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1.
J Biochem ; 2009 Jun 26.
Article in English | MEDLINE | ID: mdl-19505952

ABSTRACT

The paper has been published online in Journal of Biochemistry Advance Access and had been submitted without agreement from the co-authors. They therefore retract this paper and discourage citations of it.

2.
Arch Microbiol ; 190(6): 651-5, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18704367

ABSTRACT

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.


Subject(s)
Bacterial Proteins/biosynthesis , Cytophaga/enzymology , Starch/metabolism , Up-Regulation , Amylases/genetics , Amylases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Chaperonin 60/chemistry , Chaperonin 60/genetics , Chaperonin 60/metabolism , Cytophaga/genetics , Electrophoresis, Polyacrylamide Gel , Glutaminase/chemistry , Glutaminase/genetics , Glutaminase/metabolism , Pyruvate, Orthophosphate Dikinase/chemistry , Pyruvate, Orthophosphate Dikinase/genetics , Pyruvate, Orthophosphate Dikinase/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
3.
Curr Microbiol ; 56(6): 597-602, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18322733

ABSTRACT

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.


Subject(s)
Cloning, Molecular , Cytophaga/genetics , Membrane Glycoproteins/chemistry , Cytophaga/chemistry , Cytophaga/metabolism , Cytophaga/ultrastructure , Electrophoresis, Polyacrylamide Gel , Gene Expression , Membrane Glycoproteins/genetics , Membrane Glycoproteins/isolation & purification , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Molecular Weight , Open Reading Frames , Promoter Regions, Genetic , Sequence Analysis, DNA
4.
J Agric Food Chem ; 53(20): 7958-64, 2005 Oct 05.
Article in English | MEDLINE | ID: mdl-16190656

ABSTRACT

A novel soluble starch synthase II (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.


Subject(s)
Cloning, Molecular , Colocasia/enzymology , DNA, Complementary/genetics , Gene Expression , Plant Proteins/genetics , Plant Tubers/enzymology , Starch Synthase/genetics , Amino Acid Sequence , DNA, Plant/analysis , Escherichia coli/genetics , Molecular Sequence Data , Phylogeny , Plant Leaves/enzymology , Protein Biosynthesis , Recombinant Proteins , Sequence Alignment , Transcription, Genetic , Transfection
5.
J Agric Food Chem ; 53(20): 7985-90, 2005 Oct 05.
Article in English | MEDLINE | ID: mdl-16190660

ABSTRACT

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 2340 bp 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.


Subject(s)
Cloning, Molecular , Colocasia/enzymology , DNA, Complementary/genetics , Gene Expression , Starch Synthase/genetics , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Plant Leaves/enzymology , Plant Tubers/enzymology , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Starch Synthase/chemistry , Starch Synthase/metabolism
6.
J Agric Food Chem ; 53(16): 6301-4, 2005 Aug 10.
Article in English | MEDLINE | ID: mdl-16076110

ABSTRACT

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 alpha-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.


Subject(s)
Bacillus subtilis/enzymology , Bacillus/enzymology , Escherichia coli/enzymology , Glucosyltransferases/genetics , Catalysis , Enzyme Stability , Glucosyltransferases/chemistry , Glucosyltransferases/metabolism , Hot Temperature , Plasmids/genetics , Protein Folding , Transformation, Bacterial , Urea/pharmacology , alpha-Cyclodextrins/metabolism
7.
Curr Microbiol ; 49(3): 175-9, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15386100

ABSTRACT

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.


Subject(s)
Hemolysin Proteins/genetics , Hemolysin Proteins/physiology , Vibrio vulnificus/genetics , Vibrio vulnificus/pathogenicity , Animals , Bacterial Proteins , Base Sequence , Cloning, Molecular , Conserved Sequence , DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Expression , Genes, Bacterial , Hemolysin Proteins/toxicity , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutagenesis, Insertional , Open Reading Frames , Recombinant Proteins/genetics , Vibrio vulnificus/isolation & purification , Virulence
8.
Infect Immun ; 72(2): 629-36, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14742502

ABSTRACT

Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world. To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of V. vulnificus and screened them for hypersensitivity to human serum. Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as trkA, a gene encoding an amino acid sequence showing high identity to that of TrkA of Vibrio alginolyticus, a protein required for the uptake of potassium. A trkA isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type. At K+ concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain. In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.


Subject(s)
Blood Bactericidal Activity , Carrier Proteins/physiology , Membrane Proteins/physiology , Polymyxin B/pharmacology , Protamines/pharmacology , Receptor, trkA , Vibrio vulnificus/drug effects , Animals , Base Sequence , Carrier Proteins/genetics , Chromosome Mapping , DNA Transposable Elements , Drug Resistance, Bacterial , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Potassium/pharmacology , Rabbits , Transcription, Genetic , Vibrio vulnificus/immunology , Virulence
9.
Appl Environ Microbiol ; 69(4): 2383-5, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12676725

ABSTRACT

A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65 degrees C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca(2+)-independent mechanism.


Subject(s)
Amylases/chemistry , Cytophaga/enzymology , Hot Temperature , Mutagenesis, Site-Directed , Starch/metabolism , Amylases/genetics , Amylases/metabolism , Cytophaga/genetics , Enzyme Stability , Gene Deletion , Hydrogen-Ion Concentration
10.
Appl Environ Microbiol ; 68(7): 3651-4, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12089056

ABSTRACT

A raw-starch-digesting amylase (RSDA) gene from a Cytophaga sp. was cloned and sequenced. The predicted protein product contained 519 amino acids and had high amino acid identity to alpha-amylases from three Bacillus species. Only one of the Bacillus alpha-amylases has raw-starch-digesting capability, however. The RSDA, expressed in Escherichia coli, had properties similar to those of the enzyme purified from the Cytophaga sp.


Subject(s)
Cytophaga/enzymology , alpha-Amylases/genetics , Cloning, Molecular , Cytophaga/genetics , DNA, Bacterial/analysis , Escherichia coli/genetics , Gene Expression , Molecular Sequence Data , Recombinant Proteins/biosynthesis , alpha-Amylases/immunology
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