The characterization and genome analysis of a novel phage phiA034 targeting multiple species of Aeromonas.

Aeromonas is one of the most serious pathogens in freshwater aquaculture. Overuse of antibiotics for the treatment of fish diseases has led to the frequent occurrence of drug-resistant strains. Phage therapy is an alternative approach to overcoming the multi-drug resistance associated with antibiotics. In this study, a novel phage phiA034 targeting the host A. veronii A034 was isolated. The phage could infect 14 strains of 4 species Aeromonas. The phage phiA034 displayed head-tail structure with an icosahedral head in the TEM image. At the optimal MOI of 1, it had a latent period of nearly 20 minutes and a burst size of 286 PFU/cell. Besides, the phage phiA034 exhibited high tolerance to a wide range of temperature (30-70 °C) and acid-base (pH 6.0-10.0). The whole genome of phage phiA034 was sequenced with a size of 61,443 bp and annotated with 82 ORFs, mainly related to structure, DNA replication, and lysis. Based on the analysis and comparison of the genomes and proteomes, phage phiA034 could be classified as a novel species of an existing genus Duplodnaviria Heunggongvirae, Uroviricota, Caudoviricetes, Casjensviridae, Sharonstreetvirus. These findings have expanded the species bank and genomes library of bacterial virus and will promote the application of phage therapy in Aeromonas disease.

Genetic analysis of Vibrio alginolyticus challenged by Fructus schisandrae reveals the mechanism of virulence genes.

Due to the abusive use of antibiotics, bacterial resistance has become a global problem and poses severe threats to aquaculture. The drug-resistant diseases caused by Vibrio alginolyticus have caused significant economic losses to cultured marine fish. Fructus schisandrae is used to treat inflammatory diseases in China and Japan. There have been no reports of bacterial molecular mechanisms associated with F. schisandrae stress. In this study, the inhibiting effect of F. schisandrae on the growth of V. alginolyticus was detected to understand response mechanisms at the molecular level. The antibacterial tests were analyzed via next-generation deep sequencing technology (RNA sequencing, RNA-seq). Wild V. alginolyticus (CK) was compared with V. alginolyticus, F. schisandrae incubated for 2 h, and V. alginolyticus, F. schisandrae incubated for 4 h. Our results revealed that there were 582 genes (236 upregulated and 346 downregulated) and 1068 genes (376 upregulated and 692 downregulated), respectively. Differentially expressed genes (DEGs) were involved in the following functional categories: metabolic process, single-organism process, catalytic activity, cellular process, binding, membrane, cell part, cell, and localization. FS_2 h was compared with FS_4 h, and 21 genes (14 upregulated and 7 downregulated) were obtained. The RNA-seq results were validated by detecting the expression levels of 13 genes using quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR results matched those of the sequencing, which reinforced the reliability of the RNA-seq. The results revealed the transcriptional response of V. alginolyticus to F. schisandrae, which will provide new ideas for studying V. alginolyticus' complex virulence molecular mechanism and the possibility of developing Schisandra to prevent and treat drug-resistant diseases.

An Efficient Multi-Dimensional Resource Allocation Mechanism for Beam-Hopping in LEO Satellite Network.

Low Earth Orbit (LEO) satellite communication networks have become an important means to provide internet access services for areas with limited infrastructure. Compared with the Geostationary Earth Orbit (GEO) satellites, the LEO satellites have limited on-board communication caching and calculating resources. Furthermore, the distribution of traffic requests is dynamically changing and uneven due to the relative movement between the LEO satellites and the ground. Therefore, how to schedule the multi-dimensional resources is an important issue for the LEO satellite communication networks. Beam-hopping is an efficient approach to improve the resource utilization by dynamically allocating time, power, and frequency according to the traffic requests. This paper proposes an efficient multi-dimensional resource allocation mechanism for beam-hopping in LEO satellite networks, which simultaneously satisfies the GEO interference avoidance. First, we construct the beam-hopping model of LEO satellites, and formulate the resource optimization problem. Second, we provide the weighted greedy strategy to determine the illumination pattern. In order to reduce the search space, the cells are clustered to non-interference clusters. Then, an improved genetic algorithm is provided to jointly allocate the communication resources. Finally, we construct various simulations to evaluate our proposed mechanism. Compared with the random-BH, polling-BH and traditional genetic algorithm, our algorithm achieves better performance in terms of both system throughput, access success rate, average delay and fairness between cells. The performance improvement is more significant in scenarios where traffic demand is unevenly distributed.

Biological characteristics and genomic analysis of a novel Vibrio parahaemolyticus phage phiTY18 isolated from the coastal water of Xiamen China.

Vibrio parahaemolyticus is a common pathogen usually controlled by antibiotics in mariculture. Notably, traditional antibiotic therapy is becoming less effective because of the emergence of bacterial resistance, hence new strategies need to be found to overcome this challenge. Bacteriophages, a class of viruses that lyse bacteria, can help us control drug-resistant bacteria. In this study, a novel Vibrio parahaemolyticus phage phiTY18 isolated from the coastal water of Xiamen was explored. Transmission electron microscopy showed that phiTY18 had an icosahedral head of 130.0 ± 1.2 nm diameter and a contractile tail of length of 66.7 ± 0.6 nm. The phage titer could reach 7.2×1010 PFU/mL at the optimal MOI (0.01). The phage phiTY18 had a degree of tolerance to heat and acid and base. At the temperature of 50°C (pH7.0, 1h) the survival phages reached 1.28×106 PFU/mL, and at pH 5-9 (30°C, 1h), the survival phages was greater than 6.37×107 PFU/mL Analysis of the phage one-step growth curve revealed that it had a latent period of 10min, a rise period of 10min, and an average burst size of the phage was 48 PFU/cell. Genome sequencing and analysis drew that phage phiTY18 had double-stranded DNA (191,500 bp) with 34.90% G+C content and contained 117 open reading frames (ORFs) and 24 tRNAs. Phylogenetic tree based on major capsid protein (MCP) revealed that phage phiTY18 (MW451250) was highly related to two Vibrio phages phiKT1024 (OM249648) and Va1 (MK387337). The NCBI alignment results showed that the nucleotide sequence identity was 97% and 93%, respectively. In addition, proteomic tree analysis indicated that phage phiTY18, phiKT1024, and Va1 were belong to the same virus sub-cluster within Myoviridae. This study provides a theoretical basis for understanding the genomic characteristics and the interaction between Vibrio parahaemolyticus phages and their host.

Comparative genomics analysis of the multidrug-resistant Aeromonas hydrophila MX16A providing insights into antibiotic resistance genes.

In this paper, the whole genome of the multidrug-resistant Aeromonas hydrophila MX16A was comprehensively analyzed and compared after sequencing by PacBio RS II. To shed light on the drug resistance mechanism of A. hydrophila MX16A, a Kirby-Bauer disk diffusion method was used to assess the phenotypic drug susceptibility. Importantly, resistance against ß-lactam, sulfonamides, rifamycins, macrolides, tetracyclines and chloramphenicols was largely consistent with the prediction analysis results of drug resistance genes in the CARD database. The varied types of resistance genes identified from A. hydrophila MX16A revealed multiple resistance mechanisms, including enzyme inactivation, gene mutation and active effusion. The publicly available complete genomes of 35 Aeromonas hydrophila strains on NCBI, including MX16A, were downloaded for genomic comparison and analysis. The analysis of 33 genomes with ANI greater than 95% showed that the pan-genome consisted of 9556 genes, and the core genes converged to 3485 genes. In summary, the obtained results showed that A. hydrophila exhibited a great genomic diversity as well as diverse metabolic function and it is believed that frequent exchanges between strains lead to the horizontal transfer of drug resistance genes.

Mechanisms Underlying the Virulence Regulation of Vibrio alginolyticus ND-01 pstS and pstB with a Transcriptomic Analysis.

Vibrio alginolyticus is a common opportunistic pathogen of fish, shrimp, and shellfish, and many diseases it causes can result in severe economic losses in the aquaculture industry. Causing host disease was confirmed by several virulence factors of V. alginolyticus. To date, there have been no reports on the effect of the pstS gene on its virulence regulation of V. alginolyticus. The virulence mechanism of target genes regulating V. alginolyticus is worthy of further study. Previous studies found that Fructus schisandrae (30 mg/mL) inhibited the growth of V. alginolyticus ND-01 (OD600 = 0.5) for 4 h, while the expressions of pstS and pstB were significantly affected by F. schisandrae stress. So, we speculated that pstS and pstB might be the virulence genes of V. alginolyticus, which were stably silenced by RNAi to construct the silencing strains pstS-RNAi and pstB-RNAi, respectively. After the expression of pstS or pstB gene was inhibited, the adhesion capacity and biofilm formation of V. alginolyticus were significantly down-regulated. The chemotaxis and biofilm formation ability of pstS-RNAi was reduced by 33.33% and 68.13% compared with the wild-type strain, respectively. Sequence alignment and homology analysis showed that pstS was highly conserved, which suggested that pstS played a vital role in the secretion system of V. alginolyticus. The pstS-RNAi with the highest silencing efficiency was selected for transcriptome sequencing. The Differentially Expressed Genes (DEGs) and GO terms were mapped to the reference genome of V. alginolyticus, including 1055 up-regulated genes and 1134 down-regulated genes. The functions of the DEGs were analyzed by GO and categorized into different enriched functional groups, such as ribosome synthesis, organelles, biosynthesis, pathogenesis, and secretion. These DEGs were then mapped to the reference KEGG pathways of V. alginolyticus and enriched in commonalities in the metabolic, ribosomal, and bacterial secretion pathways. Therefore, pstS and pstB could regulate the bacterial virulence of V. alginolyticus by affecting its adhesion, biofilm formation ability, and motility. Understanding the relationship between the expressions of pstS and pstB with bacterial virulence could provide new perspectives to prevent bacterial diseases.

The temperature-dependent expression of type II secretion system controls extracellular product secretion and virulence in mesophilic Aeromonas salmonida SRW-OG1.

Aeromonas salmonicida is a typical cold water bacterial pathogen that causes furunculosis in many freshwater and marine fish species worldwide. In our previous study, the pathogenic A. salmonicida (SRW-OG1) was isolated from a warm water fish, Epinephelus coioides was genomics and transcriptomics analyzed. Type II secretion system was found in the genome of A. salmonicida SRW-OG1, while the expressions of tatA, tatB, and tatC were significantly affected by temperature stress. Also, sequence alignment analysis, homology analysis and protein secondary structure function analysis showed that tatA, tatB, and tatC were highly conservative, indicating their biological significance. In this study, by constructing the mutants of tatA, tatB, and tatC, we investigated the mechanisms underlying temperature-dependent virulence regulation in mesophilic A. salmonida SRW-OG1. According to our results, tatA, tatB, and tatC mutants presented a distinct reduction in adhesion, hemolysis, biofilm formation and motility. Compared to wild-type strain, inhibition of the expression of tatA, tatB, and tatC resulted in a decrease in biofilm formation by about 23.66%, 19.63% and 40.13%, and a decrease in adhesion ability by approximately 77.69%, 80.41% and 62.14% compared with that of the wild-type strain. Furthermore, tatA, tatB, and tatC mutants also showed evidently reduced extracellular enzymatic activities, including amylase, protease, lipase, hemolysis and lecithinase. The genes affecting amylase, protease, lipase, hemolysis, and lecithinase of A. salmonicida SRW-OG1 were identified as cyoE, ahhh1, lipA, lipB, pulA, HED66_RS01350, HED66_RS19960, aspA, fabD, and gpsA, which were notably affected by temperature stress and mutant of tatA, tatB, and tatC. All above, tatA, tatB and tatC regulate the virulence of A. salmonicida SRW-OG1 by affecting biofilm formation, adhesion, and enzymatic activity of extracellular products, and are simultaneously engaged in temperature-dependent pathogenicity.

Adaptive Access Selection Algorithm for Large-Scale Satellite Networks Based on Dynamic Domain.

The traditional satellite access selection algorithm, which is used in large-scale satellite networks, has some disadvantages, such as frequent link switching, high interrupt probability, and unable to adapt to a dynamic environment. According to the periodicity of the large-scale satellite network and the prior knowledge provided by acknowledgment packages, a dynamic domain-based adaptive access algorithm (DAA) is proposed in this paper. Firstly, this algorithm divides the large-scale satellite network into different domains according to the minimum elevation angle of the Earth station (ES) and the predictable characteristics of the trajectory of the satellite. Then, the ES selects the access satellites according to the relationship between the traffic volume and the satellites' coverage time. Finally, the ES selects the backup access satellite based on the satellites' coverage time, the traffic volume of the ES, satellite status provided by prior knowledge, and other information. When the access satellite cannot satisfy the communication demand, the ES adaptively switches the earth-satellite link to the backup access satellite. The ES first choice of access satellite does not require interaction with the satellites, reducing the consumption of communication resources. The selection strategy of backup access satellite and the concept of virtual destination address proposed in this paper can reduce the routing overhead after switching. Through theoretical analysis and simulation results in the StarLink constellation, it is proved that this paper improves the coverage time utilization of accessing satellites and reduces the switching probability compared with the traditional access algorithm, which is more suitable for ES to access large-scale satellite networks.

fliA, flrB, and fliR regulate adhesion by controlling the expression of critical virulence genes in Vibrio harveyi.

Bacteria adhesion to fish mucus is a crucial virulence mechanism. As the initial step of bacterial infection, adhesion is impacted by bacterial motility and environmental conditions. However, its molecular mechanism is yet unclear. In this study, a significant decrease in gene expression of adhesion-deficient Vibrio harveyi was observed when the bacteria were subjected by Cu2+(50 mg/L), Pb2+(100 mg/L), Hg2+(25 mg/L), and Zn2+(50 mg/L). The genes fliA, fliR, and flrB were responsible for flagellation; being crucial for adhesion, these genes were identified and silenced via RNAi. After silencing of these genes by RNAi technology, the ability of adhesion, biofilm formation, motility, and flagella synthesis of V. harveyi were considerably reduced. Compared with the control group, it was observed that the expression levels of fliS, fliD, flgH, and flrC were significant down-regulated in fliR-RNAi, flrB-RNAi, and fliA-RNAi. This data indicates that the expression levels of most virulence genes are affected by fliA, fliR, and flrB. Also, the expression of fliA, fliR, and flrB can be influenced by the salinity, temperature, and pH. The results show that: (1) fliA, fliR, and flrB have important roles in the adhesion of V. harveyi; (2) fliA, fliR, and flrB can regulate bacterial adhesion by affecting its motility, and biofilm formation; (3) fliA, fliR, and flrB can regulate adhesion ability of V. harveyi in different environments.

cheA, cheB, cheR, cheV, and cheY Are Involved in Regulating the Adhesion of Vibrio harveyi.

Diseases caused by Vibrio harveyi lead to severe economic losses in the aquaculture industry. Adhesion is an important disease-causing factor observed in bacteria with chemotactic activity. In our study, we measured the adhesion of V. harveyi by subjecting the bacteria to stress using Cu2+, Pb2+, Hg2+, and Zn2+. The genes responsible for chemotaxis (cheA, cheB, cheR, cheV, and cheY), which are also crucial for adhesion, were identified and silenced via RNAi. We observed that a decrease in chemotactic gene expression reduced the ability of the organism to demonstrate adhesion, motility, chemotaxis, and biofilm formation. Upon comparing the cheA-RNAi bacteria to the wild-type strain, we observed that the transcriptome of V. harveyi was significantly altered. Additionally, the expression of key genes and the adhesion ability were affected by the pH (pH of 5, 6, 7, 8, and 9), salinity (NaCl at concentrations of 0.8, 1.5, 2.5, 3.5, or 4.5%), and temperature (4, 15, 28, 37, and 44°C) of the medium. Based on these results, the following conclusions were made: (1) The chemotactic genes cheA, cheB, cheR, cheV, and cheY may regulate the adhesion ability of V. harveyi by affecting bacterial motility, and participate in the regulation of adhesion at different temperatures, salinities, and pH values; (2) stable silencing of cheA could alter the transcriptional landscape of V. harveyi and regulate the expression of genes associated with its adhesion mechanisms.

Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation

Background To study the relationship between intracellular anabolism and astaxanthin production, the influence of intracellular protein and fatty acids on astaxanthin production by four mutant Phaffia rhodozyma strains and their variations was investigated in this research. Results First, the content of astaxanthin in cells showed a reverse fluctuation in contrast to that of protein during the whole fermentation process. Moreover, compared with the three other strains, the astaxanthin-overproducing mutant strain of the yeast P. rhodozyma, called JMU-MVP14, had the highest specific productivity of astaxanthin as 6.8 mg/g, whereas its intracellular protein and fatty acid contents were the lowest. In addition, as a kind of sugar metabolic product, ethanol was only produced by P. rhodozyma JMU-VDL668 and JMU-7B12 during fermentation. Conclusions The results indicated that the accumulation of ethanol, intracellular protein, and fatty acids had competition effects on astaxanthin synthesis. This condition may explain why the P. rhodozyma strains JMU-VDL668 and JMU-7B12 achieved relatively lower astaxanthin production (1.7 and 1.2 mg/L) than the other two strains JMU-MVP14 and JMU-17W (20.4 and 3.9 mg/L).

[Isolation and preparation of gallic acid from Terminalia chebula Retz. with high-speed counter-current chromatography].

A separation method based on high-speed counter-current chromatography (HSCCC) has been established for the isolation and preparation of gallic acid from the ethanol extract of Terminalia chebula Retz. After comparing five kinds of solvent protocols of HSCCC, the two-phase system of n-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v) was finally chosen as the operating solvent of HSCCC for the separation of gallic acid, in which the lower phase was used as the mobile phase and the upper phase as stationary phase. The detection in the experiments was performed with an ultraviolet detector at 254 nm. Under the conditions of rotation speed of 850 r/min, lower phase flow rate of 2 mL/min, four peaks (I/II/III/IV) were displayed on HSCCC chromatogram. Among them, only peak III contained a large amount of gallic acid (about 96. 40%), which was confirmed by electrospray ionization mass spectrometry (ESI-MS) and high performance liquid chromatographic (HPLC) analysis. As much as 8. 6 mg of gallic acid was obtained from 100 mg of the ethanol extract of Terminalia chebula Retz., indicating the method is simple, robust and efficient for the preparation of gallic acid.