Risk factors for Escherichia coli O157 on beef cattle ranches located near a major produce production region.

Our goal was to identify climate variables and management practices associated with the presence of E. coli O157 in rangeland cow-calf operations located in a major leafy green production region in the California Central Coast. E. coli O157 was present in 2·6% (68/2654) of faecal, 1·5% (3/204) of water and 1·1% (1/93) of sediment samples collected on eight ranches over 2.5 years. Five (62·5%) ranches were positive at least once during the study. The odds of detecting E. coli O157 in faecal samples was higher during periods of higher maximum soil temperature, higher maximum relative humidity, and larger herd sizes, but decreased as wind speed increased. Molecular subtyping of isolates from cattle faeces and streams/sediments suggested minimal movement of strains between ranches. The findings suggest that E. coli O157 prevalence is relatively low on cow-calf ranches in this region, spatially constrained, but may vary by weather conditions and herd size.

Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens.

Turnip crinkle virus (TCV) inoculation onto TCV-resistant Arabidopsis leads to a hypersensitive response (HR) controlled by the dominant gene HRT. HRT is a member of the class of resistance (R) genes that contain a leucine zipper, a nucleotide binding site, and leucine-rich repeats. The chromosomal position of HRT and its homology to resistance gene RPP8 and two RPP8 homologs indicate that unequal crossing over and gene conversion may have contributed to HRT evolution. RPP8 confers resistance to an oomycete pathogen, Peronospora parasitica. Despite very strong similarities within the HRT/RPP8 family, HRT and RPP8 are specific for the respective pathogens they detect. Hence, the HRT/RPP8 family provides molecular evidence that sequence changes between closely related members of multigene families can generate novel specificities for radically different pathogens. Transgenic plants expressing HRT developed an HR but generally remained susceptible to TCV because of a second gene, RRT, that regulates resistance to TCV. However, several transgenic plants that overexpressed HRT produced micro-HRs or no HR when inoculated with TCV and were resistant to infection. Expression of the TCV coat protein gene in seedlings containing HRT resulted in massive necrosis and death, indicating that the avirulence factor detected by the HRT-encoded protein is the TCV coat protein.

Vacuolar H(+)-ATPase is expressed in response to gibberellin during tomato seed germination.

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA(4+7). Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V(0) membrane sector of vacuolar H(+)-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V(1) sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.

Insertional inactivation of the tomato polygalacturonase gene.

The site-selected insertion (SSI) procedure was used to generate insertional knockout mutations in the gene for tomato polygalacturonase (PG), a critical enzyme in fruit ripening. Previously, it had been shown that the Dissociation (Ds) elements in a select group of tomato plants frequently inserted into PG, at least in somatic tissues. DNA isolated from pollen produced by progeny of these plants was screened by SSI to identify plants likely to transmit the insertions in PG to progeny. These results identified one family as likely candidate for yielding germinally transmitted insertions. Four thousand progeny were screened and five were found containing germinally transmitted Ds insertions in PG, one of which contained two Ds insertions in PG. The Ds elements were stabilized by genetically removing the transposase and four of the five insertions were recovered as homozygous in the next generation. Enzymatic analysis of fruit from these individuals demonstrated that there was at least a 1000-fold reduction in polygalacturonase levels in those plants bearing Ds insertions in PG exons. Individuals with modified PG sequences due to the sequence footprint, resulting from excision of the element, were identified using the single-strand conformational polymorphism (SSCP) method. Enzymatic analysis of fruit from a plant homozygous for one such excision allele showed a significant reduction in polygalacturonase activity. Since there is no transgenic material left in PG, this demonstrates the ability to modify a gene of commercial value in planta and subsequently removing all transgenic material.

Site-selected insertional mutagenesis of tomato with maize Ac and Ds elements.

Site-selected insertion (SSI) is a PCR-based technique which uses primers located within the transposon and a target gene for detection of transposon insertions into cloned genes. We screened tomato plants bearing single or multiple copies of maize Ac or Ds transposable elements for somatic insertions at one close-range target and two long-range targets. Eight close-range Ds insertions near the right border of the T-DNA were recovered. Sequence analysis showed a precise junction between the transposon and the target for all insertions. Two insertions in separate plants occurred at the same site, but others appeared dispersed in the region of the right T-DNA border with no target specificity. However, insertions showed a preference for one orientation of the transposon. Use of plants with multiple Ac (HiAc) or Ds (HiDs) elements allowed detection of somatic insertions at two single-copy genes, PG (polygalacturonase) and DFR (dihydroflavonol 4-reductase). Certain HiDs plants showed much higher rates of insertion into PG than others. Insertions in PG and DFR were found throughout the gene regions monitored and, with the exception of one insertion in PG, the junctions between transposon and target were exact. SSI analysis of progeny from the HiDs parents revealed that in some cases the tendency to incur high levels of somatic insertions in PG was inherited. Inheritance of this character is an indication that SSI could be used to direct a search for germinal PG insertions in tomato.

Legislative Cardiovascular Health Check: a multidisciplinary health promotion experience for South Carolina lawmakers.

The South Carolina Department of Health and Environmental Control has conducted a Legislative Cardiovascular Health Check for the past 4 years. The primary purpose of the event, held in the lobby of the State Capitol Building, is to increase the awareness of State legislators about the leading causes of death in South Carolina and about community-based health promotion services that are available. The health check emphasizes the relationship between modifiable risk factors and the development of heart disease, cancer, and stroke. These legislative events are organized by State health department staff members, but they are conducted by local health department personnel from throughout South Carolina. This approach is intended to build the capacity of these local staff members to communicate more effectively with their legislators and to carry out similar events at the county or community level. The health check is staffed by a trained multidisciplinary team, including persons designated as legislative liaisons. The liaison people contact legislators prior to the event and provide them with health status data specific to their respective districts. The Legislative Cardiovascular Health Checks have been attended by members of the General Assembly and their staffs, members of the Governor's staff and the Lieutenant Governor's office, and other employees of the State Capitol. An average of 380 people have participated annually. Screening activities have included blood pressure and blood cholesterol checks, with risk factor counseling and educational materials provided to each screened participant. During the past year, activities were expanded to include a variety of interactive exhibits related to nutrition, exercise, and smoking. Feedback from participants has been positive. The Legislative Cardiovascular Health Check is encouraged and supported by the upper management of the State health department and is now established as an annual event of mutual benefit to legislators and to State and local health department staff members.

Analysis of the Ros repressor of Agrobacterium virC and virD operons: molecular intercommunication between plasmid and chromosomal genes.

The virulence genes of the Agrobacterium tumefaciens Ti plasmid are regulated both positively and negatively. The products of the genes of the virC and virD operons play an important role in host specificity and T-DNA processing. These operons are transcribed in opposite directions and therefore bear diametrically oriented promoters. These promoters are positively regulated by the VirG protein, which is believed to be activated through phosphorylation by a histidine kinase encoded by the virA gene. The virC and virD operons are also regulated by a 15.5-kDa repressor protein encoded by the ros chromosomal gene. A mutation in ros causes the constitutive expression of virC and virD in the complete absence of the VirG protein. It appears, therefore, that the Ros repressor interacts with the regulatory region of these operons. The Ros repressor is shown here to bind to an upstream sequence (Ros box) comprising 40 bp bearing a 9-bp inverted repeat, TATATTTCA/TGTAATATA, in the promoter region of these operons. The affinity for this sequence is specific and tenacious, since the addition of at least a 20,000-fold excess of competitor DNA failed to remove the Ros protein coding sequence from the Ros box. DNase I footprint analysis showed that the Ros box overlaps the binding site of VirG (Vir box). This result suggests that virC and virD transcription is modulated by Ros and VirG proteins.

Mapping of the ros virulence regulatory gene of A. tumefaciens.

Virulence functions associated with the oncogenicity of Agrobacterium tumefaciens are encoded by vir genes contained in six major operons located on the Ti plasmid. The virC and virD operons encode functions responsible for host range and T-intermediate processing. These two operons are regulated positively by the product of virG and negatively by the product of the chromosomal gene ros, which encodes a 15.5 kDa repressor. To determine the location of the ros gene we have constructed A. tumefaciens HFR strains, using transposon Tn5mob to mobilize the ros locus, and used them to map the location of ros relative to auxotrophic loci. Tight linkage was found between ros, his-34 and his-19. A linkage map is presented showing the location of ros relative to other known chromosomal genes associated with virulence functions.

The virC and virD operons of the Agrobacterium Ti plasmid are regulated by the ros chromosomal gene: analysis of the cloned ros gene.

The ros chromosomal gene is present in octopine and nopaline strains of Agrobacterium tumefaciens as well as in Rhizobium meliloti. This gene encodes a 15.5-kDa protein that specifically represses the virC and virD operons in the virulence region of the Ti plasmid. The ros gene was cloned from a genomic bank by electroporation and complementation in Agrobacterium cells. Reporter fusion to the ros gene indicates that the level of transcription is controlled in part by autoregulation. A consensus inverted repeat sequence present in the ros promoter and in the virC and virD promoters of pTiC58, pTiA6, and pRiA4b suggests that a specific Ros binding site exists in these promoters. In the virC and virD promoter region, this binding site is within a cluster of vir box consensus sequences in which the VirG protein binds. This suggests possible binding competition between Ros and VirG at the virC and virD promoters. That the Ros protein binds DNA is suggested by the presence of a 'zinc finger' consensus sequence in the protein.

Two chromosomal loci involved in production of exopolysaccharide in Agrobacterium tumefaciens.

The chromosomal locus pscA (exoC) of Agrobacterium tumefaciens LBA4301 has been cloned by complementation of the avirulent and exopolysaccharide (EPS)-deficient mutant LBA4301 pscA. We have also identified a new locus, termed psdA (polysaccharide depression) and located 16 kilobases from pscA in the A. tumefaciens chromosome, that negatively affects EPS production when it is present in more than one copy in A. tumefaciens LBA4301. Subcloning, transposon mutagenesis, and transcriptional analysis have been conducted for both loci and indicate that pscA and psdA are transcribed in the same orientation. Acidic-EPS assays showed that psdA depresses succinoglycan production and that its negative effect increases with the copy number of the gene. Virulence tests of psdA transconjugants on Datura stramonium showed no visible alteration in virulence, while LBA4301 pscA was totally avirulent.