Antibiosis plays a role in the context of direct interaction during antagonism of Paenibacillus polymyxa towards Fusarium oxysporum

Interaction of Fusarium oxysporum and Paenibacillus polymyxa starts with polar attachment of bacteria to the fungal hyphae followed by the formation of a large cluster of non-motile cells embedded in an extracellular matrix in which the bacteria develop endospores. Enumeration of fungal viable counts showed that less than one of 36,000 colony-forming units survived in paired cultures for 71 h. Effective antagonism was not observed below pH5 and was specific for the bacterial species. Development of F. oxysporum was inhibited in cell-free filtrates derived from cultures of P. polymyxa, but was much more strongly repressed in the presence of living bacteria. Furthermore, recovery of fungal growth started immediately after addition of antibiotics to paired cultures. Restoration of fungal growth was enhanced in filtrates that were supplemented with MgCl2, which suggests that anti-fungal compounds produced by the bacteria were counteracted by magnesium ions. In paired cultures, fungal counts remained very low, even in the presence of the magnesium salt. This study clearly showed that P. polymyxa antagonizes the plant pathogenic fungus F. oxysporum in liquid medium by means of an interaction process in which the presence of living bacteria is a prerequisite for continuous suppression of fungal growth.     

Optical-fiber bundles

Optical-fiber bundles have been employed as a versatile substrate for the fabrication of high-density microwell arrays. In this minireview, we discuss the application of optical-fiber-bundle arrays for a variety of biological problems. For genomics studies and microbial pathogen detection, individual beads have been functionalized with DNA probes and then loaded into the microwells. In addition, beads differentially responsive to vapors have been employed in an artificial olfaction system. Microwell arrays have also been loaded with living cells to monitor their individual response to biologically active compounds over long periods. Finally, the microwells have been sealed to enclose single enzyme molecules that can be used to measure individual molecule catalytic activity.     

Synthesis of a fluorescent ganglioside GM1 derivative and screening of a synthetic peptide library for GM1 binding sequence motifs

A ganglioside GM1 probe bearing a dark-red fluorescent dye at the sphingosine moiety of the molecule was prepared by a convenient one-pot synthesis. The labeled GM1 permitted the detection of the natural ganglioside GM1 ligand Escherichia coli heat-labile enterotoxin subunit B (EtxB) in picomole quantities on a solid support. When an epitope mapping of several ganglioside binding proteins and protein fragments was performed by screening a cellulose membrane-bound synthetic library of 64 16mer peptides with the new probe, several peptides displaying ganglioside GM1 affinity could be identified. We consider the labeled glycolipid described herein a versatile tool for manifold biochemical investigations.     

Interactions of nisin and pediocin PA-1 with closely related lactic acid bacteria that manifest over 100-fold differences in bacteriocin sensitivity.

The natural variation in the susceptibilities of gram-positive bacteria towards the bacteriocins nisin and pediocin PA-1 is considerable. This study addresses the factors associated with this variability for closely related lactic acid bacteria. We compared two sets of nonbacteriocinogenic strains for which the MICs of nisin and pediocin PA-1 differed 100- to 1,000-fold: Lactobacillus sake DSM20017 and L. sake DSM20497 and Pediococcus dextrinicus and Pediococcus pentosaccus. Strikingly, the bacteriocin-sensitive and -insensitive strains showed a similar concentration-dependent dissipation of their membrane potential (delta psi) after exposure to these bacteriocins. The bacteriocin-induced dissipation of delta psi below the MICs for the insensitive strains did not coincide with a reduction of intracellular ATP pools and glycolytic rates. This was not observed with the sensitive strains. Analysis of membrane lipid properties revealed minor differences in the phospho- and glycolipid compositions of both sets of strains. The interactions of the bacteriocins with strain-specific lipids were not significantly different in a lipid monolayer assay. Further lipid analysis revealed higher in situ membrane fluidity of the bacteriocin-sensitive Pediococcus strain compared with that for the insensitive strain, but the opposite was found for the L. sake strains. Our results provide evidence that the association of bacteriocins with the cell membrane and their subsequent insertion take place in a similar way for cells that have a high or a low natural tolerance towards bacteriocins. For insensitive strains, overall membrane constitution rather than mere membrane fluidity may preclude the formation of pores with sufficient diameters and lifetimes to ultimately cause cell death.     

Comparing Nonsynergistic Gamma Models with Interaction Models To Predict Growth of Emetic Bacillus cereus when Using Combinations of pH and Individual Undissociated Acids as Growth-Limiting Factors

A combination of multiple hurdles to limit microbial growth is frequently applied in foods to achieve an overall level of protection. Quantification of hurdle technology aims at identifying synergistic or multiplicative effects and is still being developed. The gamma hypothesis states that inhibitory environmental factors aiming at limiting microbial growth rates combine in a multiplicative manner rather than synergistically. Its validity was tested here with respect to the use of pH and various concentrations of undissociated acids, i.e., acetic, lactic, propionic, and formic acids, to control growth of Bacillus cereus in brain heart infusion broth. The key growth parameter considered was the maximum specific growth rate, μ_max, as observed by determination of optical density. A variety of models from the literature describing the effects of various pH values and undissociated acid concentrations on μ_max were fitted to experimental data sets and compared based on a predefined set of selection criteria, and the best models were selected. The cardinal model developed by Rosso (for pH dependency) and the model developed by Luong (for undissociated acid) were found to provide the best fit and were combined in a gamma model with good predictive performance. The introduction of synergy factors into the models was not able to improve the quality of the prediction. On the contrary, inclusion of synergy factors led to an overestimation of the growth boundary, with the inherent possibility of leading to underestimation of the risk under the conditions tested in this research.Consumers expect safe and sufficiently stable food within the given shelf life of a food product or component. Several growth-limiting factors, collectively referred to as hurdles, can be used to ensure food stability and safety. Examples of such hurdles are low pH, low water activity, or low temperature (12). Combining hurdles to achieve food stability and safety, known as hurdle technology, can be used to achieve an overall level of protection in food while minimizing impacts on food quality (20). When a combination of hurdles is used, generally the intensity of the hurdles may be lower, to exert a comparable preservative effect, than the intensity of those hurdles when used individually (20). Three classes of interaction can be defined when applying hurdle technology: “no interaction,” in which the effect of a combination is as expected from the response of the separate factors; “synergy,” in which the effect is greater than expected; and “antagonism,” in which the effect is less than expected (6).Though the concept of hurdle technology is rather well established, the quantification of the combined impact of hurdles on growth of microorganisms is still being developed. One significant problem is that there are two opposite views of how antimicrobial factors combine. One view states that there are interactive effects between hurdles; when they are applied together, they give a protection significantly greater than that expected on the basis of the application of the individual hurdles (synergy). The alternative view considers that the combined effect may be complex but that there are no interactive effects culminating in synergy. The latter view is called the gamma hypothesis (41) and states that inhibitory environmental factors combine in a multiplicative manner to produce the observed overall microbial inhibition. A major benefit of models based on the gamma hypothesis is a reduction in experimental work, since growth rates and, as a result, growth boundaries can be estimated upon evaluating single hurdles rather than their various combinations. This benefit can only be realized, however, when the gamma hypothesis is valid for the combination of hurdles considered. If the hypothesis is not valid and interactive effects are present, growth boundaries are estimated wrongly, which might result in fail-safe predictions.Over the years, the gamma hypothesis has been confirmed by several studies (16, 17, 26, 34, 38) that concluded that the combined effect of hurdles on growth rates is multiplicative rather than synergistic. Contrarily, Rödel and Scheuer (30) concluded that interaction occurs when various hurdles are combined, stressing the occurrence of synergy. Both Le Marc et al. (21) and Augustin and Carlier (5) developed a synergy model to take account of synergy occurring when hurdles are combined. It is prudent to conclude that the effect of combinations of hurdles is best evaluated on a case-by-case basis in order to ensure appropriate utility of hurdle technology approaches in establishing food designs that are stable and safe.This research aimed to validate or falsify the gamma hypothesis for two closely related hurdles often used in the food industry: the pH level and the undissociated acid concentration ([HA]). The approach chosen was to establish an overview of models for pH and undissociated acid from the literature. Based on predefined criteria, models were then selected to construct a new gamma model without synergy factors for the various hurdle combinations. The criteria were meant to enable evaluation of the fitting performance of all individual models to select the best-performing models for inclusion in the new gamma models. Finally, the validity of the gamma hypothesis was judged by comparing the predictive performance of the newly constructed gamma models with two gamma models, including a synergy factor reported in the literature. Bacillus cereus F4810/72, relevant for both food spoilage and poisoning (14, 19), was used as the model microorganism. Maximum specific growth rates were determined by optical density measurements combined with time to detection. This method was selected after thorough investigation of three different methods to obtain parameters for growth, as recently published (8).     

Transgenic expression in citrus of single-chain antibody fragments specific to <Emphasis Type=Italic>Citrus tristeza virus</Emphasis> confers virus resistance

Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40–60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.     

Differentiation of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica isolated from potato by Western blot and subsequent indirect ELISA

Electrotransfer of protein bands from a polyacrylamide gel to a hydrophobic poly-vinylidene difluoride (PVDF) membrane (Western blot) and their serological determination by indirect ELISA (immunoblotting) were used to differentiate Erwinia carotovora subsp. carotovora (Ecc) from Erwinia carotovora subsp. atroseptica (Eca). Ninety strains: 69 Ecc, 19 Eca and two Erwinia chrysanthemi (Echr) were examined. Eight polyclonal antisera against whole cells, glutaraldehyde fixed cells, glycopro-teins, and somatic antigens were prepared. Antisera produced with glutaraldehyde fixed cells did not recognize any band of the protein pattern. The remaining antisera recognized a limited number of bands. Two protein bands allowed differentiation of the two subspecies by the antisera against glycoproteins. One band with an estimated molecular weight of 36000 Da was present in the 19 Eca strains tested and another band with an estimated molecular weight of 35 000 Da was present in the 69 Ecc strains, except for three cases. The strains of Echr showed a band with an estimated weight of 33 000 Da.     

Generation and characterisation of functional recombinant antibody fragments against RNA replicase NIb from plum pox virus

A monoclonal antibody (mAb 2A) able to react against the RNA replicase NIb from plum pox virus (PPV) was obtained and used for generating a specific scFv fragment. The VH and VL coding sequences were cloned and expressed as a fusion scFv protein to alkaline phosphatase. This fusion protein was able to recognise viral NIb in both Western and tissue-print ELISA blots. The affinity and specificity of scFv2A for NIb was similar to that of the parental mAb and the region YLEAFY from PPV-NIb was identified by PEPSCAN assay as the putative epitope. Isolated VH domains from scFv2A were also expressed as fusion to alkaline phosphatase. However, their ability to react against NIb was greatly altered. scFv2A fragments were transiently expressed in the cytosol of Nicotiana benthamiana and although they accumulated to low levels, inhibition-ELISA results indicated that they retained antigen-binding activity.     

Comparative study on the action of S-(+)-carvone, in situ, on the potato storage fungi Fusarium solani var. coeruleum and F. sulphureum

Growth of Fusarium sulphureum was inhibited by S-(+)-carvone administered via the gas phase. Under the same conditions, the related fungus F. solani var. coeruleum was not inhibited. In liquid medium, both fungi were found to convert S-(+)-carvone with the same rate, mainly into isodihydrocarvone, isodihydrocarveol and neoisodihydrocarveol. Only a slight difference in the relative amounts of the bioconversion products was observed. Since the bioconversion products did not inhibit the growth of the fungi to the same extent as S-(+)-carvone, the process can be considered as a detoxification mechanism. The bioconversion as such cannot account for the observed difference in growth inhibition.