Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

The physical map of {\it Escherichia coli\/} K-12 strain MG1655: Construction, comparison with other strains and implications for mechanisms controlling oligonucleotide frequency

Complete NotI, SfiI, XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 were constructed. Techniques used included: CHEF pulsed field gel electrophoresis; transposon mutagenesis; fragment hybridization to the ordered $\lambda$ library of Kohara et al.; fragment and cosmid hybridization to Southern blots; correlation of fragments and cleavage sites with EcoMap, a sequence-modified version of the genomic restriction map of Kohara et al.; and correlation of cleavage sites with DNA sequence databases. In all, 105 restriction sites were mapped and correlated with the EcoMap coordinate system.^ NotI, SfiI, XbaI and BlnI restriction patterns of five commonly used E. coli K-12 strains were compared to those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the difference between the strains on the basis of simple insertions, deletions, and in one case an inversion. Insertions and deletions ranged in size from 1 kb to 86 kb. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains.^ Some aspects of the frequency and distribution of NotI, SfiI, XbaI and BlnI cleavage sites were analyzed using a method based on Markov chain theory. Overlaps of Dam and Dcm methylase sites with XbaI and SfiI cleavage sites were examined. The one XbaI-Dam overlap in the database is in accord with the expected frequency of this overlap. The occurrence of certain types of SfiI-Dcm overlaps are overrepresented. Of the four subtypes of SfiI-Dcm overlap, only one has a partial inhibitory effect on the activity of SfiI. Recognition sites for all four enzymes are rarer than expected based on oligonucleotide frequency data, with this effect being much stronger for XbaI and BlnI than for NotI and SfiI. The latter two enzyme sites are rare mainly due to apparent negative selection against GGCC (both) and CGGCCG (NotI). The former two enzyme sites are rare mainly due to effects of the VSP repair system on certain di-tri- and tetranucleotides, most notably CTAG. Models are proposed to explain several of the anomalies of oligonucleotide distribution in E. coli, and the biological significance of the systems that produce these anomalies is discussed. ^

The construction of aging and health in older African American women

Persistence of racial and ethnic health disparities and governmental policies based on outdated ideas of aging call for inclusive approaches to the study of elder African Americans. The lived experiences of aging among urban, poor African American women, who comprise a vulnerable population, are not well known, as most studies focus on mainstream populations. Gerontological studies have tended to employ methods that collapse contextual information for ease of analysis, thus failing to capture nuanced information critically relevant to health of marginalized groups. Few researchers have been successful highlighting the importance of local knowledge, resilience, and resources for health by using participatory methods with older Black women. This study utilizes participatory principles to gather discursive data from nine older African American women, engaged in three generational cohorts: those born around World War II, women born after the great depression, and those born before the great depression. Videotaped and transcribed conversations of cohorts were analyzed in search of contextual factors that influence their experience of aging and health. As women responded to general themes that provoked their talk about their lives, they helped answer the study's questions: How do older African American women make sense of their aging experience? What are some of the important social and cultural influences that shape the construction of aging and health by these women? Are generational discourse groups an effective tool for exploring changes in the experiences of aging? A key finding demonstrated rich heterogeneity of experiences with strong generational influences on the construction of aging and health. The participants' moral orders comprised of traditional values of family, reinforced by personal experiences and the church, have guided their lives through oppression and stress but appear to be failing younger women who have greater exposure to new environmental pressures. Limited time and the size of the study were weaknesses although the women's interest in the study and their participation were gratifying. The participants served to highlight the importance of recognizing generational and other contextual factors in formation of ideas of aging and likelihood of additional challenges to the experience of old age among older, poorer, African Americans. ^