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.Vibrio vulnificus is a halophilic gram-negative bacterium that has emerged as an increasingly important pathogen capable of causing both serious wound infections and fatal septicemia in humans (3,7,27,36). Primary septicemia, with a mortality rate exceeding 50%, may be acquired by consuming seafood containing this organism. Infections are associated with the exposure of wounds to seawater, with a mortality rate of about 25% (4,7,18). Although the pathogenic mechanism of V. vulnificus infection has not been fully delineated, several potential virulence factors, such as capsule polysaccharide (CPS) (37, 42, 43), iron-sequestering systems (20), and type IV leader peptidase-N-methyltransferase (28), have been described. In addition, a correlation between the presence of two exotoxins, hemolysin and metalloprotease, and the virulence of V. vulnificus strains has been reported (13,19), although these two exotoxins were not confirmed as virulence factors by genetic analysis (35,41).The bactericidal effect of serum is an important defense by the host against invading microorganisms. In response to this host defense, V. vulnificus, like many pathogenic bacteria, may evolve strategies, including encapsulating itself with CPS, to counter the bactericidal effect of serum. In addition, clinical isolates of V. vulnificus have been reported to exhibit a strong tropism for blood vessels and often spread intravascularly (4). To identify bacterial factors of V. vulnificus that are required for serum resistance, we have undertaken the isolation and characterization of mutants of V. vulnificus with defects in resistance to serum. This paper reports the isolation of these serum-susceptible mutants (designated SS mutants) and the characterization of one of them by genetic analysis. MATERIALS AND METHODSBacterial strains...

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.

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.

A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65°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.Raw starch is the most abundant source of glucose in the world. Industrially, starch granules are gelatinized in water to improve the conversion of starch molecules into maltodextrins and simple sugars by ␣-amylase and/or other amylolytic enzymes (13). This gelatinization of starch granules requires heating and a long reaction time. Therefore, finding enzymes capable of digesting raw starch directly would provide a possible replacement for such an expensive and energy-consuming process. Many organisms, including fungi, yeast, and bacteria, are known to produce raw-starch-digesting amylases (RSDAs) (1,2,3,22). Some of these RSDAs have been produced and applied to the beverage, food, paper, and textile industries as well as to dishwashing and laundry detergent production (11). However, the relatively low reaction temperature optimal for RSDA on granular starch results in not only a low reaction rate but also a high risk of bacterial contamination. Therefore, increasing the thermostability of RSDAs for industrial utilization is an important goal of protein engineering.Studies have shown that a loop area within domain B of ␣-amylase plays a crucial role in improving the thermostability of this protein (17). Deletion of an Arg-Gly dipeptide from both the liquefying ␣-amylase (LAMY) of Bacillus strain KSM-1378 and Bacillus amyloliquefaciens amylase (BAA) increased their thermostability (5, 18). In previous reports, an RSDA from a Cytophaga sp. has been shown to exhibit an excellent starch digestion activity on raw cornstarch (7,8). By comparing the amino acid sequences of this RSDA and other amylases (5,12,19,23), a loop structure within domain B in Cytophaga sp. RSDA similar to those in other amylases was also found. In this study, we demonstrated that removing Arg and Gly residues from the corresponding loop area in this RSDA improved the stability of the enzyme both at high temperatures and in stringent environments. To the best of our knowledge, this is the first thermostable RSDA that has been reported to date.The mutant RSDA, with residues 178 and 179 deleted, was generated by site-directed mutagenesis by the method of splicing by overlap extension based on PCR (4). A recombinant plasmid (unpublished data), pHR, was constructed by ligation of pHY300PLK (TaKaRa, Shing, Japan) and a DNA fragment containing the rsda gene. To generate the deletion mutant, the nucleotide sequence of the rsda gene was PCR amplified on pHR by using primer 1 (5Ј-CGGACCGTACGATTTGTACG ATCTAGG-3Ј, where the BsiWI recognition sequence is underlined) and primer 2 (5Ј-CCCATGCTTTGCCGG...

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 ␣-amylases from three Bacillus species. Only one of the Bacillus ␣-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.

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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.

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.