Putative Open Reading Frames Research Articles

Characterization of the novel temperate Staphylococcus haemolyticus phage IME1365_01

The presence of a novel functional prophage, IME1365_01, was predicted from bacterial high-throughput sequencing data and then successfully induced from Staphylococcus haemolyticus by mitomycin C treatment. Transmission electron microscopy showed that phage IME1365_01 has an icosahedral head (43nm in diameter) and a long tail (172nm long). This phage possesses a double-stranded DNA genome of 44,875 bp with a G+C content of 35.35%. A total of 63 putative open reading frames (ORFs) were identified in its genome. BLASTn analysis revealed that IME1365_01 is similar to Staphylococcus phage vB_SepS_E72, but with a genome homology coverage of only 26%. The phage genome does not have fixed termini. In ORF24 of phage IME1365_01, a conserved Toll-interleukin-1 receptor domain of the TIR_2 superfamily (accession no. c123749) is located at its N-terminus, and this might serve as a component of an anti-bacterial system. In conclusion, we developed a platform to obtain active temperate phage from prediction, identification, and induction from its bacterial host. After mass screening using this platform, numerous temperate phages and their innate anti-bacterial elements can provide extensive opportunities for therapy against bacterial (especially drug-resistant bacterial) infections.

A newly isolated bacteriophage vB8388 and its synergistic effect with aminoglycosides against multi-drug resistant Klebsiella oxytoca strain FK-8388.

The bacteriophage vB8388 can lyse multi-drug resistant Klebsiella oxytoca strain FK-8388 and maintain stability in a wide range of temperatures (from 4°C to 80°C) and pHs (3-11). Bioinformatics analysis showed that vB8388 is a linear double-stranded DNA virus that is 39,750 long with 50.65% G+C content and 44 putative open reading frames (ORFs). Phage vB8388 belongs to the family Autographviridae and possesses a non-contractile tail. The latency period of vB8388 was approximately 20min. The combination of phage vB8388 and gentamicin, amikacin, or tobramycin could effectively inhibit the growth of K. oxytoca strain FK-8388, with a decrease of more than 4 log units within 12h in vitro. Phage vB8388 showed a strong synergistic effect with gentamicin that could enhance the anti-biofilm effect of vB8388. The phage+gentamicin combination also showed synergy in vivo in the larval infection model of Galleria mellonella. In conclusion, the findings of this study suggest the potential of phage+antibiotic combination therapy to be used as an alternative therapeutic approach for treating infectious diseases caused by multidrug-resistant bacteria.

Characterization and genomic Analysis of a novel Pseudomonas phage vB_PsaP_M1, representing a new viral family, Psaeviridae

Pseudomonas is a ubiquitous and ambiguous opportunistic pathogen, and plays an important ecological role in the ocean. Here, a new species, Pseudomonas phage vB_PsaP_M1, is described, which was isolated from the surface coastal waters of Qingdao, China. vB_PsaP_M1 contains a linear, double-stranded 89,387-bp genome with a GC content of 41.04% and encoding 184 putative open reading frames (ORFs). There were 50 conservative domains were predicted with BLASTp, including two auxiliary metabolic genes (Phosphate-inducible gene phoH and signal peptide peptidase A, sppA). Phylogenetic analysis of whole genome amino acid sequence and comparative genomic analysis showed that vB_PsaP_M1 has a distant evolutionary relationship with previously isolated viruses and can be grouped into a family-level novel viral cluster (VC_61) with eleven uncultured, assembled viral genomes, named as Psaeviridae. Psaeviridae contains two ORFs (ORFs 117 and 127), which were not detected in the genomes of other viral families, confirming the proposal for a new family. Combined with its ability to infect Pseudomonas and its representation of an unstudied viral family, vB_PsaP_M1 may be an important and novel model system for the study of interactions between viruses and host cells in marine ecosystems.

The C-Terminal 300 Amino Acid Residues of the G Protein and Putative Open Reading Frame X of the G Gene of Tailam Paramyxovirus (TlmPV) Are Not Required for Replication in Tissue Culture Cells.

Tailam paramyxovirus (TlmPV) was identified in Sikkim Rats in Hong Kong, China in 2011. Its negative sense RNA genome is similar to J paramyxovirus (JPV) and Beilong paramyxovirus (BeiPV), the prototypes of the recently established genus Jeilongvirus. TlmPV genome is predicted to have eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G/X-L-5'. The predicted size of the TlmPV G protein is 1,052 amino acid (aa) residues and much larger than G proteins of typical paramyxoviruses, which are often less than 800 aa. In addition to G open reading frame (ORF) in the G gene, another ORF, termed ORF-X exists in the G gene transcript. Similar ORF-X exists in JPV and BeiPV G gene, but their expression in virus-infected cells has not been confirmed. In this study, we generated infectious TlmPV using a newly developed reverse genetics system. We have found that the G protein of TlmPV is truncated in cultured cells: stop codons emerged in the G open reading frame, resulting in deletions of amino acid residues beyond residue 732. We have obtained infectious TlmPV lacking the C-terminal 307 aa (rTlmPV-G745) and TlmPV lacking the C-terminal 306 aa and the ORF-X (rTlmPV-GΔ746-X). The recombinant TlmPVs lacking the C-terminal 300 aa reach a higher peak viral titer and have improved genome stability in tissue cultured cells. The work indicates that the C-terminal of the G protein of TlmPV and ORF-X are not required for replication in tissue culture cells, and the deletion of the C-terminal confers a growth advantage in tissue culture cells. IMPORTANCE TlmPV is a member of the recently established genus Jeilongvirus. TlmPV encodes a large G protein and its G gene contains ORF-X. In this work, infectious TlmPV was recovered using reverse genetics. Using this system, we have demonstrated that 300 aa of C-terminal of G and the ORF-X are not required for viral replication in tissue culture cells.

Mining and characterization of the PKS–NRPS hybrid for epicoccamide A: a mannosylated tetramate derivative from Epicoccum sp. CPCC 400996

BackgroundGenomic analysis indicated that the genomes of ascomycetes might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained enigmatic. The ascomycete genus Epicoccum, belonging to the family Didymellaceae, is ubiquitous that colonizes different types of substrates and is associated with phyllosphere or decaying vegetation. Species of this genus are prolific producers of bioactive substances. The epicoccamides, as biosynthetically distinct mannosylated tetramate, were first isolated in 2003 from Epicoccum sp. In this study, using a combination of genome mining, chemical identification, genetic deletion, and bioinformatic analysis, we identified the required BGC epi responsible for epicoccamide A biosynthesis in Epicoccum sp. CPCC 400996.ResultsThe unconventional biosynthetic gene cluster epi was obtained from an endophyte Epicoccum sp. CPCC 400996 through AntiSMASH-based genome mining. The cluster epi includes six putative open reading frames (epiA-epiF) altogether, in which the epiA encodes a tetramate-forming polyketide synthase and nonribosomal peptide synthetases (PKS−NRPS hybrid). Sequence alignments and bioinformatic analysis to other metabolic pathways of fungal tetramates, we proposed that the gene cluster epi could be involved in generating epicoccamides. Genetic knockout of epiA completely abolished the biosynthesis of epicoccamide A (1), thereby establishing the correlation between the BGC epi and biosynthesis of epicoccamide A. Bioinformatic adenylation domain signature analysis of EpiA and other fungal PKS-NRPSs (NRPs) indicated that the EpiA is l-alanine incorporating tetramates megasynthase. Furthermore, based on the molecular structures of epicoccamide A and deduced gene functions of the cluster epi, a hypothetic metabolic pathway for biosynthesizing compound 1 was proposed. The corresponding tetramates releasing during epicoccamide A biosynthesis was catalyzed through Dieckmann-type cyclization, in which the reductive (R) domain residing in terminal module of EpiA accomplished the conversion. These results unveiled the underlying mechanism of epicoccamides biosynthesis and these findings might provide opportunities for derivatization of epicoccamides or generation of new chemical entities.ConclusionGenome mining and genetic inactivation experiments unveiled a previously uncharacterized PKS − NRPS hybrid-based BGC epi responsible for the generation of epicoccamide A (1) in endophyte Epicoccum sp. CPCC 400996. In addition, based on the gene cluster data, a hypothetical biosynthetic pathway of epicoccamide A was proposed.

Deciphering the cytochrome P450 genes in the microbiome of a chronically polluted soil with history of agricultural activities

BackgroundCytochrome P450 monooxygenases (CYPs) are exciting biocatalysts that catalyzes diverse regio- and stereoselective reactions of a broad range of substrates. The cytochrome P450 genes (CYPomes) of a chronically polluted soil (3S) with history of agricultural activities were deciphered via functional annotation of putative ORFs (open reading frames) using KEGG KofamKOALA, PHMMER, the Cytochrome P450 Engineering Database CYPED v6.0, and the NCBI Batch Web CD-Search tool.ResultsAnnotation revealed the detection of seventy-seven CYP families and eight standalone CYPs cutting across the three domains of life. The prokaryote CYPome has 72 CYP families, 93 subfamilies, and seven standalone CYPs. The phylum Proteobacteria and the genera Streptomyces, Mycobacterium, and Bacillus with 17, 16, 24, and 5 CYP families were predominant, while the domain Archaea was represented by CYP119A1. The phylum Cyanobacteria has two families, while 23 actinobacterial CYPs (other than Streptomyces and Mycobacterium) were also detected. The detected prokaryote CYPs are responsible for biodegradation of camphor, hydroxylation of monoterpene alcohols, biosynthesis of secondary metabolites, and hydroxylation of fatty acids and steroidal compounds. The eukaryote CYPome was represented by seven fungal CYPs (CYP505A3, CYP505B1, CYP51A, CYP51C, CYP55A1, CYP55A2, and CYP55A3) from Acremonium egyptiacum, Fusarium oxysporum, Aspergillus oryzae, Gibberella moniliformis, Aspergillus flavus, and Fusarium lichenicola, respectively, and CYP524A1 from the slime mold, Dictyostelium discoideum. The fungi CYPs were involved in biosynthesis of secondary metabolites, hydroxylation of fatty acids, and nitrate reduction and denitrification.ConclusionsThis study has established the diverse roles played by CYPs in soil, its implication for soil health and resilience, and its potentials for industrial application.

Genome analysis of Psilogramma increta granulovirus and its intrapopulation diversity

The complete genome of Psilogramma increta granulovirus (PsinGV), isolated from P. increta (Lepidoptera: Sphingidae), was ultra-deep sequenced with a Novaseq PE150 platform and de novo assembled and annotated. The PsinGV genome is a circular double-stranded DNA, 103,721 bp in length, with a G+C content of 33.0%, the third lowest G+C content in present sequenced baculoviruses. It encodes 123 putative open reading frames, including 38 baculovirus core genes, 42 lepidopteran baculovirus conserved genes, 38 betabaculovirus conserved genes, and 5 genes unique to PsinGV. Meanwhile, 3 homologous repeated regions with the core sequence TTGCAA and 3 direct repeated sequences were identified within the PsinGV genome. Kimura two-parameters distance analysis confirmed that Psilogramma increta granulovirus is a representative of a prospective new species of the genus Betabaculovirus. Phylogenetic analysis based on the baculovirus core genes showed that PsinGV is closely related to Choristoneura fumiferana granulovirus, Clostera anastomosis granulovirus-B, and Erinnyis ello granulovirus. These four species therefore share a common ancestor residing in the Betabaculovirus genus. The genome of the PsinGV isolate contained two p10 copies: p10 and p10-2. Phylogenetic reconstruction of P10 suggests a transfer event of the p10-2 gene from an alphabaculovirus to the aforementioned common ancestor. Analysis of genomic diversity showed that 203 intrahost variants, including 182 single nucleotide variants and 21 short insertions/deletions, are present within the PsinGV isolate. Meanwhile, allele frequency indicated that the isolate contains three major genotypes, implying the archived isolate consists of several P. increta carcasses killed by PsinGV with different genotypes.

Characterisation of broad-spectrum phiKZ like jumbo phage and its utilisation in controlling multidrug-resistant Pseudomonas aeruginosa isolates

The emergence of highly virulent multidrug-resistant P. aeruginosa has become increasingly evident among hospital-acquired infections and has raised the need for alternative therapies. Phage therapy can be one such alternative to antibiotic therapy to combat multidrug-resistant pathogenic bacteria, but this requires the availability of phages with a broad host range. In this study, isolation and molecular characterisation of P. aeruginosa specific phages were carried out. A total of 17 phages isolated showed different spectra of activity and efficiency of lysis against 82 isolates of P. aeruginosa obtained from clinical samples (n = 13), hospital effluent (n = 46) and fish processing plant effluent (n = 23). Antibiotic susceptibility test results revealed multi-drug resistance in 61 of the total 82 isolates. Three new jumbo lytic P. aeruginosa specific broad host range phages were isolated and characterised in this present study belonged to the family Myoviridae (order Caudovirales). The genetic analysis of ɸU5 revealed that phage has a genome size of 282.6 kbp with 373 putative open reading frames (ORFs), and its genetic architecture is similar to phiKZ like jumbo phages infecting P. aeruginosa. The bacteriophages isolated in this study had lytic ability against biofilm-forming and multidrug-resistant P. aeruginosa and could be candidates for further studies towards phage therapy.

Isolation and characterization of Brochothrix phage ADU4

B. thermosphacta is a psychrotrophic bacterium that often forms the predominant part of the spoilage microflora of aerobically and anaerobically stored meats. Bacteriophages are natural enemies of bacteria and their potential for use in environmentally friendly biocontrol of specific pathogens in food is being intensively studied. In this study, we reported the isolation and characterization of the newly isolated lytic Brochothrix phage ADU4, which is capable of infecting the B. thermosphacta bacterium. For the characterization of Brochothrix phage ADU4; host range, multiplicity of infection values (MOI), phage growth parameters (latent period and burst size), stability at various temperatures and pH, reduction growth of bacteria, effect on biofilm, and molecular characterization were investigated. The spot-test analysis showed positivity with B. thermosphacta strains, while no infection was observed in any other species and genera of bacteria tested. The optimal MOI value of the phage was determined as 0.1. The phage latent period and burst sizes were 40–50 min and 311 PFU/ml per infected host cell, respectively by one-step growth curve analysis. Brochothrix phage ADU4 reduced bacteria immediately after infection, which is shown by optical density (OD) measurement and colony counting (<10 CFU/ml) for 3 days. The degradation of B. thermosphacta in biofilm by Brochothrix phage ADU4 was analyzed and it was found that high titer phage breakdown the existing biofilm and also persistently inhibited biofilm formation. Brochothrix phage ADU4 genome was found to be 127,819 bp, and GC content 41.65%. The genome contains 217 putative open reading frames (ORFs), 4 tRNAs, and additionally, no known virulence and antibiotic resistance genes (AMR) were identified. Brochothrix phage ADU4 showed a high identity (96.09%) to the A9 phage that belongs to the Herelleviridae family. Nevertheless, the assembly module and its around appeared less conserved, and some DNA fragments in Brochothrix phage ADU4 genome were not found in A9 genome and vice versa. A9 contains TnpB, a transposase accessory protein involved in lysogenicity which is absent in Brochothrix phage ADU4. In contrary Brochothrix phage ADU4 had auxiliary metabolic genes (AMG) mostly carried by lytic phages. All these results showed that the Brochothrix phage ADU4 has excellent properties such as strong antibacterial activity, short latent period, high burst size, stability in different conditions, inhibition of biofilms, and absence of virulence and AMR genes. Based on all these features, this newly isolated phage is promising to control B. thermosphacta contamination in meat and meat products, and has the potential to be used alone or in combination with phage cocktails.

Complete nucleotide sequence of hemisteptia virus A, a polero-like virus.

The complete genomic nucleotide sequence of hemisteptia virus A (HemVA) from a Hemisteptia lyrata Bunge plant in South Korea was identified by high-throughput sequencing. The HemVA genome consists of 6,122 nucleotides and contains seven putative open reading frames, ORF0-5 and ORF3a, encoding the putative proteins P0-P5 and P3a, respectively. Pairwise amino acid sequence analysis shows that the HemVA P1-P5 proteins have the highest sequence identity (23.68%-54.15%) to the corresponding proteins of members of the families Solemoviridae and Tombusviridae. Phylogenetic analysis of the P1-P2 and P3 amino acid sequences indicated that HemVA should be classified as a member of a distinct species in the genus Polerovirus.

Characterization of a Vibrio-infecting bacteriophage, VPMCC5, and proposal of its incorporation as a new genus in the Zobellviridae family

Vibrio harveyi is a Gram-negative pathogenic bacterium responsible for luminous vibriosis in shrimp and causes mass mortality of shrimp that leads to economic losses. Considering the emergence of multi-drug resistant bacteria, there is always a need for an alternative to antibiotics. In this study, we have aimed to characterize the Vibrio-infecting bacteriophage VPMCC5 (isolated from an environmental sample by using V. harveyi S2A) and evaluate its efficacy in controlling the pathogen. The bacteriophage exhibited an isometric head and short non-contractile tail. The latent period of the bacteriophage was 10 min and the burst size was 20. The genome of the bacteriophage was 48938 bp long with 40.7 mol% G+C content. A total of 71 ORFs were identified and no tRNA and antibiotic-associated genes were detected. Comparative genomic analyses (CLANS, dot plot, progressiveMauve alignment, and phylogenetic tree) strongly suggest that the bacteriophage VPMCC5 might be a new genus in the family of Zobellviridae. A distinguishing feature of this bacteriophage among the other reported Vibrio-infecting bacteriophages is the presence of putative alginate lyase family protein-coding open reading frame. The bacteriophage was found to be surviving at pH 3-9 and in a wide range of temperatures (4-45 ᵒC). In liquid culture inhibition, the bacteriophage could completely lyse the host bacteria after 3 h. This bacteriophage might be used as a biocontrol agent in the extreme environment of shrimp culture.

Isolation and Characterization of a Newly Discovered Phage, V-YDF132, for Lysing Vibrio harveyi

A newly discovered lytic bacteriophage, V-YDF132, which efficiently infects the pathogenic strain of Vibrio harveyi, was isolated from aquaculture water collected in Yangjiang, China. Electron microscopy studies revealed that V-YDF132 belonged to the Siphoviridae family, with an icosahedral head and a long noncontractile tail. The phage has a latent period of 25 min and a burst size of 298 pfu/infected bacterium. V-YDF132 was stable from 37 to 50 °C. It has a wide range of stability (pH 5–11) and can resist adverse external environments. In addition, in vitro the phage V-YDF132 has a strong lytic effect on the host. Genome sequencing results revealed that V-YDF132 has a DNA genome of 84,375 bp with a GC content of 46.97%. In total, 115 putative open reading frames (ORFs) were predicted in the phage V-YDF132 genome. Meanwhile, the phage genome does not contain any known bacterial virulence genes or antimicrobial resistance genes. Comparison of the genomic features of the phage V-YDF132 and phylogenetic analysis revealed that V-YDF132 is a newly discovered Vibrio phage. Multiple genome comparisons and comparative genomics showed that V-YDF132 is in the same genus as Vibrio phages vB_VpS_PG28 (MT735630.2) and VH2_2019 (MN794238.1). Overall, the results indicate that V-YDF132 is potentially applicable for biological control of vibriosis.

A novel epidermal growth factor‐like domain protein 6 from Apostichopus japonicus mediates coelomocyte proliferation

Epidermal growth factor-like domain protein (EGFL) 6, a member of the epidermal growth factor (EGF) domain protein superfamily, has been reported to regulate various physiological processes, including cell adhesion, proliferation, migration and inflammation in mammals. Up to date, the study of invertebrate EGFL6 expression pattern and functional analysis of EGFL6 involved in proliferation is lacking. In this study, a novel EGFL6 homologue was identified from Apostichopus japonicus (designated as AjEGFL6) by rapid amplification of cDNA ends. The full-length cDNA of AjEGFL6 was 3915 bp, with a putative open reading frame of 2490 bp encoding 829 amino acid (aa) residue proteins. Structural analysis revealed that AjEGFL6 processed characteristic five EGF repeat domains and subclass of EGF_CA domain (89–338), UPF domain (504–607) and complement control protein (CCP, 755–808) domain. Multiple sequence alignment and phylogenetic analysis supported that AjEGFL6 belongs to a new member of the EGFL6 protein subfamily. Spatial expression analysis indicated that AjEGFL6 mRNA transcripts in the polian vesicle are expressed at a high level compared with that in coelomocytes. For Vibrio splendidus-challenged sea cucumbers, the peak expression of AjEGFL6 mRNAs in coelomocytes was detected at 24 h (12.29-fold), and it remained at high levels (1.52-fold) until 72 h. Functional investigation via MTT and EdU assays revealed that 60 and 120 ng of recombinant EGFL6 administration significantly upregulated cell viability by 19.72% and 46.13% respectively. Taken together, these findings suggest that AjEGFL6 may serve as a coelomocyte proliferation regulator in the sea cucumber.

Emergence of plasmid-mediated tigecycline, β-lactam and florfenicol resistance genes tet(X), blaOXA-347 and floR in Riemerella anatipestifer isolated in China

Bacterial antimicrobial resistance (AMR) continues to develop, with the horizontal transfer of antibiotic resistance genes (ARGs) through plasmids playing a major role. Recently, the antimicrobial resistance of R. anatipestifer has become increasingly severe, jeopardizing the development of the poultry industry. In this study, we used PromethION to determine the whole genome sequence of R. anatipestifer RCAD0416, a multidrug-resistant isolate from China. We detected a plasmid in the isolate. We named the plasmid pRCAD0416RA-1; the plasmid was 37356 bp in size with 36 putative open reading frames and included the blaOXA-347, floR, tet(X), ermF, ereD, and AadS resistance genes. Most resistance genes might be obtained from R. anatipestifer HXb2. Mobile elements and floR might be transmitted by plasmid pB18–2 from Acinetobacter indicus, and the ICEPg6Chn1 mobile elements can be transmitted from Proteus genomosp. The plasmid pRCAD0416RA-1 was transferred to Escherichia coli K-12 × 7232 via electroporation. Subsequent antimicrobial sensitivity tests (AST) showed a noticeable levels of antimicrobial resistance to β-lactams (4–8 fold), tigecycline (8 fold), and florfenicol (8 fold). These types of antibiotics are in common clinical use. The purpose of this article is to elucidate the basic characteristics of pRCAD0416RA-1 and the level of resistance mediated by blaOXA-347, floR, and tet(X).

Molecular Characterization of a Novel Polerovirus Infecting Soybean in China.

Poleroviruses are positive-sense, single-stranded viruses. In this study, we describe the identification of a novel polerovirus isolated from soybean displaying curled leaves. The complete viral genome sequence was identified using high-throughput sequencing and confirmed using rapid amplification of cDNA ends (RACE), RT-PCR and Sanger sequencing. Its genome organization is typical of the members of genus Polerovirus, containing seven putative open reading frames (ORFs). The full genome is composed of single-stranded RNA of 5822 nucleotides in length, with the highest nucleotide sequence identity (79.07% with 63% coverage) for cowpea polerovirus 2 (CPPV2). Amino acid sequence identities of the protein products between the virus and its relatives are below the threshold determined by the International Committee of Taxonomy of Viruses (ICTV) for species demarcation, and this strongly supports this virus’ status as a novel species, for which the name soybean chlorotic leafroll virus (SbCLRV) is proposed. Recombination analysis identified a recombination event in the ORF5 of the 3’ portion in the genome. Phylogenetic analyses of the genome and encoded protein sequences revealed that the new virus is closely related to phasey bean mild yellows virus, CPPV2 and siratro latent polerovirus. Subsequently, we demonstrated the infectivity of SbCLRV in Nicotiana benthamiana via infectious cDNA clone generation and agroinoculation.

Complete genome sequence and phylogenetic analysis of red seabream iridovirus isolated from a cage-cultured small yellow croaker (Larimichthys polyactis) in China.

Iridoviruses are emerging pathogens that are widespread in diverse environments and hosts. Numerous members of the family Iridoviridae are known to cause severe disease in freshwater and marine organisms. Here, we report the complete genome sequence and phylogenetic analysis of iridovirus strain LPIV-ZS-2021, isolated from a small yellow croaker (Larimichthys polyactis) in China. The genome sequence comprises 110,560 bp with a G+C content of 53.42%, has 104 putative open reading frames (ORFs), and shares the highest sequence similarity with red seabream iridovirus (RSIV) isolated in Japan (98.61%). Phylogenetic analysis revealed that it belongs to RSIV clade 1. This is the first fully sequenced RSIV genome from a small yellow croaker. The host range expansion of members of the genus Megalocytivirus warrants further attention to determine its potential economic and ecological impact.

Isolation and Complete Sequence of One Novel Marine Bacteriophage PHS21 Infecting Pseudoalteromonas marina.

PHS21 against Pseudoalteromonas is isolated from Qingdao offshore seawater. The phage was characterized and identified by morphological examination, stability, whole genome sequencing, and bioinformatics analysis. Morphological analysis of PHS21 by transmission electron microscopy shows that belonged to the Siphoviridae family. PHS21 showed strong tolerance with a wide range of temperatures and pH. One-step growth assay indicates that the latent period is about 48min and the burst size is approximately 218 PFU/cell (plaque forming unit/cell). Its complete genomic sequence is 35,802-bp long with 50 putative open reading frames. Phage PHS21 and PHS3 displayed a very close evolutionary relationship; however, having different DNA packaging proteins indicates that they may have already evolved distinct ways to package DNA in host cells. This study provides the detailed description and genomic characterization of a novel Pseudoalteromonas phage.

Isolation and Characterization of a Novel Siphoviridae Phage, vB_AbaS_TCUP2199, Infecting Multidrug-Resistant Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii (MDRAB) is a pathogen recognized as antimicrobial-resistant bacteria involved in healthcare-associated infections. Resistance to antibiotics has made alternative therapies necessary. Bacteriophage therapy is considered a potential solution to treat MDRAB. In this study, we isolated and characterized the phage vB_AbaS_TCUP2199 (TCUP2199), which can infect MDRAB. Morphological analysis revealed that TCUP2199 belongs to the Siphoviridae family. TCUP2199 has a wide host range, can adsorb rapidly (68.28% in 2 min), and has a burst size of 196 PFU/cell. At least 16 distinct structural proteins were visualized by SDS polyacrylamide gel electrophoresis. A stability test showed that TCUP2199 was stable at 37 °C and pH 7. Genome analysis of TCUP2199 showed that it consists of a double-stranded DNA genome of 79,572 bp with a G+C content of 40.39%, which contains 98 putative open reading frames, none of which is closely related to the bacteriophage genome sequence that was found in the public database. TCUP2199 shows similarity in genomic organization and putative packaging mechanism with Achromobacter phage JWF and Pseudoalteromonas phage KB12-38 based on protein BLAST and phylogenetic analysis. Because of those unique characteristics, we consider TCUP2199 to be a novel phage that is suitable for inclusion in a phage cocktail to treat A. baumannii infection.

Molecular characterization of a new fusarivirus infecting Botryosphaeria dothidea, the causal agent of pear ring rot disease.

Here, a novel mycovirus, tentatively designated as "Botryosphaeria dothidea fusarivirus 2" (BdFV2), was discovered in Botryosphaeria dothidea strain JZ-3. The complete genome sequence is 6,271 nucleotides (nt) in length, excluding the poly(A) tail, and contains two putative open reading frames (ORFs). The larger ORF1 encodes a polypeptide of 1,552 amino acids (aa) with conserved RNA-dependent RNA polymerase (RdRp) domains and a viral helicase domain. The ORF1-encoded polypeptide shares 19.47-78.70% sequence identity with those of other fusariviruses and shares the highest sequence identity (78.70%) with the corresponding protein aa sequences of Neofusicoccum luteum fusarivirus 1 (NlFV1) isolate CBS110299. The small ORF2 encodes a hypothetical protein with 479 aa, which is predicted to contain a chromosome segregation protein SMC domain of unknown function. Sequence alignments and phylogenetic analysis indicated that BdFV2 is a distinct member of the recently established family Fusariviridae. BdFV2 appears to be a novel fusarivirus infecting a pathogenic B. dothidea strain that causes pear ring rot disease.

Genomic and biological characteristics of a newly isolated lytic bacteriophage PZJ0206 infecting the Enterobacter cloacae

Enterobacter cloacae is an opportunistic pathogen that widely distributes in various environments, often causing hospital and community-acquired infections. The limited phage resources and poor genomic data hinder the understanding of the lysis mechanism of Enterobacter cloacae phages. In the present study, we characterized a newly discovered lytic phage PZJ0206 that infects an E. cloacae strain isolated from sewage water into the Pearl River in Guangdong, China. The morphological characteristics of the icosahedral head and short tail of PZJ0206 were observed by transmission electron microscopy. The one-step growth curve showed that PZJ0206 has a relatively short latent period and an average burst size of approximately 124 PFU (plaque forming unit)/infected cell at an MOI of 0.1. The genome of phage PZJ0206 consists of 40,526 bp with a GC content of 48.62% and contains 45 putative open reading frames (ORFs). Based on genomic characteristics, comparative genomics and phylogenetic analysis, it was strongly demonstrated that PZJ0206 is a novel member of the genus Berlinvirus in the family Autographiviridae. Our work provides basic information on Enterobacter cloacae phage PZJ0206 and lays the foundation for the further study of the evolution of phage PZJ0206 and interactions between the phage and its host.