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The coral reef microbiome is central to reef health and resilience. Competitive interactions between opportunistic coral pathogens and other commensal microbes affect the health of coral. Despite great advances over the years in sequencing-based microbial profiling of healthy and diseased coral, the molecular mechanism underlying colonization competition has been much less explored. In this study, by examining the culturable bacteria inhabiting the gastric cavity of healthy Galaxea fascicularis, a scleractinian coral, we found that temperate phages played a major role in mediating colonization competition in the coral microbiota. Specifically, the non-toxigenic Vibrio sp. inhabiting the healthy coral had a much higher colonization capacity than the coral pathogen Vibrio coralliilyticus, yet this advantage was diminished by the latter killing the former. Pathogen-encoded LodAB, which produces hydrogen peroxide, triggers the lytic cycle of prophage in the non-toxicogenic Vibrio sp. Importantly, V. coralliilyticus could outcompete other coral symbiotic bacteria (for example, Endozoicomonas sp.) through LodAB-dependent prophage induction. Overall, we reveal that LodAB can be used by pathogens as an important weapon to gain a competitive advantage over lysogenic competitors when colonizing corals.

Background: Idiopathic ventricular tachycardia (VT) occurs in structurally normal hearts and accounts for a significant number of all types of VT. The genome-wide association study is the most effective strategy for identifying novel genetic variants for common diseases. However, no genome-wide association study has been reported for idiopathic VT. Methods: We conducted the first genome-wide association study for idiopathic VT in the Chinese Han population using a discovery population with 246 cases and 648 controls and a replication population with 222 cases and >4072 controls. Candidate VT genes were functionally characterized in zebrafish. Real-time RT-PCR analysis was used to determine the effects of candidate genes on expression of ion channels and regulators. Patch-clamping was used to record L-type calcium current from neonatal rat cardiomyocytes with overexpression of candidate genes. Results: We identified 4 significant loci represented by rs78960694 (minor allele frequency [MAF]=5.02% in cases and 1.84% in controls; P =4.30×10 − 12, odds ratio [OR]=3.91) and rs2229095 (MAF=3.25% in cases and 1.63% in controls; P =1.02×10 − 7, OR=3.44) near and in CCR7 , respectively, rs68126098 in NELL1 (MAF=40.98% in cases and 32.07% in controls; P= 2.40×10 − 8, OR=1.53), rs2390325 between PKN2 and LMO4 (MAF=21.19% in cases and 15.12% in controls; P =1.92×10 − 7, OR=1.62), and rs270065 in CSMD1 (MAF=33.63% in cases and 40.25% in controls; P =9.51×10 − 7, OR=0.69). Note that the associations of idiopathic VT for CCR7 variant rs78960694 and NELL1 variant rs68126098 reach genome-wide significance ( P − 8). Overexpression of either PKN2 or CCR7 increased the heart rate in zebrafish, and enhanced expression of CACNA1C, RYR2 , or NOS1AP in zebrafish embryos, HEK293, and AC16 cardiomyocytes. Overexpression of either PKN2 or CCR7 significantly increased L-type Ca2+ current density. Conclusions: The first genome-wide association study identifies 4 novel loci and 2 risk genes ( PKN2 and CCR7 ) for idiopathic VT. These findings identify new molecular determinants for cardiac calcium homeostasis and rhythm maintenance and provide novel targets for diagnosis and treatment for idiopathic VT.

Nitrogen cycle is critical to maintain a healthy coral reef ecosystem. Urea can provide a source of nitrogen for coral holobiont and is important for coral calcification through degradation by urease. Despite the involvement of coral-associated bacteria in nitrogen fixation, nitrification and denitrification are well recognized, to what extend urea degradation by bacteria contributing to the urea utilization by coral holobiont remains to be investigated. In this study, we demonstrate that the urea utilization is a common feature of Halomonas spp. that is a dominant genus in cultivable coral-associated bacteria. A urease operon was characterized by genome sequencing and gene knock-out technique in Halomonas meridiana SCSIO 43005, isolated from the gastric cavity of healthy scleractinian coral Galaxea fascicularis. H. meridiana showed high urease activity which was induced by urea and deletion of the urease operon reduced the capability to use urea as solo nitrogen source. Furthermore, approximately 1/3 coral-associated bacteria in the IMG/M database possess complete urease operons indicating the involvement of bacteria-derived ureases in coral holobiont. These results suggest that urease from coral-associated bacteria might be important player in the nitrogen cycling of coral reefs.

Sox2 is known to play an important role in maintaining the totipotency and self-renewal of embryonic stem cells. The purpose of this study was to prepare an anti-chicken Sox2 polyclonal antibody using prokaryotic expression techniques, to evaluate its specificity and to use it to investigate the expression and distribution of Sox2 in the chicken brain and lungs. The chicken Sox2 gene was amplified and subcloned to a pET-30a vector to construct a prokaryotic expression vector, pET-Sox2. A His-Sox2 fusion protein was expressed, purified, and used to prepare an antichicken Sox2 polyclonal antibody. Western blotting revealed that the antichicken Sox2 antibody could specifically bind not only to the purified His-Sox2 fusion protein but also to the endogenous Sox2 protein in the testes of chicken, showing a distinct dose-dependent relationship between antigen and Sox2 antibody. Indirect immunofluorescent staining of Sox2-overexpressing cells showed strong nuclear and diffuse cytoplasmic immunoreactivity for Sox2 in the antichicken Sox2 antibody-staining cells. A CRISPR/Cas9 effector system-mediated Sox2 knockdown assay indicated that Sox2 expression in HEK 293T cells was downregulated in the presence of doxycycline but upregulated in the absence of doxycycline. In addition, cryosectioning and immunohistochemical staining illustrated that most spermatogonia in the seminiferous tubules, and a small number of Sertoli and Leydig cells, were positive for Sox2. The antichicken Sox2 antibody was also successfully used to investigate the expression and distribution of Sox2 in the chicken cerebellar cortex, optic tectum, cerebral cortex, and lungs. The results of this study confirmed the specificity of the antichicken Sox2 polyclonal antibody, which will be available for the study of biological functions of the chicken Sox2 gene and the self-renewal mechanisms of chicken pluripotent stem cells.

Intraspecies diversification and niche adaptation by members of the Vibrio genus, one of the most diverse bacterial genera, is thought to be driven by horizontal gene transfer. However, the intrinsic driving force of Vibrio species diversification is much less explored. Here, by studying two dominant and competing cohabitants of the gastric cavity of corals, we found that a phenotype influencing island (named VPII) in Vibrio alginolyticus was eliminated upon coculturing with Pseudoalteromonas. The loss of VPII reduced the biofilm formation and phage resistance, but activated motility, which may allow V. alginolyticus to expand to other niches. Mechanistically, we discovered that the excision of this island is mediated by the cooperation of two unrelated mobile genetic elements harbored in Pseudoalteromonas spp., an integrative and conjugative element (ICE) and a mobilizable genomic island (MGI). More importantly, these mobile genetic elements are widespread in cohabitating Gram-negative bacteria. Altogether, we discovered a new strategy by which the mobilome is employed by competitors to increase the genomic plasticity of rivals.

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.

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 Na(v)1.5 (encoded by the SCN5A gene) and one or more auxiliary β-subunits, including Na(v)β1 to Na(v)β4 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.

Significance Since conjugative plasmids are usually large and may carry genes encoding functions that are detrimental to the bacterial host, minimizing plasmid copy number is critical for reducing the host burden. Toxin–antitoxin (TA) systems are one of the conserved modules on conjugative plasmids. Here, we demonstrate the functional significance of a large group of antitoxins on conjugative plasmids: the antitoxin acts as an unexpected player in the negative control of plasmid replication. For the plasmid-encoded PrpT/PrpA TA system, the antitoxin can control toxin production by binding to PrpT and by reducing plasmid copy number. This work shows that the antitoxin can directly regulate plasmid replication, expanding our understanding of the physiological role of TA systems., 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.

Summary 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., A proposed model of the lysis‐lysogeny switch of Pf4 in PAO1. Integration of Pf4 in host genome is maintained by MvaT/MvaU by binding to the excisionase gene xisF4. Inactivation of MvaT/MvaU upregulates xisF4 and subsequently, XisF4 activates the replication initiator gene PA0727, leading to Pf4 phage replication and release.

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.

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.

Summary Members of the marine bacterial genus Pseudoalteromonas are efficient producers of antifouling agents that exert inhibitory effects on the settlement of invertebrate larvae. The production of pigmented secondary metabolites by Pseudoalteromonas has been suggested to play a role in surface colonization. However, the physiological characteristics of the pigments produced by Pseudoalteromonas remain largely unknown. In this study, we identified and characterized a genetic variant that hyperproduces a dark-brown pigment and was generated during Pseudoalteromonas lipolytica biofilm formation. Through whole-genome resequencing combined with targeted gene deletion and complementation, we found that a point mutation within the hmgA gene, which encodes homogentisate 1,2-dioxygenase, is solely responsible for the overproduction of the dark-brown pigment pyomelanin. In P. lipolytica, inactivation of the hmgA gene led to the formation of extracellular pyomelanin and greatly reduced larval settlement and metamorphosis of the mussel Mytilus coruscus. Additionally, the extracted pyomelanin from the hmgA deletion mutant and the in vitro-synthesized pyomelanin also reduced larval settlement and metamorphosis of M. coruscus, suggesting that extracellular pyomelanin released from marine Pseudoalteromonas biofilm can inhibit the settlement of fouling organisms.

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.

Bacterial capsular polysaccharides (CPSs) participate in environmental adaptation in diverse bacteria species. However, the role and regulation of CPS production in marine bacteria have remained largely unexplored. We previously reported that both wrinkled and translucent Pseudoalteromonas lipolytica variants with altered polysaccharide production were generated in pellicle biofilm-associated cells. In this study, we observed that translucent variants were generated at a rate of ∼20% in colony biofilms of P. lipolytica cultured on HSLB agar plates for 12 days. The DNA sequencing results revealed that nearly 90% of these variants had an IS5-like element inserted within the coding or promoter regions of nine genes in the cps operon. In contrast, IS5 insertion into the cps operon was not detected in planktonic cells. Furthermore, we demonstrated that the IS5 insertion event inactivated CPS production, which leads to a translucent colony morphology. The CPS-deficient variants showed an increased ability to form attached biofilms but exhibited reduced resistance to sublethal concentrations of antibiotics. Moreover, deleting the DNA repair gene recA significantly decreased the frequency of occurrence of CPS-deficient variants during biofilm formation. Thus, IS insertion into the cps operon is an important mechanism for the production of genetic variants during biofilm formation of marine bacteria.

ObjectivesTo eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates.MethodsPlasmid 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.ResultsIn 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.ConclusionsCRISPR/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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6865-x) contains supplementary material, which is available to authorized users.

A design of 4 × 25 Gb/s parallel optical receiver analog front-end, including transimpedance amplifier (TIA) and limiting amplifier (LA), is presented in this article. It features a pseudodifferential TIA structure to cancel common mode noise and stabilize circuit. The TIA and LA use regulated cascode technique and four stages interleaving active feedback structure to acquire 23.2 GHz bandwidth and 74.3 dBΩ transimpedance gain. The parallel TIA and LA chip occupies 525 × 1835 μm2 area. It consumes 76 mW per channel and the power consumption per bit is only 2.6 mW/Gb/s. © 2015 Wiley Periodicals, Inc. Microwave Opt Technol Lett 57:974–978, 2015

Vibrio alginolyticus, an opportunistic pathogen, is commonly associated with vibriosis in fish and shellfish and can also cause superficial and ear infections in humans. V. alginolyticus ATCC 33787(T) was originally isolated from seawater and has been used as one of the type strains for exploring the virulence factors of marine bacteria and for developing vaccine against vibriosis. Here we sequenced and assembled the whole genome of this strain, and identified three megaplasmids and three Type VI secretion systems, thus providing useful information for the study of virulence factors and for the development of vaccine for Vibrio.

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, ParE

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.

Atrial fibrillation (AF) is the most common arrhythmia. In 2014, two new meta-GWAS identified 5 AF loci, including the NEURL locus, GJA1 locus, CAND2 locus, and TBX5 locus in the European ancestry populations and the NEURL locus and CUX2 locus in a Japanese population. The TBX5 locus for AF was reported by us in 2013 in the Chinese population. Here we assessed the association between AF and SNPs in the NEURL, GJA1, CAND2 and CUX2 loci in the Chinese Han population. We carried out a large case-control association study with 1,164 AF patients and 1,460 controls. Significant allelic and genotypic associations were identified between NEURL variant rs6584555 and GJA1 variant rs13216675 and AF. Significant genotypic association was found between CUX2 SNP rs6490029 and AF. No association was found between CAND2 variant rs4642101 and AF, which may be due to an insufficient power of the sample size for rs4642101. Together with our previous findings, seven of fifteen AF loci (

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.

Toxin/antitoxin (TA) systems are widespread in prokaryotic chromosomes and in mobile genetic elements including plasmids and prophages. The first characterized Type IV TA system CbtA/CbeA was found in cryptic prophage CP4-44 in Escherichia coli K-12. Two homologous TA loci of CbtA/CbeA also reside in cryptic prophages of E. coli K-12, YkfI/YafW in CP4-6 and YpjF/YfjZ in CP4-57. In this study, we demonstrated that YkfI and YpjF inhibited cell growth and led to the formation of “lemon-shaped” cells. Prolonged overproduction of YkfI led to the formation of “gourd-shaped” cells and immediate cell lysis. YafW and YfjZ can neutralize the toxicity of YkfI or YpjF. Furthermore, we found that YkfI and YpjF interacted with cell division protein FtsZ in E. coli, but ectopic expression in Pseudomonas and Shewanella did not cause the formation of “lemon-shaped” cells. Moreover, deletion of all of the three toxin genes together decreased resistance to oxidative stress and deletion of the antitoxin genes increased early biofilm formation. Collectively, these results demonstrated that the homologous Type IV TA systems in E. coli may target cell division protein FtsZ in E. coli and may have different physiological functions in E. coli.

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.

Summary Cointegrate plasmids are useful models for the study of plasmid evolution if their evolutionary processes can be replicated under laboratory conditions. pBMB0228, a 17 706 bp native plasmid originally isolated from Bacillus thuringiensis strain YBT-1518, carries two nematicidal crystal protein genes, cry6Aa and cry55Aa. In this study, we show that pBMB0228 is in fact a cointegrate of two plasmids and contains two functional replication regions and two functional mobilization regions. Upon introduction into B. thuringiensis strain BMB171, pBMB0228 spontaneously resolves into two constituent plasmids via recombination at its oriT1 and oriT2 sites. The resolution does not require conjugation but can be promoted by conjugation. We further confirm that the resolution is mediated by oriT site-specific recombination requiring Mob02281 or Mob02282. Additionally, the two constituent plasmids of pBMB0228 are mobilizable, and can fuse back via oriT site-specific integration after entering into the same cell by conjugation. Our study confirms that native plasmid can reversibly interconvert between a cointegrate structure and its constituent plasmids. This study provides insight into the evolution of cointegrate plasmids, linking plasmid evolution with conjugation and the oriT site-specific recombination function of relaxase.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0492-9) contains supplementary material, which is available to authorized users.

We have developed a strategy for isolating cry genes from Bacillus thuringiensis . The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes— cry55Aa1 , cry6Aa2 , and cry5Ba2 —were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla . The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.

Additional file 1: Figure S1. Sequence analysis of the 24bp oriT core regions before (A) and after recombination (B). Figure S2. A test of the Îą-amylase activity of amyE mutant strain BMB0260. (A) and (B), Strain BMB171 and BMB0260 were grown on LB plates containing 1% starch; One plate (B) was stained with iodine to detect the Îą-amylase activity, which was indicated by the Hydrolysis circle. 1, BMB171; 2-3, BMB0260. Figure S3. Cry5Ba and Cry2Aa expression in the unmarked cry gene integration mutants of BMB171. (A). Phase contrast microscopy graph of parasporal crystals in the unmarked cry gene integrate mutants. The arrows respectively indicates the diamond-shaped crystals encoded by cry5Ba and the round-shaped crystals encoded by cry2Aa. (B). SDS-PAGE analysis of crystal proteins in the unmarked cry gene integration mutants of BMB171. The arrows indicate the bands produced by cry5Ba and cry2Aa, respectively. 1, BMB171/cry5Ba-pHT304; 2-3, BMB0261; 4, YBT-1518; 5, BMB171/cry2Aa-pHT304; 6-7, BMB0262; 8, CT-43; 9, BMB171. M, Protein ladder.