught

Recurrent damage by lepidopteran folivores triggers repeated leaf-to-leaf electrical signaling. We found that the ability to propagate electrical signals—called slow wave potentials—was unexpectedly robust and was maintained in plants that had experienced severe damage. We sought genes that maintain tissue excitability during group insect attack. When Arabidopsis thaliana P-Type II Ca2+-ATPase mutants were mechanically wounded, all mutants tested displayed leaf-to-leaf electrical signals. However, when the auto-inhibited Ca2+-ATPase double-mutant aca10 aca12 was attacked by Spodoptera littoralis caterpillars, electrical signaling failed catastrophically, and the insects consumed these plants rapidly. The attacked double mutant displayed petiole base deformation and chlorosis, which spread acropetally into laminas and led to senescence. A phloem-feeding aphid recapitulated these effects, implicating the vasculature in electrical signaling failure. Consistent with this, ACA10 expressed in phloem companion cells in an aca10 aca12 background rescued electrical signaling and defense during protracted S. littoralis attack. When expressed in xylem contact cells, ACA10 partially rescued these phenotypes. Extending our analyses, we found that prolonged darkness also caused wound-response electrical signaling failure in aca10 aca12 mutants. Our results lead to a model in which the plant vasculature acts as a capacitor that discharges temporarily when leaves are subjected to energy-depleting stresses. Under these conditions, ACA10 and ACA12 function allows the restoration of vein cell membrane potentials. In the absence of these gene functions, vascular cell excitability can no longer be restored efficiently. Additionally, this work demonstrates that non-invasive electrophysiology is a powerful tool for probing early events underlying senescence.ISSN:0960-9822ISSN:1879-044

Bacteroides thetaiotaomicron metabolic activity decreases with polysaccharide molecular weight

ABSTRACTThe human colon hosts hundreds of commensal bacterial species, many of which ferment complex dietary carbohydrates. To transform these fibers into metabolically accessible compounds, microbes often express a series of dedicated enzymes homologous to the starch utilization system (Sus) encoded in polysaccharide utilization loci (PULs). The genome of Bacteroides thetaiotaomicron (Bt), a common member of the human gut microbiota, encodes nearly 100 PULs, conferring a strong metabolic versatility. While the structures and functions of individual enzymes within the PULs have been investigated, little is known about how polysaccharide complexity impacts the function of Sus-like systems. We here show that the activity of Sus-like systems depends on polysaccharide size, ultimately impacting bacterial growth. We demonstrate the effect of size-dependent metabolism in the context of dextran metabolism driven by the specific utilization system PUL48. We find that as the molecular weight of dextran increases, Bt growth rate decreases and lag time increases. At the enzymatic level, the dextranase BT3087, a glycoside hydrolase (GH) belonging to the GH family 66, is the main GH for dextran utilization, and BT3087 and BT3088 contribute to Bt dextran metabolism in a size-dependent manner. Finally, we show that the polysaccharide size-dependent metabolism of Bt impacts its metabolic output in a way that modulates the composition of a producer-consumer community it forms with Bacteroides fragilis. Altogether, our results expose an overlooked aspect of Bt metabolism that can impact the composition and diversity of microbiota.IMPORTANCEPolysaccharides are complex molecules that are commonly found in our diet. While humans lack the ability to degrade many polysaccharides, their intestinal microbiota contain bacterial commensals that are versatile polysaccharide utilizers. The gut commensal Bacteroides thetaiotaomicron dedicates roughly 20% of their genomes to the expression of polysaccharide utilization loci for the broad range utilization of polysaccharides. Although it is known that different polysaccharide utilization loci are dedicated to the degradation of specific polysaccharides with unique glycosidic linkages and monosaccharide compositions, it is often overlooked that specific polysaccharides may also exist in various molecular weights. These different physical attributes may impact their processability by starch utilization system-like systems, leading to differing growth rates and nutrient-sharing properties at the community level. Therefore, understanding how molecular weight impacts utilization by gut microbe may lead to the potential design of novel precision prebiotics

Distinct plastid fructose bisphosphate aldolases function in photosynthetic and non-photosynthetic metabolism in Arabidopsis

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin–Benson cycle. Three Arabidopsis genes, AtFBA1–AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.ISSN:1460-2431ISSN:0022-095

BETA-AMYLASE9 is a plastidial nonenzymatic regulator of leaf starch degradation

b-Amylases (BAMs) are key enzymes of transitory starch degradation in chloroplasts, a process that buffers the availability of photosynthetically fixed carbon over the diel cycle to maintain energy levels and plant growth at night. However, during vascular plant evolution, the BAM gene family diversified, giving rise to isoforms with different compartmentation and bio logical activities. Here, we characterized BETA-AMYLASE 9 (BAM9) of Arabidopsis (Arabidopsis thaliana). Among the BAMs, BAM9 is most closely related to BAM4 but is more widely conserved in plants. BAM9 and BAM4 share features in cluding their plastidial localization and lack of measurable a-1,4-glucan hydrolyzing capacity. BAM4 is a regulator of starch degradation, and bam4 mutants display a starch-excess phenotype. Although bam9 single mutants resemble the wild-type (WT), genetic experiments reveal that the loss of BAM9 markedly enhances the starch-excess phenotypes of mutants al ready impaired in starch degradation. Thus, BAM9 also regulates starch breakdown, but in a different way. Interestingly, BAM9 gene expression is responsive to several environmental changes, while that of BAM4 is not. Furthermore, overexpres sion of BAM9 in the WT reduced leaf starch content, but overexpression in bam4 failed to complement fully that mutant’s starch-excess phenotype, suggesting that BAM9 and BAM4 are not redundant. We propose that BAM9 activates starch degradation, helping to manage carbohydrate availability in response to fluctuations in environmental conditions. As such, BAM9 represents an interesting gene target to explore in crop species.ISSN:0032-0889ISSN:1532-254

Like Early Starvation 1 and Early Starvation 1 Promote and Stabilize Amylopectin Phase Transition in Starch Biosynthesis

Starch, the most abundant carbohydrate reserve in plants, primarily consists of the branched glucan amylopectin, which forms semi-crystalline granules. Phase transition from a soluble to an insoluble form depends on amylopectin architecture, requiring a compatible distribution of glucan chain lengths and a branch-point distribution. Here, we show that two starch-bound proteins, LIKE EARLY STARVATION 1 (LESV) and EARLY STARVATION 1 (ESV1), which have unusual carbohydrate-binding surfaces, promote the phase transition of amylopectin-like glucans, both in a heterologous yeast system expressing the starch biosynthetic machinery and in Arabidopsis plants. We propose a model wherein LESV serves as a nucleating role, with its carbohydrate-binding surfaces helping align glucan double helices to promote their phase transition into semi-crystalline lamellae, which are then stabilized by ESV1. Because both proteins are widely conserved, we suggest that protein-facilitated glucan crystallization may be a general and previously unrecognized feature of starch biosynthesis.ISSN:2375-254

Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype

<div><p>Isoamylases hydrolyse (1–6)-alpha-D-glucosidic linkages in starch and are involved in both starch granule formation and starch degradation. In plants, three isoamylase isoforms with distinct functions in starch synthesis (ISA1 and ISA2) and degradation (ISA3) have been described. Here, we created transgenic potato plants with simultaneously decreased expression of all three isoamylases using a chimeric RNAi construct targeting all three isoforms. Constitutive expression of the hairpin RNA using the 35S CaMV promoter resulted in efficient silencing of all three isoforms in leaves, growing tubers, and sprouting tubers. Neither plant growth nor tuber yield was effected in isoamylase-deficient potato lines. Interestingly, starch metabolism was found to be impaired in a tissue-specific manner. While leaf starch content was unaffected, tuber starch was significantly reduced. The reduction in tuber starch content in the transgenic plants was accompanied by a decrease in starch granules size, an increased sucrose content and decreased hexose levels. Despite the effects on granule size, only little changes in chain length composition of soluble and insoluble glucose polymers were detected. The transgenic tubers displayed an early sprouting phenotype that was accompanied by an increased level of sucrose in parenchyma cells below the outgrowing bud. Since high sucrose levels promote sprouting, we propose that the increased number of small starch granules may cause an accelerated turnover of glucan chains and hence a more rapid synthesis of sucrose. This observation links alterations in starch structure/degradation with developmental processes like meristem activation and sprout outgrowth in potato tubers.</p></div

Impact of <i>ISA</i> silencing on tuber sprouting.

<p>Tubers of 5 plants each from wild type (WT) and transgenic lines 7, 16 and 39 were stored after harvest at room temperature in darkness. A) Sprouting kinetics. To monitor the impact on dormancy length, 2–5 similar sized tubers from each plant were picked (n = 13–20) and their sprouting behaviour was regularly scored over a 15-week period until 100% sprouting had been reached in wild-type tubers. A tuber was considered to sprout when sprouts of 2 mm length became visible. B) Photographs of transgenic (lines 7, 16, 39) and control tubers taken after 13 weeks of storage showing that the transgenic lines sprout earlier than the wild-type controls (WT). C) Number of sprouts per tuber. Number of sprouts formed per tuber were counted from 13–20 individual tubers. Values represent the mean +/- SE. Significant differences to wild type were calculated using two-tailed t-test assuming equal variances and are indicated by asterisks (**p<0.01, *p<0.05).</p

Starch structure determined as relative chain length distribution.

<p>Starch from sprouting tubers was either isolated from parenchyma associated with the sprout (A) or (B) from parenchyma not associated with the sprout. Line 7 (blue), line 16 (red), line 39 (grey) and the wild-type control (WT; black). Values represent mean +/- SE of 3–4 biological replicates.</p

Schematic representation of the chimeric RNAi hairpin construct.

<p>Schematic representation of the chimeric RNAi hairpin construct.</p