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We have synthesized and structurally characterized three tetra-(p-tolyl)antimony(III)-containing heteropolytungstates, [{(p-tolyl)SbIII}4(A-α-XW9O34)2]n- [X = PV (1-P), AsV (1-As), or GeIV (1-Ge)], in aqueous solution using conventional, one-pot procedures. The polyanions 1-P, 1-As, and 1-Ge were fully characterized in the solid state and in solution and were shown to be soluble and stable in aqueous medium at pH 7. Biological studies demonstrated that all three polyanions possess significant antibacterial and antitumor activities. The minimum inhibitory concentrations of 1-P, 1-As, and 1-Ge were determined against four kinds of bacteria, including the two pathogenic bacteria strains, Vibrio parahaemolyticus and Vibrio vulnificus. The three novel polyanions also showed high cytotoxic potency in the human cell lines A549 (non-small cell lung cancer), CH1/PA-1 (ovarian teratocarcinoma), and SW480 (colon carcinoma).

World-wide increases in Vibrio-associated diseases have been reported in aquaculture and humans in co-occurrence with increased sea surface temperatures. Twelve species of Vibrio are known to cause disease in humans, but three species dominate the number of human infections world-wide: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. Fluorescent protein (FP)-labelled bacteria have been used to make great progress through in situ studies of bacterial behavior in mixed cultures or within host tissues. Currently, FP-labelling methods specific for Vibrio species are still limited by time-consuming counterselection measures that require the use of modified media and temperatures below the optimal growth temperature of many Vibrio species. Within this study, we used a previously reported R6K-based suicide delivery vector and two newly constructed transposon variants to develop a tailored protocol for FP-labelling V. cholerae, V. parahaemolyticus, and V. vulnificus environmental isolates within two days of counterselection against the donor Escherichiacoli. This herein presented protocol worked universally across all tested strains (30) with a conjugation efficiency of at least two transconjugants per 10,000 recipients.

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.

Future mining of polymetallic nodules in the Clarion Clipperton Fracture Zone (Northeastern Pacific) is expected to affect all benthic ecosystems. The diversity, distribution, and environmental functions of microorganisms inhabiting abyssal sediments are barely understood. To understand the ​consequences of deep-sea mining, experimental in vitro systems needs to be established to test hypotheses on the environmental impact of mining. For this, 40 bacterial strains, belonging to proteobacteria, actinobacteria and firmicutes were isolated from deep-sea sediments and nodules sampled at depths of  4000 m. Phenotypic characterization revealed a strong inter-species and moderate intra-species variability. Determination of minimum inhibitory concentrations indicated the presence of acute manganese-resistant bacteria such as Rhodococcus erythropolis [228.9 mM], Loktanella cinnabarina [57.2 mM], and Dietzia maris [14.3 mM] that might be suitable systems for testing the effects of release of microbes from nodules and their interactions with sediment particles in plumes generated during mining. Comparative genomic analysis indicated the presence of manganese efflux systems relevant for future transcriptomics or proteomics approaches with environmental samples and might serves in paving the way to develop model systems including representative organisms which are currently not cultivable. Monitoring deep-sea mining activity at abyssal depth is a challenge that has to be tackled. We proposed the use of API strips as a fast-on-board methodology for bacterial monitoring as an indicator for sediment plume dispersions within the water column.

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.

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.

Microalgae are a promising resource for the highly sustainable production of various biomaterials (food and feed), high-value biochemicals, or biofuels. However, factors influencing the valued lipid production from oleaginous algae require a more detailed investigation. This study elucidates the variations in lipid metabolites between a marine diatom (Cylindrotheca closterium) and a freshwater green alga (Scenedesmus sp.) under nitrogen starvation at the molecular species level, with emphasis on triacylglycerols using liquid chromatography-electrospray ionization mass spectrometry techniques. A comprehensive analysis was carried out by comparing the changes in total lipids, growth kinetics, fatty acid compositions, and glycerolipid profiles at the molecular species level at different time points of nitrogen starvation. A total of 60 and 72 triacylglycerol molecular species, along with numerous other polar lipids, were identified in Scenedesmus sp. and C. closterium, respectively, providing the most abundant triacylglycerol profiles for these two species. During nitrogen starvation, more triacylglycerol of Scenedesmus sp. was synthesized via the "eukaryotic pathway" in the endoplasmic reticulum, whereas the increase in triacylglycerol in C. closterium was mainly a result of the "prokaryotic pathway" in the chloroplasts after 96 h of nitrogen starvation. The distinct responses of lipid synthesis to nitrogen starvation exhibited by the two species indicate different strategies of lipid accumulation, notably triacylglycerols, in green algae and diatoms. Scenedesmus sp. and Cylindrotheca closterium could serve as excellent candidates for the mass production of biofuels or polyunsaturated fatty acids for nutraceutical purposes.

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.

Diverse microalgae-bacteria interactions play important roles for nutrient exchange processes and marine aggregate formation leading to the cycling, mineralization, or sedimentation of organic carbon in the Oceans. The main goal of this study is to report an alternative way to assess a distinct diatom-bacteria interaction. To study this at the cellular scale, an in vitro interaction model system consisting of the diatom, Thalassiosira weissflogii, and the gamma-proteobacterium, Marinobacter adhaerens HP15, had previously been established. HP15 is able to attach to T. weissflogii cells, to induce transparent exopolymeric particle formation, and to increase marine aggregation formation. Several bacterial genes important during this interaction have been studied thus far, but genes specifically expressed in co-culture remained unknown. To identify such bacterial genes, an in vivo Expression Technology (IVET) screen was employed. For this, a promoter-trapping vector containing a fusion between a promoter-less selection marker gene and a promoterless reporter gene was constructed. Construction of a library of plasmids carrying genomic fragments upstream of the fusion and its subsequent transformation into a selection marker mutant allowed detection of 30 bacterial promoters specifically expressed during the interactions with T. weissflogii. Their sequence analyses revealed that the corresponding genes could be involved in many processes such as biochemical detection of diatom cells, bacterial attachment, metabolic exchange of nitrogen compounds, and resistance towards heavy metals. Identification of genes potentially involved in branched-chain amino acid uptake and utilization confirmed our previous results of a proteomics analysis. Since our current approach identified several additional genes to be induced in co-culture, use of IVET might be a valuable complementing strategy to proteomic or transcriptomic analysis of the diatom-bacteria crossplay. The current IVET approach demonstrated that the interaction between M. adhaerens HP15 and T. weissflogii is multifactorial and is likely to involve a complex network of physiological processes.

It is well known that the development of chocolate flavor is initiated during cocoa bean fermentation. Storage proteins undergo the most intensive breakdown yielding peptides and free amino acids, which both serve as flavor precursors. A comprehensive analysis of cocoa proteins and oligopeptides of non-fermented and fermented beans from various geographic origins allows the assessment of systematic differences with respect to their origin as well as fermentation status. Protein quantities as well as their profiles derived from two-dimensional gel electrophoresis, showed striking differences for non-fermented beans depending on their geographical origin. From fermented beans, oligopeptides were relatively quantified by utilizing UHPLC-ESI-Q-q-TOF and annotated based on their characteristic fragmentation pattern in the positive-ion mode. With >800 unique oligopeptides, excluding di- and tri-peptides, across 25 different samples, we are herein reporting on the largest collection of cocoa oligopeptides ever observed and identified. The detected diversity of peptides could not be correlated to the geographical origin but rather to the degree of fermentation. Our findings suggest that the variability in peptide patterns depends on the fermentation method applied in the country of origin ultimately indicating diversified proteolytic activities. Furthermore, our results showed that well-fermented and fair-fermented beans can be differentiated from partially fermented and under-fermented ones by higher numbers and total amounts of oligopeptides.

Two tetra-antimony(III)-bridged, sandwich-type 18-tungsto-2-arsenates(V), [(LSb(III))4(A-α-As(V)W9O34)2](10-) (L = Ph (1), OH (2)), were prepared and fully characterized in the solid state and in solution. Both polyanions are stable in aqueous physiological medium for at least 24 h (at concentrations ≥2.5 × 10(-6) M). Despite the presence of an isostructural tetra-antimony(III) motif in 1 and 2, distinctly different antibacterial activity was observed for both polyanions. The minimum inhibitory concentrations (MIC) of 1 (7.8-62.5 μg/mL) is lower than for any other organoantimony(III)-containing polyoxometalate reported to date.

Dietary phenolic compounds are often transformed by gut microbiota prior to absorption. This transformation may modulate their biological activities. Many fundamental questions still need to be addressed to understand how the gut microbiota-diet interactions affect human health. Herein, a UHPLC-QTOF mass spectrometry-based method for the quantification of uptake and determination of intracellular bacterial concentrations of dietary phenolics from coffee and tea was developed. Quantitative uptake data for selected single purified phenolics were determined. The specific uptake from mixtures containing up to four dietary relevant compounds was investigated to assess changes of uptake parameters in a mixture model system. Indeed, perturbation of bacteria by several compounds alters uptake parameter in particular tmax. Finally, model bacteria were dosed with complex dietary mixtures such as diluted tea or coffee extracts. The uptake kinetics of the twenty most abundant phenolics was quantified and the findings are discussed. For the first time, quantitative data on in-vitro uptake of dietary phenolics from food matrices were obtained indicating a time-dependent differential uptake of nutritional compounds.

Background Rhododendron species have been traditionally used in countries like China, Nepal, Russia and North America for treating human diseases. These species are known to be a good source of polyphenolic plant secondary plant metabolites. They are known to have beneficial health properties for humans and have been used to treat diseases like asthma, skin diseases. In this contribution we investigate the phenolic profile and antibacterial activity of extracts from several plant organs including for the first time from leaves of different development stages. Methods In this study, the polyphenolic profile of fruits, flowers and leaves of different ages of Rhododendron ambiguum and Rhododendron cinnabarinum were studied by using HPLC–MS and compounds identified based on high resolution masses and identity of tandem mass spectra, UV/VIS spectra and retention times if compared to standards. Results Fifty-nine different polyphenols including isomers were identified in these species by their fragmentation pattern and high resolution data. Also, the antibacterial activity of these parts (leaves, fruits and flowers) against gram-positive bacteria was studied. Conclusion The leaves and fruits contained more polyphenols than the flowers. With the exception of flowers, the fruits and leaves of both species were also determined to have a significant antibacterial effect against four gram-positive bacteria. Electronic supplementary material The online version of this article (doi:10.1186/s13104-017-2601-1) contains supplementary material, which is available to authorized users.

A comprehensive analysis of cocoa polyphenols from unfermented and fermented cocoa beans from a wide range of geographic origins was carried out to catalogue systematic differences based on their origin as well as fermentation status. This study identifies previously unknown compounds with the goal to ascertain, which of these are responsible for the largest differences between bean types. UHPLC coupled with ultra-high resolution time-of-flight mass spectrometry was employed to identify and relatively quantify various oligomeric proanthocyanidins and their glycosides amongst several other unreported compounds. A series of biomarkers allowing a clear distinction between unfermented and fermented cocoa beans and for beans of different origins were identified. The large sample set employed allowed comparison of statistically significant variations of key cocoa constituents.

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.

In the ocean, exopolymer accumulation and the resulting aggregation of marine phytoplankton drive the flux of sinking organic matter. Little is known about the contribution of bacteria to the release and build-up of such exudates. We describe an in-depth investigation of exopolymer production under differing nutrient conditions using a diatombacteria model system. Responses of the marine diatom Thalassiosira weissflogii to different nutrient re gimes and the impact of the co-incubated bacterium Marinobacter adhaerens on algal exudation were evaluated by ana- lyzing quantity and quality of exudation products. Cul- tures of T. weissflogii were grown at nutrient-balanced conditions (Redfield ratio N:P = 16) as well as at nitro- gen- or phosphorus-depleted conditions (N:P = 1 and N:P = 95, respectively) in the presence or absence of M. adhaerens. The impact of M. adhaerens on the con- centration of dissolved organic carbon and transparent exopolymer particles (TEP), as well as on the composi- tion of amino acids and carbohydrates, depended on the nutrient regime. Under nutrient-balanced con - ditions, M. adhaerens stimulated both T. weissflogii growth and TEP production. Under nutrient-depleted conditions, TEP production was enhanced regardless of whether bacteria were present. Phosphorus limita- tion resulted in an appreciable change in the %mol composition of dissolved amino acids in xenic and axenic treatments. Differential lec tin staining revealed that the presence of M. adhae rens enhanced and modified the pro duction of specific extracellular sub- stances. A better understan ding of the effects of diatombacteria interactions on the accumulation of exudation products is crucial in modelling and predict- ing consequences of environmental changes on oceanic organic matter and nutrient cycling.

In the plant pathogenic bacterium, Pseudomonas syringae, the exopolysaccharide levan is synthesized by extracellular levansucrase (Lsc), which is encoded by two conserved 1,296-bp genes termed lscB and lscC in P. syringae strain PG4180. A third gene, lscA, is homologous to the 1,248-bp lsc gene of the bacterium Erwinia amylovora, causing fire blight. However, lscA is not expressed in P. syringae strain PG4180. Herein, PG4180 lscA was shown to be expressed from its native promoter in the Lsc-deficient E. amylovora mutant, Ea7/74-LS6, suggesting that lscA might be closely related to the E. amylovora lsc gene. Nucleotide sequence analysis revealed that lscB and lscC homologs in several P. syringae strains are part of a highly conserved 1.8-kb region containing the ORF, flanked by 450-452-bp and 49-51-bp up- and downstream sequences, respectively. Interestingly, the 450-452-bp upstream sequence, along with the initial 48-bp ORF sequence encoding for the N-terminal 16 amino acid residues of Lsc, were found to be highly similar to the respective sequence of a putatively prophage-borne glycosyl hydrolase-encoding gene in several P. syringae genomes. Minimal promoter regions of lscB and lscC were mapped in PG4180 by deletion analysis and were found to be located in similar positions upstream of lsc genes in three P. syringae genomes. Thus, a putative 498-500-bp promoter element was identified, which possesses the prophage-associated com gene and DNA encoding common N-terminal sequences of all 1,296-bp Lsc and two glycosyl hydrolases. Since the gene product of the non-expressed 1,248-bp lscA is lacking this conserved N-terminal region but is otherwise highly homologous to those of lscB and lscC, it was concluded that lscA might have been the ancestral lsc gene in E. amylovora and P. syringae. Our data indicated that its highly expressed paralogs in P. syringae are probably derived from subsequent recombination events initiated by insertion of the 498-500-bp promoter element, described herein, containing a translational start site.

Diatom aggregation is substantial for organic carbon flux from the photic zone to deeper waters. Many heterotrophic bacteria ubiquitously found in diverse marine environments interact with marine algae and thus impact organic matter and energy cycling in the ocean. In particular, Marinobacter adhaerens HP15 induces aggregate formation while interacting with the diatom, Thalassiosira weissflogii . To study this effect at the molecular level, a genetic tool system was developed for strain HP15. The antibiotic susceptibility spectrum of this organism was determined and electroporation and conjugation protocols were established. Among various plasmids of different incompatibility groups, only two were shown to replicate in M. adhaerens . 1.4 × 10 −3 transconjugants per recipient were obtained for a broad-host-range vector. Electroporation efficiency corresponded to 1.1 × 10 5 CFU per μg of DNA. Transposon and gene-specific mutageneses were conducted for flagellum biosynthetic genes. Mutant phenotypes were confirmed by swimming assay and microscopy. Successful expression of two reporter genes in strain HP15 revealed useful tools for gene expression analyses, which will allow studying diverse bacteria–algae interactions at the molecular level and hence to gain a mechanistic understanding of micro-scale processes underlying ocean basin-scale processes. This study is the first report for the genetic manipulation of a Marinobacter species which specifically interacts with marine diatoms and serves as model to additionally analyze various previously reported Marinobacter –algae interactions in depth.

The epiphyte Pseudomonas syringae pv. syringae 22d / 93 (Pss22d), isolated from soybean leaves, had been characterized as a promising and species-specific biocontrol strain in vitro and in planta against the plant pathogen P. syringae pv. glycinea (Psg), which causes bacterial blight of soybean. Three toxins are known to be produced by Pss22d: syringomycin, syringopeptin and 3-methylarginine (MeArg). In contrast to syringopeptin and syringomycin, MeArg inhibited the growth of Psg in vitro. To examine if the toxins produced by Pss22d are responsible for antagonistic effects in planta, the pathogen Psg was co-inoculated with either Pss22d wild-type, a syringopeptin/syringomycin-negative double mutant (Pss22d.ΔsypA/syrE), or a MeArg-negative mutant (Pss22d.1) into wounds of pin-pricked leaves of greenhouse-grown soybean plants, respectively. In all three cases, the wild-type Pss22d and its toxin-deficient mutants prevented development of disease symptoms normally caused by Psg. These results indicated that neither syringopeptin, nor syringomycin, nor MeArg was required for Pss22d's antagonistic activity in planta. Consequently, factors other than the three toxins may contribute to the intra-species antagonism in planta.

Erwinia amylovora causes fire blight on several plant species such as apple and pear, which produce diverse phytoalexins as defence mechanisms. An evolutionary successful pathogen thus must develop resistance mechanisms towards these toxic compounds. The E. amylovora outer membrane protein, TolC, might mediate phytoalexin resistance through its interaction with the multidrug efflux pump, AcrAB. To prove this, a tolC mutant and an acrB/tolC double mutant were constructed. The minimal inhibitory concentrations of diverse antimicrobials and phytoalexins were determined for these mutants and compared with that of a previously generated acrB mutant. The tolC and arcB/tolC mutants were considerably more susceptible than the wild type but showed similar levels as the acrB mutant. The results clearly indicated that neither TolC nor AcrAB significantly interacted with other transport systems during the efflux of the tested toxic compounds. Survival and virulence assays on inoculated apple plants showed that pathogenicity and the ability of E. amylovora to colonize plant tissue were equally impaired by mutations of tolC and acrB/tolC. Our results allowed the conclusion that TolC plays an important role as a virulence and fitness factor of E. amylovora by mediating resistance towards phytoalexins through its exclusive interaction with AcrAB.

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.

Several members of the ubiquitously found γ-proteobacterial genus Marinobacter were described or assumed to inhabit marine environments naturally enriched in heavy metals. However, direct studies that describe the ability of this genus to occupy such environments have not been conducted. To cope with heavy metal stress, bacteria possess specific efflux pumps as tools for detoxification, among which the CzcCBA type efflux system is one representative. Previous studies showed that this system plays an important role in resistance towards cadmium, zinc, and cobalt. Up to now, no study had focused on characterization of Czc pumps in Marinobacter sp. or other marine prokaryotes. Herein, we elucidated the function of two CzcCBA pumps encoded by Marinobacter adhaerens HP15's genome during exposure to cadmium, zinc, and cobalt. Single and double knock-out mutants lacking the corresponding two czcCBA operons were generated and analyzed in terms of their resistance profiles. Both operons appeared to be important for zinc resistance but had no role in tolerance towards cadmium or cobalt. One of the mutations was genetically complemented thereby restoring the wild type phenotype. In accordance with the resistance pattern, expression of the genes coding for both CzcCBA pumps was induced by zinc but neither by cadmium nor cobalt.

Background Plants are traditionally used for medicinal treatment of numerous human disorders including infectious diseases caused by microorganisms. Due to the increasing resistance of many pathogens to commonly used antimicrobial agents, there is an urgent need for novel antimicrobial compounds. Plants of the genus Rhododendron belong to the woody representatives of the family Ericaceae, which are typically used in a range of ethno-medical applications. There are more than one thousand Rhododendron species worldwide. The Rhododendron-Park Bremen grows plants representing approximately 600 of the known Rhododendron species, and thus enables research involving almost two thirds of all known Rhododendron species. Methods Twenty-six bacterial species representing different taxonomic clades have been used to study the antimicrobial potential of Rhododendron leaf extracts. Agar diffusion assay were conducted using 80% methanol crude extracts derived from 120 Rhododendron species. Data were analyzed using principal component analysis and the plant-borne antibacterial activities grouped according the first and second principal components. Results The leaf extracts of 17 Rhododendron species exhibited significant growth-inhibiting activities against Gram-positive bacteria. In contrast, only very few of the leaf extracts affected the growth of Gram-negative bacteria. All leaf extracts with antimicrobial bioactivity were extracted from representatives of the subgenus Rhododendron, with 15 from the sub-section Rhododendron and two belonging to the section Pogonanthum. The use of bacterial multidrug efflux pump mutants revealed remarkable differences in the susceptibility towards Rhododendron leaf extract treatment. Conclusions For the first time, our comprehensive study demonstrated that compounds with antimicrobial activities accumulate in the leaves of certain Rhododendron species, which mainly belong to a particular subgenus. The results suggested that common genetic traits are responsible for the production of bioactive secondary metabolite(s) which act primarily on Gram-positive organisms, and which may affect Gram-negative bacteria in dependence of the activity of multidrug efflux pumps in their cell envelope. Electronic supplementary material The online version of this article (doi:10.1186/s12906-015-0596-5) contains supplementary material, which is available to authorized users.

In the plant pathogen Pseudomonas syringae, production of the exopolysaccharide levan is mediated by extracellular levansucrase (Lsc), which is encoded by two functional genes, lscB and lscC. Comparison of extracellular protein profiles of P. syringae pv. glycinea PG4180 grown at 18 and 28 degrees C and Western blots revealed that Lsc was predominantly found in the supernatant at 18 degrees C, a temperature fostering virulence of this pathogen. Northern blot analysis indicated that transcription of lscB and lscC was temperature-dependent. Quantification of Lsc in supernatants and cellular protein samples of mutants defective in either lscB or lscC confirmed that LscB secretion at low temperature was due to a combination of thermo-regulated transcription and secretion. In contrast, LscC accumulated in the periplasmic space. LscB and LscC differ in only five amino acid residues, one of which is a cysteine residue. Temperature shift experiments suggested that de novo synthesized protein(s) at 18 degrees C might be responsible for differential LscB secretion and that the presumed secretory machinery was stable when cells were shifted to 28 degrees C. Our results imply that Lsc export and secretion may occur by yet-to-be identified novel mechanism(s).

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea infects soybean plants and causes bacterial blight. In addition to P. syringae , the human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter vinelandii produce the exopolysaccharide alginate, a copolymer of d -mannuronic and l -guluronic acids. Alginate production in P. syringae has been associated with increased fitness and virulence in planta. Alginate biosynthesis is tightly controlled by proteins encoded by the algT-muc regulatory gene cluster in P. aeruginosa and A. vinelandii . These genes encode the alternative sigma factor AlgT (σ 22 ), its anti-sigma factors MucA and MucB, MucC, a protein with a controversial function that is absent in P. syringae , and MucD, a periplasmic serine protease and homolog of HtrA in Escherichia coli . We compared an alginate-deficient algT mutant of P. syringae pv. glycinea with an alginate-producing derivative in which algT is intact. The alginate-producing derivative grew significantly slower in vitro growth but showed increased epiphytic fitness and better symptom development in planta. Evaluation of expression levels for algT , mucA , mucB , mucD , and algD , which encodes an alginate biosynthesis gene, showed that mucD transcription is not dependent on AlgT in P. syringae in vitro. Promoter mapping using primer extension experiments confirmed this finding. Results of reverse transcription-PCR demonstrated that algT , mucA , and mucB are cotranscribed as an operon in P. syringae . Northern blot analysis revealed that mucD was expressed as a 1.75-kb monocistronic mRNA in P. syringae .

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.

For fast and cost-efficient identification of bacteria we tested the Biotyper system (Bruker Daltonics) to detect Erwinia amylovora and other plant-associated bacteria by protein profiles from whole cells. Strains could also be identified by direct transfer of cells to the sample target, and the signals were compared to preceding protein extraction with formic acid. The identification scores obtained for extracted proteins were higher and more reliable than those of directly applied agar cultured cells. For verification of MALDI-TOF identification and for detection of several Erwinia species in mixed cultures we applied PCR primer sets designed from specific chromosomal sequence regions such as the gene cluster for biosynthesis of capsular EPS or the nucleotide sequences flanked by genes pstS and glmS. The novel primers readily detected E. amylovora fruit tree and raspberry strains, E. pyrifoliae, E. billingiae and E. tasmaniensis. The identification obtained by PCR agreed with the MALDI-TOF MS results. While PCR analysis is also applicable for detection of bacteria in mixed cultures obtained from plant samples, MALDI-TOF analysis identifies essentially cells from single colonies applied from agar plates or cultured in liquid medium. Both methods were combined for identification of bacteria in field samples and from artificially inoculated plant material.

Increasing Transparent Exopolymer Particle (TEP) formation during diatom blooms as a result of elevated temperature and pCO2 have been suggested to result in enhanced aggregation and carbon flux, therewith potentially increasing the sequestration of carbon by the ocean. We present experimental results on TEP and aggregate formation by Thalassiosira weissflogii (diatom) in the presence or absence of bacteria under two temperature and three pCO2 scenarios. During the aggregation phase of the experiment TEP formation was elevated at the higher temperature (20°C vs. 15°C), as predicted. However, in contrast to expectations based on the established relationship between TEP and aggregation, aggregation rates and sinking velocity of aggregates were depressed in warmer treatments, especially under ocean acidification conditions. If our experimental findings can be extrapolated to natural conditions, they would imply a reduction in carbon flux and potentially reduced carbon sequestration after diatom blooms in the future ocean.

Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae , Erwinia amylovora , and other bacterial species. The lsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene in Escherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3.0-kb Pst I fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc . Both ORFs showed high homology to the E. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring the lsc gene. Expression of lsc was found to be dependent on the vector-based P lac promoter, indicating that the native promoter of lsc was not functional in E. coli . Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli . A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.

Alga-bacterium interactions are crucial for aggregate formation and carbon cycling in aquatic systems. To understand the initiation of these interactions, we investigated bacterial chemotaxis within a bilateral model system. Marinobacter adhaerens HP15 has been demonstrated to attach to the diatom Thalassiosira weissflogii and induce transparent exopolymeric particle and aggregate formation. M. adhaerens possesses one polar flagellum and is highly motile. Bacterial cells were attracted to diatom cells, as demonstrated by addition of diatom cell homogenate or diatom culture supernatant to soft agar, suggesting that chemotaxis might be important for the interaction of M. adhaerens with diatoms. Three distinct chemotaxis-associated gene clusters were identified in the genome sequence of M. adhaerens , with the clusters showing significant sequence similarities to those of Pseudomonas aeruginosa PAO1. Mutations in the genes cheA , cheB , chpA , and chpB , which encode histidine kinases and methylesterases and which are putatively involved in either flagellum-associated chemotaxis or pilus-mediated twitching motility, were generated and mutants with the mutations were phenotypically analyzed. Δ cheA and Δ cheB mutants were found to be swimming deficient, and all four mutants were impaired in biofilm formation on abiotic surfaces. Comparison of the HP15 wild type and its chemotaxis mutants in cocultures with the diatom revealed that the fraction of bacteria attaching to the diatom decreased significantly for mutants in comparison to that for the wild type. Our results highlight the importance of M. adhaerens chemotaxis in initiation of its interaction with the diatom. In-depth knowledge of these basic processes in interspecies interactions is pivotal to obtain a systematic understanding of organic matter flux and nutrient cycling in marine ecosystems.

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump.

Revista Open Access. Artículo con licencia Creative Commons Attribution. -- 11 páginas, 4 figuras, 4 tablas., Marinobacter adhaerens HP15 is the type strain of a newly identified marine species, which is phylogenetically related to M. flavimaris, M. algicola, and M. aquaeolei. It is of special interest for research on marine aggregate formation because it showed specific attachment to diatom cells. In vitro it led to exopolymer formation and aggregation of these algal cells to form marine snow particles. M. adhaerens HP15 is a free-living, motile, rod-shaped, Gram-negative Gammaproteobacterium, which was originally isolated from marine particles sampled in the German Wadden Sea. M. adhaerens HP15 grows heterotrophically on various media, is easy to access genetically, and serves as a model organism to investigate the cellular and molecular interactions with the diatom Thalassiosira weissflogii. Here we describe the complete and annotated genome sequence of M. adhaerens HP15 as well as some details on flagella-associated genes. M. adhaerens HP15 possesses three replicons; the chromosome comprises 4,422,725 bp and codes for 4,180 protein-coding genes, 51 tRNAs and three rRNA operons, while the two circular plasmids are ~187 kb and ~42 kb in size and contain 178 and 52 protein-coding genes, respectively.

Implant-related infections are often devastating situations in orthopaedic trauma surgery particularly if multiresistant bacteria are involved. Protection of the implant surface by an antimicrobial coating exhibiting activity against multiresistant bacterial strains is of high interest. Aim of this study was to investigate the antimicrobial effects of an Ag/SiO(x)C(y) plasma polymer coating for fracture fixation devices, such as nails, plates, and external fixators, including tests against methicillin-resistant Staphylococcus aureus (MRSA) and its biocompatibility. The antimicrobial activity of the coating deposited onto 12 x 3 mm(2) stainless steel implants was tested in vitro against Staphylococcus aureus, Staphylococcus epidermidis, and MRSA using different testing methods (ASTM E-2810, JIS Z 2801, proliferation assay). Additionally, the coated devices were implanted into the paravertebral muscle of rabbits and explanted after 2, 7, 14, and 28 days to test the remaining ex vivo antimicrobial activity. For biocompatibility assessment the Ag/SiO(x)C(y) plasma polymer coating was tested in vitro according to ISO 10993-5. The Ag/SiO(x)C(y) coating exhibited excellent antimicrobial activity against all tested bacterial strains in all three in vitro tests. Ex vivo testing proved suppression of more than 99.9 % of bacterial proliferation by the coating compared to non-coated samples even after 28 days. ISO 10993-5 showed good biocompatibility of the coating without any indications of cytotoxic effects. In summary, Ag/SiO(x)C(y) plasma polymer coating showed excellent antimicrobial activity including effectiveness against MRSA and good in vitro biocompatibility. Therefore, it possesses high potential as a prophylactic agent in orthopaedic trauma surgery.

AcrAB-TolC is the major multidrug efflux system in Enterobacteriaceae recognizing structurally unrelated molecules including antibiotics, dyes, and detergents. Additionally, in Escherichia coli it mediates resistance to bile salts. In the plant pathogen Erwinia amylovora AcrAB-TolC is required for virulence and phytoalexin resistance. Exchange analysis of AcrAB-TolC was conducted by complementing mutants of both species defective in acrB or tolC with alleles from either species. The acrB and tolC mutants exhibited increased susceptibility profiles for 24 different antibiotics. All mutants were complemented with acrAB or tolC, respectively, regardless of the taxonomic origin of the alleles. Importantly, complementation of E. amylovora mutants with respective E. coli genes restored virulence on apple plants. It was concluded that AcrAB and TolC of both species could interact and that these interactions did not yield in altered functions despite the divergent ecological niches, to which E. coli and E. amylovora have adopted.

Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.

In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In Pseudomonas aeruginosa , a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens Pseudomonas syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. syringae pv. tomato DC3000 were identified, and mexAB-oprM -deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the mexAB - oprM -deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the mexAB-oprM operon in P. syringae ; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.

Genes involved in pathogenicity of several plant pathogens were shown to be induced at relatively cold temperatures. Loci from the fire blight pathogen Erwinia amylovora (Burrill) induced at 18 °C were identified using the miniTn5 transposon that contains the promoterless reporter gene gusA coding for β-glucuronidase (GUS). Certain mutants (2.7%) expressed GUS predominantly at 18 °C on minimal medium plates, indicating that the transposon had been inserted downstream of a putatively thermoregulated promoter. Those mutants were further screened with a quantitative GUS fluorometric assay. A total of 21 mutants were selected: 19 mutants had a transposon insertion in temperature-dependent genetic loci, with a 2.2- to 6.3-fold induction of gusA gene expression at 18 °C, and two mutants with impaired growth at 18 °C. Some of these genetic loci encoded (i) proteins implicated in flagella biosynthesis, biotin biosynthesis, multi-drug efflux, and type II secretion protein, and (ii) proteins of unknown function.Key words: fire blight, Erwinia amylovora, transposon mutagenesis, gene regulation, low temperature.

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.

Besides an intensively studied type III secretory system, the Hrp (hypersensitive response and pathogenicity) system, which delivers so-called avirulence gene products to the host cell causing pathogenicity, virulence factors such as phytotoxins, exopolysaccharides, plant hormones, and extracellular enzymes significantly contribute to the disease development in pathogenic plant-microbe interactions (Alfano and Collmer, 1996). It remains to be determined how and under which environmental conditions most of these virulence factors affect the disease outcome and lead to an increased fitness of the producing pathogens inside the plant. Opportunistic plant pathogens like Pseudomonas syringae preferably infect their host plants under conditions of high humidity and low temperature. Plants, as poikilothermic organisms do not maintain a given temperature but rather adjust to the environmental temperature regime they are exposed to. This complicates a direct comparison of temperature-dependent processes in plant pathogens with the well-studied thermoregulation of virulence factors in human and animal pathogens (Hurme and Rhen, 1998). While temperatures of 37–41°C signal to animal pathogens their arrival inside the warm-blooded host, numerous virulence factors of phytopathogens like Erwinia amylovora, Agrobacterium tumefaciens, E. chrysanthemi, and P. syringae are preferentially expressed, secreted, or assembled at lower temperatures, such as 18–22°C. It is therefore intriguing to assume that general principles govern thermo-adaptation of plant pathogens that are clearly different form those regulatory circuits and cascades in human and animal pathogens. Little is known about those general principles. Therefore, thermoresponsive synthesis of phytotoxins in P. syringae represents an excellent model to study them.

Pathogenic bacteria with habitats inside and outside a given host react to changes in environmental parameters by synthesizing gene products specifically needed during pathogenic or saprophytic growth. Temperature effects have been investigated in detail for pathogens of warm-blooded hosts, and major principles governing the temperature-sensing mechanism have been uncovered. Generally, transcription of virulence genes in these pathogens is induced at higher temperatures (37-41 degrees C), which are typical for body cavities and host tissues. However, effects of temperature on virulence determinants in plant pathogenic bacteria have not been focused on in detail. Interestingly, almost all virulence genes of plant pathogenic bacteria studied with respect to temperature exhibit increased transcription at temperatures well below the respective growth optima. This includes virulence determinants such as those directing bacteria-to-plant gene transfer, plant cell-wall-degrading enzymes, phytotoxins, ice nucleation activity, exopolysaccharide production, and the type III protein secretion machinery. Although many of the studied phytopathogens cause "cold-weather" diseases, the ecological rationale for this phenomenon remains to be studied in detail. This mini-review summarizes our current knowledge on thermoregulation of cellular processes taking place in bacterial phytopathogens in response to temperature changes. Since the temperature range of interest is different from that relevant to pathogens of mammals, one envisions novel principles of thermo-sensing in bacteria interacting with plants.

Plant pathogenic bacteria cause numerous devastating diseases in worldwide important crops thereby drawing increasing attention to the improved understanding of their infection processes. Phytopathogens, like our model organism, the gram-negative bacterium Pseudomonas syringae, encounter a number of environmental signals during the course of infection of their respective host plants. Foliar plant pathogens causing leaf spot diseases depend on high humidity and the proper temperature conditions to successfully infect the host plant, to rapidly overcome plant defense mechanisms, and to establish in planta population sizes which allow intense disease development. For many P. syringae pathovars high humidity and low temperatures have been shown to foster disease outcome. In analogy to human and animal pathogens that recognize the interior of their respective host organisms by a number of environmental signals, P. syringae might detect the proper conditions for bacterial infection and survival in its host plants. With respect to temperature it has been well established that human and animal pathogenic bacteria express virulence factors at 37–40°C but not at 25–30°C. It has been proposed that by means of temperature regulation - these pathogens economize gene expression: The bacteria synthesize virulence factors preferably at the time point and location when and where they are actually needed - in the interior of the warm-blooded host.

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.

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli , and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.

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)

Bacteria were isolated from leaf spots of field grown soybeans during two growing seasons. The leaf spots yielded up to 4 different species and a total population size of about 107–108 bacteria/cm2. The majority of the 192 isolates belonged to the species Pseudomonas syringae pv. glycinea (55%), causing leaf spots of bacterial blight on soybeans, and Erwinia herbicola (22%). The remaining isolates included bacteria from other genera, but occurred occasionally. The determination of biological activity of the isolates demonstrated that a high percentage of strains from the group Erwinia/Enterobacter produced biological active substances against Escherichia coli (69%) and against Chlorella pyrenoidosa (88%). The majority of P. syringae pv. glycinea strains failed to do so. None of the isolates affected the growth of Geotrichum candidum. The E. herbicola strains showed clear antagonistic properties against a wide range of isolated bacteria. Four E. herbicola strains inhibited the growth of nearly all other E. herbicola isolates and 6 other strains were active against most of the P. syringae pv. glycinea isolates. However, antagonistic interactions among strains isolated from a distinct leaf spot were very rarely.

The qualitative and quantitative composition of bacterial flora occurring inside the leaf spots of field grown soybeans was studied during the growing seasons (June to October) of 1989 and 1990. As a rule these leaf spots (necrotic lesions with chlorotic haloes) were caused by Pseudomonas syringae pv. glycinea. This pathogenic bacterium was predominantly found during the whole season in the symptomatic leaf tissue. Other species, mainly Erwinia herbicola, were also found in the same habitat. The population sizes of P. s. pv. glycinea increased from the beginning of symptom occurrence until July, stabilized until September, and then decreased a little. In general, the size of saprophytic populations was orders of magnitude lower than that of the pathogenic populations. The number of different bacterial genera per sample increased up to four genera per leaf spot by the end of the season. No significant influence of the occurring saprophytes on the population dynamics of the pathogen in planta could be observed.