Production of Novel Antibiotics Zeamines through Optimizing Dickeya zeae Fermentation Conditions

Dickeya zeae strain EC1 was recently shown to produce a new type of phytotoxins designated as zeamine and zeamine II, which are potent wide-spectrum antibiotics against Gram-positive and Gram-negative bacterial pathogens, suggesting their promising potential as clinical medicines. In this study, the optimized medium composition and culture conditions for biosynthesis of novel antibiotics zeamines have been established by using response surface methodology, largely increasing the yield of zeamines from original about 7.35 µg·mL⁻¹ in minimal medium to about 150 µg·mL⁻¹ in LS5 medium. The study identified the major factors contributing to zeamines production, which include nitrate, sucrose, asparaginate, mineral elements Mg²⁺ and K⁺, and optimized amount of phosphate. In addition, the results showed that overexpression of zmsK in D. zeae strain EC1 could further increase zeamines yield to about 180 µg·mL⁻¹ in LS5 medium. The findings from this study could facilitate further characterization and utilization of these two novel antibiotics, and also provide useful clues for understanding the regulatory mechanisms that govern D. zeae virulence.     

The complete genome sequence of Dickeya zeae EC1 reveals substantial divergence from other Dickeya strains and species

Background

Dickeya zeae is a bacterial species that infects monocotyledons and dicotyledons. Two antibiotic-like phytotoxins named zeamine and zeamine II were reported to play an important role in rice seed germination, and two genes associated with zeamines production, i.e., zmsA and zmsK, have been thoroughly characterized. However, other virulence factors and its molecular mechanisms of host specificity and pathogenesis are hardly known.

Results

The complete genome of D. zeae strain EC1 isolated from diseased rice plants was sequenced, annotated, and compared with the genomes of other Dickeya spp.. The pathogen contains a chromosome of 4,532,364 bp with 4,154 predicted protein-coding genes. Comparative genomics analysis indicates that D. zeae EC1 is most co-linear with D. chrysanthemi Ech1591, most conserved with D. zeae Ech586 and least similar to D. paradisiaca Ech703. Substantial genomic rearrangement was revealed by comparing EC1 with Ech586 and Ech703. Most virulence genes were well-conserved in Dickeya strains except Ech703. Significantly, the zms gene cluster involved in biosynthesis of zeamines, which were shown previously as key virulence determinants, is present in D. zeae strains isolated from rice, and some D. solani strains, but absent in other Dickeya species and the D. zeae strains isolated from other plants or sources. In addition, a DNA fragment containing 9 genes associated with fatty acid biosynthesis was found inserted in the fli gene cluster encoding flagellar biosynthesis of strain EC1 and other two rice isolates but not in other strains. This gene cluster shares a high protein similarity to the fatty acid genes from Pantoea ananatis.

Conlusion

Our findings delineate the genetic background of D. zeae EC1, which infects both dicotyledons and monocotyledons, and suggest that D. zeae strains isolated from rice could be grouped into a distinct pathovar, i.e., D. zeae subsp. oryzae. In addition, the results of this study also unveiled that the zms gene cluster presented in the genomes of D. zeae rice isolates and D. solani strains, and the fatty acid genes inserted in the fli gene cluster of strain EC1 were likely derived from horizontal gene transfer during later stage of bacterial evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1545-x) contains supplementary material, which is available to authorized users.     

Cyt toxin expression reveals an inverse regulation of insect and plant virulence factors of Dickeya dadantii

The plant pathogenic bacteria Dickeya dadantii is also a pathogen of the pea aphid Acyrthosiphon pisum. The genome of the bacteria contains four cyt genes, encoding homologues of Bacillus thuringiensis Cyt toxins, which are involved in its pathogenicity to insects. We show here that these genes are transcribed as an operon, and we determined the conditions necessary for their expression. Their expression is induced at high temperature and at an osmolarity equivalent to that found in the plant phloem sap. The regulators of cyt genes have also been identified: their expression is repressed by H‐NS and VfmE and activated by PecS. These genes are already known to regulate plant virulence factors, but in an opposite way. When tested in a virulence assay by ingestion, the pecS mutant was almost non‐pathogenic while hns and vfmE mutants behaved in the same way as the wild‐type strain. Mutants of other regulators of plant virulence, GacA, OmpR and PhoP, that do not control Cyt toxin production, also showed reduced pathogenicity. In an assay by injection of bacteria, the gacA strain was less pathogenic but, surprisingly, the pecS mutant was slightly more virulent. These results show that Cyt toxins are not the only virulence factors required to kill aphids, and that these factors act at different stages of the infection. Moreover, their production is controlled by general virulence regulators known for their role in plant virulence. This integration could indicate that virulence towards insects is a normal mode of life for D. dadantii.     

Genetic analysis of two phosphodiesterases reveals cyclic diguanylate regulation of virulence factors in Dickeya dadantii

Cyclic diguanylate (c‐di‐GMP) is a second messenger implicated in the regulation of various cellular properties in several bacterial species. However, its function in phytopathogenic bacteria is not yet understood. In this study we investigated a panel of GGDEF/EAL domain proteins which have the potential to regulate c‐di‐GMP levels in the phytopathogen Dickeya dadantii 3937. Two proteins, EcpB (contains GGDEF and EAL domains) and EcpC (contains an EAL domain) were shown to regulate multiple cellular behaviours and virulence gene expression. Deletion of ecpB and/or ecpC enhanced biofilm formation but repressed swimming/swarming motility. In addition, the ecpB and ecpC mutants displayed a significant reduction in pectate lyase production, a virulence factor of this bacterium. Gene expression analysis showed that deletion of ecpB and ecpC significantly reduced expression of the type III secretion system (T3SS) and its virulence effector proteins. Expression of the T3SS genes is regulated by HrpL and possibly RpoN, two alternative sigma factors. In vitro biochemical assays showed that EcpC has phosphodiesterase activity to hydrolyse c‐di‐GMP into linear pGpG. Most of the enterobacterial pathogens encode at least one T3SS, a major virulence factor which functions to subvert host defences. The current study broadens our understanding of the interplay between c‐di‐GMP, RpoN and T3SS and the potential role of c‐di‐GMP in T3SS regulation among a wide range of bacterial pathogens.     

Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 biocontrol Dickeya soft rot diseases by quenching virulence factor modulating quorum sensing signal

Virulence factor modulating (VFM) is a quorum sensing (QS) signal shared by and specific to Dickeya bacteria, regulating the production of plant cell wall degrading enzymes (PCWDEs) and virulence of Dickeya. High polarity and trace of VFM signal increase the difficulty of signal separation and structure identification, and thus limit the development of quorum quenching strategy to biocontrol bacterial soft rot diseases caused by Dickeya. In order to high-throughput screen VFM quenching bacteria, a vfmE-gfp biosensor VR2 (VFM Reporter) sensitive to VFM signal was first constructed. Subsequently, two bacterial strains with high quenching efficiency were screened out by fluorescence intensity measurement and identified as Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 using multilocus sequence analysis (MLSA). L5 and L95 supernatants reduced the expression of vfm genes, and both strains also decreased the production of PCWDEs of D. zeae MS2 and significantly reduced the virulence of D. oryzae EC1 on rice seedlings, D. zeae MS2 on banana seedlings, D. dadantii 3937 on potato and D. fangzhongdai CL3 on taro. Findings in this study provide a method to high-throughput screen VFM quenching bacteria and characterize novel functions of P. chlororaphis and E. asburiae in biocontrolling plant diseases through quenching VFM QS signal.     

Evolutionary history of the OmpR/IIIA family of signal transduction two component systems in Lactobacillaceae and Leuconostocaceae

Background  

Two component systems (TCS) are signal transduction pathways which typically consist of a sensor histidine kinase (HK) and a response regulator (RR). In this study, we have analyzed the evolution of TCS of the OmpR/IIIA family in Lactobacillaceae and Leuconostocaceae, two families belonging to the group of lactic acid bacteria (LAB). LAB colonize nutrient-rich environments such as foodstuffs, plant materials and the gastrointestinal tract of animals thus driving the study of this group of both basic and applied interest.     

Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System

Two-component signal transduction systems (TCS) are used by bacteria to sense and respond to their environment. TCS are typically composed of a sensor histidine kinase (HK) and a response regulator (RR). The Vibrio cholerae genome encodes 52 RR, but the role of these RRs in V. cholerae pathogenesis is largely unknown. To identify RRs that control V. cholerae colonization, in-frame deletions of each RR were generated and the resulting mutants analyzed using an infant mouse intestine colonization assay. We found that 12 of the 52 RR were involved in intestinal colonization. Mutants lacking one previously uncharacterized RR, VCA0566 (renamed VxrB), displayed a significant colonization defect. Further experiments showed that VxrB phosphorylation state on the predicted conserved aspartate contributes to intestine colonization. The VxrB regulon was determined using whole genome expression analysis. It consists of several genes, including those genes that create the type VI secretion system (T6SS). We determined that VxrB is required for T6SS expression using several in vitro assays and bacterial killing assays, and furthermore that the T6SS is required for intestinal colonization. vxrB is encoded in a four gene operon and the other vxr operon members also modulate intestinal colonization. Lastly, though ΔvxrB exhibited a defect in single-strain intestinal colonization, the ΔvxrB strain did not show any in vitro growth defect. Overall, our work revealed that a small set of RRs is required for intestinal colonization and one of these regulators, VxrB affects colonization at least in part through its regulation of T6SS genes.     

Articulospora sp. produces Art1, an inhibitor of bacterial histidine kinase

A two-component system (TCS) comprising a histidine kinase (HK) sensor and a response regulator (RR) plays important roles in regulating the virulence of many pathogenic bacteria. We used a new screening method to isolate novel inhibitor Art1 against bacterial sensory HK from an acetone extract of solid cultures of Articulospora sp., an aquatic hypomycete. Art1 inhibited the ATP-dependent autophosphorylation of recombinant glutathione S-transferase-fusion protein SasA, a cyanobacterial HK, with an IC50 value of 9.5 microg/ml.     

Building interacting partner predictors using co-varying residue pairs between histidine kinase and response regulator pairs of 48 bacterial two-component systems

The two‐component system (TCS) is a signal transduction system that involves a histidine kinase (HK) and a response regulator (RR). Although up to hundreds of TCSs may operate in parallel in a bacterial cell, the high‐fidelity of a TCS signaling is well maintained, minimizing irrelevant crosstalk between TCSs. When a HK gene and a RR gene in a given TCS system exist in neighboring positions, it is almost certain that their protein products (i.e., HK and RR) are interacting partners. However, large bacterial genomes often have multiple HK genes and/or cognate RR genes that are not neighboring positions. In many partially assembled genomes, some HK genes and RR genes belong to different contigs. In these cases, it is not clear which HK(s) and RR(s) interact. By combining information‐theoretic and graph‐theoretic approaches, we developed a computational method identifying co‐evolving residue pairs between HKs and cognate RRs and predicting the interacting HK:RR pairs for each TCS. In addition, we built a TCSppWWW webserver ( http://compath.org/platcom/tcs ) that takes query sequences of pairing candidates and predicts their HK:RR pairing using precomputed models. The current release of TCSppWWW provides predictors for 48 TCSs using over 20,000 protein sequences from about 900 bacterial genomes. Three different types of predictors using Random Forest, RBF Network, and Naïve Bayes are provided. Once a set of HK and RR candidate sequences are submitted, TCSppWWW aligns query sequences to the precomputed multiple sequence alignment of HK:RR pairs, extracts co‐evolving column positions, then returns prediction results with prediction margin and additional information. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Interplay of classic Exp and specific Vfm quorum sensing systems on the phenotypic features of Dickeya solani strains exhibiting different virulence levels

Bacteria from the genus Dickeya cause severe symptoms on numerous economically important plants. Dickeya solani is the Dickeya species most frequently found on infected potato plants in Europe. D. solani strains from different countries show high genetic homogeneity, but significant differences in their virulence level. Dickeya species possess two quorum sensing (QS) mechanisms: the Exp system based on classic N‐acyl‐homoserine lactone (AHL) signals and a specific system depending on the production and perception of a molecule of unknown structure, Virulence Factor Modulating (VFM). To study the interplay between these two QS systems, five D. solani strains exhibiting different virulence levels were selected. Mutants were constructed by inactivating genes coding for each QS system. Double mutants were obtained by simultaneous inactivation of genes coding for both QS systems. Most of the D. solani mutants showed an attenuation of chicory maceration and a decreased production of plant cell wall‐degrading enzymes (PCWDEs) and motility, but to different degrees depending on the strain. The VFM‐QS system seems to regulate virulence in both D. solani and Dickeya dadantii, but the AHL‐QS system has greater effects in D. solani than in D. dadantii. The inactivation of both QS systems in D. solani did not reveal any additive effect on the tested features. The inactivation of vfm genes generally has a more dominant effect relative to that of exp genes. Thus, VFM‐ and AHL‐QS systems do not work in synergy to modulate the production of diverse virulence factors and the ability to macerate plant tissue.     

The nucleoid‐associated protein Fis directly modulates the synthesis of cellulose,an essential component of pellicle–biofilms in the phytopathogenic bacterium Dickeya dadantii

Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface‐associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle–biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase‐regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid‐associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co‐ordinate basic metabolism, virulence and modifications of lifestyle.     

Dickeya dadantii pectic enzymes necessary for virulence are also responsible for activation of the Arabidopsis thaliana innate immune system

Soft‐rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions. We focused on the production of AtrbohD‐dependent reactive oxygen species (ROS), callose deposition and cell death as indicators of these reactions. We observed a significant reduction in ROS and callose formation with a bacterial mutant in which genes encoding five pectate lyases (Pels) were disrupted. Treatment of plant leaves with bacterial culture filtrates containing Pels resulted in ROS and callose production, and both reactions were dependent on a functional AtrbohD gene. ROS and callose were produced in response to treatment with a cellular fraction of a T2SS‐negative mutant grown in a Pels‐inducing medium. Finally, ROS and callose were produced in leaves treated with purified Pels that had also been shown to induce the expression of jasmonic acid‐dependent defence genes. Pel catalytic activity is required for the induction of ROS accumulation. In contrast, cell death observed in leaves infected with the wild‐type strain appeared to be independent of a functional AtrbohD gene. It was also independent of the bacterial production of pectic enzymes and the type III secretion system (T3SS). In conclusion, the work presented here shows that D. dadantii is recognized by the A. thaliana innate immune system through the action of pectic enzymes secreted by bacteria at the site of infection. This recognition leads to AtrbohD‐dependent ROS and callose accumulation, but not cell death.