Good Research Proposal On Research Hypothesis
In this study, we show that S4 gene is followed by an overlapping immunity gene and that purified S4 recognizes the FpvA receptor just as S2.
In this study, the nucleic acid sequence at the locations 4327697-4327359 of the Pseudomonas aeruginosa (POA1) genome will not be annotated. However, it has been predicted that this region encodes the immunity gene of the nearby S4 gene (PA3866) based on the current analysis of the genome sequence. RT-PCR will be used to detect the expression of the immunity gene. In addition, the downstream gene coding for the immunity protein and the PA3866 coding for S4 will be cloned and expressed in E.coli. This will also help in the isolation of His-tagged pyocin S4. Forty-three POA1 will be typed via PCR to screen for their respective ferripyoverdine receptor gene (fpvA I-III). These receptors will be tested for their sensitivity for pyocin S4. The immunity gene will then be deleted to screen for sensitivity for S4. The fpvAI in S4 sensitive strains will be deleted to test the formation of mutant S4 strains. Furthermore, the receptor binding domain (RBD) of S2 will be cloned in pET-15b vector and expressed in E.coli. It will then be purified and mixed with S4 to assess the killing activity of both S4 and S2.
Introduction (literature reviews)
Synthesized by most isolates of Pseudomonas aeruginosa, pyocins are narrow spectrum bacteriocins. They are said to play a vital role in niche establishment, as well as offer protection in mixed populations (Elfarash, Wei and Cornelis 2012). Three types of pyocins have been established. They include the R, F ans S types. The S-type pyocins have been found to be highly sensitive protease proteins. F and R-type look like bacteriophage tails. In addition, the R-type pyocins are not only contractile, but non-flexible. On the other hand, the F-type pyocins are non-contrcatile but flexible (Bodis et al. 2009; Baba and Schneewind 1998).
Pseudomonas aeruginosa is an opportunistic human pathogen. Its ability to survive in different habitats relies on the potential to adapt, as well as respond to different environmental stimuli, as well as adopt a biofilm lifestyle (de Chial et al. 2003). It can attach to surfaces for survival. They produce pyocins to kill other competing bacteria (Cascales et al. 2007; Baysse et al. 1999).
Soluble pyocins (S-type) have a C-terminal killing domain, a translocation domain, and an N-terminal receptor binding domain. Most S-type pyocins (s3, s2, s1, and AP41) kill cells by inducing cell death via DNA breakdown (Ambrosi, Leoni and Visca 2002). This killing capacity is due to the possession of an endonuclease C-terminal domain. S4 and S5 types have tRNase and pore-performing activities respectively. An immunity gene coded just adjacent to the pyocin gene is believed to protect pyocin-producing bacteria from their toxins. Iron limitation has been found to boost the killing capacity of some types of the S-type pyocins (sa, s2, and s3) (Ambrosi, Leoni and Visca 2002).
When Pseudomonas aeruginosa bacterium is grown under iron limitation, it often produces pyoverdine molecule, which is fluorescent, and serves as a high-affinity sinderophore (Denayer, Matthijis, Cornelis, 2007). Pyoverdines are differentiated by peptide chains. Pseudomonas aeruginosa strains have been found to produce three Pyoverdines that are recognized by the outer membrane receptor: FpvA I, II or III (Cornelis, Matthijs and Van Oeffelen 2009). The specific genes for each of these receptors are known and for that matter, it is possible to determine which receptor is present in a given strain (Cornelis 2010).
Ohkawa et al. (1980) were the first to report that mutants that are resistant to s2 do not produce an iron-repressed outer membrane protein and do not create a link with the ferripyoverdine receptor. Smith et al. (1992) later discovered that the Sa receptor could be same as the ferripyoverdine receptor. Another type, type I receptor, the FpvB receptor was discovered. Further studies found that the receptor for type II ferripyoverdine is employed by S3 to kill P. aeruginosa strains that are sensitive (Beare, For, Martin and Lamont 2003). In this study, we show that S4 gene is followed by an overlapping immunity gene and that purified S4 recognizes the FpvA receptor just as S2.
S4 gene is followed by an overlapping immunity gene, and that purified S4 recognizes the FpvA receptor just as S2.
Material and methods
Strains and Plasmids
PAO1 wild type, S4+im, W15Aug30, and W15Aug30 chromosomal deletion mutant, plasmid (pDM4), and E.coli (BL21-de3; S17-1 gamma pir).
RNA isolation and RT-PCR
Bacterial cells will be harvested while in the stationary phase. Consequently, bacterial RNA will be extracted using the High RNA Isolation Kit. Spectrophotometry and gel electrophoresis will be used to determine the concentration of the RNA.
Quantitative Real-time PCR
qTR-PCR will be performed in a Bio-Rad iCycler.
Overexpression and purification of the RDB of pyocin S2 and pyocin S4
Pyocin S4 gene, sa well as the immunity gene (pyoS4+im+2633 bp+ PA3386) and the RBD of pyoxin S2 (648 bp of PA1150) will be cloned from PAO1 with the help of primers. The amplified fragments will then be introduced into pET15b (+) by NdeI/XhoI double digestion, ligation, as wel as transformation into E.coli (Duport, Baysse and Michel-Briand 1995).
Pyocin sensitive assays
The sensitivity of P.aeruginosa strains to S4 will be assessed using 10 microliters of pyocin lysate that will be spotted on a bacteria layer incubated at 370C for 24 hours and containing 5x106 cells mL-1.
Strain and Plasmin construction
Amplification of the DNA regions flanking the gene to be deleted will be conducted by PCR using primers; they will then be fused in a second PCR and later ligated into pDM4 (suicide vector). Triparental matings C-coli donar strains will be conducted to introduce plasmids into P.aeruginosa (Draper, Martib, Beare and Lamont 2011).
In this experiment, we will show that pyocin S4 (pa3866) in PAO1 is followed by an immunity gene though note annotated in the genome. We will also demonstrate that S2 and S4 recognize the same FpvA I receptor. In addition, we will show that the receptor binding domain of S2 inhibits killing by S4.
An understanding of the molecular mechanisms of soluble pyocins gives limelight in the study of potential antibiotics. Characterization of the translocation, binding and killing activities of soluble pyocins can be exploited in the innovation of antibiotics. This study will help to shade more light on the same.
Ambrosi C, Leoni L, and Visca P (2002), Different responses of pyoverdine genes to autoinduction in Pseudomonas aeruginosa and the group Pseudomonas fluorescens-Pseudomonas putida, Appl. Environ. Microbiol., vol. pp.68:4122–4126.
Baba T and Schneewind O (1998), Instruments of microbial warfare: bacteriocin synthesis, toxicity and immunity. Trends Microbiol., vol. 6, pp. 66–71.
Baysse C, Meyer JM, Plesiat P, Geoffroy V, Michel-Briand Y, and Cornelis P (1999), Uptake of pyocin S3 occurs through the outer membrane ferripyoverdine type II receptor of Pseudomonas aeruginosa. J. Bacteriol., vol. 181, pp. 3849–3851.
Beare PA, For RJ, Martin LW, and Lamont IL (2003), Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis. Mol. Microbiol., vol. 47, pp.195–207.
Bodilis J, Ghysels B, Osayande J, Matthijs S, Pirnay JP, Denayer S, et al. (2009), Distribution and evolution of ferripyoverdine receptors in Pseudomonas aeruginosa. Environ. Microbiol., vol. 11, pp. 2123–2135.
Cascales E, Buchanan SK, Duche D, Kleanthous C, Lloubes R, Postle K, et al. (2007), Colicin biology. Microbiol. Mol. Biol. Rev., vol. 71, pp. 158–229.
de Chial M, Ghysels B, Beatson SA, Geoffroy V, Meyer JM, Pattery T, et al. (2003), Identification of type II and type III pyoverdine receptors from Pseudomonas aeruginosa. Microbiology, vol.149, pp. 821–831
Cornelis P (2010), Iron uptake and metabolism in pseudomonads. Appl. Microbiol. Biotechnol, vol. 86, pp.1637–1645.
Cornelis P, Matthijs S, and Van Oeffelen L (2009), Iron uptake regulation in Pseudomonas aeruginosa. Biometals, vol. 22, pp.15–22.
Denayer S, Matthijs S, and Cornelis P (2007), Pyocin S2 (Sa) kills Pseudomonas aeruginosa strains via the FpvA type I ferripyoverdine receptor. J. Bacteriol., vol.189, pp.7663–7668.
Draper RC, Martin LW, Beare PA, and Lamont IL (2011), Differential proteolysis of sigma regulators controls cell-surface signaling in Pseudomonas aeruginosa. Mol. Microbiol., vol. 82, 1444–1453.
Duport C, Baysse C, and Michel-Briand Y (1995), Molecular characterization of pyocin S3, a novel S-type pyocin from Pseudomonas aeruginosa. J. Biol. Chem., vol. 270, pp. 8920–8927.
Elfarash, A, Wei, Q and Cornelis, P (2012), The soluble pyocins S2 and S4 from Pseudomonas aeruginosa bind to the same FpvAI receptor. Microbiologyopen. 2012 Sep; 1(3): 268–275.
Ohkawa I, Shiga S, Kageyama M (1980), Effect of iron concentration in the growth medium on the sensitivity of Pseudomonas aeruginosa to pyocin S2. J. Biochem., vol. 87, pp.323–331.
Smith AW, Hirst PH, Hughes K, Gensberg K, Govan JR (1992), The pyocin Sa receptor of Pseudomonas aeruginosa is associated with ferripyoverdin uptake. J. Bacteriol., vol.174, pp. 4847–4849.
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