Abstract:[Background] Chemical control pollution is becoming more and more serious, “3R” problems are becoming more and more common. Therefore, screening new biocontrol strains and studying their antibacterial substances have become a hot spot. [Objective] To screen a strain of Bacillus velezensis that has biocontrol function against phytopathogenic bacteria such as Cochlioboluss ativus, and excavate its gene clusters that regulate the synthesis of bacteriocins and RiPPs (antibacterial peptides synthesized by ribosome pathway). [Methods] Strains were screened by methods such as separation and screening, confrontation culture, etc., and strain identification was performed by whole cell fatty acid, Biolog analysis and molecular biology methods. The Illumina Novaseq 6000 platform was used to determine the whole genome sequence, and the potential bacteriocins, RiPPs gene clusters and potential mechanisms of action were explored through BAGEL4. [Results] strain RJW-5-5 had the best inhibitory effect on Cochlioboluss ativus, and the inhibition rate reached 78.4%. Strain RJW-5-5 was identified as Bacillus velezensis. [Conclusion] strain RJW-5-5 can inhibit various crop diseases, has a variety of lantibiotic, lasso peptide, bacteriocin and other antibacterial peptide gene clusters, with broad development prospects and application potential.

Abstract:[Background] Methicillin-resistant Staphylococcus aureus (MRSA) is a common conditional pathogenic bacterium. Mastitis in dairy cows caused by MRSA infection has brought significant economic losses to dairy farmers. [Objective] To understand the genomic sequence characteristic of MRSA epidemic strain in dairy cows in Ningxia province, and provide a theoretical basis for the prevention and treatment of MRSA infection. [Methods] The isolate ld11 was tested for antimicrobial susceptibility by agar diffusion method, and the high-throughput sequencing of genomic DNA of isolate ld11 was carried out based on Illumina high-throughput sequencing platform, and the obtained sequencing sequences were processed and analyzed through network databases. [Results] The susceptibility test showed that the isolate ld11 was resistant to ceftiofur, sulfisoxazole, ampicillin, erythromycin, gentamicin, oxacillin, clindamycin, tetracycline, and doxycycline, and data analysis showed isolate ld11 carried the drug resistance genes aadD, spc, str, blaZ, mecA, cat(pC194), erm(A), norA, tet(k), and tet(M), and there was a good correlation between the two; The isolate ld11 carried more resistance genes than the MRSA reference strains, and the relationship between isolate ld11 and MRSA252 was closer. The results of COG (Clusters of orthologous groups of proteins) functional analysis and GO (Gene ontology) annotation showed that the genes involved in maintaining the basic functions of the cells and the growth and proliferation of the strains predominated. The KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis showed that the genes belonging to the metabolic pathway accounted for the most; four gene islands, nine questionable CRISPR sequences, and one complete prophage sequence were detected from the genome sequence. [Conclusion] This study revealed partial genomic sequence information of MRSA epidemic strain in dairy cows in Ningxia, which provided a reference for comparative analysis of genomic sequence information and prevention and control of MRSA infection among MRSA strains.

Abstract:[Background] The abuse of antibiotics leads to the increase and spread of drug-resistant pathogens, which has become an important factor threatening people’s health. Therefore, it is particularly important to explore new antibiotics. New species are likely to produce new bioactive substances, especially rare actinomycetes which are rarely isolated by conventional methods. [Objective] To confirm classification of two novel species of Microbispora and predict the secondary metabolic gene clusters by various molecular biological technologies, which will lay a foundation for the discovery of the medicinal actinobacteria producing novel active substances. [Methods] Taxonomic positions of isolates were preliminarily determined by the 16S rRNA gene sequence analysis. The whole genomes were sequenced and annotated by Illumina genome analyzer. On this basis, a whole-genome-based phylogenomic tree was constructed, and the ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization) values were calculated, so as to determine the taxonomic status of novel species. Based on gene annotation, COG (clusters of orthologous genes), KEGG (Kyoto Encyclopedia of Genes and Genomes), and secondary metabolic gene cluster prediction on antiSMASH were analyzed. Antimicrobial activity was tested by cylinder plate method. [Results] The ANI and dDDH values between strain H10836 and 8 species of the genus Microbispora were 85.3%?92.1% and 33.0%?44.5%, respectively. Meanwhile, the ANI and dDDH values between strain H11081 and 8 species of the genus Microbispora were 85.2%?92.1% and 31.3%?44.5%, respectively. Therefore, all the ANI and dDDH values are well below the cut-off point recommended for delineating species. In addition, a whole-genome-based phylogenomic tree showed that strains H10836 and H11081 formed an independent monophyletic branch within the genus Microbispora. Therefore, these results of phylogenetic analysis support the conclusion that strains H10836 and H11081 could be considered to represent two potential novel species of the genus Microbispora. Furthermore, there were many kinds of biosynthetic gene clusters in the genomes of both strains H10836 and H11081 by antiSMASH analysis, and the similarities with known antibiotic synthetic gene clusters were low. The metabolites of two strains exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). [Conclusion] Strains H10836 and H11081 are two novel species with antibacterial activity, so it is worth mining their novel active natural products. The results of this experiment provide reference for further research and application of the strains.

Abstract:[Background] Lactobacillus paracasei, as one of the important strains in lactic acid bacteria, is often considered as a potential resource for the development of probiotics. [Objective] L. paracasei PC-01 and L. paracasei Zhang were taken as examples to analyze the genomic differences and genetic backgrounds of different L. paracasei strains, in order to laying a foundation for the identification and development of strains. [Methods] the whole genome sequencing of L. paracasei PC-01 was sequenced by PacBio SMRT third-generation sequencing technology, combining with 2 L. paracasei model strains and the whole genome data of 36 strains which had published. through the comparative genomics methods to revealed the differences of 39 L. paracasei strains. [Results] The genome of L. paracasei PC-01 has one chromosome, with a size of 2 829 251 bp and its GC content was 46.64%; L. paracasei Zhang contained a plasmid and the genome size was 2 898 456 bp and its GC content was 46.51%. There were some differences of L. paracasei strains about genome size, the number of plasmid and GC content. L. paracasei is an open genome with a high genome diversity. Phylogenetic tree constructed by core genes has the best effect on species differentiation of L. paracasei. Whats more, L. paracasei PC-01 and L. paracasei Zhang were in different evolutionary branches. L. paracasei PC-01 and Zhang had a fragment matching rate is 91% of their genomes, and L. paracasei PC-01 has 10 specific genes related to transcriptional regulation and rhamnoglycan metabolism, such as aguA and clpB. L. paracasei Zhang contains mshA, ltrA specific genes were related to glycosyl transfer and other functions. [Conclusion] Through the comparative genomics revealed the genetic information of L. paracasei PC-01 and Zhang and found the differences of L. paracasei strains, which laid the genetic foundation for the identification and application of L. paracasei in the future.

Abstract:[Background] Streptomyces has always been the main producer of bioactive compounds. However, as the abusing of antibiotics, environmental pollution and drug resistance are becoming increasingly serious problem. The discovery of efficient bio-control factor and novel antibiotics becomes the main methods to solve these problems. [Objective] Obtain the whole genome sequence of the Streptomyces sp. SAT1 and the information about its secondary metabolite gene clusters; analyze the particularity and generality with other streptomycetes by the technology of comparative genomics and pan genomics. Form this, we could provide theoretical basis for illuminating the mechanism of bacteriostasis and growth-promoting in SAT1, and reliable data to reveal the ecological function of Streptomyces. [Methods] The sequence of SAT1 was completed by the third generation sequencing platform PacBio Sequel, then annotated and classified by bio-information technology; the software RAxML and PGAP was used to construct phylogenetic tree and analyze pan-genome, respectively. The prediction and analyze of the secondary metabolite gene clusters was achieved by antiSMASH. [Results] From the complete genome map of SAT1, the length of linear chromosome is 7.47 Mb, with 73% GC content, and four plasmids exist in the strain. Additionally, there are 7 550 genes which encoded proteins and 37 secondary metabolite gene clusters which classified by 29 types in SAT1. And the moenomycin gene cluster was highly homologous to Streptomyces ghanaesis ATCC14672 moenomycin gene cluster. In the 42 streptomycetes, it exists about 20-55 secondary metabolites gene clusters in each strain which classified into PKS, Terpene, Nrps and Heterozygous gene clusters. The dispensable genome was huge in these research objectives. [Conclusion] Streptomyces sp. SAT1 has many common points in the trait of genome and secondary gene clusters with other streptomycetes. We speculate the moenomycin and hygromycin_B gene clusters play an important role in the antibacterial activity of SAT1. In the 42 research objectives, the number of gene clusters and the size of genome has a positive correlation. In addition, the existence of abundant heterozygous gene clusters and large number of dispensable genome illustrate Streptomyces has high levels of horizon gene transfer over long periods of evolution, which possesses important environmental functions.

Abstract:[Background] Klebsiella pneumoniae (K. pneumoniae) is an important pathogen that can infect both humans and bovine. The wide spread of Klebsiella pneumoniae and its high drug resistance have caused great difficulties in the treatment of the disease. [Objective] To isolate a lytic Klebsiella pneumoniae bacteriophage. Its biological properties were studied, and the entire genome analysis of the bacteriophage was performed. [Methods] Klebsiella pneumoniae bacteriophage was isolated by the double-layer agar culture method from a dairy farm trough in Sichuan province. We used the same method to conduct host range, thermostability, pH, optimal multiplicity of infection (MOI), and one-step growth experiment. Phage morphology was observed by transmission electron microscopy (TEM). The whole genome of the bacteriophage was sequenced. [Results] A lytic Klebsiella pneumoniae bacteriophage vB_Kpn_B01 was isolated which had strong specificity. The morphology of plaque was round, transparent plaques with clear boundaries. Phage found in this study was double-stranded DNA bacterial viruses belonging to the Siphoviridae family, and the size of it was 113 227 bp. Comparative genome analysis revealed that vB_Kpn_B01 genome possesses the highest similarity to the bacteriophages in the genus of Sugarlandvirus. The genome of vB_Kpn_B01 comprises 149 coding sequences (CDS) and 25 tRNAs, but does not contain any known antibiotic resistant genes or virulent genes. [Conclusion] Phage vB_Kpn_B01 has a strong lysis ability and the necessary genes to lyse host bacteria. It has the potential to be used in the prevention and treatment of multi-drug resistant bacteria in animal husbandry.

Abstract:[Background] Dickeya zeae causes bacterial soft rot of several important crops such as banana and rice and may cause heavy losses. Canna edulis is resistant to many biotic and abiotic stressors and only a few pests are reported for this plant. Bacterial soft rot of C. edulis caused by D. zeae CE1 was first reported by our research group. [Objective] This study was conducted to sequence the whole genome of D. zeae CE1 and to compare this strain genomically with the other Guangdong strains of this pathogen from banana (strains MS1 and MS2) and rice (strains EC1, EC2 and ZJU1202), in order to explore any genetic differentiation related to the interaction between pathogenic D. zeae bacteria and their hosts. [Methods] The third-generation sequencing combined with next-generation sequencing method was used to construct the complete genome of strain CE1. Next, comparative genomics was adopted to compare the evolutionary relationships and genomic characteristics of strain CE1 with other strains of the pathogen isolated from banana and rice plants. [Results] The complete genome size of CE1 was 4 714 731 bp, with 4 052 coding genes predicted. Similarly with the taxonomic relation between C. edulis and banana, the strains from C. edulis and banana were closely related showing by the phylogenetic tree, but they were notably different from rice strains. The OrthoMCL analysis revealed that the bacterial gene clusters encoding important pathogenic factors such as: bacterial secretion systems, flagellar proteins, extracellular polysaccharides, and the clustered regularly interspaced short palindromic repeats (CRISPR), did not have obvious differences that corresponded to different types of hosts. Further analysis revealed that 80 genes were specific to the C. edulis and banana strains, while 42 genes were specific to the rice strains. According to the functional prediction, two of the gene clusters found in the specific loci of C. edulis and banana strains were related to fatty acid synthase and quorum sensing, respectively. However, more rice strain-specific genes were involved in carbohydrates transport and metabolism compared to the C. edulis and banana strains; in addition, there was a specific gene cluster from rice strains found in the adjacent genomic locus of the CRISPR array. [Conclusion] comparative genomic analysis have determined the phylogenetic relationship among C. edulis strain, banana strains, and rice strains and found several gene loci that might be involved in the interaction between these D. zeae strains and the hosts of different types. It is recommend providing an early warning to growers that crops closely related to banana and rice may be at risk of infection from these important pathogenic bacteria.

Abstract:[Background] Due to the short time of discovery of methylotrophs, only a few genomes of methylobacterium strains that can produce PQQ were sequenced. It increases the difficulty of study the genomics and biological metabolic pathways of methylobacterium. [Objective] The PQQ-producing bacteria was screened and treated with various mutagenesis methods to improve the yield of PQQ. Whole genome analysis of high-yield mutant strains was performed to provide sequence background information for studying the molecular mechanism of PQQ synthesis and subsequent molecular breeding of methylobacterium. [Methods] The wild-type PQQ production strains were subjected to ultraviolet rays mutagenesis, nitroso-guanidin mutagenesis, ethylmethylsulfone mutagenesis, diethyl sulfate mutagenesis, and ultraviolet rays-lithium chloride compound mutagenesis. The whole genome sequence of the mutant strain obtained by mutagenesis was sequenced using the PromethION sequencing platform and the MGISEQ-2000 sequencing platform. The assembled whole genome sequence was compared with the model strain Methylobacterium extorquens AM1. [Results] A mutant strain NI91 was obtained after 11 rounds of mutagenesis with PQQ yield 19.49 mg/L, which was 44.91% higher than the original strain. The genome of the mutant strain NI91 consists of a chromosome of 5 409 262 bp, encoding 4 957 proteins. Compared with the model strain M. extorquens AM1, it was found that the pqqF and pqqG genes that relate to shear processing during PQQ biosynthesis were deleted. Meanwhile, the pqqL was first discovered in methylotrophic bacteria which has a similar function to the pqqF, and the sequences of pqqC/D between the two strains were quite different. [Conclusion] This study provides basic data for functional genomics research of the methylotrophic bacterium and the study of PQQ synthesis mechanism. Comparative genomics between NI91 and the model strain M. extorquens AM1 provides a molecular basis for revealing different mechanisms of PQQ synthesis.

Abstract:[Background] Phosphorus in soil generally exists in the form of insoluble phosphate, which limits the direct absorption and utilization by plants. Phosphate-solubilizing bacteria can convert the insoluble phosphate in the environment into phosphorus that can be absorbed and utilized by plants. [Objective] To isolate and identify a highly efficient phosphate-solubilizing bacterial strain, and explore its phosphate-solubilizing capacity, plant growth-promoting effect, and related genes, so as to provide elite strain resources and theoretical support for the research and development of phosphate-solubilizing bacterial fertilizer. [Methods] The strain with significant phosphate-solubilizing effects was isolated from soil and identified based on physiological and biochemical characteristics as well as molecular evidence. The phosphate-solubilizing capability of the strain was determined by the transparent circle method and the Mo-Sb colorimetric method. Single-factor experiments were carried out to study the effects of different carbon sources, nitrogen sources, and insoluble phosphates on the phosphate-solubilizing capability of the strain. Further, the whole genome of the strain was sequenced by the next-generation sequencing platform, and the genes related to phosphate solubilizing and siderophore synthesis and transport in the genome were mined. Finally, the plant growth-promoting effect of the strain was assessed by the hydroponic experiment. [Results] Strain PSB-K was identified as Acinetobacter baumannii, with a phosphate-solubilizing index of 2.58. Strain PSB-K exhibited the highest phosphate-solubilizing capability in the case of glucose as the carbon source, potassium nitrate as the nitrogen source, and tricalcium phosphate as the insoluble phosphate, under which the soluble phosphorus concentration in the fermentation liquid reached 560.35 μg/mL. The whole genome of strain PSB-K was 3 983 883 bp in length, containing 3 849 coding genes, including various genes involved in organic acid synthesis, phosphate transport, and siderophore synthesis and transport. The results of the hydroponic experiment showed that strain PSB-K applied at 2.50×107, 2.50×107, and 1.25×107 CFU/mL demonstrated the strongest promoting effects on the leaf area, fresh weight, and root length of oilseed rape seedlings, which were increased by 72.15%, 72.08%, and 197.81%, respectively, compared with the control. [Conclusion] Strain PSB-K possessed the capability to solubilize a variety of insoluble phosphates, and carbon and nitrogen sources directly influenced the phosphate-solubilizing performance of the strain. With a growth-promoting effect on oilseed rape plants, this strain serves as a candidate for the development of microbial fertilizer. In addition, the whole-genome data provides a reference for understanding the phosphate-solubilizing mechanisms and mining functional genes of phosphate-solubilizing bacteria.

Abstract:[Background] Staphylococcus aureus is a common zoonotic opportunistic pathogen. With the emergence of multidrug resistant strains, it is urgent to develop antimicrobial agents with different modes of action from antibiotics. [Objective] To isolate S. aureus phage and identify its functional lysin components as an effective specific antimicrobial agent. [Methods] The whole genome sequence of the phage was assembled and annotated for the mining of putative lysin encoding genes. Two putative lysin genes were respectively cloned into a prokaryotic expression vector. SDS-PAGE and Western blotting were employed to confirm the expression of the target proteins. We then verified the lytic activity by spotting the expression product on the host bacterial lawn. [Results] The isolated phage in this study could lyse its host bacterium and was named vB_Sau_P68. The phage genome was 139 409 bp with the GC content of 31.0% and encoded 220 open reading frames (ORFs). Under the transmission electron microscope, the phage appeared as an icosahedron with a contractile tail, which belonged to Myoviridae. Two putative lysin genes were annotated in the phage genome. ORF161 was predicted to encode lysin with a CHAP catalytic domain and ORF163 with a SH3_5 binding domain. The results of SDS-PAGE and Western blotting showed that Lys161 was expressed successfully and had lytic activity, while the expression of Lys163 was not be detected. The Lys161 sequence had no signal peptide or transmembrane region, and random coil was its major secondary structural element. [Conclusion] In this study, two lysin genes were cloned from a Staphylococcus aureus phage genome and expressed. The results suggested that the CHAP catalytic domain had lytic activity, while the SH3_5 binding domain was not expressed. The findings provide a theoretical basis for the exploration of the acting mechanism and application of lysin.