Polymyxin P is the active principle in suppressing phytopathogenic Erwinia spp. by the biocontrol rhizobacterium Paenibacillus polymyxa M-1.

BACKGROUND: Nine gene clusters dedicated to nonribosomal synthesis of secondary metabolites with possible antimicrobial action, including polymyxin and fusaricidin, were detected within the whole genome sequence of the plant growth-promoting rhizobacterium (PGPR) Paenibacillus polymyxa M-1. To survey the antimicrobial compounds expressed by M-1 we analyzed the active principle suppressing phytopathogenic Erwinia spp. RESULTS: P. polymyxa M-1 suppressed the growth of phytopathogenic Erwinia amylovora Ea 273, and E. carotovora, the causative agents of fire blight and soft rot, respectively. By MALDI-TOF mass spectrometry and reversed-phase high-performance liquid chromatography (RP-HPLC), two antibacterial compounds bearing molecular masses of 1190.9 Da and 1176.9 Da were detected as being the two components of polymyxin P, polymyxin P1 and P2, respectively. The active principle acting against the two Erwinia strains was isolated from TLC plates and identified by postsource decay (PSD)-MALDI-TOF mass spectrometry as polymyxin P1 and polymyxin P2. These findings were corroborated by domain structure analysis of the polymyxin (pmx) gene cluster detected in the M-1 chromosome which revealed that corresponding to the chemical structure of polymyxin P, the gene cluster is encoding D-Phe in position 6 and L-Thr in position 7. CONCLUSIONS: Identical morphological changes in the cell wall of the bacterial phytopathogens treated with either crude polymyxin P or culture supernatant of M-1 corroborated that polymyxin P is the main component of the biocontrol effect exerted by strain M-1 against phytopathogenic Erwinia spp.

Botanicals to control soft rot bacteria of potato.

Extracts from eleven different plant species such as jute (Corchorus capsularis L.), cheerota (Swertia chiraita Ham.), chatim (Alstonia scholaris L.), mander (Erythrina variegata), bael (Aegle marmelos L.), marigold (Tagetes erecta), onion (Allium cepa), garlic (Allium sativum L.), neem (Azadiracta indica), lime (Citrus aurantifolia), and turmeric (Curcuma longa L.) were tested for antibacterial activity against potato soft rot bacteria, E. carotovora subsp. carotovora (Ecc) P-138, under in vitro and storage conditions. Previously, Ecc P-138 was identified as the most aggressive soft rot bacterium in Bangladeshi potatoes. Of the 11 different plant extracts, only extracts from dried jute leaves and cheerota significantly inhibited growth of Ecc P-138 in vitro. Finally, both plant extracts were tested to control the soft rot disease of potato tuber under storage conditions. In a 22-week storage condition, the treated potatoes were significantly more protected against the soft rot infection than those of untreated samples in terms of infection rate and weight loss. The jute leaf extracts showed more pronounced inhibitory effects on Ecc-138 growth both in in vitro and storage experiments.

Effect of concentration and substrate flow rate on isomaltulose production from sucrose by Erwinia sp. cells immobilized in calcium-alginate using packed bed reactor.

Isomaltulose was obtained from sucrose solution by immobilized cells of Erwinia sp. D12 using a batch and a continuous process. Parameters for sucrose conversion into isomaltulose were evaluated using both experimental design and response surface methodology. Erwinia sp. D12 cells were immobilized in different alginates, and the influence of substrate flow rate and concentration parameters to produce isomaltulose from sucrose were observed. Response surface methodology demonstrated that packed bed columns containing cells immobilized in low-viscosity sodium alginate (250 cP) presented a mean isomaltulose conversion rate of 47%. In a continuous process, both sucrose substrate concentration and substrate flow rate parameters had a significant effect (p < 0.05) and influenced the conversion of sucrose into isomaltulose. Higher conversion rates of sucrose into isomaltulose, from 53-75% were obtained using 75 g of immobilized cells at a substrate flow rate of 0.6 mL/min.

Linking environmental heterogeneity and reproductive success at single-cell resolution.

Individual-based microbial ecology (IBME) is a developing field of study in need of experimental tools to quantify the individual experience and performance of microorganisms in their natural habitats. We describe here the conception and application of a single-cell bioreporter approach with broad utility in IBME. It is based on the dilution of stable green fluorescent protein (GFP) in dividing bacteria. In the absence of de novo synthesis, GFP fluorescence of a daughter cell approximates half of that of its mother, from which follows that the fluorescence of a progeny cell is a quantitative measure for the reproductive success of its ancestor. To test this concept, we exposed GFP-filled bacteria to different degrees of environmental heterogeneity and assessed how this affected individual cells by the analysis of GFP content in their progeny. Reporter bacteria growing in rich medium in a shaking flask showed no variation in reproductive success, confirming that life in a broth is experienced much the same from one bacterium to the next. In contrast, when reporter bacteria were released onto plant leaf surfaces, representing a microscopically heterogeneous environment, clear intrapopulation differences in reproductive success were observed. Such variation suggests that individual cells in the founding population experienced different growth-permitting conditions, resulting in unequal contributions of individual bacteria to future offspring and population sizes. Being able to assess population changes bottom-up rather than top-down, the bioreporter offers opportunities to quantify single-cell competitive and facilitative interactions, assess the role of chance events in individual survivorship and reveal causes that underlie individual-based environmental heterogeneity.

Hyperproduction of 3,4-dihydroxyphenyl-L-alanine (L-Dopa) using Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR.

In the last few decades, enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) using tyrosine phenol-lyase (Tpl) has been industrialized. This method has an intrinsic problem of tyrosine contamination because Tpl is synthesized under tyrosine-induced conditions. Herein, we constructed a hyper-L-dopa-producing strain by exploiting a mutant TyrR, an activator of tpl. The highest productivity was obtained for the strain grown under non-induced conditions. It was 30-fold higher than that obtained for tyrosine-induced wild-type cells.

Erwinia toletana sp. nov., associated with Pseudomonas savastanoi-induced tree knots.

Gram-negative bacteria were isolated from knots induced by Pseudomonas savastanoi in olive trees (Olea europaea L.). A total of nine endophytic bacterial strains were isolated, each from inside a different tree knot. Biochemical characterization indicated that all the strains belong to the family Enterobacteriaceae. Phylogenetic analyses of the 16S rRNA genes of these novel isolates revealed that they formed a homogeneous cluster within Erwinia species. DNA signatures of these isolates were identical to those described for the genus Erwinia. The strains formed a homogeneous group as shown by DNA-DNA hybridization analysis and numerical analysis of phenotypic data, clearly differentiated from all species of Erwinia with validly published names. The data provide strong evidence of the differentiation of these strains from the most closely related species. Therefore, these isolates represent a novel species, for which the name Erwinia toletana sp. nov. is proposed. The isolates are available at CFBP, CECT and ATCC. The G+C content is 52+/-0.5 mol%. The type strain is CFBP 6631(T) (=A37(T)=ATCC 700880(T)=CECT 5263(T)).

Plasmid ColVBtrp maintenance in Erwinia carotovora.

Plasmid ColVBtrp maintenance in Erwinia carotovora cells was followed by measuring kinetics of elimination of plasmid genetic markers and loss of plasmid deoxyribonucleic acid. An E. carotovora mutant stably carrying plasmid ColVBtrp was isolated. Besides stable plasmid maintenance, the mutant showed altered sensitivity to male-specific phage MS2, sensitivity to drugs, and colony morphology.

Erwinia salicis: its metabolism and variability in vitro, and a method to demonstrate the pathogen in the host.

Morphological, biochemical and serological features of eleven Erwinia salicis isolates were examined. Three groups were demonstrated, one of which comprises the Dutch isolates. Serological techniques proved to be a valuable addition to conventional plating techniques for detecting the pathogen. Willow wood extract with 5% sucrose, 0.06% "Lab Lemco" broth and 1.5% agar appeared to be a suitable medium for the isolation of E. salicis, because of its selectivity.

Ultrastructure of the cell wall and cytoplasmic membrane of gram-negative bacteria with different fixation techniques.

The ultrastructure of the cytoplasmic membrane and cell wall of two strains of Escherichia coli, Proteus morganii, P. vulgaris, Acinetobacter anitratum, Moraxella lacunata, Erwinia amylovora, Acinetobacter sp., and of a plant pathogen, unclassified gram-negative, fixed by the Ryter-Kellenberger procedure, was found to be significantly affected by the use or omission of the uranyl postfixation included in that procedure, and by the presence or absence of calcium in the OsO(4) fixative. The omission of the uranyl treatment results in a less clear profile of both the outer membrane of the cell wall and of the cytoplasmic membrane. The observation of these two membranes is further limited when both uranyl and calcium are omitted. The R-layer and the material covering the surface of the cell wall appear more distinct when the uranyl postfixation is not used. Evidence is given suggesting that the influence of uranyl and calcium ions on the appearance of the outer and cytoplasmic membranes would be primarily due to their action as fixatives, whereas the influence of uranyl on the appearance of the R-layer would be due to a direct action on the peptidoglycan component of this layer. When uranyl acetate is used as a section stain after the embedding in plastic, it improves the observation of the R-layer. In this case, a well contrasted R-layer is consistently observed in all strains studied, provided that the postfixation has been omitted. The frequent difficulty in clearly observing the R-layer in many published micrographs probably results from the common use of uranyl postfixation.

Ultrastructure of the cell wall of Erwinia amylovora.

Thin sectioned cells of Erwinia amylovora revealed two electron-dense layers in their walls when fixed at 24 to 27 C and three when fixed at 4 C.