A quorum sensing-defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems.

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain.

Identification of candidate antimicrobial peptides derived from abalone hemocyanin.

Hemocyanins present in invertebrate hemolymph are multifunctional proteins, responsible for oxygen transport and contributing to innate immunity through phenoloxidase-like activity. In arthropods, hemocyanin has been identified as a source of broad-spectrum antimicrobial peptides during infection. Conversely, no hemocyanin-derived antimicrobial peptides have been reported for molluscs. The present study describes a putative antimicrobial region, termed haliotisin, located within the linking sequence between the α-helical domain and ß-sheet domain of abalone (Haliotis tuberculata) hemocyanin functional unit E. A series of synthetic peptides based on overlapping fragments of the haliotisin region were tested for their bactericidal potential. Incubating Gram-positive and Gram-negative bacteria in the presence of certain haliotisin peptides, notably peptides 3-4-5 (DTFDYKKFGYRYDSLELEGRSISRIDELIQQRQEKDRTFAGFLLKGFGTSAS) led to reductions in microbial growth. Furthermore, transmission electron micrographs of haliotisin-treated bacteria revealed damages to the microbial cell wall. Data discussed here provides the first evidence to suggest that molluscan hemocyanin may act as a source of anti-infective peptides.

[Properties of pectolitic phytopathogenic bacteria isolates obtained in Ukraine].

Bacteria obtained from potato tubers having symptoms of soft rot and grown in different regions of Ukraine are identified as Pectobacterium carotovorum subsp. carotovorum. These bacteria strains are able to produce bacteriocines. Their killer activity in respect of P. carotovorum and Esherichia coli has been studied. The sensitivity to bactericines has been shown. Purified fractions of bacteriocines having high molecular weight (MCTV) have been obtained. The difference in composition of proteins from phage tails as compared to the ones in P. carotovorum J2 has been studied by the method of electrophoresis. It was found that the composition of MCTV major proteins of studied isolates mostly corresponds to P. carotovorum J2. The set of enzyme minor fractions has some different compositions as compared to P. carotovorum J2. It has been hypothesized that this difference is responsible for killer specificity.

Ultrastructural observation on sterilization of melittin.

ABSTACT: The effects of melittin on growth and bacteriostasis of four pathogens were extensively investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results indicated that the melittin had a marked bacteriostatic effect on the four pathogenic bacteria. Among these, E. cacotowora was influenced most powerfully and quickly, the yeast and F. fulva were the second, and the S. aureus was inhibited by a low concentration but was killed by a high concentration. It was observed in the experiments that melittin killed pathogenic bacteria in three ways. One was by pore formation. After integrating melittin into the plasma membrane, a vacuole was formed then penetrated, resulting in bacterial content leakage. The vacuole also experienced plasmolysis and the growing cavity destroyed the membrane. A third effect was the formation of vacuoles in the cells which induced the pycnosis of the cytoplasm resulting in a cell death. The mechanism of melittin bacteriostasis was the result of integrating melittin with phospholipid double layers of the plasmalemma and the endomembranes.

Bacteriocin-like substance inhibits potato soft rot caused by Erwinia carotovora.

Soft rot is a major problem encountered in potatoes during postharvest storage. The soft rot bacterium Erwinia carotovora was inhibited by a novel bacteriocin-like substance (BLS) produced by Bacillus licheniformis P40. The BLS caused a bactericidal effect on E. carotovora cells at 30 microg mL(-1). Transmission electron microscopy showed that BLS-treated cells presented wrinkled bacterial surfaces and shrinkage of the whole cell, indicating plasmolysis. Erwinia carotovora cells treated with BLS were analyzed by FTIR showing differences in the 1390 cm(-1) and 1250-1220 cm(-1) bands, corresponding to assignments of membrane lipids. BLS was effective in preventing E. carotovora spoilage on potato tubers, reducing the symptoms of soft rot at 240 microg mL(-1) and higher concentrations. Soft rot development was completely blocked at 3.7 mg mL(-1). This BLS showed potential to protect potato tubers during storage.

[Defective lysogeny in Erwinia carotovora]. / Defektnaia lizogenia Erwinia carotovora.

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128-192 nm in length and 13-21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.

Bacillus subtilis BS 107 as an antagonist of potato blackleg and soft rot bacteria.

Antimicrobial substances were produced by Bacillus subtilis BS 107 in a defined medium and isolated from culture filtrate by precipitation at pH 2.5. Active fractions were extracted in ethyl acetate, acetone, and 80% ethanol and purified by thin-layer chromatography (TLC) on silica gel plates developed with an ethanol-water mixture (2:1, v/v). In each case, a band with a Rf of 0.75 formed an inhibitory zone when the TLC plates were placed in contact with agar seeded with test cultures of the Erwinia spp. The antibiotic was released into the culture medium during early stages of growth of Bacillus subtilis BS 107 but higher amounts were released in older cultures. The antibiotic was resistant to the action of nucleases, proteases, and lipase. It was stable when autoclaved twice for 35 min at 2 atm (1 atm = 101.325 kPa) in acidic, neutral, and alkaline solutions. It remained active over the pH range of 1-14 during 1 month of observation and exhibited no loss of antimicrobial activity when stored at 4 degrees C for over 1 year. Bacillus subtilis BS 107 showed activity in vitro and in vivo against Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora, the causal agents of potato blackleg and tuber soft rot. The application of an antagonist or its antibiotic to cut potato tissues prevented or reduced symptoms of the diseases. The antibiotic was active in vitro against a broad spectrum of bacterial and fungal species.