[12 Lysozyme Osmotic Shock Methods for Localization of Periplasmic Redox Proteins in Bacteria

By Victor L. Davidson and Dapeng Sun


The cell envelope of gram-negative bacteria is composed of a cytoplasmic membrane (plasma membrane), a murein peptidoglycan layer, and an outer membrane that is linked by lipoproteins to the murein layer. The compartment between the cytoplasmic and outer membrane is called the periplasm, or periplasmic space.1 Several proteins are specifically localized in the periplasm. These have long been known to include binding proteins for nutrients, and hydrolytic and degradative enzymes. More recently it has become clear that the periplasm is also home to a wide variety of redox enzymes and electron transfer

1 J. W. Costerton, J. M. Ingram, and K.-J. Cheng, Bacteriol. Rev. 39, 87 (1974).

proteins.2-5 These include c-type cytochromes, redox-active metalloproteins, thiol: disulfide oxidoreductases, and several NAD(P)+-independent dehydrogenases and reductases. In particular, a disproportionate number of soluble electron transfer proteins of gram-negative bacteria are localized in the periplasm relative to the cytoplasm. Unlike eukaryotic cells, bacteria lack internal organelles and a complex intracellular membrane system. The membrane-bound respiratory chain of bacteria is localized in the cytoplasmic membrane. From a bioenergetic perspective, the periplasm is analogous to the mitochondrial intermembrane space of eukaryotic cells.

To determine whether a bacterial protein is periplasmic or cytoplasmic, a method must be available to cleanly separate the periplasmic and cytoplasmic contents of the cell. Furthermore, to purify a periplasmic protein from the cell, it would be desirable to purify it from the isolated periplasmic fraction rather than from a whole cell extract. The most common methods for disrupting bacterial cells are ultrasonic disruption (sonication) or passage through a French press. Neither of these methods is appropriate for fractionation of bacterial cells and the localization of periplasmic proteins. These methods not only disrupt the cell wall and outer membrane, but also the plasma membrane. The resulting cell extract will contain both periplasmic and cytoplasmic proteins. These procedures will also release DNA and some membrane lipids, which will interfere with the further processing of the cell extract. Furthermore, sonication is a relatively harsh treatment that may physically damage proteins.

For Escherichia coli, several methods for the release of periplasmic proteins have been described. These include osmotic shock,6 magnesium chloride treatment,7 chloroform treatment,8 and polymyxin treatment9 of intact cells. The most widely used technique for the release of periplasmic proteins from E. coli combines treatment of cells with lysozyme and exposure to a mild osmotic shock.10 These methods for the release of the periplasmic proteins from E. coli are not, however, universally applicable to all gram-negative bacteria.

In this chapter we describe two different procedures that use the combination of lysozyme and osmotic shock for the fractionation of bacterial cells and selective

2 C. Anthony (ed.), "Bacterial Energy Transduction." Academic Press, San Diego, CA, 1988.

3 V. L. Davidson (ed.), "Principles and Applications of Quinoproteins." Marcel Dekker, New York, 1993.

4 T. E. Meyer and M. A. Cusanovich, Biochim. Biophys. Acta 975, 1 (1989).

5 R. A. Fabianek, H. Hennecke, and L. Thony-Meyer, FEMS Microbiol. Rev. 24, 303 (2000).

6 N. G. Nossal and L. A. Heppel, J. Biol. Chem. 241, 3055 (1966).

7 K. J. Cheng, J. M. Ingram, and J. W. Costerton, J. Bacteriol. 104, 748 (1970).

8 G. F.-L. Ames, C. Prody, and S. Kustu, J. Bacteriol. 160, 1181 (1984).

9 Y. Kimura, H. Matsinaga, and M. Vaarga, J. Antibiot. (Tokyo) 45, 742 (1992).

10 B. Witholt, M. Boekhout, M. Brock, J. Kingma, H. van Heerikhuizen, andL. de Leij, Anal. Biochem. 74, 160 (1976).

release of periplasmic proteins. Procedure 1 has been used in our laboratory to fractionated coli,n Paracoccus denitrificans,n andRhodobacter sphaeroides}3 Procedure 2 was used with Alcaligenes faecalis,14 which could not be fractionated by procedure 1. The development of procedure 2 required significant modification of the generally applicable technique that was developed originally for E. coli. It is likely that these procedures will require further modificaiton for optimal effectiveness with other bacteria. As such, a general protocol is also described for optimizing conditions for the fractionation of other bacterial cells that are not efficiently fractionated by either of these two procedures.

Assay of Cytoplasmic and Periplasmic Marker Proteins

To evaluate the efficiency of a particular technique for the selective release of periplasmic proteins, it is necessary to monitor the release of general markers for periplasmic and cytoplasmic proteins. The latter will also be released from cells in which the cytoplasmic membrane is inadvertently ruptured during sphero-plast formation. Spheroplasts are vesicles with an intact cytoplasmic membrane that remains after removal of the cell wall and outer membrane and release of the periplasm. Some percentage of spheroplasts is likely to rupture during any fractionation procedure. The goal of the fractionation procedure is to maximize release of the periplasmic contents while minimizing release of the cytoplasmic contents. It should be noted that not all periplasmic proteins will be released to the same extent during fractionation procedures. The extent of release will depend on the size of the protein and the degree of its association with the plasma membrane. Specific examples of cytoplasmic and periplasmic marker proteins are given below with methods by which they may be quantitated. Assay of such marker proteins provides a reasonable estimate of the efficiency of fractionation procedures.

The bacterial cytoplasm is the site of most metabolic pathways. As such, enzymes that participate in glycolysis and the citric acid cycle are reasonable cytoplasmic markers. Because NADH dehydrogenase faces the cytoplasmic side of the plasma membrane, any NAD+-dependent enzyme may be presumed to be cytoplasmic. The NAD+-dependent enzyme, malate dehydrogenase, is common to a wide range of bacteria. It is commonly used as a marker to gauge the release of cytoplasmic proteins. A method by which to assay its activity15 is given below.

11 V. L. Davidson, L. H. Jones, M. E. Graichen, F. S. Mathews, and J. P. Hosier, Biochemistry 36, 12733 (1997).

12 V. L. Davidson, Methods Enzymol. 188, 241 (1990).

13 M. E. Graichen, L. H. Jones, B. Sharma, R. J. M. van Spanning, J. P. Hosier, and V. L. Davidson, J. Bacteriol. 181,4216 (1999).

14 Z. Zhu, D. Sun, and V. L. Davidson, J. Bacteriol. 181, 6540 (1999).

15 P. R. Alefounder and S. J. Ferguson, Biochem. J. 192, 231 (1980).

Assays of several non-redox-active periplasmic marker enzymes have been described. Commonly used enzymes include acid phosphatase16 and alkaline phosphatase,17 and kits with which to assay their activities are commercially available. Soluble otype cytochromes are widely distributed in gram-negative bacteria.4 These cytochromes are localized exclusively in the periplasm, or on the periplasmic face of the plasma membrane. Cytochrome c is not an enzyme and therefore has no specific activity that may be assayed. However, cytochromes c may be readily monitored by a method described below, which allows quantitative detection of cytochromes c after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude cell extracts.18

Assay ofMalate Dehydrogenase: Cytoplasmic Marker Reagents

Potassium phosphate (pH 7.5), 0.1 M Oxaloacetate

/}-Nicotinamide adenine dinucleotide, reduced form (NADH)

Procedure. Malate dehydrogenase catalyzes the reversible reaction shown below.

The spectrophotometric assay of malate dehydrogenase is performed as follows. The reaction mixture contains 0.1 M potassium phosphate, pH 7.5, with 0.2 mM oxaloacetate and 0.27 mM NADH. The reaction is initiated by the addition of an aliquot of the fractionated cell extract. The conversion of NADH to NAD+ is monitored by the decrease in absorbance at 340 nm. An assay without oxaloacetate acid should be performed to correct for background oxidation of NADH. Preparations of malate dehydrogenase are commercially available and may be used as a positive control. This assay may also be applied to any other NADH-dependent enzyme that does not require additional cofactors by simply substituting the appropriate substrate for the enzyme in place of oxaloacetate.

Detection of Cytochromes c: Periplasmic Markers Reagents

Dimethylbenzidine (also called o-dianisidine) Sodium citrate (pH 4.4), 0.5 M

16 L. A. Hepple, D. R. Harkness, and R. J. Hilmoe, J. Biol. Chem. 237, 841 (1962).

17 H. F. Dvorak, R. W. Brockman, and L. A. Hepple, Biochemistry 6, 1743 (1967).

18 R. T. Francis and R. B. Becker, Anal. Biochem. 136, 509 (1984).

Procedure. Fractionated cell extracts are subjected to denaturing SDS-PAGE by standard methods. Sets of samples are run in duplicate on different portions of the gel or on two identical gels. One gel is stained for total protein, using standard reagents such as Coomassie blue or commercially available reagents such as Gelcode blue stain reagent (Pierce, Rockford, IL). The total protein stain is performed to ensure that the gel was run properly with the expected amount of protein present. The other set of samples is specifically stained for heme, using the protocol described below. This stain is relatively specific for c-type cytochromes. In contrast to most heme-containing proteins, the heme is covalently bound in cytochromes c, and is retained during denaturation and SDS-PAGE.18

To prepare the staining solution, 100 mg of dimethylbenzidine is dissolved in 90 ml of H20. Immediately before incubating with the gel to be stained, 10 ml of sodium citrate plus 0.2 ml of H202 are added to the solution. Immediately after SDS-PAGE, the gel to be stained is placed in 12% (w/v) trichloroacetic acid and incubated for 30 min. The gel is then rinsed with H20 and incubated in the staining solution until covalent heme-containing proteins appear as green bands (15-60 min). The gel is then rinsed with H20 to reduce background. Relative amounts of heme-stained proteins present in the gel may be quantitated by densitometry and related to the percentage of the total extract that was loaded on the gel. This will provide a reasonable estimate of the percent release of periplasmic cytochrome c. Any cytochrome c that appears in the cytoplasmic fraction reflects the percentage of the periplasmic protein that was not released by the procedure. As a positive control for the heme stain a sample of commercially available horse heart cytochrome c may be included on the gel to be stained.

Lysozyme-Osmotic Shock Procedures

The two procedures described below and summarized in Fig. 1 share the common features of treatment of cells with lysozyme plus EDTA in conjunction with the induction of a mild osmotic shock. The rationale for this method19 is as follows. Lysozyme will digest, at least partially, the murein layer of the cell wall. Divalent cations are believed to play a role in maintaining the integrity of the cell wall and outer membrane. EDTA will modify the outer cell membrane of the cell wall to make it more permeable to lysozyme and agents that are used to adjust the osmotic strength. In this way the combined actions of lysozyme and EDTA make the

19 D. C. Birdsell and E. H. Cota-Robles, J. Bacterial. 93, 427 (1967).

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