Cloning and expression of two crystal protein genes, cry30Ba1 and cry44Aa1, obtained from a highly mosquitocidal strain, Bacillus thuringiensis subsp. entomocidus INA288

Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.     

EshA accentuates ppGpp accumulation and is conditionally required for antibiotic production in Streptomyces coelicolor A3(2)

Disruption of eshA, which encodes a 52-kDa protein that is produced late during the growth of Streptomyces coelicolor A3(2), resulted in elimination of actinorhodin production. In contrast, disruption of eshB, a close homologue of eshA, had no effect on antibiotic production. The eshA disruptant accumulated lower levels of ppGpp than the wild-type strain accumulated. The loss of actinorhodin production in the eshA disruptant was restored by expression of a truncated relA gene, which increased the ppGpp level to the level in the wild-type strain, indicating that the reduced ppGpp accumulation in the eshA mutant was solely responsible for the loss of antibiotic production. Antibiotic production was also restored in the eshA mutant by introducing mutations into rpoB (encoding the RNA polymerase β subunit) that bypassed the requirement for ppGpp, which is consistent with a role for EshA in modulating ppGpp levels. EshA contains a cyclic nucleotide-binding domain that is essential for its role in triggering actinorhodin production. EshA may provide new insights and opportunities to unravel the molecular signaling events that occur during physiological differentiation in streptomycetes.     

Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.     

A retaining endo-beta-mannosidase from a dicot plant, cabbage

An endo-beta-mannosidase [EC 3.2.1.152, glycoside hydrolase family 2], which hydrolyzes the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, has been found in plant tissues [Ishimizu, T., Sasaki, A., Okutani, S., Maeda, M., Yamagishi, M., and Hase, S. (2004) J. Biol. Chem. 279, 38555-38562]. So far, this glycosidase has been purified only from a monocot plant, a lily. Here, an endo-beta-mannosidase was purified from a dicot plant, cabbage (Brassica oleracea), and characterized. The cabbage endo-beta-mannosidase consists of four polypeptides. These four polypeptides are encoded by a single gene, whose nucleotide sequence is homologous to those of the lily and Arabidopsis endo-beta-mannosidase genes. 1H NMR analysis of the stereochemistry of the hydrolysis of pyridylaminated (PA) Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc showed that the cabbage endo-beta-mannosidase is a retaining glycoside hydrolase, as are other glycoside hydrolase family 2 enzymes. The enzymatic characteristics, including substrate specificity, of the cabbage enzyme are very similar to those of the lily enzyme. These endo-beta-mannosidases specifically act on Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0 to 2). These results suggest that the endo-beta-mannosidase is present in at least the angiosperms, and has common roles, such as the degradation of N-glycans.     

Time-resolved detection of conformational changes in oat phytochrome A: time-dependent diffusion

Conformational changes in oat phytochrome A (phy) in solution after photoexcitation of the red-absorbing form (Pr) were studied in time-domain by the pulsed laser-induced transient grating technique. It was found that the diffusion coefficient (D) of far-red-absorbing form (Pfr) of large phy (1.3 x 10(-11) m(2) s(-1)) is markedly reduced compared with that of Pr (5.8 x 10(-11) m(2) s(-1)). This large reduction indicates that the conformation of Pfr is significantly changed from that of Pr, so that the intermolecular interaction with water molecules increases. This change completes within 1 ms after the photoexcitation. On the other hand, D of Pr of intact phy (4.1 x 10(-11) m(2) s(-1)) first decreases upon photoexcitation to 0.89 x 10(-11) m(2) s(-1) within 1 ms and then gradually increases with a time constant of 100 ms to the value of Pfr, 1.7 x 10(-11) m(2) s(-1). This slower phase suggests that the conformation of the N-terminal region changes with 100 ms to decrease the intermolecular interaction with water after a global change in the large phy region. The increase of D was interpreted in terms of alpha-helix formation in the Pfr form from the random coil structure in the Pr form.     

Anabolic five subunit-type pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

The thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene cluster, porEDABG, encoding pyruvate:ferredoxin oxidoreductase (POR), which plays a key role in this cycle, was cloned and sequenced. The nucleotide sequence and the gene organization were similar to those of the five subunit-type 2-oxoglutarate:ferredoxin oxidoreductase from this strain, although the anabolic POR had been previously reported to consist of four subunits. A small protein (8 kDa) encoded by porE, which had not been detected in the previous work, was identified in the purified recombinant POR expressed in Escherichia coli, indicating that the enzyme is also a five-subunit type. Incorporation of PorE in the wild-type POR enzyme was confirmed by immunological analysis. PorA, PorB, PorG, and PorE were similar to the alpha, beta, gamma, and delta subunits of the four subunit-type 2-oxoacid oxidoreductases, respectively, and had conserved specific motifs. PorD had no specific motifs but was essential for the expression of the active enzyme.     

Small molecule inhibitors of alpha-synuclein filament assembly

Alpha-synuclein is the major component of the filamentous inclusions that constitute defining characteristics of Parkinson's disease and other alpha-synucleinopathies. Here we have tested 79 compounds belonging to 12 different chemical classes for their ability to inhibit the assembly of alpha-synuclein into filaments in vitro. Several polyphenols, phenothiazines, porphyrins, polyene macrolides, and Congo red and its derivatives, BSB and FSB, inhibited alpha-synuclein filament assembly with IC(50) values in the low micromolar range. Many compounds that inhibited alpha-synuclein assembly were also found to inhibit the formation of Abeta and tau filaments. Biochemical analysis revealed the formation of soluble oligomeric alpha-synuclein in the presence of inhibitory compounds, suggesting that this may be the mechanism by which filament formation is inhibited. Unlike alpha-synuclein filaments and protofibrils, these soluble oligomeric species did not reduce the viability of SH-SY5Y cells. These findings suggest that the soluble oligomers formed in the presence of inhibitory compounds may not be toxic to nerve cells and that these compounds may therefore have therapeutic potential for alpha-synucleinopathies and other brain amyloidoses.     

Kinetic measurement of transient dimerization and dissociation reactions of Arabidopsis phototropin 1 LOV2 domain

Photochemical reaction of a plant blue-light photoreceptor, Arabidopsis phototropin 1-LOV (light-oxygen-voltage sensing) domain 2, was studied with a view to the diffusion coefficients (D) using the pulsed-laser-induced transient grating method. Although the reaction dynamics completes at a rate of several microseconds as long as it is monitored by the absorption change, the diffusion coefficient was found to be time-dependent in a time range of submilliseconds to seconds. The observed signal can be analyzed by the two-state model, which includes the D-value decrease from D of the reactant (9.8 +/- 0.4) x 10(-11) m2/s to D of the product (8.0 +/- 0.4) x 10(-11) m2/s. The D-value of the reactant implies that the dominant form in the ground state of phototropin 1 LOV2 is the monomeric form in a concentration range of 50-200 microM. According to the Stokes-Einstein relationship, the D-change can be explained by a volume increase of 1.8 times. Furthermore, the rate of the D-change was roughly proportional to the concentration of the sample. These two observations indicate that the LOV2 domain transiently forms a dimer upon photoexcitation. When the sample concentration is increased (>180 microM), a new signal component appears within a few milliseconds. This signal represents a D increase from 8.0 x 10(-11) m2/s to 9.8 x 10(-11) m2/s with a time constant of 300 micros. The completely opposite D-change from that observed in a lower concentration, as well as the concentration dependence, implies that a dimer is formed in the ground state in a higher concentration range, even though the fraction of the dimer is still minor in this range. This dimer is photodissociated, with a time constant of 300 micros. This research clearly shows that the time-resolved diffusion measurement is a very powerful tool for detecting spectrally silent association/dissociation processes during chemical reactions. The photoreaction of the LOV2 domain is discussed.