Numerous functions in pathogenic Pectobacterium are regulated by quorum sensing (QS). Two different aiiA genes isolated from Bacillus sp. A24(aiiAA24) and Bacillus sp. DMS133(aiiADMS133) were used. Both genes encode acyl-homoserine lactonase (AiiA), which disrupts QS in Pectobacterium. To investigate the effect of different AiiAs on the inhibition of Pectobacterium carotovorum pathogenicity, two aiiA genes from different Bacillus strains were cloned and the resulting plasmids pME6863 (aiiAA24) and pME7080 (aiiADMS133) were transformed into P. carotovorum EMPCC cells. The effects of different lactonases on virulence features such as enzymatic activity, twitching and swimming motilities, and production of pellicle and biofilm formation were investigated. In EMPCC/pME6863, twitching and swimming motilities, and pellicle production were significantly reduced compared with EMPCC/pME7080. Quantitative real-time PCR (qRT-PCR) was used to measure virulence gene expression in transformed cells compared with expression levels in wild-type EMPCC. The expression of peh and hrpL genes was greatly reduced in EMPCC/pME6863 compared with EMPCC/pME7080. The sequence alignment and molecular dynamic modeling of two different AiiAA24 and AiiADMS133 proteins suggested that the replacement of proline 210 from AiiAA24 to serine in AiiADMS133 caused the reduction of enzyme activity in AiiADMS133.
- MeSH
- Bacillus * genetika enzymologie MeSH
- bakteriální proteiny * genetika metabolismus MeSH
- biofilmy růst a vývoj MeSH
- karboxylesterhydrolasy * genetika metabolismus MeSH
- klonování DNA MeSH
- metaloendopeptidasy MeSH
- Pectobacterium carotovorum genetika enzymologie patogenita MeSH
- quorum sensing * MeSH
- regulace genové exprese u bakterií MeSH
- virulence MeSH
- Publikační typ
- časopisecké články MeSH
Quorum Sensing allows bacteria to sense their population density via diffusible N-acyl homoserine lactone (N-HL) signaling molecules. Upon reaching a high enough cell density, bacteria will collectively exhibit a phenotype. Until recently, methods used for detection of N-HLs have not considered the chirality of these molecules and it was assumed that only the L-enantiomer was produced by bacteria. The production and effects of D-N-HLs have rarely been studied. In this work, the temporal production of D-N-HLs by the plant pathogen Pectobacterium atrosepticum and the human pathogen Pseudomonas aeruginosa are reported. Both bacteria produced D-N-HLs in significant amounts and in some cases their concentrations were higher than other low abundance L-N-HLs. Previously unreported D-enantiomers of N-3-oxoacyl and N-3-hydroxyacyl homoserine lactones were detected in P. atrosepticum. Interestingly, L-N-HLs produced in the lowest concentrations had relatively higher amounts of their corresponding D-enantiomers. Potential sources of D-N-HLs and their significance are considered.
Two novel dsDNA bacteriophages named Pectobacterium virus CB251 (PcCB251) and Pectobacterium virus CB7V (PcCB7V) targeting plant pathogen Pectobacterium parmentieri have been isolated and sequenced. The PcCB251 genome consists of 40,557 bp with G+C content of 48.6% and contains 47 predicted genes on a single strand. The phage is classified in genus Berlinvirus, family Autographiviridae. The PcCB7V phage has a circular dsDNA genome of 146,054 bp with G+C content of 50.4% and contains 269 predicted protein genes on both strands and 13 tRNA genes. The PcCB7V phage can be classified in genus Certrevirus, subfamily Vequintavirinae. Both novel bacteriophages have narrow host ranges, but they extend the list of candidates for phage-based control of pectolytic bacteria causing soft rot disease of potato.
Confocal laser-scanning microscopy was chosen to observe the colonization and damage caused by the soft rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection, or degradation. The dual-colored pathogen and biocontrol strains were used to coinoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls, as well as a concomitant rhodococcal quorum-quenching response, at both the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.
This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer's buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.
- MeSH
- bakteriofágy izolace a purifikace fyziologie MeSH
- časové faktory MeSH
- chloridy metabolismus MeSH
- Enterobacteriaceae virologie MeSH
- mikrobiologie životního prostředí * MeSH
- Pectobacterium virologie MeSH
- rostliny virologie MeSH
- sloučeniny zinku metabolismus MeSH
- virologie metody MeSH
- Publikační typ
- časopisecké články MeSH
Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.
- MeSH
- elektroforéza kapilární metody MeSH
- Enterobacteriaceae chemie klasifikace izolace a purifikace MeSH
- Pectobacterium chemie klasifikace izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
