Functional versatility of Zur in metal homeostasis, motility, biofilm formation, and stress resistance in Yersinia pseudotuberculosis.

Zur (zinc uptake regulator) is a significant member of the Fur (ferric uptake regulator) superfamily, which is widely distributed in bacteria. Zur plays crucial roles in zinc homeostasis and influences cell development and environmental adaptation in various species. Yersinia pseudotuberculosis is a Gram-negative enteric that pathogen usually serves as a model organism in pathogenicity studies. The regulatory effects of Zur on the zinc transporter ZnuABC and the protein secretion system T6SS have been documented in Y. pseudotuberculosis. In this study, a comparative transcriptomics analysis between a ∆zur mutant and the wild-type (WT) strain of Y. pseudotuberculosis was conducted using RNA-seq. This analysis revealed global regulation by Zur across multiple functional categories, including membrane transport, cell motility, and molecular and energy metabolism. Additionally, Zur mediates the homeostasis not only of zinc but also ferric and magnesium in vivo. There was a notable decrease in 35 flagellar biosynthesis and assembly-related genes, leading to reduced swimming motility in the ∆zur mutant strain. Furthermore, Zur upregulated multiple simple sugar and oligopeptide transport system genes by directly binding to their promoters. The absence of Zur inhibited biofilm formation as well as reduced resistance to chloramphenicol and acidic stress. This study illustrates the comprehensive regulatory functions of Zur, emphasizing its importance in stress resistance and pathogenicity in Y. pseudotuberculosis. IMPORTANCE: Bacteria encounter diverse stresses in the environment and possess essential regulators to modulate the expression of genes in responding to the stresses for better fitness and survival. Zur (zinc uptake regulator) plays a vital role in zinc homeostasis. Studies of Zur from multiple species reviewed that it influences cell development, stress resistance, and virulence of bacteria. Y. pseudotuberculosis is an enteric pathogen that serves a model organism in the study of pathogenicity, virulence factors, and mechanism of environmental adaptation. In this study, transcriptomics analysis of Zur's regulons was conducted in Y. pseudotuberculosis. The functions of Zur as a global regulator in metal homeostasis, motility, nutrient acquisition, glycan metabolism, and nucleotide metabolism, in turn, increasing the biofilm formation, stress resistance, and virulence were reviewed. The importance of Zur in environmental adaptation and pathogenicity of Y. pseudotuberculosis was emphasized.

Characterization of an aspartate aminotransferase encoded by YPO0623 with frequent nonsense mutations in Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a genetically monomorphic bacterial pathogen that evolved from Yersinia pseudotuberculosis approximately 7,400 years ago. We observed unusually frequent mutations in Y. pestis YPO0623, mostly resulting in protein translation termination, which implies a strong natural selection. These mutations were found in all phylogenetic lineages of Y. pestis, and there was no apparent pattern in the spatial distribution of the mutant strains. Based on these findings, we aimed to investigate the biological function of YPO0623 and the reasons for its frequent mutation in Y. pestis. Our in vitro and in vivo assays revealed that the deletion of YPO0623 enhanced the growth of Y. pestis in nutrient-rich environments and led to increased tolerance to heat and cold shocks. With RNA-seq analysis, we also discovered that the deletion of YPO0623 resulted in the upregulation of genes associated with the type VI secretion system (T6SS) at 26°C, which probably plays a crucial role in the response of Y. pestis to environment fluctuations. Furthermore, bioinformatic analysis showed that YPO0623 has high homology with a PLP-dependent aspartate aminotransferase in Salmonella enterica, and the enzyme activity assays confirmed its aspartate aminotransferase activity. However, the enzyme activity of YPO0623 was significantly lower than that in other bacteria. These observations provide some insights into the underlying reasons for the high-frequency nonsense mutations in YPO0623, and further investigations are needed to determine the exact mechanism.

Macrophage innate immune responses delineate between defective translocon assemblies produced by Yersinia pseudotuberculosis YopD mutants.

Translocon pores formed in the eukaryotic cell membrane by a type III secretion system facilitate the translocation of immune-modulatory effector proteins into the host cell interior. The YopB and YopD proteins produced and secreted by pathogenic Yersinia spp. harboring a virulence plasmid-encoded type III secretion system perform this pore-forming translocator function. We had previously characterized in vitro T3SS function and in vivo pathogenicity of a number of strains encoding sited-directed point mutations in yopD. This resulted in the classification of mutants into three different classes based upon the severity of the phenotypic defects. To investigate the molecular and functional basis for these defects, we explored the effectiveness of RAW 264.7 cell line to respond to infection by representative YopD mutants of all three classes. Signature cytokine profiles could separate the different YopD mutants into distinct categories. The activation and suppression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to alter anti-phagocytosis and programmed cell death pathways. These analyses demonstrated that sub-optimal translocon pores impact the extent and magnitude of host cell responsiveness, and this limits the capacity of pathogenic Yersinia spp. to fortify against attack by both early and late arms of the host innate immune response.

Development of detection methods by multiplex real-time PCR for highly pathogenic Yersinia enterocolitica, low pathogenic Yersinia enterocolitica, and Yersinia pseudotuberculosis based on SYBR Green and TaqMan probes.

This study aimed to develop multiplex real-time PCR methods using SYBR Green and TaqMan probes for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis. Specific primer and probe combinations for differentiating three pathogenic Yersinia groups were designed from three chromosomally encoded genes (ail, fyuA, and inv). Twenty-six stains of pathogenic Yersinia species including 6 strains of low pathogenic Y. enterocolitica serotypes, 7 strains of highly pathogenic Y. enterocolitica serotypes, and 13 strains of pathogenic Y. pseudotuberculosis were used for specificity testing. Specific patterns of real-time amplification signals distinguished three pathogenic Yersinia groups. A detection limit of approximately 101 colony forming units (CFU) /reaction of genomic DNA was determined based on plate counts. Furthermore, the multiplex real-time PCR methods also detected Y. enterocolitica O:8 from the DNA extracted from spiked rabbit blood samples and potentially infected wild rodent fecal samples. These results demonstrated that the multiplex real-time PCR methods developed in this study are useful for rapid detection and differentiation of three pathogenic Yersinia groups. Therefore, these methods provide a new monitoring and detection capability to understand the epidemiology of pathogenic Yersinia and to diagnose three pathogenic Yersinia groups.

The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis.

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5'-untranslated region (5'-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The trxA gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5'-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with the upregulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature.

Development and validation of a bacterial gastrointestinal multiplex RT-PCR assay for use on a fully automated molecular system.

PCR-based enteric multiplex panels represent a rapid and reliable alternative to conventional "classical" phenotypic stool diagnostics. The aim of this study was to establish a laboratory-developed non-commercial multiplex Real-Time-PCR panel for the detection of the most important bacterial stool pathogens, Salmonella spp., Shigella spp., Yersinia enterocolitica/ pseudotuberculosis and Campylobacter jejuni/coli. on the "open" cobas omni Utility Channel (UC) of the cobas 6800 system (Roche). The aim was to replace the laborious phenotypical stool diagnostics with a high throughput Real-Time PCR method. The respective primers and probes were designed to cover conserved genomic regions of the pathogens and validated using Ultramer oligonucleotides, positive stool material and reference strains. To further validate the multiplex PCR-assay, the following parameters were evaluated: analytical-sensitivity and -specificity, cross-reactivity, linearity and inter- and intra-assay variance as well as limit of detection (LOD). In addition a retrospective analysis of culture positive and negative samples from daily routine was performed using 745 native stool samples. The Gastro assay was linear over a 5-log-unit and within the expected dynamic range with amplification efficiencies ranging from 94.6% to 120%. In addition, all targets showed excellent coefficients of repeatability (≤ 1.11%), intermediate precision (≤ 1.02%) and total variance (≤ 1.39%). In terms of analytical sensitivity the assay demonstrated detection limits ranging from 7.83 copies per reaction to 14.4 copies per reaction. The assay showed excellent agreement with culture methods (>95%) and a 100% sensitivity and specificity after resolution of discrepant results. The multiplex-PCR assay provides a comprehensive, rapid and sensitive alternative to conventional methods for the detection of the major bacterial stool pathogens in diagnostic laboratories.

Determination of Growth Rate and Virulence Plasmid Copy Number During Yersinia pseudotuberculosis Infection Using Droplet Digital PCR.

Pathogenic bacteria have evolved the ability to evade their host defenses and cause diseases. Virulence factors encompass a wide range of adaptations that allow pathogens to survive and proliferate in the hostile host environment during successful infection. In human pathogenic Yersinia species, the potent type III secretion system (T3SS) and other essential virulence factors are encoded on a virulence plasmid. Here, we investigated the bacterial growth rate and plasmid copy number following a Yersinia infection using droplet digital PCR (ddPCR). ddPCR is an exceptionally sensitive, highly precise, and cost-efficient method. It enables precise quantification even from very small amounts of target DNA. This method also enables analysis of complex samples with large amounts of interfering DNA, such as infected tissues or microbiome studies.

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis.

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

Inflammatory monocytes promote granuloma control of Yersinia infection.

Granulomas are organized immune cell aggregates formed in response to chronic infection or antigen persistence. The bacterial pathogen Yersinia pseudotuberculosis (Yp) blocks innate inflammatory signalling and immune defence, inducing neutrophil-rich pyogranulomas (PGs) within lymphoid tissues. Here we uncover that Yp also triggers PG formation within the murine intestinal mucosa. Mice lacking circulating monocytes fail to form defined PGs, have defects in neutrophil activation and succumb to Yp infection. Yersinia lacking virulence factors that target actin polymerization to block phagocytosis and reactive oxygen burst do not induce PGs, indicating that intestinal PGs form in response to Yp disruption of cytoskeletal dynamics. Notably, mutation of the virulence factor YopH restores PG formation and control of Yp in mice lacking circulating monocytes, demonstrating that monocytes override YopH-dependent blockade of innate immune defence. This work reveals an unappreciated site of Yersinia intestinal invasion and defines host and pathogen drivers of intestinal granuloma formation.

A secreted effector with a dual role as a toxin and as a transcriptional factor.

Bacteria have evolved multiple secretion systems for delivering effector proteins into the cytosol of neighboring cells, but the roles of many of these effectors remain unknown. Here, we show that Yersinia pseudotuberculosis secretes an effector, CccR, that can act both as a toxin and as a transcriptional factor. The effector is secreted by a type VI secretion system (T6SS) and can enter nearby cells of the same species and other species (such as Escherichia coli) via cell-cell contact and in a contact-independent manner. CccR contains an N-terminal FIC domain and a C-terminal DNA-binding domain. In Y. pseudotuberculosis cells, CccR inhibits its own expression by binding through its DNA-binding domain to the cccR promoter, and affects the expression of other genes through unclear mechanisms. In E. coli cells, the FIC domain of CccR AMPylates the cell division protein FtsZ, inducing cell filamentation and growth arrest. Thus, our results indicate that CccR has a dual role, modulating gene expression in neighboring cells of the same species, and inhibiting the growth of competitors.

Protocol for detecting Yersinia pseudotuberculosis nitric oxide exposure during in vitro growth.

Yersinia pseudotuberculosis (Yptb) is a bacterial pathogen that causes foodborne illness. Defense against the host antimicrobial gas, nitric oxide (NO), by the bacterial NO-detoxifying gene, hmp, promotes Yptb replication in mouse models of infection. Here, we detail the use of fluorescent signals as readouts for NO exposure within individual cells and subsequent detection of heterogeneity within a population, using single-cell imaging and analysis. This protocol quantifies NO exposure in culture, without capturing the full complexity of the host environment. For complete details on the use and execution of this protocol, please refer to Patel et al. (2021).

Protection Induced by Oral Vaccination with a Recombinant Yersinia pseudotuberculosis Delivering Yersinia pestis LcrV and F1 Antigens in Mice and Rats against Pneumonic Plague.

A newly attenuated Yersinia pseudotuberculosis strain (designated Yptb1) with triple mutation Δasd ΔyopK ΔyopJ and chromosomal insertion of the Y. pestis caf1R-caf1M-caf1A-caf1 operon was constructed as a live vaccine platform. Yptb1 tailored with an Asd+ plasmid (pYA5199) (designated Yptb1[pYA5199]) simultaneously delivers Y. pestis LcrV and F1. The attenuated Yptb1(pYA5199) localized in the Peyer's patches, lung, spleen, and liver for a few weeks after oral immunization without causing any disease symptoms in immunized rodents. An oral prime-boost Yptb1(pYA5199) immunization stimulated potent antibody responses to LcrV, F1, and Y. pestis whole-cell lysate (YPL) in Swiss Webster mice and Brown Norway rats. The prime-boost Yptb1(pYA5199) immunization induced higher antigen-specific humoral and cellular immune responses in mice than a single immunization did, and it provided complete short-term and long-term protection against a high dose of intranasal Y. pestis challenge in mice. Moreover, the prime-boost immunization afforded substantial protection for Brown Norway rats against an aerosolized Y. pestis challenge. Our study highlights that Yptb1(pYA5199) has high potential as an oral vaccine candidate against pneumonic plague.

NO-Stressed Y. pseudotuberculosis Has Decreased Cell Division Rates in the Mouse Spleen.

Fluorescence dilution approaches can detect bacterial cell division events and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein and then tracking partitioning of fluorescence into daughter cells. However, fluorescence can be diluted very quickly within a rapidly replicating population, such as pathogenic bacterial populations replicating within host tissues. To overcome this limitation, we have generated two revTetR reporter constructs, where either mCherry or yellow fluorescent protein (YFP) is constitutively expressed and repressed by addition of tetracyclines, resulting in fluorescence dilution within defined time frames. We show that fluorescent signals are diluted in replicating populations and that signal accumulates in growth-inhibited populations, including during nitric oxide (NO) exposure. Furthermore, we show that tetracyclines can be delivered to the mouse spleen during Yersinia pseudotuberculosis infection and defined a drug concentration that results in even exposure of cells to tetracyclines. We then used this system to visualize bacterial cell division within defined time frames postinfection. revTetR-mCherry allowed us to detect slow-growing cells in response to NO in culture; however, this strain had a growth defect within mouse tissues, which complicated results. To address this issue, we constructed revTetR-YFP using the less toxic YFP and showed that heightened NO exposure correlated with heightened YFP signal, indicating decreased cell division rates within this subpopulation in vivo. This revTetR reporter will provide a critical tool for future studies to identify and isolate slowly replicating bacterial subpopulations from host tissues.

First Description of a Yersinia pseudotuberculosis Clonal Outbreak in France, Confirmed Using a New Core Genome Multilocus Sequence Typing Method.

Yersinia pseudotuberculosis is an enteric pathogen causing mild enteritis that can lead to mesenteric adenitis in children and septicemia in elderly patients. Most cases are sporadic, but outbreaks have already been described in different countries. We report for the first time a Y. pseudotuberculosis clonal outbreak in France, that occurred in 2020. An epidemiological investigation based on food queries pointed toward the consumption of tomatoes as the suspected source of infection. The Yersinia National Reference Laboratory (YNRL) developed a new cgMLST scheme with 1,921 genes specific to Y. pseudotuberculosis that identified the clustering of isolates associated with the outbreak and allowed to perform molecular typing in real time. In addition, this method allowed to retrospectively identify isolates belonging to this cluster from earlier in 2020. This method, which does not require specific bioinformatic skills, is now used systematically at the YNRL and proves to display an excellent discriminatory power and is available to the scientific community. IMPORTANCE We describe in here a novel core-genome MLST method that allowed to identify in real time, and for the first time in France, a Y. pseudotuberculosis clonal outbreak that took place during the summer 2020 in Corsica. Our method allows to support epidemiological and microbiological investigations to establish a link between patients infected with closely associated Y. pseudotuberculosis isolates, and to identify the potential source of infection. In addition, we made this method available for the scientific community.

RNA Thermometer-coordinated Assembly of the Yersinia Injectisome.

The type III secretion system (T3SS) is indispensable for successful host cell infection by many Gram-negative pathogens. The molecular syringe delivers effector proteins that suppress the host immune response. Synthesis of T3SS components in Yersinia pseudotuberculosis relies on host body temperature, which induces the RNA thermometer (RNAT)-controlled translation of lcrF coding for a virulence master regulator that activates transcription of the T3SS regulon. The assembly of the secretion machinery follows a strict coordinated succession referred to as outside-in assembly, in which the membrane ring complex and the export apparatus represent the nucleation points. Two components essential for the initial assembly are YscJ and YscT. While YscJ connects the membrane ring complex with the export apparatus in the inner membrane, YscT is required for a functional export apparatus. Previous transcriptome-wide RNA structuromics data suggested the presence of unique intercistronic RNATs upstream of yscJ and yscT. Here, we show by reporter gene fusions that both upstream regions confer translational control. Moreover, we demonstrate the temperature-induced opening of the Shine-Dalgarno region, which facilitates ribosome binding, by in vitro structure probing and toeprinting methods. Rationally designed thermostable RNAT variants of the yscJ and yscT thermometers confirmed their physiological relevance with respect to T3SS assembly and host infection. Since we have shown in a recent study that YopN, the gatekeeper of type III secretion, also is under RNAT control, it appears that the synthesis, assembly and functionality of the Yersinia T3S machinery is coordinated by RNA-based temperature sensors at multiple levels.

Yersinia pseudotuberculosis doxycycline tolerance strategies include modulating expression of genes involved in cell permeability and tRNA modifications.

Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by tusB, and the resulting impacts on translational machinery, promotes survival during treatment with an antibiotic classically viewed as bacteriostatic. We believe this may be the first observation of bactericidal activity of doxycycline under physiological conditions, which was revealed by reversing tusB downregulation.

Osteomyelitis in a slaughter turkey flock caused by Yersinia pseudotuberculosis sequence type ST42.

A Yersinia pseudotuberculosis outbreak was diagnosed in a male turkey flock in Finland. Y. pseudotuberculosis is a quite rare zoonotic bacterium, which typically causes enteritis in humans and sudden death in animals. In this study, osteomyelitis was diagnosed in small, lame, 11- to 12-wk-old male turkeys. Lameness and slower growth among the turkeys was observed on the farm. During pathological examination, multiple lesions were found in the metaphyseal and physeal areas of the femurs, tibiotarsi, and tarsometatarsi, with multifocal to coalescing mixed heterophilic/granulomatous necrotizing osteomyelitis. Y. pseudotuberculosis was isolated from the femoral and tibiotarsal bones or from the joints of six lame turkeys sent for necropsy. The isolation required homogenizing of lesion tissue in phosphate-mannitol-peptone broth, which was cultured directly - and, if needed, after cold enrichment - on selective cefsulodin-irgasan-novobiocin agar. Whole-genome sequencing was used for identification and typing. All isolates belonged to bio/serotype 1/O:1a and sequence type ST42 (Achtman scheme), which is commonly reported in both human and animal Y. pseudotuberculosis infections in Europe. The isolates from all six turkeys showed only one to two allele differences in the core genome comparison, indicating a common source of infection. All asymptomatic turkeys were slaughtered at the age of 17 weeks. Whole and partial carcass condemnation rates at the slaughterhouse were high, but no macroscopic changes in the skeletal system were found, showing that food chain information is essential. This study confirms earlier findings that Y. pseudotuberculosis can cause osteomyelitis in fattening turkeys, leading to lameness. Food chain information is essential for slaughterhouse operations, to protect the workers and emphasize good working hygiene during slaughter.

Small Insertions and Deletions Drive Genomic Plasticity during Adaptive Evolution of Yersinia pestis.

The life cycle of Yersinia pestis has changed a lot to adapt to flea-borne transmission since it evolved from an enteric pathogen, Yersinia pseudotuberculosis. Small insertions and deletions (indels), especially frameshift mutations, can have major effects on phenotypes and contribute to virulence and host adaptation through gene disruption and inactivation. Here, we analyzed 365 Y. pestis genomes and identified 2,092 genome-wide indels on the core genome. As recently reported in Mycobacterium tuberculosis, we also detected "indel pockets" in Y. pestis, with average complexity scores declining around indel positions, which we speculate might also exist in other prokaryotes. Phylogenic analysis showed that indel-based phylogenic tree could basically reflect the phylogenetic relationships of major phylogroups in Y. pestis, except some inconsistency around the Big Bang polytomy. We observed 83 indels arising in the trunk of the phylogeny, which played a role in accumulation of pseudogenes related to key metabolism and putatively pathogenicity. We also discovered 32 homoplasies at the level of phylogroups and 7 frameshift scars (i.e., disrupted reading frame being rescued by a second frameshift). Additionally, our analysis showed evidence of parallel evolution at the level of genes, with sspA, rpoS, rnd, and YPO0624, having enriched mutations in Brazilian isolates, which might be advantageous for Y. pestis to cope with fluctuating environments. The diversified selection signals observed here demonstrates that indels are important contributors to the adaptive evolution of Y. pestis. Meanwhile, we provide potential targets for further exploration, as some genes/pseudogenes with indels we focus on remain uncharacterized. IMPORTANCE Yersinia pestis, the causative agent of plague, is a highly pathogenic clone of Yersinia pseudotuberculosis. Previous genome-wide SNP analysis provided few adaptive signatures during its evolution. Here by investigating 365 public genomes of Y. pestis, we give a comprehensive overview of general features of genome-wide indels on the core genome and their roles in Y. pestis evolution. Detection of "indel pockets," with average complexity scores declining around indel positions, in both Mycobacterium tuberculosis and Y. pestis, gives us a clue that this phenomenon might appear in other bacterial genomes. Importantly, the identification of four different forms of selection signals in indels would improve our understanding on adaptive evolution of Y. pestis, and provide targets for further physiological mechanism researches of this pathogen. As evolutionary research based on genome-wide indels is still rare in bacteria, our study would be a helpful reference in deciphering the role of indels in other species.