Abstract:Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis (TB), threatens the health of millions of people worldwide. The pattern recognition receptors (PRRs) including DNA and RNA sensors on immune cells recognize the invaded Mtb to activate the innate immune system and induce the production of interferon-beta (IFN-β). IFN-β is a major effector cytokine in innate antiviral response, while its role in the host response to Mtb infection remains controversial. IFN-β induced by Mtb can promote bacterial growth and improve the bacterial survival in the host. However, IFN-β treatment before Mtb infection can protect the host from bacterial infection. Focusing on the PRR signaling pathways that can recognize Mtb and mediate the IFN-β production, this review expounds the role of IFN-β in mediating the regulation of immune function by Mtb, especially the mutual inhibitory effect between IFN-β and IL-1β, aiming to reveal the pathogenic mechanism of Mtb and facilitate future research and development of anti-TB drugs.

Abstract:The prediction of bacteriophage host ranges is of great significance for the basic research and clinical application of bacteriophages. The conventional biological experimental methods are limited by the poor culturability of bacteriophages and strict cultivation conditions. The availability of massive genome or metagenome sequencing data provides the sequence signature of bacteriophages and bacteria. Therefore, intelligent computing serves as a feasible way to predict bacteriophage host ranges. This paper systematically reviews the studies about intelligent computing-based prediction of bacteriophage host ranges. Starting from the process of bacteriophage infecting bacteria, we describe the feature source and biological rationality of prediction models, analyze the typical intelligent models and their prediction principles, and list all the reference datasets, real-world datasets, and evaluation indicators. The review aims to improve the understanding on the mechanism of bacteriophages in invading bacterial hosts and promote the usage of bacteriophages as antibiotic substitutes in biological therapy.

Abstract:Outer membrane vesicles (OMVs) are spherical structures secreted by Gram-negative bacteria, with diameters of 10-250 nm. OMVs have gradually been recognized as a novel secretion system capable of transporting various substances such as lipids, proteins, nucleic acids, cytotoxins, and signaling molecules. OMVs possess a range of biological functions, including inter-bacterial communication, transmission of pathogenic factors, resistance to adverse external environments, and modulation of immune responses. Due to the distinct biological characteristics of OMVs, the antibiotic resistance transmission mediated by bacterial OMVs, as well as the potential applications of OMVs, has garnered increasing attention in recent years. This paper reviews the origins and substance delivery functions of bacterial OMVs and the protective effects of OMVs on the bacteria exposed to antibiotics and in adverse environments. Furthermore, this paper summarizes the potential applications of bacterial OMVs in the treatment of diseases, aiming to enrich the knowledge about bacterial OMVs.

Abstract:Guanosine tetraphosphate (ppGpp)/guanosine pentaphosphate (pppGpp) is a signaling molecule in the bacterial stringent response, whose synthesis and hydrolysis are controlled by the synthesis and hydrolysis activities of proteins in the RelA/SpoT homologue (RSH) family. The (p)ppGpp-mediated stringent response enhanced bacterial adaptation to nutrient deprivation and antibiotic resistance. In recent years, (p)ppGpp has been found to associate with bacterial growth, cell division, and antibiotic synthesis, which was an important global regulator in the bacteria. (p)ppGpp has many target sites in the cell, which allow it to regulate DNA replication, transcription, cell cycle, ribosome biosynthesis, and the expression of antibiotic synthesis gene clusters. However, how (p)ppGpp controls transcription and other metabolic processes depends on the bacterial species, and (p)ppGpp regulates the same processes in different bacteria species through different mechanisms. Therefore, this manuscript reviewed the types of (p)ppGpp synthetic and hydrolytic enzymes, the mechanisms of (p)ppGpp regulation on microbial metabolism and the cell cycle, as well as the regulation mechanisms of antibiotic synthesis and tolerance, which lays the foundation for bacterial resistance and cell physiology researches.

Abstract:The type VI secretion system (T6SS) as a dynamic multi-protein complex has a clear division of labor among its components, transporting effector proteins to compete for bacterial growth. Studies have shown that T6SS mediates the competitiveness of Acinetobacter baumannii in the microbial community and affects the drug resistance evolution and invasion in the host. Particularly, the valine-glycine repeat protein G (VgrG), the proline-alanine- alanine-arginine (PAAR), the hemolysin-coregulated protein (Hcp), and the effector-immunity (E-I) pair play a key role. Although T6SS has been extensively studied, there are few articles about its clinical application prospects, as this poses challenges to the identification, characterization, transport mechanism revealing, and other basic research on their functional proteins. We reviewed the research progress in the distribution, functional protein characteristics, and transport mechanism of T6SS in A. baumannii and provided evidence for its application based on the application cases of T6SS. This review aims to promote the research on the genes and functions of T6SS in A. baumannii and provide new targets and ideas for developing new anti-infective vaccines, screening suitable inhibitors, and producing engineered drug delivery tools.

Abstract:Terpenoids, a group of natural products possessing diverse chemical structures and biological activities, have been widely used in the production of food additives, medicines, and cosmetics. Terpenoids are mainly produced by plants. With the rapid development of synthetic biology, heterologous production of terpenoids by engineered microbial strains is more economical and environmentally friendly compared with the conventional methods of extraction from plants and chemical synthesis. The catalytic activities of terpene synthases and the structural specificity of their products are pivotal for the heterologous biosynthesis of terpenoids. Directed evolution and rational design can be adopted to optimize the catalytic properties and product profile of target terpene synthases. However, this requires a feasible high-throughput method to screen mutant libraries. In recent years, various high-throughput screening methods have been developed to boost the sensitivity and efficiency in screening terpene synthases. We review recently established high-throughput screening methods for terpene synthases and briefly outline the principles and advantages and disadvantages of each method. Eventually, we envision the future directions of the application of high-throughput screening in the engineering of terpene synthases.

Abstract:Using genomic data and bioinformatics methods has become an important approach to rapidly identify the genes and predict the phenotypes of bacterial antibiotic resistance. Dozens of antibiotic resistance databases have been established, providing information and auxiliary tools for the identification and prediction of bacterial antibiotic resistance. As the bacterial genome data and antibiotic resistance phenotype data are increasing, the correlation between them can be established via big data and machine learning. Therefore, establishing efficient models predicting antibiotic resistance phenotypes has become a research hot topic. Focusing on the gene identification and phenotype prediction of bacterial antibiotic resistance, this review discusses the related databases, the theories and methods, the machine learning algorithms, and the prediction models. In addition, we made an outlook on the future prospects in this field, aiming to provide new ideas for the related studies.

Abstract:Phytochromes play a key role in bacterial and plant development, while their roles in fungi are not fully understood. [Objective] To explore the roles of phytochrome genes PaPhy1 and PaPhy2 in the sexual reproduction and asexual development of Podospora anserina and decipher the regulatory mechanisms. [Methods] The homologous recombination method was employed to knock out the two phytochrome genes, and the resulting mutants, ΔPaPhy1 and ΔPaPhy2, were obtained. A double mutant, ΔPaPhy1ΔPaPhy2, was constructed via genetic cross. We compared the sexual production, asexual development, growth rate, and reactive oxygen metabolism between the mutants and wild-type strains under different light conditions to reveal the main roles of the phytochrome genes in P. anserina. [Results] Blue light and white light induced the formation of P. anserina ascospore shells. ΔPaPhy produced fewer ascospores in the light and showed prolonged life cycle. [Conclusion] Phytochrome genes are associated with the sexual reproduction of P. anserina. The aging delay of ΔPaPhy is related to reactive oxygen metabolism. The results of this study provide new ideas for further exploring the mechanism of light in regulating the reproduction of filamentous fungi as well as anti-aging studies.

Abstract:[Objective] To determine the functions of two key enzymes of the mevalonate pathway, phosphomevalonate kinase (CmErg8) and mevalonate diphosphate decarboxylase (CmErg19), in Cordyceps militaris and their effects on the content of ergosterol and cordycepin. [Methods] Bioinformatics analysis was conducted to identify CmErg8 and CmErg19 in C. militaris, and yeast complementation was employed to determine whether their functions were conserved. Furthermore, we employed Agrobacterium tumefaciens-mediated transformation to overexpress CmErg8 and CmErg19 in the auxotrophic mutant CmΔpyrG of C. militaris, so as to observe the effects of CmErg8 and CmErg19 on the content of ergosterol and cordycepin. [Results] CmErg8 and CmErg19 could not complement the temperature sensitivity of the erg8 and erg19 mutants of yeast. The strains overexpressing CmErg8 and CmErg19 showed increased content of ergosterol and cordycepin. Particularly, the cordycepin content increased by about 5 times in the strain overexpressing CmErg19 compared with that in the control. [Conclusion] This study revealed the functions of CmErg8 and CmErg19 in C. militaris and reported that the genes in the ergosterol synthesis pathway of C. militaris affected the content of cordycepin for the first time.

Abstract:[Objective] Pseudomonas aeruginosa is a major Gram-negative pathogen that can exacerbate lung infections in the patients with cystic fibrosis, which can ultimately lead to death. The emerging multi-drug resistant and pan-drug resistant strains pose challenges to the prevention and control of P. aeruginosa infection. [Methods] A phage capable of lysing multi-drug resistant P. aeruginosa was isolated from farm sewage. The morphological characteristics, biological characteristics, host range, genomic features, and in vitro inhibitory ability of the isolate were studied. The biofilm inhibition test was conducted with both the phage and antibiotics. [Results] The phage PH826 was identified to be a member of the genus Nankokuvirus and it can keep stability within a wide temperature range from 4℃ to 60℃ and pH range (pH 3.0-11.0). The host range test showed that PH826 could lyse 13 P. aeruginosa strains from human and animals. The antibacterial test in vitro showed that PH826 was strongly lytic against P. aeruginosa at multiplicity of infection (MOI) of 10, 1, and 0.1. The genome of PH826 was 87 956 bp in length, with the G+C content of 54.70%, encoding 165 open reading frames (ORFs) including an endolysin gene. The combination of PH826 at 1×MIC (minimum inhibitory concentration, MIC), 2×MIC, or 4×MIC and ciprofloxacin reduced biofilm formation by more than 80% at the time points of 24 h and 48 h. The combination of PH826 at 2×MIC or 4×MIC and meropenem reduced biofilm formation by more than 80% at the time points of 24 h and 48 h. [Conclusion] PH826 may be a potential disinfectant and therapeutic agent that can be used alone or in combination with antibiotics for the prevention and treatment of pathogenic P. aeruginosa.

Abstract:[Objective] To construct the γ-aminobutyrate-producing recombinant strains of Escherichia coli and investigate their fermentation characteristics. [Methods] We constructed two recombinant plasmids pTrc99a-gadB and pTrc99a-gadB-SNO1-SNZ1 and then respectively transformed them into the gene-knockout strain E. coli K12/ΔgabTΔgabPΔpuuE. We investigated and optimized the fermentation process of the recombinant strains for producing γ-aminobutyrate. [Results] The target proteins were highly expressed in the recombinant strains harboring the constructed plasmids. The highest concentration of γ-aminobutyrate was 4.6 g/L in the fermentation broth of E. coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB cultured in the medium containing 10 g/L l-monosodium glutamate and was 21.9 times higher than that in the fermentation broth of the wild type strain. At the l-monosodium glutamate concentration of 20 g/L, the conversion rate of substrate was the highest and the concentration of γ-aminobutyrate reached 8.4 g/L. The concentration of γ-aminobutyrate was slightly lower in the fermentation broth of the recombinant strain E. coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB-SNO1-SNZ1, probably due to the excessive consumption of energy. The highest concentration of γ-aminobutyrate was 9.4 g/L when E. coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB was cultured in 1 L fermentation medium containing 20 g/L l-monosodium glutamate. [Conclusion] We obviously increased the yield of γ-aminobutyrate produced by the recombinant strain. This finding lays a foundation for the industrial production of γ-aminobutyrate.

Abstract:[Objective] Cytochrome P450 can catalyze the oxidation of unactivated C-H bonds in complex compounds with high regio- and stereoselectivity under mild conditions. They are multifunctional biocatalysts which play important roles in the synthesis of chemical raw materials, the degradation of environmental pollutants, and drug synthesis. This paper probed into the functions of a novel cytochrome P450 enzyme named CYP154C34 from Streptomyces nanshensis by heterologous expression and whole-cell biotransformation. [Methods] We constructed two recombinant BL21(DE3) strains for whole-cell biotransformation harboring pET28a-CYP154C34-RhFRED and pET28a-CYP154C34+pACYCDuet-Pdx/PdR, respectively. Additionally, we generated a recombinant BL21(DE3) strain for heterologous expression containing pET28a-CYP154C34. We employed whole-cell biotransformation to screen the substrates and analyzed product structures using standard methods. We then compared the substrate conversion rates of the two strains of whole-cell biotransformation with those of enzyme reactions. Finally, we analyzed the substrate affinity and analogues of CYP154C34.[Results] Nine steroids hydroxylated at the 16α position by CYP154C34 were identified, including progesterone, testosterone, and androstenedione. The BL21(DE3) strain carrying pET28a-CYP154C34-RhFRED showed the highest substrate conversion rate, achieving over 90% conversion rates for seven substrates including progesterone, testosterone, and androstenedione. In addition, CYP154C34 had stronger affinity to its substrates than the analogues. [Conclusion] This study constructed two recombinant strains of CYP154C34 for whole-cell biotransformation and identified their catalytic functions. CYP154C34 can efficiently hydroxylate a wide range of steroid substrates at the 16α position with high conversion rates and excellent regio- and stereoselectivity, serving as a promising candidate for industrial applications.

Abstract:[Objective] To investigate the effect of supplementing a probiotic mixture (Bacillus licheniformis, B. subtilis, and Clostridium butyricum) in drinking water on the meat quality of broilers and explore the underlying mechanism. [Methods] A total of 360 1-day-old white feather broilers were randomly selected and assigned to control (CON, normal drinking water), low-dose probiotic (LG, drinking water supplemented with 0.2% probiotic mixture), and high-dose probiotic (HG, drinking water supplemented with 0.4% probiotic mixture) groups. The experiment lasted for 42 days. [Results] Compared with the CON group, the LG and HG groups showed increased average body weight on days 7, 35, and 42. The HG groups increased average daily gain during days 21-28, 28-35, and 35-42, while the LG groups increased average daily gain during days 35-42. LG and HG significantly increased the chest muscle redness at the time points of 45 min, 24 h, and 48 h and the chest muscle yellowness at the time point of 24 h, decreased the brightness at 24 h and 48 h and the dripping and cooking loss at 48 h. The HG group had higher content of crude protein and crude fat in chest muscle than the CON group and no significant difference compared with the LG group. Both LG and HG groups reduced the ash content of chest muscle. The addition of the probiotic mixture significantly increased total antioxidant capacity (T-AOC), while the levels of total-superoxide dismutase (T-SOD) and catalase (CAT) had an increasing trend. The expression levels of CAT, SOD-1, Keap1, and Nrf2 were significantly up-regulated, and the HG group had higher expression levels of SOD-2 and HO-1 than the LG group. The concentrations of acetic acid, propionic acid, butyric acid, and valeric acid in cecum on days 21 and 42 significantly increased in LG and HG groups. [Conclusion] The probiotic mixture enhanced the growth performance and meat quality of broilers by increasing the levels of antioxidant activity and short-chain fatty acids, and the HG group outperformed the LG group.

Abstract:[Objective] The increasing antibiotic resistant bacteria (ARB) caused by the horizontal transfer of antibiotic resistance genes (ARGs) have aroused increasing concern, and it is particularly important to monitor the ARB in the environment. [Methods] We collected seawater and sand samples from three different bathing beaches in Zhanjiang. The plate coating method and agar diffusion method were employed to study the bacterial counts, diversity, and susceptibility to antibiotics. [Results] The seawater samples had higher content of inorganic nitrogen than the normal value and the bacterial counts increased with the increase in the visitor flow. The bacteria counts in the sand samples were higher than those in the seawater samples. The bacteria from the bathing beaches belonged into 18 genera, 12 families of 3 phyla. Proteobacteria (49.64%) was dominant in the seawater samples, while Firmicutes (54.74%) was dominant in the sand samples. The bacteria showed stronger resistance to beta-lactams, with the resistance rates of 23.25%, 20.53%, and 17.42% to penicillin, vancomycin, and ceftriaxone, respectively. The resistant bacteria were mainly Bacillus, Vibrio, Pseudomonas, Streptomyces, and Enterobacter. The multidrug-resistant bacterial strains from seawater were significantly more than those from sand, and increased with the increase in the visitor flow. [Conclusion] There are diverse drug-resistant bacteria from bathing beaches, and continuous surveillance is required to assess its potential impact on public health.

Abstract:[Objective] To study the effects of different water and fertilizer conditions on the biomass of safflower and the phosphorus and microorganisms in the rhizosphere soil, and isolate the strains with high phosphorus-solubilizing ability from the rhizosphere soil samples, so as to provide a theoretical basis for the reasonable water and fertilizer management of safflower and excellent strains for the research on safflower growth and rhizosphere microenvironment. [Methods] The safflower plants were treated with phosphorus fertilizer at different gradients, and the plant biomass was determined at the rosette, jointing, blooming, and seed maturity stages. At the same time, the microbial community composition, total phosphorus, available phosphorus, and phosphatase activity in the rhizosphere soil were determined. The strains with high phosphorus-solubilizing ability were screened by soil shaking and dilution coating methods and preliminarily identified by comparative analysis of 16S rRNA gene sequences. The phosphorus-solubilizing abilities of the strains in different media were determined by the molybdenum antimony colorimetric method. Furthermore, we inoculated the dominant strains by using the root irrigation method and dilution coating method to analyze the abilities of the strains to colonize the rhizosphere and promote the growth of safflower. [Results] W3-P2 water and fertilizer treatments were conducive to the biomass accumulation of safflower. As the application amount of phosphorus fertilizer increased, the available phosphorus content and phosphatase activity showed a trend of first increasing and then decreasing. The effects of water on soil total phosphorus, available phosphorus, and phosphatase activity were correlated with the safflower development stages. Bacteria were the dominant microorganisms in the rhizosphere soil of safflower, with the highest counts of 3.017×10⁷ CFU/g and 3.021×10⁷ CFU/g at the seed maturity stage in the water treatment and phosphorus fertilizer treatment groups, respectively, which were much higher than those of fungi and actinomycetes in the same treatment group. Five efficient phosphorus-solubilizing strains (accession number C1:OR493125; C2:OR493126; C5:OR493127; C6:OR493128; C7:OR493129) were screened out from the rhizosphere soil of safflower. All of them had the ability to solubilize phosphorus and lowered the pH of the medium with inorganic and organic phosphorus as the only phosphorus source. Strain C6 had the strongest ability to solubilize phosphorus, with the amounts of phosphorus solubilized in the inorganic phosphorus media of tricalcium phosphate, aluminum phosphate, ferric phosphate, and calcium phytate being 380.00, 269.32, 7.15, and 48.16 mg/L, respectively. It solubilized 18.19 mg/L phosphorus in the medium with organic phosphorus (lecithin) as the only phosphorus source. C6 was identified by 16S rRNA gene sequencing analysis as Pseudomonas sp. and C1, C2, C5, and C7 as Sinorhizobium sp. After inoculation of 2% suspensions (10⁸ CFU/mL) of the dominant phosphorus-solubilizing bacterial strains C1, C5, and C6 around safflower plants, the cell count remained 10⁵ CFU/g on day 21, and C6 demonstrated stronger colonization. The strains increased the leaf number, plant height, stem diameter, stem weight, and root length of safflower at the blooming stage, and the C6 strain group presented stronger effects, with the indicators above being 122 tablets, 115.96 cm, 12.49 mm, 43.36 g, and 21.17 cm, respectively. [Conclusion] Water and fertilizer management affects the available phosphorus content and microbial community structure in the rhizosphere microenvironment of safflower and promotes the growth and development of safflower roots, thus directly or indirectly affecting the biomass of safflower. The irrigation at 3 000 m³/hm² and phosphorus fertilizer application 375 kg/hm² were suitable for the growth of safflower. C6 is an efficient phosphorus-solubilizing strain, capable of decomposing insoluble organic and inorganic phosphorus. Pot experiments showed that C6 could colonize the rhizosphere of safflower and significantly promoted the growth of safflower.

Abstract:[Objective] We isolated bacteria from the rhizosphere soils of three blueberry varieties, explored the bacterial diversity, and screened out the strains with acid-producing, plant growth-promoting, and antifungal properties, aiming to provide high-quality strain resources and a theoretical basis for the research on blueberry-specialized microbial fertilizers. [Methods] Five different media were used to isolate the bacteria from rhizosphere soils, and 16S rRNA gene sequencing and phylogenetic analysis were performed. The strains capable of producing acid, indole-3-acetic acid (IAA), and siderophores, fixing nitrogen, solubilizing phosphorus, and inhibiting the growth of Botrytis cinerea were screened out. The suitable strains with excellent properties were selected and then inoculated in blueberry seedlings cultivated in pots. The effects on the growth and element absorption of the seedlings and fertility of rhizosphere soil were examined. [Results] A total of 124 strains were isolated from the rhizosphere soils of three blueberry varieties. Seventy representative strains were selected for 16S rRNA gene sequencing, belonging to 21 genera of 3 phyla, among which Bacillus, Pseudomonas, Streptomyces, and Rhodococcus were the dominant bacteria. Among the representative strains, 21.4%, 21.4%, 47.1%, 65.7%, and 14.3% could produce acids, produce IAA, fix nitrogen, solubilize phosphorus, and secrete siderophores, respectively. A few strains displayed the abilities of producing acids and IAA, fixing nitrogen, solubilizing phosphorus, and inhibiting B. cinerea simultaneously. Pseudomonas chlororaphis CSM-70 and Pseudomonas piscium CSM-129 with acid-producing and growth-promoting characteristics were selected and inoculated in blueberry seedlings. Both strains significantly promoted the growth and development of blueberry seedlings and regulated the pH of the rhizosphere soil. In addition, strain CSM-70 significantly promoted the absorption of nitrogen and phosphorus in blueberry leaves and increased the content of available potassium and available nitrogen in the soil. [Conclusion] The blueberry rhizosphere soil has high bacterial diversity and harbors abundant plant growth-promoting strains. P. chlororaphis CSM-70 and P. piscium CSM-129 can promote the growth of blueberry seedlings, regulate rhizosphere soil pH and fertility, and promote nutrient absorption, demonstrating the potential of serving as blueberry-specific microbial fertilizers.

Abstract:[Objective] The long-term intense continuous cropping and abuse of mineral fertilizers result in the degradation of upland red soil and the accumulation of soil-borne plant pathogens. Fungi are the microorganisms closely related to soil health in agroecosystems. We investigated the changes of soil fungal community to explore the effects of hairy vetch (Vicia villosa Roth L.) application on the agroecosystem with upland red soil. [Methods] We employed quantitative polymerase chain reaction (qPCR) and high-throughput sequencing (Illumina MiSeq) to investigate the responses of fungal communities to mineral fertilizer (hereinafter referred to as NPK) alone and mineral fertilizer combined with hairy vetch (hereinafter referred to as NPKG) in upland red soil. [Results] Compared with NPK, NPKG increased the soil fertility, peanut yield, and fungal abundance and decreased soil pH and soil fungal diversity. Different treatments significantly altered the soil fungal community composition. Compared with NPK, NPKG increased the relative abundance of saprophytic fungi by 37.42% and decreased the relative abundance of Cercospora arachidicola and Lasidiplodia theobromae by 89.11% and 88.10%, respectively. [Conclusion] The application of hairy vetch significantly increased soil fertility, reduced the risk of peanut exposure to soil-borne diseases, and increased peanut yield in the upland red soil. Therefore, the application of hairy vetch was conducive to the sustainable development of upland red soil in southern China.

Abstract:[Objective] To study the transcriptional regulation of type VI secretion system 1 (T6SS1) genes by QsvR in Vibrio parahaemolyticus. [Methods] Total RNA was extracted from the wild type (WT) and qsvR mutant (ΔqsvR). Quantitative real-time PCR (qPCR) was employed to investigate the transcriptional regulation of target genes by QsvR. Primer extension was carried out to detect the transcription initiation site and core promoter for each target gene and calculate the transcriptional variations between WT and ΔqsvR. The regulatory DNA region of each target gene was cloned into the restriction endonuclease sites of pHRP309 harboring a promoterless gene lacZ, and then each recombinant plasmid was transferred into WT and ΔqsvR, respectively. A β-Galactosidase Enzyme Assay System (Promega) was used to measure the β-galactosidase activity in cell lysates. The recombinant pHRP309 vector containing the regulatory DNA region of one of the target gene was transferred into Escherichia coli 100λpir harboring an empty pBAD33 or pBAD33-qsvR to test whether QsvR can regulate the target genes in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and His-QsvR was over-expressed and then purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay (EMSA) was employed to determine the DNA-binding activity of His-QsvR to each target DNA fragment in vitro. [Results] The mRNA levels of T6SS1-associated genes, VP1388 (the first gene of VP1388-1390 operon) and hcp1 (the first gene of VP1393-1406 operon), were significantly up-regulated in ΔqsvR relative to those in WT, indicating that QsvR activated the transcription of VP1388 and hcp1. Only one transcription initiation site was detected for VP1388 or hcp1, locating at 64 bp upstream of VP1388 and 62 bp upstream of hcp1, respectively, and their transcriptional activities were all repressed by QsvR. QsvR repressed the promoter activities of VP1388 and hcp1 in both V. parahaemolyticus and E. coli 100λpir. His-QsvR was able to bind to the regulatory DNA regions of VP1388 and hcp1. [Conclusion] QsvR directly repressed the transcription of T6SS1-associated operons, VP1388-1390 and VP1393-1406, in V. parahaemolyticus.

Abstract:Salinization is a global soil problem. Plant growth-promoting rhizobacteria (PGPR) have unique advantages in remediating saline-alkali soils and promoting plant growth. Screening the rhizosphere microorganisms of Tamarix chinensis, a typical halophyte, and revealing the growth-promoting effect and mechanism of these microorganisms are of great significance for developing microbial fertilizers. [Objective] To screen out the microorganisms from the rhizosphere soil of saline-alkaline tolerant T. chinensis and measure their basic characteristics, salt and alkali tolerance, and plant growth-promoting effects. [Methods] A saline-alkali tolerant strain Bachu 26 was screened out from the rhizosphere soil of wild T. chinensis in Bachu, Xinjiang and identified based on the morphological, physiological, and biochemical characteristics and 16S rRNA gene sequence. We cultured the strain Bachu 26 in the media with different salt concentrations (0%-20%) and pH values (7.0-13.0) to evaluate its salt and alkali tolerance. We then used multiple functional media to examine the growth-promoting function of Bachu 26 and measured the production of indole-3-acetic acid (IAA). The petri dishes with two compartments were used to verify the ability of Bachu 26 to produce acidic volatile substances. Arabidopsis thaliana seedlings were co-cultured with strain Bachu 26 in common culture dishes, based on which the growth-promoting effect of Bachu 26 on A. thaliana seedlings was analyzed. Furthermore, A. thaliana seedlings and Bachu 26 were cultured dividually in the petri dishes with two compartments, based on which the growth-promoting effect of volatile acidic substances produced by Bachu 26 on the seedlings was analyzed. The growth-promoting effect of Bachu 26 on maize seedlings was analyzed by pot experiments. [Results] Strain Bachu 26 was identified as a species of Halomonas and named Halomonas sp. Bachu 26, with the maximum tolerance to 20% salt and pH 11.0. Halomonas sp. Bachu 26 could solubilize organic phosphorus, fix nitrogen, and produce ACC deaminase, IAA, and volatile acidic substances, with the IAA yield reaching 45.885 6 mg/L. Halomonas sp. Bachu 26 significantly increased the fresh weight, lateral roots, taproot length, and leaves of A. thaliana seedlings under saline-alkali stress. In pot experiments, Halomonas sp. Bachu 26 significantly increased the fresh weight of aboveground part, fresh weight of underground part, and plant height of maize under saline-alkali stress. [Conclusion] The newly isolated PGPR strain Bachu 26 has a significant plant growth-promoting effect. The findings provide material and theoretical support for the remediation of saline-alkali soil and development of saline-alkali tolerant plant growth-promoting microbial fertilizers. Furthermore, the results will promote the applied and basic research on Halomonas.

Abstract:[Objective] The cell wall structure characteristics make it a challenge to obtain the whole proteins of Paenibacillus sp. as a Gram-positive bacterium. We employed three representative lysis methods:lysozyme combined with ultrasonic treatment (method 1), lysozyme combined with SDS heat treatment (method 2), and liquid nitrogen combined with ultrasonic treatment (method 3), to obtain the samples for mass spectrometry-based proteomics research on Paenibacillus sp. [Methods] We compared the protein extraction efficiency of the three methods based on protein yield and SDS-PAGE. Furthermore, we analyzed the obtained protein samples by mass spectrometry. [Results] The protein extraction efficiency of the three methods was similar. The number of proteins identified in the sample prepared with method 3 was the highest (2 638 proteins), followed by that with method 1 (2 452 proteins), and that with method 2 was the lowest (2 003 proteins). The Venn diagram was established to compare the protein identification flux of the samples prepared with different methods. Considering the protein extraction efficiency and the shortcomings of method 3, we finally chose method 1 as the most suitable method to extract the proteins for mass spectrometry-based proteomic analysis. Finally, the basic properties of proteins identified by mass spectrometry, including molecular weight, isoelectric point, and hydrophobicity, were analyzed. The proportion of each component of the proteins obtained by the three methods was the same as that of the predicted proteins of the model strain Paenibacillus polymyxa. [Conclusion] For the proteomics research on Gram-positive Paenibacillus sp. by mass spectrometry, lysozyme combined with ultrasonic treatment is the most suitable method for sample preparation.