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CO2-Responsive Expression and Gene Organization of Three Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Enzymes and Carboxysomes in Hydrogenovibrio marinus Strain MH-110. Yoichi Yoshizawa, 2004.Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, fixes CO2 by the Calvin-Benson-Bassham cycle . Strain MH-110 possesses three different sets of genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO): CbbLS-1 and CbbLS-2, which belong to form I (L8S8), and CbbM, which belongs to form II (Lx) . In this paper, we report that the genes for CbbLS-1 (cbbLS-1) and CbbM (cbbM) are both followed by the cbbQO genes and preceded by the cbbR genes encoding LysR-type regulators . In contrast, the gene for CbbLS-2 (cbbLS-2) is followed by genes encoding carboxysome shell peptides . We also characterized the three RubisCOs in vivo by examining their expression profiles in environments with different CO2 availabilities . Immunoblot analyses revealed that when strain MH-110 was cultivated in 15% CO2, only the form II RubisCO, CbbM, was expressed . When strain MH-110 was cultivated in 2% CO2, CbbLS-1 was expressed in addition to CbbM . In the 0.15% CO2 culture, the expression of CbbM decreased and that of CbbLS-1 disappeared, and CbbLS-2 was expressed . In the atmospheric CO2 concentration of approximately 0.03%, all three RubisCOs were expressed . Transcriptional analyses of mRNA by reverse transcription-PCR showed that the regulation was at the transcriptional level . Electron microscopic observation of MH-110 cells revealed the formation of carboxysomes in the 0.15% CO2 concentration . The results obtained here indicate that strain MH-110 adapts well to various CO2 concentrations by using different types of RubisCO enzymes . Activity of the New Triazole Derivative Albaconazole against Trypanosoma (Schizotrypanum) cruzi in Dog Hosts. Paulo Marcos da Matta Guedes, 2004.Albaconazole is an experimental triazole derivative with potent and broad-spectrum antifungal activity and a remarkably long half-life in dogs, monkeys, and humans . In the present work, we investigated the in vivo activity of this compound against two strains of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, using dogs as hosts . The T . cruzi strains used in the study were previously characterized (murine model) as susceptible (strain Berenice-78) and partially resistant (strain Y) to the drugs currently in clinical use, nifurtimox and benznidazole . Our results demonstrated that albaconazole is very effective in suppressing the proliferation of the parasite and preventing the death of infected animals . Furthermore, the parasitological, PCR, serological, and proliferative assay results indicated parasitological cure indices of 25 and 100% among animals inoculated with T . cruzi strain Y when they were treated with albaconazole at 1.5 mg/kg of body weight/day for 60 and 90 days, respectively . On the other hand, although albaconazole given at 1.5 mg/kg/day was very effective in suppressing the proliferation of the parasite in animals infected with the Berenice-78 T . cruzi strain, no parasitological cure was observed among them, even when a longer treatment period (150 doses) was used . In conclusion, our results demonstrate that albaconazole has trypanocidal activity in vivo and is capable of inducing radical parasitological cure, although natural resistance to this compound was also indicated . Furthermore, the compound can be used in long-term treatment schemes (60 to 150 days) with minimal toxicity and thus represents a potentially useful candidate for the treatment of human Chagas' disease . Genetic Instability of Heterozygous, Hybrid, Natural Wine Yeasts. Manuel Ramírez, 2004.We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae . Spontaneous cyh2R/cyh2R mutants resistant to high levels of cycloheximide can be directly isolated from cyh2S/cyh2S wine yeasts . Heterozygous cyh2R/cyh2S hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2 . There were two main classes of hybrids . The lawn hybrids have high genetic instability and generally become cyh2R/cyh2R homozygotes and lose the killer phenotype under nonselective conditions . The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype . The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported . Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity . This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations . Characterization of Indigoidine Biosynthetic Genes in Erwinia chrysanthemi and Role of This Blue Pigment in Pathogenicity. Sylvie Reverchon, 2002.In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins . One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine . Since production of this pigment is cryptic in the wild-type strain, E . chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant . These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM . Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis . No specific function could be assigned to IndA . In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS) . The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS . These data suggest that glutamine is the precursor of indigoidine . We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated . Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis . DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions . The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E . chrysanthemi pathogenicity . Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha . Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response . The Cellulosome System of Acetivibrio cellulolyticus Includes a Novel Type of Adaptor Protein and a Cell Surface Anchoring Protein. Qi Xu, 2003.A scaffoldin gene cluster was identified in the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. The previously described scaffoldin gene, cipV, encodes an N-terminal family 9 glycoside hydrolase, a family 3b cellulose-binding domain, seven cohesin domains, and a C-terminal dockerin . The gene immediately downstream of cipV was sequenced and designated scaB . The protein encoded by this gene has 942 amino acid residues and a calculated molecular weight of 100,358 and includes an N-terminal signal peptide, four type II cohesions, and a C-terminal dockerin . ScaB cohesins 1 and 2 are very closely linked . Similar, but not identical, 39-residue Thr-rich linker segments separate cohesin 2 from cohesin 3 and cohesin 3 from cohesin 4, and an 84-residue Thr-rich linker connects the fourth cohesin to a C-terminal dockerin . The scaC gene downstream of scaB codes for a 1,237-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module . A long, ca . 550-residue linker separates the third cohesin and the SLH module of ScaC and is characterized by an 18-residue Pro-Thr-Ala-Ser-rich segment that is repeated 27 times . The calculated molecular weight of the mature ScaC polypeptide (excluding the signal peptide) is 124,162 . The presence of the cohesins and the conserved SLH module implies that ScaC acts as an anchoring protein . The ScaC cohesins are on a separate branch of the phylogenetic tree that is close to, but distinct from, the type I cohesins . Affinity blotting with representative recombinant probes revealed the following specific intermodular interactions: (i) an expressed CipV cohesin binds selectively to an enzyme-borne dockerin, (ii) a representative ScaB cohesin binds to the CipV band of the cell-free supernatant fraction, and (iii) a ScaC cohesin binds to the ScaB dockerin . The experimental evidence thus indicates that CipV acts as a primary (enzyme-recognizing) scaffoldin, and the protein was also designated ScaA . In addition, ScaB is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC) . The cellulosome system of A . cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the ScaB adaptor protein . The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit . At least 96 enzymes can apparently be incorporated into an individual A . cellulolyticus cellulosome . The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose . Comparison of the emerging organization of the A . cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture . ATP-Dependent Interactions between Escherichia coli Min Proteins and the Phospholipid Membrane In Vitro. Laura L. Lackner, 2003.Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor . MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles . To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions . We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg2+, whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked . The results support and extend recent work by Z . Hu et al . (Z . Hu, E . P . Gogol, and J . Lutkenhaus, Proc . Natl . Acad . Sci . USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP) . The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide . Secondary Metabolites of Flustra foliacea and Their Influence on Bacteria. Lars Peters, 2003.The North Sea bryozoan Flustra foliacea was investigated to determine its secondary metabolite content . Gas chromatography-mass spectrometry analysis of a dichloromethane extract of the bryozoan enabled 11 compounds to be identified . Preparative high-performance liquid chromatography of the extract resulted in the isolation of 10 brominated alkaloids (compounds 1 to 10) and one diterpene (compound 11) . All of these compounds were tested to determine their activities in agar diffusion assays against bacteria derived from marine and terrestrial environments . Compounds 1, 3 to 7, 10, and 11 exhibited significant activities against one or more marine bacterial strains originally isolated from F . foliacea but only weak activities against all of the terrestrial bacteria . By using the biosensors Pseudomonas putida(pKR-C12), P . putida(pAS-C8), and Escherichia coli(pSB403) the antagonistic effect on N-acyl-homoserine lactone-dependent quorum-sensing systems was investigated . Compounds 8 and 10 caused reductions in the signal intensities in these bioassays ranging from 50 to 20% at a concentration of 20 µg/ml .
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