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The exceptional diversity of the genus Rhododendron has a strong potential for identification, characterization, and production of bioactive lead compounds for health purposes. A particularly relevant field of application is the search for new antibiotics. Here, we present a comparative analysis of nearly 90 Rhododendron species targeted towards the search for such candidate substances. Through a combination of phytochemical profiles with antimicrobial susceptibility and cytotoxicity, complemented by phylogenetic analyses, we identify seven potentially antimicrobial active but non-cytotoxic compounds in terms of mass-to-charge ratios and retention times. Exemplary bioactivity-guided fractionation for a promising Rhododendron species experimentally supports in fact one of these candidate lead compounds. By combining categorical correlation analysis with Boolean operations, we have been able to investigate the origin of bioactive effects in further detail. Intriguingly, we discovered clear indications of systems effects (synergistic interactions and functional redundancies of compounds) in the manifestation of antimicrobial activities in this plant genus.

The science of metabolomics offers the possibility to measure full secondary plant metabolomes with limited experimental effort to allow identification of metabolome differences using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of liquid chromatography mass spectrometry (LC-MS) data.To demonstrate a bioinformatics driven hypothesis generator for identification of biologically active compounds in plant crude extracts, which is validated by activity guided fractionation.Crude extracts of Rhododendron leaves were tested for their antibacterial activity using agar diffusion and minimum inhibitory concentration assays. Extracts were profiled by LC-MS. PCA and PLS-DA were used for differentiation of bioactive and inactive extracts and their metabolites. Preparative-high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy were used for separation and structure elucidation of pure compound(s) respectively.An antibacterial Rhododendron collettianum was compared to a series of inactive extracts. Three metabolites were found to distinguish R. collettianum from other species indicating the ability of PCA and PLS-DA to suggest potential bioactive substances. An activity-guided fractionation of R. collettianum extracts was carried out and cannabiorcichromenic acid (CCA) was identified as antibacterial compound thereby validating the PCA and PLS-DA generated hypothesis. Four mammalian cell lines were used to estimate possible cytotoxicity of CCA.It was shown that bioinformatics tools facilitate early stage identification of a biologically active compound(s) using LC-MS data, which reduce complexity and number of separation steps in bioactive screening. Copyright © 2017 John WileySons, Ltd.

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.

The exceptional diversity of the genus Rhododendron has a strong potential for identification, characterization, and production of bioactive lead compounds for health purposes. A particularly relevant field of application is the search for new antibiotics. Here, we present a comparative analysis of nearly 90 Rhododendron species targeted toward the search for such candidate substances. Through a combination of phytochemical profiles with antimicrobial susceptibility and cytotoxicity, complemented by phylogenetic analyses, we identify seven potentially antimicrobial active but non-cytotoxic compounds in terms of mass-to-charge ratios and retention times. Exemplary bioactivity-guided fractionation for a promising Rhododendron species experimentally supports in fact one of these candidate lead compounds. By combining categorical correlation analysis with Boolean operations, we have been able to investigate the origin of bioactive effects in further detail. Intriguingly, we discovered clear indications of systems effects (synergistic interactions and functional redundancies of compounds) in the manifestation of antimicrobial activities in this plant genus.

BACKGROUND Complex polysaccharides are important in the pharmaceutical industry, yet, due to their large molecular weight and reduced charges, their purification is a highly demanding process that requires binding matrices with unique properties. This work demonstrates for the first time that complex polysaccharides biosynthesized by microalga Porphyridium purpureum can be adsorbed onto Q fibrous anion exchangers. RESULTS When the polysaccharides were characterized, the extent of sulfation was higher in native polysaccharides than in ethanol- or alkali-extracts. The zeta potentials increased with increasing pH and the highest charge was observed at pH 8, while the Z-average diameters of the polysaccharide at pH 6 were highest for alkali-extracts. Instead of pellicular resins, Q fibrous adsorbents were used to determine Langmuir thermodynamic properties and dynamic binding capacities. The parameters included static binding capacity and dissociation constant of 13.47 ± 1.02 mg g-1 and 0.141 ± 0.027 mg mL-1, and 10 and 50% breakthrough capacities of 4.46 ± 0.22 and 5.51 ± 0.28 mg g-1, respectively. The antiviral activity of the polysaccharides was demonstrated by minimizing bacteriophage lysis of Streptococcus thermophilus. CONCLUSION This work demonstrates that polysaccharide extraction can be optimized and the adsorption and desorption of a complex polysaccharide onto Q fibrous matrix is feasible. These parameters could be exploited for up-scaling of polysaccharides for nutraceutical and pharmaceutical applications. © 2013 Society of Chemical Industry

We have synthesized and structurally characterized the first discrete thallium-containing polyoxometalate, [Tl

The fermentation of cocoa beans is essential for the generation of flavour precursors that are required later on to form the flavour components of chocolate. From the many different precursors that are generated, oligopeptides and free amino acids comprise a significant proportion as some of them form Maillard reaction products during the roasting process. Therefore, the diversity of peptides is an important contributing factor to the quality of a fermentation which is in turn controlled by proteolytic activity within the cocoa bean, and is driven by changes in the presence of fermentation by-products as a result of microbial activity outside the bean. Being able to control proteolytic activity within the bean using only the presence of fermentation by-products would prove a valuable tool in the study of these proteases and the processing of cocoa storage proteins. Thus, this tool would help elucidate key mechanisms that generate the components responsible for flavour. In this study, we describe an artificial fermentation system, free from microbial activity, which is able to replicate proteolytic degradation of protein as well as to generate similar peptide fragments as seen during a commercial fermentation. It was also found that acidification is a main contributor to protein degradation.

DOI: 10.1021/acs.inorgchem.6b01921 URL: http://pubs.acs.org/doi/abs/10.1021/acs.inorgchem.6b01921 Filiació URV: SI Inclòs a la memòria: SI We have synthesized and structurally characterized the first discrete thallium-containing polyoxometalate, [Tl2{B-¿-SiW8O30(OH)}2]12- (1). Polyanion 1 was characterized in the solid-state and shown to be solution-stable by 203/205Tl NMR, electrospray ionization mass spectrometry, and electrochemical studies. The antibacterial activity of 1 was also investigated.

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.

Background Rhododendron leaf extracts were previously found to exert antimicrobial activities against a range of Gram-positive bacteria. In this study, we investigated which of the extracts with these antimicrobial properties would be best suited for further exploitation. Specifically, the project aims to identify biologically active compounds that affect bacterial but not mammalian cells when applied in medical treatments such as lotions for ectopic application onto skin, or as orally administered drugs. Methods Different concentrations of DMSO-dissolved remnants of crude methanol Rhododendron leaf extracts were incubated for 24 h with cultured epidermal keratinocytes (human HaCaT cell line) and epithelial cells of the intestinal mucosa (rat IEC6 cell line) and tested for their cytotoxic potential. In particular, the cytotoxic potencies of the compounds contained in antimicrobial Rhododendron leaf extracts were assessed by quantifying their effects on (i) plasma membrane integrity, (ii) cell viability and proliferation rates, (iii) cellular metabolism, (iv) cytoskeletal architecture, and (v) determining initiation of cell death pathways by morphological and biochemical means. Results Extracts of almost all Rhododendron species, when applied at 500 μg/mL, were potent in negatively affecting both keratinocytes and intestine epithelial cells, except material from R. hippophaeoides var. hippophaeoides. Extracts of R. minus and R. racemosum were non-toxic towards both mammalian cell types when used at 50 μg/mL, which was equivalent to their minimal inhibitory concentration against bacteria. At this concentration, leaf extracts from three other highly potent antimicrobial Rhododendron species proved non-cytotoxic against one or the other mammalian cell type: Extracts of R. ferrugineum were non-toxic towards IEC6 cells, and extracts of R. rubiginosum as well as R. concinnum did not affect HaCaT cells. In general, keratinocytes proved more resistant than intestine epithelial cells against the treatment with compounds contained in Rhododendron leaf extracts. Conclusions We conclude that leaf extracts from highly potent antimicrobial R. minus and R. racemosum are safe to use at 50 μg/mL in 24-h incubations with HaCaT keratinocytes and IEC6 intestine epithelial cells in monolayer cultures. Extracts from R. rubiginosum as well as R. concinnum or R. ferrugineum are applicable to either keratinocytes or intestinal epithelial cells, respectively. Beyond the scope of the current study, further experiments are required to identify the specific compounds contained in those Rhododendron leaf extracts that exert antimicrobial activity while being non-cytotoxic when applied onto human skin or gastrointestinal tract mucosa. Thus, this study supports the notion that detailed phytochemical profiling and compound identification is needed for characterization of the leaf extracts from specific Rhododendron species in order to exploit their components as supplementary agents in antimicrobial phyto-medical treatments. Electronic supplementary material The online version of this article (doi:10.1186/s12906-015-0860-8) contains supplementary material, which is available to authorized users.

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.

Background Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight on soybean plants and enters the leaf tissue through stomata or open wounds, where it encounters a sucrose-rich milieu. Sucrose is utilized by invading bacteria via the secreted enzyme, levansucrase (Lsc), liberating glucose and forming the polyfructan levan. P. syringae PG4180 possesses two functional lsc alleles transcribed at virulence-promoting low temperatures. Results We hypothesized that transcription of lsc is controlled by the hexose metabolism repressor, HexR, since potential HexR binding sites were identified upstream of both lsc genes. A hexR mutant of PG4180 was significantly growth-impaired when incubated with sucrose or glucose as sole carbon source, but exhibited wild type growth when arabinose was provided. Analyses of lsc expression resulted in higher transcript and protein levels in the hexR mutant as compared to the wild type. The hexR mutant’s ability to multiply in planta was reduced. HexR did not seem to impact hrp gene expression as evidenced by the hexR mutant’s unaltered hypersensitive response in tobacco and its unmodified protein secretion pattern as compared to the wild type under hrp-inducing conditions. Conclusions Our data suggested a co-regulation of genes involved in extra-cellular sugar acquisition with those involved in intra-cellular energy-providing metabolic pathways in P. syringae. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0349-0) contains supplementary material, which is available to authorized users.

Kopf, Anna ... et. al.-- 5 pages, 1 figure.-- This manuscript is NOAA-GLERL contribution number 1763, Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world’s oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits, This work was supported by the Micro B3 project, which is funded from the European Union’s Seventh Framework Programme (FP7; Joint Call OCEAN.2011‐2: Marine microbial diversity – new insights into marine ecosystems functioning and its biotechnological potential) under the grant agreement no 287589

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 .

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.

Plasmid pPSR1 is a conjugative plasmid originally isolated from Pseudomonas syringae pv. syringae A2, and is a member of the recently described pPT23A plasmid family. We have determined the complete sequence of pPSR1 and found the plasmid to be 72,601 bp in length, encoding 55 ORFs. Putative functions were assigned to 49 ORFs; of these, 24 (49.0%) are involved in plasmid replication, maintenance or conjugation, 17 (34.7%) have roles in virulence or ecological fitness, and eight (16.3%) encode transposase functions as part of mobile elements. pPSR1 carries the effector gene orf34, the mutagenic DNA repair operon rulAB which confers tolerance to ultraviolet radiation, and two genes for methyl-accepting chemotaxis proteins, one of which was located within the novel transposon Tn 5395. The streptomycin resistance transposon Tn 5393a, which carries a strA-strB determinant, was found inserted immediately downstream of the pPSR1 repA gene. Functional analysis of the replication region of pPSR1 indicated that the repA gene and flanking upstream and downstream sequences are required for autonomous replication in P. syringae. Hybridization analyses of the distribution of 11 of the pPSR1 ORFs indicated that many of the ecologically important ORFs were confined to the pathovar P. syringae pv. syringae -either to strains from the local population from which pPSR1 was originally isolated, or strains from a worldwide collection. Conjugative transfer genes and a gene encoding a transcriptional regulator were more widely distributed among several P. syringae pathovars. The sequence analysis of pPSR1 suggests that pPT23A-family plasmids evolve by accumulating genes that are important for host-pathogen interactions or growth on plant hosts, which are incorporated onto a conserved backbone encoding conjugation and stability determinants.

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 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.

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.

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.

The indigenous plasmids, pPSR1 and pPSR5, were each shown to confer resistance to ultraviolet light (UV) in Pseudomonas syringae (Ps) pv. syringae FF5. The UV-resistance (UVR) determinant was subcloned from a cosmid library of pPSR1, and sequence analysis revealed the presence of two ORFs, designated rulAB which are homologous to the Escherichia coli umuDC mutagenic DNA repair systems and other plasmid-encoded UVR operons. Amino acid (aa) alignments indicated that RulAB are most closely related to the RumAB proteins from plasmid R391, sharing 40.5% and 48.6% aa identity with RumA and RumB, respectively. UV sensitivity assays with the cloned rulAB genes indicated that the expression of UVR in Ps required a functional recA gene.

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.

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.

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.

The use of naturally occurring microbial antagonists to suppress plant diseases offers a favorable alternative to classical methods of plant protection. The soybean epiphyte Pseudomonas syringae pv. syringae strain 22d/93 shows great potential for controlling P. syringae pv. glycinea, the causal agent of bacterial blight of soybean. Its activity against P. syringae pv. glycinea is highly reproducible even in field trials, and the suppression mechanisms involved are of special interest. In this work we demonstrated that P . syringae pv. syringae 22d/93 produced a significantly larger amount of siderophores than the pathogen P. syringae pv. glycinea produced. While P . syringae pv. syringae 22d/93 and P. syringae pv. glycinea produce the same siderophores, achromobactin and pyoverdin, the regulation of siderophore biosynthesis in the former organism is very different from that in the latter organism. The epiphytic fitness of P . syringae pv. syringae 22d/93 mutants defective in siderophore biosynthesis was determined following spray inoculation of soybean leaves. The population size of the siderophore-negative mutant P . syringae pv. syringae strain 22d/93ΔSid was 2 orders of magnitude lower than that of the wild type 10 days after inoculation. The growth deficiency was compensated for when wound inoculation was used, indicating the availability of iron in the presence of small lesions on the leaves. Our results suggest that siderophore production has an indirect effect on the biocontrol activity of P . syringae pv. syringae 22d/93. Although siderophore-defective mutants of P . syringae pv. syringae 22d/93 still suppressed development of bacterial blight caused by P. syringae pv. glycinea, siderophore production enhanced the epiphytic fitness and thus the competitiveness of the antagonist.

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.

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.

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.

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.

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.