Vol. 160, No. 1JOURNAL OF BACTERIOLOGY, OCt. 1984, p. 107-1110021-9193/84/100107-05$02.00/0Copyright C 1984, American Society for Microbiology

Protein on the Cell Surface of the Moderately HalophilicPhototrophic Bacterium Rhodospirillum salexigens

DIERK EVERS, JURGEN WECKESSER,* AND GERHART DREWSInstitut far Biologie II, Mikrobiologie, der Albert-Ludwigs-Universitdt Freiburg, D-7800 Freiburg, Federal Republic of

Germany

Received 27 December 1983/Accepted 5 May 1984

A cell surface protein (Mr 68,000) of the moderately but obligately halophilic phototrophic bacteriumRhodospirillum salexigens was identified by two independent methods: first, by labeling the cell surface withradioactive iodine and lactoperoxidase, and second, by washing cells in 30% sucrose to remove proteinsattached to the cell surface by ionic bonds. The identified protein very likely represents the outermost layer ofthe cell envelope of R. salexigens as observed by electron microscopy. The protein was isolated. Its isoelectricpoint was determined to be 4.4; the excess of acidic over basic amino acids was found to be 18.3 mol %; and itsaverage hydrophobicity was 2.26 kJ per residue.

Although extremely halophilic bacteria and their cell wallshave been the focus of extensive work (for a review, seereference 10), moderately halophilic bacteria have not beenstudied in such detail yet. The latter are found in alltaxonomic groups of bacteria (2, 17, 27). Moderately halo-philic bacteria are eubacteria, i.e., their cell walls containmurein; therefore, in this respect, they are comparable toother typical gram-negative or gram-positive bacteria. Onthe other hand, the environment they live in implies thatmajor adaptations to life in high salt concentrations are to beexpected. Although most of these bacteria are salt tolerant,Rhodospirillum salexigens (5), the bacterium which waschosen for study, is salt dependent. This phototrophicbacterium grows at sodium chloride concentrations between5 and 20%. The cells react negatively on Gram staining, theircell walls contain peptidoglycan in surprisingly smallamounts, and proteins seem to be firmly associated with thispeptidoglycan (25). Electron microscopic studies showed anoutermost layer outside a very thin so-called "outer mem-brane" bilayer (7).Thin layers of regularly arranged particles outside the

outer membrane have been observed in many gram-negativebacteria (6, 24). Similar structures also occur in extremelyhalophilic Archaebacteria (11). Although the chemical com-position of most of these layers observed by electron micros-copy remains unknown, some were found to consist ofprotein (3). The present paper deals with a protein located onthe cell surface of R. salexigens.

MATERIALS AND METHODSR. salexigens WS 68 (German Collection of Microorga-

nisms, DSM 2132) was grown in a salt medium (7) anaerobi-cally in the light in batch cultures of 50 ml to 1 liter. Cellswere harvested in the exponential-growth phase by centrifu-gation at 2,500 x g for 20 min.Sucrose wash treatment. Freshly harvested cells were first

washed in medium (as described above) and then suspendedin 30% sucrose (9). This suspension was kept on a rotaryshaker at 30°C for 60 min. Intact cells were removed bycentrifugation at 20,000 x g for 30 min. The pink supernatantwas dialyzed to remove sucrose and was then centrifuged at

* Corresponding author.

250,000 x g for 1 h. The sediment was suspended in 1 ml ofdistilled water and then, to remove membrane material ofcells that had been disrupted during the first steps ofpreparation, was applied to a 20 to 60% sucrose densitygradient and centrifuged at 28,000 x g for 16 h. A paleyellowish band near the bottom of the gradient, below all redmembrane bands, was isolated; sucrose was removed bydialysis; and the material was freeze-dried.

lodination. lodination of surface proteins was performedby the method of Mescher and Strominger (21). Freshlyharvested and washed cells (wet weight, 1 g) were suspendedin 5 ml of 0.76 mM phosphate buffer (pH 6.9) containing 8%NaCl and 0.015% MgCl2. Lactoperoxidase (100 jig; SigmaChemical Co., St. Louis, Mo.) was added, and then 1 mCi ofNa125I (Amersham Buchler, Braunschweig, Federal Repub-lic of Germany). lodination was started by adding 10 LI of0.03% H202. Another 10 ,ul of 0.03% H202 was added every5 min. After 30 min, the iodination was stopped by adding 10RI of 10 mM Na2SO3. To remove excessive Na125I, weapplied the preparation to a small column (40 ml) of coarseSephadex G-25, and this column was centrifuged at 500 x gfor 3 min. The eluate contained the iodinated cells, whereasmost of the unbound iodine was retained by the column.

Stabilization of the cell wall by cross-linking. Freshlyharvested and washed cells (wet weight, 30 mg) were sus-pended in 1 ml of 0.5 M triethanolamine buffer, pH 7.6,containing 8% NaCl and 0.015% MgCl2. Every 15 min for atotal of 2 h, 1.25 mg of dimethyl-3,3'-dithiobispropionimi-date dihyrochloride (DDPI) (Pierce Chemical Co., Rockford,Ill.) was added (final concentration, 50 mM). Cross-linkingwas stopped after 2 h by adding 50 ,u1 of 0.75 M Tris buffer.Before gel electrophoretic separation, cross-linking bridgeswere cleaved by f-mercaptoethanol (sample buffer for sodi-um dodecyl sulfate [SDS]-polyacrylamide gel electrophore-sis contained 6.6% ,-mercaptoethanol).SDS-polyacrylamide gel electrophoresis and autoradiogra-

phy. Polyacrylamide electrophoresis was performed in verti-cal slab gels with continuous gradients of 7 to 16.5% acryl-amide. Solutions were prepared by the method of Laemmli(14). Apparent molecular weights were determined by com-parisons with the low-molecular-weight protein standard ofPharmacia Fine Chemicals AB, Uppsala, Sweden.

Polyacrylamide gels were dried on filter paper under mildvacuum. The gel surface was brought in direct contact with a

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108 EVERS, WECKESSER, AND DREWS

Kodak X-Omat X-ray film (Eastman Kodak Co., Rochester,N.Y.) for 4 days.

Protein extraction. Protein bands were extracted from anSDS-polyacrylamide gel by several additions of smallamounts of 70%, trichloroacetic acid to the cut-out bands.Trichloroacetic acid was then removed by dialysis, and theextracted protein was freeze-dried.Amino acid analysis. Samples were hydrolyzed in 6 N HCl

at 110°C for 24 h. Analyses were carried out on a Durrum D-500 automatic amino acid analyzer.

Isoelectric focusing. Isoelectric focusing was performed onhorizontal polyacrylamide gels (4). Servalyt AG 2-11 and AG2-4 (Serva, Heidelberg, Federal Republic of Germany) am-pholytes were used. Samples were applied to the gel surfaceby filter paper. The pH gradient was determined by aid ofprotein bands of test mixture no. 9 (Serva) and by directmeasurement on the gel surface with a surface pH electrode(LKB Instruments, Inc., Bromma, Sweden).

Malate dehydrogenase assay. Oxalacetic acid (10 mg) and200 Fg of NADH (Sigma) were suspended in 750 [L of 100mM phosphate buffer, pH 7.5; the test was started by adding250 [I of the enzyme-containing sample. The decrease inNADH concentration was calculated from the absorptiondecrease at 340 nm.

Electron microscopy. Freeze-etch preparations and elec-tron microscopic observations were carried out as previous-ly described (7).

RESULTSSucrose wash treatment. When obligately halophilic bacte-

ria are exposed to nonionic media, at least some of theirmacromolecules will lose their stability because of changesin the strength of noncovalent bonds. In an isotonic orhypertonic but nonionic medium, it is therefore to be expect-ed that structures outside the osmotic barrier of the cytoplas-mic membrane will be destabilized and, if not covalentlybound, will dissociate, whereas under such conditions, cellswill not burst in large quantities.

Sucrose is well suited for this purpose because cytoplas-mic membranes are almost impermeable to this sugar (28).Exposing whole cells of R. salexigens to 30% sucrose at 30°Cfor 60 min led to the dissociation of two proteins which werethen isolated by density gradient centrifugation. In SDS-polyacrylamide gel electrophoresis, these two proteinsshowed apparent molecular masses (Mr) of ca. 100,000 and68,000, respectively (Fig. 1, lane d; obtained from iodinatedcells).A control experiment was done to test how many cells

burst during the sucrose wash treatment by determining howmuch malate dehydrogenase was released. Of freshly har-vested cells, one-half were used untreated, and the other half(the controls) were disrupted sonically. Both samples wereexposed to 30% sucrose for 1 h and then centrifuged (20,000x g). The supernatant of the sample with disrupted cellsshowed a malate dehydrogenase activity of 806.7 pkat/ml,and that of the sample with undisrupted cells was 39.9 pkat/ml, 4.9% of the enzyme activity of the control sample. Thus,less than 5% of the cells burst during the sucrose washtreatment.

Cells that had been exposed to 30% sucrose were studiedby electron microscopy. Whereas in intact cells an outer-most layer can be observed in freeze-etch preparations (7),this layer was not detectable in sucrose-treated cells (Fig. 2).

lodination. An attractive method for identification of sur-face proteins is iodination of proteins with 1251- by means ofa catalyst which either is insoluble or else is unable topenetrate the surface to be examined. Lactoperoxidase has

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a b c d eFIG. 1. SDS-polyacrylamide gel electropherogram (lanes a, b,

and d) and corresponding autoradiogram (lanes c and e). (a) Molecu-lar weight standards (top to bottom) were 94,000, 67,000, 43,000,30,000, 20,100, and 14,400 Mr; (b) whole cells (their cell walls notchemically cross-linked) iodinated with lactoperoxidase; (c) autora-diogram of the gel in lane b; (d) whole cells (their cell walls notchemically cross-linked) iodinated with lactoperoxidase, then sus-pended in 30% sucrose and shaken for 1 h at 30°C, after whichdissociated proteins were isolated by centrifugation and densitygradient centrifugation and applied to the gel; (e) autoradiogram ofthe gel in lane d.

frequently been used to iodinate surface proteins of bacterialcells (8, 20, 21), and the lactoperoxidase system is known tobe operable at high salt concentrations (21). This enzymecatalyzes the iodination of tyrosine and histidine in thepresence of HO, (22). Due to the large size of this enzyme,it will not pass through cytoplasmic membranes and entercells (23).

Since cells of R. salexigens are easily disrupted even byminor unfavorable conditions, applying the lactoperoxidasesystem for iodination often causes a considerable percentageof cells to burst. As a result, all major cell proteins will showat least some level of labeling. This problem was avoided bystabilizing cell walls before iodination by treating cells withthe cleavable, hydrophilic cross-linking agent DDPI. TheDDPI concentration used (50 mM) was less than half theconcentration needed to prevent the cells from burstingwhen suspended in distilled water. lodinated stabilized cellswere solubilized, and their proteins were separated by SDS-polyacrylamide gel electrophoresis. Figure 3 shows a gel(lane b) and corresponding autoradiogram (lane c). Only oneprotein contained nearly all of the radioactivity. This poly-peptide had an approximate Mr of 68,000.

lodination of stabilized cell walls did not lead to artifactssince under optimal conditions identical results were ob-tained from cells not pretreated with cross-linking agents.

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CELL SURFACE PROTEIN OF R. SALEXIGENS 109

FIG.2. 3 W M wereFIG. 2. Freeze-etch and freeze-fracture preparations of R. salexigens. Cells that had been exposed to sucrose wash treatments were

prepared in a buffer containing sucrose (a and d). Panels b, c, and e were reprinted from reference 7 by permission of the publisher. Arrows inthe upper right corner, direction of shadowing; bars, 100 nm. (a) Freeze-etch preparation of sucrose-treated cells exposing exoplasmicfracture faces (EF) and plasmic fracture faces (PF) of the cytoplasmic membrane (CM). Notice the lack of any layer outside the "outermembrane" (OM); (b and c) preparations of untreated cells corresponding to (a). Notice the outermost layer (OL) outside the "outermembrane" lacking in picture (a). (d) Freeze-etch preparation of sucrose-treated cells exposing the outer surface (ES) of the cell. Here, theouter cell surface is the outer membrane (OM). (e) Preparation of untreated cells corresponding to (d). Here, the outer cell surface (ES) showsa hexagonal pattern of subunits forming the outermost layer (OL) lacking in picture (d).

Figure 1 shows such a gel and corresponding autoradiogram(lanes b and c). Again, the polypeptide of Mr 68,000 con-tained most of the radioactivity. Some background activityin the upper part of the gel was observed, and a few otherproteins, probably from disrupted cells, showed very littleradioactivity.

Combination of the above methods. The proteins of Mr68,000 obtained by sucrose wash treatment and by iodinationshowed the same apparent molecular mass (Mr) in gelelectrophoresis (Fig. 1, lanes d and c), suggesting that thesetwo proteins are identical. To prove this assumption, cellslabeled with 1251- were exposed to 30% sucrose (methods asabove; cell walls were not cross-linked). Polypeptides disso-ciated by this procedure and isolated by density gradientcentrifugation were separated by SDS-polyacrylamide gelelectrophoresis (Fig. 1). Two proteins were observed (laned), one of Mr 100,000 and the other of Mr 68,000, and the

corresponding autoradiogram (lane e) clearly shows thesmaller one (Mr 68,000) to be iodinated.Thus the protein of Mr 68,000 obtained after sucrose wash

treatment and the protein of the same apparent molecularmass labeled after iodination of whole cells appear to beidentical.

Isoelectric focusing. The two proteins isolated by sucrosewash treatment, including the protein of Mr 68,000 labeledwith radioactive iodine, were separated by isoelectric focus-ing. The labeled protein of Mr 68,000 was identified byautoradiography, and its isoelectric point was measured tobe 4.4.Amino acid analysis. The band of protein of Mr 68,000 was

cut out of an SDS-polyacrylamide gel and extracted withtrichloroacetic acid. The amino acid analysis (Table 1) showsa high excess of acidic over basic amino acids (24.2 mol% ofthe amino acids are acidic; 5.9% are basic) and a very low

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110 EVERS, WECKESSER, AND DREWS

FIG. 3. SDS-polyacrylamide gel electropherogram (lanes a andb) and corresponding autoradiogram (lane c). (a) Molecular weightstandards (top to bottom) were 94,000, 67,000, 43,000, 30,000, and20,100 M,; (b) whole cells (their cell walls cross-linked with DDPI)iodinated with lactoperoxidase; (c) autoradiogram of the gel in lane b.

content of nonpolar amino acids. The average hydrophobic-ity of the protein of Mr 68,000 calculated by the method ofBigelow (1) was found to be 2.26 kJ per residue.

DISCUSSION

Washing cells in sucrose is a method which in severalcases led to the isolation of whole outer membranes ofmarine bacteria. With other species, no such effects wereobserved; others lost only part of their outer membrane (12,13). Applying this method to cells of R. salexigens, we

isolated only two proteins.For several years it was taken for granted that lactoperoxi-

dase will catalyze iodination of cell surfaces only, since itcannot penetrate cytoplasmic membranes. Loeb and Smith(19) recently reported that in Haemopliillis in flueizie, pro-teins of both outer and inner membranes are labeled bylactoperoxidase, supposedly because the outer membrane ofthis organism contains pores large enough for lactoperoxi-dase to enter the periplasmic space. This problem does notseem to arise with R. sialexigen.s, for we found only one

protein to be labeled (Fig. 3, lane c).This one iodinated protein (M, 68,000) obviously is ex-

posed on the cell surface. By repeating the sucrose washtreatment with iodinated cells, we were able to show that theiodinated polypeptide and the protein of Mr 68,000 obtainedby sucrose wash treatment are identical. Thus, this proteinwas identified by two independent methods which theoreti-

cally both lead to isolation and labeling of surface proteins.This shows that the protein of Mr 68,000 is part of the cellsurface of R. salexigens and, considering electron micro-scopic observations, most likely is identical with the outer-most layer of the cell wall. Freeze-etching electron micro-graphs of the outermost layer of R. salexigens (7) showregularly arranged particles comparable to the outermostlayers of other bacteria (6, 24), and this layer has disap-peared from the cell surface after sucrose wash treatmentswhich lead to the dissociation of the two proteins of Mr68,000 and 100,000 from the cells (Fig. 2).The protein having an Mr of ca. 100,000 that was isolated

after sucrose wash treatment was not labeled by lactoperoxi-dase iodination of whole cells with stabilized cell walls (Fig.3) and shows a very low level of labeling after iodination ofcells not treated with cross-linking agents (Fig. 1). Controlexperiments-iodination of mechanically disrupted cells ofR. salexigens-showed that this protein is iodinatable bylactoperoxidase. This finding can be explained by assumingthat this protein is not located on the immediate cell surface,or it might be folded in such a way that, even though it is partof the cell surface, all tyrosine and histidine residues areinaccessible for lactoperoxidase. Since less than 5%G of thecells burst during the sucrose wash treatment, it does notseem to be likely that a protein isolated by this methodoriginates from the cytoplasm.A very thin bilayer, called the outer membrane (7), just

below the outermost surface layer has been observed infreeze-etch preparations of R. salexigens (7), the chemicalcomposition of which is not known. It remains to be seenwhether the noniodinated protein of Mr 100,000 is part of thismembrane.High concentrations of ions in the solvent have significant

effects on proteins; negatively charged groups are beingscreened by cations, and ionic bonds are weakened (26).Small anions decrease the size of water clusters, and thus,hydrophobic bonds are stabilized considerably (16). It istherefore to be expected, and has been observed (9, 15, 17,18), that proteins from halophilic bacteria are more acidicthan their counterparts from nonhalophilic species. Also, ifthe single hydrophobic bond is stronger, fewer such bondsare needed to maintain the structure of a given protein.Proteins from halophilic bacteria will and do (15) show less

TABLE 1. Amino acid analysis of the isolated cell surfaceprotein (Mr 68.000) of R. sailexvigenis

Amino acid mol%

Alanine .... ... .. 11.78Arginine ... 0.00Aspartic acid ... ... .. 7.70Cysteine ..... ... .. 0.51Glutamic acid ... 16.51Glycine ... ..... 20.66Histamine ... 2.75Isoleucine ... 3.72Leucine ... 4.87Lysine ... 3.22Methionine .... ... .. 0.21Phenylalanine... ... .. 2.26Proline......... 0.00Serine ... 15.84Threonine ... 4.88Tryptophan.......... N D"Tyrosine ..... ... .. 0.00Valine ..... ... .. 5.09

" ND, Not determined.

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CELL SURFACE PROTEIN OF R. SALEXIGENS 111

average hydrophobicity than those of nonhalophilic species.In fact, the average hydrophobicity of the isolated protein ofMr 68,000 of R. salexigens was calculated to be as low as2.26 kJ per residue. In addition, the excess of acidic overbasic amino acids is high (18.3 mol%), which is in accordwith the low isoelectric point of 4.4. When compared withthe values of proteins from several halophilic and nonhalo-philic bacteria (15), the figures for the protein of Mr 68,000isolated from R. salexigens are in excellent agreement withtheoretical considerations for proteins from halophilic bacte-ria.

ACKNOWLEDGMENTSWe thank J. R. Golecki for taking the electron micrographs. The

amino acid analysis was kindly performed by M. Wiesner.This work was supported by the Deutsche Forschungsgemein-

schaft.Soli Deo Gloria.

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