Motility and thermotactic responses of Thermotoga maritima.

Thermotoga maritima, a thermophilic eubacterium, is motile at temperatures ranging from 50 to 105 degrees C. The cells are propelled by a single flagellum which most of the time spins clockwise. Changes in the swimming direction ("tumbles") are achieved by short reversals of the direction of filament rotation. The average speed of swimming cells depends on the temperature, reaching a maximum value of about 60 microns/s at 85 degrees C. The cells show a thermotactic response to temporal temperature changes. When the temperature is raised, the rate of tumbles is increased, while decreasing temperature decreases the tumbling rate.     

Relevance of three-dimensional reconstructions of stain distributions for structural analysis of biomolecules.

The relevance of a three-dimensional model of a protein molecule is discussed in the context of artefacts which inevitably occur during the preparation and imaging process. It is emphasized that even under optimistic assumptions, there-construction of a structure complementary to a given spatial stain distribution cannot, at resolutions below 10-15 A, be regarded as being identical with the real protein structure.     

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies.

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.     

Two-dimensional crystallization of a bacterial surface protein on lipid vesicles under controlled conditions.

The solubilized surface protein of the Gram-negative bacterium Comamonas acidovorans was reconstituted on lipid vesicles by means of controlled dialysis. To this end, a multichamber dialysis apparatus was built which allows one to control the temperature and the dialysis rate, to apply various temperatures or buffer systems and sample conditions in a single experiment, and to monitor the turbidity of the sample by means of light scattering. The reconstitution conditions were optimized such that the surface protein formed two-dimensional crystals suitable for electron crystallography. The recrystallized surface protein arrays gave a resolution of approximately 1.3 nm in projection after correlation averaging of negatively stained preparations. The surface protein assembled into partially self-contained two-dimensional crystals which possess a strong shape-determining effect and formed cylinders and various cone-shaped vesicles. The development of the various vesicle forms is described in a model.     

Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor.

Each of 22 amino acids in the proposed alpha-helix-turn-alpha-helix operator binding motif of the Tn10 encoded Tet repressor was replaced by alanine and one residue was replaced by valine to determine their role in tet operator recognition by a 'loss of contact' analysis with 16 operator variants. One class of amino acids consisting of T27 and R28 in the first alpha-helix and L41, Y42, W43 and H44 in the recognition alpha-helix are quantitatively most important for wild-type operator binding. These residues are probably involved in the structural architecture of the motif. A second class of residues is quantitatively less important for binding, but determines specificity by forming base pair specific contacts to three positions in tet operator. This property is most clearly demonstrated for Q38 and P39 and to a lesser extent for T40 at the N-terminus of the recognition alpha-helix. The contacted operator base pairs indicate that the N-terminus of the recognition alpha-helix is located towards the palindromic center in the repressor-operator complex. Although the orientation of the recognition alpha-helix in the Tet repressor-tet operator complex is inversed as compared with the lambda- and 434 repressor-operator complexes, the reduced operator binding of the TA27 mutation in the first alpha-helix suggests that the hydrogen bonding networks connecting the two alpha-helices may be similar in these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface structure variants inDeinococcus radiodurans

The cell wall morphology and the polypeptide composition of two different strains as well as of two spontaneous mutants ofDeinococcus radiodurans have been compared. The two strains differ with respect to the density of their carbohydrate coat. One of the mutants lacks the surface (HPI) layer; the other one is devoid of a carbohydrate coat.     

A threonine to alanine exchange at position 40 of Tet repressor alters the recognition of the sixth base pair of tet operator from GC to AT.

The tet operators of two naturally evolved tetracycline resistance determinants differ by a G.C to A.T transition at the sixth base pair. This mutation prevents heterologous recognition of these tet operators by their respective two Tet repressor proteins. The amino acid side chains responsible for this sequence-specific distinction of operators were determined. For this purpose in vitro recombinants of the two tetR genes were constructed. Restriction sites were introduced by oligonucleotide-directed mutagenesis in both genes followed by the exchange of different coding segments between them. The encoded chimeric Tet repressor proteins were expressed and their operator recognition specificity was scored in vivo. Exchanging gradually smaller coding segments led finally to a single amino acid exchange in both genes at position 40 of the primary structures. Each Tet repressor containing Thr at this position recognizes the G.C operator while those with Ala recognize the A.T operator regardless of the rest of the sequences. This result demonstrates clearly that the amino acid 40 of Tet repressor contacts and recognizes base pair 6 of tet operator. Sterical interference of the large Thr side chain with the methyl group of A.T and a possible involvement of the hydroxyl in hydrogen bonding to the operator are discussed as the molecular basis of this differentiation between A.T and G.C base pairs.     

Three-dimensional structure of an open form of the surface layer from the fish pathogen Aeromonas salmonicida.

Cell-free culture supernatants of a lipopolysaccharide (LPS) O-polysaccharide-deficient, single-insertion transposon mutant of the tetragonal surface protein array (S layer)-containing fish pathogen Aeromonas salmonicida were examined by electron microscopy. Negative staining showed that the S layer was released as sheets of tetragonal material, indicating that although surface retention of assembled S layer requires the presence of wild-type LPS oligosaccharides, initial assembly of S-layer subunits into sheets does not require the presence of O-polysaccharide chains. The three-dimensional structure of the S layer was reconstructed from tilted micrographs of the released sheets. Horizontal sections through this reconstruction showed that the released sheets were composed of two identical S layers that were perfectly in register. The reconstructed layer had a lattice constant of 12.5 nm. At a resolution of 1.6 nm, the layer consisted of a major tetragon at one fourfold axis of symmetry and a minor tetragon at the second fourfold axis of symmetry. The core, composed of four of the major domains, contained a large depression and was located toward the inside of the layer. The minor tetragon provided connectivity within the layer and was located toward the outer surface of the layer. Projections through the double layer gave a type I (closed) pattern (M. Stewart, T. J. Beveridge, and T. J. Trust, J. Bacteriol. 166:120-127, 1986), yet projections through the single layer indicated that the type II (open) pattern was present. This open pattern was indistinguishable from that seen in S layer released from the surfaces of wild-type cells.