Complementation Of Strain Research Articles

Identification and Functional Analysis of a Novel Hydrophobic Protein VdHP1 from Verticillium dahliae.

ABSTRACTVerticillium dahliae could cause destructive vascular wilt disease on hundreds of plant species around the world, including cotton. In this study, we characterized the function of a hydrophobin gene VdHP1 in pathogen development and pathogenicity. Results showed that VdHP1 could induce cell death and activate plant immune responses. The VdHP1 deletion mutants (ΔVdHP1) and the complement mutants (C-ΔVdHP1) were obtained by the homologous recombination method. The VdHP1 deletion mutants exhibited increased hydrophilicity, inhibited microsclerotial formation, and reduced spore smoothness. In addition, the deletion mutants were more sensitive to NaCl, while relatively insensitive to KCl and sorbitol. Mutants also had greater resistance to Congo red, UV radiation, and high temperature, which suggested that ΔVdHP1 strains have stronger resistance to abiotic stress in general. Different carbon source assays showed that the utilization ability of skim milk, cellulose, and starch was greatly enhanced in ΔVdHP1, compared with that of WT and complemented strains. Furthermore, VdHP1 did not affect mycelium penetration on cellophane but contributed to mycelium growth on surface of the living plant cells. The pathogenicity test found that the crude toxin content, colonization, and dispersal of ΔVdHP1 was significantly increased compared with the WT and complementary strains. In addition, cotton seedlings showed more severe wilting symptoms after inoculation with ΔVdHP1 strains. These results suggested that the hydrophobin VdHP1 negatively regulated the virulence of V. dahliae, and played an important role in development, adaptability, and pathogenicity in V. dahliae, which maybe provide a new viewpoint to further understand the molecular mechanisms of pathogen virulence.IMPORTANCE Verticillium dahliae is a soilborne fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, it was illustrated that the hydrophobin VdHP1 could induce cell death and activate plant immune responses. VdHP1 affected the hydrophobicity of V. dahliae, and negatively regulated the strains resistant to stress, and the utilization ability of different carbon sources. In addition, VdHP1 did not affect mycelium penetration on cellophane but contributed to mycelium growth on surface of the living plant cells. The VdHP1 gene negatively regulated the total virulence, colonization, and dispersal of V. dahliae, with enhanced pathogenicity of mutant strains in this gene. These results suggested that the hydrophobin VdHP1 played an importance in development, adaptability, and pathogenicity in V. dahliae, and would provide a new viewpoint to further understand the molecular mechanisms of pathogen virulence.

The AdcR-regulated AdcA and AdcAII contribute additively to zinc acquisition and virulence in Streptococcus suis

Metals are necessary elements for bacteria. Typically, vertebrate hosts restrict invading bacterial pathogens from accessing metals. Therefore, bacteria have evolved high-affinity metal importers to acquire metals. Streptococcus suis is a major swine pathogen and an emerging zoonotic agent that endangers the swine industry and human health worldwide. Herein, we aimed to identify the zinc acquisition systems in S. suis and evaluate their roles in bacterial virulence. Bioinformatic analyses revealed that S. suis encodes homologues of AdcA and AdcAII, two well-characterised Zn-binding lipoproteins in certain streptococci. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that the expressions of adcA and adcAII were significantly upregulated in response to Zn limitation, with a higher expression level of adcAII than adcA. Gene deletion mutants and complementation strains were constructed; their growth characteristics under Zn-deficient and Zn-replete conditions indicated that AdcA and AdcAII have overlapping functionality in Zn acquisition. A mouse infection model was used to evaluate the roles of AdcA and AdcAII in S. suis virulence. Mice infected with the double mutant ΔadcAΔadcAII exhibited a significantly higher survival rate, decreased bacterial burden, and lower production of inflammatory cytokines compared to those infected with the wild type (WT) strain. Furthermore, ΔadcAΔadcAII showed reduced competitiveness in infection establishment compared with the WT strain. RNA sequencing, qRT-PCR, and electrophoretic mobility shift assays revealed that AdcR negatively regulates the expressions of adcA and adcAII. Collectively, our results demonstrated that AdcA and AdcAII, which are negatively regulated by AdcR, contribute additively to zinc acquisition and virulence in S. suis.

Oxadiazon Derivatives Elicit Potent Intracellular Growth Inhibition against Toxoplasma gondii by Disrupting Heme Biosynthesis.

Infections of Toxoplasma gondii can cause severe and sometimes fatal diseases in immunocompromised individuals. The de novo heme biosynthesis pathway is required for intracellular growth and pathogenesis, making it an appealing therapeutic target. We synthesized a small library of derivatives of the herbicide oxadiazon, a known inhibitor of the penultimate reaction within the heme biosynthesis pathway in plants, catalyzed by protoporphyrinogen oxidase (PPO). Seven of the 18 analogs exhibit potent intracellular growth inhibition of wild-type T. gondii (IC50 = 1 to 2.4 μM). An assay of the compounds against Toxoplasma PPO knockout and complementation strains confirmed the mode of action to be due to the potent inhibition of PPO. The most potent compounds have no detectable cytotoxicity against human foreskin fibroblast cells up to 100 μM. This study suggests that oxadiazon derivatives may represent a new molecular scaffold for the effective treatment of T. gondii infections.

A LysR Transcriptional Regulator Manipulates Macrophage Autophagy Flux During Brucella Infection.

Brucella, the intracellular bacteria, have evolved subtle strategies to efficiently survive and replicate in macrophages. However, the virulence effector proteins involved are still unclear. LysR-type transcriptional regulators (lttrs) are the largest regulator family with diverse function in prokaryotes. However, very little is known about the role of LysR regulators in the Brucella spp. Here, a BSS2_II0858 gene, encoded as one of the LysR-type regulators, was studied. We successfully constructed a BSS2_II0858 deletion mutant, Δ0858, and complementation strain CΔ0858 in Brucella suis S2. The cell apoptosis induced by B. suis S2 and its derivatives were detected by flow cytometry. The autophagy was then assessed by immunoblot analysis using the IL3I/II and p62 makers. In addition, the autophagy flux was evaluated by double fluorescent labeling method for autophagy marker protein LC3. Our studies demonstrated that B. suis S2 and its derivatives inhibited the programmed cell death in early stage and promoted apoptosis in the later stage during infection in RAW264.7 cells. The BSS2_II0858 gene was found to play no role during apoptosis according to these results. Compared with the wild-type strain, Δ0858 mutant can stimulate the conversion of LC3-I to LC3-II and markedly inhibited the autophagy flux at early stage leading to obvious autophagosome accumulation. This study explored the function of BSS2_II0858 gene and may provide new insights for understanding the mechanisms involved in the survival of Brucella in macrophages.

AroC, a chorismate synthase, is required for the formation of Edwardsiella tarda biofilms

Biofilms contribute to the resistance of Edwardsiella tarda to antibiotics and host immunity. AroC in the shikimate pathway produces chorismate to synthesize crucial intermediates such as indole. In this study, the differences between biofilms produced by aroC mutants (△aroC), wild-type (WT) strains, and △aroC complementary strains (C△aroC) were detected both in vitro with 96-well plates, tubes, or coverslips and in vivo using a mouse model of subcutaneous implants. When examining potential mechanisms, we found that the diameters of the movement rings in soft agar plates and the flagellar sizes and numbers determined by silver staining were all lower for △aroC than for WT and C△aroC. Moreover, qRT-PCR showed that the transcription levels of flagellar synthesis genes, fliA and fliC, were reduced in △aroC. AroC, FliC, or FliA may accompany the motility of △aroC strains. In addition, compared with the WT and C△aroC, the amounts of indole in △aroC were significantly decreased. Notably, the formation of biofilms by these strains could be promoted by exogenous indole. Therefore, the aroC gene could affect the biofilm formation of E. tarda concerning its impact on flagella and indole.

The Transcription Factor VpxlnR Is Required for the Growth, Development, and Virulence of the Fungal Pathogen Valsa pyri.

Pears (Pyrus sp.) are widely cultivated in China, and their yield accounts for more than 60% of global pear production. The fungal pathogen Valsa pyri is a major causal agent of pear canker disease, which results in enormous losses of pear production in northern China. In this study, we characterized a Zn2Cys6 transcription factor that contains one GAL4 domain and a fungal-trans domain, which are present in VpxlnR. The vpxlnR gene expression was upregulated in the invasion stage of V. pyri. To investigate its functions, we constructed gene deletion mutants and complementary strains. We observed that the growth of the vpxlnR mutants was reduced on potato dextrose agar (PDA), Czapek plus glucose or sucrose compared with that of the wild-type strain. Additionally, vpxlnR mutants exhibited loss of function in fruiting body formation. Moreover, vpxlnR mutants were more susceptible to hydrogen peroxide (H2O2) and salicylic acid (SA) and were reduced in their virulence at the early infection stage. According to a previous study, VpxlnR-interacting motifs containing NRHKGNCCGM were searched in the V. pyri genome, and we obtained 354 target genes, of which 148 genes had Clusters of Orthologous Groups (COG) terms. PHI-BLAST was used to identify virulence-related genes, and we found 28 hits. Furthermore, eight genes from the 28 PHI-BLAST hits were further assessed by yeast one-hybrid (Y1H) assays, and five target genes, salicylate hydroxylase (VP1G_09520), serine/threonine-protein kinase (VP1G_03128), alpha-xylosidase (VP1G_06369), G-protein beta subunit (VP1G_02856), and acid phosphatase (VP1G_03782), could interact with VpxlnR in vivo. Their transcript levels were reduced in one or two vpxlnR mutants. Taken together, these findings imply that VpxlnR is a key regulator of growth, development, stress, and virulence through controlling genes involved in signaling pathways and extracellular enzyme activities in V. pyri. The motifs interacting with VpxlnR also provide new insights into the molecular mechanism of xlnR proteins.

FrtR Gene Affects Acid Production and Demineralization Ability of Streptococcus mutans

To study the effect of the frtR gene of TetR family on the acid production ability of Streptococcus mutans( S. mutans) and the bacteria's ability to induce tooth demineralization . The growth of two strains of S. mutans UA159, Δ frtR, the frtR gene in-frame deletion strain, and Δ frtR/pDL278- frtR, the complement strain, was examined. The structure of biofilm was observed by laser scanning confocal microscopy (LSCM). The quantitative determination of water-insoluble extracellular polysaccharide (EPS) in the bacterial biofilms was done by anthrone-sulfuric acid method. The acid production capacity of S. mutans was measured by glycolytic pH drop. The demineralization-inducing ability of the strains on bovine teeth was determined by transverse microradiography (TMR). The growth curves of the strains showed that frtR did not affect the growth of S. mutans. According to the findings of LSCM observation, frtR did not affect the biofilm formation. According to the findings of the anthrone-sulfuric acid method, frtR did not have any significant impact on the EPS synthesis of S. mutans. The results of the glycolytic pH drop assay showed that the deletion of frtR delayed the rate of acid production by S. mutans when sucrose was the only carbon source. In addition, according to the TMR results, knocking out frtR reduced the depth and amount of demineralization induced by S. mutans on the surface of bovine teeth. The deletion of frtR can weaken the acid production ability and the demineralization ability of S. mutans.

Involvement of PhoP/PhoQ two-component system in biofilm formation in Cronobacter sakazakii

The aim of this study was to investigated the effect of PhoP/PhoQ two component system on the biofilm formation of Cronobacter sakazakii BAA-894. Crystal violet staining assay and XTT assay showed that the biofilm biomass and the viability of biofilm cells were significantly reduced in ΔphoPQ strain, while these defects were reversed in the complementation strain ΔphoPQC. Moreover, the phoPQ gene deletion weakened the ability of BAA-894 strain to form biofilms on glass and silicon wafers, and affected the content of extracellular polymeric substance (EPS) in biofilms formed on stainless-steel plates. In addition, qRT-PCR results showed that the PhoP/PhoQ system of C. sakazakii played roles in regulating the expression of several genes associated with flagella and cellulose biosynthesis, which are both involved in biofilm formation. These finding suggest that the PhoP/PhoQ system plays important roles in biofilm formation in C. sakazakii and the system could be considered as a potential target for developing novel alternative strategies for controlling biofilms.

Mycobacterium tuberculosis Rv0309 Dampens the Inflammatory Response and Enhances Mycobacterial Survival.

To reveal functions of novel Mycobacterium tuberculosis (M. tb) proteins responsible for modulating host innate immunity is essential to elucidation of mycobacterial pathogenesis. In this study, we aimed to identify the role of a putative protein Rv0309 encoded within RD8 of M. tb genome in inhibiting the host inflammatory response and the underlying mechanism, using in-vitro and in-vivo experiments. A recombinant M. smegmatis strain Ms_rv0309 expressing Rv0309 and a mutant Bacillus Calmette-Guérin (BCG)ΔRS01790 strain with deletion of BCG_RS01790, 100% homologue of Rv0309 in BCG, were constructed. Rv0309 was found to localize in the cell wall and be able to decrease cell wall permeability. Purified recombinant rRv0309 protein inhibited lipopolysaccharide-induced IL-6 release in RAW264.7 cells. BCG_RS01790 in BCG or Rv0309 in Ms_rv0309 strain greatly inhibited production of IL-6, IL-1β, and TNF-α in RAW264.7 cells. Similarly, BCGΔRS01790 strongly induced expression of these cytokines compared with wild-type BCG and complement strain, cBCGΔRS01790::RS01790. Further BCG_RS01790 or Rv0309 suppressed cytokine production through NF-κB p65/IκBα and MAPK ERK/JNK signaling. Importantly, BCG_RS01790 in BCG and Rv0309 in Ms_rv0309 strain enhanced mycobacterial survival in macrophages. Mice infected with BCGΔRS01790 exhibited high levels of IFN-γ, TNF-α and IL-1β, and large numbers of neutrophils and lymphocytes in the early stage, and minimal lung bacterial load and inflammatory damage in late stage of the experiment. In conclusion, the cell wall protein Rv0309 or BCG_RS01790 enhanced mycobacterial intracellular survival after infection likely through inhibition of the pro-inflammatory response and decrease of bacterial cell wall permeability, thereby contributing to mycobacterial pathogenesis.

Insights Into the Impact of Small RNA SprC on the Metabolism and Virulence of Staphylococcus aureus

AimOur previous proteomic analysis showed that small RNA SprC (one of the small pathogenicity island RNAs) of Staphylococcus aureus possesses the ability to regulate the expression of multiple bacterial proteins. In this study, our objective was to further provide insights into the regulatory role of SprC in gene transcription and metabolism of S. aureus.MethodsGene expression profiles were obtained from S. aureus N315 wild-type and its sprC deletion mutant strains by RNA-sequencing (RNA-seq), and differentially expressed genes (DEGs) were screened by R language with a |log2(fold change)| ≥1 and a false discovery rate (FDR) ≤ 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out to understand the significance of the DEGs. The quality of RNA-seq was further verified by quantitative real-time PCR (qRT-PCR), mRNA target prediction, metabolomics analysis and transcript-level expression analysis of genes of sprC complementation strain.ResultsA total of 2497 transcripts were identified, of which 60 transcripts expressions in sprC knockout strain were significantly different (37 up-regulated and 23 down-regulated DEGs). GO analysis showed that the functions of these DEGs were mainly concentrated in the biological process and molecular function related to metabolism and pathogenesis, and a higher number of genes were involved in the oxidation-reduction process, catalytic activity and binding. KEGG pathways enrichment analysis demonstrated that metabolism and pathogenesis were the most affected pathways, such as metabolic pathways, biosynthesis of secondary metabolites, purine metabolism, fructose and mannose metabolism and S. aureus infection. The qRT-PCR results of the DEGs with defined functions in the sprC deletion and complementation strains were in general agreement with those obtained by RNA-seq. Metabolomics analysis revealed 77 specific pathways involving metabolic pathways. Among them, many, such as metabolic pathways, biosynthesis of secondary metabolites and purine metabolism, were consistent with those enriched in the RNA-seq analysis.ConclusionThis study offered valuable and reliable information about the regulatory roles of SprC in S. aureus biology through transcriptomics and metabolomics analysis. These results may provide clues for new potential targets for anti-virulence adjuvant therapy on S. aureus infection.

Effect of Hcp Iron Ion Regulation on the Interaction Between Acinetobacter baumannii With Human Pulmonary Alveolar Epithelial Cells and Biofilm Formation.

Acinetobacter baumannii is a type of bacterial nosocomial infection with severe drug resistance. Hemolysin co-regulated protein (Hcp) is a marker of activated type VI secretion system (T6SS), a key secretory system that promotes Gram-negative bacteria colonization, adhesion, and invasion of host cells. Hcp is also regulated by iron ions (Fe). In this study, an ATCC17978 hcp deletion strain (ATCC17978Δhcp), an hcp complement strain (ATCC17978Δhcp+ ), and an A. baumannii–green fluorescent protein (GFP) strain were constructed and used to investigate the role of hcp in bacterial adhesion to cells (human pulmonary alveolar epithelial cells (HPAEpiC)) and biofilm formation. Our results indicate that the inhibitory concentrations of the three A. baumannii strains (ATCC17978 wild type, ATCC17978Δhcp, and ATCC17978Δhcp +) were drug-sensitive strains. A. baumannii hcp gene and iron ions might be involved in promoting the formation of a biofilm and host–bacteria interaction. Iron ions affected the ability of A. baumannii to adhere to cells, as there was no significant difference in the bacterial numbers when assessing the adhesion of the three strains to HPAEpiC in the presence of iron ion concentrations of 0 μM (F = 3.1800, p = 0.1144), 25 μM (F = 2.067, p = 0.2075), 100 μM (F = 30.52, p = 0.0007), and 400 μM (F = 17.57, p = 0.0031). The three strains showed significant differences in their ability to adhere to HPAEpiC. The numbers of bacteria adhesion to HPAEpiC were ATCC17978Δhcp>ATCC17978Δhcp +>ATCC17978 in descending order. Hcp gene was positively regulated by iron ions in the bacteria–cells’ co-culture. It is speculated that the effect of iron ions on the interaction between A. baumannii and HPAEpiC might be related to the transport function of hcp and bacterial immune escape mechanisms.

The Transcription Factor Lrp of Pantoea stewartii subsp. stewartii Controls Capsule Production, Motility, and Virulence Important for in planta Growth.

The bacterial phytopathogen Pantoea stewartii subsp. stewartii causes leaf blight and Stewart’s wilt disease in susceptible corn varieties. A previous RNA-Seq study examined P. stewartii gene expression patterns during late-stage infection in the xylem, and a Tn-Seq study using a P. stewartii mutant library revealed genes essential for colonization of the xylem. Based on these findings, strains with in-frame chromosomal deletions in the genes encoding seven transcription factors (NsrR, IscR, Nac, Lrp, DSJ_00125, DSJ_03645, and DSJ_18135) and one hypothetical protein (DSJ_21690) were constructed to further evaluate the role of the encoded gene products during in vitro and in planta growth. Assays for capsule production and motility indicate that Lrp plays a role in regulating these two key physiological outputs in vitro. Single infections of each deletion strain into the xylem of corn seedlings determined that Lrp plays a significant role in P. stewartii virulence. In planta xylem competition assays between co-inoculated deletion and the corresponding complementation or wild-type strains as well as in vitro growth curves determined that Lrp controls functions important for P. stewartii colonization and growth in corn plants, whereas IscR may have a more generalized impact on growth. Defining the role of essential transcription factors, such as Lrp, during in planta growth will enable modeling of key components of the P. stewartii regulatory network utilized during growth in corn plants.

Type II toxin–antitoxin system, RatAB, contributes to oxidative resistance, biofilm formation and virulence of Edwardsiella piscicida

Since the diversity and importance of its role, the type II TA system is attracting increasing attention from researchers. Edwardsiella piscicida is becoming a leading pathogen that threatens seawater and fresh aquaculture, and has been supposed to be a model organism for the study of intracellular infections. Currently, the pathogenesis of E. piscicida remains unclear, and some new pathogenic factors need to be identified. Type II TA system is largely unknown in E. piscicida. In this study, a type II TA system, RatAB, was identified and characterized in E. piscicida. RatA shares high sequence homology with RatA family toxin, and RatB shares high sequence homology with RnfH family protein. ratA and ratB form a bicistronic operon. The biological roles of RatAB were studied by mutants TX01ΔratA and TX01ΔratAB, and corresponding complementary strains. The growths of TX01ΔratA and TX01ΔratAB were similar to that of TX01 in normal LB medium. However, TX01ΔratA, especially TX01ΔratAB, were more sensitive to oxidation pressure than wild strain TX01. Two mutants' abilities to form persistent bacteria were markedly reduced compared with TX01. The deletions of ratA and ratAB reduced bacterial biofilm formation. Cell infection test indicated that the ratA and ratAB mutations damaged E. piscicida′s ability to adhere and invade host non-phagocyte and weaken the ability of bacterial to survive and reproduce in host phagocyte. Consistently, in vivo infection tests confirmed that ratA and ratAB were required for bacterial dissemination in host tissues. Taken together, our findings demonstrate for the first time that RatAB is requisite for resistance to adversity and full virulence of E. piscicida.

The response regulator OmpR contributes to the pathogenicity of avian pathogenic Escherichia coli

Avian colibacillosis is a serious systemic infectious disease in poultry and caused by avian pathogenic Escherichia coli (APEC). Previous studies have shown that 2-component systems (TCSs) are involved in the pathogenicity of APEC. OmpR, a response regulator of OmpR/EnvZ TCS, plays an important role in E. coli K-12. However, whether OmpR correlates with APEC pathogenesis has not been established. In this study, we constructed an ompR gene mutant and complement strains by using the CRISPR-Cas9 system and found that the inactivation of the ompR gene attenuated bacterial motility, biofilm formation, and the production of curli. The resistance to environmental stress, serum sensitivity, adhesion, and invasion of DF-1 cells, and pathogenicity in chicks were all significantly reduced in the mutant strain AE17ΔompR. These phenotypes were restored in the complement strain AE17C-ompR. The qRT-PCR results showed that OmpR influences the expression of genes associated with the flagellum, biofilm formation, and virulence. These findings indicate that the regulator OmpR contributes to APEC pathogenicity by affecting the expression and function of virulence factors.

Regulation of adult neurogenesis by the endocannabinoid-producing enzyme diacylglycerol lipase alpha (DAGLa)

The endocannabinoid system modulates adult hippocampal neurogenesis by promoting the proliferation and survival of neural stem and progenitor cells (NSPCs). This is demonstrated by the disruption of adult neurogenesis under two experimental conditions: (1) NSPC-specific deletion of cannabinoid receptors and (2) constitutive deletion of the enzyme diacylglycerol lipase alpha (DAGLa) which produces the endocannabinoid 2-arachidonoylglycerol (2-AG). However, the specific cell types producing 2-AG relevant to neurogenesis remain unknown. Here we sought to identify the cellular source of endocannabinoids in the subgranular zone of the dentate gyrus (DG) in hippocampus, an important neurogenic niche. For this purpose, we used two complementary Cre-deleter mouse strains to delete Dagla either in neurons, or in astroglia and NSPCs. Surprisingly, neurogenesis was not altered in mice bearing a deletion of Dagla in neurons (Syn-Dagla KO), although neurons are the main source for the endocannabinoids in the brain. In contrast, a specific inducible deletion of Dagla in NPSCs and astrocytes (GLAST-CreERT2-Dagla KO) resulted in a strongly impaired neurogenesis with a 50% decrease in proliferation of newborn cells. These results identify Dagla in NSPCs in the DG or in astrocytes as a prominent regulator of adult hippocampal neurogenesis. We also show a reduction of Daglb expression in GLAST-CreERT2-Dagla KO mice, which may have contributed to the neurogenesis phenotype.

Effect of csn2 gene deficiency on the starvation tolerance and the synthesis of extracellular polysaccharides in oligotrophic environment of Streptococcus mutans

Objective: To explore the effect of csn2 gene deficiency on starvation tolerance and extracellular polysaccharides (EPS) synthesis in an oligotrophic environment of Streptococcus mutans (Sm). Methods: The csn2 gene deletion strains and complementary strains of Sm were cultivated and then an oligotrophic growth environment for Sm growth by setting different concentration gradient media were created. Cell growth in oligotrophic environment was detected by growth curve. Biofilm volume was measured by crystalline violet staining. Scanning electron microscopy (SEM) and laser confocal microscope were performed to observe the biofilm structure of Sm. The synthesis of EPS was measured by the anthrone-sulfuric acid method. The expression of genes related to EPS synthesis was evaluated by quantitative real-time PCR (qRT-PCR). Results: The growth curve results showed that the deletion of csn2 gene inhibited the growth of Sm under starvation stress. Furthermore, the results of laser confocal microscope showed that the biofilm EPS/bacteria ratios produced by the wild-type strain, csn2 gene-deficient strain and complement strains under nutrient sufficient culture conditions were 0.44±0.07, 1.05±0.13 and 0.57±0.08 respectively, while the ratios of EPS/bacteria in an oligotrophic environment were 0.93±0.24, 3.05±0.21 and 1.32±0.46 respectively, indicating that the deletion of csn2 gene enhanced the ability of extracellular polysaccharide synthesis of Sm in the oligotrophic environment. The expression levels of EPS synthesis-related genes gtfB and gtfC were up-regulated by 2.5 fold and 1.8 fold respectively and the expression level of gtfD was down-regulated by two-thirds. Conclusions: The csn2 gene deficiency showed multiple effects on the physiological functions and virulence characteristics of Sm, including starvation tolerance and EPS synthesis. These changes might be related to the shift of the complex regulative network caused by csn2 gene deletion.

Manipulation of viral protein production using the PCNA of halovirus фCh1 via alternative start codon usage

• Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. • Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing majorly in the presence of an alternate GTG start codon in the фCh1 variant 5’ of the ATG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. • Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behavior, viral protein levels, and virus titer. • Deletion of фCh1-ORF59, encoding PCNA ΦCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. • Complementation and overexpression with WT, and mutant variants of ORF59revealed that the 5’ GTG to ATG sequence enables фCh1 to control its equilibrium with the host. The different variants have a broad range of variable effects on each of the phenotypes observed. • Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of φCh1 modulated by the 5’ GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the behavior of the virus фCh1. The 5’ region upstream of the primary ATG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship. Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing in the presence of an alternate GUG start codon 5’ of the AUG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behaviour, viral protein levels, and virus titer. Deletion of фCh1 ORF59, encoding PCNA ФCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. Complementation and overexpression with WT and mutant variants of ORF59 revealed that modification of the GTG start codon to ATG changes the life cycle in terms of production of progeny virus particles. The different variants have a broad range of variable effects on each of the phenotypes observed. Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of фCh1 modulated by the 5’GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the life cycle of the virus фCh1. The 5’ region upstream of the primary AUG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship.

Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host.

Background Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified.ObjectivesTo verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles.MethodsConstructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro.ResultsBspJ gene deletion reduced the survival and intracellular proliferation of Brucella at the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1β, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella.ConclusionsBspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

A Novel LysR Family Factor STM0859 is Associated with The Responses of Salmonella Typhimurium to Environmental Stress and Biofilm Formation.

Salmonella enterica subsp. enterica serovar Typhimurium (ST) is an intracellularly parasitic bacterium. This zoonotic pathogen causes food poisoning and thus imposes a severe threat to food safety. Here, to understand the regulatory roles of the novel transcription factor STM0859 on the response of ST to environmental stress and biofilm formation, the STM0859 gene-deficient strain and the complementation strain ΔSTM0859/STM0859 were generated, respectively. Then, its capacity of responding to environmental stresses and biofilm (BF) formation ability under different stresses, including acid, alkali, high salt, cholate, and oxidative stresses was tested. We further analyzed the interaction between the STM0859 protein and the promoter of the acid stress response-related gene rcsB by performing an electrophoresis mobility shift assay (EMSA). The results showed that acid resistance and BF formation capacities of ST-ΔSTM0859 strain were significantly weaker, as compared with those of Salmonella Typhimurium SL1344 (ST-SL1344) wild strain (p < 0.01). Quantitative qRT-PCR analysis showed that the expression levels of acid stress and BF formation-related genes, rcsB and rpoS, of ST-ΔSTM0859 strain were significantly reduced at the transcription levels, while the transcription levels of these genes were fully restored in complementation strain ST-ΔSTM0859/STM0859. The results of EMSA showed that STM0859 was capable of binding the promoter DNA fragments of the rcsB gene, suggesting that STM0859 can promote the transcription of the rcsB gene through interaction with its promoter, thereby exerting an indirectly regulatory role on the adaptive responses to acid stress and BF formation of ST. This study provided new insights into the regulatory mechanisms of the LysR family factors on the tolerances of ST under adverse environmental stresses.

LncRsp1, a long noncoding RNA, influences Fgsp1 expression and sexual reproduction in Fusarium graminearum.

Long noncoding RNAs (lncRNAs) are crucial regulators of gene expression in many biological processes, but their biological functions remain largely unknown, especially in fungi. Fusarium graminearum is an important pathogen that causes the destructive disease Fusarium head blight (FHB) or head scab disease on wheat and barley. In our previous RNA sequencing (RNA‐Seq) study, we discovered that lncRsp1 is an lncRNA that is located +99 bp upstream of a putative sugar transporter gene, Fgsp1, with the same transcription direction. Functional studies revealed that ΔlncRsp1 and ΔFgsp1 were normal in growth and conidiation but had defects in ascospore discharge and virulence on wheat coleoptiles. Moreover, lncRsp1 and Fgsp1 were shown to negatively regulate the expression of several deoxynivalenol (DON) biosynthesis genes, TRI4, TRI5, TRI6, and TRI13, as well as DON production. Further analysis showed that the overexpression of lncRsp1 enhanced the ability of ascospore release and increased the mRNA expression level of the Fgsp1 gene, while lncRsp1‐silenced strains reduced ascospore discharge and inhibited Fgsp1 expression during the sexual reproduction stage. In addition, the lncRsp1 complementary strains lncRsp1‐LC‐1 and lncRsp1‐LC‐2 restored ascospore discharge to the level of the wild‐type strain PH‐1. Taken together, our results reveal the distinct and specific functions of lncRsp1 and Fgsp1 in F. graminearum and principally demonstrate that lncRsp1 can affect the release of ascospores by regulating the expression of Fgsp1.