Isolation and characterization of drosocrystallin, a lens crystallin gene of Drosophila melanogaster.
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We have cloned the drosocrystallin gene (dcy) of Drosophila melanogaster, which encodes a major protein of the corneal lens, previously described in part by Komori et al. (1992, J. Cell Sci. 102, 191-201). Synthesis of the DCY protein starts weakly in 2-day-old pupae, reaches a peak at day 3 and day 4 of pupal development, and decreases very fast in young adults. The dcy mRNA is detected in the compound eyes as well as in the ocelli. The presence of a putative signal peptide and the extracellular location of DCY suggest that DCY is a secreted protein. Interestingly, the dcy gene shows sequence similarities to some insect cuticular proteins and is detected as well in two closely related Drosophila species, D. sechellia and D. simulans, and in one more distantly related species, D. virilis. This finding supports the hypothesis that Drosophila used the same strategy as vertebrates and mollusks, namely, recruiting a multifunctional protein for refraction in the lens, by a gene-sharing mechanism. Furthermore, it supports our intercalary evolution hypothesis, which suggests that the development of an elaborate structure (for example, a compound eye) from an original primitive form (an ancestral photoreceptor organ) can be achieved by recruiting novel genes into the original developmental pathway. (+info)
Electron microscopy studies of cell-wall-anchored cellulose (Avicel)-binding protein (AbpS) from Streptomyces reticuli.
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Streptomyces reticuli produces a 35-kDa cellulose (Avicel)-binding protein (AbpS) which interacts strongly with crystalline cellulose but not with soluble types of cellulose. Antibodies that were highly specific for the NH2-terminal part of AbpS were isolated by using truncated AbpS proteins that differed in the length of the NH2 terminus. Using these antibodies for immunolabelling and investigations in which fluorescence, transmission electron, or immunofield scanning electron microscopy was used showed that the NH2 terminus of AbpS protrudes from the murein layer of S. reticuli. Additionally, inspection of ultrathin sections of the cell wall, as well as biochemical experiments performed with isolated murein, revealed that AbpS is tightly and very likely covalently linked to the polyglucane layer. As AbpS has also been found to be associated with protoplasts, we predicted that a COOH-terminal stretch consisting of 17 hydrophobic amino acids anchors the protein to the membrane. Different amounts of AbpS homologues of several Streptomyces strains were synthesized. (+info)
The nuclear ribosomal DNA intergenic spacer as a target sequence to study intraspecific diversity of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum directly on pinus root systems.
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Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was studied to evaluate whether this sequence could be used in field studies to estimate the diversity of strains forming mycorrhizas on individual Pinus pinaster root systems. This sequence was amplified by PCR from 125 haploid homokaryotic strains collected in 14 P. pinaster stands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of the amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose frequencies differed. Nine of these alleles were found only once, whereas about 60% of the strains contained four of the alleles. Local populations could be almost as diverse as the entire population along a 150-km stretch of coastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequence data were used to design oligonucleotides which allowed separate PCR amplification of three different segments of the IGS. Most polymorphisms observed among the full-length IGS regions resulted from polymorphisms in an internal ca. 1,500-bp-long sequence characterized by length variations that may have resulted from variable numbers of a T2AG3 motif. This internal polymorphic sequence could not be amplified from the genomes of nine other Hebeloma species. Analysis of this internal sequence amplified from the haploid progenies of 10 fruiting bodies collected in a 70-m2 area resulted in identification of six allelic forms and seven distinct diplotypes out of the 21 possible different combinations. Moreover, optimization of the PCR conditions resulted in amplification of this sequence from more than 80% of the DNA samples extracted from individual H. cylindrosporum infected P. pinaster mycorrhizal root tips, thus demonstrating the usefulness of this sequence for studying the below-ground diversity of mycorrhizas formed by genets belonging to the same fungal species. (+info)
Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199.
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Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants. (+info)
Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations.
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Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences. (+info)
Anaerobic oxidation of o-xylene, m-xylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria.
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Various alkylbenzenes were depleted during growth of an anaerobic, sulfate-reducing enrichment culture with crude oil as the only source of organic substrates. From this culture, two new types of mesophilic, rod-shaped sulfate-reducing bacteria, strains oXyS1 and mXyS1, were isolated with o-xylene and m-xylene, respectively, as organic substrates. Sequence analyses of 16S rRNA genes revealed that the isolates affiliated with known completely oxidizing sulfate-reducing bacteria of the delta subclass of the class Proteobacteria. Strain oXyS1 showed the highest similarities to Desulfobacterium cetonicum and Desulfosarcina variabilis (similarity values, 98.4 and 98.7%, respectively). Strain mXyS1 was less closely related to known species, the closest relative being Desulfococcus multivorans (similarity value, 86.9%). Complete mineralization of o-xylene and m-xylene was demonstrated in quantitative growth experiments. Strain oXyS1 was able to utilize toluene, o-ethyltoluene, benzoate, and o-methylbenzoate in addition to o-xylene. Strain mXyS1 oxidized toluene, m-ethyltoluene, m-isoproyltoluene, benzoate, and m-methylbenzoate in addition to m-xylene. Strain oXyS1 did not utilize m-alkyltoluenes, whereas strain mXyS1 did not utilize o-alkyltoluenes. Like the enrichment culture, both isolates grew anaerobically on crude oil with concomitant reduction of sulfate to sulfide. (+info)
Diversity of dissimilatory bisulfite reductase genes of bacteria associated with the deep-sea hydrothermal vent polychaete annelid Alvinella pompejana.
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A unique community of bacteria colonizes the dorsal integument of the polychaete annelid Alvinella pompejana, which inhabits the high-temperature environments of active deep-sea hydrothermal vents along the East Pacific Rise. The composition of this bacterial community was characterized in previous studies by using a 16S rRNA gene clone library and in situ hybridization with oligonucleotide probes. In the present study, a pair of PCR primers (P94-F and P93-R) were used to amplify a segment of the dissimilatory bisulfite reductase gene from DNA isolated from the community of bacteria associated with A. pompejana. The goal was to assess the presence and diversity of bacteria with the capacity to use sulfate as a terminal electron acceptor. A clone library of bisulfite reductase gene PCR products was constructed and characterized by restriction fragment and sequence analysis. Eleven clone families were identified. Two of the 11 clone families, SR1 and SR6, contained 82% of the clones. DNA sequence analysis of a clone from each family indicated that they are dissimilatory bisulfite reductase genes most similar to the dissimilatory bisulfite reductase genes of Desulfovibrio vulgaris, Desulfovibrio gigas, Desulfobacterium autotrophicum, and Desulfobacter latus. Similarities to the dissimilatory bisulfite reductases of Thermodesulfovibrio yellowstonii, the sulfide oxidizer Chromatium vinosum, the sulfur reducer Pyrobaculum islandicum, and the archaeal sulfate reducer Archaeoglobus fulgidus were lower. Phylogenetic analysis separated the clone families into groups that probably represent two genera of previously uncharacterized sulfate-reducing bacteria. The presence of dissimilatory bisulfite reductase genes is consistent with recent temperature and chemical measurements that documented a lack of dissolved oxygen in dwelling tubes of the worm. The diversity of dissimilatory bisulfite reductase genes in the bacterial community on the back of the worm suggests a prominent role for anaerobic sulfate-reducing bacteria in the ecology of A. pompejana. (+info)
Isolation from estuarine sediments of a Desulfovibrio strain which can grow on lactate coupled to the reductive dehalogenation of 2,4, 6-tribromophenol.
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Strain TBP-1, an anaerobic bacterium capable of reductively dehalogenating 2,4,6-tribromophenol to phenol, was isolated from estuarine sediments of the Arthur Kill in the New York/New Jersey harbor. It is a gram-negative, motile, vibrio-shaped, obligate anaerobe which grows on lactate, pyruvate, hydrogen, and fumarate when provided sulfate as an electron acceptor. The organism accumulates acetate when grown on lactate and sulfate, contains desulfoviridin, and will not grow in the absence of NaCl. It will not utilize acetate, succinate, propionate, or butyrate for growth via sulfate reduction. When supplied with lactate as an electron donor, strain TBP-1 will utilize sulfate, sulfite, sulfur, and thiosulfate for growth but not nitrate, fumarate, or acrylate. This organism debrominates 2-, 4-, 2,4-, 2,6-, and 2,4,6-bromophenol but not 3- or 2,3-bromophenol or monobrominated benzoates. It will not dehalogenate monochlorinated, fluorinated, or iodinated phenols or chlorinated benzoates. Together with its physiological characteristics, its 16S rRNA gene sequence places it in the genus Desulfovibrio. The average growth yield of strain TBP-1 grown on a defined medium supplemented with lactate and 2,4,6-bromophenol is 3.71 mg of protein/mmol of phenol produced, and the yield was 1.42 mg of protein/mmol of phenol produced when 4-bromophenol was the electron acceptor. Average growth yields (milligrams of protein per millimole of electrons utilized) for Desulfovibrio sp. strain TBP-1 grown with 2,4,6-bromophenol, 4-bromophenol, or sulfate are 0.62, 0.71, and 1.07, respectively. Growth did not occur when either lactate or 2,4,6-bromophenol was omitted from the growth medium. These results indicate that Desulfovibrio sp. strain TBP-1 is capable of growth via halorespiration. (+info)