Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans

Abstract

We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces fividans. This was accomplished by using the vector pU7O2 and BgM-BclI libraries of M. viridifaciens chromosomal inserts created in S. fividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BgI fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M, of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-XThr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. fividans. Neuraminidase (EC 3.2.1.18) is a glycosidase which cleaves an a-ketosidic linkage between sialic acid and the adjacent sugar residue on glycoproteins, glycolipids, and oligosaccharides. It is widely distributed in microorganisms such as viruses, bacteria, and actinomycetes and in various tissues from fowls and mammals. For myxovirus it is an important surface antigen and also plays a role in facilitating the movement of the virus both to and from the site of infection (1). Bacteria and actinomycetes which produce neuraminidase can release sialic acids from glycoconjugates for use as carbon and energy sources. In pathogenic microorganisms, neuraminidase activity has a strong correlation with pathogenesis (27).Recently, neuraminidase genes were cloned from several pathogenic microorganisms, including Clostridium sordellii G12 (25), Clostridium perfringens A99 (24), Salmonella typhimurium LT-2 (23), Vibrio cholerae 395 (29), and Bacteroides fragilis TAL2480 (26). From examination of their deduced amino acid sequences, a conserved sequence of 12 amino acids (Asp box) which was repeated at four or five positions was found (23). This conserved sequence shows similarity to the neuraminidase of influenza virus A H7N1 and H13N9. The structures of neuraminidases from nonpathogenic bacteria have not been characterized. In a recent report, neuraminidase was widely found in culture fluids of actinomycetes (3). For Micromonospora spp., 10 of the 39 strains examined exhibited neuraminidase activity. Micromonospora viridifaciens showed the highest neuraminidase activity among this group. Neuraminidase from M.viridifaciens was purified, and its properties were analyzed (2). Neuraminidase activity in M. viridifaciens was detected in the culture broth only when a substrate such as colominic acid or the product N-acetylneuraminic acid was present in the culture medium (2).We would like to understand the regulation of neuraminidase gene (ned4) expression and the properties of its * Corresponding author. product in M. viridifaciens and ...

Cloning, Expression, and Characterization of a Neuraminidase Gene fromArcanobacterium pyogenes

“…The 107-kDa S. pneumoniae NanA is observed as an enzymatically active 86-kDa species following purification (32). The size of the M. viridifaciens NedA protein varies between 41 and 68 kDa depending on the culture conditions (45). Indeed, upon prolonged storage at 4°C, His-NanH converted to a species of 62 kDa, with no loss of specific activity (data not shown).…”

Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme

“…2). The sequence (5Ј-TTGCCA-18 bp-TAACGT-3Ј) resembles the E. coli consensus promoter sequence (5Ј-TTGACA-17 bp-TATAAT-3Ј) (20). A long inverted repeat sequence, which may act as a transcription terminator, was observed downstream of the termination codon of the aphA gene (Fig.…”

Characterization of a novel Vibrio pathogenicity island (VPI-2) encoding neuraminidase (nanH) among toxigenic Vibrio cholerae isolates

“…Similarly, in Clostridium perfringens, the nanH gene is located near a phage attachment site on the chromosome and has a GjC content of 31n9 mol % compared to the chromosomal DNA GjC content of 24-27 mol % (Canard & Cole, 1989). In M. viridifaciens it has been observed that a region flanking the nanH gene shows significant homology to a transposase (Sakurada et al, 1992).…”