Genetic manipulation of Patescibacteria provides mechanistic insights into microbial dark matter and the epibiotic lifestyle.

Patescibacteria, also known as the candidate phyla radiation (CPR), are a diverse group of bacteria that constitute a disproportionately large fraction of microbial dark matter. Its few cultivated members, belonging mostly to Saccharibacteria, grow as epibionts on host Actinobacteria. Due to a lack of suitable tools, the genetic basis of this lifestyle and other unique features of Patescibacteira remain unexplored. Here, we show that Saccharibacteria exhibit natural competence, and we exploit this property for their genetic manipulation. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth, and a transposon-insertion sequencing (Tn-seq) genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii, as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

Bifunctional Immunity Proteins Protect Bacteria against FtsZ-Targeting ADP-Ribosylating Toxins.

ADP-ribosylation of proteins can profoundly impact their function and serves as an effective mechanism by which bacterial toxins impair eukaryotic cell processes. Here, we report the discovery that bacteria also employ ADP-ribosylating toxins against each other during interspecies competition. We demonstrate that one such toxin from Serratia proteamaculans interrupts the division of competing cells by modifying the essential bacterial tubulin-like protein, FtsZ, adjacent to its protomer interface, blocking its capacity to polymerize. The structure of the toxin in complex with its immunity determinant revealed two distinct modes of inhibition: active site occlusion and enzymatic removal of ADP-ribose modifications. We show that each is sufficient to support toxin immunity; however, the latter additionally provides unprecedented broad protection against non-cognate ADP-ribosylating effectors. Our findings reveal how an interbacterial arms race has produced a unique solution for safeguarding the integrity of bacterial cell division machinery against inactivating post-translational modifications.

A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

Human gut bacteria contain acquired interbacterial defence systems.

The human gastrointestinal tract consists of a dense and diverse microbial community, the composition of which is intimately linked to health. Extrinsic factors such as diet and host immunity are insufficient to explain the constituents of this community, and direct interactions between co-resident microorganisms have been implicated as important drivers of microbiome composition. The genomes of bacteria derived from the gut microbiome contain several pathways that mediate contact-dependent interbacterial antagonism1-3. Many members of the Gram-negative order Bacteroidales encode the type VI secretion system (T6SS), which facilitates the delivery of toxic effector proteins into adjacent cells4,5. Here we report the occurrence of acquired interbacterial defence (AID) gene clusters in Bacteroidales species that reside within the human gut microbiome. These clusters encode arrays of immunity genes that protect against T6SS-mediated intra- and inter-species bacterial antagonism. Moreover, the clusters reside on mobile elements, and we show that their transfer is sufficient to confer resistance to toxins in vitro and in gnotobiotic mice. Finally, we identify and validate the protective capability of a recombinase-associated AID subtype (rAID-1) that is present broadly in Bacteroidales genomes. These rAID-1 gene clusters have a structure suggestive of active gene acquisition and include predicted immunity factors of toxins derived from diverse organisms. Our data suggest that neutralization of contact-dependent interbacterial antagonism by AID systems helps to shape human gut microbiome ecology.

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 µg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

Cross-species comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei quorum-sensing regulons.

Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei.

Genetic manipulation of candidate phyla radiation bacteria provides functional insights into microbial dark matter.

The study of bacteria has yielded fundamental insights into cellular biology and physiology, biotechnological advances and many therapeutics. Yet due to a lack of suitable tools, the significant portion of bacterial diversity held within the candidate phyla radiation (CPR) remains inaccessible to such pursuits. Here we show that CPR bacteria belonging to the phylum Saccharibacteria exhibit natural competence. We exploit this property to develop methods for their genetic manipulation, including the insertion of heterologous sequences and the construction of targeted gene deletions. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth and a transposon insertion sequencing genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their Actinobacteria hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii , as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Identification of a p-coumarate degradation regulon in Rhodopseudomonas palustris by Xpression, an integrated tool for prokaryotic RNA-seq data processing.

High-throughput sequencing of cDNA prepared from RNA, an approach known as RNA-seq, is coming into increasing use as a method for transcriptome analysis. Despite its many advantages, widespread adoption of the technique has been hampered by a lack of easy-to-use, integrated, open-source tools for analyzing the nucleotide sequence data that are generated. Here we describe Xpression, an integrated tool for processing prokaryotic RNA-seq data. The tool is easy to use and is fully automated. It performs all essential processing tasks, including nucleotide sequence extraction, alignment, quantification, normalization, and visualization. Importantly, Xpression processes multiplexed and strand-specific nucleotide sequence data. It extracts and trims specific sequences from files and separately quantifies sense and antisense reads in the final results. Outputs from the tool can also be conveniently used in downstream analysis. In this paper, we show the utility of Xpression to process strand-specific RNA-seq data to identify genes regulated by CouR, a transcription factor that controls p-coumarate degradation by the bacterium Rhodopseudomonas palustris.

Genome Capture Sequencing Selectively Enriches Bacterial DNA and Enables Genome-Wide Measurement of Intrastrain Genetic Diversity in Human Infections.

Within-host evolution produces genetic diversity in bacterial strains that cause chronic human infections. However, the lack of facile methods to measure bacterial allelic variation in clinical samples has limited understanding of intrastrain diversity's effects on disease. Here, we report a new method termed genome capture sequencing (GenCap-Seq) in which users inexpensively make hybridization probes from genomic DNA or PCR amplicons to selectively enrich and sequence targeted bacterial DNA from clinical samples containing abundant human or nontarget bacterial DNA. GenCap-Seq enables accurate measurement of allele frequencies over targeted regions and is scalable from specific genes to entire genomes, including the strain-specific accessory genome. The method is effective with samples in which target DNA is rare and inhibitory and DNA-degrading substances are abundant, including human sputum and feces. In proof-of-principle experiments, we used GenCap-Seq to investigate the responses of diversified Pseudomonas aeruginosa populations chronically infecting the lungs of people with cystic fibrosis to in vivo antibiotic exposure, and we found that treatment consistently reduced intrastrain genomic diversity. In addition, analysis of gene-level allele frequency changes suggested that some genes without conventional resistance functions may be important for bacterial fitness during in vivo antibiotic exposure. GenCap-Seq's ability to scalably enrich targeted bacterial DNA from complex samples will enable studies on the effects of intrastrain and intraspecies diversity in human infectious disease. IMPORTANCE Genetic diversity evolves in bacterial strains during human infections and could affect disease manifestations and treatment resistance. However, the extent of diversity present in vivo and its changes over time are difficult to measure by conventional methods. We developed a novel approach, GenCap-Seq, to enrich microbial DNA from complex human samples like sputum and feces for genome-wide measurements of bacterial allelic diversity. The approach is inexpensive, scalable to encompass entire targeted genomes, and works in the presence of abundant untargeted nucleic acids and inhibiting substances. We used GenCap-Seq to investigate in vivo responses of diversified bacterial strains to antibiotic treatment. This method will enable new ideas about the effects of intrastrain diversity on human infections to be tested.

Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism.

Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that Pseudomonas aeruginosa possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.

Genome-wide protein-DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase.

DNA-protein interactions are central to fundamental cellular processes, yet widely implemented technologies for measuring these interactions on a genome scale in bacteria are laborious and capture only a snapshot of binding events. We devised a facile method for mapping DNA-protein interaction sites in vivo using the double-stranded DNA-specific cytosine deaminase toxin DddA. In 3D-seq (DddA-sequencing), strains containing DddA fused to a DNA-binding protein of interest accumulate characteristic mutations in DNA sequence adjacent to sites occupied by the DNA-bound fusion protein. High-depth sequencing enables detection of sites of increased mutation frequency in these strains, yielding genome-wide maps of DNA-protein interaction sites. We validated 3D-seq for four transcription regulators in two bacterial species, Pseudomonas aeruginosa and Escherichia coli. We show that 3D-seq offers ease of implementation, the ability to record binding event signatures over time and the capacity for single-cell resolution.

An interbacterial DNA deaminase toxin directly mutagenizes surviving target populations.

When bacterial cells come in contact, antagonism mediated by the delivery of toxins frequently ensues. The potential for such encounters to have long-term beneficial consequences in recipient cells has not been investigated. Here, we examined the effects of intoxication by DddA, a cytosine deaminase delivered via the type VI secretion system (T6SS) of Burkholderia cenocepacia. Despite its killing potential, we observed that several bacterial species resist DddA and instead accumulate mutations. These mutations can lead to the acquisition of antibiotic resistance, indicating that even in the absence of killing, interbacterial antagonism can have profound consequences on target populations. Investigation of additional toxins from the deaminase superfamily revealed that mutagenic activity is a common feature of these proteins, including a representative we show targets single-stranded DNA and displays a markedly divergent structure. Our findings suggest that a surprising consequence of antagonistic interactions between bacteria could be the promotion of adaptation via the action of directly mutagenic toxins.

A population-level strain genotyping method to study pathogen strain dynamics in human infections.

A hallmark of chronic bacterial infections is the long-term persistence of 1 or more pathogen species at the compromised site. Repeated detection of the same bacterial species can suggest that a single strain or lineage is continually present. However, infection with multiple strains of a given species, strain acquisition and loss, and changes in strain relative abundance can occur. Detecting strain-level changes and their effects on disease is challenging because most methods require labor-intensive isolate-by-isolate analyses, and thus, only a few cells from large infecting populations can be examined. Here, we present a population-level method for enumerating and measuring the relative abundance of strains called population multi-locus sequence typing (PopMLST). The method exploits PCR amplification of strain-identifying polymorphic loci, next-generation sequencing to measure allelic variants, and informatic methods to determine whether variants arise from sequencing errors or low-abundance strains. These features enable PopMLST to simultaneously interrogate hundreds of bacterial cells that are cultured en masse from patient samples or are present in DNA directly extracted from clinical specimens without ex vivo culture. This method could be used to detect epidemic or super-infecting strains, facilitate understanding of strain dynamics during chronic infections, and enable studies that link strain changes to clinical outcomes.

Combining Ivacaftor and Intensive Antibiotics Achieves Limited Clearance of Cystic Fibrosis Infections.

Drugs called CFTR modulators improve the physiologic defect underlying cystic fibrosis (CF) and alleviate many disease manifestations. However, studies to date indicate that chronic lung infections that are responsible for most disease-related mortality generally persist. Here, we investigated whether combining the CFTR modulator ivacaftor with an intensive 3.5-month antibiotic course could clear chronic Pseudomonas aeruginosa or Staphylococcus aureus lung infections in subjects with R117H-CFTR, who are highly ivacaftor-responsive. Ivacaftor alone improved CFTR activity, and lung function and inflammation within 48 h, and reduced P. aeruginosa and S. aureus pathogen density by ∼10-fold within a week. Antibiotics produced an additional ∼10-fold reduction in pathogen density, but this reduction was transient in subjects who remained infected. Only 1/5 P. aeruginosa-infected and 1/7 S. aureus-infected subjects became persistently culture-negative after the combined treatment. Subjects appearing to clear infection did not have particularly favorable baseline lung function or inflammation, pathogen density or antibiotic susceptibility, or bronchiectasis scores on CT scans, but they did have remarkably low sweat chloride values before and after ivacaftor. All persistently P. aeruginosa-positive subjects remained infected by their pretreatment strain, whereas subjects persistently S. aureus-positive frequently lost and gained strains. This work suggests chronic CF infections may resist eradication despite marked and rapid modulator-induced improvements in lung infection and inflammation parameters and aggressive antibiotic treatment. IMPORTANCE Recent work shows that people with CF and chronic lung infections generally remain persistently infected after treatment with drugs that target the CF physiological defect (called CFTR modulators). However, changes produced by modulators could increase antibiotic efficacy. We tested the approach of combining modulators and intensive antibiotics in rapid succession and found that while few subjects cleared their infections, combined treatment appeared most effective in subjects with the highest CFTR activity. These findings highlight challenges that remain to improve the health of people with CF.

Targeted Depletion of Bacteria from Mixed Populations by Programmable Adhesion with Antagonistic Competitor Cells.

Selective and targeted removal of individual species or strains of bacteria from complex communities can be desirable over traditional, broadly acting antibacterials in several contexts. However, generalizable strategies that accomplish this with high specificity have been slow to emerge. Here we develop programmed inhibitor cells (PICs) that direct the potent antibacterial activity of the type VI secretion system (T6SS) against specified target cells. The PICs express surface-displayed nanobodies that mediate antigen-specific cell-cell adhesion to effectively overcome the barrier to T6SS activity in fluid conditions. We demonstrate the capacity of PICs to efficiently deplete low-abundance target bacteria without significant collateral damage to complex microbial communities. The only known requirements for PIC targeting are a Gram-negative cell envelope and a unique cell surface antigen; therefore, this approach should be generalizable to a wide array of bacteria and find application in medical, research, and environmental settings.

Human and Extracellular DNA Depletion for Metagenomic Analysis of Complex Clinical Infection Samples Yields Optimized Viable Microbiome Profiles.

Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens.

Conditional toxicity and synergy drive diversity among antibacterial effectors.

Bacteria in polymicrobial habitats contend with a persistent barrage of competitors, often under rapidly changing environmental conditions 1 . The direct antagonism of competitor cells is thus an important bacterial survival strategy 2 . Towards this end, many bacterial species employ an arsenal of antimicrobial effectors with multiple activities; however, the benefits conferred by the simultaneous deployment of diverse toxins are unknown. Here we show that the multiple effectors delivered to competitor bacteria by the type VI secretion system (T6SS) of Pseudomonas aeruginosa display conditional efficacy and act synergistically. One of these effectors, Tse4, is most active in high-salinity environments and synergizes with effectors that degrade the cell wall or inactivate intracellular electron carriers. We find Tse4 synergizes with these disparate mechanisms by forming pores that disrupt the ΔΨ component of the proton motive force. Our results provide evidence that the concomitant delivery of a cocktail of effectors serves as a bet-hedging strategy to promote bacterial competitiveness in the face of unpredictable and variable environmental conditions.

Ixodes scapularis does not harbor a stable midgut microbiome.

Hard ticks of the order Ixodidae serve as vectors for numerous human pathogens, including the causative agent of Lyme Disease Borrelia burgdorferi. Tick-associated microbes can influence pathogen colonization, offering the potential to inhibit disease transmission through engineering of the tick microbiota. Here, we investigate whether B. burgdorferi encounters abundant bacteria within the midgut of wild adult Ixodes scapularis, its primary vector. Through the use of controlled sequencing methods and confocal microscopy, we find that the majority of field-collected adult I. scapularis harbor limited internal microbial communities that are dominated by endosymbionts. A minority of I. scapularis ticks harbor abundant midgut bacteria and lack B. burgdorferi. We find that the lack of a stable resident midgut microbiota is not restricted to I. scapularis since extension of our studies to I. pacificus, Amblyomma maculatum, and Dermacentor spp showed similar patterns. Finally, bioinformatic examination of the B. burgdorferi genome revealed the absence of genes encoding known interbacterial interaction pathways, a feature unique to the Borrelia genus within the phylum Spirochaetes. Our results suggest that reduced selective pressure from limited microbial populations within ticks may have facilitated the evolutionary loss of genes encoding interbacterial competition pathways from Borrelia.