Aims: Particulate methane monooxygenase (pMMO) is an integral membrane protein that converts methane to methanol as the first step in the metabolic pathway of methanotroph bacteria. Methanotroph have a slow growth rate that make researcher have to develop an alternative approach by expressing the pMMO genes in Escherichia coli. However, it was very difficult to express all the pMMO encoded genes in E. coli and it is suspected that the protein might be toxic to E. coli. Therefore, this research tried another approach by expressing the active site of pMMO enzyme; cupredoxin domain of pmoB subunit encoded by spmoB gene. Methodology and results: The spmoB gene from Methylococcus capsulatus (Bath) was expressed in E. coli BL21 (DE3) under T7 promoter and pET15b as the expression vector. Several modifications were made so this gene would be expressed in the cytoplasm. Expression analysis with SDS-PAGE showed that overexpression of this gene could be done at several concentrations of IPTG and incubation temperature. The spmoB gene expression produced a recombinant protein with a size approximately 38.9 kDa. Assay of spmoB protein activity showed that the amount of methanol accumulated during methane oxidation by the recombinant strain was 0.114 mmol/mL culture.h. Conclusion, significance and impact study: We successfully expressed spmoB gene in E. coli BL21 (DE3) without high production of toxic compounds and it has methane oxidation activity. This result allowed further characterization of its potential applications.
Escherichia coli

OBJECTIVE: The study aimed to determine the physico-chemical and microbiological quality of ice blocks sold in selected wet markets located in the city of Manila.

METHODS: Twenty-eight samples were collected from three markets. Microbiological quality was analyzed in terms of the presence or absence of coliforms with E. coli as indicator organism. Physico-chemical quality was determined by measuring turbidity, apparent color, pH, iron, and residual chlorine. Measurements obtained were compared with the 2007 Philippine National Standards for Drinking Water (PNSDW).

RESULTS: Results showed that all samples tested positive for coliforms while 25 out of the 28 samples were positive for E. coli. Mean turbidity was 2.74 ± 3.68 NTU; for both apparent color and iron tests, all samples complied with the PNSDW standard limit set; mean pH was 6.15 ± 0.64; and mean residual chlorine was 0.06 ± 0.02 mg/L. Average values of apparent color and iron comply with the PNSDW standards. Six out of 28 samples had turbidity values exceeding the standards. All samples were found to have residual chlorine levels below the standards.

CONCLUSION: Ice in markets do not comply with key 2007 PNSDW standards and findings warrant strict compliance of ice quality from manufacturers to the point of distribution to protect consumer health.

No abstract available.
Escherichia coli* ; Escherichia*

No abstract available.
Escherichia coli* ; Escherichia* ; Humans*

No abstract available.
Escherichia coli* ; Escherichia*

No abstract available.
Escherichia coli* ; Escherichia*

No abstract available.
Escherichia coli ; Escherichia

Background: aaP, aggR, and astA have been found to play important roles in diarrheal pathogenecity of EAEC. They may be exist in other diarrheagenic E.coli (DEC). Objectives: To determine the distribution of aaP, aggR, and astA in ETEC, EPEC, EIEC and non-diarrheagenic E.coli. Subjects and method: 75 strains of ETEC, EPEC, EIEC and 100 non-DEC have been screen by PCR with primers specific toaaP, aggR, and astA. Results: aaP, aggR, and astA have been seen in DEC with the prevence from 7 to 72,7%. The highest prevence was in EIEC, 72,7% for aap; 45,5% in EIEC for aggR; and 50% in ETEC for astA. 14% of non-DEC harbored aggR and more than 30% harbored aap and astA. Conclusion: This finding has contributed to understanding the distribution of aap, aggR and astA in ETEC, EPEC, EIEC and non-DEC as well.
Enterotoxigenic Escherichia coli ; Enteropathogenic Escherichia coli ; Escherichia coli ;

OBJECTIVE: To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7. METHODS: The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains. RESULTS: At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion. CONCLUSIONS We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.
Escherichia coli O157 ; Escherichia coli Proteins ; Polyphosphates

No abstract available.
Animals* ; Escherichia coli* ; Escherichia* ; Humans*