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Prevalence and number of invertons per genome are enriched in host-associated species. (A and B) The percentage of genomes identified with invertons (A) and the number of invertons per genome (B) from aquatic, terrestrial, and host-associated isolates. (C and D) In the phylum Bacteroidetes, the percentage of genomes identified with invertons (C) and the number of invertons per genome (D) from aquatic, terrestrial, host sites other than gut, and host gut-associated isolates. **P ≤ 0.01, ***P ≤ 0.001.
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Switching ON resistance
Clonal bacterial colonies will often grow dissimilar patches, similar to a tortoiseshell pattern. These differing phenotypes arise by reversible mechanisms called phase variation. Jiang et al. developed an algorithm to survey bacterial genomes for invertible promoters that cause phase variation. Inverted repeats signal the p...
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... predominantly found in one orientation within an individual with little or no fluctuation (mean > 95% and min > 75% for either the F or R ori- entation) (Fig. 4A); the orientations of 59 were relatively stable (max-min %R ≤ 50%) within an individual (Fig. 4B), whereas the orientations of 42 were unstable (max-min %R > 50%) within an individual (Fig. 4C and table ...
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... the orientations of 90% of invertons in the same individual were relatively stable over time, the orientations between individuals varied extensively ( Fig. 4D and fig. S10). The mean %R orientation of 214 out of 423 of the invertons varied by more than 50% between individuals. In 122 examples, averaging across time, the inver- ton was predominantly (>95%) in the F orienta- tion in at least one individual and predominantly (>95%) in the R orientation in another. Addi- tionally, 119 out of 238 invertons ...
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... we identified strains from the donor that engrafted into the patient's microbiome. To identify engraftment, we found cases where the same strain was present in the donor and in at least one patient after FMT, but absent in the same patient(s) before FMT (Fig. 4, E, G, and I, and fig. S11). We found three high-abundance species with invertons from the donor that en- grafted in patients: B. fragilis, B. vulgatus, and B. ...
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... we compared the orientations of in- vertons from the donor and patient after en- graftment and found that the orientations of 42.8% of invertons (48/112) diverged from the donor orientation (Wilcoxon rank sum test, FDR P < 0.05) (Fig. 4, F, H, and J, and fig. S12). In B. fragilis, two invertons (IBP183 and IBP198) engrafted in the opposite orientation of the donor strain and remained predominantly (87.2% and 87.1%) in the R orientation, but drifted toward F near the end of the sampling (Fig. 4F). For B. ovatus, a strain existed in Patient 014 before FMT but was replaced by the donor B. ovatus ...
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... (90.1% and 100%) in the R orientation, whereas the newly engrafted strain was oriented entirely in the F orientation and remained in the F orienta- tion over the course of sampling (Fig. 4H). In B. vulgatus, an inverton was initially present completely in the F orientation but over the course of 145 days completely reversed to the R orientation (Fig. 4J, IBP121). In addition to the invertons that reversed their orientations, we also identified examples of invertons that main- tained their orientations (Fig. 4F, IBP189; Fig. 4H, IBP166; Fig. 4J, ...
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... the course of sampling (Fig. 4H). In B. vulgatus, an inverton was initially present completely in the F orientation but over the course of 145 days completely reversed to the R orientation (Fig. 4J, IBP121). In addition to the invertons that reversed their orientations, we also identified examples of invertons that main- tained their orientations (Fig. 4F, IBP189; Fig. 4H, IBP166; Fig. 4J, ...
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... (Fig. 4H). In B. vulgatus, an inverton was initially present completely in the F orientation but over the course of 145 days completely reversed to the R orientation (Fig. 4J, IBP121). In addition to the invertons that reversed their orientations, we also identified examples of invertons that main- tained their orientations (Fig. 4F, IBP189; Fig. 4H, IBP166; Fig. 4J, ...
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... B. vulgatus, an inverton was initially present completely in the F orientation but over the course of 145 days completely reversed to the R orientation (Fig. 4J, IBP121). In addition to the invertons that reversed their orientations, we also identified examples of invertons that main- tained their orientations (Fig. 4F, IBP189; Fig. 4H, IBP166; Fig. 4J, ...
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We assumed that the enrichment of successful gut microbes by lantibiotic/antibiotic resistance genes can be related to gut microbiota colonization resistance by third-party microbe phenomena and resistance to bacterium-derived or host-derived antimicrobial substances. According to this assumption, competition between the donor-derived and recipient...
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... A key evolved feature of these bacteria is the ability of each strain to produce numerous (eight or more) distinct capsular polysaccharides (CPS) 1,2 that are tightly regulated so that only one CPS is typically produced per bacterial cell. This bet-hedging strategy generates Bacteroides populations with great surface variability that protect from phage [3][4][5] and mediate immune modulation, biofilm formation, antibiotic resistance, and inflammation [6][7][8][9][10][11] . CPS diversity is achieved by regulating both transcription initiation and elongation of CPS biosynthesis operons. ...
The genomes of human gut bacteria in the genus Bacteroides include numerous operons for biosynthesis of diverse capsular polysaccharides (CPSs). The first two genes of each CPS operon encode a locus-specific paralog of transcription elongation factor NusG (called UpxY), which enhances transcript elongation, and a UpxZ protein that inhibits noncognate UpxYs. This process, together with promoter inversions, ensures that a single CPS operon is transcribed in most cells. Here, we use in-vivo nascent-RNA sequencing and promoter-less in-vitro transcription (PIVoT) to show that UpxY recognizes a paused RNA polymerase via sequences in both the exposed non-template DNA and the upstream duplex DNA. UpxY association is aided by ‘pause-then-escape’ nascent RNA hairpins. UpxZ binds non-cognate UpxYs to directly inhibit UpxY association. This UpxY-UpxZ hierarchical regulatory program allows Bacteroides to generate subpopulations of cells producing diverse CPSs for optimal fitness.
... Since then, computational approaches have enabled higher-throughput discovery of these 'invertons' across the genomes of a small subset of specific bacterial species 12,13 . In 2019, Jiang et al. developed a method that facilitated broad-scale identification of 4,686 intergenic invertons (invertons between genes) in 54,875 bacterial reference genomes 14 , using short-read mapping as evidence. Similarly, in early 2023, Milman et al. 15 used a computational model to predict more than 11,000 potential invertons that partially overlap with genes (partial intergenic) in more than 35,000 bacterial species. ...
... Within these complex samples, we first chose to examine invertons in organisms within the taxon Bacteroidetes, because they are prevalent and abundant in the human gut and they are known to have intergenic invertons 14 . So that we could orthogonally confirm sequencing-based observations in subsequent microbiological and genetic experiments, we focused our analysis on B. thetaiotaomicron VPI-5482 (BTh), a genetically tractable strain that is suitable for downstream experimental manipulation. ...
... Of note, compared with Jiang et al. 14 , we find invertons at a higher rate per sequencing dataset (0.15 versus 0.07) and per individual genome (1.15 versus 0.09), highlighting the increased sensitivity of long reads for detecting this type of structural variation. Similar to Jiang et al. 14 , we found that Bacteroidetes have a relatively large number of intergenic invertons (673; Fig. 2a) and intergenic invertons per genome (2.18; Fig. 2b). Fusobacteria, Gammaproteobacteria and Verrucomicrobia also have high numbers of intergenic invertons per genome (Fig. 2b), with Verrucomicrobia having the highest number per genome overall at 3.13 intergenic invertons per genome. ...
Bacterial populations that originate from a single bacterium are not strictly clonal and often contain subgroups with distinct phenotypes¹. Bacteria can generate heterogeneity through phase variation—a preprogrammed, reversible mechanism that alters gene expression levels across a population¹. One well-studied type of phase variation involves enzyme-mediated inversion of specific regions of genomic DNA². Frequently, these DNA inversions flip the orientation of promoters, turning transcription of adjacent coding regions on or off². Through this mechanism, inversion can affect fitness, survival or group dynamics3,4. Here, we describe the development of PhaVa, a computational tool that identifies DNA inversions using long-read datasets. We also identify 372 ‘intragenic invertons’, a novel class of DNA inversions found entirely within genes, in genomes of bacterial and archaeal isolates. Intragenic invertons allow a gene to encode two or more versions of a protein by flipping a DNA sequence within the coding region, thereby increasing coding capacity without increasing genome size. We validate ten intragenic invertons in the gut commensal Bacteroides thetaiotaomicron, and experimentally characterize an intragenic inverton in the thiamine biosynthesis gene thiC.
... Bacterial populations display phenotypic heterogeneity, an evolutionarily conserved phenomenon that facilitates complex community behaviors despite minimal genetic diversity and potentially small population sizes of just a few dozen individuals. This phenotypic diversity is especially critical during infection, enabling monoclonal pathogen populations to persist [120], thrive [116], and adapt [121] within challenging and variable environmental conditions prior to and during antibiotic treatment. ...
Bacterial nanomachines represent remarkable feats of evolutionary engineering, showcasing intricate molecular mechanisms that enable bacteria to perform a diverse array of functions essential to persist, thrive, and evolve within ecological and pathological niches. Injectosomes and bacterial flagella represent two categories of bacterial nanomachines that have been particularly well studied both at the molecular and functional levels. Among the diverse functionalities of these nanomachines, bistability emerges as a fascinating phenomenon, underscoring their dynamic and complex regulation as well as their contribution to shaping the bacterial community behavior during the infection process. In this review, we examine two closely related bacterial nanomachines, the type 3 secretion system, and the flagellum, to explore how the bistability of molecular-scale devices shapes the bacterial eco-pathological life cycle.
... The average time for any pressure to flip would thus be approximately every 2 years, or less frequently if adaptive events occur in bursts (e.g., upon transmission to a new host). While 55 loci under distinct selective pressures may seem high, Bacteroidetes genomes are known to have dozens of invertible promoters (up to 47 in B. fragilis; Jiang et al., 2019). Invertible promoters are restricted out of the genome and re-ligated in the opposite direction to turn gene expression on or off. ...
When examining bacterial genomes for evidence of past selection, the results depend heavily on the mutational distance between chosen genomes. Even within a bacterial species, genomes separated by larger mutational distances exhibit stronger evidence of purifying selection as assessed by d N /d S , the normalized ratio of nonsynonymous to synonymous mutations. Here, we show that the classical interpretation of this scale dependence, weak purifying selection, leads to problematic mutation accumulation when applied to available gut microbiome data. We propose an alternative, adaptive reversion model with opposite implications for dynamical intuition and applications of d N /d S . Reversions that occur and sweep within-host populations are nearly guaranteed in microbiomes due to large population sizes, short generation times, and variable environments. Using analytical and simulation approaches, we show that adaptive reversion can explain the d N /d S decay given only dozens of locally fluctuating selective pressures, which is realistic in the context of Bacteroides genomes. The success of the adaptive reversion model argues for interpreting low values of d N /d S obtained from long timescales with caution as they may emerge even when adaptive sweeps are frequent. Our work thus inverts the interpretation of an old observation in bacterial evolution, illustrates the potential of mutational reversions to shape genomic landscapes over time, and highlights the importance of studying bacterial genomic evolution on short timescales.
... Over the past decades, tremendous effort, including ours (Jia et al., 2021;Liu et al., 2022;Liu et al., 2017), has been devoted to understanding the impact of microbial strains or more complex communities on their hosts. In fact, interactions between the host and microbiome are mutually bidirectional (Bäckhed et al., 2005;Jiang et al., 2019), conferring benefits to the host and microbial sides. Nevertheless, the knowledge of the effect of the host on the resident microbial community is still in its infancy. ...
Host-microbe interactions are virtually bidirectional, but how the host affects their microbiome is poorly understood. Here, we report that the host is a critical modulator to regulate the lifestyle switch and pathogenicity heterogeneity of the opportunistic pathogens Serratia marcescens utilizing the Drosophila and bacterium model system. First, we find that Drosophila larvae efficiently outcompete S. marcescens and typically drive a bacterial switch from pathogenicity to commensalism toward the fly. Furthermore, Drosophila larvae reshape the transcriptomic and metabolic profiles of S. marcescens characterized by a lifestyle switch. More importantly, the host alters pathogenicity and heterogeneity of S. marcescens in the single-cell resolution. Finally, we find that larvae-derived AMPs are required to recapitulate the response of S. marcescens to larvae. Altogether, our findings provide an insight into the pivotal roles of the host in harnessing the life history and heterogeneity of symbiotic bacterial cells, advancing knowledge of the reciprocal relationships between the host and pathogen.
... Mutation-driven switching is commonly relegated only to genomic elements with elevated mutation rates (e.g. simple sequence repeats [37][38][39] ). Our work challenges this expectation by presenting an in situ case of bacteria undergoing mutation-mediated phenotypic switching and calls for theoretical models that consider mutation-mediated reversion. ...
... Mutation-driven adaptations could be considered less likely for commensal and other organisms with prolonged tenure in mammalian environments which have had ample time to evolve more complex regulatory mechanisms 42,43 . However, recent observations suggest that mutation-mediated phenotypic switching might be common even for species with ample time to evolve gene regulation: E. coli undergoes mutation-mediated switching in laboratory mice 10 and B. fragilis' in-human adaptive mutations target conserved genes 44 , despite these same genes possessing complex regulatory mechanisms 38 . ...
Bacteria evolving within human hosts encounter selective tradeoffs that render mutations adaptive in one context and deleterious in another. Here, we report that the cystic fibrosis-associated pathogen Burkholderia dolosa overcomes in-human selective tradeoffs by acquiring successive point mutations that alternate phenotypes. We sequenced the whole genomes of 931 respiratory isolates from two recently infected patients and an epidemiologically-linked, chronically-infected patient. These isolates are contextualized using 112 historical genomes from the same outbreak strain. Within both newly infected patients, diverse parallel mutations that disrupt O-antigen expression quickly arose, comprising 29% and 63% of their B. dolosa communities by 3 years. The selection for loss of O-antigen starkly contrasts with our previous observation of parallel O-antigen-restoring mutations after many years of chronic infection in the historical outbreak. Experimental characterization revealed that O-antigen loss increases uptake in immune cells while decreasing competitiveness in the mouse lung. We propose that the balance of these pressures, and thus whether O-antigen expression is advantageous, depends on tissue localization and infection duration. These results suggest that mutation-driven alternation during infection may be more frequent than appreciated and is underestimated without dense temporal sampling.
... [6][7][8] Analysis of the orientations of bacterial invertible regions in various host disease states can provide new insights into bacterial adaptation and functional contributions to the disease pathogenesis or its resolution. Phase variation in the gut Bacteroidales often modulates the production of components presented on the bacterial surface 9 dictating which surface molecules interact, for example, with neighboring microbes (e.g., bacteria and bacteriophages) or with the host. ...
... Phase variations, prevalent in host-associated species, especially the gut Bacteroidales, 9 contribute to bacterial fitness in changing ecosystems. Reversible DNA inversions lead to phase variable synthesis of numerous molecules (e.g., surface, regulatory, and other molecules), and as such, confer functional plasticity. ...
Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory T cells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented “OFF” in IBD patients, which correlated with increased B. fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B. fragilis, induction of colitis caused a decline of PSA in the “ON” orientation that reversed as inflammation resolved. Monocolonization of mice with B. fragilis revealed that bacteriophage infection increased the frequency of PSA in the “OFF” orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease.
... To entirely avoid reference genomes and MAG creation, two pattern-driven methods have been developed. PhaseFinder (16) was created for detection of inversions in bacterial genomes from genomic or metagenomic data, by detecting regions flanked by inverted repeats where sequencing reads support both orientations. DIVE (1) was developed in 2023 to identify sequences surrounding genetic diversification such as transposable elements, within MGE variability hotspots, or CRISPR repeats, by detecting constant k-mers with diverse flanking sequences to define MGE bounding sequences and transposon arms. ...
Bacterial genome dynamics are vital for understanding the mechanisms underlying microbial adaptation, growth, and their broader impact on host phenotype. Structural variants (SVs), genomic alterations of 10 base pairs or more, play a pivotal role in driving evolutionary processes and maintaining genomic heterogeneity within bacterial populations. While SV detection in isolate genomes is relatively straightforward, metagenomes present broader challenges due to absence of clear reference genomes and presence of mixed strains. In response, our proposed method rhea, forgoes reference genomes and metagenome-assembled genomes (MAGs) by encompassing a single metagenome coassembly graph constructed from all samples in a series. The log fold change in graph coverage between subsequent samples is then calculated to call SVs that are thriving or declining throughout the series. We show rhea to outperform existing methods for SV and horizontal gene transfer (HGT) detection in two simulated mock metagenomes, which is particularly noticeable as the simulated reads diverge from reference genomes and an increase in strain diversity is incorporated. We additionally demonstrate use cases for rhea on series metagenomic data of environmental and fermented food microbiomes to detect specific sequence alterations between subsequent time and temperature samples, suggesting host advantage. Our innovative approach leverages raw read patterns rather than references or MAGs to include all sequencing reads in analysis, and thus provide versatility in studying SVs across diverse and poorly characterized microbial communities for more comprehensive insights into microbial genome dynamics.
... [58][59][60] Phase variation of surface structures involves the reversible inversion of DNA regions containing promoters, resulting in the expression or suppression of downstream genes depending on the orientation. 61 Invertible promotor regions are characteristic of Bacteroidales species residing in the human gut and are generally not conserved among Bacteroidales occupying other niches. 59,61 This highlights the importance of host factors in influencing phage-host interactions. ...
... 61 Invertible promotor regions are characteristic of Bacteroidales species residing in the human gut and are generally not conserved among Bacteroidales occupying other niches. 59,61 This highlights the importance of host factors in influencing phage-host interactions. Further investigation, such as transcriptomics analysis, may provide insights into the role of these features and help understand the mechanisms mediating the interaction between φPDS1 and P. distasonis APCS2/PD. ...
The human gut microbiome plays a significant role in health and disease. The viral component (virome) is predominantly composed of bacteriophages (phages) and has received significantly less attention in comparison to the bacteriome. This knowledge gap is largely due to challenges associated with the isolation and characterization of novel gut phages, and bioinformatic hurdles such as the lack of a universal phage marker gene and the absence of sufficient numbers of homologs in viral databases. Here, we describe the isolation from human feces of a novel lytic phage with siphovirus morphology, φPDS1, infecting Parabacteroides distasonis APCS2/PD, and classified within a newly proposed Sagittacolavirus genus. In silico and biological characterization of this phage is presented in this study. Key to the isolation of φPDS1 was the antibiotic-driven selective enrichment of the bacterial host in a fecal fermenter. Despite producing plaques and lacking genes associated with lysogeny, φPDS1 demonstrates the ability to coexist in liquid culture for multiple days without affecting the abundance of its host. Multiple studies have shown that changes in Parabacteroides distasonis abundance can be linked to various disease states, rendering this novel phage-host pair and their interactions of particular interest.
... Invertons are invertible promoters under the control of site-specific recombinases and flanked by inverted repeats. These genomic structures are thought to be constrained to Bacteroidetes (Bacteroidota), where they control the expression of antimicrobial resistance genes 31 and help maintain capsid polysaccharide heterogeneity in isogenic populations 32 . We identify instances where a strong PRO-seq signal originates within an inverton but terminates before downstream https://doi.org/10.1038/s41564-023-01558-w ...
... Invertons were identified with PhaseFinder (v.1.0) 31 . ...
Bacteria respond to environmental stimuli through precise regulation of transcription initiation and elongation. Bulk RNA sequencing primarily characterizes mature transcripts, so to identify actively transcribed loci we need to capture RNA polymerase (RNAP) complexed with nascent RNA. However, such capture methods have only previously been applied to culturable, genetically tractable organisms such as E. coli and B. subtilis. Here we apply precision run-on sequencing (PRO-seq) to profile nascent transcription in cultured E. coli and diverse uncultured bacteria. We demonstrate that PRO-seq can characterize the transcription of small, structured, or post-transcriptionally modified RNAs, which are often absent from bulk RNA-seq libraries. Applying PRO-seq to the human microbiome highlights taxon-specific RNAP pause motifs and pause-site distributions across non-coding RNA loci that reflect structure-coincident pausing. We also uncover concurrent transcription and cleavage of CRISPR guide RNAs and transfer RNAs. We demonstrate the utility of PRO-seq for exploring transcriptional dynamics in diverse microbial communities.
