Histone-like Protein Research Articles

Characterization of Medusavirus encoded histones reveals nucleosome-like structures and a unique linker histone

The organization of DNA into nucleosomes is a ubiquitous and ancestral feature that was once thought to be exclusive to the eukaryotic domain of life. Intriguingly, several representatives of the Nucleocytoplasmic Large DNA Viruses (NCLDV) encode histone-like proteins that in Melbournevirus were shown to form nucleosome-like particles. Medusavirus medusae (MM), a distantly related giant virus, encodes all four core histone proteins and, unique amongst most giant viruses, a putative acidic protein with two domains resembling eukaryotic linker histone H1. Here, we report the structure of nucleosomes assembled with MM histones and highlight similarities and differences with eukaryotic and Melbournevirus nucleosomes. Our structure provides insight into how variations in histone tail and loop lengths are accommodated within the context of the nucleosome. We show that MM-histones assemble into tri-nucleosome arrays, and that the putative linker histone H1 does not function in chromatin compaction. These findings expand our limited understanding of chromatin organization by virus-encoded histones.

Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development

ABSTRACT Candida albicans is an opportunistic fungal pathogen known for surviving in various nutrient-limited conditions within the host and causing infections. Our prior research revealed that Hfl1p, an archaeal histone-like or Hap5-like protein, is linked to mitochondrial ATP generation and yeast-hyphae morphogenesis. However, the specific roles of Hfl1p in these virulence behaviours, through its function in the CBF/NF-Y complex or as a DNA polymerase II subunit, remain unclear. This study explores Hfl1p’s diverse functions in energy metabolism and morphogenesis. By combining proteomic analysis and phenotypic evaluations of the hfl1Δ/hfl1Δ mutant with ChIP data, we found that Hfl1p significantly impacts mitochondrial DNA-encoded CI subunits, the tricarboxylic acid (TCA) cycle, and morphogenetic pathways. This influence occurs either independently or alongside other transcription factors recognizing a conserved DNA motif (TAXXTAATTA). These findings emphasize Hfl1p’s critical role in linking carbon metabolism and mitochondrial respiration to the yeast-to-filamentous form transition, enhancing our understanding of C. albicans’ metabolic adaptability during morphological transition, an important pathogenic trait of this fungus. This could help identify therapeutic targets by disrupting the relationship between energy metabolism and cell morphology in C. albicans.

Molecular docking, MD simulation, and MMGBSA-binding free energy estimation study identify antibiotic analogs as potential antimalarials targeting housekeeping proteins of Plasmodium falciparum apicoplast

ABSTRACT Plasmodium falciparum exclusively harbours a non-photosynthetic plastid-like organelle called ‘apicoplast,’ crucial for survival. The apicoplast genome resembles chloroplasts, albeit much reduced at 35 kb. The housekeeping functions of the apicoplast are similar to prokaryotes, and several antibiotics have been reported to kill the malaria parasite by targeting its housekeeping functions. However, a significant structural difference between bacterial and apicoplast housekeeping proteins promotes the discovery of antibiotic analogs specifically targeting apicoplast proteins. In this study, we used a high-throughput virtual screening approach to analyze 80% of structurally similar antibiotic analog libraries against five protein targets, including histone-like protein (PfHU), gyrase A subunit (PfGyrA), RNA polymerase β subunit (PfRpoβ), elongation factor G (PfEF-G), and elongation factor Tu (PfEF-Tu) involved in different housekeeping functions of the apicoplast. This computational screening identified molecules with better binding affinities than those antibiotics shown to have antimalarial activities, suggesting they may be more potent antimalarial compounds. The molecular dynamics simulations study revealed stable conformational changes with strong hydrogen bonds between the protein–ligand complexes. The top-hit antibiotic analogs exhibit favourable drug-like characteristics, making them promising candidates for future clinical studies. We propose further biochemical and phenotypic testing of these antibiotic analogs to develop more effective apicoplast-targeting antimalarials.

TagP, a PAAR-domain containing protein, plays roles in the fitness and virulence of Acinetobacter baumannii.

Type VI secretion system (T6SS) is widely present in Gram-negative bacteria and directly mediates antagonistic prokaryote interactions. PAAR (proline-alanine-alanine-arginine repeats) proteins have been proven essential for T6SS-mediated secretion and target cell killing. Although PAAR proteins are commonly found in A. baumannii, their biological functions are not fully disclosed yet. In this study, we investigated the functions of a PAAR protein termed TagP (T6SS-associated-gene PAAR), encoded by the gene ACX60_RS09070 outside the core T6SS locus of A. baumannii strain ATCC 17978. In this study, tagP null and complement A. baumannii ATCC 17978 strains were constructed. The influence of TagP on T6SS function was investigated through Hcp detection and bacterial competition assay; the influence on environmental fitness was studied through in vitro growth, biofilm formation assay, surface motility assay, survivability in various simulated environmental conditions; the influence on pathogenicity was explored through cell adhesion and invasion assays, intramacrophage survival assay, serum survival assay, and G. melonella Killing assays. Quantitative transcriptomic and proteomic analyses were utilized to observe the global impact of TagP on bacterial status. Compared with the wildtype strain, the tagP null mutant was impaired in several tested phenotypes such as surface motility, biofilm formation, tolerance to adverse environments, adherence to eukaryotic cells, endurance to serum complement killing, and virulence to Galleria melonella. Notably, although RNA-Seq and proteomics analysis revealed that many genes were significantly down-regulated in the tagP null mutant compared to the wildtype strain, there is no significant difference in their antagonistic abilities. We also found that Histone-like nucleoid structuring protein (H-NS) was significantly upregulated in the tagP null mutant at both mRNA and protein levels. This study enriches our understanding of the biofunction of PAAR proteins in A. baumannii. The results indicates that TagP involved in a unique modulation of fitness and virulence control in A. baumannii, it is more than a classic PAAR protein involved in T6SS, while how TagP play roles in the fitness and virulence of A. baumannii needs further investigation to clarify.

A REVIEW ON EPIGENETIC REGULATION IN BACTERIAL PATHOGENESIS AND RESISTANCE

The study of bacterial Bacterial epigenetics has become important ,because it help to understanding the mechanisms used by bacteria to regulate gene expression with keeping their DNA sequence. This regulation has been known to play an important role in bacterial pathogenesis and resistance mechanisms, which assit the bacteria to easily adapt to the environmental changes and withstand hard conditions, including antibiotic exposures. In this review we clarify the essential mechanism of epigenetic regulation of the bacteria that include, DNA methylation, histone-like protein modifications, and non-coding RNAs. We focus into their importance to bacterial virulence and resistance, focusing on understanding how to use ne therapeutic strategies against antibiotic resistance and invasive diseases

The histone-like nucleoid-structuring protein encoded by the plasmid pMBL6842 regulates both plasmid stability and host physiology of Pseudoalteromonas rubra SCSIO 6842

Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host metabolism, creating a competitive advantage for plasmid-free cells. Plasmid stability hinges on efficient replication and partition mechanisms. While plasmids commonly encode histone-like nucleoid-structuring (H-NS) family proteins, the precise influence of plasmid-encoded H-NS proteins on stability remains elusive. In this study, we examined the conjugative plasmid pMBL6842, harboring the hns gene, and observed its positive regulation of parAB transcription, critical for plasmid segregation. Deletion of hns led to rapid plasmid loss, which was remedied by hns complementation. Further investigations unveiled adverse effects of hns overexpression on the bacterial host. Transcriptome analysis revealed hns's role in regulating numerous bacterial genes, impacting both host growth and swimming motility in the presence of the hns gene. Therefore, our study unveils the multifaceted roles of H-NS in both plasmid stability and host physiology, underscoring its biological significance and paving the way for future inquiries into the involvement of H-NS in horizontal gene transfer events.

Nε-lysine acetylation of the histone-like protein HBsu influences antibiotic survival and persistence in Bacillus subtilis.

Nε-lysine acetylation is recognized as a prevalent post-translational modification (PTM) that regulates proteins across all three domains of life. In Bacillus subtilis, the histone-like protein HBsu is acetylated at seven sites, which regulates DNA compaction and the process of sporulation. In Mycobacteria, DNA compaction is a survival strategy in response antibiotic exposure. Acetylation of the HBsu ortholog HupB decondenses the chromosome to escape this drug-induced, non-growing state, and in addition, regulates the formation of drug-tolerant subpopulations by altering gene expression. We hypothesized that the acetylation of HBsu plays similar regulatory roles. First, we measured nucleoid area by fluorescence microscopy and in agreement, we found that wild-type cells compacted their nucleoids upon kanamycin exposure, but not exposure to tetracycline. We analyzed a collection of HBsu mutants that contain lysine substitutions that mimic the acetylated (glutamine) or unacetylated (arginine) forms of the protein. Our findings indicate that some level of acetylation is required at K3 for a proper response and K75 must be deacetylated. Next, we performed time-kill assays of wild-type and mutant strains in the presence of different antibiotics and found that interfering with HBsu acetylation led to faster killing rates. Finally, we examined the persistent subpopulation and found that altering the acetylation status of HBsu led to an increase in persister cell formation. In addition, we found that most of the deacetylation-mimic mutants, which have compacted nucleoids, were delayed in resuming growth following removal of the antibiotic, suggesting that acetylation is required to escape the persistent state. Together, this data adds an additional regulatory role for HBsu acetylation and further supports the existence of a histone-like code in bacteria.

Investigation of heterotrophs reveals new insights in dinoflagellate evolution

Dinoflagellates are diverse and ecologically important protists characterized by many morphological and molecular traits that set them apart from other eukaryotes. These features include, but are not limited to, massive genomes organized using bacterially-derived histone-like proteins (HLPs) and dinoflagellate viral nucleoproteins (DVNP) rather than histones, and a complex history of photobiology with many independent losses of photosynthesis, numerous cases of serial secondary and tertiary plastid gains, and the presence of horizontally acquired bacterial rhodopsins and type II RuBisCo. Elucidating how this all evolved depends on knowing the phylogenetic relationships between dinoflagellate lineages. Half of these species are heterotrophic, but existing molecular data is strongly biased toward the photosynthetic dinoflagellates due to their amenability to cultivation and prevalence in culture collections. These biases make it impossible to interpret the evolution of photosynthesis, but may also affect phylogenetic inferences that impact our understanding of character evolution. Here, we address this problem by isolating individual cells from the Salish Sea and using single cell, culture-free transcriptomics to expand molecular data for dinoflagellates to include 27 more heterotrophic taxa, resulting in a roughly balanced representation. Using these data, we performed a comprehensive search for proteins involved in chromatin packaging, plastid function, and photoactivity across all dinoflagellates. These searches reveal that 1) photosynthesis was lost at least 21 times, 2) two known types of HLP were horizontally acquired around the same time rather than sequentially as previously thought; 3) multiple rhodopsins are present across the dinoflagellates, acquired multiple times from different donors; 4) kleptoplastic species have nucleus-encoded genes for proteins targeted to their temporary plastids and they are derived from multiple lineages, and 5) warnowiids are the only heterotrophs that retain a whole photosystem, although some photosynthesis-related electron transport genes are widely retained in heterotrophs, likely as part of the iron-sulfur cluster pathway that persists in non-photosynthetic plastids.

Characterization of the monoclonal antibody and the immunodominant B-cell epitope of African swine fever virus pA104R by using mouse model.

The African swine fever virus (ASFV) structural protein pA104R is the only histone-like protein encoded by eukaryotic viruses. pA104R is an essential DNA-binding protein required for DNA replication and genome packaging of ASFV, which are vital for pathogen survival and proliferation. pA104R is an important target molecule for diagnosing, treating, and immune prevention of ASFV. This study characterized monoclonal antibodies (mAbs) against pA104R and found them to recognize natural pA104R in ASFV strains with different genotypes, showing high conservation. Confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Furthermore, the pA104R protein functions through the polar interactions between the binding amino acid sites; however, these interactions may be blocked by the recognition of generated mAbs. Characterizing the immunodominant B-cell epitope of the ASFV critical proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and vaccine candidate targets.IMPORTANCEAfrican swine fever (ASF) is a highly pathogenic, lethal, and contagious viral disease affecting domestic pigs and wild boars. As no effective vaccine or other treatments have been developed, the control of African swine fever virus (ASFV) relies heavily on virus detection and diagnosis. A potential serological target is the structural protein pA104R. However, the molecular basis of pA104R antigenicity remains unclear, and a specific monoclonal antibody (mAb) against this protein is still unavailable. In this study, mAbs against pA104R were characterized and found to recognize natural pA104R in ASFV strains with different genotypes. In addition, confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Characteristics of the immunodominant B-cell epitope of ASFV proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and identifying vaccine candidate targets.

The role of cryptic ancestral symmetry in histone folding mechanisms across Eukarya and Archaea.

Histones compact and store DNA in both Eukarya and Archaea, forming heterodimers in Eukarya and homodimers in Archaea. Despite this, the folding mechanism of histones across species remains unclear. Our study addresses this gap by investigating 11 types of histone and histone-like proteins across humans, Drosophila, and Archaea through multiscale molecular dynamics (MD) simulations, complemented by NMR and circular dichroism experiments. We confirm and elaborate on the widely applied "folding upon binding" mechanism of histone dimeric proteins and report a new alternative conformation, namely, the inverted non-native dimer, which may be a thermodynamically metastable configuration. Protein sequence analysis indicated that the inverted conformation arises from the hidden ancestral head-tail sequence symmetry underlying all histone proteins, which is congruent with the previously proposed histone evolution hypotheses. Finally, to explore the potential formations of homodimers in Eukarya, we utilized MD-based AWSEM and AI-based AlphaFold-Multimer models to predict their structures and conducted extensive all-atom MD simulations to examine their respective structural stabilities. Our results suggest that eukaryotic histones may also form stable homodimers, whereas their disordered tails bring significant structural asymmetry and tip the balance towards the formation of commonly observed heterotypic dimers.

In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.

Targeted mutagenesis of the β-strand DNA binding region of African swine fever virus histone-like protein (pA104R) impairs DNA-binding activity and antibody recognition

African Swine Fever (ASF) is a highly contagious disease caused by a double-stranded DNA virus (ASFV). Despite significant advances made over the last decade, issues such as residual virulence and absence of differentiating infected from vaccinated animals (DIVA) capacity remain an obstacle in the development of live attenuated vaccines (LAVs) against ASFV. It is, therefore, necessary to identify novel strategies to improve vaccine safety, by rational mutagenesis of virulence associated genes and generation of DIVA markers. ASFV encodes a HU (histone-like protein from E. coli strain U93) homolog protein, pA104R, which is involved in viral genome assembly and host immune recognition. A phylogenetic analysis revealed that pA104R is highly conserved among ASFV isolates, suggesting that it can be a good target for vaccine design. Thus, we selectively mutated the β-strand DNA binding region (BDR) of pA104R to attenuate its enzymatic activity, and identified and mutated several B-cell epitopes present in pA104R to generate a negative marker. Residues K64, K66, and R69 in the BDR were identified as relevant for pA104R activity, with double mutation of the first two showing additive attenuation. pA104R-reactive IgM and IgG epitopes were also identified in the bottom of the BDR, with selective mutagenesis drastically reducing antibody recognition and, when combined with mutations in the arm of the BDR, leading to a further reduction of DNA-binding activity. Interestingly, the immunodominant pA104R-reactive IgG epitope was mainly recognized by IgG1 suggesting that pA104R induces a dominant Th2 response. In sum, the rational mutagenesis can reduce pA104R-DNA binding activity and immune reactivity, providing a rationale for the development of an ASFV pA104R-based DIVA vaccine.

Lysine acetylation regulates the AT-rich DNA possession ability of H-NS.

H-NS, the histone-like nucleoid-structuring protein in bacteria, regulates the stability of the bacterial genome by inhibiting the transcription of horizontally transferred genes, such as the type III and type VI secretion systems (T3/T6SS). While eukaryotic histone posttranslational modifications (PTMs) have been extensively studied, little is known about prokaryotic H-NS PTMs. Here, we report that the acetylation of H-NS attenuates its ability to silence horizontally transferred genes in response to amino acid nutrition and immune metabolites. Moreover, LC-MS/MS profiling showed that the acetyllysine sites of H-NS and K120 are indispensable for its DNA-binding ability. Acetylation of K120 leads to a low binding affinity for DNA and enhances T3/T6SS expression. Furthermore, acetylation of K120 impairs the AT-rich DNA recognition ability of H-NS. In addition, lysine acetylation in H-NS modulates in vivo bacterial virulence. These findings reveal the mechanism underlying H-NS PTMs and propose a novel mechanism by which bacteria counteract the xenogeneic silencing of H-NS.

Dynamic action of an intrinsically disordered protein in DNA compaction that induces mycobacterial dormancy.

Mycobacteria are the major human pathogens with the capacity to become dormant persisters. Mycobacterial DNA-binding protein 1 (MDP1), an abundant histone-like protein in dormant mycobacteria, induces dormancy phenotypes, e.g. chromosome compaction and growth suppression. For these functions, the polycationic intrinsically disordered region (IDR) is essential. However, the disordered property of IDR stands in the way of clarifying the molecular mechanism. Here we clarified the molecular and structural mechanism of DNA compaction by MDP1. Using high-speed atomic force microscopy, we observed that monomeric MDP1 bundles two adjacent DNA duplexes side-by-side via IDR. Combined with coarse-grained molecular dynamics simulation, we revealed the noveldynamic DNA cross-linking model of MDP1 in which a stretched IDR cross-links two DNA duplexes like double-sided tape. IDR is able to hijack HU function, resulting in the induction of strong mycobacterial growth arrest. This IDR-mediated reversible DNA cross-linking is a reasonable model for MDP1 suppression of the genomic function in the resuscitable non-replicating dormant mycobacteria.

Comparative Structural Investigation of Histone-Like HU Proteins by Small-Angle X-ray Scattering

Comparative Structural Investigation of Histone-Like HU Proteins by Small-Angle X-ray Scattering

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.

Comparative Structural Investigation of Histone-Like HU Proteins by Small-Angle X-ray Scattering

Nucleoid-associated proteins (NAPs) control the structure and functions of bacterial nucleoid. Histone-like HU proteins are most abundant NAPs in dividing bacterial cells. Previously, structural ensembles of conformations of HU proteins from pathogenic mycoplasmas Spiroplasma melliferum and Mycoplasma gallisepticum were obtained using NMR spectroscopy. A structural study of these mycoplasma proteins is performed by small-angle X-ray scattering (SAXS). The occurrence of individual conformations from the ensemble, obtained by NMR, is estimated from the scattering data on HU protein solutions. In particular, an approach based on characterization of equilibrium mixtures in terms of volume fractions of their components was applied. The general shape of the proteins and their oligomeric state are independently confirmed using ab initio bead modelling. The flexibility of DNA-binding protein domains is analyzed by the ensemble optimization method, which is based on comparison of the structural characteristics of conformations fitting the SAXS data to the distribution of these characteristics in a randomly generated set. The results obtained give a new insight on the variability of the structure of HU proteins, which is necessary for their functioning.

Z-Form Extracellular DNA in Pediatric CRS May Provide a Mechanism for Recalcitrance to Treatment.

We examined sinus mucosal samples recovered from pediatric chronic rhinosinusitis (CRS) patients for the presence of Z-form extracellular DNA (eDNA) due to its recently elucidated role in pathogenesis of disease. Further, we immunolabeled these specimens for the presence of both members of the bacterial DNA-binding DNABII protein family, integration host factor (IHF) and histone-like protein (HU), due to their known role in converting common B-DNA to the rare Z-form. Sinus mucosa samples recovered from 20 patients during functional endoscopic sinus surgery (FESS) were immunolabelled for B- and Z-DNA, as well as for both bacterial DNABII proteins. Nineteen of 20 samples (95%) included areas rich in eDNA, with the majority in the Z-form. Areas positive for B-DNA were restricted to the most distal regions of the mucosal specimen. Labeling for both DNABII proteins was observed on B- and Z-DNA, which aligned with the role of these proteins in the B-to-Z DNA conversion. Abundant Z-form eDNA in culture-positive pediatric CRS samples suggested that bacterial DNABII proteins were responsible for the conversion of eukaryotic B-DNA that had been released into the luminal space by PMNs during NETosis, to the Z-form. The presence of both DNABII proteins on B-DNA and Z-DNA supported the known role of these bacterial proteins in the B-to-Z DNA conversion. Given that Z-form DNA both stabilizes the bacterial biofilm and inactivates PMN NET-mediated killing of trapped bacteria, we hypothesize that this conversion may be contributing to the chronicity and recalcitrance of CRS to treatment. NA Laryngoscope, 134:1564-1571, 2024.

Whole Genome Sequencing and Comparative Genomic Analysis of Chlamydia gallinacea Field Strains Isolated from Poultry in Poland.

Chlamydia gallinacea is an intracellular bacterium belonging to the Chlamydiaceae family. Poultry is considered to be the major reservoir of this agent, which has worldwide distribution and a particularly consistent worldwide occurrence in chicken flocks. The bacterium has been linked to respiratory disease in humans but without definitive confirmation; nevertheless, while it has not been proved to be the cause of human respiratory disease, a recent report from Italy verified its bird-to-human transmission. This aspect being significant for public health, more research is needed to gain insight into the infection biology of C. gallinacea. In this study, the genomes of eleven novel C. gallinacea field strains from different regions of Poland were analyzed comparatively. It was confirmed that C. gallinacea strains are closely related, with at least 99.46% sequence identity. They possess a conservative genome structure involving the plasticity zone with a complete cytotoxin, the type three secretion system, inclusion membrane proteins, polymorphic membrane proteins, hctA and hctB histone-like proteins, and the chlamydial protease-like activating factor exoenzyme, as well as plasmids. Genetic diversity seems to be restricted. However, some genetic loci, such as ompA and multi-locus sequence typing target genes, are diverse enough to enable high-resolution genotyping and epidemiological tracing.

Temperature-dependent carrier state mediated by H-NS promotes the long-term coexistence of Y. pestis and a phage in soil.

The study of carrier state phages challenged the canonical lytic-lysogenic binary, and carrier state appears to be ubiquitous and ecologically important. However, the mechanisms of the carrier state are not well elucidated due to the limited phage models. Herein, we reported phage HQ103, similar to Escherichia coli phage P2. In contrast to the temperate P2 phage, the HQ103 phage does not insert its genome into the bacterial chromosome and displays a dual behavior depending on the temperature. At 37°C, HQ103 lyses the host and forms clear plaques due to the truncation of repressor CI and mutation of promoter Pc. In contrast, HQ103 maintains a carrier state lifestyle with Y. pestis at an environmental temperature (21°C). Mechanistically, we found that the host-encoded histone-like nucleoid-structuring protein H-NS, which is highly expressed at 21°C to silence the Cox promoter Pe and inhibits the phage lytic cycle. Subsequently, the HQ103 carrier state Y. pestis could grow and co-exist with the phage in the soil at 21°C for one month. Thus, this study reveals a novel carrier state lifestyle of phage HQ103 due to the H-NS mediated xenogeneic silencing and demonstrates that the carrier state lifestyle could promote long-term phage-host coexist in nature.