Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 14-740, México, DF 07000, Mexico1
Departamento de Ingeniería Genética de Plantas, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 629, Irapuato, Gto. 36500, Mexico2
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas IPN, Prolongación de Carpio y Plan de Ayala s/n, México, DF 11340, Mexico3
División de Investigación Básica, Instituto Nacional de Cancerología, San Fernando No 22, Tlalpan, DF 14000, Mexico4
Author for correspondence: Magdalena Segura-Nieto. Tel: +52 46 23 96 00 ext. 665/413. Fax: +52 46 24 58 49. e-mail: msegura{at}irapuato.ira.cinvestav.mx
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ABSTRACT |
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Keywords: prokaryotic actin-related proteins, actin-related proteins, actin-binding proteins, cytoskeleton-like structure, Actinobacillus pleuropneumoniae
Abbreviations: ABP, actin-binding proteins; ARP, actin-related proteins; CD, cytochalasin D; Hsc, heat-shock cognate protein; Hsp, heat-shock protein; PARP, prokaryotic actin-related proteins; S1, myosin subfragment-1; TRITC, tetramethylrhodamine isothiocyanate; Ap, Actinobacillus pleuropneumoniae; Av, Anabaena variabilis
a Present address: Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3 CP 62250, Cuernavaca, Mor, Mexico.
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INTRODUCTION |
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The prokaryotic actin-related proteins (PARP) from Anabaena species and E. coli share epitopes with smooth muscle actin (Guerrero-Barrera et al., 1995 , 1996
), and actin-like proteins from Synechocystis spp. share antigenic reactivity with distinct antibodies that recognize actin regions known to interact with proteins cross-linking actin filaments (Usmanova et al., 1998
). Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia is a Gram-negative bacterium that has been intensively investigated. Special attention has been paid to A. pleuropneumoniae virulence factors and the production of toxin-neutralizing antibodies to protect pigs against porcine pleuropneumonia (Haesebrouck et al., 1997
; Prideaux et al., 1999
). However, no studies have been done on the cell biology of this pathogen. To study the putative cytoskeleton of A. pleuropneumoniae, we searched for the presence of molecules which behave like actin. Here we report on the purification and characterization of three actin-related proteins (ARP) present in A. pleuropneumoniae. These proteins show similar behaviour to eukaryotic actin in several of their biochemical and immunological properties.
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METHODS |
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Bacteria.
A. pleuropneumoniae strain 35 (serotype 1) was isolated from a lung abscess of a pig with acute porcine pleuropneumonia (Negrete-Abascal et al., 1994 ). Cultures were maintained on brain heart agar (Difco) with 10 µg NAD ml-1. Bacteria for extracts were grown to stationary phase in brain heart infusion broth with 10 µg NAD ml-1 for 48 h at 37 °C with constant shaking (150 r.p.m.). A. pleuropneumoniae cells from a 500 ml culture were harvested and washed twice with PBS (0·145 M sodium chloride, 0·15 M sodium phosphate, pH 7·4) by centrifugation at 10000 g at 4 °C for 10 min. Cells were washed five times by centrifugation with cold acetone (-20 °C) and dried to a powder at room temperature.
A. pleuropneumoniae PARP (Ap-PARP) depolymerization and polymerization.
The method of Pardee & Spudich (1982) was used with some modifications. Acetone-dried cells (500 mg) were resuspended in 500 µl protease inhibitor cocktail (62·5 mM Tris/HCl, pH 7·4, 5 mM diisopropyl fluorophosphate, 2 mM N-ethylmaleimide, 10 mM p-hydroxymercuribenzoate, 5 mM tosyl-L-lysine chloromethyl ketone and 2 µM leupeptin). This cell suspension was diluted in 10 ml actin depolymerization buffer (2 mM Tris/HCl, pH 7·4, 0·2 mM Na2ATP, 2 mM CaCl2 and 0·5 mM ß-mercaptoethanol) and incubated for 2 h at 4 °C with gentle stirring. Following incubation, the suspension was centrifuged at 25000 g at 4 °C for 30 min and the pellet was discarded. A range of 15 mM Na2ATP was used to optimize Ap-PARP polymerization. PARP were polymerized by adjusting the supernatant to contain 50 mM KCl, 2 mM MgCl2, 5 mM EGTA and 5 mM Na2ATP (polymerization buffer A) and gently stirring for 2 h at 37 °C. The preparation was then centrifuged at 180000 g for 1·5 h at 4 °C and the supernatant was discarded. The pellet contained polymerized Ap-PARP filaments. Ap-PARP filaments were solubilized in 100 µl 1% SDS, 2% ß-mercaptoethanol, 4 M urea. Precipitation according to Wessel & Flügge (1984)
resulted in a detergent-free Ap-PARP pellet.
Ap-PARP purification by binding to DNase I.
Ap-PARP from the detergent-free pellet were purified by affinity chromatography on a DNase I column (Guerrero-Barrera et al., 1996 ).
Ap-PARP purification by electroelution.
The detergent-free Ap-PARP pellet was also fractionated by preparative Tricine/SDS-PAGE (15% gels) as described by Schägger & von Jagow (1987) . Part of the gel was used to identify Ap-PARP by Western blot analysis with different antibodies against actin and actin-binding proteins (ABP). The rest of the gel was stained with Coomassie blue and the corresponding PARP bands were cut out and electroeluted in an Electro-Eluter model 422 (Bio-Rad). The electroelution buffer was 50 mM NH4HCO3, 0·1% SDS. Electroelution was performed for 5 h at 10 mA per tube as described in the Bio-Rad manual.
Electrophoretic analysis.
Two methods were used for one-dimensional SDS-PAGE: the first was the Laemmli (1970) method using 10% gels; the second was the Tricine/SDS-PAGE method of Schägger & von Jagow (1987)
using 15% gels. Electrophoresis was performed in a Mighty-Small II SE250 Cell (Hoefer) at 100 V for 1 or 2·5 h. Gels were stained with 0·2% Coomassie blue R250 in destaining solution (10% methanol, 10% acetic acid in water).
Two-dimensional (2D) gel electrophoresis was done according to OFarrell (1975) .
Western blotting.
Ap-PARP were separated by Tricine/SDS-PAGE as described above and transferred to Immobilon-P (Millipore) or nitrocellulose membranes in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) at 253 mA for 45 min. The transfer buffer contained 10 mM CAPS, 0·4 mM DTT and 10% methanol (pH 11). For immunodetection, the following antibodies were used: polyclonal mouse antibody against Anabaena variabilis PARP (Av-PARP) and monoclonal mouse anti-rat brain actin (Guerrero-Barrera et al., 1996 ), polyclonal rabbit anti-C-terminal actin (A 2066, synthetic peptide SGPSIVHRKCF; Sigma), monoclonal mouse anti-
-smooth muscle actin (A 2547; Sigma), polyclonal rabbit anti-profilin
-actin complex from calf spleen (M. Segura-Nieto & U. Lindberg, unpublished data), monoclonal mouse anti-Hsc70 (Sigma), polyclonal rabbit anti-
-actinin and polyclonal rabbit anti-spectrin (kit Cyto-2; Sigma). In all cases peroxidase-labelled protein A was used to detect antibody binding. The immune reaction was revealed with 4-chloronaphthol in a Mini-Protean II Multiscreen Apparatus (Bio-Rad). All procedures were done at room temperature.
Phalloidin binding
Whole cells.
A. pleuropneumoniae cells were treated with phalloidin labelled with tetramethylrhodamine isothiocyanate (TRITC) as described previously (Guerrero-Barrera et al., 1996 ). For the assays, 10 ml of a stationary-phase culture was used. Readings were taken in a spectrofluorometer (Perkin-Elmer Luminiscence Spectrometer L5 50B) at an excitation wavelength of 550 nm and emission wavelength of 574 nm. Non-treated cells were used as negative control.
Ap-PARP.
DNase I-affinity-purified Ap-PARP (100 µg) was incubated for 2 h at 37 °C in 500 µl polymerization buffer A with 50 ng TRITCphalloidin; the drug excess was removed following precipitation of Ap-PARP using the method of Wessel & Flügge (1984) . The TRITCphalloidin filamentous Ap-PARP (F-Ap-PARP) precipitate was resuspended in 1 ml PBS. As negative control, PARP were incubated with unlabelled phalloidin, and as positive control skeletal muscle F-actin was incubated with TRITCphalloidin. The interaction was measured with a spectrofluorometer as described above.
Electron microscopy
Negative staining of PARP.
The Ap-PARP detergent-free pellet (100 µg) was resuspended in 500 µl polymerization buffer A and incubated for 2 h at 37 °C. Ten microlitres of this reaction mixture containing F-Ap-PARP was laid on nickel grids covered with Formvar and settled for 10 min at room temperature. Then the samples were fixed with 2·5% glutaraldehyde and washed with polymerization buffer A. Samples were contrasted with 0·5% uranyl acetate, pH 7·4. Skeletal muscle actin was used as positive control. The observations were made with a JEOL 2000 EX transmission electron microscope at 80 kV.
Decoration of F-Ap-PARP with myosin subfragment-1 (S1).
Ten microlitres of the above polymerized F-Ap-PARP mixture was deposited on a carbon Formvar-coated nickel grid and allowed to settle for 10 min at room temperature. Then, 10 µl S1 (25 mg ml-1) in polymerization buffer A (without Na2ATP) was added before incubation overnight at 4 °C. Samples were fixed and washed, and observations were made with the electron microscope as described above.
Amino acid composition.
The electroeluted Ap-PARP were hydrolysed in 6 M HCl for 24 h at 110 °C under vacuum and the amino acid composition was determined as described by Bidlingmeyer et al. (1984) .
RESULTS AND DISCUSSION
The presence of PARP in A. pleuropneumoniae was investigated as an initial step in the study of its putative cytoskeletal structure. To identify and characterize ARP, the following five classical criteria for actin identification in eukaryotic cells were used.
1. Enrichment by one depolymerization/polymerization cycle and purification by binding to DNase I
One of the ways to obtain and enrich functional eukaryotic actin is through depolymerization/polymerization cycles. This method was used with A. pleuropneumoniae cell extracts, and a polypeptide band with an Mr of 43000 in SDS-PAGE was enriched as shown in Fig. 1(a), lane 3. This Mr is similar to that of skeletal muscle actin (Fig. 1a
, lane 1). Using this method it was possible to identify and purify Ap-PARP from cell extracts that had polymerization capability similar to eukaryotic actin. The Ap-PARP were also enriched from the cell-free culture filtrate of exponential-phase viable cells (data not shown), apparently as a product of secretion from the bacteria.
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2. Biochemical characterization
(a) Mr and pI. Ap-PARP have an Mr of 43000 (Fig. 1a, b), distinct from that reported for other actin-like proteins, e.g. 60000 for Streptococcus pyogenes (Barnett & Cunningham, 1992
) and E. coli (Guerrero-Barrera et al., 1996
), and 56000 for Synechococcus spp. (Labbé et al., 1996
). The Ap-PARP displayed three isoforms with pI values of 5·80, 6·15 and 6·20 (Fig. 1b
). These isoforms are more acidic than PARP from Anabaena variabilis and E. coli (Table 1
), but less acidic than actins from other species (Garrels & Gibson, 1976
; Herman, 1993
). The Ap-PARP pI values are closer to those of Arp2 and Arp3 reported by the ExPASy WWW server (April, 1998). Arp1 is closer in pI, structure and amino acid sequence to skeletal muscle actin isoform (Herman, 1993
; Schroer et al., 1994
; Mullins et al., 1996
).
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(c) Toxin binding. Phalloidin binding to polymerized actin (F-actin) is a biochemical characteristic that all actins share. This is a stoichiometric interaction of one molecule of phalloidin to two actins. This toxin stabilizes actin filaments and prevents its depolymerization by agents such as DNase I, ultrasonication and proteolytic degradation (Vandekerckhove et al., 1985 ; Miyamoto et al., 1986
; Sheterline et al., 1998
). This binding is inhibited by cytochalasin D (CD), a toxin which binds both to monomeric actin and to the barbed end of actin filaments. CD binds at a stoichiometry of one molecule per filament, thus inhibiting monomer exchange and resulting in depolymerization (Sheterline et al., 1998
). These two drugs were used to test for F-Ap-PARP both in permeabilized cells and in vitro. Table 3
shows the results of specific TRITCphalloidin binding to permeabilized A. pleuropneumoniae cells. This interaction is blocked when these cells are preincubated with either unlabelled phalloidin or CD. The specific binding of F-Ap-PARP to TRITCphalloidin was also measured in vitro with proteins purified by DNase I affinity chromatography, using the same strategy as above. This specific interaction is inhibited by preincubation of F-Ap-PARP with either unlabelled phalloidin or CD and measured by spectrofluorometry (Table 4
). As a positive control skeletal muscle F-actin was incubated with TRITCphalloidin and this interaction was also inhibited by prior incubation with CD or unlabelled phalloidin (Table 4
). These data corroborate the observations made with permeabilized cells.
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Actin and myosin form one of the major motor protein complexes in all eukaryotic cells. Most of the studies on myosin interaction with actin during force generation involve the use of mammalian fast muscle myosin II or its functional proteolytic fragments S1 and HMM, together with mammalian skeletal muscle actin. Each myosin head has similar actin-binding ATPase domains but different tail regions. Also, each myosin head may bind to two adjacent molecules of actin in the filament during the cross-bridge cycle. The most stable conformation is the rigor conformation which exists at the end of the cross-bridge cycle. This conformation can be experimentally frozen by removal of ATP necessary to release the myosin head (Andreev & Borejdo, 1991 ).
S1 binds to filaments of Ap-PARP (Fig. 3b) in a similar fashion to skeletal muscle actin (Fig. 3a
). This interaction shows that filaments of Ap-PARP expose similar S1-binding sites to vertebrate actin. This observation suggests that Ap-PARP filaments might have the ability to interact with myosin-like molecules from the bacteria in vivo, probably participating in intrabacterial motility, bacterial cell division and nucleoid segregation, as has been suggested by Casaregola et al. (1990)
. Although in A. pleuropneumoniae there have been no studies describing the presence of motor-like proteins as yet, the evidence from studies in different bacteria suggests that motor-like proteins are ubiquitous (Melby et al., 1998
).
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Recent studies have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, there appears to exist an active, possibly mitotic-like machinery (Sharpe & Errington, 1999 ; Melby et al., 1998
). MukB and SMC proteins from Bacillus subtilis function in chromosome condensation and some other aspects of DNA processing (Niki et al., 1992
; Lockhart & Kendrick-Jones, 1998
; Melby et al., 1998
). These findings support the idea that prokaryotes might have a cytoskeletal-like structure involved in fundamental cellular functions as in eukaryotes.
5. Immunological recognition of Ap-PARP by different antibodies
Ap-PARP have several properties shown above which should be sufficient to consider them as members of the actin family. To reinforce this statement, other criteria were used, e.g. their recognition with different anti-actin antibodies and anti-ABP antibodies.
Ap-PARP obtained after one polymerization cycle were fractionated in a Tricine/SDS-PAGE gel, blotted and reacted with polyclonal antibodies against Av-PARP (Fig. 4, lane 2) and four different anti-actin antibodies (Fig. 4
, lanes 3, 4, 5 and 8). All antibodies gave a positive reaction, showing that Ap-PARP have epitopes similar to those present in different actins. On the other hand, the positive recognition of Ap-PARP by two anti-ABP antibodies against
-actinin and spectrin, both from chicken gizzard (Fig. 4
, lanes 6 and 7), suggests structural similarities to eukaryotic actin molecules as well. This cross-reaction between actin and ABP was not non-specific, since antibodies against filamin, vimentin, tropomyosin and myosin did not recognize any epitope on Ap-PARP (data not shown). On the other hand, the immunoelectron microscopic studies of prokaryotes of various taxonomic affiliations (archaea and bacteria) have shown a network made up of protein fibrils. These fibrils are composed of proteins which exhibit cross-reactivity with the anti-actin antibody used in Fig. 4
, lane 5 (C terminus of actin). The network seems to be located around the cytoplasm close to the periphery of all the cells (Mayer et al., 1998
). These authors propose the existence of a prokaryotic cytoskeleton that might function to preserve the shape of different prokaryotes.
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Other cytoskeleton-like components have been found in prokaryotic cells; e.g. myosin-like protein from E. coli (Nakamura & Watanabe, 1978 ), M protein in S. pyogenes, which has amino acid sequence homology with coiled-coil proteins similar to tropomyosin and myosin-like proteins (Manjula & Fiscetti, 1986
; Manjula et al., 1985
, 1991
; Mische et al., 1987
; Barnett & Cunningham, 1992
), and
-actinin-like proteins in cyanobacterium Spirulina platensis (Usmanova et al., 1998
). Thus, PARP as well as ABP might have existed before the emergence of eukaryotic cells. It is clear that many more cytoskeletal-like proteins in bacteria remain to be found and studied at the biochemical, genetic and subcellular level to understand their organization and participation in the dynamics of the prokaryotic cell.
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ACKNOWLEDGEMENTS |
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Received 22 June 1999;
accepted 22 July 1999.
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