CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.

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CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against invasive phages through general, non-specific RNA degradation upon complementary target transcript binding. Despite being RNA nucleases, Cas13 effectors are capable of inhibiting the infection of dsDNA phages but have only been investigated across a relatively small sampling of phage diversity. Here, we employ a systematic, phage-centric approach to determine the anti-phage capacity of Cas13 and find LbuCas13a to be a remarkably potent phage inhibitor. LbuCas13a confers robust, consistent antiviral activity regardless of gene essentiality, gene expression timing or target sequence location. Furthermore, after challenging LbuCas13a with eight diverse E. coli phages distributed across E. coli phage phylogenetic groups, we find no apparent phage-encoded limits to its potent antiviral activity. In contrast to other Class 2 CRISPR-Cas proteins, these results suggest that DNA phages are generally vulnerable to Cas13a targeting. Leveraging this effective anti-phage activity, LbuCas13a can be used seamlessly as a counter-selection agent for broad-spectrum phage editing. Using a two-step phage editing and enrichment approach, we show that LbuCas13a enables markerless genome edits in phages with exceptionally high efficiency and precision, including edits as small as a single codon. By taking advantage of the broad vulnerability of RNA during viral infection, Cas13a enables a generalizable strategy for editing the most abundant and diverse biological entities on Earth.

Biological sulfate reduction (BSR) is an attractive approach for the bioremediation of sulfate-rich wastewater streams. Many sulfate-reducing microorganisms (SRM), which facilitate this process, have been well-studied in pure culture. However, the role of individual members of microbial communities within BSR bioreactors remains understudied. In this study we investigated the performance of two up-flow anaerobic packed bed reactors (UAPBRs) supplemented primarily with acetate and with lactate, respectively, during a hydraulic retention time (HRT) study set up to remediate sulfate-rich synthetic wastewater over the course of 1,000 + days. Plug-flow hydrodynamics led to a continuum of changing volumetric sulfate reduction rates (VSRRs), available electron donors, degrees of biomass retention and compositions of microbial communities throughout these reactors. Microbial communities throughout the successive zones of the reactors were resolved using 16S rRNA gene amplicon sequencing which allowed the association of features of performance with discrete microorganisms. The acetate UAPBR achieved a maximum VSRR of 23.2 mg.L−1. h−1 at a one-day HRT and a maximum sulfate conversion of the 1 g/L sulfate of 96% at a four-day HRT. The sulfate reduction reactions in this reactor could be described with a reaction order of 2.9, an important observation for optimisation and future scale-up. The lactate UAPBR achieved a 96% sulfate conversion at one-day HRT, corresponding with a VSRR of 40.1 mg.L−1. h−1. Lactate was supplied in this reactor at relatively low concentrations necessitating the subsequent use of propionate and acetate, by-products of lactate fermentation with acetate also a by-product of incomplete lactate oxidation, to achieve competitive performance. The consumption of these electron donors could be associated with specific SRM localised within biofilms of discrete zones. The sulfate reduction rates in the lactate UAPBR could be modelled as first-order reactions, indicating effective rates were conferred by these propionate- and acetate-oxidising SRM. Our results demonstrate how acetate, a low-cost substrate, can be used effectively despite low associated SRM growth rates, and that lactate, a more expensive substrate, can be used sparingly to achieve high VSRR and sulfate conversions. We further identified the preferred environment of additional microorganisms to inform how these microorganisms could be enriched or diminished in BSR reactors.

Biological sulfate reduction (BSR) represents a promising bioremediation strategy, yet the impact of metabolic interactions on performance has been largely unexplored. Here, genome-resolved metagenomics was used to characterise 17 microbial communities associated with reactors operated with defined sulfate-contaminated solutions. Pairs of reactors were supplemented with lactate or with acetate plus a small amount of fermentable substrate. At least thirty draft quality genomes, representing all the abundant bacteria, were recovered from each metagenome. All of the 22 SRB genomes encode genes for H2 consumption. And of the total 163 genomes recovered, 130 encode 321 NiFe and FeFe hydrogenases. The lactate-supplemented packed-bed bioreactor was particularly interesting as it resulted in stratified microbial communities that were distinct in their predominant metabolisms. Pathways for fermentation of lactate and hydrogen production were enriched towards the inlet whereas increased autotrophy and acetate-oxidizing SRB were evident towards the end of the flow path. We hypothesized that high sulfate removal towards the end of the flow path, despite acetate being an electron donor that typically sustains low SRB growth rates, was stimulated by H2 consumption. This hypothesis was supported by sustained performance of the predominantly acetate-supplemented stirred-tank reactor, which was dominated by diverse fermentative, hydrogen-evolving bacteria and low-abundance SRB capable of acetate and hydrogen consumption. We conclude that the performance of BSR reactors supplemented with inexpensive acetate can be improved by the addition of a low concentration of fermentable material due to stimulation of syntrophic relationships among hydrogen-producing non-SRB and dual hydrogen- and acetate-utilising SRB.

Microbial communities are fundamental components of most ecosystems but little is known about the interactions that structure them. Metagenomic data have revolutionized our understanding of complex consortia, yet predicted interactions are rarely explored experimentally. We developed an organism abundance correlation network to investigate factors that control community organization in soil-derived laboratory microbial consortia grown under dozens of conditions. The network was overlaid with metagenomic information about functional capacities to generate testable hypotheses. We developed a metric to predict the importance of each node within its local network environments relative to correlated vitamin auxotrophs and predicted that a Variovorax species is a hub because it is a highly important source of thiamine. Quantification of thiamine during the growth of Variovorax in minimal media conditions showed extraordinarily high levels of production of thiamine, up to 100 mg/L. This over-production of thiamine explains why the auxotrophs correlated with Variovorax are reliant on this organism for thiamine, despite the presence of other predicted thiamine producers in the community. A few of these thiamine auxotrophs are predicted to produce pantothenate that we show that Variovorax requires to grow, implying that a subset of vitamin-dependent interactions are mutualistic. We found that Cryptococcus produces the B-vitamin pantothenate, and co-cultures led to a 90-130-fold fitness increase for both Cryptococcus and Variovorax. This study demonstrates the predictive power of metagenome-informed, microbial consortia-based network analyses for identifying microbial interactions that underpin the structure and functioning of microbial communities.

Biological sulfate reduction represents an alternative and sustainable option to reduce the high sulfate load, precipitate heavy metals and neutralise the acidity associated with acid rock drainage (ARD). Sulfate-reducing enrichment cultures have been developed on simple and complex electron donors from several environmental samples and used to inoculate three reactor configurations, namely a continuous stirred tank bioreactor, up-flow anaerobic packed bed reactor and a linear flow channel reactor, with varying degrees of biomass retention provided by carbon microfibres and polyurethane foam. These matrices are included to enhance microbial attachment and colonisation, allowing for the decoupling of hydraulic retention time and biomass retention time. The bioreactor systems are operated under increasingly stringent conditions through the reduction in the hydraulic residence time. The biological sulfate reduction performance and the biomass concentration of planktonic, matrix-attached and matrix-associated communities are routinely monitored. This investigation makes use of biomass quantification of the planktonic community and, following detachment, the matrix-associated community to investigate the resultant microbial communities in these reactor systems. Evaluation of these mixed microbial communities, and their link to process performance, provides an opportunity to impact the design and operation of pilot- and industrial-scale bioprocesses.