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PubMed2001

Modeling the bacterial photosynthetic reaction center. 4. The structural, electrochemical, and hydrogen-bonding properties of 22 mutants of Rhodobacter sphaeroides.

Site-directed mutagenesis has been employed by a number of groups to produce mutants of bacterial photosynthetic reaction centers, with the aim of tuning their operation by modifying hydrogen-bond patterns in the close vicinity of the "special pair" of bacteriochlorophylls P identical with P(L)P(M). Direct X-ray structural measurements of the consequences of mutation are rare. Attention has mostly focused on effects on properties such as carbonyl stretching frequencies and midpoint potentials to infer indirectly the induced structural modifications. In this work, the structures of 22 mutants of Rhodobacter sphaeroides have been calculated using a mixed quantum-mechanical molecular-mechanical method by modifying the known structure of the wild type. We determine (i) the orientation of the 2a-acetyl groups in the wild type, FY(M197), and FH(M197) series mutants of the neutral and oxidized reaction center, (ii) the structure of the FY(M197) mutant and possible water penetration near the special pair, (iii) that significant protein chain distortions are required to assemble some M160 series mutants (LS(M160), LN(M160), LQ(M160), and LH(M160) are considered), (iv) that there is competition for hydrogen-bonding between the 9-keto and 10a-ester groups for the introduced histidine in LH(L131) mutants, (v) that the observed midpoint potential of P for HL(M202) heterodimer mutants, including one involving also LH(M160), can be correlated with the change of electrostatic potential experienced at P(L), (vi) that hydrogen-bond cleavage may sometimes be induced by oxidation of the special pair, (vii) that the OH group of tyrosine M210 points away from P(M), and (viii) that competitive hydrogen-bonding effects determine the change in properties of NL(L166) and NH(L166) mutants. A new technique is introduced for the determination of ionization energies at the Koopmans level from QM/MM calculations, and protein-induced Stark effects on vibrational frequencies are considered.

Hydrogen Bonding↗
PubMed1991

Differential handling of bacterial antigens in macrophages infected with Mycobacterium leprae as studied by immunogold labeling of ultrathin sections.

Mycobacterium leprae were purified from the livers of experimentally infected armadillos, and the purity of the bacterial preparation was established by electron microscopy, immunoelectrophoresis of purified bacilli with rabbit serum raised against liver tissues from a noninfected armadillo, and gas chromatography. Such purified and intact bacilli were fixed and embedded by a gelatin-Lowicryl method for electron microscopy which preserved the mycobacterial antigens. Ultrathin sections were labeled with antisera raised in rabbits against the total antigens of the following species of mycobacteria: M. leprae, M. bovis BCG, M. avium, and a rapid-growing, nonpathogenic species, M. fallax. Bacteria were also labeled using serum raised against 2,3-diacyl-trehalose-2'-sulfate (sulfolipid-IV or SLIV) isolated and purified from M. tuberculosis. The immunolabeling was visualized under the electron microscope (EM) by using a secondary probe (goat-antirabbit IgG, H+L, coupled to 5 nm gold particles; GAR-5). EM results showed that M. leprae bacilli were highly labeled with all of the antisera used except SLIV, which was present only in discrete amounts. All of the antisera used labeled the bacterial "capsule," showing that this structure was not an artifact since it contained mycobacterial antigens. In parallel experiments, the murine J-774 macrophage cell line was infected with purified M. leprae, and fixed for EM at various time intervals for 1 week. Although the phagocytized bacteria did not multiply during the 1-week experiment, macrophages were unable to lyse them. Immunogold labeling of bacterial antigens in ultrathin sections of infected macrophages helped us to conclude: a) bacterial death and/or lysis is not a prerequisite for processing of antigens by infected macrophages; b) there was conclusive evidence for a differential antigen handling, i.e., some antigens were rapidly released (within 2 days, mostly capsular antigens) inside infected macrophages and transported to the macrophage surface, whereas others (the majority of them located in the cell-wall skeleton and in deeper bacterial structures) remained unreleased even after 4 to 7 days of infection; c) although relatively fewer epitopes reacting with anti-SLIV antibodies were found, they were rapidly released (within 2 days) inside macrophages, and exocytized to the macrophage surface. These novel findings are discussed in relation to leprosy and the current knowledge about the processing of bacterial antigens.

Animals↗
PubMed1976

[Intracellular membrane structures of Bdellovibrio bacteriovorus during development in the bacterial host cell].

Electron microscope investigation of intracellular membrane structures of Bd. bacteriovorous during its intracellular growth and development has been carried out. Increased amount of membrane structures has been stated. Along with simply organized invaginations of plasmalemma, complicated membrane structures resembling mesosomes of grampositive bacteria are observed. Localization of these structures testifies to their involvement in the synthesis of exotoxins and exoenzymes.

Bdellovibrio↗
PubMed1990

Structural resemblance between the families of bacterial signal-transduction proteins and of G proteins revealed by graph theoretical techniques.

The first application of a novel technique for the identification of common folding motifs in proteins is presented. Using techniques derived from graph theory, developed in order to compare secondary structure motifs in proteins, we have established that there is a striking resemblance in the tertiary fold of the Salmonella typhimurium Che Y chemotaxis protein and that of the GDP-binding domain of Escherichia coli elongation factor Tu (EF Tu). These two protein structures are representatives of two major macromolecular classes: CheY is a signal-transduction protein with sequence homologies to a wide range of bacterial proteins involved in regulation of chemotaxis, membrane synthesis and sporulation; whilst EF Tu is one of a family of guanosine-nucleotide-binding proteins which include the ras oncogene proteins and signal-transducing G proteins. The similarity we have found extends far beyond the previously recognized resemblances of each protein's fold to that of a generic nucleotide-binding domain. The lack of significant sequence homology between the two classes of proteins may mean that the common fold of the two proteins constitutes a particularly stable folding motif. However, an alternative possibility is that the strong three-dimensional structural resemblance may be indicative of a remote shared common ancestry between the bacterial signal-transduction proteins and the GDP-binding proteins.

Algorithms↗
PubMed2003

Probing the pore of ClC-0 by substituted cysteine accessibility method using methane thiosulfonate reagents.

ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged MTS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl- selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species.

Amino Acid Substitution↗
PubMed1996

Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism.

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems.

Bacteria↗
PubMed2003

Structure and activity of multiple nitrifying bacterial populations co-existing in a biofilm.

A biofilm from a nitrifying pilot-scale sequencing batch reactor was investigated for effects of varying process conditions on its microscale activity and structure. Microsensor measurements of oxygen, substrates and products of nitrification were applied under incubation at different ammonium and oxygen concentrations which reflected various situations during a treatment cycle. A high net N loss was observed under high ammonium (HA) concentrations in contrast to low ones. Additionally, results indicated inhibition of nitrite-oxidizing bacteria (NOB), but not of ammonia-oxidizing bacteria (AOB) by free ammonia under HA conditions. Diversity, spatial distribution, and abundance of nitrifying bacteria as analysed by fluorescence in situ hybridization (FISH) revealed six different nitrifying populations with heterogeneous distributions. Nitrosococcus mobilis formed conspicuous microcolonies locally surrounded by cells of the dominating N. europaea/eutropha-related AOB population. A third less abundant population was affiliated to N. oligotropha. Nitrite-oxidizing bacteria of the genera Nitrobacter and Nitrospira (with at least two distinct populations) showed a large scale heterogeneity in their distribution. Nitrospira spp. were also found in deeper inactive layers where they might persist rather than thrive, and act as seed population when detached. Results of functional and structural analyses are discussed with respect to specific niches of individual populations in this system.

Ammonia↗
PubMed1988

Site-directed cross-linking. Establishing the dimeric structure of the aspartate receptor of bacterial chemotaxis.

Cysteine residues introduced at specific locations in the aspartate receptor of Salmonella typhimurium provide anchor points for cross-linking and serve as chemical markers for structural studies of this oligomeric receptor. These markers have been used to measure the rate of subunit exchange between oligomeric receptors and to show that ligand binding inhibits this exchange. The cysteine-containing receptors can be oxidatively cross-linked to completion within the oligomeric receptor, indicating that the receptor has an even number of subunits. Based on this observation, a technique has been developed that can be used to determine the oligomeric structure of proteins under a variety of experimental conditions. The technique involves the measurement of the effect of dilution by "cysteineless" receptor subunits on cross-linking and reveals that the aspartate receptor is dimeric in detergent solution, in a mixed-micelle system, and in reconstituted membrane vesicles. Binding of aspartate does not change the oligomeric structure of the receptor, indicating that transmembrane signaling occurs within an oligomeric receptor of constant size.

Algorithms↗
PubMed1998

Crystal structure of glycosylasparaginase from Flavobacterium meningosepticum.

The crystal structure of recombinant glycosylasparaginase from Flavobacterium meningosepticum has been determined at 2.32 angstroms resolution. This enzyme is a glycoamidase that cleaves the link between the asparagine and the N-acetylglucosamine of N-linked oligosaccharides and plays a major role in the degradation of glycoproteins. The three-dimensional structure of the bacterial enzyme is very similar to that of the human enzyme, although it lacks the four disulfide bridges found in the human enzyme. The main difference is the absence of a small random coil domain at the end of the alpha-chain that forms part of the substrate binding cleft and that has a role in the stabilization of the tetramer of the human enzyme. The bacterial glycosylasparaginase is observed as an (alphabeta)2-tetramer in the crystal, despite being a dimer in solution. The study of the structure of the bacterial enzyme allows further evaluation of the effects of disease-causing mutations in the human enzyme and confirms the suitability of the bacterial enzyme as a model for functional analysis.

Aspartylglucosylaminase↗
PubMed1981

[Structural features of the elementary bodies of bacterial L-forms].

Elementary bodies have been detected in the L-forms of various bacteria, irrespective of their strain, the phase of development and stabilization of their culture. Elementary bodies have been found to have all organoids peculiar to other L-culture cells (the unit membrane, the nucleoid, ribosomes, more seldom mesosome-like structures and the outer membrane of the cell wall), which is indirectly indicative of their possible viability. The cytoplasmic membrane of elementary bodies contains dehydrogenases (detected in the NBT test), has the hydrophobic layer structure typical of bacterial membranes and, judging by the results of the ferritin test, possesses the same antigenic properties as the cytoplasmic membrane of other L-culture cells. Nevertheless, the above-mentioned properties are far from being the attribute of every elementary body, and the completeness of their manifestation is not directly related with the size of elementary bodies.

Cell Membrane↗
PubMed1996

High-resolution structures of the ligand binding domain of the wild-type bacterial aspartate receptor.

The high-resolution structures of the wild-type periplasmic domain of the bacterial aspartate receptor have been determined in the absence and presence of bound aspartate to 1.85 and 2.2 A resolution, respectively. As we reported earlier, in the refined structure of the complexed form of the crosslinked cysteine mutant receptor, the binding of the aspartate at the first site was mediated through four bridging water molecules while the second site showed an occupant electron density that best fit a sulfate group, which was present in the crystallization solution at high concentration. In the wild-type periplasmic domain structure two aspartate residues are bound per dimer, but with different occupancies. There exists a "strong" aspartate-binding site whose binding is again mediated by four water molecules while the second site contains aspartate whose B-factor is about 10% higher, signifying weaker binding. The interaction between the second, "weaker" aspartate with the three ligand-binding arginine side-chains is slightly different from the first site. The major difference is that there are three water molecules mediating the binding of aspartate at the second site, whereas in the first site there are four bridging water molecules. The fact that aspartate-complexed crystals of the wild-type were grown with a large excess aspartate while the cross-linked crystals were grown with equal molar aspartate may explain the difference in the stoichiometry observed. The conservation of the four bridging water molecules in the strong aspartate site of both the cross-linked and wild-type periplasmic domain may reflect an important binding motif. The periplasmic domain in the apo form is a symmetrical dimer, in which each of the subunits is equivalent, and the two aspartate binding sites are identical. Upon the binding of aspartate, the subunits are no longer symmetrical. The main difference between the aspartate-bound and unbound forms is in a small, rigid-body rotation between the subunits within a dimer. The rotation is similar in both direction and magnitude in the crosslinked and wild-type periplasmic domains. The presence of the second aspartate in the wild-type structure does not make any additional rotation compared to the single-site binding. The conservation of the small angular change in vitro suggests that the inter-subunit rotation may have relevance to the understanding of the mechanism of transmembrane signal transduction in vivo.

Aspartic Acid↗
PubMed1989

Ganglioside epitope recognized by K99 fimbriae from enterotoxigenic Escherichia coli.

The receptor structure recognized by Escherichia coli possessing K99 fimbriae and by isolated K99 fimbrial fractions was examined by using an equine erythrocyte hemagglutination inhibition test. Both K99-positive organisms (strain B41) and fimbrial preparations reacted with N-glycolylneuraminyl-lactosyl-ceramide (NeuGcLacCer) purified from equine erythrocytes with very high potency. Fimbrial preparations were 253 times more potent than intact organisms, indicating that isolated fimbriae more precisely recognize the structure of NeuGcLacCer than do fimbriae located on the bacterial cell wall. Structurally, the N-glycolyl group of the sialic acid was shown to be essential because substitution of the N-acetyl group for the N-glycolyl group caused the reactivity to completely disappear. The substitution of the O-acetyl group for the C4 hydroxyl group of the sialic acid (4-O-Ac-NeuGcLacCer) also diminished the reactivity by about 500 times, indicating that the fine structure of NeuGc is necessary for recognition. N-Glycolylneuraminyl-neolactotetraosyl-ceramide (NeuGcnLc4Cer) and N-glycolylneuraminyl-neolactohexaosyl-ceramide (NeuGcnLc6Cer), both of which have identical disaccharides at the nonreducing terminal and longer carbohydrate chains, showed reduced reactivity, indicating that the ceramide of NeuGcLacCer is also involved in the recognition. Indeed, NeuGcLac oligosaccharides altered by cleavage of the ceramide or the terminal sialic acid (NeuGc) showed dramatically reduced reactivities. Ten other E. coli strains (isolated from diseased calves) and two strains (isolated from diseased piglets) which possessed the same K99 antigen and various O antigens were used for the recognition test. The results obtained were similar to those mentioned above.

Animals↗
PubMedDate not supplied

The primary structures of the core antenna polypeptides from Rhodopseudomonas marina.

The antenna complex B880 of Rp. marina has been isolated by applying ion-exchange chromatography on Whatman DE-52 resin and sucrose density centrifugation of LDAO-solubilized photosynthetic membranes. The antenna polypeptides B880-alpha and B880-beta were prepared by organic solvent extraction of extensively dialyzed and freeze-dried B880 antenna complex material or photosynthetic membranes. Gel filtration on Sephadex LH-60 and ion-exchange chromatography on Whatman DE-32 resin in the presence of organic solvents and an additional step on a C-8 reversed phase column yielded pure alpha- and beta-apoproteins. Their complete primary structures have been elucidated using automated Edman degradation and carboxypeptidase digestion. According to quantitative Edman degradation the ratio of B880-alpha and B880-beta has been determined as 1:1 in the isolated antenna complex as well as in the photosynthetic membrane. B880-alpha of Rp. marina, presumably N-formylated, consists of 52 amino acid residues and is 75, 56, 52 and 44% homologous to the corresponding core antenna polypeptides of Rs. rubrum, Rp. viridis, Rb. capsulatus and Rb. sphaeroides. In contrast, B880-beta (56 amino acid residues) is less homologous to the corresponding core beta-antenna polypeptides of the same strains (57, 51, 41 and 42%). It shows an extended N-terminal domain as compared to the B880-alpha polypeptide. Apart from the typical structural features of bacterial membrane-bound antenna polypeptides (three domain structure. His-residue in the hydrophobic stretch) the antenna polypeptides of Rp. marina are structurally related to polypeptides of core antenna complexes with strong near infrared circular dichroism signals.

Amino Acid Sequence↗
PubMed1987

Structure of the porin from a bacterial stalk.

The stalks (hyphae) of a prosthecate bacterium, directly sampled from the water surface of a hot pond, show extended regular patterns on their envelope in the electron microscope. Image processing revealed a structure of the crystalline complexes which is very similar to the gross morphology of the Escherichia coli porins OmpC and OmpF. The natural two-dimensional crystal of the outer membrane protein has p3 symmetry and a lattice constant of 7.95 nm. The three-dimensional structure of the stalk porin has been determined to an almost isotropic resolution of 1.7 nm. The reconstruction revealed a complex network of channels within the membrane matrix with a triplet of pores merging into a common outlet, similar to the structure of the E. coli porin OmpF in reconstituted membranes. In addition, a blindly ending pore exists which appears to be connected to the continuous pores via small channels. The significance of the regularly arrayed porin cylinders with respect to the shape and function of the stalks is discussed.

Bacterial Outer Membrane Proteins↗
PubMed1997

Langmuir-Blodgett and X-ray diffraction studies of isolated photosystem II reaction centers in monolayers and multilayers: physical dimensions of the complex.

The photosystem II (PSII) reaction center (RC) is a hydrophobic intrinsic protein complex that drives the water-oxidation process of photosynthesis. Unlike the bacterial RC complex, an X-ray crystal structure of the PSII RC is not available. In order to determine the physical dimensions of the isolated PSII RC complex, we applied Langmuir techniques to determine the cross-sectional area of an isolated RC in a condensed monolayer film. Low-angle X-ray diffraction results obtained by examining Langmuir-Blodgett multilayer films of alternating PSII RC/Cd stearate monolayers were used to determine the length (or height; z-direction, perpendicular to the plane of the original membrane) of the complex. The values obtained for a PSII RC monomer were 26 nm2 and 4.8 nm, respectively, and the structural integrity of the RC in the multilayer film was confirmed by several approaches. Assuming a cylindrical-type RC structure, the above dimensions lead to a predicted volume of about 125 nm3. This value is very close to the expected volume of 118 nm3, calculated from the known molecular weight and partial specific volume of the PSII RC proteins. This same type of comparison was also made with the Rhodobacter sphaeroides RC based on published data, and we conclude that the PSII RC is much shorter in length and has a more regular solid geometric structure than the bacterial RC. Furthermore, the above dimensions of the PSII RC and those of PSII core (RC plus proximal antenna) proteins protruding outside the plane of the PSII membrane into the lumenal space as imaged by scanning tunneling microscopy (Seibert, Aust. J. Pl. Physiol. 22, 161-166, 1995) fit easily into the known dimensions of the PSII core complex visualized by others as electron-density projection maps. From this we conclude that the in situ PSII core complex is a dimeric structure containing two copies of the PSII RC.

Photosynthetic Reaction Center Complex Proteins↗
PubMed2001

New insights into the thermostability of bacterial ferredoxins: high-resolution crystal structure of the seven-iron ferredoxin from Thermus thermophilus.

The crystal structure of the seven-iron ferredoxin from Thermus thermophilus (FdTt) has been determined at 1.64 A resolution, allowing us to unveil the common mechanisms of thermostabilization within "bacterial-type" ferredoxins. FdTt and other homologous thermophilic seven-iron ferredoxins are smaller than their mesophilic counterparts. Thermostabilizing features are optimized in a minimal structural and functional unit, with an extensive cross-linking of secondary structure elements mediated by improved polar and hydrophobic interactions. Most of the potentially stabilizing features are focused on the vicinity of the functional [3Fe-4S] cluster. The structural [4Fe-4S] cluster is shielded in thermophilic FdTt by an increased number of polar interactions involving the two N-terminal residues. Comparisons with the hyperthermostable ferredoxin from Thermotoga maritima reveal that (1) a reduction in the number of non-glycine residues in strained conformations, (2) improved polar interactions within the common iron-sulfur cluster binding (betaalphabeta)2 motif, and (3) an optimized charge distribution at the protein surface, constitute a common strategy for increasing the thermal stability of these ferredoxins.

Amino Acid Sequence↗
PubMed1979

Spatio-temporal structure of migrating chemotactic band of Escherichia coli. I. Traveling band profile.

We developed a rapid-scanning, light-scattering densitometer by which extensive measurements of band migration speeds and band profiles of chemotactic bands of Escherichia coli in motility buffer both with and without serine have been made. The purpose is to test the applicability of the phenomenological model proposed by Keller and Segel (J. Theor. Biol. 1971. 30:235) and to determine the motility (mu) and chemotactic (delta) coefficients of the bacteria. We extend the previous analytical solution of the simplified Keller-Segel model by taking into account the substrate diffusion which turns out to be significant in the case of oxygen. We demonstrate that unique sets of values of mu and delta can be obtained for various samples at different stages of migration by comparing the numerical solution of the model equation and the experimental data. The rapid-scanning technique also reveals a hitherto unobserved time-dependent fine structure in the bacterial band. We give a qualitative argument to show that the fine structure is an example of the dissipative structure that arises from a nonlinear coupling between the bacterial density and the oxygen concentration gradient. Implications for a further study of the dissipative structure in testing the Keller-Segel model of chemotaxis are briefly discussed.

Chemotaxis↗