Acidithiobacillus strains SP5/1 and HV2/2 are rod-shaped, Gram-negative, aerobically pyrite-oxidizing, acidophilic γ-proteobacteria they thrive optimally at 30°C and 45°C, respectively (Klingl, Moissl-Eichinger et al. 2011). The similarity of S-layer molecular structure in symmetry in closely related species can be used as a taxonomic feature distinguishing the phylogenetic position of the organism (Baumeister and Lembcke 1992, Klingl, Moissl-Eichinger et al. 2011). The S-layer protein wrapped around the surface of the bacteria, with the potential to assemble. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essay The protein is contained in a 2-D pseudo-crystalline form with a variation in symmetry (p1, p2, p3, p4, or p6) (Eichler 2003 ). Two strains mentioned possess the S-layer protein with p2 symmetry (Klingl 2007). The surf layer is identified for the first time in the cell wall of a Spirillum sp. (Houwink and Le Poole 1952, Houwink 1953), and today it has been found in almost all the taxonomy of bacteria and archaeological shells (Rachel, Pum et al. 1997, Sleytr 1997). S-layer subunits are linked to the peptidoglycan layer or pseudomurein in archaea and gram-positive bacteria. The subunits are associated with lipopolysaccharide (LPS) components in gram-negative bacteria (Sára and Sleytr 2000). The only cell wall component bound to the cytoplasmic membrane in archaea is supposed to be Slayer (König, Rachel et al. 2007). It represents the simplest protein of archaeological envelopes and bacteria (Sára and Sleytr 2000); furthermore, since they possess approximately 10% of protein cells in bacteria and archaea, they introduce fascinating prototypes for cellular investigation during cell growth and division and evolutionary associations (Whitman 1998, Sleytr, Huber et al. 2007). Extracellular polymeric substances (EPS) mediate contact between the cell and the hydrogen sulfide energy source, playing a key role in organic film formation and bacterial base interactions (Savage and Fletcher 1985). , composed of polysaccharides, proteins, lipids and nucleic acids, of varying quantity and composition depending on the energy source (Gehrke, Telegdi et al. 1998). A. ferrooxidans ATCC 23270T was reported to grow more EPS in a medium containing pyrite or sulfur than in ferrous iron (Gehrke, Telegdi et al. 1998). Extracellular polysaccharides (EPS) are linked to the complexation of iron(III) ions (Sand and Gehrke 2006, Klingl, Moissl-Eichinger et al. 2011). The amount of adhesion forces and attached cells is reduced in the absence of EPSin A. ferrooxidans ATCC 23270 (Li, Wang et al. 2016). Factors such as pH and energy source affect cell surface properties; therefore, different cultures showed different adhesion strengths to the same substrate (Li, Wang et al. 2016). Iron binding does not occur randomly (Sand and Gehrke 2006), a correlation between the amount of iron(III) ions complexed within the EPS and FeS2 cell attachment has been stated for A. ferrooxidans strains R1 and SPIII/3 (Sand and Gehrke 2006 ). Different electron microscopy methods such as negative staining, freeze-etching method provide considerable information on the network structure of the S layer (Messner, Pum et al. 1986, Rachel 1999). As a phylogenetic approach, common characteristics of the surface layer such as symmetry and size can be used to clarify the taxonomic position of the organism by electron microscopy (König, Rachel et al. 2007, Klingl, Moissl-Eichinger et al.2011). High resolution freeze-etching techniques as the best method to consider S-layer structure and flagella in fresh, unwashed intact bacteria (Sleytr, Schuster et al. 2014). Isolation of S-layer bacteria by hydrogen bond-breaking agent proteins leads to recrystallization into monomolecular or bilayer matrices in suspension, represented in the form of flat sheets or open-ended cylinders (Sleytr, Huber et al. 2007) , (Jarosch, Egelseer et al. 2001); Furthermore, some native bacterial strains lose the ability to produce S-layers due to plasmid loss during prolonged laboratory cultivation (Jarosch, Egelseer et al. 2001, Blecha, Zarschler et al. 2005). To overcome these difficulties, S-layer proteins will be heterologously expressed in eukaryotic systems such as [PIY3 (Ahmad, Hirz et al. 2014). The stability of expressed proteins has been demonstrated in yeast (Blecha, Zarschler et al. 2005). ObjectivesThe surface layer proteins of Acidithiobacillus strains SP5/1 and HV2/2 will be isolated and localized in:Keep in mind: this is only a sample.Receive a personalized paper from our expert writers now.Receive a personalized essayClarify the phylogenetic position of these two strainsComparison of the mutual characters of both strains with other Acidithiobacillus species. The description provided by (Kelly and Wood 2000) Acidithiobacillus ferrooxidans SP5/1 is a member of the species Acidithiobacillus caldus; however, growing in a medium with Fe2+ as the sole energy substrate and oxidizing pyrite, suggesting classifying strain SP5/1 into an appropriate new species “Candidatus Acidithiobacillus striatothermus”. The existence of the S layer was demonstrated in the SP5/1 strain (Klingl, Moissl-Eichinger et al. 2011). Furthermore, we hypothesize that the Afe_2303 protein has putative similarities to a surface layer protein in A. ferrooxidans ATCC 23270 (Klingl, Moissl-Eichinger et al. 2011). By investigating the structure of the S layer, the phylogenetic position of both strains will be clarified, however, by studying the nature of the stripes in these two strains and comparing each other's characters with other same species more detailed information could be obtained to classify Aciditiobacillus species. Research design, methods and procedures The bacterial strain will be used in this study as follows: The strain Acidithiobacillus ferrooxidans SP5/1 was isolated in 1984 from Solfatara volcano, Pisciarelli area in Italy and the sampling of Acidithiobacillus sp HV2/2 was from Hveravellier , Isola and was isolated in 1986 by Dr. med. Harald Huber at the University of Regensburg. Strains and cultivation conditions Both Acidithiobacillus strains SP5/1 and HV2/2 will be grown in 9-K medium (Silverman and Lundgren 1959) containing 125 g of pyrite in Erlenmeyer flasks. The pH medium will be adjusted to 2.5 with 50% sulfuric acid and will be incubated on a rotary incubator at 150 rpm at the optimal growth temperature of 30°C for the SP5/1 strain and 45°C for the SP5/1 strain HV2/2 for 72 hours A. ferrooxidans ATCC 23270, A. caldus DSM 8584 will be used in this study. A deficient laboratory strain of A. ferrooxidans SP5/1, however, will be used as a control. The growth and morphology of both strains will be checked by light microscopy and will be counted with the Thoma cell counting chamber. Electron microscopy. The appendix of the samples will be analyzed by negative staining and freeze-etching method. Cells will be plated onto carbon-coated gold grids and negatively stained with 2% (w/v) uranyl acetate pH 4. 5. Bacterial cells will be fixed with 1.25% glutaraldehyde for 15 minutes, followed by centrifugation at 20,000 × g for 10 minutes and resuspended in 50 μl of residual medium. The.