Identification of Pathogenicity-Related Genes in Biofilm-Defective Acidovorax citrulli by Transposon Tn5 Mutagenesis.

Biofilm formation is important for virulence of a large number of plant pathogenic bacteria. Indeed, some virulence genes have been found to be involved in the formation of biofilm in bacterial fruit blotch pathogen Acidovorax citrulli. However, some virulent strains of A. citrulli were unable to format biofilm, indicating the complexity between biofilm formation and virulence. In this study, virulence-related genes were identified in the biofilm-defective strain A1 of A. citrulli by using Tn5 insertion, pathogenicity test, and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). Results from this study indicated that 22 out of the obtained 301 mutants significantly decreased the virulence of strain A1 compared to the wild-type. Furthermore, sequence analysis indicated that the obtained 22 mutants were due to the insertion of Tn5 into eight genes, including Aave 4244 (cation diffusion facilitator family transporter), Aave 4286 (hypothetical protein), Aave 4189 (alpha/beta hydrolase fold), Aave 1911 (IMP dehydrogenase/GMP reductase domain), Aave 4383 (bacterial export proteins, family 1), Aave 4256 (Hsp70 protein), Aave 0003 (histidine kinase, DNA gyrase B, and HSP90-like ATPase), and Aave 2428 (pyridoxal-phosphate dependent enzyme). Furthermore, the growth of mutant Aave 2428 was unaffected and even increased by the change in incubation temperature, NaCl concentration and the pH of the LB broth, indicating that this gene may be directly involved in the bacterial virulence. Overall, the determination of the eight pathogenicity-related genes in strain A1 will be helpful to elucidate the pathogenesis of biofilm-defective A. citrulli.

Protein profiling analyses of the outer membrane of Burkholderia cenocepacia reveal a niche-specific proteome.

Outer membrane proteins (OMPs) are integral ß-barrel proteins of the Gram-negative bacterial cell wall and are crucial to bacterial survival within the macrophages and for eukaryotic cell invasion. Here, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to comprehensively assess the outer membrane proteome of Burkholderia cenocepacia, an opportunistic pathogen causing cystic fibrosis (CF), in conditions mimicking four major ecological niches: water, CF sputum, soil, and plant leaf. Bacterial cells were harvested at late log phase, and OMPs were extracted following the separation of soluble proteins by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE). Protein bands were excised and identified by LC-MS/MS analysis. The proteins identified under various growth conditions were further subjected to in silico analysis of gene ontology (subcellular localization, structural, and functional analyses). Overall, 72 proteins were identified as common to the four culture conditions, while 33, 37, 20, and 10 proteins were exclusively identified in the water, CF sputum, soil, and plant leaf environments, respectively. The functional profiles of the majority of these proteins revealed significant diversity in protein expression between the four environments studied and may indicate that the protein expression profiles are unique for every condition. Comparison of OMPs from one strain in four distinct ecological niches allowed the elucidation of proteins that are essential for survival in each niche, while the commonly expressed OMPs, such as RND efflux system protein, TonB-dependent siderophore receptor, and ABC transporter-like protein, represent promising targets for drug or vaccine development.

Deciphering the role of Burkholderia cenocepacia membrane proteins in antimicrobial properties of chitosan.

Chitosan, a versatile derivative of chitin, is widely used as an antimicrobial agent either alone or mixed with other natural polymers. Burkholderia cenocepacia is a multidrug-resistant bacteria and difficult to eradicate. Our previous studies shown that chitosan had strong antibacterial activity against B. cenocepacia. In the current study, we have investigated the molecular aspects for the susceptibility of B. cenocepacia in response to chitosan antibacterial activity. We have conducted RNA expression analysis of drug efflux system by RT-PCR, membrane protein profiling by SDS-PAGE, and by LC-MS/MS analysis following the validation of selected membrane proteins by real-time PCR analysis. By RT-PCR analysis, it was found that orf3, orf9, and orf13 were expressed at detectable levels, which were similar to control, while rest of the orf did not express. Moreover, shotgun proteomics analysis revealed 21 proteins in chitosan-treated cells and 16 proteins in control. Among them 4 proteins were detected as shared proteins under control and chitosan-treated cells and 17 proteins as uniquely identified proteins under chitosan-treated cells. Among the catalog of uniquely identified proteins, there were proteins involved in electron transport chain and ATP synthase, metabolism of carbohydrates and adaptation to atypical conditions proteins which indicate that utilization and pattern of chitosan is diverse which might be responsible for its antibacterial effects on bacteria. Moreover, our results showed that RND drug efflux system, which display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents, was not determined to play its role in response to chitosan. It might be lipopolysaccharides interaction with chitosan resulted in the destabilization of membrane protein to membrane lyses to cell death. Membrane proteome analysis were also validated by RT-qPCR analysis, which corroborated our results that of membrane proteins.