Poly(indole-5-carboxylic acid)/reduced graphene oxide/gold nanoparticles/phage-based electrochemical biosensor for highly specific detection of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is an enteric bacterium causing yersiniosis in humans. The existing Yersinia pseudotuberculosis detection methods are time-consuming, requiring a sample pretreatment step, and are unable to discriminate live/dead cells. The current work reports a phage-based electrochemical biosensor for rapid and specific detection of Yersinia pseudotuberculosis. The conductive poly(indole-5-carboxylic acid), reduced graphene oxide, and gold nanoparticles are applied for surface modification of the electrode. They possess ultra-high redox stability and retain 97.7% of current response after performing 50 consecutive cycles of cyclic voltammetry.The specific bacteriophages vB_YepM_ZN18 we isolated from hospital sewage water were immobilized on modified electrodes by Au-NH2 bond between gold nanoparticles and phages. The biosensor fabricated with nanomaterials and phages were utilized to detect Yersinia pseudotuberculosis successfully with detection range of 5.30 × 102 to 1.05 × 107 CFU mL-1, detection limit of 3 CFU mL-1, and assay time of 35 min. Moreover, the biosensor can specifically detect live Yersinia pseudotuberculosis without responding to phage-non-host bacteria and dead Yersinia pseudotuberculosis cells. These results suggest that the proposed biosensor is a promising tool for the rapid and selective detection of Yersinia pseudotuberculosis in food, water, and clinical samples.

Complete genome sequence of the novel phage vB_BthS-HD29phi infecting Bacillus thuringiensis.

The phage vB_BthS-HD29phi infecting Bacillus thuringiensis strain HD29 was isolated and purified. The morphology of the phage showed that it belongs to the family Siphoviridae. The phage genome was 32,181 bp in length, comprised linear double-stranded DNA with an average G + C content of 34.9%, and exhibited low similarity to known phage genomes. Genomic and phylogenetic analysis revealed that vB_BthS-HD29phi is a novel phage. In total, 50 putative ORFs were predicted in the phage genome, and only 18 ORFs encoded proteins with known functions. This article reports the genome sequence of a new tailed phage and increases the known genetic diversity of tailed phages.

Characterization of rhizosphere bacterial community and berry quality of Hutai No.8 (Vitis vinifera L.) with different ages, and their relations.

BACKGROUND: Rhizosphere soil microbial communities play an important role in grapevine growth. However, the relationship of the rhizosphere soil bacterial community and berry quality of Hutai No.8 grape with different tree-ages is unclear. In this work, the Biolog-ECO technique was used to explore the functional diversity of the rhizosphere soil bacterial communities of Hutai No.8 grape with five ages (3, 5, 7, 9 and 11 years old). Meanwhile, grape berry quality indicators related to berry appearance, flavor and functional substance quality was also examined. RESULTS: Principal component analysis of grape berry quality mainly separated 3-year-old (first bear fruit) and the other tree-ages. In particular, peel weight and total soluble solid content of 3-year-old grape berry was significantly less than that of others. Furthermore, average well color development, species richness and Shannon's diversity index increased significantly with grapevine age. Moreover, the metabolic activities and functional diversity of soil microbial communities in using carbon sources were significantly increasing with grapevine age. Moreover, there were significant correlation between physicochemical indices of grape berry quality and six functional categories of carbon sources. CONCLUSION: Tree-ages could greatly affect the rhizosphere microbial community structure and richness, and then affect the grape berry quality. © 2019 Society of Chemical Industry.

Characteristics and complete genome analysis of a novel jumbo phage infecting pathogenic Bacillus pumilus causing ginger rhizome rot disease.

Tailed phages with genomes larger than 200 kbp are classified as jumbo phage and exhibit extremely high diversity. In this study, a novel jumbo phage, vB_BpuM_BpSp, infecting pathogenic Bacillus pumilus, the cause of ginger rhizome rot disease, was isolated. Notable features of phage vB_BpuM_BpSp are the large phage capsid of 137 nm and baseplate-attached curly tail fibers. The genome of the phage is 255,569 bp in size with G+C content of 25.9 %, and it shows low similarity to known biological entities. The phage genome contains 318 predicted coding sequences. Among these predicted coding sequences, 26 genes responsible for nucleotide metabolism were found, and seven structural genes could be identified. The findings of this study provide new understanding of the genetic diversity of phages.

Construction of an environmental safe Bacillus thuringiensis engineered strain against Coleoptera.

Cloning of new toxic genes from Bacillus thuringiensis (Bt) and construction of Bt engineered strains are two key strategies for bio-control of coleopteran pests in agriculture and forestry. In this study, we cloned a new cry3Aa-type gene, cry3Aa8, from wild Bt strain YC-03 against coleopteran, and constructed a Bt engineered strain, ACE-38, containing insecticidal protein-encoding gene cry3Aa8. The engineered strain, with almost four times of Cry3Aa yield compared with strain YC-03, was an antibiotic marker-free strain. Though no selective pressure was presented in the medium, cry3Aa8 in the engineered strain ACE-38 remained stable. The yield of Cry3Aa by strain ACE-38 reached 2.09 mg/ml in the optimized fermentation medium. The activity of strain ACE-38 against Plagiodera versicolora was tested, and the LC50 of ACE-38 cultures in the optimized fermentation medium was 1.13 µl/ml. Strain ACE-38 is a non-antibiotic Bt engineered strain with high Chrysomelidae toxicity and exhibits good fermentation property. The modified indigenous site-specific recombination system constructed in this study might be useful for the construction of Bt engineered strains containing genes that cannot be expressed in the indigenous site-specific recombination system using plasmid pBMB1205R.

Effects of actin-like proteins encoded by two Bacillus pumilus phages on unstable lysogeny, revealed by genomic analysis.

We characterized two newly isolated myoviruses, Bp8p-C and Bp8p-T, infecting the ginger rhizome rot disease pathogen Bacillus pumilus GR8. The plaque of Bp8p-T exhibited a clear center with a turbid rim, suggesting that Bp8p-T could transform into latent phage. Lysogeny assays showed that both the two phages could form latent states, while Bp8p-T could form latent phage at a higher frequency and stability than Bp8p-C. The genomes of Bp8p-C and Bp8p-T were 151,417 and 151,419 bp, respectively; both encoded 212 putative proteins, and only differed by three nucleotides. Moreover, owing to this difference, Bp8p-C encoded a truncated, putative actin-like plasmid segregation protein Gp27-C. Functional analysis of protein Gp27 showed that Gp27-T encoded by Bp8p-T exhibited higher ATPase activity and assembly ability than Gp27-C. The results indicate that the difference in Gp27 affected the phage lysogenic ability. Structural proteome analysis of Bp8p-C virion resulted in the identification of 14 structural proteins, among which a pectin lyase-like protein, a putative poly-gamma-glutamate hydrolase, and three proteins with unknown function, were firstly identified as components of the phage virion. Both phages exhibited specific lytic ability to the host strain GR8. Bp8p-C showed better control effect on the pathogen in ginger rhizome slices than Bp8p-T, suggesting that Bp8p-C has a potential application in bio-control of ginger rhizome rot disease.

Genomic characteristics and comparative genomics analysis of Penicillium chrysogenum KF-25.

BACKGROUND: Penicillium chrysogenum has been used in producing penicillin and derived ß-lactam antibiotics for many years. Although the genome of the mutant strain P. chrysogenum Wisconsin 54-1255 has already been sequenced, the versatility and genetic diversity of this species still needs to be intensively studied. In this study, the genome of the wild-type P. chrysogenum strain KF-25, which has high activity against Ustilaginoidea virens, was sequenced and characterized. RESULTS: The genome of KF-25 was about 29.9 Mb in size and contained 9,804 putative open reading frames (orfs). Thirteen genes were predicted to encode two-component system proteins, of which six were putatively involved in osmolarity adaption. There were 33 putative secondary metabolism pathways and numerous genes that were essential in metabolite biosynthesis. Several P. chrysogenum virus untranslated region sequences were found in the KF-25 genome, suggesting that there might be a relationship between the virus and P. chrysogenum in evolution. Comparative genome analysis showed that the genomes of KF-25 and Wisconsin 54-1255 were highly similar, except that KF-25 was 2.3 Mb smaller. Three hundred and fifty-five KF-25 specific genes were found and the biological functions of the proteins encoded by these genes were mainly unknown (232, representing 65%), except for some orfs encoding proteins with predicted functions in transport, metabolism, and signal transduction. Numerous KF-25-specific genes were found to be associated with the pathogenicity and virulence of the strains, which were identical to those of wild-type P. chrysogenum NRRL 1951. CONCLUSION: Genome sequencing and comparative analysis are helpful in further understanding the biology, evolution, and environment adaption of P. chrysogenum, and provide a new tool for identifying further functional metabolites.

Genomic analysis of a phage and prophage from a Bacillus thuringiensis strain.

Bacteriophages have been found to be the most abundant and also potentially most diverse biological entities on Earth. In the present study, Bacillus phages were isolated rapidly and shown to have a high degree of diversity. The genomes of a newly isolated phage, phiCM3, and a prophage, proCM3, from the Bacillus thuringiensis strain YM-03 were sequenced and characterized. Comparative genome analysis showed that the phiCM3 genome is highly similar to the genomes of eight other Bacillus phages and seven of these phages were classified as the Wß group of phages. Analysis of the differential evolution of the genes in the Wß-group phages indicated that the genes encoding the antirepressor and tail fibre protein were more highly conserved than those encoding the major capsid protein, DNA replication protein, and RNA polymerase σ factor, which might have diverged to acquire mechanisms suitable for survival in different microbial hosts. Genome analysis of proCM3 revealed that proCM3 might be a defective phage because of mutations in the minor structural protein, and it was not inducible by mitomycin C treatment. The proCM3 genome was similar to those of two lytic Bacillus phages in sequence, but had a different genomic structure, composed of three regions in a different order. These data suggest that the three phages might have had a common ancestor and that genome rearrangement might have occurred during evolution. The findings of this study enrich our current knowledge of Bacillus phage diversity and evolution, especially for the Wß-group and TP21-L-like phages, and may help the development of practical applications of Bacillus phages.

Bacillus pumilus, a Novel Ginger Rhizome Rot Pathogen in China.

Ginger rhizome rot is a major factor limiting the yield and marketability of ginger in Shandong Province, China. In order to identify the pathogen causing ginger rhizome rot, evaluate its pathogenicity, and explore its pathogenesis, diseased ginger rhizomes and surrounding soils were collected. A gram-positive, spore-forming, rod-shaped bacterium, designated GR8, was frequently isolated from the ginger rhizome samples. The bacterium was identified as Bacillus pumilus based on physio-biochemical and molecular biology characteristics. Pathogenicity studies with GR8 showed that it could cause disease of the tested rhizomes slices and the entire rhizome when wounded but no disease occurred when the rhizome was not wounded. Preliminary pathogenicity studies demonstrated that cell-free cultures of GR8 could not cause any disease symptoms, whereas the bacterial suspensions caused severe symptoms. The pathology studies revealed that infection of GR8 could cause starch grains to shrink from normal size, and destroy the parenchyma cells by invading and propagating in them. This is the first report of B. pumilus causing ginger rhizome rot.

Machine learning and statistical models for analyzing multilevel patent data.

A recent surge of patent applications among public hospitals in China has aroused significant research interest. A country's healthcare innovation capacity can be measured by its number of patents. This paper explores the link between the number of patents and ten independent variables. Multicollinearity was carefully detected and removed by using the variable selection method and LASSO regression, respectively. The Poisson model and the negative binomial model were proposed to analyze the patent data. Three goodness of fit tests, the Pearson test, the deviance test, and the DHARMa non-parametric dispersion test, were conducted to investigate if the model has a good fit. After discovering four clusters by conducting agglomerative hierarchical clustering, these two models were replaced by the negative binomial mixed model. The likelihood ratio test was used to determine which model is more appropriate and the results reveal that the negative binomial mixed model outperforms both the Poisson model and the negative binomial model. Three variables, number of health technicians per 10,000 people, financial expenditure on science and technology as well as number of patent applications per 10,000 health personnel, have a significantly positive relationship with the number of patents in Chinese tertiary public hospitals.

Characteristics of a broad lytic spectrum endolysin from phage BtCS33 of Bacillus thuringiensis.

BACKGROUND: Endolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33). RESULTS: Bioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50 °C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60 °C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins. CONCLUSIONS: PlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain.

Chromate reduction by a chromate-resistant bacterium, Microbacterium sp.

Heavy-metal chromium [Cr(VI)] is a ubiquitous environmental pollutant. Comparing with chemical reduction, microbiological reduction is considered to be a friendly and cheaper way to decrease the damage caused by chromate. A bacterial strain, CR-07, which is resistant to and capable of reducing chromate was isolated from a mud sample of iron ore and identified as a Microbacterium sp. The bacterium had a high degree of tolerance to chromate, and could grow in LB medium containing 4.08 mM of K(2)Cr(2)O(7). It also had a degree of resistance to other heavy metals, e.g. Cd(2+), Pb(2+), Zn(2+), Cu(2+), Co(2+), Hg(2+) and Ag(+). The bacterium could remove 1.02 mM of Cr(VI) from LB medium within 36 h of incubation. Chromate removal was achieved in the supernatant from the bacterial cultures, and corresponded to chromate reduction. The activity of chromate reduction by the bacterium was not related to enzymes or reducing sugars, while fluorometric assay suggested that glutathione, a chromate-reducing substance which was produced by the bacterium, was one of the factors that contributed to the reduction of Cr(VI).

Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis.

A novel δ-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel ß-sheets (domain II); and 134 residues forming a ß-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC50s) were 8.98 µg/ml for the expressed Cry7Ca1, 0.87 µg/ml for the activated toxin 1, and 4.43 µg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.

Isolation and phosphate-solubilizing ability of a fungus, Penicillium sp. from soil of an alum mine.

The use of microorganisms to solubilize elemental phosphorus from insoluble rock phosphate is a promising method to greatly reduce not only environmental pollution but also production costs. Phosphate-solubilizing microorganisms were isolated from soils in China, and a fungus strain (PSM11-5) from a soil sample from an alum mine, with the highest phosphate solubilization potential, was selected and identified as a Penicillium sp. Strain PSM11-5 could grow in buffered medium with pH values between 3.0 and 8.0 and showed phosphate solubilizing activity at pH values from 5.0 to 8.0. It also exhibited a degree of tolerance to the heavy metal ions, Cd(2+), Co(2+), and Cr(6+). PSM11-5 could rapidly solubilize tricalcium phosphate, and a high phosphate-solubilizing efficiency of 98% was achieved in an optimized medium. The strain could solubilize rock phosphate and aluminum phosphate with a solubilizing efficiency of approximately 74.5%, but did not solubilize iron phosphate. Solubilization of phosphate depended on acidification. Analysis of PSM11-5 culture supernatants by capillary electrophoresis showed that tricalcium phosphate was solubilized to PO(4) (3-) and Ca(2+) , and that the organic acid produced by the fungus was mainly gluconic acid at approximately ca. 13 g l(-1). In addition, PSM11-5 produced ca. 830 mg l(-1) of citric acid when it was used to solubilize rock phosphate. These excellent properties of strain PSM11-5 suggest that the fungus has potential for agricultural and industrial utilization.

Genomic, Antimicrobial, and Aphicidal Traits of Bacillus velezensis ATR2, and Its Biocontrol Potential against Ginger Rhizome Rot Disease Caused by Bacillus pumilus.

Ginger rhizome rot disease, caused by the pathogen Bacilluspumilus GR8, could result in severe rot of ginger rhizomes and heavily threaten ginger production. In this study, we identified and characterized a new Bacillus velezensis strain, designated ATR2. Genome analysis revealed B. velezensis ATR2 harbored a series of genes closely related to promoting plant growth and triggering plant immunity. Meanwhile, ten gene clusters involved in the biosynthesis of various secondary metabolites (surfactin, bacillomycin, fengycin, bacillibactin, bacilysin, difficidin, macrolactin, bacillaene, plantazolicin, and amylocyclicin) and two clusters encoding a putative lipopeptide and a putative phosphonate which might be explored as novel bioactive compounds were also present in the ATR2 genome. Moreover, B. velezensis ATR2 showed excellent antagonistic activities against multiple plant pathogenic bacteria, plant pathogenic fungi, human pathogenic bacteria, and human pathogenic fungus. B. velezensis ATR2 was also efficacious in control of aphids. The antagonistic compound from B. velezensis ATR2 against B.pumilus GR8 was purified and identified as bacillomycin D. In addition, B. velezensis ATR2 exhibited excellent biocontrol efficacy against ginger rhizome rot disease on ginger slices. These findings showed the potential of further applications of B. velezensis ATR2 as a biocontrol agent in agricultural diseases and pests management.

Genomic Analysis of a Novel Phage Infecting the Turkey Pathogen Escherichia coli APEC O78 and Its Endolysin Activity.

Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of maintaining a healthy digestive tract. However, some E. coli strains can cause serious zoonotic diseases, including diarrhea, pneumonia, urinary tract infections, and hemolytic uremic syndrome. In this study, we characterized a newly isolated Myoviridae phage, vB_EcoM_APEC. The phage vB_EcoM_APEC was able to infect E. coli APEC O78, which is the most common MDR E. coli serotype in turkeys. Additionally, the phage's host range included Klebsiella pneumoniae and other E. coli strains. The genome of phage vB_EcoM_APEC (GenBank accession number MT664721) was 35,832 bp in length, with 52 putative open reading frames (ORFs) and a GC content of 41.3%. The genome of vB_EcoM_APEC exhibited low similarity (79.1% identity and 4.0% coverage) to the genome of Acinetobacter phage vB_AbaM_IME284 (GenBank no. MH853787.1) according to the nucleotide Basic Local Alignment Search Tool (BLASTn). Phylogenetic analysis revealed that vB_EcoM_APEC was a novel phage, and its genome sequence showed low similarity to other available phage genomes. Gene annotation indicated that the protein encoded by orf11 was an endolysin designated as LysO78, which exhibited 64.7% identity (91.0% coverage) with the putative endolysin of Acinetobacter baumannii phage vB_AbaM_B9. The LysO78 protein belongs to glycoside hydrolase family 19, and was described as being a chitinase class I protein. LysO78 is a helical protein with 12 α-helices containing a large domain and a small domain in terms of the predicted three-dimensional structure. The results of site-directed mutagenesis indicated that LysO78 contained the catalytic residues E54 and E64. The purified endolysin exhibited broad-spectrum bacteriolytic activity against Gram-negative strains, including the genera Klebsiella, Salmonella, Shigella, Burkholderia, Yersinia, and Pseudomonas, as well as the species Chitinimonas arctica, E. coli, Ralstonia solanacearum, and A. baumannii. An enzymatic assay showed that LysO78 had highly lytic peptidoglycan hydrolases activity (64,620,000 units/mg) against E. coli APEC O78, and that LysO78 had lytic activity in the temperature range of 4-85 °C, with an optimal temperature of 28 °C and optimal pH of 8.0, and was active at pH 3.0-12.0. Overall, the results suggested that LysO78 might be a promising therapeutic agent for controlling MDR E. coli APEC O78 and nosocomial infections caused by multidrug-resistant bacteria.

Reliability and Validity Study of the Children's Eating Behavior Questionnaire in Chinese School-Age Children.

Children's Eating Behavior Questionnaire (CEBQ) was widely used in western countries to measure children's eating behavior and played an important role in the research on the risk of childhood obesity, but it is rarely used in China yet. This study aimed to examine the Chinese version of CEBQ's reliability and validity in a pilot study, then applied it to bigger population. Data was collected with CEBQ in two districts of Shanghai, respectively. Using stratified cluster sampling method, a total of 2,520 children were included in the study. The questionnaires were filled out by children's parents. It was proved that the Cronbach's coefficient α of the questionnaire was greater than 0.7, indicating that the questionnaire had good internal consistency. Aside from the subscales 'Emotional undereating' and 'Food fussiness', the Pearson's correlation coefficients of the other subscales were all greater than 0.5. It showed that test-retest reliability of CEBQ was acceptable. We found that boys and girls had different eating behavior characteristics. And there was a graded association between eating behavior and nutritional status of school-age children. It suggested that appetitive traits of school-age children could be used as indicators of susceptibility to weight gain. In conclusion, the Chinese version of CEBQ was of good reliability and validity, so it is considered as a reliable tool that can be applied to Chinese school-age children's eating behavior and related studies.

Diagnostic efficacy of intravascular ultrasound combined with Gd2O3-EPL contrast agent for patients with atherosclerosis.

Atherosclerosis is a cardiovascular disease that is pathologically associated with the growth of atherosclerotic plaques and vascular vulnerability. Intravascular ultrasound (IVUS) has been used to evaluate and treat cardiovascular diseases. Accumulating evidence has demonstrated that Gd2O3-doped nanoparticles contrast can be applied for the diagnosis of human diseases. In the present study, eplerenone (EPL), a mineralocorticoid receptor antagonist, was first doped with Gd2O3 nanoparticles (Gd2O3-EPL), following which its diagnostic efficacy for use in IVUS measurements (Gd2O3-EPL-IVUS) was evaluated for patients suspected with atherosclerosis. Gd2O3-EPL-IVUS presented with higher accuracy and sensitivity compared with IVUS in diagnosing 188 patients with suspected atherosclerosis. Gd2O3-EPL-IVUS exhibited stronger signals associated with plaque morphology compared with aloe IVUS for patients with atherosclerosis. In addition, Gd2O3-EPL-IVUS application resulted in clearer arterial plaque images compared with IVUS by binding mineralocorticoid receptors. Atherosclerosis was subsequently confirmed in all patients using computerized tomography-coronary angiography. Gd2O3-EPL-IVUS showed more accuracy in measuring vessel size, plaque burden and minimal lumen area compared with IVUS analysis alone. In conclusion, these outcomes suggest that Gd2O3-EPL-IVUS is a reliable tool for the evaluation of coronary lesions in patients with atherosclerosis.

Novel spore lytic enzyme from a Bacillus phage leading to spore killing.

Bacterial spores maintain metabolic dormancy and have high resistance to external pressure. Germination requires degradation of the spore cortex and the participation of germination-specific cortex-lytic enzymes (GSLEs). Previously reported GSLEs have been identified in bacteria and facilitate germination. In this study, we have characterized a novel spore lytic enzyme, Ply67, from Bacillus pumilus phage vB_BpuM_BpSp. Ply67 had a similar cortex-lytic activity to GSLEs but disrupted the inner membranes (IMs) of spores, leading to spore killing rather than germination. The amino acid sequence of the complete protein, Ply67FL, exhibited 40% homology to the GSLE SleB. Domain prediction showed that Ply67FL was composed of three domains: a signal peptide, N-terminal domain protein and C-terminal domain protein. Ply67FL rapidly caused E. coli cells lysis when it was expressed in E. coli. The protein containing the C-terminal domain protein, Ply67C, could kill B. pumilus spores. The protein containing the N-terminal domain protein, Ply67N, could combine with the decoated B. pumilus spores, indicating that N-terminal was the binding domain and C-terminal was the hydrolase domain. The protein lacking the signal peptide but containing the N-terminal and C-terminal domain proteins, Ply67, had activity against spores of various Bacillus species. The surface of spores treated with Ply67 shrank and the permeability barrier was disrupted, and the inner contents leaked out. Immunoelectron microscopic observation showed that Ply67 was mainly acted on the spore cortex. Overall, Ply67 is a novel spore lytic enzyme that differs from other GSLEs not only in amino acid sequence but also in activity against spores, and Ply67 might have the potential to kill spores of pathogenic Bacillus species, e.g., B. cereus and B. anthracis.