Multivariate Optimization of the FLC-dc-APGD-Based Reaction-Discharge System for Continuous Production of a Plasma-Activated Liquid of Defined Physicochemical and Anti-Phytopathogenic Properties.

To the present day, no efficient plant protection method against economically important bacterial phytopathogens from the Pectobacteriaceae family has been implemented into agricultural practice. In this view, we have performed a multivariate optimization of the operating parameters of the reaction-discharge system, employing direct current atmospheric pressure glow discharge, generated in contact with a flowing liquid cathode (FLC-dc-APGD), for the production of a plasma-activated liquid (PAL) of defined physicochemical and anti-phytopathogenic properties. As a result, the effect of the operating parameters on the conductivity of PAL acquired under these conditions was assessed. The revealed optimal operating conditions, under which the PAL of the highest conductivity was obtained, were as follows: flow rate of the solution equaled 2.0 mL min-1, the discharge current was 30 mA, and the inorganic salt concentration (ammonium nitrate, NH4NO3) in the solution turned out to be 0.50% (m/w). The developed PAL exhibited bacteriostatic and bactericidal properties toward Dickeya solani IFB0099 and Pectobacterium atrosepticum IFB5103 strains, with minimal inhibitory and minimal bactericidal concentrations equaling 25%. After 24 h exposure to 25% PAL, 100% (1-2 × 106) of D. solani and P. atrosepticum cells lost viability. We attributed the antibacterial properties of PAL to the presence of deeply penetrating, reactive oxygen and nitrogen species (RONS), which were, in this case, OH, O, O3, H2O2, HO2, NH, N2, N2+, NO2-, NO3-, and NH4+. Putatively, the generated low-cost, eco-friendly, easy-to-store, and transport PAL, exhibiting the required antibacterial and physicochemical properties, may find numerous applications in the plant protection sector.

Antibacterial activity and mode of action of potassium tetraborate tetrahydrate against soft-rot bacterial plant pathogens.

Bacterial soft rot caused by the bacteria Dickeya and Pectobacterium is a destructive disease of vegetables, as well as ornamental plants. Several management options exist to help control these pathogens. Because of the limited success of these approaches, there is a need for the development of alternative methods to reduce losses. In this study, we evaluated the effect of potassium tetraborate tetrahydrate (PTB) on the growth of six Dickeya and Pectobacterium spp. Disc diffusion assays showed that Dickeya spp. and Pectobacterium spp. differ in their sensitivity to PTB. Spontaneous PTB-resistant mutants of Pectobacterium were identified and further investigation of the mechanism of PTB resistance was conducted by full genome sequencing. Point mutations in genes cpdB and supK were found in a single Pectobacterium atrosepticum PTB-resistant mutant. Additionally, point mutations in genes prfB (synonym supK) and prmC were found in two independent Pectobacterium brasiliense PTB-resistant mutants. prfB and prmC encode peptide chain release factor 2 and its methyltransferase, respectively. We propose the disruption of translation activity due to PTB leads to Pectobacterium growth inhibition. The P. atrosepticum PTB-resistant mutant showed altered swimming motility. Disease severity was reduced for P. atrosepticum-inoculated potato stems sprayed with PTB. We discuss the potential risk of selecting for bacterial resistance to this chemical.

Phage cocktail containing Podoviridae and Myoviridae bacteriophages inhibits the growth of Pectobacterium spp. under in vitro and in vivo conditions.

Globally, there is a high economic burden caused by pre- and post-harvest losses in vegetables, fruits and ornamentals due to soft rot diseases. At present, the control methods for these diseases are limited, but there is some promise in developing biological control products for use in Integrated Pest Management. This study sought to formulate a phage cocktail which would be effective against soft rot Pectobacteriaceae species affecting potato (Solanum tuberosum L.), with potential methods of application in agricultural systems, including vacuum-infiltration and soil drench, also tested. Six bacteriophages were isolated and characterized using transmission electron microscopy, and tested against a range of Pectobacterium species that cause soft rot/blackleg of potato. Isolated bacteriophages of the family Podoviridae and Myoviridae were able to control isolates of the Pectobacterium species: Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum. Genomic analysis of three Podoviridae phages did not indicate host genes transcripts or proteins encoding toxin or antibiotic resistance genes. These bacteriophages were formulated as a phage cocktail and further experiments showed high activity in vitro and in vivo to suppress Pectobacterium growth, potentially indicating their efficacy in formulation as a microbial pest control agent to use in planta.

Protein profiling analysis based on matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry and its application in typing Streptomyces isolates.

Marine Streptomyces is a potential source of novel bioactive natural products in medicine and agriculture. The current discrimination and screening method of Streptomyces isolates is not accurate and time-consuming, and a novel method is necessary. In this study, a protein profiling method based on an ultrahigh resolution 15 T Matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) was established and applied for differentiation and bioactivity screening of marine Streptomyces isolates. To obtain robust protein profiling, the effects of the protein extraction method, the matrix-solvent, the sample deposition mode, and the culture time of isolates on protein profiling were thoroughly studied, the optimal conditions were obtained. To evaluate the performance of the developed MALDI-FTICR MS method, MALDI-time of flight (TOF) MS and 16S rRNA were applied in parallel to analyze 25 marine Streptomyces isolates. We found that the clustering result of MALDI-FTICR MS was more similar to that of 16S rRNA than MALDI-TOF MS. And MALDI-FTICR MS could effectively indicate the antibacterial activity of Streptomyces isolates against three plant pathogenic bacteria including Xanthomonas campestris, Xanthomonas oryzae and Erwinia carotovora. Furthermore, a differential protein/peptide was defined and successfully applied to predict antibacterial activity of blind samples. This study demonstrated that MALDI-FTICR MS has great potential to discriminate and screen complex microorganisms, especially those closely related strains.

Threat of establishment of non-indigenous potato blackleg and tuber soft rot pathogens in Great Britain under climate change.

Potato blackleg and soft rot caused by Pectobacterium and Dickeya species are among the most significant bacterial diseases affecting potato production globally. In this study we estimate the impact of future temperatures on establishment of non-indigenous but confirmed Pectobacterium and Dickeya species in Great Britain (GB). The calculations are based on probabilistic climate change data and a model fitted to disease severity data from a controlled environment tuber assay with the dominant potato blackleg and soft rot-causing species in GB (P. atrosepticum), and three of the main causative agents in Europe (P. carotovorum subsp. brasiliense, P. parmentieri, Dickeya solani). Our aim was to investigate if the European strains could become stronger competitors in the GB potato ecosystem as the climate warms, on the basis of their aggressiveness in tubers at different temperatures. Principally, we found that the tissue macerating capacity of all four pathogens will increase in GB under all emissions scenarios. The predominant Pectobacterium and Dickeya species in Europe are able to cause disease in tubers under field conditions currently seen in GB but are not expected to become widely established in the future, at least on the basis of their aggressiveness in tubers relative to P. atrosepticum under GB conditions. Our key take-home messages are that the GB potato industry is well positioned to continue to thrive via current best management practices and continued reinforcement of existing legislation.

The viability of lytic bacteriophage ΦD5 in potato-associated environments and its effect on Dickeya solani in potato (Solanum tuberosum L.) plants.

Dickeya solani is one of the most important pectinolytic phytopathogens responsible for high losses in potato, especially in seed potato production in Europe. Lytic bacteriophages can affect the structure of the host population and may influence spread, survival and virulence of the pathogen and in consequence, infection of the plant. In this study, we aimed to acquire information on the viability of the broad host lytic bacteriophage ΦD5 on potato, as well as to apprehend the specific effect of this bacteriophage on its host D. solani type-strain in different settings, as a preliminary step to target co-adaptation of phages and host bacteria in plant environment. Viability of the ΦD5 phage in tuber extract, on tuber surface, in potting compost, in rainwater and on the leaf surface, as well as the effect of copper sulfate, were examined under laboratory conditions. Also, the interaction of ΦD5 with the target host D. solani in vitro and in compost-grown potato plants was evaluated. ΦD5 remained infectious in potato tuber extract and rain water for up to 72 h but was inactivated in solutions containing 50 mM of copper. The phage population was stable for up to 28 days on potato tuber surface and in potting compost. In both, tissue culture and compost-grown potato plants, ΦD5 reduced infection by D. solani by more than 50%. The implications of these findings are discussed.

Biocontrol of the Potato Blackleg and Soft Rot Diseases Caused by Dickeya dianthicola.

Development of protection tools targeting Dickeya species is an important issue in the potato production. Here, we present the identification and the characterization of novel biocontrol agents. Successive screenings of 10,000 bacterial isolates led us to retain 58 strains that exhibited growth inhibition properties against several Dickeya sp. and/or Pectobacterium sp. pathogens. Most of them belonged to the Pseudomonas and Bacillus genera. In vitro assays revealed a fitness decrease of the tested Dickeya sp. and Pectobacterium sp. pathogens in the presence of the biocontrol agents. In addition, four independent greenhouse assays performed to evaluate the biocontrol bacteria effect on potato plants artificially contaminated with Dickeya dianthicola revealed that a mix of three biocontrol agents, namely, Pseudomonas putida PA14H7 and Pseudomonas fluorescens PA3G8 and PA4C2, repeatedly decreased the severity of blackleg symptoms as well as the transmission of D. dianthicola to the tuber progeny. This work highlights the use of a combination of biocontrol strains as a potential strategy to limit the soft rot and blackleg diseases caused by D. dianthicola on potato plants and tubers.

Microbial population dynamics in response to Pectobacterium atrosepticum infection in potato tubers.

Endophytes are microbes and fungi that live inside plant tissues without damaging the host. Herein we examine the dynamic changes in the endophytic bacterial community in potato (Solanum tuberosum) tuber in response to pathogenic infection by Pectobacterium atrosepticum, which causes soft rot in numerous economically important crops. We quantified community changes using both cultivation and next-generation sequencing of the 16S rRNA gene and found that, despite observing significant variability in both the mass of macerated tissue and structure of the endophytic community between individual potato tubers, P. atrosepticum is always taken over by the endophytes during maceration. 16S rDNA sequencing revealed bacteria from the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Fusobacteria, Verrucomicrobia, Acidobacteria, TM7, and Deinococcus-Thermus. Prior to infection, Propionibacterium acnes is frequently among the dominant taxa, yet is out competed by relatively few dominant taxa as the infection proceeds. Two days post-infection, the most abundant sequences in macerated potato tissue are Gammaproteobacteria. The most dominant genera are Enterobacter and Pseudomonas. Eight days post-infection, the number of anaerobic pectolytic Clostridia increases, probably due to oxygen depletion. These results demonstrate that the pathogenesis is strictly initiated by the pathogen (sensu stricto) and proceeds with a major contribution from the endophytic community.

Penicillin V acylase from Pectobacterium atrosepticum exhibits high specific activity and unique kinetics.

Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria.

Effect of overhead spray and brush roller treatment on the survival of Pectobacterium and Salmonella on tomato surfaces.

Overhead spray and brush roller (OSBR) treatment has been shown to remove significantly more Salmonella from tomato surfaces than flume treatment. However, OSBR is not widely used in tomato packing facilities compared with other commodities, and little is known about whether brushing causes microabrasions or other physical damage. Bacteria such as Pectobacterium, a soft rot-producing plant pathogen, and Salmonella, a human pathogen, show increased survival and growth on damaged tomato surfaces. This study evaluated whether OSBR treatment had a negative effect on the safety and/or marketability of tomatoes by examining its effect on Pectobacterium and Salmonella survival. Pectobacterium survival was evaluated on inoculated tomatoes that were OSBR treated with water or sanitizer (100 ppm of NaOCl, 5 ppm of ClO2, or 80 ppm of peracetic acid). A 15-s OSBR treatment using water or sanitizer achieved a 3-log CFU/ml reduction in Pectobacterium levels. Survival of Pectobacterium and Salmonella on OSBR-treated, untreated, and puncture-wounded tomatoes stored at 25°C and 75 to 85 % relative humidity for 7 days was also assessed. Both Pectobacterium and Salmonella populations declined rapidly on OSBR-treated and untreated tomatoes, indicating that brushing does not damage tomato fruit to the extent of promoting better pathogen survival. In contrast, the survival of both organisms was significantly (P ≤ 0.05) higher on artificially wounded fruit. These results indicate that OSBR treatment does not increase the survival and growth of Pectobacterium or Salmonella on tomato surfaces and that it is effective in reducing Pectobacterium levels on the surface of inoculated tomatoes. These results suggest that, if used properly, an OSBR system in packinghouses is effective in removing surface contamination and does not affect tomato quality or safety.

Antibacterial activity of carob (Ceratonia siliqua L.) extracts against phytopathogenic bacteria Pectobacterium atrosepticum.

Acetone and ethanol extracts of carob (Ceratonia siliqua L.) leaf and pods were evaluated for their in vitro inhibitory ability against the pectinolytic Gram negative Pectobacterium atrosepticum (Pca, CFBP-5384) bacteria, the causal agent of potato soft rot. Potato (Solanum tuberosum, var nicola) tuber rot tissues obtained after 5 day bacterial inoculation was analyzed by LC-MS and GC-MS to study Pca pathogenicity. Trans/cis N-feruloylputrescine was identified in potato tuber after 5-day inoculation with Pca in a dark moist chamber. Although glycoalkoloid (α-chaconine and α-solanine) production increased due to Pca soft rot infection, it was not a resistance-determining factor. Many secondary metabolites were identified including the phytoalexins solavetivone and fatty acids responsible for plant defence responses. Acetone extract of carob leaf (FCA) exhibited the strongest inhibitory effect (IC50 = 1.5 mg/ml) and displayed synergistic antimicrobial effect in the presence of infected potato tuber extract (Pdt-Pca extract) against Pca. This synergy could be used in an integrated control program against potato soft rot pathogens, thereby reducing chemical treatments.

Postharvest application of organic and inorganic salts to control potato (Solanum tuberosum L.) storage soft rot: plant tissue-salt physicochemical interactions.

Soft rot caused by Pectobacterium sp. is a devastating disease affecting stored potato tubers, and there is a lack of effective means of controlling this disease. In this study, 21 organic and inorganic salts were tested for their ability to control soft rot in potato tubers. In the preventive treatment, significant control of soft rot was observed with AlCl3 (≥66%) and Na2S2O3 (≥57%) and to a lesser extent with Al lactate and Na benzoate (≥34%) and K sorbate and Na propionate (≥27%). However, only a moderate control was achieved by curative treatment with AlCl3 and Na2S2O3 (42%) and sodium benzoate (≥33%). Overall, the in vitro inhibitory activity of salts was attenuated in the presence of plant tissue (in vivo) to different degrees. The inhibitory action of the salts in the preventive treatment, whether effective or otherwise, showed an inverse linear relationship with water ionization capacity (pK') of the salt ions, whereas in the curative treatment, only the effective salts showed this inverse linear relationship. Salt-plant tissue interactions appear to play a central role in the attenuated inhibitory activity of salts in potato tuber through reduction in the availability of the inhibitory ions for salt-bacteria interactions. This study demonstrates that AlCl3, Na2S2O3, and Na benzoate have potential in controlling potato tuber soft rot and provides a general basis for understanding of specific salt-tissue interactions.

Dissociation of a population of Pectobacterium atrosepticum SCRI1043 in tobacco plants: formation of bacterial emboli and dormant cells.

The population dynamics of Pectobacterium atrosepticum SCRI1043 (Pba) within tobacco plants was monitored from the time of inoculation until after long-term preservation of microorganisms in the remnants of dead plants. We found and characterised peculiar structures that totally occlude xylem vessels, which we have named bacterial emboli. Viable but non-culturable (VBN) Pba cells were identified in the remnants of dead plants, and the conditions for resuscitation of these VBN cells were established. Our investigation shows that dissociation of the integrated bacterial population during plant colonisation forms distinct subpopulations and cell morphotypes, which are likely to perform specific functions that ensure successful completion of the life cycle within the plant.

Stress response in Pectobacterium atrosepticum SCRI1043 under starvation conditions: adaptive reactions at a low population density.

The adaptive reactions of plant pathogenic bacterium Pectobacterium atrosepticum SCRI1043 under starvation conditions were studied. The main emphasis was given to the peculiarities of stress responses depending on the bacterial population densities. When bacteria were subjected to starvation at high population densities (10(7)-10(9) CFU ml(-1)), their adaptive reactions conformed to the conventional conception of bacterial adaptation related to autolysis of part of the population, specific modification of cell ultrastructure, activation of expression of stress responsive genes and acquiring cross protection against other stress factors. In contrast, at low initial population densities (10(3)-10(5) CFU ml(-1)), as described in our recent work, the cell density increased due to multiple cell division despite the absence of exogenous growth substrate. Here we present data that demonstrate that such unconventional behavior is part of a stress response, which provides increased stress tolerance while retaining virulence. Cell morphology and gene expression in high- and low-cell-density starving Pba cultures were compared. Our investigation demonstrates the existence of alternative adaptive strategies enabling pathogenic bacteria to cope with a variety of stress factors, including starvation, especially necessary when residing outside of their host.

The endophytic lifestyle of Escherichia coli O157:H7: quantification and internal localization in roots.

The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants.

Lack of RsmA-mediated control results in constant hypervirulence, cell elongation, and hyperflagellation in Pectobacterium wasabiae.

The posttranscriptional regulator RsmA controls the production of plant cell wall degrading enzymes (PCWDE) and cell motility in the Pectobacterium genus of plant pathogens. In this study the physiological role of gene regulation by RsmA is under investigation. Disruption of rsmA gene of the Pectobacterium wasabiae strain, SCC3193 resulted in 3-fold decrease in growth rate and increased virulence. The comparison of mRNA levels of the rsmA(-) mutant and wild-type using a genome-wide microarray showed, that genes responsible for successful infection, i.e. virulence factors, motility, butanediol fermentation, various secretion systems etc. were up-regulated in the rsmA(-) strain. The rsmA(-) strain exhibited a higher propensity to swarm and produce PCWDE compared to the wild-type strain. Virulence experiments in potato tubers demonstrated that in spite of its more efficient tissue maceration, the rsmA(-) strain's ability to survive within the host is reduced and the infection site is taken over by resident bacteria. Taken together, in the absence of RsmA, cells revert to a constitutively infective phenotype characterized by expression of virulence factors and swarming. We hypothesize that lack of control over these costly energetic processes results in decreased growth rate and fitness. In addition, our findings suggest a relationship between swarming and virulence in plant pathogens.

Quorum sensing signaling molecules produced by reference and emerging soft-rot bacteria (Dickeya and Pectobacterium spp.).

BACKGROUND: Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. METHODOLOGY/PRINCIPAL FINDINGS: Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. CONCLUSIONS/SIGNIFICANCE: Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates.

Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in Pectobacterium spp.

In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium Pectobacterium carotovorum carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of Pectobacterium carotovorum and Pectobacterium atrosepticum with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that Pectobacterium spp. carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of Pectobacterium carotovorum and atrosepticum that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells.

Chemical structure of the O-polysaccharide isolated from Pectobacterium atrosepticum SCRI 1039.

The lipopolysaccharide (LPS) of the bacterium Pectobacterium atrosepticum SCRI 1039 was hydrolyzed and the products were separated. A study of the obtained O-polysaccharide by means of chemical methods, GLC, GLC-MS, and NMR spectroscopy allowed us to identify a branched polymer with a pentasaccharide repeating unit of the structure shown below, in which the fucose residue was partially O-acetylated at C-2, C-3 or C-4.

N-Acetylglucosamine-dependent biofilm formation in Pectobacterium atrosepticum is cryptic and activated by elevated c-di-GMP levels.

The phytopathogenic bacterium Pectobacterium atrosepticum (Pba) strain SCRI1043 does not exhibit appreciable biofilm formation under standard laboratory conditions. Here we show that a biofilm-forming phenotype in this strain could be activated from a cryptic state by increasing intracellular levels of c-di-GMP, through overexpression of a constitutively active diguanylate cyclase (PleD*) from Caulobacter crescentus. Randomly obtained Pba transposon mutants defective in the pga operon, involved in synthesis and translocation of poly-ß-1,6-N-acetyl-D-glucosamine (PGA), were all impaired in this biofilm formation. The presence of the PGA-degrading enzyme dispersin B in the growth media prevented biofilm formation by Pba overexpressing PleD*, further supporting the importance of PGA for biofilm formation by Pba. Importantly, a pga mutant exhibited a reduction in root binding to the host plant under conditions of high intracellular c-di-GMP levels. A modest but consistent increase in pga transcript levels was associated with high intracellular levels of c-di-GMP. Our results indicate tight control of PGA-dependent biofilm formation by c-di-GMP in Pba.