Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.

Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.

Conjugative plasmid-encoded toxin-antitoxin system PrpT/PrpA directly controls plasmid copy number.

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Benzo[a]pyrene exposure disrupts the organelle distribution and function of mouse oocytes.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon compound that is generated during combustion processes, and is present in various substances such as foods, tobacco smoke, and burning emissions. BaP is extensively acknowledged as a highly carcinogenic substance to induce multiple forms of cancer, such as lung cancer, skin cancer, and stomach cancer. Recently it is shown to adversely affect the reproductive system. Nevertheless, the potential toxicity of BaP on oocyte quality remains unclear. In this study, we established a BaP exposure model via mouse oral gavage and found that BaP exposure resulted in a notable decrease in the ovarian weight, number of GV oocytes in ovarian, and oocyte maturation competence. BaP exposure caused ribosomal dysfunction, characterized by a decrease in the expression of RPS3 and HPG in oocytes. BaP exposure also caused abnormal distribution of the endoplasmic reticulum (ER) and induced ER stress, as indicated by increased expression of GRP78. Besides, the Golgi apparatus exhibited an abnormal localization pattern, which was confirmed by the GM130 localization. Disruption of vesicle transport processes was observed by the abnormal expression and localization of Rab10. Additionally, an enhanced lysosome and LC3 fluorescence intensity indicated the occurrence of protein degradation in oocytes. In summary, our results suggested that BaP exposure disrupted the distribution and functioning of organelles, consequently affecting the developmental competence of mouse oocytes.

Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system.

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO-K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.

Prophage Tracer: precisely tracing prophages in prokaryotic genomes using overlapping split-read alignment.

The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. However, the identification of prophages that are highly divergent from known phages remains challenging. In this study, by taking advantage of the lysis-lysogeny switch of temperate phages, we designed Prophage Tracer, a tool for recognizing active prophages in prokaryotic genomes using short-read sequencing data, independent of phage gene similarity searching. Prophage Tracer uses the criterion of overlapping split-read alignment to recognize discriminative reads that contain bacterial (attB) and phage (attP) att sites representing prophage excision signals. Performance testing showed that Prophage Tracer could predict known prophages with precise boundaries, as well as novel prophages. Two novel prophages, dsDNA and ssDNA, encoding highly divergent major capsid proteins, were identified in coral-associated bacteria. Prophage Tracer is a reliable data mining tool for the identification of novel temperate phages and mobile genetic elements. The code for the Prophage Tracer is publicly available at https://github.com/WangLab-SCSIO/Prophage_Tracer.

SNP rs2243828 in MPO associated with myeloperoxidase level and atrial fibrillation risk in Chinese Han population.

Previous studies shown that myeloperoxidase (MPO) level is higher in patients with atrial fibrillation (AF); however, no genetic evidence between MPO and AF risk in human population was observed. Therefore, the present study was aimed to investigate the association between rs2243828, a variant in promoter region of MPO and the risk of AF in Chinese GeneID population. The results demonstrated that the minor G allele of rs2243828 showed a significant association with AF in two independent population (GeneID-north population with 694 AF cases and 710 controls, adjusted P-adj = 6.25 × 10-3 with an odds ratio was 0.77, GeneID-central population with 1106 cases and 1501 controls, P-adj = 9.88 × 10-5 with an odds ratio was 0.75). The results also showed G allele was significantly associated with lower plasma concentration of myeloperoxidase in general population. We also observed a significant difference of odds ratio between subgroups of hypertension and non-hypertension. Therefore, our findings identified variant in MPO associated with risk of AF and it may give strong evidence to link the inflammation with the incidence of AF.

Excisionase in Pf filamentous prophage controls lysis-lysogeny decision-making in Pseudomonas aeruginosa.

Pf filamentous prophages are prevalent among clinical and environmental Pseudomonas aeruginosa isolates. Pf4 and Pf5 prophages are integrated into the host genomes of PAO1 and PA14, respectively, and play an important role in biofilm development. However, the genetic factors that directly control the lysis-lysogeny switch in Pf prophages remain unclear. Here, we identified and characterized the excisionase genes in Pf4 and Pf5 (named xisF4 and xisF5, respectively). XisF4 and XisF5 represent two major subfamilies of functional excisionases and are commonly found in Pf prophages. While both of them can significantly promote prophage excision, only XisF5 is essential for Pf5 excision. XisF4 activates Pf4 phage replication by upregulating the phage initiator gene (PA0727). In addition, xisF4 and the neighboring phage repressor c gene pf4r are transcribed divergently and their 5'-untranslated regions overlap. XisF4 and Pf4r not only auto-activate their own expression but also repress each other. Furthermore, two H-NS family proteins, MvaT and MvaU, coordinately repress Pf4 production by directly repressing xisF4. Collectively, we reveal that Pf prophage excisionases cooperate in controlling lysogeny and phage production.

Significant association of rare variant p.Gly8Ser in cardiac sodium channel ß4-subunit SCN4B with atrial fibrillation.

Atrial fibrillation (AF) affects 33.5 million individuals worldwide. It accounts for 15% of strokes and increases risk of heart failure and sudden death. The voltage-gated cardiac sodium channel complex is responsible for the generation and conduction of the cardiac action potential, and composed of the main pore-forming α-subunit Nav 1.5 (encoded by the SCN5A gene) and one or more auxiliary ß-subunits, including Nav ß1 to Nav ß4 encoded by SCN1B to SCN4B, respectively. We and others identified loss-of-function mutations in SCN1B and SCN2B and dominant-negative mutations in SCN3B in patients with AF. Three missense variants in SCN4B were identified in sporadic AF patients and small nuclear families; however, the association between SCN4B variants and AF remains to be further defined. In this study, we performed mutational analysis in SCN4B using a panel of 477 AF patients, and identified one nonsynonymous genomic variant p.Gly8Ser in four patients. To assess the association between the p.Gly8Ser variant and AF, we carried out case-control association studies with two independent populations (944 AF patients vs. 9,81 non-AF controls in the first discovery population and 732 cases and 1,291 controls in the second replication population). Significant association was identified in the two independent populations and in the combined population (p = 4.16 × 10-4 , odds ratio [OR] = 3.14) between p.Gly8Ser and common AF as well as lone AF (p = 0.018, OR = 2.85). These data suggest that rare variant p.Gly8Ser of SCN4B confers a significant risk of AF, and SCN4B is a candidate susceptibility gene for AF.

Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system.

OBJECTIVES: To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates. METHODS: Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing. RESULTS: In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing. CONCLUSIONS: CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.

Type II toxin/antitoxin system ParESO /CopASO stabilizes prophage CP4So in Shewanella oneidensis.

Toxin/antitoxin (TA) loci are commonly found in mobile genetic elements such as plasmids and prophages. However, the physiological functions of these TA loci in prophages and cross-regulation among these TA loci remain largely unexplored. Here, we characterized a newly discovered type II TA pair, ParESO /CopASO , in the CP4So prophage in Shewanella oneidensis. We demonstrated that ParESO /CopASO plays a critical role in the maintenance of CP4So in host cells after its excision. The toxin ParESO inhibited cell growth, resulting in filamentous growth and eventually cell death. The antitoxin CopASO neutralized the toxicity of ParESO through direct protein-protein interactions and repressed transcription of the TA operon by binding to a DNA motif in the promoter region containing two inverted repeats [5'-GTANTAC (N)3 GTANTAC-3']. CopASO also repressed transcription of another TA system PemKSO /PemISO in megaplasmid pMR-1 of S. oneidensis through binding to a highly similar DNA motif in its promoter region. CopASO homologs are widely spread in Shewanella and other Proteobacteria, either as a component of a TA pair or as orphan antitoxins. Our study thus illustrated the cross-regulation of the TA systems in different mobile genetic elements and expanded our understanding of the physiological function of TA systems.

Molecular Basis of Gene-Gene Interaction: Cyclic Cross-Regulation of Gene Expression and Post-GWAS Gene-Gene Interaction Involved in Atrial Fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia at the clinic. Recent GWAS identified several variants associated with AF, but they account for <10% of heritability. Gene-gene interaction is assumed to account for a significant portion of missing heritability. Among GWAS loci for AF, only three were replicated in the Chinese Han population, including SNP rs2106261 (G/A substitution) in ZFHX3, rs2200733 (C/T substitution) near PITX2c, and rs3807989 (A/G substitution) in CAV1. Thus, we analyzed the interaction among these three AF loci. We demonstrated significant interaction between rs2106261 and rs2200733 in three independent populations and combined population with 2,020 cases/5,315 controls. Compared to non-risk genotype GGCC, two-locus risk genotype AATT showed the highest odds ratio in three independent populations and the combined population (OR=5.36 (95% CI 3.87-7.43), P=8.00×10-24). The OR of 5.36 for AATT was significantly higher than the combined OR of 3.31 for both GGTT and AACC, suggesting a synergistic interaction between rs2106261 and rs2200733. Relative excess risk due to interaction (RERI) analysis also revealed significant interaction between rs2106261 and rs2200733 when exposed two copies of risk alleles (RERI=2.87, P<1.00×10-4) or exposed to one additional copy of risk allele (RERI=1.29, P<1.00×10-4). The INTERSNP program identified significant genotypic interaction between rs2106261 and rs2200733 under an additive by additive model (OR=0.85, 95% CI: 0.74-0.97, P=0.02). Mechanistically, PITX2c negatively regulates expression of miR-1, which negatively regulates expression of ZFHX3, resulting in a positive regulation of ZFHX3 by PITX2c; ZFHX3 positively regulates expression of PITX2C, resulting in a cyclic loop of cross-regulation between ZFHX3 and PITX2c. Both ZFHX3 and PITX2c regulate expression of NPPA, TBX5 and NKX2.5. These results suggest that cyclic cross-regulation of gene expression is a molecular basis for gene-gene interactions involved in genetics of complex disease traits.

A minireplicon of plasmid pBMB26 represents a new typical replicon in the megaplasmids of Bacillus cereus group.

A new minireplicon (rep26 minireplicon) from pBMB26, the 188 kb indigenous plasmid related to spore-crystal association (SCA) phenotype in Bacillus thuringiensis strain YBT-020, was characterized. A 12 kb EcoRI fragment, which encoded 10 putative open reading frames (ORFs), was capable of supporting replication when cloned in a replication probe vector. Deletion and frame shift mutation analysis showed that a 4.1 kb region encompassing two putative ORFs (orf21 and orf22) was essential for the plasmid replication in B. thuringiensis. Gene orf21 encoding a 49.8 kDa protein (named Rep26) with a helix-turn-helix motif showed no homology with known replication proteins and gene orf22 encoding a protein of 82.6 kDa showed homology to bacterial PcrA helicase. The replication origin of rep26 minireplicon was proved to be located in the coding region of orf21. Plasmid stability experiments indicated that the recombinant plasmid containing rep26 minireplicon has excellent segregational stability. BLASTP analysis revealed that amino acid sequences of ORF21 and ORF22 were well conserved among Bacillus cereus group strains. The rep26 minireplicon was widely distributed and could be defined as a new typical replicon in the megaplasmids of B. cereus group.

Dissemination and loss of a biofilm-related genomic island in marine Pseudoalteromonas mediated by integrative and conjugative elements.

Acquisition of genomic islands (GIs) plays a central role in the diversification and adaptation of bacteria. Some GIs can be mobilized in trans by integrative and conjugative elements (ICEs) or conjugative plasmids if the GIs carry specific transfer-related sequences. However, the transfer mechanism of GIs lacking such elements remains largely unexplored. Here, we investigated the transmissibility of a GI found in a coral-associated marine bacterium. This GI does not carry genes with transfer functions, but it carries four genes required for robust biofilm formation. Notably, this GI is inserted in the integration site for SXT/R391 ICEs. We demonstrated that acquisition of an SXT/R391 ICE results in either a tandem GI/ICE arrangement or the complete displacement of the GI. The GI displacement by the ICE greatly reduces biofilm formation. In contrast, the tandem integration of the ICE with the GI in cis allows the GI to hijack the transfer machinery of the ICE to excise, transfer and re-integrate into a new host. Collectively, our findings reveal that the integration of an ICE into a GI integration site enables rapid genome dynamics and a new mechanism by which SXT/R391 ICEs can augment genome plasticity.

Mob/oriT, a mobilizable site-specific recombination system for unmarked genetic manipulation in Bacillus thuringiensis and Bacillus cereus.

BACKGROUND: Bacillus thuringiensis and Bacillus cereus are two important species in B. cereus group. The intensive study of these strains at the molecular level and construction of genetically modified bacteria requires the development of efficient genetic tools. To insert genes into or delete genes from bacterial chromosomes, marker-less manipulation methods were employed. RESULTS: We present a novel genetic manipulation method for B. thuringiensis and B. cereus strains that does not leave selection markers. Our approach takes advantage of the relaxase Mob02281 encoded by plasmid pBMB0228 from Bacillus thuringiensis. In addition to its mobilization function, this Mob protein can mediate recombination between oriT sites. The Mob02281 mobilization module was associated with a spectinomycin-resistance gene to form a Mob-Spc cassette, which was flanked by the core 24-bp oriT sequences from pBMB0228. A strain in which the wild-type chromosome was replaced with the modified copy containing the Mob-Spc cassette at the target locus was obtained via homologous recombination. Thus, the spectinomycin-resistance gene can be used to screen for Mob-Spc cassette integration mutants. Recombination between the two oriT sequences mediated by Mob02281, encoded by the Mob-Spc cassette, resulted in the excision of the Mob-Spc cassette, producing the desired chromosomal alteration without introducing unwanted selection markers. We used this system to generate an in-frame deletion of a target gene in B. thuringiensis as well as a gene located in an operon of B. cereus. Moreover, we demonstrated that this system can be used to introduce a single gene or an expression cassette of interest in B. thuringiensis. CONCLUSION: The Mob/oriT recombination system provides an efficient method for unmarked genetic manipulation and for constructing genetically modified bacteria of B. thuringiensis and B. cereus. Our method extends the available genetic tools for B. thuringiensis and B. cereus strains.

Curing of plasmid pBMB28 from Bacillus thuringiensis YBT-020 using an unstable replication region.

Bacillus thuringiensis serovar finitimus strain YBT-020 is the well-studied spore-crystal association (SCA) phenotypic strain, whose parasporal crystals adhere to spore after lysis of the mother cell. Its endogenous plasmids pBMB26 and pBMB28 were proved essential for this SCA phenotype. In our previous study, using conventional methods, pBMB26 cured derivative and both pBMB26 and pBMB28 cured derivative of YBT-020 were obtained. However, YBT-020 solely cured of pBMB28 could not be obtained. In this study, an unstable replication region of pBMB28 was identified and was used to construct an incompatible plasmid pRep28B. This incompatible plasmid was successfully used to cure plasmid pBMB28 and was easily eliminated through segregational instability under the optimum growth temperature of YBT-020. Therefore, an endogenous plasmid was cured from the B. thuringiensis strain utilizing plasmid incompatibility. Moreover, using an unstable replication region instead of a temperature sensitive (Ts) replication region is better to cure the incompatible plasmid because it can avoid culturing at higher temperature. This method provides an efficient method for plasmid curing in B. thuringiensis and other bacteria.

Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas.

Pseudoalteromonas is commonly found throughout the world's oceans, and has gained increased attention due to the ecological and biological significance. Although over fifty Pseudoalteromonas genomes have been sequenced with an aim to explore the adaptive strategies in different habitats, in vivo studies are hampered by the lack of effective genetic manipulation systems for most strains in this genus. Here, nine Pseudoalteromonas strains isolated from different habitats were selected and used as representative strains to develop a universal genetic manipulation system. Erythromycin and chloramphenicol resistance were chosen as selection markers based on antibiotics resistance test of the nine strains. A conjugation protocol based on the RP4 conjugative machinery in E. coli WM3064 was developed to overcome current limitations of genetic manipulation in Pseudoalteromonas. Two mobilizable gene expression shuttle vectors (pWD2-oriT and pWD2Ery-oriT) were constructed, and conjugation efficiency of pWD2-oriT from E. coli to the nine Pseudoalteromonas strains ranged from 10(-6) to 10(-3) transconjugants per recipient cells. Two suicide vectors, pK18mobsacB-Cm and pK18mobsacB-Ery (with sacB for counter-selection), were constructed for gene knockout. To verify the feasibility of this system, we selected gene or operon that may lead to phenotypic change once disrupted as targets to facilitate in vivo functional confirmation. Successful deletions of two genes related to prodigiosin biosynthesis (pigMK) in P. rubra DSM 6842, one biofilm related gene (bsmA) in P. sp. SM9913, one gene related to melanin hyperproduction (hmgA) in P. lipolytica SCSIO 04301 and two flagella-related genes (fliF and fliG) in P. sp. SCSIO 11900 were verified, respectively. In addition, complementation of hmgA using shuttle vector pWD2-oriT was rescued the phenotype caused by deletion of chromosomal copy of hmgA in P. lipolytica SCSIO 04301. Taken together, we demonstrate that the vectors and the conjugative protocol developed here have potential to use in various Pseudoalteromonas strains.

Characterization of self-generated variants in Pseudoalteromonas lipolytica biofilm with increased antifouling activities.

Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12±5%) and translucent (frequency of 5±3%), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00_08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00_17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling.

ApnI, a transmembrane protein responsible for subtilomycin immunity, unveils a novel model for lantibiotic immunity.

Subtilomycin was detected from the plant endophytic strain Bacillus subtilis BSn5 and was first reported from B. subtilis strain MMA7. In this study, a gene cluster that has been proposed to be related to subtilomycin biosynthesis was isolated from the BSn5 genome and was experimentally validated by gene inactivation and heterologous expression. Comparison of the subtilomycin gene cluster with other verified related lantibiotic gene clusters revealed a particular organization of the genes apnI and apnT downstream of apnAPBC, which may be involved in subtilomycin immunity. Through analysis of expression of the apnI and/or apnT genes in the subtilomycin-sensitive strain CU1065 and inactivation of apnI and apnT in the producer strain BSn5, we showed that the single gene apnI, encoding a putative transmembrane protein, was responsible for subtilomycin immunity. To our knowledge, evidence for lantibiotic immunity that is solely dependent on a transmembrane protein is quite rare. Further bioinformatic analysis revealed the abundant presence of ApnI-like proteins that may be responsible for lantibiotic immunity in Bacillus and Paenibacillus. We cloned the paeI gene, encoding one such ApnI-like protein, into CU1065 and showed that it confers resistance to paenibacillin. However, no cross-resistance was detected between ApnI and PaeI, even though subtilomycin and paenibacillin share similar structures, suggesting that the protection provided by ApnI/ApnI-like proteins involves a specific-sequence recognition mechanism. Peptide release/binding assays indicated that the recombinant B. subtilis expressing apnI interacted with subtilomycin. Thus, ApnI represents a novel model for lantibiotic immunity that appears to be common.

A nuclease domain fused to the Snf2 helicase confers antiphage defence in coral-associated Halomonas meridiana.

The coral reef microbiome plays a vital role in the health and resilience of reefs. Previous studies have examined phage therapy for coral pathogens and for modifying the coral reef microbiome, but defence systems against coral-associated bacteria have received limited attention. Phage defence systems play a crucial role in helping bacteria fight phage infections. In this study, we characterized a new defence system, Hma (HmaA-HmaB-HmaC), in the coral-associated Halomonas meridiana derived from the scleractinian coral Galaxea fascicularis. The Swi2/Snf2 helicase HmaA with a C-terminal nuclease domain exhibits antiviral activity against Escherichia phage T4. Mutation analysis revealed the nickase activity of the nuclease domain (belonging to PDD/EXK superfamily) of HmaA is essential in phage defence. Additionally, HmaA homologues are present in ~1000 bacterial and archaeal genomes. The high frequency of HmaA helicase in Halomonas strains indicates the widespread presence of these phage defence systems, while the insertion of defence genes in the hma region confirms the existence of a defence gene insertion hotspot. These findings offer insights into the diversity of phage defence systems in coral-associated bacteria and these diverse defence systems can be further applied into designing probiotics with high-phage resistance.

Efficacy and safety of nalbuphine for epidural labor analgesia at high altitude: An observational study.

Xining is located at the eastern edge of the Qinghai-Tibet Plateau, with an average altitude of >7000 feet (>2000 m). Nalbuphine is a kappa-opioid receptor agonist that can provide analgesia with fewer side effects than other opioid analgesics. This study aimed to evaluate pain control, side effects, and neonatal outcomes from combining nalbuphine with sufentanil and ropivacaine in 600 women during epidural anesthesia while giving birth at a high altitude in Xining, China. A total of 600 parturients receiving epidural labor analgesia were randomly divided into 2 groups, each group 300 parturients. The nalbuphine group received nalbuphine, sufentanil, and ropivacain, the control group only received sufentanil and ropivacain. The analgesic effect was evaluated through the Visual Analogue Scale scores. Neonatal outcomes were mainly evaluated through the Apgar Scores. Compared to the control group, the nalbuphine group showed lower Visual Analogue Scale scores at all time points after analgesia (P < .05). In comparison with the control group, parturients in the nalbuphine group showed lower incidence rates of fever at delivery, 24-hour postpartum bleeding, and pruritus (P < .05). However, between the 2 groups, there were no statistically significant differences in the remaining maternal and infant outcomes and neonatal outcomes (P > .05). Moreover, no adverse effects on neonatal outcomes were observed. The findings from this study support findings from previous studies that nalbuphine provided safe epidural analgesia without significant side effects for the mother and infant, and showed both safety and efficacy when used during labor at high altitude.