[Structure of the intracellular part of the motility apparatus of halobacteria].

The electron microscopic study of the structure of the motility apparatus of the archaea Halobacterium salinarium 4W12 and Natronobacterium magadii confirmed our earlier observation that the motility apparatus of halobacteria contains an intracellular disk-shaped lamellar structure (DLS). Polar cap structures (PCSs) isolated from the halobacterium were preliminarily identified as the DLSs. The PCSs in complexes with flagella were also isolated from the haloalkaliphilic bacterium N. magadii. The specific structure of the archaeal motility apparatus is discussed.

Bacterial and archaeal flagella as prokaryotic motility organelles.

The properties and molecular organization of flagella--the bacterial and archaeal motility organelles--are reviewed. The organization of these functional motility elements of prokaryotic organisms belonging to different kingdoms is compared. A mechanism for both in vivo and in vitro assembly of bacterial flagellum filaments (BFFs) is discussed, and similarity is supposed between flagellin and actin with regard to their polymeric forms (BFF and F-actin). Our own data on intracellular fixation of the Halobacterium salinarium flagellum are presented. Comparative characteristics of intracellular fixation of bacterial and archaeal flagella are also described.

Interaction of bacterial flagellum filaments with skeletal muscle myosin.

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+ -ATP. Addition of BFF inhibited both K+ -EDTA- and Ca2+ -ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+ -ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.

[Halophilic archaea flagella: biochemical and genetic analysis]. / Zhgutiki galofil'nykh arkhei: biokhimicheskii i geneticheskii analiz.

The protein compositions of archaebacteria (Halobacterium salinarium, Halobacterium volcanii, Halobacterium saccharovorum and Natronobacterium pharaonis 12) flagella have been studied. It was found that flagella of these archaebacterial species are made up of flagellins. The flagellins of H. salinarium, H. volcanii and H. saccharovorum are glycosylated. Based on the known primary sequences of Halobacterium halobium R1M1 flagellin genes, oligonucleotides to the 5'- and 3'-ends of locus A containing two out of five such genes have been synthesized. The amplified by primers fragment of chromosomal DNA coding for H. halobium flagellins A1 and A2 was used as a probe for detecting homologous sites in archaebacterial DNA. Southern blotting hybridization revealed that the DNA of all archaebacterial species tested in this study contains sequences that are homologous to genes flg A1 and flg A2 of H. halobium.

A study on the mechanism of polymerisation of Bacillus brevis flagellin.

Optical properties of intact flagellin and of modified-on-tyrosine flagellin incapable of self-assembly have been studied by the circular dichroism (CD) method. The CD spectra of both flagellins do not change and virtually coincide within the pH range from 2.9 to 10.0. In the presence of polyethylene glycol (PEG) and ammonium sulfate which accelerate flagellin polymerization, the character of the CD spectra changes and depends on pH. The increase in the PEG content to 20% and that in the ammonium sulfate content to 1 M over the pH range from 4.3 to 10.0 results in a significant rise of the molar ellipticity at 222 nm ([theta]222) of both flagellins. However, [theta]222 does not reach values typical for bacterial flagella. The results obtained are discussed with respect to conformational changes in the flagellin molecule during polymerization.

[Existence of an additional structural component in the basal bodies of Escherichia coli and Vibrio alginolyticus flagellae]. / Sushchestvovanie dopolnitel'nogo strukturnogo komponenta v bazal'nykh telakh zhgutikov Escherichia coli i Vibrio alginolyticus.

The structure of Escherichia coli and Vibrio alginolyticus flagella was studied using electron microscopy. An additional protein structure was shown to exist in the basal bodies of intact flagella in these organisms. It is possible that this structure involves three proteins important for the assembly of flagella, energy transduction, and a change-over in the direction of flagellar rotation.

[Similarity of Vibrio alginolyticus, V. cholerae and other Vibrio species with respect to the structure of their flagellar apparatus and ribosomal 5S-RNA]. / Skhodstvo Vibrio alginolyticus, V. cholerae i nekotorykh drugikh vibrionov po stroeniiu flagelliarnogo motora i strukture ribosomal'nykh 5S-RNK.

Electron microscopic analysis of basal bodies of the flagella Vibrio alginolyticus revealed a structure composed of four discs. The diameters of two discs localized in the cytoplasmic membrane appeared to be twice as little as those of the other two discs. In this respect the basal body of V. alginolyticus resembles that of V. cholerae. The 5S sequence of ribosomal RNA from V. alginolyticus appeared to be similar to those of V. cholerae, V. harveyi and some other vibrios. Comparison of 5S-RNA sequence culminated in a dendrogram of evolutionary relationships of various bacterial species, suggesting that V. alginolyticus is a typical representative of the Vibrionacea family. The data obtained are discussed in terms of the role of Na+ energy metabolism in living cells.

The sodium cycle. III. Vibrio alginolyticus resembles Vibrio cholerae and some other vibriones by flagellar motor and ribosomal 5S-RNA structures.

An electron microscopic study of the basal bodies of the Vibrio albinolyticus flagellum revealed a four-disc structure. The diameters of the two discs localized closer to the cytoplasmic membrane proved to be about 2-fold shorter than those of the two others. In this respect the basal body of V. alginolyticus resembles very much that of V. cholerae described by Ferris and co-workers. The sequence of the V. alginolyticus ribosomal 5S-RNA showed that it is similar to those of V. cholerae, V. harveyi and some other vibriones. On the basis of the 5S-RNA sequences, a dendrogram of prokaryotes is presented. It confirmed the suggestion that V. alginolyticus is a typical representative of Vibrionaceae rather than a 'monster' greatly differing from other vibriones. Possible evolutionary relation of various bacterial species possessing the primary Na+ pumps is discussed.

[Structural study of the basal bodies of the flagella of Bacillus brevis var. G.-B. P+]. / Izuchenie struktury bazal'nykh tel zgutikov Bacillus brevis var. G.-B. P+.

The structural organisation of the flagellum basal body was studied in Bacillus brevis var. G.-B. P+ by electron microscopy. It was compared with that of Escherichia coli MS 1350. The basal body of a B. brevis flagellum contains, in addition to two pairs of rings on a rod, another ring-like structure (d = 13.6 nm, h = 4.3 nm) which we referred to as a "collar". The collar makes the basal body of B. brevis different from that of B. subtilis, another Gram-positive bacterium. The collar seems to fasten the flagellum of B. brevis to the cell wall. We have concluded that the basal body can differ not merely among bacterial systematic groups, but also among bacteria belonging to one and the same genus. The role of individual elements in the structure of the basal body of bacterial flagella is discussed.

[Interaction of bacterial flagella with myosin]. / Vzaimodeistvie nitei bakterial'nykh zhgutikov s miozinom.

The interaction of isolated flagellar filaments of Bacillus brevis var. G.-B. P+ with skeletal muscle myosin has been investigated. Bacterial flagellar filaments co-precipitate with myosin at low ionic strength (at the conditions of myosin aggregation). Addition of bacterial flagellar filaments to myosin led to inhibition of its K+-EDTA- and Ca2+-ATPase activity, but had no influence on Mg2+-ATPase. Monomeric protein of bacterial flagella filaments (flagellin) did not co-precipitate with myosin and had no influence on its ATPase activity. The flagella filaments did not co-precipitate with myosin in the presence of F-actin if it was mixed with myosin before the filaments. If the flagella filaments were added to myosin solution before the addition of F-actin the amount of filaments and actin in myosin precipitate were comparable. In this case the presence of flagella filaments decreased activation of myosin Mg2+-ATPase by actin to 25-30%. Thus the bacterial flagellar filaments are able to interact with myosin and modify its ATPase activity. Probably, these properties of filaments are caused by resemblance of flagellin and actin. For instance, the unique origin of these proteins may be the reason of such resemblance.

[Mechanism of flagellin polymerization in Bacillus brevis]. / Izuchenie mekhanizma polimerizatsii flagellina Bacillus brevis.

The optical properties of acid-dissociated and tetranitromethane-modified flagellin has been studied by circular dichroism (CD) techniques. The flagellins have the same CD spectra which do not change over the 2.9-10.0 range. The spectra character depends on pH in the presence of polyethylene glycol (PEG) and ammonium sulphate which accelerate polymerization. As the content of PEG goes up to 20% and ammonium sulphate to 1 M, the value of the molecular ellipticity at 222 nm ([O]222) of the both flagellins considerably increases at pH 4.3-10.0, however [O]222 does not achieve the value for bacterial flagella. PEG and ammonium sulphate addition at pH 2.9-3.9 gives less dramatic increase of the [O]222 value. It has been concluded that the changes in the CD spectra at pH 4.3-10.0 is a result of conformational rearrangements in flagellin before it incorporates into the flagella.

[Polymerization of bacterial flagellar flagellin in the presence of polyethyleneglycol]. / Polimerizatsiia flagellina bakteriial'nykh zhgutikov v prisutstvii polietilenglikolia.

Polymerization of flagellin from the flagella of Bacillus brevis in the presence of polyethyleneglycol (PEG) with molecular weight varying from 400 to 40 000 was studied. When 10% PEG2000-6000 was used, polymerization was completed within 5 min. When PEG with higher molecular weights were used, their higher concentrations were necessary. Polymerization of flagellin in the presence of PEG400-600 did not practically differ from that without PEG. At flagellin concentrations up to 0.1 mg/ml the polymerization process with PEG also occurred at a very high rate. Polymerization of flagellin obtained by heating of bacterial flagella in the presence of PEG did not require exogenous primer. A possible mechanism of flagellin polymerization in the presence of PEG is discussed.

[Structure and physico-chemical properties of bacterial flagella]. / Struktura i fiziko-khimicheskie svoistva bakterial'nykh zhgutikov.

The structural organization of bacterial flagella was studied with Bacillus brevis and Escherichia coli MS 1350. The presence of a spherical body at the basis of a flagellum was confirmed. The structural organization of ingredients of the flagellar appratus, its inner and outer part, was investigated. The molecular weight of protein subunits in the filamentous portion of the flagellum was assayed as well as their amino acid composition. The mode of attachment of the flagellum to the cell is discussed.

Immobilization of actin and myosin.

Using glutaric dialdehyde, the muscle proteins myosin, actin, actomyosin and heavy meromyosin subfragment-1 (S-1) have been immobilized on capron fibers. The ATPase activity of myosin and its capability to interact with actin have been preserved whereas the ATPase activity of its subfragment decreased significnatly. Immobilization on capron fibers changes the pH dependence of the ATPase activity of myosin and of S-1 shifting the maximum towards the acid zone (pH 5.5) and increases the thermal stability of the enzyme. Calcium ions produce a stimulatory effect on ATPase; Mg2+ions yield no effect on myosin and S-1 but enhance the activity in the case of immobilized actomyosin though to a lesser degree than the ions of Ca2+. Immobilized actin retains its ability to form actomyosin complex.

[Chemical suturing of myosin and actin to capron fiber]. / Khimicheskaia prishivka miozina i aktina k kapronovomu voloknu

Immobilization of myosin, actin, actomyosin and subfragment S1 on kapron fibre was achieved with the help of glutaric aldehyde. The ATPase activity of myosin and its ability to interact with actin is preserved; while the ATPase activity of S1 subfragment decreases considerably. The immobilization on kapron fibre changes the pH-dependance of ATPase activity of myosin and that of subfragment S1, shifting the maximum to low pH zone (pH 5.5), and increases the thermostability of the enzyme. The ions of Ca++ in all cases act as an activating agent on ATPase while the ions of Mg++ either do not affect myosin and subfragment S1 at all, or increase the activity in the case of the immobilized of actomyosin but to a lesser degree than the ions of Ca++. The immobilized actin preserves its ability to form actomyosin complex.