Identification of three proteins up-regulated by raw starch in Cytophaga sp

Raw starch-digesting amylases (RSDAs) in many microorganisms convert starch granules into maltodextrins and simple sugars. We cloned and sequenced from Cytophaga sp. an RSDA with an excellent raw starch digestion activity. This RSDA was highly inducible by raw starch, but not by other sugars, suggesting that an unknown signal transduction mechanism is involved in the degradation of raw starch. We used a proteomic approach to investigate the effect of raw starch on protein expression in Cytophaga sp. Using MALDI-TOF MS protein analysis, we have identified three proteins up-regulated by raw starch, i.e., a 60-kDa chaperonin (cpn60), glutaminase, and pyruvate phosphate dikinase (PPDK). Subsequent time-course studies detected an increased expression of RSDA as well as the highest expression of PPDK occurring 6 h post-incubation with raw corn starch, implying that the latter enzyme may work along with RSDA on the digestion of raw starch. Finding these proteins up-regulated by raw starch may provide an insight into how Cytophaga sp. cells respond to raw starch stimulation

Characterization, identification, and cloning of the S-layer protein from Cytophaga sp

We characterized, identified, and cloned a major protein which comprised 16% of the total proteins from Cytophaga sp. cell lysate. After French pressing, the fraction of cell envelope was treated with 0.2% Triton X-100 to remove cell membranes. Subsequent SDS-PAGE analysis of the Triton X-100-insoluble cell wall revealed a protein of 120 kDa with a pI of 5.4, which was identified by gold immunostaining as the surface (S)-layer protein of this soil bacterium. The nucleotide sequence of the cloned S-layer protein gene (slp) encoding this protein consisted of 3144 nucleotides with an ORF for 1047 amino acids, which included a typical 32-amino acid leader peptide sequence. Amino acid sequence alignment revealed 29-48% similarity between this protein and the S-layer proteins from other prokaryotic organisms. The 120-kDa protein from the Cytophaga sp. cell lysate has been characterized as a member of the S-layer proteins, and the slp gene was cloned and expressed in Escherichia coli. E. coli harboring the plasmid containing the 600- or 800-bp DNA fragment upstream of the initiation codon of the slp gene, in the presence of the reporter gene rsda (raw starch digesting amylase), showed amylase activity in starch containing plate. The putative promoter region of slp located 600 bp upstream of the initiation codon might be used for foreign gene expression

Characterization and virulence of hemolysin III from Vibrio vulnificus

Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the word. A gene (hlyIII) encoding a hemolysin was cloned and sequenced from V. vulnificus. Nucleotide sequence analysis predicted an open reading frame of 642 bp encoding a 214 amino acid polypeptide that showed 48% sequence identity to the hemolysin III of Bacillus cereus. When HlyIII of V. vulnificus was expressed in Escherichia coli, crude extracts exhibited hemolytic activity similar to that of hemolysin III from Bacillus cereus. A hlyIII isogenic mutant was constructed via insertional inactivation and showed an attenuated virulence compared with the wild-type strain when this mutant was administered intraperitoneally in mice

cDNA cloning, expression, and characterization of taro SSII: A novel member of starch synthase II family

A novel soluble starch synthase 11 (SSII) gene was isolated from taro (Colocasia esculenta var. esculenta) tubers. This 2939 bp SSII transcript encodes 804 amino acids with a putative transit peptide of 52 residues. It displays 58-63% identity and 63-69% similarity with SSIIs from other sources. Alignment and phylogenetic analyses showed that taro SSII is more closely related with dicot SSIIs than with the monocot ones, though taro is a monocot. The identification of taro SSII clone as starch synthase was confirmed by the expression of its enzymatic activity in Escherichia coli. Genomic DNA blot analysis revealed a single copy or low number copies of SSII in taro. Expression profile showed that more transcript and protein were accumulated in tubers of 597 37 g fresh weight, that is, a stage of rapid starch synthesis, than tubers of other stages. By Western blot analysis, SSII was found in both soluble and granule bound portions of tuber extracts, and more SSII protein was found in aged leaves than in leaves of other stages. These results suggest that taro SSII is a novel starch synthase for the synthesis of both transit and storage starch

Cloning, expression, and characterization of soluble starch synthase I cDNA from taro (Colocasia esculenta var. esculenta)

Soluble starch synthase I (SSSI) cDNA was isolated from taro (Colocasia esculenta var. esculenta) by RT-PCR and rapid amplification of cDNA ends reaction. The transcript of this single-copy gene is 2340bp and encodes 642 amino acids protein containing a putative transit peptide of 54 residues. Recombinant SSSI protein displayed both primer-dependent and primer-independent activities of starch synthase. More SSSI transcript was expressed in taro leaves than in tubers, with no evident expression in petioles; and more transcript and protein were found in tubers of 597 37 g of fresh weight than in smaller or larger ones. Two forms of SSSI, i.e., 72 and 66 kDa, exist in leaves, and only the 66 kDa form was found in tubers. The taro SSSI, proposed as a novel member, was located only in the soluble fraction of tuber extract, while SSSI from other sources exist in both soluble and granule-bound forms

Characterization of cyclodextrin glycosyltransferase of the same gene expressed from Bacillus macerans, Bacillus subtilis, and Escherichia coli

The plasmid pHG contains a cyclodextrin glycosyltransferase (CGTase) gene (cgt) derived from Bacillus macerans. Two transformants, Bacillus subtilis (pHG) and Escherichia coli (pHG), were found to produce CGTases with the same primary structure as the enzyme from B. macerans. However, the beta-cyclodextrin coupling activity of the CGTase from E coli (pHG) was 14-fold higher than that of the enzymes from the other strains. By contrast, no differences in a-cyclodextrin coupling activities were observed among these CGTases. CGTase from E coli (pHG) was found to be less thermostable than the other CGTases. When the CGTase produced by B. subtilis was treated with increasing urea concentrations (10-1000 mM) to promote increasing degrees of protein unfolding, a bell-shaped beta-cyclodextrin coupling activity profile was obtained. Subtle differences in the conformation of the CGTase produced by E coli are therefore proposed to be responsible for the markedly increased beta-cyclodextrin coupling activity of this enzyme

Cloning and characterization of a cDNA encoding the endo-1,4-beta-D-glucanase gene expressed in rapidly growing tuber and leaf of taro (Colocasia esculenta var. esculenta)

A cDNA for endo-beta-1,4-glucanase (EGase) was isolated by RT-PCR, and rapid amplification of cDNA ends reaction from taro leaves (Colocasia esculenta var. esculenta). This single copy gene has 2,185 bps and an open reading frame (ORF) of 1,854 bps. A polypeptide of 618 residues was deduced from the ORF, with a calculated molecular mass of 68,434 Da and theoretical pI of 8.8. The remarked activities of the EGase on CMC plate revealed this enzyme to be involved in cellulose metabolism in taro. Taro EGase was identified as a kind of membrane-anchored EGase. Phylogenetic analysis showed it is a member of the gamma subfamily and an ortholog of the Arabidopsis KORRIGAN gene (KOR), proposed to be involved in cytokinesis, pectin metabolism in the primary cell wall, and cell elongation. Transcripts of taro EGase are highly accumulated in 300 g tubers, in the upper part of 1,000 g tubers, and in rolled leaves undergoing rapid growth, indicating that taro EGase is involved in the regulation of plant growth. The expression pattern of soluble starch synthase III was similar to that of EGase during rapid growth of taro plant tubers. These results suggested that taro EGase, an ortholog of KOR in Arabidopsis, may play an important role in the rapid growth in taro

Accumulation of the inner nuclear envelope protein Sun1 is pathogenic in progeric and dystrophic laminopathies

10.1016/j.cell.2012.01.059Cell1493565-577CELL