Bacterial communities usually develop biofilms abound in nature niche. The development of biofilm is a highly dynamic and complex process coordinated by multiple mechanisms, of which two-component system and quorum sensing are two well-defined systems. Biofilm is involved in the virulence of many pathogens. Therefore, targeting the key factors involved in the biofilm formation represents a novel and promising avenue for developing better antibiotics.
Acyl-Butyrolactones ; metabolism ; Bacteria ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biofilms ; growth & development ; Drug Delivery Systems ; Gene Expression Regulation, Bacterial ; Homoserine ; analogs & derivatives ; metabolism ; Lactones ; metabolism ; Quorum Sensing ; Signal Transduction

N-acyl-homoserine lactones (AHLs), are widely conserved signal molecules present in quorum-sensing systems of many Gram-negative bacteria. AHLs molecules mediate the expression of virulence genes of a range of bacterial pathogens. Recently, it has been reported that AiiA protein, which widely exists in Bacillus species, can inactivate the AHLs by hydrolyzing the lactone bond of AHLs, thus attenuate the diseases caused by the expression of virulence genes of bacterial pathogens. Bacillus thuringiensis, a type of Gram-positive bacteria, has been used extensively as a microbial insecticide in the last few decades. However, most of important insecticidal B. thuringiensis strains have not been exploited for bacterial disease control because they usually do not produce antibiotics that are effective against bacteria and fungi. The discovery of AiiA protein in B. thuringiensis shows the application potential of B. thuringiensis on biocontrol against bacterial diseases. In this study, in order to construct the B. thuringiensis recombinant strain that has high expression of AiiA protein, the promoter of insecticidal crystal protein coding gene cry3Aa of B. thuringiensis was selected. The promoter of gene cry3Aa is a non-sporulation promoter, it promotes the transcription earlier and longer than the promoters of other cry genes. The promoter of AiiA protein coding gene aiiA was replaced with the promoter of gene cry3Aa by overlapping PCR, resulting fusion gene pro3A-aiiA. The gene pro3A-aiiA was inserted into shuttle vector pHT304 at site BamH I / Sph I , resulting recombinant plasmid pBMB686. The plasmid pBMB686 was introduced into B. thuringiensis acrystalliferous strain BMB171, the resulting strain BMB686 had a higher and more stable expression level of protein AiiA comparing with the parental strain BMB171. Furthermore, the strain BMB686 exhibited stronger ability of AHLs inactivation and much more effective restraint to the potato's soft rot disease caused by Erwinia carotovora than those of the parental strain BMB171. From these results, it was concluded that the B. thuringiensis strain harvesting the fusion gene pro3A-aiiA may be utilized in the future to control bacterial diseases which are mediated by the AHL quorum-sensing signals.
Acyl-Butyrolactones ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Daucus carota ; microbiology ; Endotoxins ; genetics ; metabolism ; Hemolysin Proteins ; genetics ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Models, Genetic ; Pectobacterium carotovorum ; pathogenicity ; Plant Diseases ; microbiology ; prevention & control ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; genetics ; metabolism

We isolated a new strain of endophytic Pseudomonas G5 from the stems of Chinese parsley (Coriandrum sativum L.), and it is tentatively identified as Pseudomonas aurantiaca according to analysis of the entire substrate utilization profiles using BIOLOG Microstation system (BIOLOG, Inc, Hayward CA). An array of evidence established that many Gram-negative bacteria employ Quorum sensing (QS) system to regulate gene expression in response to cell density using small diffusible signal molecules, N-acyl homoserine lactones (AHLs), and control diverse phenotypic traits in plant-associated bacteria. In this study, we showed that Pseudomonas sp. strain G5 can produce several types of AHLs at a detectable level using Thin Layer Chromatography (TLC) analysis combined with bioreporter Chromobacterium violaceum CV026 bioassay, and N-hexanoyl-homoserine lactone (HHL, C6-HSL) with Rf value 0.4 is the major signal molecule. Furthermore, we have identified its quorum sensing system composed of PhzI and PhzR by cloning and sequencing of phzI-phzR. PhzI is responsible for synthesis of AHLs signal molecules, and PhzR is a transcriptional regulator. Finally, we heterologously expressed the recombinant plasmid pMD-phzIR in Escherichia coli JM109 and verified it using C. violaceum CV026 bioassay. The phylogenetic analysis using MEGA4 revealed highly similarities exist among the phzIR homologs, suggesting it is evolutionary well conserved in the genus Pseudomonas.
4-Butyrolactone ; analogs & derivatives ; metabolism ; Acyl-Butyrolactones ; metabolism ; Amino Acid Sequence ; Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Phylogeny ; Pseudomonas ; classification ; genetics ; isolation & purification ; Trans-Activators ; biosynthesis ; genetics