Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.Biological and Environmental Research/[DE-FC02-07ER64494]/BER/Estados UnidosNational Science Foundation/[DGE-1256259]/NSF/Estados UnidosNational Science Foundation/[DEB-0747002]/NSF/Estados UnidosNational Science Foundation/[MCB-0702025]/NSF/Estados UnidosNational Institutes of Health/[T32 GM07215]/NIH/Estados UnidosUniversidad de Costa Rica/[]/UCR/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[]/MICITT/Costa RicaUniversity of Wisconsin-Madison's Hilldale Undergraduate Faculty Research Fellowship/[]//Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Aerobic deconstruction of cellulosic biomass by an insect-associated Streptomyces

Abstract: Streptomyces are best known for producing antimicrobial secondary metabolites, but they are also recognized for their contributions to biomass utilization. Despite their importance to carbon cycling in terrestrial ecosystems, our understanding of the cellulolytic ability of Streptomyces is currently limited to a few soil-isolates. Here, we demonstrate the biomass-deconstructing capability of Streptomyces sp. SirexAA-E (ActE), an aerobic bacterium associated with the invasive pine-boring woodwasp Sirex noctilio. When grown on plant biomass, ActE secretes a suite of enzymes including endo- and exo-cellulases, CBM33 polysaccharide-monooxygenases, and hemicellulases. Genome-wide transcriptomic and proteomic analyses, and biochemical assays have revealed the key enzymes used to deconstruct crystalline cellulose, other pure polysaccharides, and biomass. The mixture of enzymes obtained from growth on biomass has biomass-degrading activity comparable to a cellulolytic enzyme cocktail from the fungus Trichoderma reesei, and thus provides a compelling example of high cellulolytic capacity in an aerobic bacterium

Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection

Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes

Evolution of High Cellulolytic Activity in Symbiotic <i>Streptomyces</i> through Selection of Expanded Gene Content and Coordinated Gene Expression

<div><p>The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in <i>Streptomyces</i>, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the <i>Streptomyces</i> genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in <i>Streptomyces</i>, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in <i>Streptomyces</i>, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic <i>Streptomyces</i> for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.</p></div

PCoA clustering of Morisita-Horn Diversity Index.

<p>Sample shape indicates colony. Sample color indicates degradation (<b>A</b>) or layer (<b>B</b>). Panel <b>C</b> shows the correlation analysis. The vectors indicate the correlation of each OTU and the percentage of cellulose degradation with the principal coordinates shown.</p

Morisita-Horn Beta Diversity Clustering of Samples.

<p>The corresponding percentage of cellulose degradation, colony, layer, and taxonomic classification of OTUs are shown for each sample.</p

Correlation between number of reads and cellulose degradation by the community for OTU1 (<i>Acidovorax</i>) and OTU9 (<i>Ferruginibacter</i>) across sequenced samples.

<p>Correlation between number of reads and cellulose degradation by the community for OTU1 (<i>Acidovorax</i>) and OTU9 (<i>Ferruginibacter</i>) across sequenced samples.</p

Comparison of degradation ability across colonies and layers.

<p><b>(A and B</b>) Qualitative Assay Data. Test tubes containing carbon-free minimal media and a strip of cellulosic filter paper were used to enrich for cellulolytic communities. Failure plots, indicating when the filter paper broke apart in each culture, were fit with Kaplan Meier curves and analyzed using the Wilcoxon method to determine significant differences among colonies (indicated by letters A-C) and layers (no significant differences). (<b>C and D)</b> Quantitative Assay Data. Pre-weighed, submerged cellulosic filter paper allowed quantification of cellulose degradation after 10 days. Samples are grouped by colony or dump layer. Error bars represent one standard error from the mean. Significant differences were determined using Tukey’s HSD test and are indicated above the data. <b>Photo credits:</b> Gina Lewin (A, C).</p