The genes of the bacterial sialidases from Clostridium sordellii G12, C. perfringens A99, Salmonella typhimurium LT-2 and Vibrio cholerae 395 sequenced so far were examined for homologies and were compared with sequences of viral sialidases. Each of the bacterial sialidases contains a short sequence of twelve amino-acids, which is repeated at four positions in the protein. All these sequences exhibit significant similarities. Comparing the repeated sequences of the four sialidases, five amino-acids were found to be highly conserved at defined positions: Ser-X-Asp-X-Gly-X-Thr-Trp. Additionally, most of the distances between the four repeated regions are also conserved among the different sialidases. The conserved bacterial sequences show similarity with sialidases of influenza A H7N1 and H13N9.
Escherichia coli was transformed with pUC vectors containing Sau3A restriction fragments (RF) of Clostridium perfringens DNA. Two clones expressed sialidase activity when assayed with the fluorogenic substrate 4‐methylumbelliferyl‐α‐D‐N‐acetylneuraminic acid. A synthetic oligonucleotide representing the N‐terminus of the expressed enzyme hybridized with the clostridial insert and with a corresponding 2.1 kb Sau3A RF of the C. perfringens genome. The insert reduced to 1.4 kb, which still encoded active sialidase, has been sequenced. The structural gene encodes 382 amino acids representing an M r of 42 770. A hydrophobic leader sequence is absent. Upstream from the initiation codon ATG, a GA‐rich region is found and considered as the Shine‐Dalgarno sequence. Homology with the N‐terminus of the Vibrio cholerae sialidase gene and with viral sialidase sequences was not found.
The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K. Due to the environmental significance of its chlorinated derivatives, the metabolism of dibenzofuran has been studied intensively in the last few years (10, 14, 16-18, 48, 58, 59, 65). Accordingly, bacterial mineralization of dibenzofuran is initiated by dibenzofuran 4,4a-dioxygenase, catalyzing the angular dioxygenation of dibenzofuran. This gives rise to the chemically labile hemiacetal 4,4a-dihydro-4,4a-dihydroxydibenzofuran, which, after spontaneous rearomatization, yields 2,2Ј, 3-trihydroxybiphenyl (16, 59). A multicomponent dioxygenase of this angular dioxygenase type was purified from Sphingomonas sp. strain RW1 (9) as a four-component class IIA dioxygenase system. Ring cleavage of 2,2Ј,3-trihydroxybiphenyl was shown to be catalyzed by extradiol dioxygenases yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid (16, 59), which is subsequently hydrolyzed to 2-oxopent-4-enoate and salicylate (8,16,59). From Sphingomonas sp. strain RW1, a 2,2Ј,3-trihydroxybiphenyl-cleaving extradiol dioxygenase which was suggested to be involved in dibenzofuran mineralization was cloned and characterized (24). In this paper, three distinct extradiol dioxygenases from Terrabacter sp. strain DPO360 are shown to be involved in total mineralization of dibenzofuran and characterized according to their function in the degradative pathway.- MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. Terrabacter sp. strain DPO360 was isolated from tar-contaminated soil in Germany (57), and Terrabacter sp. strain DPO1361 was isolated from Rhine water (58). Both grampositive isolates were catalase positive and cytochrome oxidase negative. Biochemical characterization of these strains using the API-CH50 and API-20NE test kits (Biomerieux) gave identical results, apart from positive reactions for esculin and 5-ketogluconate with Terrabacter sp. strain DPO1361, reactions which were negative for Terrabacter sp. strain DPO360. Both strains were cultivated as described earlier (58) with dibenzofuran as a source of carbon. Yeast extract (0.005% [wt/vol]) was added to the minimal medium to supply vitamins. L broth (42) was used as a complex medium for Terrabacter sp. and Escherichia coli strains.[47] was used as a recipient for library construction and screening, and E. coli JM109 [recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ⌬(lac-proAB) FЈ(traD36 proAB ϩ lacI q lacZ⌬M15) [69] was used in all other cloning experiments. The cloning vectors pUC20 and pUC21 (identical with pUC20, except for an inverted polylinker) were obtained from Boehringer Mannheim Biochemicals. Plasmids constructed in the present study are shown in Fig. 3.Chemicals. Chemicals were of the highest purity commercially available (Merck, Darmstadt, Germany; Sigma-Aldrich, Steinheim, Germany). 3-Phenylcatechol was obtained from Wako Chemicals (Neuss, Germany). 3-C...
An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110,000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.