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The growing interest in the therapeutic application of bacteriophages leads to a drastic increase in the number of sequenced genomes. Luckily, recent insights in phage taxonomy facilitate the classification of phages in a comprehensive and data-driven manner as recently proposed by the International Committee on Taxonomy of Viruses. In this research, we present the taxonomical classification of a novel, narrow host range Xanthomonas phage FoX4, isolated from a Brussels sprouts field in Belgium infested with Xanthomonas campestris pv. campestris. The phage has a limited ability to lyse a bacterial culture, yet adsorbs efficiently to its host. Based on its genome sequence and low similarity to previously described phages, the phage comprises the novel phage genus Foxquatrovirus.

Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recognition of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthora thermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activity towards xanthan was only observed when xanthan molecules were in the disordered conformation. Optimal performance towards xanthan was observed at 53 °C in the complete absence of salt, a condition favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulases and analyzed for its repeating units using UPLC–HILIC–ELSD/ESI–MS. This showed that AXE3 specifically removes the acetyl groups positioned on the inner mannose and that acetyl groups positioned on the outer mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of all acetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed.

Five xanthans were hydrolyzed to their repeating units using cellulases. Hydrophilic interaction chromatography with online electrospray ionization ion trap mass spectrometry and evaporative light scattering detection was used to analyze the oligomers released. It was concluded that six different pentamer repeating units (RUs) exists within a xanthan sample. The most abundant RU shows acetylation on the inner mannose and pyruvylation on the outer mannose. The second most abundant RU shows acetylation on both the inner and the outer mannose. It becomes clear that more variations in the xanthan structure exist than generally recognized. Comparison of five different xanthan samples revealed that, although the molecular composition of xanthan samples can be exactly the same, the ratio in which the RUs occur can differ significantly. It is, therefore, concluded that xanthan samples should be characterized for both, their molecular composition and the relative abundance of the RUs present.

Background Pseudomonas fuscovaginae (Pfv) is an emerging plant pathogen of rice and also of other gramineae plants. It causes sheath brown rot disease in rice with symptoms that are characterized by brown lesions on the flag leaf sheath, grain discoloration and sterility. It was first isolated as a high altitude pathogen in Japan and has since been reported in several countries throughout the world. Pfv is a broad host range pathogen and very little is known about its virulence mechanisms. Results An in planta screen of 1000 random independent Tn5 genomic mutants resulted in the isolation of nine mutants which showed altered virulence. Some of these isolates are mutated for functions which are known to be virulence associated factors in other phytopathogenic bacteria (eg. pil gene, phytotoxins and T6SS) and others might represent novel virulence loci. Conclusions Being an emerging pathogen worldwide, the broad host range pathogen Pfv has not yet been studied for its virulence functions. The roles of the nine loci identified in the in planta screen are discussed in relation to pathogenicity of Pfv. In summary, this article reports a first study on the virulence of this pathogen involving in planta screening studies and suggests the presence of several virulence features with known and novel functions in the Pseudomonas group of bacteria. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0274-7) contains supplementary material, which is available to authorized users.

[EN] Hydroxycinnamic acid amides (HCAA) are secondary metabolites involved in plant development and defense that have been widely reported throughout the plant kingdom. These phenolics show antioxidant, antiviral, antibacterial, and antifungal activities. Hydroxycinnamoyl-CoA: tyramine N-hydroxycinnamoyl transferase (THT) is the key enzyme in HCAA synthesis and is induced in response to pathogen infection, wounding, or elicitor treatments, preceding HCAA accumulation. We have engineered transgenic tomato plants overexpressing tomato THT. These plants displayed an enhanced THT gene expression in leaves as compared with wild type (WT) plants. Consequently, leaves of THT-over-expressing plants showed a higher constitutive accumulation of the amide coumaroyltyramine (CT). Similar results were found in flowers and fruits. Moreover, feruloyltyramine (FT) also accumulated in these tissues, being present at higher levels in transgenic plants. Accumulation of CT, FT and octopamine, and noradrenaline HCAA in response to Pseudomonas syringae pv. tomato infection was higher in transgenic plants than in the WT plants. Transgenic plants showed an enhanced resistance to the bacterial infection. In addition, this HCAA accumulation was accompanied by an increase in salicylic acid levels and pathogenesis-related gene induction. Taken together, these results suggest that HCAA may play an important role in the defense of tomato plants against P. syringae infection., The authors are grateful to C. Torres, A. Sauri, and T. Caballero for technical support. We also thank C. Gomez-Mena for her assistance in tomato flower sampling. This work was supported by Grant BIO2012-33419 from the Direccion General de Programas y Transferencia de Conocimiento, from the Spanish Ministry of Science and Innovation. L. Campos was the recipient of a predoctoral fellowship ACIF/2010/231 from Generalitat Valenciana (Spain). The work presented here was carried out in collaboration between all authors. J. M. Belles defined the research theme. L. Campos, L. Zacares and M. P. Lopez-Gresa carried out the laboratory experiments. P. Lison and I. Rodrigo contributed to the experimental design and the interpretation of the data. L. Campos drafted the article. M. P. Lopez-Gresa, P. Lison and I. Rodrigo participated in revising it critically for important intellectual content. J. M. Belles and V. Conejero gave the final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for the content.

Xanthans occurring in different levels of disordered conformation were enzymatically hydrolyzed to their six pentamer repeating units (RUs). The RUs present in the enzyme digests were analyzed using LC–MS. As only disordered xanthan segments are degraded by cellulases, the influence of the six different RUs on the transitional behavior of xanthan could be studied. The results indicate that especially xanthan segments rich in RUs that are acetylated on the outer mannose unit stabilize the xanthan conformation. Acetylation of the inner mannose did not show to have a clear stabilizing effect on the xanthan conformation. As the enzymatic release of pyruvylated RUs gradually increased with increasing levels of disordered xanthan segments, it is concluded that the distribution of these RUs is random. On the contrary, single and double acetylated RUs are distributed over specific xanthan segments as these RUs were instantly hydrolyzed.

Differently modified xanthans, varying in degree of acetylation and/or pyruvylation were incubated with the experimental cellulase mixture C1-G1 from Myceliophthora thermophila C1. The ionic strength and/or temperature of the xanthan solutions were varied, to obtain different xanthan conformations. The exact conformation at the selected incubation conditions was determined by circular dichroism. The xanthan degradation was analyzed by size exclusion chromatography. It was shown that at a fixed xanthan conformation, the backbone degradation by cellulases is equal for each type of xanthan. Complete backbone degradation is only obtained at a fully disordered conformation, indicating that only the secondary xanthan structure influences the final degree of hydrolysis by cellulases. It is thereby shown that, independently on the degree of substitution, xanthan can be completely hydrolyzed to oligosaccharides. These oligosaccharides can be used to further investigate the primary structure of different xanthans and to correlate the molecular structure to the xanthan functionalities.

Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS) circuits. These QS systems either use N-acyl homoserine lactones (AHL) or cis-2-dodecenoic acid ("Burkholderia diffusible signal factor'', BDSF) as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870), CciI (BCAM0239a) and the BDSF synthase (BCAM0581) were inactivated, and also constructed double (Delta cepI Delta BCAM0581, Delta cciI Delta BCAM0581 and Delta cepI Delta cciI) mutants and a triple (Delta cepI Delta cciI Delta BCAM0581) mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans) and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The Delta BCAM0581 mutant and the Delta cepI Delta BCAM0581 and Delta cciI Delta BCAM0581 double mutants produced significantly less AHL compared to the WT strain and the Delta cepI and Delta cciI single mutant, respectively. The expression of cepI and cciI in Delta BCAM0581, was approximately 3-fold and 7-fold (p < 0.05) lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the Delta BCAM0581 mutant could (at least partially) be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a lesser extent the C8-HSL synthase CepI) is partially controlled by BDSF, it seems likely that the BDSF QS systems controls AHL production through this system.

International audience; Many plant bacteriologists, if not all, feel that their particular microbe should appear in any list of the most important bacterial plant pathogens. However, to our knowledge, no such list exists. The aim of this review was to survey all bacterial pathologists with an association with the journal Molecular Plant Pathology and ask them to nominate the bacterial pathogens they would place in a Top 10 based on scientific/economic importance. The survey generated 458 votes from the international community, and allowed the construction of a Top 10 bacterial plant pathogen list. The list includes, in rank order: (1) Pseudomonas syringae pathovars; (2) Ralstonia solanacearum; (3) Agrobacterium tumefaciens; (4) Xanthomonas oryzae pv. oryzae; (5) Xanthomonas campestris pathovars; (6) Xanthomonas axonopodis pathovars; (7) Erwinia amylovora; (8) Xylella fastidiosa; (9) Dickeya (dadantii and solani); (10) Pectobacterium carotovorum (and Pectobacterium atrosepticum). Bacteria garnering honourable mentions for just missing out on the Top 10 include Clavibacter michiganensis (michiganensis and sepedonicus), Pseudomonas savastanoi and Candidatus Liberibacter asiaticus. This review article presents a short section on each bacterium in the Top 10 list and its importance, with the intention of initiating discussion and debate amongst the plant bacteriology community, as well as laying down a benchmark. It will be interesting to see, in future years, how perceptions change and which bacterial pathogens enter and leave the Top 10.

Background Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H2O2 and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria. Results In this study we report the characterisation of organic hydroperoxide resistance gene ohr and its regulator ohrR in S. meliloti. The inactivation of ohr affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide or menadione in vitro. The expression of ohr and ohrR genes is specifically induced by organic peroxides. OhrR binds to the intergenic region between the divergent genes ohr and ohrR. Two binding sites were characterised. Binding to the operator is prevented by OhrR oxidation that promotes OhrR dimerisation. The inactivation of ohr did not affect symbiosis and nitrogen fixation, suggesting that redundant enzymatic activity exists in this strain. Both ohr and ohrR are expressed in nodules suggesting that they play a role during nitrogen fixation. Conclusions This report demonstrates the significant role Ohr and OhrR proteins play in bacterial stress resistance against organic peroxides in S. meliloti. The ohr and ohrR genes are expressed in nodule-inhabiting bacteroids suggesting a role during nodulation.

A novel and more comprehensive formulation of the optimal control problem that reflects the operational requirements of a typical industrial fermentation has been proposed in this work. This formulation has been applied to a fed-batch bioreactor with three control variables, i.e., feed rates of carbon source, nitrogen source, and an oxygen source, to result in a 148.7% increase in product formation. Xanthan gum production using Xanthomonas campestris has been used as the model system for this optimization study, and the liquid-phase oxygen supply strategy has been used to supply oxygen to the fermentation. The formulated optimization problem has several constraints associated with it due to the nature of the system. A robust stochastic technique, differential evolution, has been used to solve this challenging optimization problem. The infinite dimensional optimization problem has been approximated to a finite dimensional one by control vector parametrization. The state constraints that are path constraints have been addressed by using penalty functions and by integrating them over the total duration to ensure a feasible solution. End point constraints on final working volume of the reactor and on the final residual concentrations of carbon and nitrogen sources have been included in the problem formulation. Further, the toxicity of the oxygen source, H(2)O(2), has been addressed by imposing a constraint on its maximum usable concentration. In addition, the initial volume of the bioreactor contents and feed concentrations have been handled as decision variables, which has enabled a well-grounded choice for their values from the optimization procedure; adhoc values are normally used in the industry. All results obtained by simulation have been validated experimentally with good agreements between experimental and simulated values.

Intracellular reactive oxygen species (iROS) induction by HOCl was used as a novel strategy to improve enzyme productivities in Aspergillus niger growing in a bioreactor. With induced iROS, the specific intracellular activities of alpha-amylase, protease, catalase, and glucose oxidase were increased by about 170%, 250%, 320%, and 260%, respectively. The optimum specific iROS level for achieving maximum cell concentration and enzyme production was about 15 mmol g cell(-1). The type of iROS inducing the enzyme production was identified to be a derivative of the superoxide radical.

Cultivations of Aspergillus niger cells in which oxygen was provided through the liquid-phase oxygen supply strategy (which involves the need-based decomposition of H 2 O 2 pulses to yield the necessary oxygen) were studied. Concentrations of H 2 O 2 in the range of 1 to 5 mM were found to be suitable for use in cultivations. The growth rate constant of 0.17 h -1 obtained in the H 2 O 2 -based cultivation, was comparable to that obtained in the cultivation with aeration; however, the maximum cell concentration in the H 2 O 2 -based cultivation was 124% of that obtained in cultivation with aeration. Maximum concentrations of catalase, protease and glucose oxidase obtained in the H 2 O 2 -based cultivation, were 240%, 172%, and 124% respectively, of those obtained in the cultivation with aeration. Further, the specific enzyme levels (units per gram cell) of catalase and protease obtained in the H 2 O 2 -based cultivation were 172% and 156% of those obtained in the cultivation with aeration, whereas, the specific glucose oxidase levels were comparable. In addition, the oxygen profiles inside the pellets of A. niger during both the modes of oxygen supply were described using mathematical models. Studies to elucidate the mechanism of oxygen availability showed that the decomposition of the H 2 O 2 supplied in the extracellular space occurred intracellularly, as well as extracellularly, to yield oxygen. Also, the proton motive force (PMF) was found to be involved in the process of oxygen availability from H 2 O 2 to A. niger cells.

The dielectric and conductometric properties of aqueous solutions of xanthan, an ionic polysaccharide consisting of a linear cellulosic backbone with three-sugar side chains, have been extensively investigated over two frequency ranges, from 1 kHz to 10 MHz and from 1 MHz to 1 GHz by means of frequency domain dielectric spectroscopy, at different polyion concentrations and at two different temperatures, above room temperature, in the range where an order-disorder transition occurs. The dielectric spectra have been analyzed on the basis of two contiguous dielectric dispersions described by a Cole-Cole relaxation function, and the parameters have been discussed on the basis of currently accepted dielectric relaxation theories of polyelectrolyte solutions. For the majority of the polyion concentrations employed, a semidilute regime occurs and the two observed dielectric relaxation are ascribed to counterion fluctuations on two different scale lengths, the first associated to the polymer contour length and the second one to an average distance between charged groups of adjacent polyions. Moreover, these measurements suggests that a conformational transition occurs at temperatures above 40 degrees C with the consequent formation of an extended three-dimensional network. This transition results in a particular behavior of the dielectric relaxation on the polymer concentration.