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Motifs found in the inverted repeats of Bacteroidetes and Akkermansia invertons consist of five to seven base pair palindromes with two or three intervening base pairs. (A) Functional profiling of operons regulated by invertons reveals specializations of each inverted repeat (IR) motif. The heat map represents the number of operons per functional class regulated by invertons with either global or local invertases. CPS, capsular polysaccharide; Fimb, fimbriae; OmpA, outer membrane protein A; SusCD, starch utilization system proteins C and D. Superscript I indicates the presence of a local invertase. The absence of local invertases directly upstream suggests that IR motifs 2 and 4 are likely regulated by global invertases. (B) Promoter motif identified from invertons from Bacteroidetes species. (C) The inverted repeat motif found in all identified invertons from Akkermansia spp. (D) Promoter motif identified from invertons from Akkermansia spp.
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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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... searched Genomes with invertons Percentage of genomes with invertons Total invertons inverted repeat, which we call motif 0 ( fig. S5). We found conserved promoter consensus mo- tifs in 98% (231/235) of invertons with IR mo- tifs 1 to 4 (Fig. 2B) (20). In contrast, promoter motifs were not observed in any of the inver- tible regions of IR motif 0-containing sequences (table S9). Because of the lack of promoter mo- tifs combined with their location downstream of operons, invertons containing motif 0 may function as another type of regulatory element, such as a phase-variable ...
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... promoter consensus motif in relation to surrounding genes, we determined which genes or operons were regulated by invertible promoters and whether the promoter was in the ON or OFF orientation with respect to the downstream gene (tables S7 and S8). In both species of Akkermansia, all invertons are flanked by inverted repeats with the same motif (Fig. 2C), contain a promoter motif (Fig. 2D), and lack upstream invertases, which suggests that they are all invertible promoters co-regulated by a single, master invertase (EAJ16_05345 in Akkermansia muciniphila; MK095134 in Ak- kermansia sp. ...
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... to surrounding genes, we determined which genes or operons were regulated by invertible promoters and whether the promoter was in the ON or OFF orientation with respect to the downstream gene (tables S7 and S8). In both species of Akkermansia, all invertons are flanked by inverted repeats with the same motif (Fig. 2C), contain a promoter motif (Fig. 2D), and lack upstream invertases, which suggests that they are all invertible promoters co-regulated by a single, master invertase (EAJ16_05345 in Akkermansia muciniphila; MK095134 in Ak- kermansia sp. ...
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... functional annotation, we found that 73% (228/312) of invertible promoters regulate genes involved in the biosynthesis of polysac- charides, fimbriae, outer membrane proteins, and autotransporters; genes involved in the utilization of polysaccharides; or genes encoding PEP-CTERM domain-containing proteins ( Fig. 2A, tables S7 and S8, and fig. S4). All of these functional categories except polysaccharide uti- lization are enriched in genes regulated by in- vertible promoters (tables S10 and S11). Genes regulated by invertible promoters are enriched for cell surface products (e.g., GO:0016020, mem- brane, P = 1.93 × 10 -14 ) (table S11). The most enriched functional class is ...
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Fecal microbiota transplantation (FMT) is currently used in medicine to treat recurrent clostridial colitis and other intestinal diseases. However, neither the therapeutic mechanism of FMT nor the mechanism that allows the donor bacteria to colonize the intestine of the recipient has yet been clearly described. From a biological point of view, FMT...
Citations
... ICEs, IMEs, AICEs and CIMEs) are important hotspots for the accretion of defense systems, helping to protect the host from superinfection by other MGEs. Considering the known evolutionary relationship MGEs have with defense systems (2,4,(18)(19)(20), AMR (5,(20)(21)(22) and virulence genes (23), I also explore if these three functions are positively or negatively correlated across ciMGEs from multiple phyla. I found that (i) IMEs and ICEs are widespread across different phyla; (ii) ICEs are the most common ciMGEs for the accumulation of defense systems, AMR and virulence genes; (iii) these three functions are negatively correlated across ICEs and IMEs and (iv) ICEs and IMEs from multiple phyla share high genetic similarity and challenge interphylum barriers. ...
Mobile genetic elements (MGEs) are key promoters of microbial evolution. These elements can be located extrachromosomally or integrated into the chromosome. Well-known examples of chromosomally integrated MGEs (ciMGEs) are integrative and conjugative/mobilizable elements (ICEs and IMEs), and most studies to date have focused on the biological mechanisms that shape their lifestyle. It is crucial to profile the diversity and understand their distribution across the microbial community, as the number of genome sequences increases exponentially. Herein, I scanned a collection of >20 000 bacterial and archaeal non-redundant genomes and found over 13 000 ciMGEs across multiple phyla, representing a massive increase in the number of ciMGEs available in public databases (<1000). Although ICEs are the most important ciMGEs for the accretion of defense systems, virulence, and antimicrobial resistance (AMR) genes, IMEs outnumbered ICEs. Moreover, defense systems, AMR, and virulence genes were negatively correlated in both ICEs and IMEs. Multiple ciMGEs form heterogeneous communities and challenge inter-phylum barriers. Finally, I observed that the functional landscape of ICEs was populated by uncharacterized proteins. Altogether, this study provides a comprehensive catalog of nucleotide sequences and associated metadata for ciMGEs from 34 phyla across the bacterial and archaeal domains.
... One such mechanism is phase variation, which generates frequent and reversible modifications in protein expression within individual cells of a clonal population. 1 The molecular mechanisms governing phase variations range from epigenetic modifications via DNA methylation, to genetic alterations involving slipped strand mispairing or site-specific recombination within hypermutable loci. [2][3][4][5][6] In this regard, simple sequence repeats (SSRs) in specific loci can serve as mediators of hypermutations through slipped strand mispairing that can occur in these regions during DNA replication. [7][8][9][10] Accordingly, several studies have demonstrated enhanced adaptation to unstable environmental conditions through insertion or deletion (indels) of nucleotides generated by SSRs. ...
Microbial adaptation to changing environmental conditions is frequently mediated by hypermutable sequences. Here we demonstrate that such a hypermutable hotspot within a gene encoding a flagellar unit of Paenibacillus glucanolyticus generated spontaneous non-swarming mutants with increased stress resistance. These mutants, which survived conditions that eliminated wild-type cultures, could be carried by their swarming siblings when the colony spread, consequently increasing their numbers at the spreading edge. Of interest, the hypermutable nature of the aforementioned sequence enabled the non-swarming mutants to serve as “seeds” for a new generation of wild-type cells through reversion of the mutation.Using a mathematical model, we examined the survival dynamics of P. glucanolyticus colonies under fluctuating environments. Our experimental and theoretical results suggest that the non-swarming, stress-resistant mutants can save the colony from extinction. Notably, we identified this hypermutable sequence in flagellar genes of additional Paenibacillus species, suggesting that this phenomenon could be wide-spread and ecologically important.
... The production of type 1 fimbriae is subject to phase variation, with fimbriate and afimbriate cells coexisting in the same population [9,12]. This behaviour has been interpreted as a bet-hedging strategy that balances the risks of producing these highly immunogenic fimbriae (detection by the host immune system; the physiological cost of making, exporting and assembling the structures) with the benefits (biofilm-based community living; colonization of a host or another environmental niche) [13][14][15][16]. The invertible fimS genetic element is the basis of phase-variable fim operon expression. ...
The structural genes expressing type 1 fimbriae in Escherichia coli alternate between expressed (phase ON) and non-expressed (phase OFF) states due to inversion of the 314 bp fimS genetic switch. The FimB tyrosine integrase inverts fimS by site-specific recombination, alternately connecting and disconnecting the fim operon, encoding the fimbrial subunit protein and its associated secretion and adhesin factors, to and from its transcriptional promoter within fimS. Site-specific recombination by the FimB recombinase becomes biased towards phase ON as DNA supercoiling is relaxed, a condition that occurs when bacteria approach the stationary phase of the growth cycle. This effect can be mimicked in exponential phase cultures by inhibiting the negative DNA supercoiling activity of DNA gyrase. We report that this bias towards phase ON depends on the presence of the Fis nucleoid-associated protein. We mapped the Fis binding to a site within the invertible fimS switch by DNase I footprinting. Disruption of this binding site by base substitution mutagenesis abolishes both Fis binding and the ability of the mutated switch to sustain its phase ON bias when DNA is relaxed, even in bacteria that produce the Fis protein. In addition, the Fis binding site overlaps one of the sites used by the Lrp protein, a known directionality determinant of fimS inversion that also contributes to phase ON bias. The Fis-Lrp relationship at fimS is reminiscent of that between Fis and Xis when promoting DNA relaxation-dependent excision of bacteriophage λ from the E. coli chromosome. However, unlike the co-binding mechanism used by Fis and Xis at λ attR, the Fis-Lrp relationship at fimS involves competitive binding. We discuss these findings in the context of the link between fimS inversion biasing and the physiological state of the bacterium.
... As different phenotypes are often better equipped to overcome different challenges, such intra-population heterogeneity might allow the bacterial population a bet-hedging strategy to better survive sudden environmental challenges. Indeed, phase variation was observed in various bacterial processes (1)(2)(3)(4) and was shown to be important for survival in major environmental challenges faced by bacteria, including bacteriophages (5,6), antibiotic drugs (7,8) and virulence (9)(10)(11). ...
... The genomic signature of inverted repeats flanking programmed inversions, as well as the rapidly growing amount of publicly available DNA sequence data, provide an opportunity for computational identification of programmed inversions. Indeed, multiple methods to identify programmed inversions were developed and successfully deployed (7,8,12,17,18,(23)(24)(25)(26)(27). Yet, only a few of these methods were applied widely across the bacterial domain, specifically searching for programmed inversions that target promoters (7,8), or searching for gene-altering programmed inversions targeting a specific gene family, the Type I RM specificity subunit HsdS (17,18). ...
... Indeed, multiple methods to identify programmed inversions were developed and successfully deployed (7,8,12,17,18,(23)(24)(25)(26)(27). Yet, only a few of these methods were applied widely across the bacterial domain, specifically searching for programmed inversions that target promoters (7,8), or searching for gene-altering programmed inversions targeting a specific gene family, the Type I RM specificity subunit HsdS (17,18). A more comprehensive gene-independent search for programmed inversions was developed based on abnormally aligned shortreads and manual curation, and was used to identify programmed inversions, regardless of target, in over 200 genomes (27). ...
Programmed chromosomal inversions allow bacteria to generate intra-population genotypic and functional heterogeneity, a bet-hedging strategy important in changing environments. Some programmed inversions modify coding sequences, producing different alleles in several gene families, most notably in specificity-determining genes such as Type I restriction-modification systems, where systematic searches revealed cross phylum abundance. Yet, a broad, gene-independent, systematic search for gene-altering programmed inversions has been absent, and little is known about their genomic sequence attributes and prevalence across gene families. Here, identifying intra-species variation in genomes of over 35 000 species, we develop a predictive model of gene-altering inversions, revealing key attributes of their genomic sequence attributes, including gene-pseudogene size asymmetry and orientation bias. The model predicted over 11,000 gene-altering loci covering known targeted gene families, as well as novel targeted families including Type II restriction-modification systems, a protein of unknown function, and a fusion-protein containing conjugative-pilus and phage tail domains. Publicly available long-read sequencing datasets validated representatives of these newly predicted inversion-targeted gene families, confirming intra-population genetic heterogeneity. Together, these results reveal gene-altering programmed inversions as a key strategy adopted across the bacterial domain, and highlight programmed inversions that modify Type II restriction-modification systems as a possible new mechanism for maintaining intra-population heterogeneity.
... Identical genomic regions can give rise to different phenotypes depending on their orientation and association with other genomic regions (Evans et al., 2013;Jiang et al., 2019). To capture such paired interactions, we developed the 'paired regions' feature in PRAWNS: each feature corresponds to two conserved regions, r 1 and r 2 , and their relative orientations such that r 1 and r 2 are separated by at most a small distance D (user-defined, default: 50) nucleotides in ! ...
Motivation:
Scientists seeking to understand the genomic basis of bacterial phenotypes, such as antibiotic resistance, today have access to an unprecedented number of complete and nearly-complete genomes. Making sense of these data requires computational tools able to perform multiple-genome comparisons efficiently, yet currently available tools cannot scale beyond several tens of genomes.
Results:
We describe PRAWNS, an efficient and scalable tool for multiple-genome analysis. PRAWNS defines a concise set of genomic features (metablocks), as well as pairwise relationships between them, which can be used as a basis for large-scale genotype-phenotype association studies. We demonstrate the effectiveness of PRAWNS by identifying genomic regions associated with antibiotic resistance in Acinetobacter baumannii.
Availability:
PRAWNS is implemented in C ++ and Python3, licensed under the GPLv3 license, and freely downloadable from GitHub (https://github.com/KiranJavkar/PRAWNS.git).
Supplementary information:
Supplementary data are available at Bioinformatics online.
... Though it is costly, bacteria can ameliorate the fitness costs of maintaining ARGs through different strategies 9 , such as no-cost, low-cost or gain-of-fitness mutations 10,11 , compensatory mutations at a second site [12][13][14] , or genetic coselection of resistance genes in genetic linkage 15,16 . Phase-variable antibiotic resistance, which was only recently reported 17 , is a newly identified mechanism for antibiotic resistant bacteria to mitigate the fitness cost of encoding ARGs. ...
... Phase variation refers to a reversible change that generates phenotypic variation that helps bacteria adapt to rapidly changing environments 18,19 . Phase variation often manifests through reversible inversion of DNA regions containing promoters such that in one orientation, a downstream gene is expressed, while in the alternate orientation, the downstream gene is not expressed 17 . Such DNA inversions are generally mediated by invertases, which recognize inverted repeats flanking the invertible region and catalyze the reversible inversion [20][21][22] . ...
... Recent advances in computational methods have contributed to the effective identification of the intergenic invertible DNA regions in microbial genomes 17,28 . ARGs were found to be regulated by invertible promoters in certain human gut bacteria 17 . ...
Antibiotic-resistance genes (ARGs) regulated by invertible promoters can mitigate the fitness cost of maintaining ARGs in the absence of antibiotics and could potentially prolong the persistence of ARGs in bacterial populations. However, the origin, prevalence, and distribution of these ARGs regulated by invertible promoters remains poorly understood. Here, we sought to assess the threat posed by ARGs regulated by invertible promoters by systematically searching for ARGs regulated by invertible promoters in the human gut microbiome and examining their origin, prevalence, and distribution. Through metagenomic assembly of 2227 human gut metagenomes and genomic analysis of the Unified Human Gastrointestinal Genome (UHGG) collection, we identified ARGs regulated by invertible promoters and categorized them into three classes based on the invertase-regulating phase variation. In the human gut microbiome, ARGs regulated by invertible promoters are exclusively found in Bacteroidales species. Through genomic analysis, we observed that ARGs regulated by invertible promoters have convergently originated from ARG insertions into glycan-synthesis loci that were regulated by invertible promoters at least three times. Moreover, all three classes of invertible promoters regulating ARGs are located within integrative conjugative elements (ICEs). Therefore, horizontal transfer via ICEs could explain the wide taxonomic distribution of ARGs regulated by invertible promoters. Overall, these findings reveal that glycan-synthesis loci regulated by invertible promoters in Bacteroidales species are an important hotspot for the emergence of clinically-relevant ARGs regulated by invertible promoters.
... They facilitate pathogenic processes that include the establishment of urinary tract infections and they play a founding role in biofilm formation (Anderson et al., 2003;Justice et al., 2004;Wright et al., 2007). In addition to their utility in promoting an attached lifestyle, type 1 fimbriae are highly immunogenic and therefore pose a threat to the bacterium in the context of interactions with the host (Jiang et al., 2019;Sánchez-Romero & Casadesús, 2021). It is thought that, for this reason, bacteria employ a battery of molecular mechanisms that render stochastic the production of type 1 fimbriae, ensuring that populations of bacteria contain both fimbriate and afimbriate members. ...
Bacteria deal with an unpredictable, and often hostile, environment by being unpredictable themselves. This article will link some contributions made by variable DNA topology and nucleoid-associated proteins to the generation of stochasticity in bacterial gene expression and describe how the associated mechanistic insights can elucidate the means by which diversity in antibody and neuronal cell development might be produced in humans and other higher organisms. The focus here will not be on mutation; instead, the article will address epigenetic effects on gene expression brought about by the modulation of topoisomerase activity in both prokaryotes and eukaryotes.
... ; https://doi.org/10.1101/2022.11.18.517082 doi: bioRxiv preprint AICEs) are important hotspots for the accretion of defense systems, helping to protect the host from superinfection by other MGEs. Considering the known evolutionary relationship MGEs have with defense systems (2,11,17,18), AMR (12,19,20) and virulence genes (21), I also explore if these three functional groups are positively or negatively correlated across ciMGEs from multiple phyla. I found that i) IMEs and ICEs are widespread across different phyla; ii) ICEs are the most important ciMGEs for the accumulation of defense systems, AMR, and virulence genes; iii) these three functional groups are negatively correlated across ICEs and IMEs; and iv) ICEs and IMEs from multiple phyla share high genetic similarity and challenge interphylum barriers. ...
Mobile genetic elements (MGEs) are key promoters of bacterial and archaeal genome evolution. These elements can be located extrachromosomally (such as plasmids) or integrated within the chromosome. Well-known examples of chromosomally integrated MGEs (ciMGEs) are the integrative and conjugative/mobilizable elements (ICEs and IMEs), and most studies so far have focused on the biological mechanisms that shape the lifestyle of these elements. It is crucial to illustrate the overall diversity and understand the distribution of circulating ciMGEs in the microbial community as the number of genome sequences increases exponentially. Herein, I scanned a publicly available collection of more than 20000 bacterial and archaeal non-redundant genomes and found more than 13000 ciMGEs across multiple phyla, representing a massive increase in the number of ciMGEs currently available in public databases (<1000). Although ICEs are the most important ciMGEs for the accretion of defensive systems, virulence genes, and antimicrobial resistance (AMR) genes, IMEs outnumbered ICEs. Moreover, I discovered that defense systems, AMR, and virulence genes are negatively correlated in both ICEs and IMEs. Multiple representatives of these ciMGEs form heterogeneous communities and challenge interphylum barriers. Finally, I observed that the functional landscape of ICEs is populated by uncharacterized proteins. Taken together, this study provides a comprehensive catalog compiling the nucleotide sequence and associated metadata for ciMGEs from 34 distinct phyla across the bacterial and archaeal domains.
... With regard to the functional annotation of the representative genes/proteins of the pangenome likely acquired by HGT events, 134 were annotated as resistance-related proteins (55.4% of the total number of resistance-related proteins detected). Moreover, several invertases (proteins that switch antibiotic-resistance regulatory genes, among other functions [39]) appear to be acquired from Klebsiella species. Similarly, 850 of 3,245 CAZys would have been horizontally acquired. ...
Microbes play important roles in the health of living beings and in the environment. The knowledge of these functions may be useful for the development of new clinical and biotechnological applications and the restoration and preservation of natural ecosystems.
... These high rates of cell turnover create enormous opportunities for rapid evolutionary change: the trillions of bacteria within a single human colon produce more than a billion new mutations every day (7), and tens of thousands of bacterial generations will typically elapse within a single human lifetime (1,8). Consistent with these estimates, a growing number of empirical studies, ranging from experimental evolution in mouse models (9-12) to longitudinal sequencing of human subjects (7,(13)(14)(15)(16)(17)(18)(19)(20), have shown that genetic variants can sweep through resident populations of gut bacteria on timescales of weeks and months. The causes and consequences of this genetic turnover could play an important role in mediating the stability and resilience of this diverse microbial ecosystem (21). ...
... These high rates of cell turnover create enormous opportunities for rapid evolutionary change: the trillions of bacteria within a single human colon produce more than a billion new mutations every day (7), and tens of thousands of bacterial generations will typically elapse within a single human lifetime (1,8). Consistent with these estimates, a growing number of empirical studies, ranging from experimental evolution in mouse models (9)(10)(11)(12) to longitudinal sequencing of human subjects (7,(13)(14)(15)(16)(17)(18)(19)(20), have shown that genetic variants can sweep through resident populations of gut bacteria on timescales of weeks and months. The causes and consequences of this genetic turnover could play an important role in mediating the stability and resilience of this diverse microbial ecosystem (21). ...
... By applying our results to recent estimates from the human colon, we found that continuous fluid flow (and simple forms of wall growth) are unlikely to lower effective population sizes to the point where we would expect large fluctuations in genetic composition within a host lifetime. This suggests that additional factors, such as natural selection or temporal bottlenecks, are necessary to explain the rapidly changing mutation frequencies that have been observed in sequenced human fecal samples (7,(13)(14)(15)(16)(17)(18)(19)(20). We also found that the spatial gradients generated by fluid flow can cause the effective generation time to be about 5 to 10 times shorter than the traditional ∼1/d estimates obtained from the average growth rate across the colon (60,61). ...
The genetic composition of the gut microbiota is constantly reshaped by ecological and evolutionary forces. These strain-level dynamics are challenging to understand because they depend on complex spatial growth processes that take place within a host. Here we introduce a population genetic framework to predict how stochastic evolutionary forces emerge from simple models of microbial growth in spatially extended environments like the intestinal lumen. Our framework shows how fluid flow and longitudinal variation in growth rate combine to shape the frequencies of genetic variants in simulated fecal samples, yielding analytical expressions for the effective generation times, selection coefficients, and rates of genetic drift. We find that over longer timescales, the emergent evolutionary dynamics can often be captured by well-mixed models that lack explicit spatial structure, even when there is substantial spatial variation in species-level composition. By applying these results to the human colon, we find that continuous fluid flow and simple forms of wall growth alone are unlikely to create sufficient bottlenecks to allow large fluctuations in mutant frequencies within a host. We also find that the effective generation times may be significantly shorter than expected from traditional average growth rate estimates. Our results provide a starting point for quantifying genetic turnover in spatially extended settings like the gut microbiota and may be relevant for other microbial ecosystems where unidirectional fluid flow plays an important role.
