[Objective] Riemerella anatipestifer (RA) is a Gram-negative pathogen that can cause duck serositis. The type Ⅸ secretion system (T9SS) of this bacterium is involved in sliding and pathogenic processes. The previous study showed that the expression ofB739_0093 inR.anatipestifer CH-1 was significantly up-regulated under the iron-limited condition. The sequence analysis showed that the protein encoded byB739_0093 contained a conserved C-terminal domain of the proteins secreted by T9SS, while its function remained unknown. This study aims to identify whether the protein encoded byB739_0093 is secreted by T9SS and the role of this protein in the pathogenesis of this bacterium. [Methods] qPCR was conducted to determine whether the transcription ofB739_0093 was regulated by iron and the ferric uptake regulator (Fur). The recombinant truncated B739_0093 protein was expressed inEscherichia coli, and the polyclonal antibody was prepared. Western blotting was employed to detect whether the protein was secreted by T9SS. Furthermore, we deletedB739_0093 from RA CH-1 and identified the role ofB739_0093 in the pathogenicity ofR.anatipestifer by virulence and colonization tests in ducklings. [Results] The expression ofB739_0093 was significantly up-regulated in the iron-restricted medium, which was mediated by Fur. Western blotting results showed that the protein encoded byB739_0093 was localized in the secretion of the parent strain RA CH-1, while it was localized in the bacterial lysate and not detected in the secretion of the T9SS-deleted strain. Compared with the parent strain RA CH-1, RA CH-1ΔB739_0093 demonstrated attenuated pathogenicity and reduced colonization ability in various tissues and organs of ducklings. [Conclusion] The protein encoded byB739_0093 is secreted by T9SS and involved in the pathogenicity ofR.anatipestifer, and its expression is regulated by iron and Fur.

[Objective] To construct a recombinant food-and-mouth disease virus (FMDV) strain carrying the genes encoding three topotypes of immunodominant structural proteins of serotype O FMDV by reverse genetic manipulation and evaluate the potential of the recombinant strain serving as a vaccine candidate for porcine food-and-mouth disease (FMD) type O. [Methods] Based on the gene of the recombinant FMDV with the replacement of the VP1 structural protein of O/NXYCh/CHA/2018 epidemic strain, the recombinant full-length plasmid featuring substitution of G-H loop genes of the structural protein VP1 of O/TUR/5/2009 vaccine strain was constructed by gene synthesis. The recombinant virus was rescued after transfection of the linearized recombinant plasmid into BSR/T7 cells expressing T7 RNA polymerase, and then identified by RT-PCR, sequencing, and indirect immunofluorescence. The plaque assay and one-step growth curve building were employed to characterize the recombinant virus. Pigs were vaccinated with the vaccines prepared from the recombinant virus and the parental virus, and then virus neutralization tests were carried out to examine the cross-reactive responses against the epidemic serotype O FMDV isolates of three topotypes. [Results] The recombinant FMDV strain carrying the structural protein genes of three topotypes was successful rescued. The recombinant strain showed similar biological properties to the parental virus. Pigs vaccinated with the vaccines prepared from the recombinant virus and the parental virus produced protective neutralizing antibodies with the mean titer of > 1.65log₁₀ against the viruses of the Middle East-South Asia (ME-SA) and South-East Asia (SEA) topotypes. The pigs did not produce protective neutralizing antibodies against the Cathay topotype (< 1.65log₁₀). The substitution of O/TUR/5/2009 G-H loop gene improved the cross-reactivity against the viruses of ME-SA and SEA topotypes compared with the parental virus (P < 0.05). [Conclusion] This study has guiding significance for the design of FMD vaccines in the future.

The proposal of ban on antibiotics in feed is a turning point for the animal husbandry, marking that the feed industry and animal husbandry have entered a new stage of transformation and upgrading. [Objective] To investigate the effects of addingBacillus licheniformis HDTN to feed on the growth performance and intestinal flora of '817' broilers. [Methods] B.licheniformis HDTN powder (7.0×10¹⁰ CFU/g) was added to the feed for '817' broilers. The effects of high-, medium-, and low-dose (1 000, 500, and 250 g/t) bacterial addition on the growth performance, serum biochemical indexes, intestinal morphology, and intestinal flora structure of the '817' broilers during 1–56 days were studied, with the group without bacterial addition as the control. [Results] Compared with the control group, the addition of low-doseB.licheniformis HDTN increased the average body weight by 105.47 g (P < 0.01) and reduced the feed-to-weight ratio by 0.25 (P < 0.05) during days 1–35. The antioxidant indexes in the broiler serum were positively correlated with the dose of HDTN, which reduced oxidative stress in broilers. The content of malondialdehyde was negatively correlated with the dose of HDTN, which suggested that HDTN reduced cellular damage in broilers. The ratio of villus height to crypt depth in the duodenum of the low-dose group was higher than that of the control group (P < 0.01). In addition, compared with the control group, medium-dose (P < 0.01) and low-dose (P < 0.05) groups showed increased relative abundance ofFirmicutes, and the medium-dose group showed increased relative abundance ofBacteroides (P < 0.05). [Conclusion] Addition of low-doseB.licheniformis HDTN for '817' broilers can improve the growth performance, reduce the feed-to-weight ratio, improve the intestinal morphology, and accelerate the assembly of intestinal flora during days 1–35.

[Objective] To reveal the composition and functions of endophytic bacterial communities in maize under different patterns of tillage combined with straw returning in the Tumochuan Plain, identify the endophytic bacterial resources that promote maize straw degradation under different patterns, and lay a foundation for the selective isolation, cultivation, and functional verification. [Methods] We employed Illumina MiSeq high-throughput sequencing to compare the diversity and community structure of endophytes during the mature stage of maize under different patterns of tillage combined with straw returning in the continuous positioning experiment in the irrigation area of Tumochuan Plain, Inner Mongolia Autonomous Region. [Results] No tillage and deep tillage demonstrated significant effects on the endophytic bacterial diversity of maize. Tillage methods exerted stronger effects on the composition and structure of endophytic bacterial community than straw returning. The structures of endophytic bacterial communities in maize can be classified into two categories: no tillage combined with straw returning and the other seven patterns. The dominant endophytic bacterial genera shared by the nine patterns of tillage combined with straw returning werePseudomonas, unclassified_f__Enterobacteriaceae,Pantoea,Raoultella, andRahnella1. Straw returning increased the abundance ofRaoultella and unclassified_f__Enterobacteriaceae. [Conclusion] Different tillage practices alter the diversity, composition, and structure of endophytic bacterial community in maize. Straw returning can increase the relative abundance ofRaoultella andLactococcus, which have positive effects on the degradation of maize straw.

Bacterial cellulose, a natural biopolymer with higher purity and better mechanical properties than plant cellulose, is expected to be widely used as a new green polymer material. A variety of bacteria have now been proven to have the ability to produce cellulose, in which bacterial cellulose synthase plays a crucial role. Therefore, understanding the catalysis mechanism of bacterial cellulose synthase is a key to the mass production and broad utilization of bacterial cellulose. This paper reviews the basic properties of bacterial cellulose synthase, including the screening of strains, the enhancement of yield, and the cellular localization of the synthase, aiming to promote the research on the catalysis mechanism of cellulose synthase. Further, based on the mechanism of cellulose synthase, this paper detail the influencing factors ofin vitro synthesis and review the research progress in the roles of each subunit of this synthesis method. We explore the catalysis mechanism of bacterial cellulose synthase, point out the problems in the current research, and envision the future research directions in this field, with a view to providing a theoretical basis for the large-scale application of bacterial cellulose by deciphering the synthesis mechanism.

[Objective] To alleviate the soil microecological imbalance caused by continuous cropping and improve the quality and yield ofGynostemmapentaphyllum, we studied the effects ofStreptomycesrochei D74 and the newly developed compound microbial agent T3 on the yield, quality, and rhizosphere bacterial community composition ofG.pentaphyllum, aiming to determine the suitable microbial agent for this medicinal plant in continuous cropping and rotational cropping. [Methods] The five-point sampling method was used to determine the yield ofG.pentaphyllum per unit area in the field. The content of main active constituents including flavonoids, polysaccharides, and saponins was determined by UV-VIS spectrophotometry and high performance liquid chromatography. The 16S rRNA gene high-throughput sequencing was employed to reveal the rhizosphere bacterial community structure ofG.pentaphyllum. [Results] In the Z-zone of rotational cropping, the dry weight, total flavonoids, and total polysaccharides of the T3 group increased by 63.44%, 12.50%, and 32.90%, respectively, compared with those in the control group, and T3 outperformed D74 in improving the yield and quality. In the P-zone of continuous cropping, the dry weight, total flavonoids, and total polysaccharides of the D74 group increased by 77.41%, 112.50%, and 23.10%, respectively, compared with those in the control group, and D74 outperformed T3. The differential microorganisms enriched in the T3 group compared with the control group were beneficial microorganisms such asNovosphingobium andRhodanbacter, and those enriched in the D74 group wereBradyrhizobium andNitrospira. [Conclusion] Both T3 and D74 could optimize the microbial community structure in rhizosphere soil and improve the micro-environment for plant growth by recruiting beneficial microorganisms in the soil, thus increasing the content and yield of the active constituents ofG.pentaphyllum. D74 and T3 demonstrate better performance in the fields of continuous cropping and rotational cropping, respectively.

[Objective] The biosafety of silver nanoparticles (AgNPs) has been a subject of concern due to the narrow therapeutic window. Expanding the therapeutic window could facilitate the application of AgNPs in the treatment of multi-drug resistant bacterial infections in humans and animals. This study aimed to enhance the biosafety of AgNPs by modifying their surface with alpha-ketoglutaric acid (AKG), a crucial component of the tricarboxylic acid cycle. [Methods] Silver ion was reduced to AgNPs by rutin at room temperature, and then AgNPs were stabilized with 1 mmol/L polyvinylpyrrolidone (PVP) solution to generate PVP-AgNPs. AKG (10 mmol/L) was added to generate PVP-AgNPs@AKG. The prepared AgNPs were characterized by a full-wavelength spectrophotometer, a particle size analyzer, and a transmission electron microscope. The antibacterial activities of PVP-AgNPs and PVP-AgNPs@AKG were evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill curve, and post-antibiotic effect. The cytotoxicity of the prepared AgNPs to human cervical epithelial cells (HCerEpic) was examined by the MTT assay and flow cytometry. Furthermore, the effects of the prepared AgNPs on the energy metabolism, oxidative stress, and expression of genes involved in anaerobic respiration ofEscherichia coli BW25113 were studied. [Results] The MIC and MBC of PVP-AgNPs@AKG against Gram-positive and Gram-negative bacteria were 50% or above 50% lower than those of PVP-AgNPs. PVP-AgNPs@AKG and PVP-AgNPs showed no significant difference in the cytotoxicity to HCerEpic cells. Compared with PVP-AgNPs, PVP-AgNPs@AKG at the MIC showed significantly enhanced inhibitory effect on the α-ketoglutarate dehydrogenase inEscherichia coli, increased accumulation of AKG, lowered ATP level, and elevated reactive oxygen species level. Moreover, PVP-AgNPs@AKG significantly up-regulated the expression ofsoxS and down-regulated the expression of genes involved in anaerobic respiration, such asarcA,fnr, andfdnH. [Conclusion] The findings suggested that PVP-AgNPs@AKG disrupted the energy metabolism by targeting α-ketoglutarate dehydrogenase, rending bacteria more vulnerable to oxidative damage. Modifying with AKG would be a potential method to expand the therapeutic window of AgNPs.

[Objective] Riemerella anatipestifer is a Gram-negative bacterium infecting ducks, causing serious economic losses to the duck industry. After infection,R.anatipestifer regulates gene expression to adapt to the 42 ℃ body temperature of ducks. To identify the adaptation mechanism ofR.anatipestifer CH-1 in ducks, we sequenced and compared the transcriptomes ofR.anatipestifer CH-1 at 37 ℃ and 42 ℃. [Methods] R.anatipestifer CH-1 was cultured to the exponential growth phase at 37 ℃ and then subjected to heat stress at 37 ℃ and 42 ℃, respectively, for 1 h. The cells were then collected for the extraction of total RNA. The raw transcriptome data of the bacteria cultured at 37 ℃ and 42 ℃ were obtained by transcriptome sequencing, and differentially expressed genes (DEGs) were screened. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were carried out for the DEGs. The genednaK involved in the response to heat stress was selected for preliminary functional identification. [Results] A total of 234 DEGs were screened out, including 169 genes with up-regulated expression and 65 genes with down-regulated expression. The GO enrichment analysis showed that the DEGs were mainly enriched in the nucleotide metabolic process, glycosyl compound metabolic process, and core RNA polymerase binding transcription factor activity. The KEGG enrichment analysis indicated that the DEGs were mainly involved in oxidative phosphorylation, ribosomes, and bacterial secretion systems. The deletion ofdnaK impaired the growth ofR.anatipestifer CH-1 at 42 ℃, compared with that at 37 ℃. [Conclusion] Compared with that at 37 ℃, the growth ofR.anatipestifer CH-1 was not affected at 42 ℃. The strain up-regulated or down-regulated the expression of heat shock response proteins and other factors to cope with heat stress.

Phenolic acids autotoxic substances secreted by plant roots, such as p-hydroxybenzoic acid (PHBA), are the main factors causing continuous cropping obstacles in plants. [Objective] In order to obtain PHBA degrading bacteria and improve its degradation efficiency. [Methods] A PHBA-degrading strain was isolated by screening medium and inorganic salt medium, and it was identified asMicrobacterium aurantiacum. The initial content of PHBA, culture temperature, pH and nitrogen source were optimized by single factor experiment and response surface methodology. [Results] The highest degradation rate was obtained when the PHBA content was 0.4 g/L, the temperature was 30 ℃, the pH was 8.0, and the nitrogen source was ammonium sulfate. The optimal degradation conditions were temperature 30.2 ℃, pH 8.3, and PHBA concentration 0.18 g/L, and the degradation rate reached 100%. Combined with pot experiments and high performance liquid chromatography, the results showed that the strain could effectively mitigate the PHBA stress effect in cucumber rhizosphere. [Conclusion] The strain ofMicrobacterium aurantiacum screened in this study has a high ability to degrade PHBA, and has the potential value in continuous cropping obstacles.

[Objective] To establish a more efficient knockout method for the serine protease coding gene (cp40) ofCorynebacteriumpseudotuberculosis and evaluate the role of this gene in the pathogenicity ofC.pseudotuberculosis. [Methods] A vector pEC-cp40gRNA-HDarm, with guide RNA, upstream and downstream sequences flankingcp40 ofC.pseudotuberculosis Xuanhan strain (XH02), and spacer, was constructed from pECXK99E. The recombinant vector pEC-cp40gRNA-HDarm was transferred intoC.pseudotuberculosis competent cells carrying pCas9gRNA-ccdB to form the CRISPR/Cas9 gene editing system for the deletion ofcp40. The roles ofcp40 in the pathogenicity ofC.pseudotuberculosis were evaluated by comparison of the colony morphology and growth curves between thecp40-deleted (Δcp40) strain and wild type (WT) strain, the viability and interleukin (IL)-1β secretion of J774A.1 macrophages infected with Δcp40 and WTinvitro, and the mortality and organ bacterial loads in mice infected with Δcp40 and WTinvivo. [Results] We successfully constructed thecp40-deleted strain XH02Δcp40 by using the established dual-plasmid CRISPR/Cas9 editing system. Compared with WT (XH02), XH02Δcp40 showed no obvious difference in the colony morphology or growth curve. However, the J774A.1 cells infected with XH02Δcp40 showed decreased lactate dehydrogenase (LDH) release (P=0.06) and propidium iodide (PI) staining ratio (P < 0.01) compared with those infected with XH02. The mortality of XH02Δcp40-infected mice reduced by 50% and the bacterial loads in the liver and kidney of XH02Δcp40-infected mice significantly reduced compared with those of XH02-infected mice (P < 0.001). [Conclusion] The CRISPR/Cas9 gene editing system established in this study can effectively deletecp40 ofC.pseudotuberculosis. The results confirm thatcp40 is a virulence-related gene, providing a foundation for subsequent research on the infection ofC.pseudotuberculosis based on this gene.