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Here we report on the synthesis and structural characterization of the dithallium(III)-containing 30-tungsto -4-phosphate [Tl2Na2(H2O)2{P2W15O56}2]16- (1) by a multitude of solid-state and solution techniques. Polyanion 1 comprises two octahedrally coordinated Tl3+ ions sandwiched between two trilacunary {P2W15} Wells-Dawson fragments and represents only the second structurally characterized, discrete thallium-containing polyoxometalate to date. The two outer positions of the central rhombus are occupied by sodium ions. The title polyanion is solution-stable as shown by 31P and 203/205Tl NMR. This was also supported by Tl NMR spectra simulations including several spin systems of isotopologues with half-spin nuclei (203Tl, 205Tl, 31P, 183W). 23Na NMR showed a time-averaged signal of the Na+ counter cations and the structurally bonded Na+ ions. 203/205Tl NMR spectra also showed a minor signal tentatively attributed to the trithallium-containing derivative [Tl3Na(H2O)2(P2W15O56)2]14-, which could also be identified in the solid state by single-crystal X-ray diffraction. The bioactivity of polyanion 1 was also tested against bacteria and Leishmania.

The production and extraction of antibacterial substances from Phaeodactylum tricornutum biomass were tested on a pilot scale in a photo-bioreactor greenhouse installation. Using column chromatography two antibacterial active fractions were obtained from the algal biomass: a pure fraction of eicosapentaenoic acid (EPA) and a potentially new antibacterial substance(s) (identified as fraction B) produced by P. tricornutum. Both fractions exhibited antibacterial activities against three human pathogens: Vibrio vulnificus, V. parahaemolyticus, and V. cholerae. Minimal inhibitory concentrations against Bacillus subtilis of active fractions and one antibacterial commercial product were determined revealing a higher antibacterial activity of EPA in comparison to the commercial product. Consequently, EPA as well as the raw algal biomass with both EPA and the fraction B compounds could be used as food additives and feeds in aquaculture, poultry, and livestock breeding. Two types of phytohormones, methyl jasmonate and salicylic acid, as well as one phytotoxin, coronatine, were tested for induction of the defense system of P. tricornutum. However, only a minor change in the fatty acids profile and antibacterial effects of the treated cultures indicated that P. tricornutum must employ another strategy to regulate its antibacterial defense system.

Key cocoa-specific aroma precursors are generated during the fermentation of cocoa beans via the proteolysis of the vicilin-like globulin. Previous studies had shown that degradation of this particular 566 amino acid-long storage protein leads to three distinct subunits with different molecular masses. Although oligopeptides generated from the proteolysis of vicilin-like globulin have been studied previously, changes occurring to vicilin at different stages of fermentation have not yet been explored in detail. The aim of this study was to investigate the fate of vicilin protein from the non-fermented stage up to the dried cocoa beans. Our results showed a remarkable shift in the electrophoretic mobility of vicilin towards higher pI during the onset of fermentation. The pI-shifted subunit was found susceptible to further degradation into a lower-molecular-weight vicilin subunit. The observed pI shift correlated with, but did not depend on protein phosphorylation. Glycosylation of some but not all vicilin subunits occurred at different stages of the fermentation process. Peptides generated from vicilin throughout fermentation were analyzed by UHPLC-ESI-MS/MS revealing an initial increase and subsequent decrease in the diversity of peptides with an increasing degree of fermentation. We furthermore describe the rate of degradation of different vicilin subunits. The detected diversity and dynamics of vicilin peptides will help to define biochemical markers of distinct steps of the fermentation process.

A liquid chromatography-mass spectrometry-based method for the quantification of intracellular concentrations of dietary phenolics in bacteria was developed. Using this assay, the time-dependent uptake of quercetin and two of its glycosides into gut microbiota model organisms (Escherichia coli and Bifidobacterium bifidum) under aerobic and anaerobic conditions was studied. For the first time, quantitative data on the uptake of dietary phenols were obtained indicating a time-dependent differential uptake of the dietary compounds. The shape of the uptake curve and the comparative use of bacterial mutants lacking porins OmpFC or the multi-drug efflux pump AcrAB, respectively, along with the identification and quantification of selected bacterial metabolites provided a mechanistic insight into the uptake process.

Cocoa bean fermentation still remains a rather empirical process. The research presented here employed an artificial system of fermentation, using controlled incubations, in order to achieve greater control over the external influences that cocoa beans are exposed to, with the aim of experimentally modelling changes to bean components (responses). Experimental design was used, in a first-ever attempt, to study the effects of five factors and their interactions on the profiles of pH, peptides, and flavanols in the bean during the incubations. Temperature, incubation time and the concentration of acetic acid were the main factors influencing the three responses. Moreover, there was a significant amount of factor interaction, revealing the process to be more complex than initially thought, especially with respect to the role of ethanol. Using the model, one was also able to accurately predict the response of the bean to the exposure to specific factors.

This study encompassed the lab-scale fermentation of cocoa beans in 300-g heaps under controlled laboratory conditions, in order to replicate the microbial dynamics and metabolomic changes that usually occur in large-scale spontaneous fermentations. Growth profiles of yeast and acetic acid bacteria (AAB) with the native assortment of microbes as well as with the use of a starter culture were very similar to those observed in literature. Greater production of acetic acid by AAB not only led to more acidic-tasting liquor but also contributed to bitterness, due to polyphenol preservation. It also brought about a drastic drop in pH leading to greater proteolytic activity. Peptides generated through proteolysis also showed incredible similarity to those reported in literature, in particular, those speculated to be involved in cocoa-specific flavour. A closer look at the naturally occurring peptide repertoires of our fermentation trials, generated by the breakdown of cocoa storage protein, pointed to a potential peptide responsible for cocoa-specific aroma.

Background Marine diatoms have a higher fucoxanthin content in comparison to macroalgae. Fucoxanthin features many potent bioactive properties, particularly anti-obesity properties. Despite the great potential for harvesting larger amounts of fucoxanthin, the impacts of light quality (light source, intensity, and photoperiod) on fucoxanthin production and the essential proteins involved in fucoxanthin biosynthesis in marine diatoms remain unclear. Results In the present study, Cylindrotheca closterium was selected from four different species of diatoms based on its high fucoxanthin content and productivity. Optimal light conditions (light source, intensity, and regime) were determined by a “Design of Experiment” approach (software MODDE Pro 11 was used). The model indicated that an 18/6 light/darkness regime increased fucoxanthin productivity remarkably as opposed to a 12/12 or 24/0 regime. Eventually, blue light-emitting diode light, as an alternative to fluorescent light, at 100 μmol/m2/s and 18/6 light/darkness regime yielded maximum fucoxanthin productivity and minimal energy consumption. The fucoxanthin production of C. closterium under the predicted optimal light conditions was assessed both in bottle and bag photobioreactors (PBRs). The high fucoxanthin content (25.5 mg/g) obtained from bag PBRs demonstrated the feasibility of large-scale production. The proteomes of C. closterium under the most favorable and unfavorable fucoxanthin biosynthesis light/darkness regimes (18/6 and 24/0, respectively) were compared to identify the essential proteins associated with fucoxanthin accumulation by matrix-assisted laser desorption/ionization-time of flight–mass spectrometry. Six proteins that were up-regulated in the 18/6 regime but down-regulated in the 24/0 were identified as important chloroplastic proteins involved in photosynthesis, energy metabolism, and cellular processes. Conclusions Blue light-emitting diode light at 100 μmol/m2/s and 18/6 light/darkness regime induced maximum fucoxanthin productivity in C. closterium and minimized energy consumption. The high fucoxanthin production in the bag photobioreactor under optimal light conditions demonstrated the possibility of commercialization. Proteomics suggests that fucoxanthin biosynthesis is intimately associated with the photosynthetic efficiency of the diatom, providing another technical and bioengineering outlook on fucoxanthin enhancement. Electronic supplementary material The online version of this article (10.1186/s12934-018-0957-0) contains supplementary material, which is available to authorized users.

The nutritional value of cocoa butter is mainly determined by the composition of triacylglycerols (TAGs). In this paper we have developed a non-aqueous reversed-phase liquid chromatographic method, using ethanol as the mobile phase, coupled to electrospray ionization (ESI) tandem mass spectrometry to identify TAGs in raw cocoa beans from six different origins. Tandem mass spectrometry was adopted to facilitate the identification of TAGs using unique diacylglycerol product ions and neutral losses. Additionally, two-dimensional m/z retention time maps aided the identification of entire homologous series of TAGs. A total of 83 different TAGs were identified in unfermented cocoa beans, 58 of which were not previously reported in cocoa. Thirty-one of these compounds represent a new class of TAGs characterized by the presence of one to three hydroxyl groups on the unsaturated fatty acid chain. To date, this represents the largest number of TAGs identified in cocoa.

Background: Ziziphus spina-christi is a tropical evergreen tree of Sudanese origin. The plant is commonly used in ethno-medicine for the treatment of many diseases such as malaria, digestive disorders, weakness, hepatic diseases, obesity, urinary problems, diabetes, skin infections, fever, diarrhea and insomnia. However, the pharmaceutical properties and bioactive compounds present in Z. spina-christi are still unknown. Therefore, the aim of the study was to significantly improve and deepen our knowledge about the phenolic composition of Z. spina-christi leaf extracts as well as their antibacterial and potential cytotoxic effects. Methods: The phytochemical profile of the known compounds in the methanol/water (7:3 v/v) extract of Z. spinachristi leaves were tentatively assigned on the basis of reported analytical data from three to four pieces of independent methods including retention time (tR), UV/Vis spectroscopy, high resolution mass spectrometry (HR-MS) and tandem mass spectrometry (MSn). Four bioactive compounds were purified via preparative-HPLC and identified by means of HPLC-ESI-TOF, HPLC-ESI-MSn, 1H-NMR, 13C-NMR, literature data and authentic reference standards. Antibacterial activities of aqueous and methanol leaf extracts were carried out with six bacterial strains (Bacillus subtilis, Bacillus aquimaris, Clavibacter michiganensis, Escherichia coli, Erwinia amylovora, and Pseudomonas syringae) using an agar diffusion assay. Cytotoxic effects towards the human epidermal keratinocyte cell line HaCaT and rat intestine epithelial IEC-6 cells was assess by cytoskeletal and plasma membrane integrity or analyses of mitochondrial and proliferative activities. Results: We tentatively assigned fifty-seven phenolic compounds (fifty-two of them are known) to their regioisomeric level in the methanol/water extract of Z. spina-christi leaves. To our knowledge 45 of them were not reported previously in Z. spina-christi and five for the first time in nature. Highly glycosylated flavonoids, proanthocyanidins, and chlorogenic acids were identified as the major components. Preparative-HPLC let to the isolation of four known phytochemicals including quercetin 3-O-(6-O-rhamnosyl-glucoside) (rutin) 26, quercetin 3-O-(2-O-rhamnosyl-arabinoside) 27, phloretin 3',5' di-C-glucoside 39 and quercetin 3-O-(2,6-di-O-rhamnosyl-glucoside) 50. Regarding the antimicrobial assays, the methanol extracts of the plant were found to show higher activity than the aqueous extracts indicating hydrophobic substances of Ziziphus leaves exerting antibacterial activity. The extracts revealed no cytotoxic effects towards the HaCaT or IEC-6 cells. Conclusions: Several previously known and unknown compounds were indentified in Z. spina-christi leaf extracts. The plant possesses strong antimicrobial activity and no toxicity. Our results therefore suggest that Z. spina-christi extracts might be potentially suitable for topical applications and support the use of the plant in traditional medicine to treat infectious diseases.

Several plant pathogenic bacteria belonging to the species Pseudomonas syringae produce the phytotoxin coronatine to enhance their virulence. Pseudomonas syringae pv. glycinea PG4180 synthesizes coronatine at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C, its optimal growth temperature. In contrast, temperature has virtually no effect on coronatine synthesis in P. syringae pv. tomato strain DC3000. A modified two-component system controlling coronatine synthesis and consisting of the histidine protein kinase (HPK), CorS, the response regulator, CorR, and a third essential component, CorP, had been identified previously in both strains. CorS had been identified previously as a potential thermo-sensor. Comparison of the amino acid sequences of the HPKs from the two organisms revealed distinct differences. Site-directed mutagenesis of CorS from PG4180 was used to identify amino acyl residues potentially important for temperature signal perception. Point mutations and combinations of these were introduced into corS of PG4180 to generate corS variants with increased similarities to the respective allele from strain DC3000. These mutations resulted in either loss of activity, increase of thermoresponsiveness, or had no effect on CorS activity. Although none of the introduced mutations resulted in a clear conversion of CorS activity from thermo-responsive to temperature-independent, amino acyl residues important for temperature-dependent CorS activity and coronatine biosynthesis were identified.

Coronafacic acid, the polyketide component of the phytotoxin coronatine, is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the coronafacate ligase (cfl) gene product. In the present study, cfl was fused to the carboxy terminus of malE, which encodes the maltose-binding protein (MBP), and overexpressed in Escherichia coli. Immunoblot analysis indicated that Cfl contained an ATP-binding region, a motif conserved in enzymes which activate their substrates by adenylation. MBP-Cfl was overproduced and purified from Pseudomonas syringae and the protein fusion was used to generate antisera. Anti-MBP-Cfl antibodies and a transcriptional fusion of the cfl promoter to a promoterless glucuronidase gene were used to follow the temporal expression of coronafacate ligase. The results indicated that transcription of cfl is temperature-sensitive. Furthermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.

Plant-pathogenic bacteria may sense variations in environmental factors, such as temperature, to adapt to plant-associated habitats during pathogenesis or epiphytic growth. The bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, preferentially produces the phytotoxin coronatine at 18 degrees C and infects the host plant under conditions of low temperature and high humidity. A miniTn5-based promoterless glucuronidase (uidA) reporter gene was used to identify genetic loci of PG4180 preferentially expressed at 18 or 28 degrees C. Out of 7500 transposon mutants, 61 showed thermoregulated uidA expression as determined by a three-step screening procedure. Two-thirds of these mutants showed an increased reporter gene expression at 18 degrees C whilst the remainder exhibited higher uidA expression at 28 degrees C. MiniTn5-uidA insertion loci from these mutants were subcloned and their nucleotide sequences were determined. Several of the mutants induced at 18 degrees C contained the miniTn5-uidA insertion within the 32.8 kb coronatine biosynthetic gene cluster. Among the other mutants with increased uidA expression at 18 degrees C, insertions were found in genes encoding formaldehyde dehydrogenase, short-chain dehydrogenase and mannuronan C-5-epimerase, in a plasmid-borne replication protein, and in the hrpT locus, involved in pathogenicity of P. syringae. Among the mutants induced at 28 degrees C, insertions disrupted loci with similarities to a repressor of conjugal plasmid transfer, UV resistance determinants, an isoflavanoid-degrading enzyme, a HU-like DNA-binding protein, two additional regulatory proteins, a homologue of bacterial adhesins, transport proteins, LPS synthesis enzymes and two proteases. Genetic loci from 13 mutants did not show significant similarities to any database entries. Results of plant inoculations showed that three of the mutants tested were inhibited in symptom development and in planta multiplication rates. Temperature-shift experiments suggested that all of the identified loci showed a rather slow induction of expression upon change of temperature.

Coronatine (COR) is a nonhost-specific phytotoxin that substantially contributes to the virulence of several pathovars (pvs.) of Pseudomonas syringae. The COR gene cluster in P. syringae is generally plasmid-encoded in pvs. atropurpurea, glycinea, morsprunorum, and tomato but chromosomally encoded in pv. maculicola. In the present study, we investigated whether the COR plasmids in four pathovars shared other traits including self-transmissibility, conserved oriV/par loci, and insertion sequences (ISs) known to reside on other plasmids in P. syringae. Three COR plasmids were shown to be self-transmissible, and all COR plasmids shared a related oriV/par region. Two COR plasmids hybridized to IS801, an IS element widely distributed in P. syringae. Further analysis of p4180A, a 90-kb COR plasmid in P. syringae pv. glycinea, indicated that multiple copies of IS801 were present on this plasmid, and all copies mapped outside the COR gene cluster. Sequence analysis of the region adjacent to the COR gene cluster in p4180A indicated the presence of additional IS elements including IS870, IS51, and IS1240. The IS elements borne on p4180A may have contributed to horizontal transfer of the COR gene cluster and the evolution of the COR biosynthetic pathway.

A modified two-component regulatory system consisting of the histidine protein kinase CorS and two highly homologous response regulators, CorR and CorP, controls biosynthesis of the polyketide phytotoxin coronatine (COR) by Pseudomonas syringae pv. glycinea PG4180 in a temperature-dependent manner. COR synthesis is maximal at 18 degrees C but does not occur at 28 degrees C. Fusions of CorR and CorP to the maltose-binding protein (MBP) were overproduced in Escherichia coli and P. syringae PG4180, and tested for functionality by complementation of corR and corP mutants of PG4180, respectively. The cmaABT promoter region was defined by deletion mapping, and the DNA-binding capability of CorR and CorP was examined by gel retardation assays. When overproduced in P. syringae at 18 degrees C and purified, MBP-CorR was shown to bind specifically to a 218-bp DNA fragment corresponding to positions -841 to -623 bp upstream of the transcriptional start site of the cmaABT operon. In contrast, MBP-CorP and MBP itself, when overproduced in P. syringae and E. coli at 18 degrees C and 28 degrees C, respectively, did not bind to the 218-bp fragment or to any other DNA fragment analyzed. The CorP protein lacks typical DNA-binding motifs, suggesting that it might modulate the function of CorR. However, addition of purified MBP-CorP did not alter the DNA-binding activity of MBP-CorR. On the other hand, this activity was completely abolished when MBPCorR was overproduced at 28 degrees C or in a corS mutant, indicating that the binding of CorR depended on the growth temperature at which it was produced and was controlled by CorS. In addition, overproduction of MBP-CorR in a corP mutant of PG4180 also yielded inactive protein, underlining the importance of CorP for CorR activation. We propose that CorR is activated by CorS at low temperature and that CorP is required for this activation before CorR can bind to DNA.

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae . The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3′ end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated cfa8 , cfa9 , and tnp1 ; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of cfa8 and cfa9 were overproduced in Escherichia coli BL21; however, tnp1 was not translated in these experiments. Mutagenesis and complementation analysis indicated that cfa8 is required for the production of CFA and COR. Analysis of a cfa9 mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tnp1 , however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a P. syringae strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.

The plant-pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18°C, whereas no detectable toxin is produced at 28°C. Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression of cmaB , which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for the cmaABT operon during bacterial growth at 18 and 28°C. Promoter activity and transcription rates were maximal when cells were incubated at 18°C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18°C than at 28°C. Western blot analysis indicated that CmaB accumulated in the late stationary phase of P. syringae cultures grown at 18°C but not in cultures incubated at 28°C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18°C than at 28°C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.

(Received 25 September 1997/Accepted 12 November 1997)Several pathovars of Pseudomonas syringae produce the polyketide phytotoxin coronatine (COR). Inthe bacterial blight pathogen of soybean, P. syringae pv. glycinea PG4180, COR is produced at highlevels at 18 °C whereas no toxin is synthesized at 28 °C. Previously, activation of three promotersinside the COR biosynthetic gene cluster by a modified two-component regulatory system was shownto influence thermoregulation of COR biosynthesis. Using phenotypic determination of COR synthe-sis, a transcriptional reporter gene fusion, and Western blot analysis, we screened a representativenumber of natural isolates of P. syringae for effects of temperature on expression of cmaA, cmaB,and cmaT, which encode enzymes involved in the biosynthesis of COR. Thermoregulation ofcmaABT expression was frequent among the tested strains. However, intensities of the temperatureeffects varied widely. Coronatine sysnthesis was found to differ at up to six-fold among CORproducing strains. There was no strain which synthesized COR at 28 °C although some of themshowed increased basal cmaABT promoter activities at this temperature. Transcriptional fusionsbetween the cmaABT promoter and a promoterless reporter gene were found to be down regulated at28 °C only in COR producing strains but not in the non-producing strains tested. The geographicorigin of the bacterial strains did not influence the occurrence of temperature-dependent gene expres-sion.

Coronatine (COR) is a non-host-specific phytotoxin that is produced by several different pathovars in the species Pseudomonas syringae. COR consists of two distinct components: coronafacic acid (CFA), which is synthesized via the polyketide pathway, and coronamic acid (CMA), a cyclized derivative of isoleucine. Both CFA and CMA function as intermediates in the pathway to COR and must be joined together by an amide bond to form the phytotoxin. Although the mode of action for COR remains obscure, the CFA moiety is a structural and functional analogue of jasmonic acid, a compound that is produced in a variety of plants in response to stress. The COR biosynthetic gene cluster generally occurs on large plasmids in P. syringae, an observation that helps to explain the production of COR by multiple pathovars. Mutagenesis, feeding studies, and complementation analyses have been used to divide the COR biosynthetic gene cluster into functional regions. Nucleotide sequencing of the regions involved in CFA and CMA biosynthesis has revealed relatedness to genes encoding polyketide and peptide synthetases, respectively. The deduced amino acid sequence of the gene responsible for catalyzing amide bond formation between CMA and CFA shows relatedness to enzymes that activate cyclic carboxylic acids by adenylation. Coronatine biosynthesis has been shown to be temperature-sensitive and regulated by a modified two-component regulatory system.

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn 5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn 5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S -adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized ( K m , 7 mM; k cat , 85 min −1 ). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

The plant pathogenic bacterium Pseudomonas syringae PG4180 synthesizes high levels of the phytotoxin coronatine (COR) at the virulence-promoting temperature of 18 °C, but negligible amounts at 28 °C. Temperature-dependent COR gene expression is regulated by a modified two-component system, consisting of a response regulator, CorR, the histidine protein kinase CorS, and a third component, termed CorP. We analyzed at transcriptional and translational levels the expression of corS and the cma operon involved in COR biosynthesis after a temperature downshift from 28 to 18 °C. Expression of cma was induced within 20 min and increased steadily whereas corS expression was only slightly temperature-dependent. Accumulation of CmaB correlated with accumulation of cma mRNA. However, cma transcription was suppressed by inhibition of de novo protein biosynthesis. A transcriptional fusion of the cma promoter to a promoterless egfp gene was used to monitor the cma expression in vitro and in planta. A steady induction of cma::egfp by temperature downshift was observed in both environments. The results indicate that PG4180 responds to a temperature decrease with COR gene expression. However, COR gene expression and protein biosynthesis increased steadily, possibly reflecting adaptation to long-term rather than rapid temperature changes.

Two closely related phytopathogenic bacterial strains, Pseudomonas syringae pv. glycinea PG4180 and P. syringae pv. tomato DC3000, produce the chlorosis-inducing phytotoxin coronatine (COR) in a remarkably divergent manner. PG4180 produces COR at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C. In contrast, temperature has no effect on COR synthesis in DC3000. A modified two-component system consisting of the histidine protein kinase (HPK), CorS, the response regulator (RR), CorR, and a third component, CorP, governs COR biosynthesis in both strains. A plasmid-based component and domain swapping approach was used to introduce different combinations of RRs, HPKs and hybrid HPKs into corS mutants of both strains. Subsequently, expression levels of the COR biosynthetic cma operon were determined using RNA dot-blot analysis, suggesting that CorRSP of PG4180 mediates a thermoresponsive phenotype dependent on the genomic background of each strain. The reciprocal experiment demonstrated a loss of temperature dependence in the corS mutant of PG4180. The presence of corR from PG4180 led to more pronounced cma expression in DC3000 and was associated with thermoresponsiveness, while corS of PG4180 did not mediate a temperature-dependent phenotype in the DC3000 mutant containing native corR and corP. These findings were substantiated by RT-PCR experiments. The C-terminal domain of CorS of PG4180 mediated thermosensing, while the N terminus did not respond to temperature changes, suggesting cytosolic perception of the temperature signal.

Exopolysaccharides play important roles in attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and the impact of the exopolysaccharides, levan and alginate, on biofilm formation, we compared biofilms of Pseudomonas syringae PG4180 strains with different exopolysaccha- ride patterns. The mucoid strain PG4180 (PG4180.muc), which produced levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow-chambers. We applied confocal laser scanning microscopy (CLSM) with fluorescently labelled lectins to investigate the spatial distribution of levan and an additional yet-unknown exopolysaccharide in flow chamber biofilms. The lectin Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centers of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose. Production of a yet-to-be characterized additional exopolysaccharide might be more important for biofilm formation than the syntheses of levan and alginate.

The plant pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180 synthesizes high levels of the phytotoxin coronatine (COR) at the virulence- promoting temperature of 18°C, but only low amounts at 28°C, the optimal growth temperature. The temperature-dependent COR gene expression is regulated by a modified two-component system, consisting of the histidine protein kinase CorS, a response regulator, CorR, and a third functionally essential protein, CorP. We ana- lyzed at the transcriptional and translational level the expression of corS and the cma operon involved in COR biosynthesis, after a temperature downshift from 28°C to 18°C. The synthesis of cma mRNA was induced within 20 min and then increased steadily and gradually in the 14 h following the shift to 18°C. The synthe- sis of corS mRNA was induced to a lesser extent by the temperature downshift. The induction of cma expression was a result of accelerated transcription rather than increased stability of the cma transcript at 18°C. Accumulation of the COR bio- synthetic protein CmaB correlated with accumulation of cma mRNA. However, cma transcription was suppressed by inhibition of de novo protein biosynthesis.

Two-component systems provide a means for bacteria to sense and adapt to environmental signals in order to survive in a continuously changing environment. Understanding of the mechanism by which these systems function is important in combating bacterial infections because many bacterial two-component systems are associated with virulence. The plant pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180 synthesizes high levels of the phytotoxin coronatine at the virulence-promoting temperature of 18 degrees , but not at 28 degrees , the optimal growth temperature. Temperature-dependent coronatine biosynthesis is regulated by a modified two-component system, consisting of the response regulator, CorR, the histidine protein kinase CorS, and a third component, CorP. To elucidate the mechanism by which CorRSP functions, genetic, transcriptional, and biochemical analyses were applied, including in vitro and in planta reporter gene analysis, mRNA quantification, protein expression, mutagenesis, and membrane topology analysis. A combination of these techniques helped to elucidate, to a considerable extent, the temperature-sensing activity of CorS, which seems to act as a membrane-bound molecular thermometer.

Coronatine (COR) is a chlorosis-inducing phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Confocal laser scanning microscopy was used to investigate in vitro and in planta expression of COR genes by two model organisms, P. syringae pv. glycinea PG4180, a pathogen of soybean, and P. syringae pv. tomato DC3000, a pathogen of tomato and crucifers. Previously, it was shown in vitro that the cma operon involved in COR synthesis in PG4180 is expressed in a temperature-dependent manner, with maximal rates at 18°C and low activity at 28°C. However, nothing was known about the influence of temperature on the expression of COR biosynthetic genes in planta. Therefore, transcriptional fusions of the PG4180 and DC3000 cma promoter regions to a promoterless egfp gene were constructed and expressed in both P. syringae strains. The fluorescence patterns in response to temperature during growth of a strain in vitro were consistent with its COR production and the cma transcript abundance as revealed by RNA dot blot hybridization. Quantification of fluorescence indicated that cma promoter activity was dependent on the genetic background of the host strain. Expression of cma∷egfp in PG4180 was temperature-dependent in minimal medium as well as inside the plant tissue. In contrast, transcription of the cma operon was not significantly affected by temperature in DC3000. However, cells of DC3000 harboring the cma∷egfp fusions showed higher levels of fluorescence when recovered from infected host plants compared with cells grown in minimal medium. These results indicate that the signals for induction of COR biosynthesis differ significantly in PG4180 and DC3000.

A modified two-component regulatory system consisting of two response regulators, CorR and CorP, and the histidine protein kinase CorS, regulates the thermoresponsive production of the phytotoxin coronatine (COR) in Pseudomonas syringae PG4180. COR is produced at the virulence-promoting temperature of 18 °C, but not at 28 °C, the optimal growth temperature of PG4180. Assuming that the highly hydrophobic N-terminus of CorS might be involved in temperature-signal perception, the membrane topology of CorS was determined using translational phoA and lacZ fusions, leading to a topological model for CorS with six transmembrane domains (TMDs). Interestingly, three PhoA fusions located downstream of the sixth TMD showed a thermoresponsive phenotype. Enzymic activity, immunoblot, and protease-sensitivity assays were performed to localize the CorS derivatives, to analyse the expression level of hybrid proteins and to examine the model. In-frame deletions of the last four, or all six TMDs gave rise to non-functional CorS. The results indicated that the transmembrane region is important for CorS to function as a temperature sensor, and that the membrane topology of CorS might be involved in signal perception.

A modified two-component regulatory system consisting of two response regulators, CorR and CorP, and the histidine protein kinase CorS, regulates the thermoresponsive production of the phytotoxin coronatine (COR) in Pseudomonas syringae PG4180, which is the causal agent of bacterial blight of soybean. COR is produced at 18°C but not at 28°C. It is assumed that CorS responds to a temperature change and subsequently transducer the signal to its cognate response regulator(s), which in turn facilitates transcriptional activation of COR biosynthetic genes. Based on results of experiments with translational fusions between CorS and either alkaline phosphatase (PhoA) or (3-galactosidase (LacZ), CorS possesses six membrane-spanning domains and the location inside or outside of the membrane of certain regions of CorS is altered in dependence of temperature. The genome sequencing project of P. syringae DC3000 has revealed the presence of the CorRSP system in this strain as well. However, DC3000 produces significantly less COR than does PG4180, and the production is temperature-independent. We analysed COR production of various P. syringae strains at 18°C and 28°C and also deduced amino acid sequences of CorS from COR-producing strains. We suggest that PG4180-specific sequence alterations in the sequence of the temperature sensor kinase-encoding gene corS might be responsible for thermoresponsive production. We performed site-directed mutagenesis of CorS from PG4180 by introducing mutations identified in CorS of DC3000. Each of these mutations resulted in a change of the thermoresponsive phenotype for CorS of PG4180.

Coronatine (COR) is a chlorosis-inducing phytotoxin produced by the plant pathogenic bacterium Pseudomonas syringae. A variety of environmental factors affect COR production. Previously, it was shown in vitro that temperature had a highly significant effect on COR synthesis in P. s. pv. glycinea PG4180, with maximal production rates at 18°C and low activity at 28°C. However, little was known about the actual expression of biosynthetic genes required for production of COR in planta. Confocal laser scanning microscopy was used to investigate in vitro and in planta expression of COR genes by two model organisms, P. s. pv. glycinea PG4180, a pathogen of soybean, and P. s. pv. tomato DC3000, a pathogen of tomato. The results indicate that the signals for induction of COR biosynthesis differ in these strains. COR biosynthesis by P. s. pv. glycinea PG4180 was temperature-dependent in minimal medium as well as inside the plant tissue. However, transcription of COR biosynthetic genes was not significantly affected by temperature in P. s. pv. tomato DC3000. In contrast, DC3000 showed higher levels of COR gene expression when inoculated into host plants as compared to DC3000 grown in minimal medium.

Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight of soybean and produces the phytotoxin coronatine (COR) in a temperature-dependent manner. COR consists of a polyketide, coronafacic acid (CFA), and an amino acid derivative, coronamic acid, and is produced optimally at 18 degrees C whereas no detectable synthesis occurs at 28 degrees C. We investigated the impact of temperature on PG4180 during compatible and incompatible interactions with soybean and tobacco plants, respectively. After spray inoculation, PG4180 caused typical bacterial blight symptoms on soybean plants when the bacteria were grown at 18 degrees C prior to inoculation but not when derived from cultures grown at 28 degrees C. The disease outcome was quantified by determination of bacterial populations in planta. The temperature effect was not observed when PG4180 was artificially infiltrated into soybean leaves, indicating that the pre-inoculation temperature and phytotoxin synthesis were important for bacterial invasion via natural plant openings. In the incompatible interaction, PG4180 elicited the hypersensitive response (HR) on tobacco plants regardless of the bacterial pre-inoculation temperature. However, the HR was significantly delayed when tobacco plants were treated with cells of the CFA-overproducing derivative, PG4180.N9, which were derived from cultures grown at 18 degrees C, compared with parallels incubated at 28 degrees C. CFA biosynthesis by PG4180.N9 was optimal at 18 degrees C and negligible at 28 degrees C. The impact of CFA synthesis on the HR was studied with different growth media, mutants, and transconjugants of PG4180, indicating that the amount of synthesized CFA but not that of COR influenced the outcome of the HR. Feeding experiments with purified coronafacoyl compounds suggested that the observed delay of the HR was mediated by CFA, shedding further light on CFA's putative role as a molecular mimic of the plant signaling molecule, jasmonic acid.

Biosynthesis of the Phytotoxin coronatine (COR) in Pseudomonas syringae pv. glycinea PG4180 is regulated by temperature at the transcriptional level. A 3.4-kb DNA fragment from the coronatine biosynthetic gene cluster restored temperature-regulated Phytotoxin production to Tn5 mutants defective in COR production. Nucleotide sequence analysis of this fragment revealed three genes, corS, corP, and corR, which encode a modified two-component regulatory system consisting of one sensor protein, CorS, and two response regulator proteins, CorP and CorR, Although only one response regulator, CorR, had a DNA-binding domain, the phosphate-receiving domains of both response regulator proteins were highly conserved. Transcriptional fusions of the corP and corR promoters to a promoteriess glucuronidase (uidA) gene indicated that these two genes are expressed constitutiveiy at 18 and 28°C. In contrast, a corSv::uidA fusion exhibited the temperature-dependence previously observed for COR biosynthetic promoters and exhibited maximal transcriptional activity at 18°C and low activity at 28°C. Furthermore, glucuronidase activity for corSv::uidA was decreased in corP, corR, and corS mutants relative to the levels observed for PG4180(corS::uidA). This difference was not observed for corPv.uidA and corRv.uidA transcriptional fusions since expression of these fusions remained low and constitutive regardless of the genetic background. The three regulatory genes functioned in a COR background to achieve temperature-dependent activation of a COR biosynthetic promoter indicating that this triad of genes is the primary control for coronatine biosynthesis and responsible for thermoregulation. Our data suggest that the modified two-component regulatory system described in this study might transduce and amplify a temperature signal which results in transcriptional activation of COR biosynthetic genes.

Biosynthesis of the phytotoxin coronatine (COR) in Pseudomonas syringae pv. glycinea PG4180 is regulated by temperature at the transcriptional level. A 3.4-kb DNA fragment from the COR biosynthetic gene cluster restored temperature-regulated phytotoxin production to Tn5 mutants defective in COR production. Nucleotide sequence analysis of this fragment revealed three genes, corS, corP, and corR, which encode a modified two-component regulatory system consisting of one sensor protein, CorS, and two response regulator proteins, CorP and CorR. Although only one response regulator, CorR, had a DNA-binding domain, the phosphate-receiving domains of both response regulator proteins were highly conserved. Transcriptional fusions of the corP and corR promoters to a promoterless glucuronidase gene (uidA) indicated that these two genes are expressed constitutively at 18 and 28 degrees C. In contrast, a corS::uidA fusion exhibited the temperature dependence previously observed for COR biosynthetic promoters and exhibited maximal transcriptional activity at 18 degrees C and low activity at 28 degrees C. Furthermore, glucuronidase activity for corS::uidA was decreased in corP, corR, and corS mutants relative to the levels observed for PG4180(corS::uidA). This difference was not observed for corP::uidA and corR::uidA transcriptional fusions since expression of these fusions remained low and constitutive regardless of the genetic background. The three regulatory genes functioned in a P. syringae strain lacking the COR gene cluster to achieve temperature-dependent activation of an introduced COR biosynthetic promoter, indicating that this triad of genes is the primary control for COR biosynthesis and responsible for thermoregulation. Our data suggest that the modified two-component regulatory system described in this study might transduce and amplify a temperature signal which results in transcriptional activation of COR biosynthetic genes.

Coronafacic acid (CFA), the polyketide component of the phytotoxin coronatine (COR), is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the CFA ligase (cfl) gene product. The COR biosynthetic gene cluster in Pseudomonas syringae pv. glycinea PG4180 is located within a 32-kb region of a 90-kb plasmid designated p4180A. In the present study, a cloned region of p4180A complemented all CFA- mutants spanning an 18.8-kb region of the COR biosynthetic cluster. The genetic evidence presented in this study indicates that cfl and the CFA biosynthetic gene cluster are encoded by a single transcript and that transcription of all of the genes in this operon is directed by the cfl promoter. The cfl promoter was localized to a 0.37-kb region upstream of the transcriptional start site by progressive subcloning in pRG960sd, a vector containing a promoterless glucuronidase gene. Transcription of the cfl/CFA operon was temperature sensitive and showed maximal glucuronidase activity at 18 degrees C. Furthermore, transcription of the cfl/CFA operon was dependent on the functional activity of a modified two-component regulatory system located within the COR biosynthetic gene cluster. Thermoregulation of the cfl/CFA operon and the coronamic acid biosynthetic gene cluster via the modified two-component regulatory system is discussed.

Coronamic acid (CMA), an ethylcyclopropyl amino acid derived from isoleucine, functions as an intermediate in the biosynthesis of coronatine, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. The DNA required for CMA biosynthesis (6.9 kb) was sequenced, revealing three distinct open reading frames (ORFs) which share a common orientation for transcription. The deduced amino acid sequence of a 2.7-kb ORF designated cmaA contained six core sequences and two conserved motifs which are present in a variety of amino acid-activating enzymes, including nonribosomal peptide synthetases. Furthermore, CmaA contained a spatial arrangement of histidine, aspartate, and arginine residues which are conserved in the ferrous active site of some nonheme iron(II) enzymes which catalyze oxidative cyclizations. The deduced amino acid sequence of a 1.2-kb ORF designated cmaT was related to thioesterases of both procaryotic and eucaryotic origins. These data suggest that CMA assembly is similar to the thiotemplate mechanism of nonribosomal peptide synthesis. No significant similarities between a 0.9-kb ORF designated cmaU and other database entries were found. The start sites of two transcripts required for CMA biosynthesis were identified in the present study. pRG960sd, a vector containing a promoterless glucuronidase gene, was used to localize and study the promoter regions upstream of the two transcripts. Data obtained in the present study indicate that CMA biosynthesis is regulated at the transcriptional level by temperature.

Production of the chlorosis-inducing phytotoxin coronatine in the Pseudomonas syringae pathovars atropurpurea, glycinea, maculicola, morsprunorum, and tomato has been previously reported. DNA hybridization studies previously indicated that the coronatine biosynthetic gene cluster is highly conserved among P. syringae strains which produce the toxin. In the present study, two 17-bp oligonucleotide primers derived from the coronatine biosynthetic gene cluster of P. syringae pv. glycinea PG4180 were investigated for their ability to detect coronatine-producing P. syringae strains by PCR analysis. The primer set amplified diagnostic 0.65-kb PCR products from genomic DNAs of five different coronatine-producing pathovars of P. syringae. The 0.65-kb products were not detected when PCR experiments utilized nucleic acids of nonproducers of coronatine or those of bacteria not previously investigated for coronatine production. When the 0.65-kb PCR products were digested with ClaI, PstI, and SmaI, fragments of identical size were obtained for the five different pathovars of P. syringae. A restriction fragment length polymorphism was detected in the amplified region of P. syringae pv. atropurpurea, since this pathovar lacked a conserved PvuI site which was detected in the PCR products of the other four pathovars. The 0.65-kb PCR products from six strains comprising five different pathovars of P. syringae were cloned and sequenced. The PCR products from two different P. syringae pv. glycinea strains contained identical DNA sequences, and these showed relatedness to the sequence obtained for the pathovar morsprunorum. The PCR products obtained from the pathovars maculicola and tomato were the most similar to each other, which supports the hypothesis that these two pathovars are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)

Pseudomonas syringae pv. glycinea PG4180 produces a chlorosis-inducing phytotoxin, coronatine (COR), which consists of a polyketide component, coronafacic acid (CFA), which is coupled via amide bond formation to coronamic acid (CMA), an ethylcyelopropyl amino acid (aa) derived from isoleucine. P. syringae pv. syringae strains PS51 and PS61, which do not synthesize coronafacoyl compounds (conjugates between CFA and aa), acquired the ability to produce CFA and COR when transformed with p4180A, a 90-kb indigenous plasmid in PG4180. Tn5 mutagenesis indicated that the COR biosynthetic genes in PG4180 are clustered within a 30-kb region on p4180A. The phenotype of selected COR-defective mutants was determined by supplying them with CFA and CMA and by complementation studies with cloned DNA from the COR biosynthetic cluster. Using this approach, the regions encoding CFA and CMA synthesis and coupling activity were localized to the 24-, 12.5- and 2.3-kb regions of the cluster, respectively. Mutants in a 6-kb region required the addition of both CFA and CMA for COR synthesis, which may indicate a regulatory role for this part of the cluster. PS51 and PS61 transconjugants containing cloned DNA from the coupling region produced COR when supplied with CFA and CMA, indicating that coupling activity was cloned and expressed in bacteria lacking the COR biosynthetic cluster.

A simple approach is described for the production and purification of proteins in Pseudomonas syringae. The strategy involves the use of the tac promoter, the maltose-binding protein, and the broad-host-range vector, pRK415. This approach was used to partially purify two proteins involved in coronatine biosynthesis from P. syringae. The activity of the fusions was demonstrated in vivo in complementation experiments using the appropriate mutants.