Proc. Natl. Acad. Scd. USA Vol. 74, No. 9, pp. 4041-4045, September 1977 Microbiology

Extraction of an actin-like protein from the prokaryote Mycoplasma pneumonlae * (gliding motility/electron microscopy) HAROLD C. NEIMARK Department of Microbiology and Immunology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203 Communicated by Lewis Thomas, June 29,1977

ABSTRACT An actin-like protein has been identified in pneumoniae, an important cause of respiratory disease in cell extracts from the prokaryote Mycoplasma pneumoniae. children and young adults (14), was selected for examination This protein bears a striking resemblance to actin from verte- because it posse gliding motility (8), maintains a specific cell brates: (i) the solubility of the protein during isolation is anal- and has an ogous to that of actin bound to myosin (soluble in high ionic shape, accessible cell membrane. strength salt solution and insoluble at low ionic strength), (ii) sodium dodecyl sulfate treatment of the partially purified M. MATERIALS AND METHODS pneumoniae extract produces a protein with an electrophoretic mobility very close to that of vertebrate actin in sodium dodecyl Organisms and Growth Conditions. A recently isolated sulfate/polyacrylamide gels, (iii) treatment of preparations with virulent strain of M. pneumoniae received from W. Clyde was ATP-Mg2+ allows separation of long curvilinear filaments, 5-6 used for this study. Strains passaged in the laboratory, however, nm wide, that closely resemble eukaryotic filamentous actin, and (iv) the prokaryotic filamentous actin binds vertebrate are known to retain motility (8). The media and growth con- heavy meromyosin fragments to form hybrid complexes with ditions were essentially as described (15) except that 20% horse the characteristic shape of periodic repeating arrowheads, and serum (unheated) was used. The strain was adapted to grow also no heavy meromyosin is bound in the presence of ATP. in medium containing 3% PPLO serum fraction (Difco) instead of horse serum. The final glucose concentration was 0.5%. Proteins similar to the muscle contractile proteins actin and Penicillin (300 units/ml) was added in some instances. Cell myosin occur in a wide variety of nonmuscle cells, where they proteins were labeled by growth in media supplemented with are believed to function in the fundamental cellular processes [FsSimethionine (New England Nuclear Corp.) (specific ac- of motility and maintainance of cell shape. (for a review see ref. tivity, 295 Ci/mmol; 5 ,uCi/ml). Organisms were collected 1). In nonmuscle cells, the contractility and motility processess when a confluent layer of adherent cells formed on the bottom include amoeboid movement and cytoplasmic streaming (2), of the flasks. Cells were scraped off the flasks, centrifuged phagocytosis (3), and cytokinesis (4) as well as clot retraction (23,000 X g for 45 min), and washed once with 0.145 M by platelets (5). The widespread occurrence of actomyosin-like NaCI/0.02 M potassium phosphate buffer, pH 7.5. proteins in organisms representing broadly divergent eukaryotic Preparation of the Actin-Like Protein. The isolation pro- phyla suggests that these proteins provide a general mechanism cedure was based on those developed for vertebrate platelet for cell motility and contractility and that muscle contraction actomyosin (16,17). All steps were carried out at 4°. The packed may be but a specialized case of-a very general form of cell cell pellet was resuspended in 4 volumes of 0.6 M KCI/0.015 motility (1). M Tris-HCI, pH 8.5, 25 gl of butanol was added for each mil- Among the prokaryotes are various bacteria that lack flagella liliter of cell suspension, and the cells were extracted by stirring or other recognized organelles for locomotion but are never- for 16 hr. The supernatant (SI) was separated by centrifugation theless capable of movement. Organisms in the orders Myxo- (30,000 X g for 1 hr), made to approximately 0.07 M KCI by bacterales and Cytophagales as well as most cyanobacteria diluting with 6 volumes of water, and adjusted to pH 6.3 by (blue-green algae) display a directional gliding motility on solid addition of 0.125 M sodium acetate buffer, pH 4.9. After 40 min surfaces (6, 7). The molecular basis for movement in these at 40, the precipitate (P2) containing the actin-like protein was bacteria is presently unknown. Indeed, the precise basis for collected by centrifugation (30,000 X g for 30 min) and dis- movement of bacterial flagella also is not understood. solved in a small volume of 0.6 M KCI (S3). For some prepara- Certain members of the order Mycoplasmatales are also ca- tions, solutions contained 1 mM sodium sulfite as a proteinase pable of gliding motility (8, 9), and the recently discovered inhibitor (R. Siemankowski and P. Dreizen, unpublished data) spiral-shaped mycoplasmas exhibit flexing movement (10, 11). and 5 mM dithiothreitol. Preparations were usually used within In contrast to most bacteria, the mycoplasmas lack cell walls yet 1 week. still are able to maintain specific cell shapes. In addition, my- Gel Electrophoresis and Autoradiography. Sodium dodecyl coplasmas are notable for possessing the smallest genomes of sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was any organisms known to be capable of growth on cell-free carried out as described by Laemmli (18) or by a modification media (12, 13). The relative structural simplicity of the myco- of the procedure of Weber and Osborn (19). For the latter, gel plasmas suggested that the basis for motility in these organisms buffer was either 0.075 M Tris-acetate, pH 7.9 (20), or 0.02 M could be sought directly at the molecular level and might be phosphate, pH 7.1, containing 0.1% NaDodSO4 and 0.1% 2- a contractile protein, possibly resembling actomyosin. A search mercaptoethanol; 8% gels cast the day before use were prerun for a contractile protein was therefore undertaken. Mycoplosma Abbreviations: NaDodSO4, sodium dodecyl sulfate; HMM, heavy The costs of publication of this article were defrayed in part by the meromyosin. payment of page charges. This article must therefore be hereby marked * A preliminary report of this work was presented at the Annual "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate Meeting of the American Society for Microbiology, 1976, p. 61, ab- this fact. stract D 62. 4041 Downloaded by guest on September 27, 2021 4042 Microbiology: Neimark Proc. Nati. Acad. Sci. USA 74 (1977) at 5 mA per gel for 30 min. Samples dissolved in 0.6 M KCI were dialyzed against 1% NaDodSO4/1% 2-mercaptoethanol before sample preparation. Protein samples were dissolved in gel buffer containing 1% NaDodSO4 and 1% 2-mercaptoethanol and denatured by heating in boiling water for 3 min. Gels were ... _ sliced, dried on cellophane film, and autoradiographed as de- ..f.. scribed (21). Preparation of Mycoplasma Filamentous Actin-Like

Protein. The actin-like protein was obtained in a sedimentable, .. filamentous form by treatment of the preparation with ATP and Mg2+. Samples of once-precipitated and redissolved my- _r. coplasma actin preparations in 0.6 M KCI were brought to 5 ._-g'i.J.:i.' _ mM ATP, 5 mM MgCl2, and 3 mM dithiothreitol in 8 mM ..... ::_;:_ Tris-HCl buffer, pH 7.5; ATP and Mg2+ were omitted from - controls. The samples were then centrifuged at 143,000 X g for .:. :..:: :..:.i7 ._ _*:_. 1 hr, and the pellets werestaken up in a small volume of 0.1 M :: ,: _ KCI/5 mM imidazole-HCI buffer, pH 7.5, and dialyzed against the same buffer. The supernatants and pellet preparations were

examined by gel electrophoresis. Pellet preparations were also _ examined by electron microscopy. I _ _ Reaction of Actin with Heavy Meromyosin (HMM), and Electron Microscopy. Pellet preparations taken up in and di- alyzed against 0.1 M KCI/5 mM imidazole.HCI buffer, pH 7.5, were mixed with rabbit muscle HMM (approximately 60 ug A B C D or 100 ,ug/ml of reaction mixture) in buffer or in buffer con- taining 10 mM ATP. Electron microscopy was done essentially FIG. 1. _NaDodSO4/polyacrylamide gels (18) of extracts of M. as described by Huxley (22). Samples placed on carbon-coated pneumoniae actin. (A) Rabbit muscle actin. (B) M. pneumoniae extract (P2) containing actin and other proteins insoluble in low ionic Formvar-covered grids were stained with 1% uranyl acetate strength salt solution (0.07 M KCl). (C) Soluble proteins remaining and examined in a Siemens Elmiskop IA. in low-salt solution after removal by centrifugation of insoluble pro- Biochemical Determinations. ATPase (ATP phosphohy- teins (supernatant was dialyzed, lyophilized, and reconstituted in gel drolase, EC 3.6.1.3) activity was determined as described (18) buffer). Note low protein content in region ofactin band (arrow). (D) by measuring release of inorganic phosphate in reaction mix- Proteins (S3) soluble in high ionic strength salt solution (0.6 M KCl) tures incubated at 370 for 30 min. Protein concentration was after precipitation in low-salt solution. a, Actin band; d, position of estimated by the method of Lowry et al. (23). the tracking dye. Materials. Chicken muscle myosin, rabbit muscle myosin (24), and column-purified rabbit muscle myosin (25) were gifts than 15 nmol of inorganic phosphate released per mg of protein from E. McGowan, P. Dreizen, and C. Moos, respectively. per 30 min) could be demonstrated in the partially purified Rabbit muscle actin was prepared by the method of Spudich preparation. and Watt (26) and HMM was prepared by the procedure of The actin content of M. pneumoniae, estimated by densi- Lowey et al. (27) from partially purified myosin (24) or col- tometry of Coomassie brilliant blue-stained gels, was near 6% No actin of the total stainable proteins soluble in NaDodSO4 sample umn-purified myosin (25). filaments were ever seen buffer. This estimate is preliminary because it was based on a in any of the HMM preparations. method subject to several sources of error. An average of approximately 150 mg (wet weight) of M. pneumoniae cells was obtained per liter of medium. Because RESULTS of the low yields, the actin-like protein was further character- Identificati,on of an Actin-Like Protein. The extraction ized by using small-scale procedures and electron microsco- procedure described yielded a protein from M. pneumoniae py. that possessed characteristic properties that identified it as Separation of Filamentous Actin-Like Protein. Treatment actin-like. Similar properties characterize actin from vertebrate of the once-precipitated preparation from M. pneumoniae with skeletal muscle and the actin-like proteins identified in a wide ATP-Mg2+ resulted in the appearance of a sedimentable, fila- variety of nonmuscle cell types (1). The behavior of the protein mentous, actin-like protein. After centrifugation of the ATP- during isolation indicated that it possessed solubility properties Mg2+-treated samples and untreated control samples, the su- analogous to those characteristic of actomyosin, in that it was pernatants were dialyzed and examined by gel electrophoresis soluble in high ionic strength salt solution (0.6 M KCl) and in- to determine which proteins remained in the supernatant. soluble at low ionic strength (0.07 M KCl). NaDodSO4 treat- Centrifugation after ATP-Mg2+ treatment nearly eliminated ment of the partially purified cell extract released a protein with the actin-like protein from the supernatant as estimated by the an electrophoretic migration rate very close to that of actin near disappearance of the actin-like band from acrylamide gels. (molecular weight taken as 45,000) from vertebrate skeletal In contrast, the untreated controls were not visibly altered (data muscle (Fig. 1). Electrophoresis in the Laemmli system (18) not shown). Electron microscopic search (with binoculars) of acrylamide gels revealed that the bacterial actin migrated the resuspended pellet material obtained from ATP-Mg2+- slightly faster than rabbit actin. This small difference in mi- treated samples revealed the presence of long, fine filaments gration rate of M. pneumoniae actin was not readily discernible (Fig. 2A). The filaments were curvilinear and were observed in Tris-acetate buffer gels at pH 7.9. The enrichment of actin scattered singly and occasionally in clusters of a few filaments. was achieved by utilizing the characteristic solubility properties The width of the filaments was estimated to be approximately of the mycoplasma actin. Only very low ATPase activity (less 5-6 nm. The filaments did not appear to be of uniform thickness Downloaded by guest on September 27, 2021 Microbiology: Neimark Proc. Natl. Acad. Sci. USA 74 (1977) 4043 C P

m -

r~~~~~~~~~~~~~~~~~~~~~~~1

;.... -...;.

S~~~~'.."WO

FIG. 2. Electron micrographs of M. pneumoniae actin filaments negatively stained with 1% uranyl acetate. (A) Actin-like filaments obtained by treating a mycoplasma actin preparation with 5 mM ATP/5 mM MgCl2, centrifuging, and dialyzing against 0.1 M KCI/5 mM imidazole-HCl buffer, pH 7.5. (B) Hybrid arrowhead complexes formed between mycoplasma actin-like filaments and rabbit muscle I HMM. Periodicity of repeating arrowheads is 36-37 nm (indicated by short bars). The arrowhead complexes did not form in the presence of 10 mM ATP. The repeating arrowhead pattern can be seen more readily by viewing the filament along its axis at a low angle. (X90,000; scale bar represents 100 nm.)

along their lengths but the narrow points were not seen with sufficient regularity in these preparations to ascertain whether periodicity was present. These filaments were presumed to be F-actin-like and subsequently were shown to behave similarly to eukaryotic F-actin (see below). Several grids were prepared from pellets from untreated samples and extensive searches did not reveal any filaments. Thus, treatment of the partially pu- rified preparation with ATP-Mg2+ allowed the actin-like protein to be separated as a filament. Decoration of the Actin-Like Protein with HMM. The presumptive actin-like filaments were decorated with heavy FIG. 3. NaDodSO4polyacrylamide gel electrophoresis (19) and meromyosin, the specific proteolytic cleavage product of my- autoradiography of [35S]methionine-labeled proteins from M. osin that interacts with actin (22). The mycoplasma actin fila- pneumoniae. (P) X-ray film image of an unstained dried gel slice containing an actin-like protein preparation from [35S]methionine- ments formed characteristic repeating arrowhead-shaped labeled cells ofM. pneumoniae. (C) Coomassie brilliant blue-stained structures (22) when mixed with rabbit muscle HMM (Fig. 2B). dried gel slice containing partially purified chick muscle actomyosin. These structures appeared to be similar to the homologous a, Actin band; m, myosin band. The stained gel (C) slice and the un- complexes formed between rabbit muscle actin and rabbit stained gel (P) slice were dried together on cellophane and taped to x-ray was to the gel slice for 37 days. HMM. The direction of the arrowheads on the decorated fila- film, and the film exposed ments was the same along the entire length of the filament in all cases seen; the periodicity of the arrowhead spacing was ture medium which adsorbed to the organisms. First, neither estimated to be 36-37 nm. The specificity of the reaction was actin nor myosin could be demonstrated by gel electrophoresis shown by the failure of arrowheads to form in the presence of in samples of horse serum, PPLO serum fraction, or autoclaved ATP, which is an inhibitor of actin-myosin interaction. Thus, yeast extract. Also, no actin could be detected in PPLO serum a hybrid complex could be formed between prokaryotic F-actin fraction by affinity chromatography on DNase agarose (28). and HMM from eukaryotic muscle myosin. More importantly, when M. pneumoniae was grown in the Evidence that the Actin-Like Protein Was Synthesized by presence of [a5S]methionine of high specific activity, radioau- the Mycoplasma. Two independent lines of evidence pointed tographs of gel electrophoretograms of extracts from the my- to the conclusion that the actin was actually synthesized by M. coplasmas (Fig. 3) revealed the typical gel pattern for myco- pneumoniae and was not a preformed component of the cul- plasma extract proteins. All the proteins in the region of actin Downloaded by guest on September 27, 2021 4044 Microbiology: Neimark Proc. Natl. Acad. Sci. USA 74 (1977) were labeled including the protein (gel P) congruent with the If the actin standard (gel plasmas (29). prokaryotic actin also functions in other C). Thus, there was no evidence for the contractile processes, it might be found to occur widely among presence of actin in the culture medium, whereas active syn- bacteria. thesis by M. pneumoniae of a protein with the physical prop- Conjecture that actomyosin might be present in motile erties of an actin-like protein was clearly demonstrated. Fur- bacteria is not novel (30, 31). Earlier workers observed fibrillar thermore, the slightly faster electrophoretic mobility of the structures in the envelopes of nonflagellated motile bacteria and mycoplasma actin-like protein evident in Laemmli gels also speculated that actomyosin might be present, but no evidence suggested that the actin found was not of eukaryotic origin. has been forthcoming. More recently, Henrichsen (32) sought However, the possibility cannot yet be eliminated that a small evidence for an association between contractile activity and amount of eukaryotic actin or actomyosin undetectable by motility in gliding and twitching bacteria by treating these electrophoresis was present in the preparations. bacteria with cytochalasin B, but no inhibition of movement was detected. M. pneumoniae possesses a specialized terminal DISCUSSION structure containing an electron-dense rod (33), and workers The discovery of an actin-like protein in a prokaryote has broad have suggested that the structure might function in reproduc- biochemical and evolutionary implications. First, this finding tion or locomotion (33, 34). This structure and the one in M. suggests that actin arose and evolved in prokaryotes. Second, gallisepticum (35) are present in "tiplike" projections that if one accepts the proposition that actin arose only once during adhere to the surface and proceed in the direction of movement the course of evolution, then prokaryotic actin would appear in motile mycoplasmas (8). However, an attempt to demon- to have been transmitted to eukaryotic cells. The results may strate an effect on motility by cytochalasin B in M. pneumoniae also have a bearing on our understanding of bacterial evolution, similarly was unsuccessful (8). particularly on phylogenetic relationships between myco- Actin conceivably could have a role in the pathogenicity of plasmas and other prokaryotes. M. pneumoniae. This organism must penetrate a mucous layer The state of the mycoplasma actin in the cell extract is not to reach the respiratory epithelium where it is known to attach clear, but it is improbable that the actin could be present either to ciliated respiratory epithelial cells by its specialized terminal as a monomer or in the form of free filaments. Rather, the sol- structure in a characteristic orientation and cause cytopathic ubility and sedimentation behavior observed during isolation changes that result in cell death (34). Also, it would be inter- suggest that the actin may be bound in a complex that possibly esting to know whether patients are able to develop antibody contains membrane material. The solubility of the bacterial to prokaryotic actin. It has been found that, for unknown rea- actin during extraction is analogous to that of actin associated sons, some patients with chronic active hepatitis develop anti- with myosin and suggests that the actin could be associated with bodies that react with actin (36). a protein analogous to myosin. The finding that ATP-Mg2+ A particularly significant result reported here is the striking treatment allowed separation of bacterial actin filaments also similarity found between prokaryotic actin and its eukaryotic suggests an association of actin with a protein functionally counterpart, because this has implications for the evolutionary analogous to myosin. Conceivably, the cofactors could be history of actin itself. In particular, the ability of mycoplasma causing polymerization of a monomeric actin-like protein, but filamentous actin to substitute for rabbit muscle actin and to the solubility and sedimentation behavior of the actin-like form hybrid arrowhead complexes with rabbit muscle HMM protein during extraction are not consistent with its being in a (Fig. 2) indicates a degree of homology between prokaryotic monomeric form, at least not one with properties similar to actin and actin from higher vertebrate cells. This suggests that those of monomeric actin from higher cells. The finding in the little structural change has occurred in actin over a long span untreated control of this experiment differs, however, from the of evolution. It is already known from studies in tryptic digest result that would be expected for eukaryotic actomyosin in that fingerprints that actins from various eukaryotic organisms show free actomyosin would be expected to sediment under the ex- considerable conservation of primary structure (37). Similar perimental conditions,The ATP-Mg2+ could be acting here by studies on prokaryotic actin should reveal to what degree the dissociating the actin-like protein from a complex whose structural similarity that allows for hybrid arrowhead formation properties prevent it from sedimenting; for example, the extends to amino acid sequence homology. In any event, actin complex could be associated with lipid membrane material appears to form a connecting bridge across the gap in evolution which would cause it to float. Attachment of bacterial actin to that separates prokaryotes and eukaryotes, and consequently membrane material could provide anchoring points that pre- the finding reported here bears on a larger evolutionary ques- sumably are necessary for a contractile system. tion, the origin of the eukaryotic cell. The very existence of an actin-like protein in prokaryotes The discovery of an actin-like protein in a prokaryote raises suggests a need early in evolution for the cellular functions the possibility of applying the powerful methods of bacterial performed by actin. No evidence is yet available to indicate the genetics to the investigation of the structure, function, and function of prokaryotic actin, but one can infer by analogy to regulation of actin-like proteins. higher cells that it is involved in contractile processes such as Note Added in Proof. Early in this study a survey was made of known motility and possibly in maintenance of cell shape. It should be bacterial proteins having molecular weight near that of actin. A protein recognized, however, that even in higher cells there is only a involved in protein synthesis, elongation factor Tu, does have a mo- large body of circumstantial evidence and no direct proof that lecular weight near that of actin (the author thanks E. Lazarides for actomyosin generates the force for cell motility (1). If pro- directing my attention to a protein synthetic factor). Recently, a karyotic actin is responsible for motility, then the results pre- 42,000-dalton protein was identified in the membrane-DNA complex sented here suggest that actin-like proteins may be present in of Bacillus subtilis (38). After this report was communicated, I learned other mycoplasmas that are capable of gliding motility, in that Rosenbusch et al. (39) had raised the question of whether Esche- spiroplasmas, and also in other groups of gliding bacteria in- richia coli elongation factor Tu and actin share a common protein ancestor. Minkoff and Damadian (40) independently reported a protein cluding cyanobacteria (blue-green algae). Finding actin in any fraction from E. coli "with characteristics reminiscent of muscle actin." of the bacterial groups would be important in understanding Also, Weltman and Dowben (41) have suggested that a K+-dependent phylogenetic relationships among walled bacteria and myco- membrane ATPase from Streptococcus faecalis has a high degree of Downloaded by guest on September 27, 2021 Microbiology: Neimark Proc. Nati. Acad. Sci. USA 74 (1977) 4045 relatedness to several membrane proteins and contractile proteins, 18. -Laemmli, U. K. (1970) Nature 227,680-6. including actin. 19. Weber, K. & Osborn, M. (1969) J. Blot. Chem. 244, 4406- 4412. 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Bacteriol. 125, 353- Acad. Sci. USA 68,2703-2707. 365. 17. Malik, M. N., Abramowitz, J., Detweiler, T. C. & Stracher, A. 41. Weltman, J. & Dowben, R. M. (1973) Proc. Natl. Acad. Sci. USA (1974) Arch. Biochem. Biophys. 161, 268-274. 70,3230-3234. Downloaded by guest on September 27, 2021