Background and Objectives: RPOS is a bacterial sigma factor of RNA polymerase which is involved in the expression of the genes which control regulons and play a critical role in survival against stresses. Few suitable vectors are available which could be maintained successfully in Flexibacter chinesis cells and could in particular be used as a suicide vector to make mutation in the RPOS gene. The aim of this study was to investigate if RPOS mutagenesis has impact on bacterial morphology in addition to cell division. Materials and Methods: A 0. 603 kb BamHI-PstI fragment subclone of pICRPOS38Ω was cloned into linearized pLYLO3. The final construct, pLRPOS38 suicide vector, was introduced into Flexibacter chinesis. Then the cytoplasm of mutant strain and wild-type were investigated by transmission electron microscopy. Results: After successful subcloning of suicide vector into F. chinesis, based on TEM study, it was demonstrated that mutation in RPOS gene leads to decomposition of outer membrane of F. chinesis. Conclusion: A suitable vector to make suicide mutation in RPOS was constructed for F. chinesi.

Background: Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The RPOS (RNA polymerase, sigma S) gene encodes sigma-38 (σ38, or RPOS), a 37.8 kDa protein inPseudomonas aeruginosa (P. aeruginosa) strains. RPOS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection).Methods: The significance of RPOS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RPOS ofP. aeruginosa PAO1 using bioinformatics tools.Results: The results showed that RPOS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D) structure was modeled using SWISS-MODEL Workspace.Conclusion: Determination of sequence, function, structure, and predicted epitopes of RPOS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR) strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions.

Due to the emergence of antibiotic-resistant clinical strains of Pseudomonas aeruginosa, there is an urgent need for a suitable and affordable alternative that is a non-antibiotic antimicrobial agent and creates a new generation of infectious disease treatment. In this regard, this study aims to investigate the antimicrobial potential of ellagic acid against clinical strains of P. aeruginosa under physiological growth conditions and oxidative stress. P. aeruginosa isolates from clinical samples were identified by biochemical tests and their antibiotic susceptibility was tested by disc diffusion method. The inhibitory effect of ellagic acid against multiple drug resistance isolates was investigated by disc diffusion and broth microdilution methods and its effect on bacterial survival under physiological conditions and oxidative stress was investigated by Time-Kill assay. The effect of ellagic acid on RPOS gene expression was also investigated by the Real time-PCR method. In this study, ellagic acid showed an inhibitory effect on the growth of all MDR isolates of P. aeruginosa tested. The minimum inhibitory concentration (MIC) of ellagic acid in 5 isolates varied between 250 and 2000 µg/ml. Treatment with ellagic acid rendered drug-resistant clinical strains of P. aeruginosa sensitive to oxidative stress. It reduced the survival of P. aeruginosa, while treatment with 1/4MIC concentration of ellagic acid resulted in a 4.7-fold decrease in log10 CFU/mL compared to the control. In the present study, treatment with a sub-inhibitory concentration of ellagic acid also caused a significant decrease in RPOS gene expression (P˂0.05). The results of this study indicate that ellagic acid inhibits the growth of P. aeruginosa and increases its sensitivity to oxidative stress conditions. Conducting in vivo studies may clarify the possibility of its clinical application in the control of infections caused by resistant isolates of P. aeruginosa.

A one kb portion of the RPOS gene from Flexibacter chinensis was isolated by PCR, sequenced and compared to the RPOS gene of a variety of other organisms. The gene was found to be 98% similar to previously sequenced genes. Mutation of the RPOS gene with tri-parental mating produced strain JR101 and the growth rate of the mutant was compared with that of the wild-type. The mutant grew slower, and produced longer cells in the stationary phase. The cell size of the mutant was not reduced during the stationary phase, suggesting that cell division was inhibited during the stationary phase. The mutant also showed a much reduced survival under starvation conditions compared to the wild-type strain.  

Background: In this study, spore-forming probiotics were employed to eradicate Staphylococcus epidermidis biofilms and the presence and expression of genes involved in stress response was examined. Methods: Polymerase chain reaction (PCR) assay was used to detect RPOS, relA and mazF genes in S. epidermidis ATCC 12228. Biofilm production was investigated by microtiter plate (MTP) assay. 100X minimum inhibitory concentration (MIC) of gentamycin was used to induce persister cells in planktonic and biofilm bacterial cells. The expression of RPOS, relA, and mazF genes was assessed at different time intervals of 2, 8, and 24 h using real-time PCR assay. Then, dilutions of 1, 0.5, and 0.25 µg/ml of the supernatant of Bacillus coagulans culture was used to eradicate the persister cells and the number of colonies was determined. Results: Persister cells of S. epidermidis were formed after 7 h in planktonic and 5 h in the biofilm structure after exposure to 50 µg/ml of gentamycin. The expression of mazF and RPOS in biofilm structure and the expression of RPOS and relA in persister cells were significantly higher compared to the control (p< 0.05). The number of persister cells showed a reduction of log 2.4 and log 0.8 after exposure to 1 and 0.5 µg/ml B. coagulans supernatant, respectively, but no reduction was observed at the concentration of 0.25 µg/ml. Conclusions: The results showed that the supernatant of probiotics containing their secretive metabolites can be used as a novel approach to combat persister cells.