T6SS and T4SS redundantly secret effectors to govern the virulence and bacterial competition in Pectobacterium PccS1.

The previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either T6SS or T4SS. This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with stains defective in T6SS or/and T4SS confirm that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose-deposition in the course of infection. Notably, this redundant secretion mechanism between T6SS and T4SS is believed to be the first of its kind in bacteria.

A genome-wide association study reveals that epistasis underlies the pathogenicity of Pectobacterium.

Pectobacterium spp. are important bacterial pathogens that cause soft rot symptoms in various crops. However, their mechanism of pathogenicity requires clarity to help control their infections. Here, genome-wide association studies (GWAS) were conducted by integrating genomic data and measurements of two phenotypes (virulence and cellulase activity) for 120 various Pectobacterium strains in order to identify the genetic basis of their pathogenicity. An artificial intelligence-based software program was developed to automatically measure lesion areas on Chinese cabbage, thereby facilitating accurate and rapid data collection for virulence phenotypes for use in GWAS analysis. The analysis discovered 428 and 158 loci significantly associated with Pectobacterium virulence (lesion area) and cellulase activity, respectively. In addition, 1,229 and 586 epistasis loci pairs were identified for the virulence and cellulase activity phenotypes, respectively. Among them, the AraC transcriptional regulator exerted epistasis effects with another three nutrient transport-related genes in pairs contributing to the virulence phenotype, and their epistatic effects were experimentally confirmed for one pair with knockout mutants of each single gene and double gene. This study consequently provides valuable insights into the genetic mechanism underlying Pectobacterium spp. pathogenicity. IMPORTANCE Plant diseases and pests are responsible for the loss of up to 40% of food crops, and annual economic losses caused by plant diseases reach more than $220 billion. Fighting against plant diseases requires an understanding of the pathogenic mechanisms of pathogens. This study adopted an advanced approach using population genomics integrated with virulence-related phenotype data to investigate the genetic basis of Pectobacterium spp., which causes serious crop losses worldwide. An automated software program based on artificial intelligence was developed to measure the virulence phenotype (lesion area), which greatly facilitated this research. The analysis predicted key genomic loci that were highly associated with virulence phenotypes, exhibited epistasis effects, and were further confirmed as critical for virulence with mutant gene deletion experiments. The present study provides new insights into the genetic determinants associated with Pectobacterium pathogenicity and provides a valuable new software resource that can be adapted to improve plant infection measurements.

Predation of oomycetes by myxobacteria via a specialized CAZyme system arising from adaptive evolution.

As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora. These enzymes include three specialized ß-1,3-glucanases (AcGlu13.1, -13.2 and -13.3) that act as a cooperative consortium to target ß-1,3-glucans of Phytophthora. However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain ß-1,3-glucans. Heterologous expression of AcGlu13.1, -13.2 or -13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae, conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora, and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection.

Myxobacterial Outer Membrane ß-1,6-Glucanase Induced the Cell Death of Fusarium oxysporum by Destroying the Cell Wall Integrity.

The ß-1,6-glucan is the key linker between mannoproteins in the outermost part of the cell wall and ß-1,3-glucan/chitin polysaccharide to maintain the rigid structure of the cell wall. The ß-1,6-glucanase GluM, which was purified from the fermentation supernatant of Corallococcus sp. EGB, was able to inhibit the germination of Fusarium oxysporum f. sp. cucumerinum conidia at a minimum concentration of 2.0 U/mL (0.08 µg/mL). The survival rates of GluM-treated conidia and monohyphae were 10.4% and 30.7%, respectively, which were significantly lower than that of ß-1,3-glucanase treatment (Zymolyase, 20.0 U/mL; equate to 1.0 mg/mL) (72.9% and 73.9%). In contrast to ß-1,3-glucanase treatment, the high-osmolarity glycerol (HOG) pathway of F. oxysporum f. sp. cucumerinum cells was activated after GluM treatment, and the intracellular glycerol content was increased by 2.6-fold. Moreover, the accumulation of reactive oxygen species (ROS) in F. oxysporum f. sp. cucumerinum cells after GluM treatment induced apoptosis, but it was not associated with the increased intracellular glycerol content. Together, the results indicate that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents. IMPORTANCE Phytopathogenic fungi are the most devastating plant pathogens in agriculture, causing enormous economic losses to global crop production. Biocontrol agents have been promoted as replacements to synthetic chemical pesticides for sustainable agriculture development. Cell wall-degrading enzymes (CWDEs), including chitinases and ß-1,3-glucanases, have been considered as important armaments to damage the cell wall. Here, we found that F. oxysporum f. sp. cucumerinum is more sensitive to ß-1,6-glucanase GluM treatment (0.08 µg/mL) than ß-1,3-glucanase Zymolyase (1.0 mg/mL). The HOG pathway was activated in F. oxysporum f. sp. cucumerinum cells after GluM treatment, and the intracellular glycerol content was significantly increased. Moreover, the decomposition of F. oxysporum f. sp. cucumerinum cell wall by GluM induced the burst of intracellular ROS and apoptosis, which eventually leads to cell death. Therefore, we suggest that the ß-1,6-glucan of the fungal cell wall may be a better antifungal target compared to the ß-1,3-glucan.

OxyR contributes to virulence of Acidovorax citrulli by regulating anti-oxidative stress and expression of flagellin FliC and type IV pili PilA.

In many bacteria, OxyR acts as a transcriptional regulator that facilitates infection via degrading hydrogen peroxide (H2O2) generated by the host defense response. Previous studies showed that OxyR also plays an important role in regulating biofilm formation, cell motility, pili relate-genes expression, and surface polysaccharide production. However, the role of OxyR has not been determined in Acidovorax citrulli strain xjl12. In the current study, the qRT-PCR and western blot assays revealed that the expression level of oxyR was significantly induced by H2O2. The oxyR deletion mutant of A. citrulli was significantly impaired bacterial tolerance to oxidative stress and reduced catalase (CAT) activity. In addition, oxyR mutant resulted in reduced swimming motility, twitching motility, biofilm formation, virulence, and bacterial growth in planta by significantly affecting flagellin and type IV pili-related gene (fliC and pilA) expression. The qRT-PCR assays and western blot revealed that OxyR positively regulated the expression of fliC and pilA. Furthermore, bacterial one-hybrid assay demonstrated that OxyR directly affected pilA and fliC promoter. Through bacterial two-hybrid assay, it was found that OxyR can directly interact with PilA and FliC. These results suggest that OxyR plays a major role in the regulating of a variety of virulence traits, and provide a foundation for future research on the global effects of OxyR in A. citrulli.

Effects of Natural Rheum tanguticum on the Cell Wall Integrity of Resistant Phytopathogenic Pectobacterium carotovorum subsp. Carotovorum.

The abuse of agricultural antibiotics has led to the emergence of drug-resistant phytopathogens. Rifampicin and streptomycin and streptomycin resistance Pectobacterium carotovorum subsp. carotovorum (PccS1) was obtained from pathological plants in a previous experiment. Rheum tanguticum, derived from the Chinese plateau area, exhibits excellent antibacterial activity against PccS1, yet the action mode has not been fully understood. In present text, the cell wall integrity of the PccS1 was tested by the variation of the cellular proteins, SDS polyacrylamide gel electrophoresis (SDS-PAGE), scanning electron microscopy (SEM) and Fourier transform infrared spectrophotometer (FTIR) characteristics. Label-free quantitative proteomics was further used to identify the DEPs in the pathogen response to treatment with Rheum tanguticum Maxim. ex Balf. extract (abbreviated as RTMBE). Based on the bioinformatics analysis of these different expressed proteins (DEPs), RTMBE mainly inhibited some key protein expressions of beta-Lactam resistance, a two-component system and phosphotransferase system. Most of these membrane proteins were extraordinarily suppressed, which was also consistent with the morphological tests. In addition, from the downregulated flagellar motility related proteins, it was also speculated that RTMBE played an essential antibacterial role by affecting the swimming motility of the cells. The results indicated that Rheum tanguticum can be used to attenuate the virulence of the drug-resistant phytopathogenic bacteria.

Lysobacter enzymogenes antagonizes soilborne bacteria using the type IV secretion system.

Soil microbiome comprises numerous microbial species that continuously interact with each other. Among the modes of diverse interactions, cell-cell killing may play a key role in shaping the microbiome composition. Bacteria deploy various secretion systems to fend off other microorganisms and Type IV Secretion System (T4SS) in pathogenic bacteria was shown to function as a contact-dependent, inter-bacterial killing system only recently. The present study investigated the role played by T4SS in the killing behaviour of the soilborne biocontrol bacterium Lysobacter enzymogenes OH11. Results showed that L. enzymogenes OH11 genome encompasses genes encoding all the components of T4SS and effectors potentially involved in inter-bacterial killing system. Generation of knock-out mutants revealed that L. enzymogenes OH11 uses T4SS as the main contact-dependent weapon against other soilborne bacteria. The T4SS-mediated killing behaviour of L. enzymogenes OH11 decreased the antibacterial and antifungal activity of two Pseudomonas spp. but at the same time, protected carrot from infection by Pectobacterium carotovorum. Overall, this study showed for the first time the involvement of T4SS in the killing behaviour of L. enzymogenes and its impact on the multiple interactions occurring in the soil microbiome.

Pectobacterium atrosepticum KDPG aldolase, Eda, participates in the Entner-Doudoroff pathway and independently inhibits expression of virulence determinants.

Pectobacterium carotovorum has an incomplete Entner-Doudoroff (ED) pathway, including enzyme 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda) but lacking phosphogluconate dehydratase (Edd), while P. atrosepticum (Pba) has a complete pathway. To understand the role of the ED pathway in Pectobacterium infection, mutants of these two key enzymes, Δeda and Δedd, were constructed in Pba SCRI1039. Δeda exhibited significant decreased virulence on potato tubers and colonization in planta and was greatly attenuated in pectinase activity and the ability to use pectin breakdown products, including polygalacturonic acid (PGA) and galacturonic acid. These reduced phenotypes were restored following complementation with an external vector expressing eda. Quantitative reverse transcription PCR analysis revealed that expression of the pectinase genes pelA, pelC, pehN, pelW, and pmeB in Δeda cultured in pyruvate, with or without PGA, was significantly reduced compared to the wild type, while genes for virulence regulators (kdgR, hexR, hexA, and rsmA) remained unchanged. However, Δedd showed similar phenotypes to the wild type. To our knowledge, this is the first demonstration that disruption of eda has a feedback effect on inhibiting pectin degradation and that Eda is involved in building the arsenal of pectinases needed during infection by Pectobacterium.

Transcriptome of Pectobacterium carotovorum subsp. carotovorum PccS1 infected in calla plants in vivo highlights a spatiotemporal expression pattern of genes related to virulence, adaptation, and host response.

Bacterial pathogens from the genus Pectobacterium cause soft rot in various plants, and result in important economic losses worldwide. We understand much about how these pathogens digest their hosts and protect themselves against plant defences, as well as some regulatory networks in these processes. However, the spatiotemporal expression of genome-wide infection of Pectobacterium remains unclear, although researchers analysed this in some phytopathogens. In the present work, comparing the transcriptome profiles from cellular infection with growth in minimal and rich media, RNA-Seq analyses revealed that the differentially expressed genes (log2 -fold ratio ≥ 1.0) in the cells of Pectobacterium carotovorum subsp. carotovorum PccS1 recovered at a series of time points after inoculation in the host in vivo covered approximately 50% of genes in the genome. Based on the dynamic expression changes in infection, the significantly differentially expressed genes (log2 -fold ratio ≥ 2.0) were classified into five types, and the main expression pattern of the genes for carbohydrate metabolism underlying the processes of infection was identified. The results are helpful to our understanding of the inducement of host plant and environmental adaption of Pectobacterium. In addition, our results demonstrate that maceration caused by PccS1 is due to the depression of callose deposition in the plant for resistance by the pathogenesis-related genes and the superlytic ability of pectinolytic enzymes produced in PccS1, rather than the promotion of plant cell death elicited by the T3SS of bacteria as described in previous work.

A predatory myxobacterium controls cucumber Fusarium wilt by regulating the soil microbial community.

BACKGROUND: Myxobacteria are micropredators in the soil ecosystem with the capacity to move and feed cooperatively. Some myxobacterial strains have been used to control soil-borne fungal phytopathogens. However, interactions among myxobacteria, plant pathogens, and the soil microbiome are largely unexplored. In this study, we aimed to investigate the behaviors of the myxobacterium Corallococcus sp. strain EGB in the soil and its effect on the soil microbiome after inoculation for controlling cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC). RESULTS: A greenhouse and a 2-year field experiment demonstrated that the solid-state fermented strain EGB significantly reduced the cucumber Fusarium wilt by 79.6% (greenhouse), 66.0% (2015, field), and 53.9% (2016, field). Strain EGB adapted to the soil environment well and decreased the abundance of soil-borne FOC efficiently. Spatiotemporal analysis of the soil microbial community showed that strain EGB migrated towards the roots and root exudates of the cucumber plants via chemotaxis. Cooccurrence network analysis of the soil microbiome indicated a decreased modularity and community number but an increased connection number per node after the application of strain EGB. Several predatory bacteria, such as Lysobacter, Microvirga, and Cupriavidus, appearing as hubs or indicators, showed intensive connections with other bacteria. CONCLUSION: The predatory myxobacterium Corallococcus sp. strain EGB controlled cucumber Fusarium wilt by migrating to the plant root and regulating the soil microbial community. This strain has the potential to be developed as a novel biological control agent of soil-borne Fusarium wilt. Video abstract.

A novel outer membrane ß-1,6-glucanase is deployed in the predation of fungi by myxobacteria.

Myxobacterial predation on bacteria has been investigated for several decades. However, their predation on fungi has received less attention. Here, we show that a novel outer membrane ß-1,6-glucanase GluM from Corallococcus sp. strain EGB is essential for initial sensing and efficient decomposition of fungi during predation. GluM belongs to an unstudied family of outer membrane ß-barrel proteins with potent specific activity up to 24,000 U/mg, whose homologs extensively exist in myxobacteria. GluM was able to digest fungal cell walls efficiently and restrict Magnaporthe oryzae infection of rice plants. Genetic complementation with gluM restored the fungal predation ability of Myxococcus xanthus CL1001, which was abolished by the disruption of gluM homolog oar. The inability to prey on fungi with cell walls that lack ß-1,6-glucans indicates that ß-1,6-glucans are targeted by GluM. Our results demonstrate that GluM confers myxobacteria with the ability to feed on fungi, and provide new insights for understanding predator-prey interactions. Considering the attack mode of GluM, we suggest that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents.

Optimization of culture conditions for promoting heat-stable antifungal factor production level in Lysobacter enzymogenes.

Heat-stable antifungal factor (HSAF), which was first isolated from Lysobacter enzymogenes, exhibits inhibitory activities against a wide range of pathogens; however, a low level of HSAF was obtained from L. enzymogenes cultured in 0.1 × tryptic soy broth (TSB), an amount that does not satisfy HSAF application in disease control. In this study, the optimization of media components and environmental conditions were examined for improving the production of HSAF from L. enzymogenes OH11. The one factor at a time method was used to screen optimal nitrogen and carbon sources and inorganic salt. Then the orthogonal matrix method was used to determine the optimal concentration of the media components and environmental factors. The results showed that the maximum level of HSAF (23361 mAU·s) was achieved when OH11 cultured in the media of 0.7% (w/v) soybean powder, 0.5% (w/v) glucose and 0.08% CaCl2 at 200 rpm at 30°C for 60 h, which is much higher than that cultured in 0.1 × TSB. This opens up the possibility of HSAF or L. enzymogenes utilization for biological control of plant disease.

Hfq, a RNA Chaperone, Contributes to Virulence by Regulating Plant Cell Wall-Degrading Enzyme Production, Type VI Secretion System Expression, Bacterial Competition, and Suppressing Host Defense Response in Pectobacterium carotovorum.

Hfq is a RNA chaperone and participates in a wide range of cellular processes and pathways. In this study, mutation of hfq gene from Pectobacterium carotovorum subsp. carotovorum PccS1 led to significantly reduced virulence and plant cell wall-degrading enzyme (PCWDE) activities. In addition, the mutant exhibited decreased biofilm formation and motility and greatly attenuated carbapenem production as well as secretion of hemolysin coregulated protein (Hcp) as compared with wild-type strain PccS1. Moreover, a higher level of callose deposition was induced in Nicotiana benthamiana leaves when infiltrated with the mutant. A total of 26 small (s)RNA deletion mutants were obtained among a predicted 27 sRNAs, and three mutants exhibited reduced virulence in the host plant. These results suggest that hfq plays a key role in Pectobacterium virulence by positively impacting PCWDE production, secretion of the type VI secretion system, bacterial competition, and suppression of host plant responses.

Characterization of Pectobacterium carotovorum proteins differentially expressed during infection of Zantedeschia elliotiana in vivo and in vitro which are essential for virulence.

The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria-Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar 'Black Magic' (in vitro) and in plant tissues (in vivo) by two-dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5-fold) were identified (up-regulated or down-regulated in vitro, in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro, or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which (clpP, mreB, flgK and eda) led to a loss of virulence on Z. elliotiana, although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP, flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda, which encodes 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, a key enzyme in Entner-Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors.

The Ribosomal Protein RplY Is Required for Pectobacterium carotovorum Virulence and Is Induced by Zantedeschia elliotiana Extract.

Pectobacterium carotovorum subsp. carotovorum strain PccS1, a bacterial pathogen causing soft rot disease of Zantedeschia elliotiana (colored calla), was investigated for virulence genes induced by the host plant. Using a promoter-trap transposon (mariner), we obtained 500 transposon mutants showing kanamycin resistance dependent on extract of Z. elliotiana. One of these mutants, PM86, exhibited attenuated virulence on both Z. elliotiana and Brassica rapa subsp. pekinensis. The growth of PM86 was also reduced in minimal medium (MM), and the reduction was restored by adding plant extract to the MM. The gene containing the insertion site was identified as rplY. The deletion mutant ΔrplY, exhibited reduced virulence, motility and plant cell wall-degrading enzyme production but not biofilm formation. Analysis of gene expression and reporter fusions revealed that the rplY gene in PccS1 is up-regulated at both the transcriptional and the translational levels in the presence of plant extract. Our results suggest that rplY is induced by Z. elliotiana extract and is crucial for virulence in P. carotovorum subsp. carotovorum.

Dickeyafangzhongdai sp. nov., a plant-pathogenic bacterium isolated from pear trees (Pyrus pyrifolia).

Gram-stain-negative, pectinolytic bacteria were repeatedly isolated from pear trees displaying symptoms of bleeding canker in China. Three strains, JS5T, LN1 and QZH3, had identical 16S rRNA gene sequences that shared 99 % similarity to the type strain of Dickeya dadantii. Phylogenetic analysis of strains JS5T, LN1 and QZH3 with isolates representing all species of the genus Dickeya and related Pectobacterium species supported their affiliation to Dickeya. Multi-locus sequence typing employing concatenated sequences encoding recA, fusA, gapA, purA, rplB, dnaX and the intergenic spacer illustrated a phylogeny which placed strains JS5T, LN1 and QZH3 as a distinct clade, separate from all other species of the genus Dickeya. Average nucleotide identity values obtained in comparison with all species of the genus Dickeya supported the distinctiveness of strain JS5T within the genus Dickeya. Additionally, all three strains were phenotypically distinguished from other species of the genus Dickeya by failing to hydrolyse casein, and by producing acids from (-)-d-arabinose, (+)melibiose, (+)raffinose, mannitol and myo-inositol, but not from 5-keto-d-gluconate or ß-gentiobiose. The name Dickeya fangzhongdai sp. nov. is proposed to accommodate these strains; the type strain is JS5T (=CGMCC 1.15464T=DSM 101947T).

Proteomic Analysis Reveals Resistance Mechanism Against Chlorpyrifos in Frankliniella occidentalis (Thysanoptera: Thripidae).

The western flower thrips is an economically important worldwide pest of many crops, and chlorpyrifos has been used to control western flower thrips for many years. To develop a better resistance-management strategy, a chlorpyrifos-resistant strain of western flower thrips (WFT-chl) was selected in the laboratory. More than 39-fold resistance was achieved after selected by chlorpyrifos for 19 generations in comparison with the susceptible strain (WFT-S). Proteome of western flower thrips (WFT-S and WFT-chl) was investigated using a quantitative proteomics approach with isobaric tag for relative and absolute quantification technique and liquid chromatography-tandem mass spectrometry technologies. According to the functional analysis, 773 proteins identified were grouped into 10 categories of molecular functions and 706 proteins were presented in 213 kinds of pathways. Comparing the proteome of WFT-chl with that of WFT-S, a total of eight proteins were found up-regulated and three down-regulated. The results from functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that the differentially expressed protein functions in binding, catalyzing, transporting, and enzyme regulation were most important in resistance development. A list of proteins functioning in biological processes of metabolism, biological regulation, and response to stimulus was found in WFT-chl, suggesting that they are possibly the major components of the resistance mechanism to chlorpyrifos in western flower thrips. Notably, several novel potential resistance-related proteins were identified such as ribosomal protein, Vg (vitellogenin), and MACT (muscle actin), which can be used to improve our understanding of the resistance mechanisms in western flower thrips. This study provided the first comprehensive view of the complicated resistance mechanism employed by WFT-S and WFT-chl through the isobaric tag for relative and absolute quantification coupled with liquid chromatography-tandem mass spectrometry technologies.

The mitochondrial genome of Frankliniella intonsa: insights into the evolution of mitochondrial genomes at lower taxonomic levels in Thysanoptera.

Thrips is an ideal group for studying the evolution of mitochondrial (mt) genomes in the genus and family due to independent rearrangements within this order. The complete sequence of the mitochondrial DNA (mtDNA) of the flower thrips Frankliniella intonsa has been completed and annotated in this study. The circular genome is 15,215bp in length with an A+T content of 75.9% and contains the typical 37 genes and it has triplicate putative control regions. Nucleotide composition is A+T biased, and the majority of the protein-coding genes present opposite CG skew which is reflected by the nucleotide composition, codon and amino acid usage. Although the known thrips have massive gene rearrangements, it showed no reversal of strand asymmetry. Gene rearrangements have been found in the lower taxonomic levels of thrips. Three tRNA genes were translocated in the genus Frankliniella and eight tRNA genes in the family Thripidae. Although the gene arrangements of mt genomes of all three thrips species differ massively from the ancestral insect, they are all very similar to each other, indicating that there was a large rearrangement somewhere before the most recent common ancestor of these three species and very little genomic evolution or rearrangements after then. The extremely similar sequences among the CRs suggest that they are ongoing concerted evolution. Analyses of the up and downstream sequence of CRs reveal that the CR2 is actually the ancestral CR. The three CRs are in the same spot in each of the three thrips mt genomes which have the identical inverted genes. These characteristics might be obtained from the most recent common ancestor of this three thrips. Above observations suggest that the mt genomes of the three thrips keep a single massive rearrangement from the common ancestor and have low evolutionary rates among them.

Lysobacter enzymogenes uses two distinct cell-cell signaling systems for differential regulation of secondary-metabolite biosynthesis and colony morphology.

Lysobacter enzymogenes is a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown in L. enzymogenes. In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time from L. enzymogenes. The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis in L. enzymogenes. Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology of L. enzymgenes. Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions in L. enzymogenes, and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis of L. enzymogenes.

Proteomic analysis reveals novel extracellular virulence-associated proteins and functions regulated by the diffusible signal factor (DSF) in Xanthomonas oryzae pv. oryzicola.

Quorum sensing (QS) in Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is mediated by the diffusible signal factor (DSF). DSF-mediating QS has been shown to control virulence and a set of virulence-related functions; however, the expression profiles and functions of extracellular proteins controlled by DSF signal remain largely unclear. In the present study, 33 DSF-regulated extracellular proteins, whose functions include small-protein mediating QS, oxidative adaptation, macromolecule metabolism, cell structure, biosynthesis of small molecules, intermediary metabolism, cellular process, protein catabolism, and hypothetical function, were identified by proteomics in Xoc. Of these, 15 protein encoding genes were in-frame deleted, and 4 of them, including three genes encoding type II secretion system (T2SS)-dependent proteins and one gene encoding an Ax21 (activator of XA21-mediated immunity)-like protein (a novel small-protein type QS signal) were determined to be required for full virulence in Xoc. The contributions of these four genes to important virulence-associated functions, including bacterial colonization, extracellular polysaccharide, cell motility, biofilm formation, and antioxidative ability, are presented. To our knowledge, our analysis is the first complete list of DSF-regulated extracellular proteins and functions in a Xanthomonas species. Our results show that DSF-type QS played critical roles in regulation of T2SS and Ax21-mediating QS, which sheds light on the role of DSF signaling in Xanthomonas.