Identification and Localization of The S-layer Proteins of Two Acidithiobacillus 

Acidithiobacillus strains SP5/1 and HV2/2 are rod-shaped, Gram-negative, γ-proteobacterium, aerobically pyrite-oxidizing and Acidophilic bacteria that thrives optimally at 30°C and 45°C respectively (Klingl, Moissl-Eichinger et al. 2011). S-layer molecular structure similarity in symmetry in the closely related species can be used as a taxonomic characteristic that distinguishes phylogenetic position of the organism (Baumeister and Lembcke 1992, Klingl, Moissl-Eichinger et al. 2011). S-layers protein has enveloped the bacteria surface, with the potential to assemble itself.

The protein is contained 2-D pseudo-crystalline with a variation in (p1, p2, p3, p4 or p6) symmetry (Eichler 2003). Two mentioned strains are possessed S-layer protein with p2-symmetry (Klingl 2007). Surf-layer first identify in the cell wall of a Spirillum sp. (Houwink and Le Poole 1952, Houwink 1953), and from today it has been found in nearly all taxonomic of bacteria and archaeal envelopes (Rachel, Pum et al. 1997, Sleytr 1997). The S-layer subunits are connected to the peptidoglycan layer or to the pseudomurein in archaea and gram-positive bacteria. Subunits are associated to components of Lipopolysaccharides (LPS) in gram negative bacteria (Sára and Sleytr 2000).

The only cell wall component linked to the membrane of cytoplasmic in archaea supposed to be Slayer (König, Rachel et al. 2007). It represents the simplest protein of archaeal envelopes and bacteria (Sára and Sleytr 2000); furthermore, due to possession about 10% of protein cells in Bacteria and Archaea, they introduce fascinating prototype for cell investigating during cell growth and division and evolutionary associations (Whitman 1998, Sleytr, Huber et al. 2007). Extracellular polymeric substances (EPS) mediate the contact between the cell, and the sulfidic energy source, playing a key role in organic film formation and bacterium basis interactions (Savage and Fletcher 1985). , composed of polysaccharides, proteins, lipids and nucleic acids, with vary an amount and composition in various energy source (Gehrke, Telegdi et al. 1998). A. ferrooxidans ATCC 23270Tmore EPS growth was reportedin a medium containing pyrite or sulfur in a compare to ferrous iron (Gehrke, Telegdi et al. 1998). The extracellular polysaccharides (EPSs) are connected to the iron(III) ions complexation (Sand and Gehrke 2006, Klingl, Moissl-Eichinger et al. 2011). The amount of adhesion forces and attached cells are reduced in absence of EPSin A. ferrooxidans ATCC 23270 (Li, Wang et al. 2016). The factors, such as pH, energy source, affects the properties of cell surface; therefore, the different cultures shown varied adhesion forces 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 declared for A. ferrooxidans strains R1 and SPIII/3 (Sand and Gehrke 2006).

Different method of electron microscopy such as negative staining, freeze-etching method is provided considerable information on meshworks structure of S-layer (Messner, Pum et al. 1986, Rachel 1999). As phylogenetic approach, surface layer common features like Symmetry and dimension can be used to elucidate taxonomic position of the organism by electron microscopy (König, Rachel et al. 2007, Klingl, Moissl-Eichinger et al. 2011). High resolution of freeze-etching techniques as best method to consider s-layer structure and flagella in fresh, unwashed intact bacteria (Sleytr, Schuster et al. 2014). Isolating S-layer bacteria by hydrogen bond-breaking agent proteins leads to recrystallize into monomolecular or double layers arrays in suspension, it represents either in the shape of flat sheets or open-ended cylinders (Sleytr, Huber et al. 2007), (Jarosch, Egelseer et al. 2001); Furthermore, some native bacterial strains are lost their ability to produce S-layers due to loos of plasmids during prolonged laboratory cultivation (Jarosch, Egelseer et al. 2001, Blecha, Zarschler et al. 2005), to overcome these difficulties, S-layer proteins will be heterologous expressed in an eukaryotic systems like[PIY3 (Ahmad, Hirz et al. 2014). The stability of expressed protein, has been proved in yeast (Blecha, Zarschler et al. 2005). ObjectivesSurface layer proteins of Acidithiobacillus strains SP5/1 and HV2/2 will be isolated and localized to:

• Clarify phylogenetic position of these two strains
• Comparison of both strains’ mutual characters with other Acidithiobacillus species

AS description given by (Kelly and Wood 2000) Acidithiobacillus ferrooxidans SP5/1 be a member of species Acidithiobacillus caldus; however, growing in a medium with Fe2+ as sole energy substrate and oxidizing pyrite, suggesting categorize strain SP5/1 in an appropriate new species “Candidatus Acidithiobacillus striatothermus”. Existing of S-layer have been proved in strain SP5 / 1 (Klingl, Moissl-Eichinger et al. 2011). In addition, we hypothesize that Afe_2303 protein have putative similarities to a Surface layer protein in A. ferrooxidans ATCC 23270 (Klingl, Moissl-Eichinger et al. 2011). Investigating the S-Layer structure, the phylogenetic position of both strain will be clarified, however, by studying the nature of strip-likes in these two strains, and comparing the mutual characters with other same species there would be able to more details information to classify Acidithiobacillus species. Research Design, Methods, and Procedures

1. The bacterial strain will be used in this study as follows: The Acidithiobacillus ferrooxidans strains SP5/1 was isolated in 1984 from Solfatara volcanic, Pisciarelli area in Italy and Acidithiobacillus sp HV2/2 sampling was from Hveravellier, Island and was isolated in 1986 by Dr. med. Harald Huber at University of Regensburg.
2. Strains and cultivation conditionBoth Acidithiobacillus strains SP5/1 and HV2/2 strains will be cultivated in 9-K medium (Silverman and Lundgren 1959) containing with 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 temperatures of 30°C for strain SP5/1 and 45°C for strain HV2/2 for 72 h. A. ferrooxidans ATCC 23270, A. caldus DSM 8584 will be used in this study. A deficient laboratory strain of A. ferrooxidans SP5/1, meanwhile, will be used as control. Growth and morphology of both strain will be controlled by Light microscopy, will be counted with Thoma cell counting chamber.
3. Electron microscopy. The appendage of samples will be investigated by negative staining, freeze-etching method. The cells will be deposited on carbon coated gold grids and negatively stained with 2% (w/v) uranyl acetate pH 4. 5. Bacteria cells will be fixed with 1. 25% glutaraldehyde for 15 min, will be followed by centrifugation at 20000 × g for 10 min and resuspended in 50 μl residual medium. The cells will be harvested by a centrifuge freeze-etching method, will be followed by high-pressure freezing on gold carriers in freeze-etching method according to (Klingl, Moissl-Eichinger et al. 2011).
4. Preparation of genomic DNA and DNA-cloningThe protein sequences and gene codes will be obtained from NCBI the UniProt, JGI, and NCBI databases. Chromosomal DNA will be isolated from Acidithiobacillus strains SP5/1 and HV2/2, and DNA manipulations, plasmid preparations, electroporations and DNA sequencing will be performed according to (Sambrook, Fritsch et al. 1989). Isolated DNA will be used as a template for polymerase chain reaction. The Ynce gene will be isolated by PCR with following primer: forward primer (5'-CGGCGAAAATACTGGAGCCTA-3') and the reverse primer (5'-ATGAGAATCGCTCAATCGGGTT-3'). The amplification products will be investigated by electrophoresis on a 1% agarose gel containing ethidium bromide. The PCR product will be gel purified.
5. Cloning into vector E. coli vectorE. coli will be grown in Liquid and on agar plates of LB media containing 50 µg/mL of kanamycin at 37 °C. The gene will be cloned into E. coli vector possessing AOX1 Promotor and of Bgl II and Bam HI restriction sites according to kit instructions. The constructed plasmids will be named P-YncE. All materials will be used according to manufactured. The expression vector will be digested with EcoRI and Not I, and obtained gene YncE fragments. The Competent cells, will be prepared chemically. The expressed gene will be confirmed by Fluorescence microscopy.
6. Cloning into Pichia pastoris vectorPichia pastoris strain GS115 will be grown in pre-cultural YPD medium in Erlenmeyer flasks containing 1% w/v yeast extract, 2% w/v tryptone, and 2% w/v dextrose at 30 °C at 140 rpm for 48 h. The yeast will be inoculated BMGY and BMMY supplemented by 2% w/v peptone, 1% w/v yeast extract, 100 mM potassium phosphate buffer pH 6. 0, 1. 34% w/v yeast nitrogen base and 1% v/v glycerol (BMGY) or 1% v/v methanol (BMMY) for growth and protein production, respectively (Wang, Zhang et al. 2017). P. pastoris cells grown and transformed cells on YPD, YPM or YPO medium will be investigated by an electron microscopic. The electroporation will be adjusted settings as follows: 1,500 V, 25 µF, 600 Ω. The transformed cells with OD 600 of will be spread on YPD plate containing 0. 1 or 0. 25 mg/mL geneticin. The bigger colonies with the higher copies of interested DNA grow on plates YPD agar geneticin will be chosen for next step (Logez, Alkhalfioui et al. 2012). The expression of S-layer protein will be investigated by Fluorescence microscopy in Yeast.
7. Isolation protein Afe-2303 The transformed cells grown on YPM or YPO media for 26 h to the late logarithmic phase will be harvest. To obtain highly purified, 1. 5 ml sample will be pelleted in an Eppendorf Microfuge and suspended in 200 µl of breaking buffer containing of 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl and 1 mM EDTA pH 8. Glass beads 200 µlwill be added, it will be followed by the addition of 200 µl of 25:24:1 phenol-chloroform isoamyl alcohol. The mixture will be vortexed at high speed for 2 min. Lysates will be centrifuged at high speed for 5 min (Faber, Heyman et al. 1998). The validity of protein will be confirmed by protein sequencing.
8. Protein analysisThe protein will be denatured on SDS-PAGE and preparation of sample will be carried out as described by Laemmli(Laemmli 1970). Concentration of protein will be determined according to (Bradford 1976). On 7,5% (w/v) acrylamide gels, 10 μg of protein will be separated. It will be followed by transferring onto a PVDF membrane and will be probed with antibody of monoclonal mouse directed against the GFP epitope. The antibody bounds will be detected by horseradish peroxidase conjugated secondary antibodies. As positive control, immunoreactivity of anti-GFP antibodies whole cell extract of Afe-2303 expressing Pichia pastoris transformed cells will be used.
9. Electron microscopyThe structure of Afe-2303 will be analyzed with Transmission Electron Microscopy (TEM) by negative staining and freeze-drying. The spheroplasts will be prepared according to (Waterham, Titorenko et al. 1994). The GFP antibodies in vivo will be studied by immunolabeling according to (Slot and Geuze 1984).
10. Phylogenetic tree of protein Afe_2303A homology search using the BLASTP program will be created to compare Afe-2303 protein with other related Acidithiobacillus strains. It will be constructed by pairwise alignment of Afe_2303 in this BLAST search.