Comparative transcriptome revealed the molecular responses of Aconitum carmichaelii Debx. to downy mildew at different stages of disease development.

BACKGROUND: Aconitum carmichaelii Debx. has been widely used as a traditional medicinal herb for a long history in China. It is highly susceptible to various dangerous diseases during the cultivation process. Downy mildew is the most serious leaf disease of A. carmichaelii, affecting plant growth and ultimately leading to a reduction in yield. To better understand the response mechanism of A. carmichaelii leaves subjected to downy mildew, the contents of endogenous plant hormones as well as transcriptome sequencing were analyzed at five different infected stages. RESULTS: The content of 3-indoleacetic acid, abscisic acid, salicylic acid and jasmonic acid has changed significantly in A. carmichaelii leaves with the development of downy mildew, and related synthetic genes such as 9-cis-epoxycarotenoid dioxygenase and phenylalanine ammonia lyase were also significant for disease responses. The transcriptomic data indicated that the differentially expressed genes were primarily associated with plant hormone signal transduction, plant-pathogen interaction, the mitogen-activated protein kinase signaling pathway in plants, and phenylpropanoid biosynthesis. Many of these genes also showed potential functions for resisting downy mildew. Through weighted gene co-expression network analysis, the hub genes and genes that have high connectivity to them were identified, which could participate in plant immune responses. CONCLUSIONS: In this study, we elucidated the response and potential genes of A. carmichaelii to downy mildew, and observed the changes of endogenous hormones content at different infection stages, so as to contribute to the further screening and identification of genes involved in the defense of downy mildew.

RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid-carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence plasmid in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

TADB 3.0: an updated database of bacterial toxin-antitoxin loci and associated mobile genetic elements.

TADB 3.0 (https://bioinfo-mml.sjtu.edu.cn/TADB3/) is an updated database that provides comprehensive information on bacterial types I to VIII toxin-antitoxin (TA) loci. Compared with the previous version, three major improvements are introduced: First, with the aid of text mining and manual curation, it records the details of 536 TA loci with experimental support, including 102, 403, 8, 14, 1, 1, 3 and 4 TA loci of types I to VIII, respectively; Second, by leveraging the upgraded TA prediction tool TAfinder 2.0 with a stringent strategy, TADB 3.0 collects 211 697 putative types I to VIII TA loci predicted in 34 789 completely sequenced prokaryotic genomes, providing researchers with a large-scale dataset for further follow-up analysis and characterization; Third, based on their genomic locations, relationships of 69 019 TA loci and 60 898 mobile genetic elements (MGEs) are visualized by interactive networks accessible through the user-friendly web page. With the recent updates, TADB 3.0 may provide improved in silico support for comprehending the biological roles of TA pairs in prokaryotes and their functional associations with MGEs.

ICEberg 3.0: functional categorization and analysis of the integrative and conjugative elements in bacteria.

ICEberg 3.0 (https://tool2-mml.sjtu.edu.cn/ICEberg3/) is an upgraded database that provides comprehensive insights into bacterial integrative and conjugative elements (ICEs). In comparison to the previous version, three key enhancements were introduced: First, through text mining and manual curation, it now encompasses details of 2065 ICEs, 607 IMEs and 275 CIMEs, including 430 with experimental support. Secondly, ICEberg 3.0 systematically categorizes cargo gene functions of ICEs into six groups based on literature curation and predictive analysis, providing a profound understanding of ICEs'diverse biological traits. The cargo gene prediction pipeline is integrated into the online tool ICEfinder 2.0. Finally, ICEberg 3.0 aids the analysis and exploration of ICEs from the human microbiome. Extracted and manually curated from 2405 distinct human microbiome samples, the database comprises 1386 putative ICEs, offering insights into the complex dynamics of Bacteria-ICE-Cargo networks within the human microbiome. With the recent updates, ICEberg 3.0 enhances its capability to unravel the intricacies of ICE biology, particularly in the characterization and understanding of cargo gene functions and ICE interactions within the microbiome. This enhancement may facilitate the investigation of the dynamic landscape of ICE biology and its implications for microbial communities.

Mobilizable plasmids drive the spread of antimicrobial resistance genes and virulence genes in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence genes. In comparison to self-transmissible conjugative plasmids, mobilizable plasmids have received much less attention due to their defects in conjugative elements. However, the contribution of mobilizable plasmids to the horizontal transfer of antimicrobial resistance genes and virulence genes of K. pneumoniae remains unclear. In this study, the transfer, stability, and cargo genes of the mobilizable plasmids of K. pneumoniae were examined via genetic experiments and genomic analysis. METHODS: Carbapenem-resistant (CR) plasmid pHSKP2 and multidrug-resistant (MDR) plasmid pHSKP3 of K. pneumoniae HS11286, virulence plasmid pRJF293 of K. pneumoniae RJF293 were employed in conjugation assays to assess the transfer ability of mobilizable plasmids. Mimic mobilizable plasmids and genetically modified plasmids were constructed to confirm the cotransfer models. The plasmid morphology was evaluated through XbaI and S1 nuclease pulsed-field gel electrophoresis and/or complete genome sequencing. Mobilizable plasmid stability in transconjugants was analyzed via serial passage culture. In addition, in silico genome analysis of 3923 plasmids of 1194 completely sequenced K. pneumoniae was performed to investigate the distribution of the conjugative elements, the cargo genes, and the targets of the CRISPR-Cas system. The mobilizable MDR plasmid and virulence plasmid of K. pneumoniae were investigated, which carry oriT but lack other conjugative elements. RESULTS: Our results showed that mobilizable MDR and virulence plasmids carrying oriT but lacking the relaxase gene were able to cotransfer with a helper conjugative CR plasmid across various Klebsiella and Escherichia coli strains. The transfer and stability of mobilizable plasmids rather than conjugative plasmids were not interfered with by the CRISPR-Cas system of recipient strains. According to the in silico analysis, the mobilizable plasmids carry about twenty percent of acquired antimicrobial resistance genes and more than seventy-five percent of virulence genes in K. pneumoniae. CONCLUSIONS: Our work observed that a mobilizable MDR or virulence plasmid that carries oriT but lacks the relaxase genes transferred with the helper CR conjugative plasmid and mobilizable plasmids escaped from CRISPR-Cas defence and remained stable in recipients. These results highlight the threats of mobilizable plasmids as vital vehicles in the dissemination of antibiotic resistance and virulence genes in K. pneumoniae.

[Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP].

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.

DeepSecE: A Deep-Learning-Based Framework for Multiclass Prediction of Secreted Proteins in Gram-Negative Bacteria.

Proteins secreted by Gram-negative bacteria are tightly linked to the virulence and adaptability of these microbes to environmental changes. Accurate identification of such secreted proteins can facilitate the investigations of infections and diseases caused by these bacterial pathogens. However, current bioinformatic methods for predicting bacterial secreted substrate proteins have limited computational efficiency and application scope on a genome-wide scale. Here, we propose a novel deep-learning-based framework-DeepSecE-for the simultaneous inference of multiple distinct groups of secreted proteins produced by Gram-negative bacteria. DeepSecE remarkably improves their classification from nonsecreted proteins using a pretrained protein language model and transformer, achieving a macro-average accuracy of 0.883 on 5-fold cross-validation. Performance benchmarking suggests that DeepSecE achieves competitive performance with the state-of-the-art binary predictors specialized for individual types of secreted substrates. The attention mechanism corroborates salient patterns and motifs at the N or C termini of the protein sequences. Using this pipeline, we further investigate the genome-wide prediction of novel secreted proteins and their taxonomic distribution across ~1,000 Gram-negative bacterial genomes. The present analysis demonstrates that DeepSecE has major potential for the discovery of disease-associated secreted proteins in a diverse range of Gram-negative bacteria. An online web server of DeepSecE is also publicly available to predict and explore various secreted substrate proteins via the input of bacterial genome sequences.

Ethyl ferulate suppresses post-myocardial infarction myocardial fibrosis by inhibiting transforming growth factor receptor 1.

BACKGROUND: With an increasing number of myocardial infarction (MI) patients, myocardial fibrosis is becoming a widespread health concern. It's becoming more and more urgent to conduct additional research and investigations into efficient treatments. Ethyl ferulate (EF) is a naturally occurring substance with cardioprotective properties. However, the extent of its impact and the underlying mechanism of its treatment for myocardial fibrosis after MI remain unknown. PURPOSE: The goal of this study was to look into how EF affected the signaling of the TGF-receptor 1 (TGFBR1) in myocardial fibrosis after MI. METHODS: Echocardiography, hematoxylin-eosin (HE) and Masson trichrome staining were employed to assess the impact of EF on heart structure and function in MI-affected mice in vivo. Cell proliferation assay (MTS), 5-Ethynyl-2'-deoxyuridine (EdU), and western blot techniques were employed to examine the influence of EF on native cardiac fibroblast (CFs) proliferation and collagen deposition. Molecular simulation and surface plasmon resonance imaging (SPRi) were utilized to explore TGFBR1 and EF interaction. Cardiac-specific Tgfbr1 knockout mice (Tgfbr1ΔMCK) were utilized to testify to the impact of EF. RESULTS: In vivo experiments revealed that EF alleviated myocardial fibrosis, improved cardiac dysfunction after MI and downregulated the TGFBR1 signaling in a dose-dependent manner. Moreover, in vitro experiments revealed that EF significantly inhibited CFs proliferation, collagen deposition and TGFBR1 signaling followed by TGF-ß1 stimulation. More specifically, molecular simulation, molecular dynamics, and SPRi collectively showed that EF directly targeted TGFBR1. Lastly, knocking down of Tgfbr1 partially reversed the inhibitory activity of EF on myocardial fibrosis in MI mice. CONCLUSION: EF attenuated myocardial fibrosis post-MI by directly suppressing TGFBR1 and its downstream signaling pathway.

CpxR promotes the carbapenem antibiotic resistance of Klebsiella pneumoniae by directly regulating the expression and the dissemination of blaKPC on the IncFII conjugative plasmid.

Klebsiella pneumoniae is an important human pathogen known for its resistance to carbapenem antibiotics, especially the increasing carbapenem-resistant hypervirulent variants. The carbapenem resistance is mainly caused by the carbapenemase gene blaKPC which was commonly found on the IncFII transferable plasmids in K. pneumoniae ST11 isolates in regions of China. However, the mechanisms of the plasmid-carrying blaKPC regulation by the host strain are not clear. To investigate the chromosome-encoded two-component system (TCS) that regulates the carbapenem resistance of K. pneumoniae caused by blaKPC, twenty-four TCSs of a carbapenem-resistant classical K. pneumoniae ST11 clinical isolate were knocked out. The deletion mutation of the TCS regulator cpxR exhibited increased sensitivity to carbapenem, which could be restored by complementation with cpxR in trans. Electrophoretic mobility shift, isothermal titration calorimetry and DNase I footprinting results revealed that CpxR directly bound to the promoter DNA of blaKPC and the binding was abolished by disrupting the DNA-binding domain in CpxR. The subsequent in vivo assays using the lacZ reporter system and qPCR showed that CpxR upregulates the transcription of blaKPC. Notably, CpxR was also found to activate the transfer of the blaKPC-carrying IncFII plasmid between the hypervirulent K. pneumoniae and E. coli isolates, in which CpxR promoted the transcription of the tra operon via binding to its promoter region. These results provide an important insight into the regulation of the host factor CpxR in the plasmid-carrying carbapenemase gene in the classical and hypervirulent K. pneumoniae.

Antibiotic-induced degradation of antitoxin enhances the transcription of acetyltransferase-type toxin-antitoxin operon.

BACKGROUND: Bacterial toxin-antitoxin (TA) modules respond to various stressful conditions. The Gcn5-related N-acetyltransferase-type toxin (GNAT) protein encoded by the GNAT-RHH TA locus is involved in the antibiotic tolerance of Klebsiella pneumoniae. OBJECTIVES: To investigate the transcriptional mechanism of the GNAT-RHH operon kacAT under antibiotic stress. METHODS: The transcriptional level of the kacAT operon of K. pneumoniae was measured by quantitative real-time (qRT) PCR assay. The degradation of antitoxin KacA was examined by western blot and fluorescent protein. The ratio of [KacA]:[KacT] was calculated by the fluorescence intensity of KacA-eGFP and mCherry-KacT. Mathematical modelling predicted protein and transcript synthesis dynamics. RESULTS: A meropenem-induced increase in transcript levels of kacA and kacT resulted from the relief from transcriptional autoregulation of the kacAT operon. Meropenem induces the degradation of KacA through Lon protease, resulting in a reduction in the ratio of [KacA]:[KacT]. The decreased ratio causes the dissociation of the KacAT complex from its promoter region, which eliminates the repression of kacAT transcription. In addition, our dynamic model of kacAT expression regulation quantitatively reproduced the experimentally observed reduction of the [KacA]:[KacT] ratio and a large increase in kacAT transcript levels under the condition of strong promoter autorepression by the KacAT complex. CONCLUSIONS: Meropenem promotes the degradation of antitoxin by enhancing the expression of Lon protease. Degradation of antitoxin reduces the ratio of intracellular [antitoxin]:[toxin], leading to detachment of the TA complex from its promoter, and releasing repression of TA operon transcription. These results may provide an important insight into the transcriptional mechanism of GNAT-RHH TA modules under antibiotic stress.

Analysis of CRISPR-Cas Loci and their Targets in Levilactobacillus brevis.

The CRISPR‒Cas system acts as a bacterial defense mechanism by conferring adaptive immunity and limiting genetic reshuffling. However, under adverse environmental hazards, bacteria can employ their CRISPR‒Cas system to exchange genes that are vital for adaptation and survival. Levilactobacillus brevis is a lactic acid bacterium with great potential for commercial purposes because it can be genetically manipulated to enhance its functionality and nutritional value. Nevertheless, the CRISPR‒Cas system might interfere with the genetic modification process. Additionally, little is known about the CRISPR‒Cas system in this industrially important microorganism. Here, we investigate the prevalence, diversity, and targets of CRISPR‒Cas systems in the genus Levilactobacillus, further focusing on complete genomes of L. brevis. Using the CRISPRCasFinder webserver, we identified 801 putative CRISPR-Cas systems in the genus Levilactobacillus. Further investigation focusing on the complete genomes of L. brevis revealed 54 putative CRISPR-Cas systems. Of these, 46 were orphan CRISPRs, and eight were CRISPR‒Cas systems. The type II-A CRISPR‒Cas system is the most common in Levilactobacillus and L. brevis complete genomes. Analysis of the spacer's target showed that the CRISPR‒Cas systems of L. brevis mainly target the enterococcal plasmids. Comparative analysis of putative CRISPR-Cas loci in Levilactobacillus brevis.

SecReT6 update: a comprehensive resource of bacterial Type VI Secretion Systems.

Type VI Secretion System (T6SS) plays significant roles in microbial activities via injecting effectors into adjacent cells or environments. T6SS increasingly gained attention due to its important influence on pathogenesis, microbial competition, etc. T6SS-associated research is explosively expanding on numerous grounds that call for an efficient resource. The SecReT6 version 3 provides comprehensive information on T6SS and the interactions between T6SS and T6SS-related proteins such as T6SS regulators and T6SS effectors. To assist T6SS researches like microbial competition and regulatory mechanisms, SecReT6 v3 developed online tools for detection and analysis of T6SS and T6SS-related proteins and estimation of T6SS-dependent killing risk. We have identified a novel T6SS regulator and T6SS-dependent killing capacity in Acinetobacter baumannii clinical isolates with the aid of SecReT6 v3. 17,212 T6SSs and plentiful T6SS-related proteins in 26,573 bacterial complete genomes were also detected, analyzed and incorporated into the database. The database is freely available at https://bioinfo-mml.sjtu.edu.cn/SecReT6/ .

Transmission of Nonconjugative Virulence or Resistance Plasmids Mediated by a Self-Transferable IncN3 Plasmid from Carbapenem-Resistant Klebsiella pneumoniae.

Klebsiella pneumoniae poses a critical challenge to clinical and public health. Along with conjugative plasmids, nonconjugative resistance or virulence plasmids associated with carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKP), and even carbapenem-resistant and hypervirulent K. pneumoniae (CR-hvKP) strains have been spreading globally. In this study, a clinical CRKP strain KP2648 was isolated, and the transferability of its plasmids was assessed using conjugation experiments. The transconjugants were characterized by polymerase chain reaction (PCR) detection, XbaI and S1-pulsed-field gel electrophoresis (PFGE), and/or whole-genome sequencing. Genetically modified IncN3 plasmids were employed to elucidate the self-transferability and the mobilization mechanisms. KP2648 has three natural plasmids: a nonconjugative IncFIB/IncHI3B virulence plasmid, a nonconjugative IncFII/IncR carbapenem-resistant plasmid, and a self-transferable IncN3 plasmid with a high conjugation frequency (7.54 ± 1.06) × 10-1. The IncN3 plasmid could mobilize the coexisting nonconjugative virulence/resistance plasmids either directly or by employing intermediate E. coli with two forms: a hybrid plasmid fused with IncN3 or a cotransfer with the helper plasmid, IncN3. Various mobile genetic elements, including ISKpn74, ISKpn14, IS26, ISShes11, ISAba11, and Tn3, are involved in the genetic transposition of diverse hybrid plasmids and the cotransfer process during the intra/interspecies transmission. IMPORTANCE Nowadays, the underlying mobilization mechanism and evolutionary processes of nonconjugative virulence or resistance plasmids in Klebsiella pneumoniae remain poorly understood. Our study revealed the high conjugation ability of IncN3 plasmid isolated from carbapenem-resistant K. pneumoniae and confirmed its capability to mobilize the nonconjugative virulence or resistance plasmids. The self-transferable IncN3 plasmid could facilitate the transmission of pathogenicity and genetic evolution of carbapenem-resistant and hypervirulent K. pneumoniae, including hv-CRKP (virulence plasmid obtained by carbapenem-resistant K. pneumoniae) and CR-hvKP (resistance plasmid obtained by hypervirulent K. pneumoniae), warranting further monitoring.

Comparative Analysis of Diverse Acetyltransferase-Type Toxin-Antitoxin Loci in Klebsiella pneumoniae.

Toxin-antitoxin (TA) modules containing a Gcn5-related N-acetyltransferase (GNAT) toxin domain regulate bacterial physiology under adverse environmental stresses. Multiple GNAT-ribbon-helix-helix domain (RHH) TA loci have been identified in single bacterial genomes. However, their diversity and interactions are still obscure. Our previous analysis showed that the GNAT toxin of Klebsiella pneumoniae, KacT, introduces antibiotic tolerance and the toxicity of GNAT is neutralized by KacA, an RHH antitoxin. We here present a phylogenetic analysis of GNAT toxins of more than 1,000 GNAT-RHH pairs detected in completely sequenced K. pneumoniae genomes, revealing that the GNAT toxins are diverse and grouped into four distinct clades. Overexpression of GNAT toxins representative of each of the four clades halts the cell growth of K. pneumoniae, while the coexpression of the cognate RHH antitoxin neutralizes GNAT toxicity. We also identify point mutations that inactivate the GNAT toxins. Moreover, we observe a cross-interaction between GNAT-RHH pairs encoded by different replicons, where a chromosomal toxin (KacT2) can be neutralized by its cognate RHH antitoxin (KacA2 on a chromosome) and another antitoxin (KacA3 on a plasmid). Finally, statistical analysis of the distribution of GNAT-RHH loci in K. pneumoniae strains shows pronounced deviation from random distribution within the same clades. Moreover, we also obtain statistically significant correlations between different clades, which we discuss in terms of the experimental results. IMPORTANCE Elucidating the roles of multifaceted GNAT-RHH TA loci is essential for understanding how these TAs interact among themselves. Recently, the reaction mechanisms and structures of several GNAT-RHH pairs have been reported. While bacterial strains can carry multiple GNAT-RHH loci with diverse origins, studies on the possible cross-interactions of these TA pairs are still limited. Here, we find that 1,000 predicted GNAT toxins of K. pneumoniae can be grouped into four distinct clades. The distributions of TA loci within these clades in K. pneumoniae strains are highly nonrandom, with the presence of a single locus of each clade per strain being highly overrepresented. Moreover, the toxicity of a GNAT toxin encoded by a chromosome was alleviated by a noncognate RHH antitoxin on a plasmid. These results might yield a profound understanding of the widespread GNAT-RHH TA pairs and the cross-interactions between noncognate TA pairs located on different replicons.

VRprofile2: detection of antibiotic resistance-associated mobilome in bacterial pathogens.

VRprofile2 is an updated pipeline that rapidly identifies diverse mobile genetic elements in bacterial genome sequences. Compared with the previous version, three major improvements were made. First, the user-friendly visualization could aid users in investigating the antibiotic resistance gene cassettes in conjunction with various mobile elements in the multiple resistance region with mosaic structure. VRprofile2 could compare the predicted mobile elements to the collected known mobile elements with similar architecture. A new mobilome indicator was proposed to give an overall estimation of the mobilome size in individual bacterial genomes. Second, the relationship between antibiotic resistance genes, mobile elements, and host strains would be efficiently examined with the aid of predicted strain's sequence typing, the incompatibility group and the transferability of plasmids. Finally, the updated back-end database, MobilomeDB2, now collected nearly a thousand active mobile elements retrieved from literature or based on prediction. The pre-computed results of the antibiotic resistance gene-carrying mobile elements of >5500 ESKAPEE genomes were also provided. We expect that VRprofile2 will provide better support for researchers interested in bacterial mobile elements and the dissemination of antibiotic resistance. VRprofile2 is freely available to all users without any login requirement at https://tool2-mml.sjtu.edu.cn/VRprofile.

ICEO, a biological ontology for representing and analyzing bacterial integrative and conjugative elements.

Bacterial integrative and conjugative elements (ICEs) are highly modular mobile genetic elements critical to the horizontal transfer of antibiotic resistance and virulence factor genes. To better understand and analyze the ongoing increase of ICEs, we developed an Integrative and Conjugative Element Ontology (ICEO) to represent the gene components, functional modules, and other information of experimentally verified ICEs. ICEO is aligned with the upper-level Basic Formal Ontology and reuses existing reliable ontologies. There are 31,081 terms, including 26,814 classes from 14 ontologies and 4128 ICEO-specific classes, representing the information of 271 known experimentally verified ICEs from 235 bacterial strains in ICEO currently and 311 predicted ICEs of 272 completely sequenced Klebsiella pneumoniae strains. Three ICEO use cases were illustrated to investigate complex joins of ICEs and their harboring antibiotic resistance or virulence factor genes by using SPARQL or DL query. ICEO has been approved as an Open Biomedical Ontology library ontology. It may be dedicated to facilitating systematical ICE knowledge representation, integration, and computer-assisted queries.

Identification and characterization of a central replication origin of the mega-plasmid pSCATT of Streptomyces cattleya.

Streptomyces linear plasmids often contain an internal replication origin. In this study, a new replication origin was identified and confirmed in the 1.8-Mb plasmid pSCATT of Streptomyces cattleya DSM46488. The real-time qPCR results indicated that the copy number of pSCATT was one copy per chromosome. The identified replication origin oriC1-II was found to locate in the central region of pSCATT and was 2 kb in size. This replication origin consists of a protein-coding gene SCATT_p08010 with an unknown function and the upstream non-coding sequence. Deletion or disruption analysis of SCATT_p08010 or the upstream non-coding sequence revealed that both SCATT_p08010 and the non-coding sequence were essential for replication. However, the identified replication origin was shown to endow the plasmid with the ability to replicate in a circular model but not in a linear model in S. lividans. Interestingly, the knockout of the replication origin did not result in the curing of pSCATT, indicating that there might be other replication origins present in the mega-plasmid. The experimental validation of the central replication origin oriC1-II might be helpful for the investigation of the replication mechanism of the mega-plasmid and the genome evolution of Streptomyces.

T4SEfinder: a bioinformatics tool for genome-scale prediction of bacterial type IV secreted effectors using pre-trained protein language model.

Bacterial type IV secretion systems (T4SSs) are versatile and membrane-spanning apparatuses, which mediate both genetic exchange and delivery of effector proteins to target eukaryotic cells. The secreted effectors (T4SEs) can affect gene expression and signal transduction of the host cells. As such, they often function as virulence factors and play an important role in bacterial pathogenesis. Nowadays, T4SE prediction tools have utilized various machine learning algorithms, but the accuracy and speed of these tools remain to be improved. In this study, we apply a sequence embedding strategy from a pre-trained language model of protein sequences (TAPE) to the classification task of T4SEs. The training dataset is mainly derived from our updated type IV secretion system database SecReT4 with newly experimentally verified T4SEs. An online web server termed T4SEfinder is developed using TAPE and a multi-layer perceptron (MLP) for T4SE prediction after a comprehensive performance comparison with several candidate models, which achieves a slightly higher level of accuracy than the existing prediction tools. It only takes about 3 minutes to make a classification for 5000 protein sequences by T4SEfinder so that the computational speed is qualified for whole genome-scale T4SEs detection in pathogenic bacteria. T4SEfinder might contribute to meet the increasing demands of re-annotating secretion systems and effector proteins in sequenced bacterial genomes. T4SEfinder is freely accessible at https://tool2-mml.sjtu.edu.cn/T4SEfinder_TAPE/.

Identification of Type II Toxin-Antitoxin Loci in Levilactobacillus brevis.

Levilactobacillus brevis are present in various environments, such as beer, fermented foods, silage, and animal host. Like other lactic acid bacteria, L. brevis might adopt the viable but nonculturable (VBNC) state under unfavorable conditions. The toxin-antitoxin (TA) system, known to regulate cell growth in response to environmental stresses, is found to control the dynamic of the VBNC state. Here, we investigate the type II TA locus prevalence and compare the TA diversity in L. brevis genomes. Using the TAfinder software, we identified a total of 273 putative type II TA loci in 110 replicons of 21 completely sequenced genomes. Genome size does not appear to correlate with the amount of putative type II TA in L. brevis. Besides, type II TA loci are distributed differently among the chromosomes and plasmids. The most prevalent toxin domain is MazF-like in the chromosomes, and RelE/RelE-like in the plasmids; while for antitoxin, Xre-like and Phd-like domains are the most common in the chromosomes and plasmids, respectively. We also observed a unique GNAT-like/ArsR-like TA pair that presents only in the L. brevis chromosome. Detection of 273 putative type II TA loci in 21 complete genomes of Levilactobacillus brevis.