Abstract:The studies on lactic acid bacteria as live vehicles for expression and display of heterologous proteins or antigens have gained great progress in the past decades. Recently, a novel display system called Bacterium-like particles was designed and described. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, generally the innocuous bacterium Lactococcus lactis pretreated by hot acids. The peptidoglycan-binding domain of lactococcal AcmA protein has been used as the protein anchor for heterologous surface display of various proteins on lactic acid bacteria. Compared to the living lactic acid bacteria, Bacterium-like particles have a higher binding capacity, safety, delivering efficiency, and less anticarrier response. They have been widely used in the development of mucosal vaccines and adjuvants, purification of viral antigens, and preparation of biocatalysts. In this review, we focus on the construction, unique advantages of Bacterium-like particles, and successful application in many fields. Finally, we will discuss the broad application prospects and problems to be solved in the nearly future.

Abstract:Syrbactins are microbial natural products, mainly composed of syringolins, glidobactins, cepafunins and luminmycins. At present, several biosynthetic gene clusters of syringolins and other compounds have been cloned, sequenced and heterologously expressed. These studies revealed that, despite the structural differences between syrbactins, they are synthesized in vivo in a similar mode and share a NRPS-PKS hybrid assembly biosynthetic pathway for the formation of their core skeletons. They have drawn continuous research attentions due to the irreversible proteasome inhibition activity. This review focuses on the research advances in syrbactins and highlights their structure, biosynthesis and mode-of-action.

Abstract:Neuraminidase hydrolyzes the terminal neuraminic acid residue of glycoconjugations. It is used in bacteria for adhesion to host cells, plasma proteins, hemocyte and important barriers and for further colonization, penetration and spread. Thus, the enzyme is an important virulence factor for various bacteria. Streptococci are widely spread pathogens for zoonoses. The activity of neuraminidase could be detected in many Streptococci spp. Streptococcus pneumonia produces 3 neuraminidases (NanA, NanB, NanC). NanA not only catalyzes the sialic residue covering the adhesion receptor, but also accelerates the bacterial infection on host cells. NanB provides carbon source for the bacteria. NanC helps S. pneumonia invading into the brain. As for group B streptococcus and S. suis, the activity of neuraminidase has not been certified yet. Researchers tend to hold the view that the neuraminidase activity has lost through evolution as both kinds of bacteria contain sialic acid in their capsules. The preference for neuraminidase of S. pyogenes and group C, G and L streptococcus is similar with strong catalytic activity for saliva mucin which avails the proliferation in organizations containing saliva mucin. The neuraminidase in S. oralis and S. sanguis destroys the sialic chain in component of blood. In all, the functions of neuraminidase are closely related to the location of bacterial colonization. In the test of enzyme activity, thiobarbital is used in the 1960s. Now the fluorogenic substrate and spectrophotometric method, the more convenient and sensitive methods, are the main ways measuring the neuraminidase activity. This paper summarizes the category, properties, mechanism of neuraminidase produced by Streptococci as well as the relationship between the enzyme and virulence. The assays for detection of neuraminidase activity are also reviewed.

Abstract:Estrogens are major substances of environmental endocrine disruptors, and biodegradation is the most economical, greenest and most suitable method to remove them. We elaborated the research status and progress of estrogen biodegradation by analyzing the major sources and hazards of estrogen, the isolation and identification of estrogen-degrading bacteria, the expression and detection of steroidal estrogen-degrading enzymes, the genomics study of estrogen-degrading bacteria and the estrogen degradation pathways. We also address future works of biodegradation of environmental estrogens.

Abstract:Quorum sensing (QS) is cell-density dependent cell-to-cell communication in bacteria. In this process, bacteria secrete and recognize the QS signals, and then coordinate the gene expressions in group level for gene transferring, virulence factor secretion, spore production, biofilm formation and so on. Interfering with any part of the process could block QS. Quorum quenching (QQ) is one of the important strategies for the control of pathogenicity, prevention of spoilage and inhibition of biofilm pollution. In this paper, according to the principal axis of secretion-recognition-response of quorum-sensing signals, bacterial QS network was divided into three types:hierarchical, parallel and competitive ones. Firstly, their characteristics were elaborated. Then, the strategies of QQ containing signal competition, signal degradation, and receptors/key proteins activation or inhibition as well as their applications were discussed. Finally, the advance for QS research was prospected. This review was beneficial to enrich the knowledge of bacteria QS and promote the application of QQ.

Abstract:[Objective] The two-component system (TCS) Rcs is involved in the regulation of bacterial adaptation to external environment and survival. The aim of this study is to elucidate effects of sensor kinase RcsC on associated biological characteristics and virulence of avian pathogenic Escherichia coli (APEC).[Methods] We constructed the rcsC gene mutant and complementary strain of APEC by the Red recombination system and complementation plasmid. Then we compared the growth curve, motility, biofilm formation, agglutination, pathogenicity and virulence gene transcription levels of mutant, wild-type and complementary strains.[Results] rcsC gene deletion had no influence on bacterial growth. However, inactivation of rcsC gene resulted in enhanced motility, decreased biofilm formation and increased agglutination. Bacterial adherence and invasion assays showed that RcsC contribute to the invasion of APEC to DF-1 cells, whereas, it did not affect the adhesion capacity. Moreover, rcsC gene mutant strain exhibited attenuated virulence in ducks. Quantitative real-time PCR analysis demonstrated that the transcription levels of virulence genes ompA, aatA, fyuA and luxS were significantly decreased in mutant strain. Whereas, the fimC and tsh transcription levels were significantly upregulated.[Conclusion] These data indicated that RcsC play roles in regulation of bacterial motility, biofilm formation, agglutination and virulence of APEC.

Abstract:[Objective] To characterize the translesion synthesis of different damages by DNA polymerase IV from thermophilic archaea Sulfolobus acidocaldarius. [Methods] We detected the translesion synthesis ability of Sulfolobus acidocaldarius polymerase IV (SacpolIV) using fluorescence labeled oligonucleotides as substrates through denaturing polyacrylamide gel electrophoresis. [Results] We successfully purified SacpolIV from induced E. coli and confirmed SacpolIV possessed strong translesion synthesis ability to DNA damages on purines and pyrimidines. The TLS ability depends on whether damaged bases can pair normal bases perfectly. Besides, we found SacpolIV can incorporate rNMP into DNA strand. [Conclusion] We confirmed that SacpolIV is a typical TLS DNA polymerase with the abilities of bypassing damaged purines and pyrimidines on DNA, and incorporating rNMP into DNA.

Abstract:[Objective] The aim of this study is to analyze bacterial community and antibiotic resistant genes (ARGs) in organic, fertilized and wild Houttuynia cordata Thunb, and to reveal the relationship between bacterial community and ARGs. [Methods] The bacterial community structure was investigated by high-throughput 16S rRNA V3-V4 variable region sequencing. The qualitative and quantitative analysis of 29 ARGs was determined by PCR and qPCR. The redundancy analysis was used to reveal the relationship between bacterial community and ARGs. [Results] A total of 35 genera of bacteria were detected. The bacterial diversity in organic H. cordata Thunb was lower than that in fertilized and wild H. cordata Thunb. Fourteen ARGs were detected on H. cordata Thunb. All detected ARGs were found in the organic H. cordata Thunb, while only part was found in the fertilized and wild H. cordata Thunb. Redundancy analysis showed ARGs were significantly affected by bacterial diversity and abundance, and Lactococcus, Escherichia, Fluviicola, Enterococcus, Sanguibacter and Acidovorax were main bacteria which affected on the diversity and abundance of ARGs on H. cordata Thunb. [Conclusion] Organic planting affects bacterial community on H. Cordata Thunb, and increases diversity and abundance of ARGs, suggesting a potential food safety risk. Therefore, it is necessary to bring ARGs contamination into the scope of food safety assessment for organic H. cordata Thunb.

Abstract:[Objective] To study the function of MCHK-3535 gene in symbiotic nitrogen fixation between Mesorhizobium huakuii 7653R and Astragalus sinicus. [Methods] We constructed the deletion mutant Δ3535, overexpression strains OV3535 and complementary strains C3535, followed by identifying symbiotic phenotypes, detecting the growth curve, swimming motility and biofilm formation Besides, we used qRT-PCR and promoter-GUS fusion vector to detect the expression characteristics of MCHK-3535 in the symbiotic process. [Results] Compared with the wild type 7653R, mutant Δ3535 grew faster, decreased motility, and increased biofilm formation, moreover, Δ3535 significantly increased the ability of nitrogen fixation and the fresh weight of plants, while the number and weight of root nodules were not affected, in addition, the complementary strain C3535 partially compensated to the wild-type phenotype, whereas OV3535 had no significant difference in these aspects. The results of qRT-PCR and the promoter-GUS reporter system showed that MCHK-3535 expressed in the infection zone, transitional zone and nitrogen fixation zone of the nodules, and the gene expressed lasted for the entire period of nitrogen fixation. [Conclusion] MCHK-3535 functions in the symbiotic nitrogen fixation process and may participates in the normal development of nodules and negatively regulates nitrogenase activity.

Abstract:[Objective] The purpose of this research is to study the effects of herbicide "starane" on the structure and diversity of bacterial communities in different micro-environments. [Methods] We used the high-throughput sequencing technique Illumina Miseq to determine the V4-V5 variable region sequence of 16S rRNA in endophytic, rhizosphere and non-rhizosphere soils of maize roots. The herbicides were sprayed in different growth stages, and we researched the herbicide effect on the structure and diversity of maize root endophytes as well as soil bacteria. [Results] We obtained a total of 544393 effective sequences and 333565 high-quality sequences from 15 samples. The multi-sample OTU analysis showed that the community structure of non-rhizosphere soil and rhizosphere soil was similar, which indicated that the colonization of maize-related bacteria was selective and the gradual specialization from rhizosphere to root. The results of rank abundance curve and Alpha diversity showed that the richness and uniformity of non-rhizosphere and rhizosphere soil were higher, while the richness and uniformity of endophytic community in maize roots of mature stage were lower. The abundance of endophytic community in maize roots was significantly reduced by the application of herbicides. The composition analysis of the community showed that the distribution of bacteria in the maize roots was with a great change at both the Phylum and Genus level. The prediction of metabolic function of the flora indicated that the growth pressure of the microorganisms was gradually increased from the seeding stage to the mature stage of the corn, which required more energy for bacterial metabolism and environmental adaptation. [Conclusion] The application of herbicide "starane" can reduce the diversity of bacterial community in the roots of maize, and the effect on the endophytic community of maize roots is more obvious.

Abstract:[Objective] It is to identify and explore the genetic variation of an infectious bronchitis virus isolated from 11-day-old cherry valley ducks that developed suspected respiratory infection in Guangxi province. [Methods] The virus was identified by hemagglutinin test, chicken embryo inoculation and gene sequence analysis of the 3ʹ untranslated region (UTR). The S1, E, M, and N genes of the virus were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), cloned, sequenced, similarity comparison, phylogenetic tree and serotype analyais was conducted. [Results] The hemagglutination test was negative and dwarf embryo appeared after 5 passages of inoculation of chicken embryos. The sequencing result of 3ʹUTR gene indicated the virus was IBV. The sequence of cleavage site within S protein of the virus was RRSRR. The nucleotide similarities of S1, E, M, and N genes with those of H120, 4/91 and LTD3 reference strains were 78.6%-99.7%, 85.4%-100.0%, 91.6%-93.2% and 86.7%-91.7%, respectively. Compared with the reference strains, there is no insertion or deletion in N gene of the virus except mutations. However, substitutions, insertions and (or) deletions were found in S1, E, M genes of the isolated strain. According to the phylogenetic tree analysis, it was clustered into 4/91-type based on S1 gene and LDT3-type based on E, M and N genes. The serotype analysis showed that the serotype of the isolated strain was different from vaccine strains H120 and 4/91. [Conclusion] The strain isolated from ducks was IBV and the isolated strain existed variation both in structural genes and serotype. The present study suggests that the prevention and control of IB in poultry is facing more severe challenges.

Abstract:[Objective] Lycium ruthenicum is a halophyte and used to improve saline lands in northwest China. However, little is known about the bacterial community structural dynamics with growth stage.[Methods] We investigated the dynamics of rhizosphere bacterial community structure in three growth stages using Illumina MiSeq high-throughput sequencing. [Results] We obtained a total of 317467 16S rDNA reads, corresponding to 7028 bacterial/archaeal operational taxonomic units. The alpha diversity was higher in the rhizosphere than in bulk soil. The diversity and richness of rhizosphere bacteria were much lower in senescence stage than that in vegetative and flowering/fruiting stages. The relative abundances of Proteobacteria and Acidobacteria gradually decreased, whereas the abundance of Cyanobacteria increased along with growth cycle. The phylum Firmicutes abundance was significantly higher in senescence stage than in other stages. The abundant genera composition also changed with growth stage. Seventeen genera (i.e. Corynebacterium, Acidovorax, Elizabethkingia, Albirhodobacter and Pseudomonas) were abundant at vegetative stage; Sixteen bacterial genera were enriched in flowering/fruiting stage, including Rhodoligotrophos, Geminicoccus, Gracilimonas and Thioprofundum. Four bacterial genera, Exiguobacterium, Citrobacter, Acinetobacter and Pseudomonas, were abundant in senescence stage. In vegetative and flowering/fruiting stages, the rhizosphere bacterial community was of high similarity, and the similarities between rhizosphere communities were higher than that between rhizosphere and bulk soil communities. However, in senescence stage, the rhizosphere bacterial community composition was more different from the communities in previous stages, but turned to be more similar with that of bulk soil. [Conclusion] The rhizosphere bacterial community diversity and composition were changing with growth stage, and great difference was found between senescence stage and previous two stages. Plant growth stage had important effects on structuring the rhizosphere bacterial community of L. ruthenicum.

Abstract:[Objective] To explore the function of type Ⅲ secretion system of bradyrhizbial strains in peanut-rhizobia interaction. [Methods] We constructed the ttsI mutant of type Ⅲ secretion system in Bradyrhizobium sp. MZ5 by the way of homologous recombination and three parental conjugative transfer. The different expression of ttsI in MZ5 and MZ5ΔttsI were analyzed by real-time PCR by using the cDNA as templates after addition of Daidzein and Genistein. The vermiculite nodulation experiment was performed to comparative analysis of the nodulation ability of Bradyrhizobium sp. MZ5 and its ttsI mutant on Arachis hypogea (S523 and Y45).[Results] We found that daidzein and genistein had a significant inhibiting effect on the ttsI gene of the strain MZ5 (P<0.05). In the vermiculite nodulation experiment, we found that there was a significant reduction in the number of nodules and the shoot dry weight on peanut S523 and Y45 for MZ5ΔttsI mutant (P<0.05) comparing with the wild-type strain MZ5. The semi-thin nodule section showed that the MZ5ΔttsI mutant contained fewer cells in the nodules than the wild-type strain. [Conclusion] The type Ⅲ secretion system of Bradyrhizobium sp. MZ5 has a positive effect on peanut-rhizobia interaction.

Abstract:[Objective] To explore the diversity of bacterial endophyte communities in the seeds of Palmae plants species.[Methods] We investigated and characterized bacterial endophytes communities in the seeds of 7 different plant species within Palmae, including Livistona chinensis, Trachycarpus fortuneii, Phoenix canariensis, Arenga engleri, P. roebelenii, Caryota mitis and Areca triandra, by a MiSeq high throughput sequencing method followed by bioinformatics analysis targeted at V3-V4 regions of the 16S rRNA gene. [Results] We recovered a total of 2341-40671 V3-V4 valid sequences of 16S rRNA gene from the tested seeds, which clustered into 97-590 distinct operational taxonomic units (OTUs). And the Shannon index was calculated to be varying in the range from 1.35 to 2.94, with the highest detected from P. canariensis seed and the lowest form A. engleri seed. The results of taxonomic assignment showed that the total number of taxonomic taxa and population composition detected in different seeds was different. At the genus level, the genus Enterococcus belonging to Firmicutes was found to be the highest in total abundance across all seeds, followed by Bacillus belonging to the same bacterial phylum. The most dominant bacterial genera detected from the tested seeds were as follow:L. chinensis seed (Bacillus, 45.8%), T. fortuneii seed (Enterococcus, 51.2%), A. engleri seed (Enterococcus, 12.1%), C. mitis seed (Enterococcus, 32.6%) A. triandra seed (Enterococcus, 42.9%), P. roebelenii seed (Enterococcus, 28.3%), P. canariensis seed (Saccharopolyspora, 31.2%). And the sub-dominant bacterial genera were: Lactococcus (L. chinensis seed), Paenibacillus (T. fortunei seed, A. engleri seed, and A. triandra seed), Goodfellowiella (P. canariensis seed), Sphingomonas (P. roebelenii seed, and C. mitis seed). Furthermore, PICRUSt was used to determine potential functional profiles associated with the recovered sequences. Several KEGG orthology (KO) terms related to the human organs and human diseases were inferred from this analysis. In addition, the KO terms related to biosynthesis of terpenoids, polyketides, and glycan, as well as xenobiotics biodegradation showed relative high abundance in the predicted KO terms of the recovered sequences from each seed.[Conclusion] Seeds of Palmae plants colonize diverse bacterial endophytes, whose population compositions were different among the tested seeds. The bacterial endophyte communities of tested Palmae seeds consisted of many human-associated bacterial genera, and some potential functions related to human diseases were also included in the predicted KO terms of the seeds bacterial endophytes communities. Each tested seed also colonized some bacterial genera with beneficial function properties, being worth for further study.

Abstract:[Objective] We expressed and purified a Pif1 helicase from Anaerobaculum hydrogeniforman to assess its unwinding characteristics.[Methods] Recombinant vector pET21a-Ana.Pif1-TEV/SUMO was constructed and expressed in E. coli BL21(DE3), and Ana.Pif1 protein was purified using Ni-NTAs and Superdex 200 column chromatography. We then evaluated the unwinding polarity, optimal ATP concentration, optimal metal cofactor, optimal unwinding temperature, and substrate specificity of Ana.Pif1 by stopped-flow FRET assay. [Results] The molecular weight of Ana.Pif1 helicase was 59 kDa with no tags, protein yield was 9.5 mg/L, and purity was about 97%. We confirmed the unwinding polarity of Ana.Pif1 was from DNA 5' to 3' end. And we found the optimal ATP concentration for Ana.Pif1 unwinding to be 2 mmol/L, the optimal metal cofactor to be Mg²⁺, and the optimal reaction temperature to be 55℃. The substrate specificity of Ana.Pif1 unwinding DNA replication intermediates indicated that the Y-S fork substrate had the highest unwinding rate, 0.127 s^-1, and 12 nt-bubble substrate had the largest unwinding amplitude, 78.8%, indicating these two substrates could be natural replication intermediates for Ana.Pif1. [Conclusion] This study is the first to systematically analyze the unwinding characteristics of Ana.Pif1 helicase. These findings facilitate elucidation of the molecular mechanisms of these thermophilic bacteria Pif1-family helicases.

Abstract:[Objective] To understand the route and mechanism of tick borne encephalitis virus transmitting across the blood brain barrier, we established an in vitro blood brain barrier model, and studied the difference of the virus cultured from two different cell lines for transmitting across the blood brain barrier. [Methods] We used human brain microvascular endothelial cells (hCMEC/D3) to establish the vitro blood brain barrier model. First, we detected the infection and replication of tick borne encephalitis virus in hCMEC/D3 using real time PCR and plague assay. Then, we conducted the transmitting assay by adding tick borne encephalitis virus into the transwell inserts of the blood brain barrier model and detected virus titer leaking from the upper inserts into the bottom wells using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and plaque assay. Meanwhile, we added the THP-1 cells infected with tick borne encephalitis virus into the transwell inserts and observed the bottom wells under the optical microscope to confirm if the monocyte THP-1 could leak across the vito blood brain barrier. The virus titer leaked into bottom wells were detected by using qRT-PCR and plaque assay. The permeability change of blood brain barrier at different time post infection was tested by using Bovine Serum Albumin conjugated evens blue.[Results] We found that no replication of tick borne encephalitis virus in hCMEC/D3 cells, and tick borne encephalitis virus cultured in BHK-21 was unable to transmigrate across the blood brain barrier directly. However, the monocyte cell line THP-1 infected with tick borne encephalitis virus could cause its transmitting across the blood brain barrier and increase the permeability of the blood brain barrier, though THP-1 did not transmit across the vitro blood brain barrier directly.[Conclusion] Tick borne encephalitis virus can transmigrate across the blood brain barrier in assistance of monocyte THP-1 cells.