The genetic structure of bacterial species is most often interpreted in terms of demographic processes such as clonal descent, but can also reflect natural selection and hence give functional and ecological insight. Klebsiella pneumoniae (KP) disperses effectively around the world and has high recombination rates, which should result in the species having a well-mixed gene pool. Nevertheless, phylogenies based on diverse KP strains contain a "backbone." This structure reflects a component of variation where the first component in Principal Components Analysis (PCA), PC1, explains 16.8% of the total variation. We propose that the component reflects a "bacterial ecocline" generated by diversifying selection on a quantitative genetic trait. We simulated the evolution of a bacterial population with a polygenic quantitative trait, where strains with the most extreme trait values have a small advantage. These simulations can recapitulate our KP PCA results and other features of its genetic diversity. As well as providing an explanation for the phylogenetic backbone, our results provide insight into how species such as KP can speciate, via stronger selection on the trait or a reduction in gene flow. Our hypothesis that there is a bacterial ecocline in KP raises two questions, namely what the trait is underlying it and why is the trait under diversifying selection? The genes that are most strongly associated with PC1 provide some hints, with the top locus encoding Kpa fimbriae. Identification of the trait, if it exists, should facilitate insight into selection on quantitative genetic traits in natural bacterial populations, which have largely been unstudied in microbiology, except in the atypical context of antibiotic resistance.

Abstract Bacterial sRNAs are pervasive post-transcriptional regulators, yet how they arise, evolve, and decay remains poorly understood. Here, we provide a high-resolution transcriptome map and curated sRNA set for the pathogen Vibrio parahaemolyticus . We identify over 100 sRNAs, including broadly conserved, lineage-specific, and previously unidentified transcripts, as well as dual-function regulatory/coding sRNAs. Functional analysis of several examples highlights conserved and lineage-specific regulators of metabolism and flagella. Broadly conserved VcrX represses chitin utilization genes and may regulate Vibrio Spot 42, which we confirm is translated. We expand on FlaX regulation of polar flagella across the genus by demonstrating that the sRNA differentially activates/represses downstream flagellins, with a potential FlaX sponge mediating feedback in specific clades. We further show that V. parahaemolyticus , but not V. cholerae , RyhB is translated into a Cys-rich small protein that could regulate related pathways. Together, these findings establish a resource for Vibrio and a platform for comparative studies of post-transcriptional regulation, enabling investigation of how sRNAs and their regulatory networks evolve.

Understanding the reservoirs and transmission dynamics of foodborne pathogens is critical for developing effective surveillance and control strategies. Here, we investigate the strain-level diversity and temporal dynamics of Vibrio parahaemolyticus, the leading cause of seafood-associated infections worldwide, through a three-month longitudinal sampling of a single retail seafood stall. Whole-genome sequencing of 238 isolates (2-38 per item) from individual oysters and clams revealed exceptionally high intra-seafood strain diversity, with a median of eight (range: 2-32) distinct sequence types per item. Clonal groups (CGs), defined as clusters of closely related strains sharing recent ancestry, were rarely observed across sampling time points, indicating a highly dynamic and transient population structure in the retail environment. Only a small subset of CGs was shared between seafood items or aquaculture water, suggesting that cross-contamination within the stall occurs less frequently than anticipated. Together, these findings provide the first direct evidence of pronounced intra-seafood strain diversity, rapid temporal turnover, and possibly limited cross-item transmission of V. parahaemolyticus in retail seafood, highlighting the complexity of the ecological reservoirs and transmission dynamics of foodborne pathogens.

In contrast to transcriptome maps, bacterial small protein (≤50-100 aa) coding landscapes, including overlapping genes, are poorly characterized. However, an emerging number of small proteins have crucial roles in bacterial physiology and virulence. Here, we present a Ribo-seq-based high-resolution translatome map for the major foodborne pathogen Campylobacter jejuni. Besides conventional Ribo-seq, we employed translation initiation site (TIS) profiling to map start codons and also developed a translation termination site (TTS) profiling approach, which revealed stop codons not apparent from the reference genome in virulence loci. Our integrated approach combined with independent validation expanded the small proteome by two-fold, including CioY, a new 34 aa component of the CioAB oxidase. Overall, our study generates a high-resolution annotation of the C. jejuni coding landscape, provided in an interactive browser, and showcases a strategy for applying integrated Ribo-seq to other species to enrich our understanding of small proteomes.

Macrogenetics investigates the patterns and predictors of intraspecific genetic variation across diverse taxa, offering a framework to address longstanding evolutionary hypotheses. Here, we present a comprehensive macrogenetic atlas of prokaryotes (MAP), integrating genomic data (summarized by 30 parameters in 12 categories) from 15,235 prokaryotic species and population genetic data (35 parameters in 7 categories) from 786 species with phylogenetic, phenotypic, and ecological data. We illustrate the power and potential of the MAP by addressing a series of fundamental questions. For example, we provide evidence that genome size in prokaryotes depends on a balance between deletion bias and the beneficial effects of additional genes. Moreover, we show that long-range and short-range linkage disequilibrium represent quite different features of evolutionary dynamics and contribute independently to genetic diversity. Notably, we show that as within-species diversity increases, selection becomes an increasingly strong force structuring diversity, for example creating convergent ecospecies structures in Streptococcus mitis and S. oralis. Overall, our atlas represents a widely applicable resource and offers novel insights into the drivers of macroevolution and the life-cycle of prokaryotic species. ### Competing Interest Statement The authors have declared no competing interest.

Bacterial ecospecies are a recently recognized genetic structure that is hypothesized to arise by complex adaptation to a new niche. Using coadaptation analysis as a foundation for laboratory experiments, we find that the ecological strategy of the “Molassodon” ecospecies of Vibrio parahaemolyticus is to hunt, kill and devour bacterial prey in viscous liquids. Instead of swarming on surfaces, Molassodon strains have differentiated and repurposed their lateral flagella to enhance swimming. We functionally link Type VI secretion and nutrient uptake to this motility phenotype and reconstruct the evolution of Molassodon, showing that several key genes have been imported from other Vibrio species. Our analysis confirms the functional coherence of bacterial ecospecies and provides a window into adaptation for environmental microbes, where characterizing relevant microhabitats and evolutionary strategies remains a considerable challenge. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 32250610209, 92478118, 32270064, 32270003, 32350710791 Shanghai Municipal Science and Technology Commission, 24ZR1493200

While spontaneous mutation and gene acquisition are well-established drivers of pathogen adaptation, the role of gene loss remains underexplored. Here we investigated the emergence and diversification of the pandemic clone of Vibrio parahaemolyticus through large-scale phylogenomic analysis of 8,684 global isolates. The pandemic clone rapidly acquired multiple marker genes and genomic islands, subsequently diverging into successive sublineages mediating independent waves of cross-country transmission, as also observed in Vibrio cholerae. Wave succession in the last two decades was driven by loss of putrescine utilization (Puu) genes, conferring phenotypic advantages for environmental adaptation (enhanced biofilm formation) and human transmission (increased cell adhesion and intestinal colonization and reduced virulence), consistent with the virulence trade-off hypothesis. We identified Puu-gene loss in several bacterial genera, with effects on biofilm and adhesion replicated in V. cholerae and Escherichia coli, suggesting convergent evolution and universal phenotypic effects. Our results highlight the indispensable role of gene loss in bacterial pathogen adaptation.

Are quantitative traits important for bacteriology? The quantitative trait models used in animal and plant genetics assume that the trait takes continuous values and is influenced by many variants circulating in the population, each of which acts approximately independently. Amongst the many continuous-valued bacterial traits such as cell size or antibiotic minimum inhibitory concentration, we do not know of any for which these assumptions have been validated. We propose that such traits can be identified if they are the targets of selection and this selection structures overall genetic variation of the species. The phylogenetic trees of global strain collections of Klebsiella pneumoniae (KP) have a backbone which has not been explained. We propose that this backbone reflects a genome-wide component of variation, which is well-captured by Principal Components Analysis (PCA) applied to diverse KP genomes. We show that this structure can be recapitulated in simulations of diversifying selection on a quantitative trait and cannot be explained by neutral models. An implication is that variation in KP forms an ecocline, i.e. a cline generated by natural selection rather than geography but the trait underlying the cline, if it exists, remains to be determined. ### Competing Interest Statement The authors have declared no competing interest.

The increasing antibiotic resistance of Helicobacter pylori has had a serious impact on gastric cancer prevention. Our study aimed to profile the genomic characteristics and explore variants associated with resistance in H. pylori strains from a high-risk area of gastric cancer in China. We isolated 153 strains from a community-based cohort and assessed their susceptibility to six antibiotics by MIC Test Strip and genomic characteristics by whole-genome sequencing. Phylogenetic analysis identified the strains as an independent cluster within H. pylori East Asian population (hpEastAsia). HefA, an efflux pump gene, showed the highest differentiation in the Linqu strains compared with the other Chinese strains. Bacterial genome-wide association study (GWAS) identified 86 resistance variants covering 44 genes. Novel resistance variants were found in lon and babA for metronidazole, HP1168 for clarithromycin, hcpC for levofloxacin, and sabA for rifamycin. Two newly identified hefA mutations (R229K and A283V) showed significant associations with metronidazole (P = 0.012) and tetracycline (P = 0.044) resistance, respectively. HefA mutations and GWAS variants were integrated with the significant literature-reported mutations to optimize the prediction models for metronidazole, levofloxacin, clarithromycin, and tetracycline resistance with area under the receiver operating characteristic curves of 0.82-0.93. Double-antibiotic resistance models were established for clinical applicability. Furthermore, hefA expression may play a potential mediating role in the associations between mutations and resistance. This study identified genetic independence in the representative H. pylori strains from a high-risk area of gastric cancer. Optimized resistance prediction panels, including novel hefA mutations and GWAS variants, may provide preliminary guidance for localized precise treatment and helpful experiences for the similar high-risk populations.IMPORTANCEHelicobacter pylori is a remarkable pathogen due to its virulence in gastric cancer and high genetic plasticity. Linqu County in China, a high-risk area of gastric cancer, faces serious antibiotic resistance issues and necessitates genomic profiling of local H. pylori strains. Phylogenetic analysis revealed the Linqu strains as a relatively independent cluster within the hpEastAsia population. Novel antibiotic resistance-associated hefA mutations and variants from our bacterial genome-wide association study in the Linqu strains were optimized to improve the prediction performances for single antibiotic and double-drug combination resistance compared with traditional literature-reported mutations. This study identified relative genetic independence and high differentiation in the representative H. pylori strains from a population with high risk of gastric cancer and high prevalence of antibiotic resistance. The optimized panels with novel variants improve antibiotic resistance prediction models compared with literature-reported mutations, providing guidance for localized precise treatment and suggesting prevention strategies for similar high-risk populations.

Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings, indirect evidence of negative epistasis. However, multiple HGT events enable recovery of the genome's co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred, and independently reinstated in a new genetic background-facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.IMPORTANCEGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox: 0n one hand, promoting adaptation by conferring novel functionality; on the other, potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyze natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in functional networks in a recipient genome. The positive impact of two-gene interactions appears to be adaptive by expanding metabolic capacity and facilitating niche shifts through interspecific hybridization. This challenges conventional ideas and highlights the possibility of multiple-step evolution of multi-gene traits by interspecific introgression.

Salmonella enterica causes severe food-borne infections through contamination of the food supply chain. Its evolution has been associated with human activities, especially animal husbandry. Advances in intensive farming and global transportation have substantially reshaped the pig industry, but their impact on the evolution of associated zoonotic pathogens such as S. enterica remains unresolved. Here we investigated the population fluctuation, accumulation of antimicrobial resistance genes and international serovar Choleraesuis transmission of nine pig-enriched S. enterica populations comprising more than 9,000 genomes. Most changes were found to be attributable to the developments of the modern pig industry. All pig-enriched salmonellae experienced host transfers in pigs and/or population expansions over the past century, with pigs and pork having become the main sources of S. enterica transmissions to other hosts. Overall, our analysis revealed strong associations between the transmission of pig-enriched salmonellae and the global pork trade.

While the importance of spontaneous mutation and gene acquisition in the emergence and adaptive evolution of microbial pathogens is well recognized, the role of gene loss remains underexplored. Here, by large-scale phylogenomic analysis of 8,684 global isolates, we reconstructed the origin and evolutionary dynamics of the pandemic clone (PC) of Vibrio parahaemolyticus , the leading cause of seafood-associated infections worldwide. As has also been observed for the pandemic lineage of V. cholerae , there was an ‘explosive’ burst of gene and genomic island acquisition during the emergence of the clone followed by divergence into distinct, successive sublineages mediating waves of infections. In V. parahaemolyticus, a recent global wave succession event has been driven by gene-loss in the putrescine utilization (Puu) pathway, which confers phenotypic advantages related to environmental adaptation (enhanced biofilms) and human transmission (increased cell adhesion and intestinal colonization, reduced virulence in an animal model and milder clinical symptoms in humans), aligning with the trade-off hypothesis of pathogen virulence evolution. We identify similar lineage-specific Puu-gene-loss across multiple bacterial genera, with effects on biofilm and adhesion replicated in V. cholerae and E. coli , suggesting convergent evolution and universal mechanisms across bacteria. Our findings provide a novel paradigm of adaptative evolution by gene loss in a pandemic pathogen. ### Competing Interest Statement The authors have declared no competing interest.

Helicobacter pylori disturbs the stomach lining during long-term colonization of its human host, with sequelae including ulcers and gastric cancer1,2. Numerous H. pylori virulence factors have been identified, showing extensive geographic variation1. Here we identify a 'Hardy' ecospecies of H. pylori that shares the ancestry of 'Ubiquitous' H. pylori from the same region in most of the genome but has nearly fixed single-nucleotide polymorphism differences in 100 genes, many of which encode outer membrane proteins and host interaction factors. Most Hardy strains have a second urease, which uses iron as a cofactor rather than nickel3, and two additional copies of the vacuolating cytotoxin VacA. Hardy strains currently have a limited distribution, including in Indigenous populations in Siberia and the Americas and in lineages that have jumped from humans to other mammals. Analysis of polymorphism data implies that Hardy and Ubiquitous coexisted in the stomachs of modern humans since before we left Africa and that both were dispersed around the world by our migrations. Our results also show that highly distinct adaptive strategies can arise and be maintained stably within bacterial populations, even in the presence of continuous genetic exchange between strains.

AbstractThe colonization of our stomachs byHelicobacter pyloriis believed to predate the oldest splits between extant human populations. We identify a “Hardy” ecospecies ofH. pyloriassociated with indigenous groups, isolated from people in Siberia, Canada, USA and Chile. The ecospecies shares the ancestry of “Ubiquitous”H. pylorifrom the same geographical region in most of the genome but has nearly fixed SNP differences in 100 genes, many of which encode outer membrane proteins and host interaction factors. For these parts of the genome, the ecospecies has a separate, independently evolving gene pool with a distinct evolutionary history.H. acinonychis, found in big cats, and a newly identified primate-associated lineage both belong to the Hardy ecospecies and both represent human to animal host jumps. Most strains from the ecospecies encode an additional iron-dependent urease that is shared byHelicobacterfrom carnivorous hosts, as well as a tandem duplication ofvacA, encoding the vacuolating toxin. We conclude thatH. pylorisplit into two highly distinct ecospecies in Africa and that both dispersed around the globe with humans, but the Hardy ecospecies has gone extinct in most parts of the world. Our analysis also pushes back the likely length of the association betweenH. pyloriand humans.

Bacterial genome data are accumulating at an unprecedented speed due to the routine use of sequencing in clinical diagnoses, public health surveillance, and population genetics studies. Genealogical reconstruction is fundamental to many of these uses; however, inferring genealogy from large-scale genome data sets quickly, accurately, and flexibly is still a challenge. Here, we extend an alignment- and annotation-free method, PopPUNK, to increase its flexibility and interpretability across data sets. Our method, iterative-PopPUNK, rapidly produces multiple consistent cluster assignments across a range of sequence identities. By constructing a partially resolved genealogical tree with respect to these clusters, users can select a resolution most appropriate for their needs. We showed the accuracy of clusters at all levels of similarity and genealogical inference of iterative-PopPUNK based on simulated data and obtained phylogenetically concordant results in real data sets from seven bacterial species. Using two example sets of Escherichia/Shigella and Vibrio parahaemolyticus genomes, we show that iterative-PopPUNK can achieve cluster resolutions ranging from phylogroup down to sequence typing (ST). The iterative-PopPUNK algorithm is implemented in the "PopPUNK_iterate" program, available as part of the PopPUNK package.

Helicobacter pylori , a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project ( Hp GP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the Hp GP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori . The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the Hp GP dataset and the corresponding strains to become a major asset for H. pylori genomics.

The Helicobacter pylori genome is more thoroughly mixed by homologous recombination than by any other organism that has been investigated, leading to apparent "free recombination" within populations. A recent mBio article by F. Ailloud, I. Estibariz, G. Pfaffinger, and S. Suerbaum (mBio 13:e01811-22, 2022, https://doi.org/10.1128/mbio.01811-22) helps to elucidate the cellular machinery that is used to achieve these unusual rates of genetic exchange. Specifically, they show that the UvrC gene, which is part of the repair machinery for DNA damage caused by ultraviolet light, has evolved an additional function in H. pylori, allowing very short tracts of DNA-with a mean length of only 28 bp-to be imported into the genome during natural transformation.

The East Asian region, including China, Japan and Korea, accounts for half of gastric cancer deaths. However, different areas have contrasting gastric cancer incidences and the population structure of Helicobacter pylori in this ethnically diverse region is yet unknown. We aimed to investigate genomic differences in H. pylori between these areas to identify sequence polymorphisms associated with increased cancer risk. We analysed 381 H . pylori genomes collected from different areas of the three countries using phylogenetic and population genetic tools to characterize population differentiation. The functional consequences of SNPs with a highest fixation index (Fst) between subpopulations were examined by mapping amino acid changes on 3D protein structure, solved or modelled. Overall, 329/381 genomes belonged to the previously identified hspEAsia population indicating that import of bacteria from other regions of the world has been uncommon. Seven subregional clusters were found within hspEAsia, related to subpopulations with various ethnicities, geographies and gastric cancer risks. Subpopulation-specific amino acid changes were found in multidrug exporters ( hefC ), transporters ( frpB-4 ), outer membrane proteins ( hopI ) and several genes involved in host interaction, such as a catalase site, involved in H 2 O 2 entrance, and a flagellin site mimicking host glycosylation. Several of the top hits, including frpB-4 , hefC , alpB / hopB and hofC, have been found to be differentiated within the Americas in previous studies, indicating that a handful of genes may be key to local geographic adaptation. H. pylori within East Asia are not homogeneous but have become differentiated geographically at multiple loci that might have facilitated adaptation to local conditions and hosts. This has important implications for further evaluation of these changes in relation to the varying gastric cancer incidence between geographical areas in this region.

Helicobacter pylori lives in the human stomach and has a population structure resembling that of its host. However, H. pylori from Europe and the Middle East trace substantially more ancestry from modern African populations than the humans that carry them. Here, we use a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations, implying selection to remove them during admixture. Simulations show that population fitness can be restored after bottlenecks by migration and subsequent admixture of small numbers of bacteria from non-bottlenecked populations. We conclude that recent spread of African DNA has been driven by deleterious mutations accumulated during the original out-of-Africa bottleneck.