Escherichia coli O157:H7, which causes hemorrhagic colitis and hemolytic uremic syndrome, has been responsible for several outbreaks associated with consumption of unpasteurized and improperly processed pasteurized milk, and yogurt. Studies were conducted to determine the survival and growth characteristics of this pathogen in unpasteurized milk and pasteurized milk (3.5% fat, 2% fat, skim) at 5,8, 15, and 22°C for up to 28 days. Two levels of inocula (103 and 105 CFU/ml) of a mixture of five nalidixic acid-resistant E. coli O157:H7 strains were used. E. coli O157:H7 did not grow at 5°C and decreased by 1.6 to 2.0 log CFU/ml in 28 days. Growth occurred at 8°C, with an approximately 1- to 2-log CFU/ml increase within the first 4 days. About a 3- to 5-log CFU/ml increase in E. coli O157:H7 populations was observed at 15°C within the first 3 days. In 3 pasteurized milk samples, E. coli O157:H7 continued to grow to populations of greater than 1.0 × 108 CFU/ml at day 7 and remained constant during the remainder of the incubation period. At 22°C, the pH decreased rapidly to less than 4.0 within 4 days and E. coli O157:H7 decreased to undetectable populations within 14 days. E. coli O157:H7 grew more slowly (P < 0.01) in unpasteurized milk, which had a higher initial microbial population, than in pasteurized milks at 8, 15, or 22°C, likely because of antagonistic activity from preexisting bacteria. No significant differences (P> 0.05) in survival or growth of E. coli O157:H7 were observed among the pasteurized milk samples, regardless of fat concentration, at all temperatures throughout the study. The data indicate that temperature abuse during shipping and handling can result in significant growth of E. coli O157:H7. Holding milk at ≤5°C is recommended to prevent growth of this pathogen.

Bioactive gibberellins (GAs, diterpenes) are essential hormones in land plants, controlling many aspects of plant growth and developments. In flowering plants, 13-OH (low bioactivity; such as GA1) and 13-H GAs (high bioactivity; such as GA4) frequently coexist. However, the bona fide GA 13-hydroxylase and its physiological functions in Arabidopsis remain unknown. Here, we report that novel cytochrome P450 genes (CYP72A9 and its homologs) encode active GA 13-hydroxylases in Brassicaceae plants. CYP72A9-overexpressing plants exhibited semi-dwarfism, which was caused by significant reduction in GA4 levels. Biochemical assays revealed that recombinant CYP72A9 protein catalyzed the conversion from 13-H GAs to the corresponding 13-OH GAs. CYP72A9 was expressed predominantly in developing seeds in Arabidopsis. Freshly harvested seeds of cyp72a9 mutants germinated more quickly than wild-type, while long-term storage and stratification-treated seeds did not. The evolutionary origin of GA 13-oxidases from the CYP72A subfamily also was investigated and discussed here.

The Arabidopsis (Arabidopsis thaliana) CLAVATA2 (CLV2) gene encodes a leucine-rich repeat receptor-like protein (RLP) that is involved in controlling the stem cell population size in the shoot apical meristem. Our previous genome-wide functional analysis of 57 AtRLP genes revealed only a few phenotypes for mutant alleles, despite screening a wide range of growth and developmental stages and assaying sensitivity to various stress responses, including susceptibility toward pathogens. To gain further insight into the biological role of AtRLPs, in particular CLV2-related AtRLP genes, we tested their ability to complement the clv2 mutant phenotype. We found that out of four close CLV2 homologs tested, AtRLP2 and AtRLP12 could functionally complement the clv2 mutant when expressed under the control of the CLV2 promoter. This indicates that the functional specificity of these three genes is determined at the level of their transcriptional regulation. Single and double mutant combinations with impaired AtRLP2 and/or AtRLP12 did not show an aberrant phenotype, suggesting that other genes are redundant with these CLV2-like genes. To understand which protein domains are essential for CLV2 function and which parts are interchangeable between related CLV2-like proteins, we performed domain-deletion and domain-swap experiments. These experiments revealed that CLV2 remains functional without the island domain, whereas the C1 and C3 regions of the leucinerich repeat domain are essential for functionality. Analysis of domain-swap constructs showed that the C3-G region of CLV2 can be replaced by that of AtRLP38, although it could not complement the clv2 mutant under control of the CLV2 promoter. This suggests that the C3-G region is conserved among related AtRLP members, whereas the C1 domain may determine the functional specificity of CLV2.