Abstract
Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.
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Abbreviations
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- IPTG:
-
Isopropyl thio-galactopyranoside
- AcsD:
-
Acetobacter cellulose synthase operon protein D
References
Anwar H, Brown MR, Cozens RM, Lambert PA (1983) Isolation and characterization of the outer and cytoplasmic membranes of Pseudomonas cepacia. J Gen Microbiol 129(2):499–507
Benziman M, Haigler CH, Brown RM, White AR, Cooper KM (1980) Cellulose biogenesis: polymerization and crystallization are coupled processes in Acetobacter xylinum. Proc Natl Acad Sci USA 77(11):6678–6682
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brown RM Jr, Willison JH, Richardson CL (1976) Cellulose biosynthesis in Acetobacter xylinum: visualization of the site of synthesis and direct measurement of the in vivo process. Proc Natl Acad Sci USA 73(12):4565–4569
Bureau TE, Brown RM (1987) In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum. Proc Natl Acad Sci USA 84(20):6985–6989
de Maagd RA, Lugtenberg B (1986) Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components. J Bacteriol 167(3):1083–1085
Eitan B (2007) The periplasm: co- and posttranslational protein targetting to the SecYEG translocon in Escherichia coli. ASM Press, Washington DC
Fox BG, Borneman JG, Wackett LP, Lipscomb JD (1990) Haloalkene oxidation by the soluble methane monooxygenase from Methylosinus trichosporium ob3b- mechanistic and environmental implications. Biochemistry-Us 29(27):6419–6427
Gardy JL, Laird MR, Chen F, Rey S, Walsh CJ, Ester M, Brinkman FS (2005) Psortb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 21(5):617–623
Garen A, Levinthal C (1960) A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim Biophys Acta 38:470–483
Graham LL, Beveridge TJ, Nanninga N (1991a) Periplasmic space and the concept of the periplasm. Trends Biochem Sci 16(9):328–329
Graham LL, Harris R, Villiger W, Beveridge TJ (1991b) Freeze-substitution of gram-negative eubacteria: general cell morphology and envelope profiles. J Bacteriol 173(5):1623–1633
Haigler CH, White AR, Brown RM Jr, Cooper KM (1982) Alteration of in vivo cellulose ribbon assembly by carboxymethylcellulose and other cellulose derivatives. J Cell Biol 94(1):64–69
Halford SE (1971) Escherichia coli alkaline phosphatase. An analysis of transient kinetics. Biochem J 125(1):319–327
Hestrin S, Schramm M (1954) Synthesis of cellulose by acetobacter xylinum. Ii. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose. Biochem J 58(2):345–352
Hu XM, Zhang J, Xiao J, Li Y (2008) Protein folding in hydrophobic-polar lattice model: a flexible ant-colony optimization approach. Protein Peptide Lett 15(5):469–477
Lamb JR, Tugendreich S, Hieter P (1995) Tetratrico peptide repeat interactions: to tpr or not to tpr? Trends Biochem Sci 20(7):257–259
Lin FC, Brown RM Jr, Drake RR Jr, Haley BE (1990) Identification of the uridine 5′-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc. J Biol Chem 265(9):4782–4784
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193(1):265–275
Myers CR, Myers JM (1992) Localization of cytochromes to the outer membrane of anaerobically grown shewanella putrefaciens mr-1. J Bacteriol 174(11):3429–3438
Ohad I, Danon D (1964) On dimensions of cellulose microfibrils. J Cell Biol 22(1):302–305
Olsson RT, Kraemer R, Lopez-Rubio A, Torres-Giner S, Ocio MJ, Lagaron JM (2010) Extraction of microfibrils from bacterial cellulose networks for electrospinning of anisotropic biohybrid fiber yarns. Macromolecules 43(9):4201–4209
Palmer T (2007) The periplasm: the Tat protein export pathway. ASM Press, Washingtom DC
Ponting CC, Phillips C (1996) Rapsyn’s knobs and holes: eight tetratrico peptide repeats. Biochem J 314(Pt 3):1053–1054
Robinson PA, Anderton BH, Loviny TLF (1988) Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries. J Immunol Methods 108(1–2):115–122
Romling U (2002) Molecular biology of cellulose production in bacteria. Res Microbiol 153(4):205–212
Ruebush SS, Brantley SL, Tien M (2006) Reduction of soluble and insoluble iron forms by membrane fractions of Shewanella oneidensis grown under aerobic and anaerobic conditions. Appl Environ Microbiol 72(4):2925–2935
Saxena IM, Lin FC, Brown RM Jr (1990) Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum. Plant Mol Biol 15(5):673–683
Saxena IM, Kudlicka K, Okuda K, Brown RM Jr (1994) Characterization of genes in the cellulose-synthesizing operon (acs operon) of acetobacter xylinum: implications for cellulose crystallization. J Bacteriol 176(18):5735–5752
Sha Z, Stabel TJ, Mayfield JE (1994) Brucella abortus catalase is a periplasmic protein lacking a standard signal sequence. J Bacteriol 176(23):7375–7377
Streeter JG, Le Rudulier D (1990) Release of periplasmic enzymes from Rhizobium leguminosarum bv phaseoli bacteroids by lysozyme is enhanced by pretreatment of cells at low ph. Curr Microbiol 21(3):169–173
Thomas JD, Daniel RA, Errington J, Robinson C (2001) Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (tat) pathway in Escherichia coli. Mol Microbiol 39(1):47–53
Wong HC, Fear AL, Calhoon RD, Eichinger GH, Mayer R, Amikam D, Benziman M, Gelfand DH, Meade JH, Emerick AW et al (1990) Genetic organization of the cellulose synthase operon in acetobacter xylinum. Proc Natl Acad Sci USA 87(20):8130–8134
Woodman ME (2008) Direct PCR of intact bacteria (colony PCR). Curr Protoc Microbiol Appendix 3:Appendix 3D
Yahr TL, Wickner WT (2001) Functional reconstitution of bacterial tat translocation in vitro. EMBO J 20(10):2472–2479
Zaar K (1979) Visualization of pores (export sites) correlated with cellulose production in the envelope of the gram-negative bacterium Acetobacter xylinum. J Cell Biol 80(3):773–777
Acknowledgments
The work was supported in part by the US Department of Energy, Office of Basic Energy Sciences as part of an Energy Frontier Research Center award number DE-SC0001090. Support for Prashanti Iyer was provided by USDA National Needs Graduate Fellowship Competitive Grant No. 2007-38420-17782 from the National Institute of Food and Agriculture and the Penn State College of Agricultural Sciences Dean’s Scholars fund. We thank Molly Hanlon for preparation of Fig. 4. The mass spectrometric analysis of AcsD was performed by Hasan Koc of The Pennsylvania State University.
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Iyer, P.R., Catchmark, J., Brown, N.R. et al. Biochemical localization of a protein involved in synthesis of Gluconacetobacter hansenii cellulose. Cellulose 18, 739–747 (2011). https://doi.org/10.1007/s10570-011-9504-4
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DOI: https://doi.org/10.1007/s10570-011-9504-4