FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine–Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric–pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor–ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric–pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric–pyoverdines, i.e. the chromophore, iron, and its chelating groups.     

Sporicidal Activity of Synthetic Antifungal Undecapeptides and Control of Penicillium Rot of Apples

The antifungal activity of cecropin A(2-8)-melittin(6-9) hybrid undecapeptides, previously reported as active against plant pathogenic bacteria, was studied. A set of 15 sequences was screened in vitro against Fusarium oxysporum, Penicillium expansum, Aspergillus niger, and Rhizopus stolonifer. Most compounds were highly active against F. oxysporum (MIC < 2.5 μM) but were less active against the other fungi. The best peptides were studied for their sporicidal activity and for Sytox green uptake in F. oxysporum microconidia. A significant inverse linear relationship was observed between survival and fluorescence, indicating membrane disruption. Next, we evaluated the in vitro activity against P. expansum of a 125-member peptide library with the general structure R-X¹KLFKKILKX¹⁰L-NH₂, where X¹ and X¹⁰ corresponded to amino acids with various degrees of hydrophobicity and hydrophilicity and R included different N-terminal derivatizations. Fifteen sequences with MICs below 12.5 μM were identified. The most active compounds were BP21 {Ac,F,V} and BP34 {Ac,L,V} (MIC < 6.25 μM), where the braces denote R, X¹, and X¹⁰ positions and where Ac is an acetyl group. The peptides had sporicidal activity against P. expansum conidia. Seven of these peptides were tested in vivo by evaluating their preventative effect of inhibition of P. expansum infection in apple fruits. The peptide Ts-FKLFKKILKVL-NH₂ (BP22), where Ts is a tosyl group, was the most active with an average efficacy of 56% disease reduction, which was slightly lower than that of a commercial formulation of the fungicide imazalil.The discovery of antimicrobial compounds to treat plant diseases of economical importance in agriculture remains a major scientific challenge (1). Antimicrobial peptides are being considered as a good alternative to current fungicides and a great deal of scientific effort has been invested in studying their application in plant disease control (29, 34, 35).Antimicrobial peptides have been reported to display interesting activities against pathogenic microbes that are resistant to conventional antibiotics and to exhibit a broad spectrum of activity against bacteria, fungi, enveloped viruses, parasites, and tumor cells (7-10, 19, 20, 40, 49). The mechanism of action of these peptides against fungi consists of cell lysis by binding to the membrane surface and disrupting its structure, interference with the synthesis of essential cell wall components, or interaction with specific internal targets (12, 13, 15, 23, 29).Despite their good lytic activity, major concerns about the use of antimicrobial peptides as pesticides in plant protection are the high production cost associated with synthetic procedures and their low stability toward protease degradation. Several design strategies have been devised in order to find shorter and more stable peptides, while maintaining or increasing the activity with a low cytotoxicity. These strategies include the juxtaposition of fragments of natural antimicrobial peptides, the modification of natural peptides, and the de novo design of sequences maintaining the crucial features of native antimicrobial peptides (2, 3, 11, 24, 32, 38, 42). However, the process involved in the development of lead candidates is time consuming and limited by the number of individual compounds that can be synthesized. Combinatorial chemistry has allowed the rapid preparation of synthetic libraries and their screening has led to the identification of peptides with high activity against selected phytopathogenic bacteria and fungi (4, 26, 27, 33).During our current research oriented to the development of new antimicrobial agents for use in plant protection, we designed linear undecapeptides (CECMEL11) derived from the cecropin A-melittin hybrid peptide WKLFKKILKVL-NH₂ (Pep3) (5, 17). Using a combinatorial approach, we identified peptides with high activity against plant pathogenic bacteria, such as Erwinia amylovora, Xanthomonas vesicatoria, and Pseudomonas syringae, and with low susceptibility to protease degradation (4, 5).In order to broaden the study, we decided to test the CECMEL11 peptides against the plant pathogenic fungi Fusarium oxysporum, Aspergillus niger, Rhizopus stolonifer, and Penicillium expansum. The fungus F. oxysporum causes Fusarium wilt in more than a hundred species of plants, and it is an important pathogen in horticultural crops (44). Several Rhizopus and Penicillium species cause soft rot and blue mold rot, respectively, which are important postharvest diseases in stone and pome fruits (6, 18, 22, 39). Apart from the economic losses, Aspergillus and Penicillium species are also of interest from a public health point of view due to the production of mycotoxins (45, 47). The importance of Penicillium species in the postharvest of fruits emphasizes the interest to develop antimicrobial peptides to control this fungus.Taking into account the relevance of these pathogens, the aim of the present study was the analysis of the antifungal activity profile of the CECMEL11 peptides in order to identify sporicidal sequences against the above fungi. As a proof of concept, the feasibility of using such peptides to protect fruits from fungal spoilage was evaluated using a P. expansum/apple model.     

Loss of plasmids of Borrelia burgdorferi sensu lato during prolonged in vitro cultivation

In the present study we analyzed stability of plasmid content in 34 Borrelia strains of three different species (13 Borrelia afzelii, 10 Borrelia garinii and 11 Borrelia burgodorferi sensu stricto) using pulse field gel electrophoresis (PFGE). During long-term in vitro cultivation consisting of 50 passages, plasmid loss was established in 46% of B. afzelii, 40% of B. garinii and 36% of B. burgdorferi sensu stricto strains. Loss of plasmids occurred as early as between the 5th and 10th passage, affected only plasmids in the range 9-41 kb but not plasmids in the range 50-68 kb and manifested with the loss of one to up to three plasmids.     

Detection of wood boring insects by measurement of oxygen consumption

A possible method for detection of wood-boring insects in wood is the measurement of oxygen consumption. In order to develop such a method, several parameters have to be determined first. The most important one is to determine the respiration rate of possible pest species. To obtain the data for establishing the method mentioned above, the respiration rates of the following species were determined: the old house borer, Hylotrupes bajulus, common furniture beetle, Anobium punctatum and subterranean termite, Reticulitermes lucifugus. We compared the O₂ consumption between different species as well as between adults and larvae of one species in different environmental conditions (temperature, day/night and light/dark regime). The most intensive respiration rates were found for the larvae of Hylotrupes bajulus (4.0 ml/g h O₂) and Anobium punctatum (3.9 ml/g h O₂). Less intensive breathing was measured by Reticulitermes lucifugus (workers 2.9 ml/g h, nymphs 2.6 ml/g.h and soldiers 2.0 ml/g h O₂). These results indicate that it is possible to detect the presence of wood-boring insects respirometrically. To detect the presence of an individual insect in the wood by means of respirometry, the sensitivity of the instrument for oxygen measurement at the optimal conditions must be around 0.2 ml/h.     

Long-term changes in wood density and radial growth of Quercus petraea Liebl. in northern France since the middle of the nineteenth century

Long-term changes in sessile oak (Quercus petraea Liebl.) growth and wood density were studied using cores collected from 99 even-aged high forest stands between 56 and 187 years old, located in northeastern and north-central France. Growth and density trends were tested by analysis of variance and covariance. Two models were applied to two samples, sample A and sample B (sample B being a sub-sample with limited cambial age and calendar date ranges). Model 1 showed a significant increase in radial growth: +35%, +87% and +66% in earlywood width, latewood width and ring width, respectively, from 1811 to 1993 for sample A. Consequently, there was a positive trend in latewood ratio (+14%). A slight decrease in wood density was found: -3.3% and -5.4% for earlywood and latewood density, respectively. Despite an increase in latewood percentage, mean ring density showed a -2.0% decrease. Model 1 applied to a biomass indicator (density2ring width) showed a 62% increase from 10.4 to 16.8 kg m^-3 between 1811 and 1993 for sample A. Results for sample B were slightly different: the increase in latewood ratio was not detected. Model 2 showed a change with time in the positive hyperbolic relationship between mean density and ring width. The results are discussed. The decrease in wood density cannot be explained by N atmospheric deposition or by long-term changes in average temperature. Increasing atmospheric CO₂ levels cannot be invoked owing to the present lack of studies. Finally, hypotheses concerning long-term changes in wood anatomical characteristics are proposed.     

Stomoxys calcitrans,mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re‐emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re‐examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non‐infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.     

Binuclear manganese(III) complexes of an unsymmetric pyrazolate-based compartmental ligand with hard donor set

The synthesis, crystal structure and magnetic properties of manganese(III) binuclear complexes [Mn^III₂(L-3Н)₂(CH₃ОH)₄]·2CH₃ОH (1) and [Mn^III₂(L-3Н)₂(Py)₄]·2Py (2) (L = 3-[(1E)-N-hydroxyethanimidoyl]-4-methyl-1H-pyrazole-5-carboxylic acid) are reported. The ligand contains two distinct donor compartments formed by the pyrazolate-N and the oxime or the carboxylic groups. The complexes were characterized by X-ray single crystal diffraction, revealing that both 1 and 2 consist of dinuclear units in which the two metal ions are linked by double pyrazolate bridges with a planar {Mn₂N₄} core. Cryomagnetic measurements show antiferromagnetic interaction with g = 1.99, J = −3.6 cm⁻¹, Θ = −2.02 K for 1 and g = 2.00, J = −3.7 cm⁻¹, Θ = 1.43 K for 2.     

Measurement of Ice Nucleation-Active Bacteria on Plants and in Precipitation by Quantitative PCR

Ice nucleation-active (INA) bacteria may function as high-temperature ice-nucleating particles (INP) in clouds, but their effective contribution to atmospheric processes, i.e., their potential to trigger glaciation and precipitation, remains uncertain. We know little about their abundance on natural vegetation, factors that trigger their release, or persistence of their ice nucleation activity once airborne. To facilitate these investigations, we developed two quantitative PCR (qPCR) tests of the ina gene to directly count INA bacteria in environmental samples. Each of two primer pairs amplified most alleles of the ina gene and, taken together, they should amplify all known alleles. To aid primer design, we collected many new INA isolates. Alignment of their partial ina sequences revealed new and deeply branching clades, including sequences from Pseudomonas syringae pv. atropurpurea, Ps. viridiflava, Pantoea agglomerans, Xanthomonas campestris, and possibly Ps. putida, Ps. auricularis, and Ps. poae. qPCR of leaf washings recorded ∼10⁸ina genes g⁻¹ fresh weight of foliage on cereals and 10⁵ to 10⁷ g⁻¹ on broadleaf crops. Much lower populations were found on most naturally occurring vegetation. In fresh snow, ina genes from various INA bacteria were detected in about half the samples but at abundances that could have accounted for only a minor proportion of INP at −10°C (assuming one ina gene per INA bacterium). Despite this, an apparent biological source contributed an average of ∼85% of INP active at −10°C in snow samples. In contrast, a thunderstorm hail sample contained 0.3 INA bacteria per INP active at −10°C, suggesting a significant contribution to this sample.