Resistance of Gram-positive bacteria to nisin is not determined by Lipid II levels

Lipid II is essential for nisin-mediated pore formation at nano-molar concentrations. We tested whether nisin resistance could result from different Lipid II levels, by comparing the maximal Lipid II pool in Micrococcus flavus (sensitive) and Listeria monocytogenes (relatively insensitive) and their nisin-resistant variants, with a newly developed method. No correlation was observed between the maximal Lipid II pool and nisin sensitivity, as was further corroborated by using spheroplasts of nisin-resistant and wild-type strains of M. flavus, which were equally sensitive to nisin. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved

A Functional Genomic Yeast Screen to Identify Pathogenic Bacterial Proteins

Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor ∼1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture

A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data

BACKGROUND: In research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed. RESULTS: A number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance. CONCLUSION: Even in cases where 90 % of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25 %. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality

The shdA Gene Is Restricted to Serotypes of Salmonella enterica Subspecies I and Contributes to Efficient and Prolonged Fecal Shedding

Little is known about factors which enable Salmonella serotypes to circulate within populations of livestock and domestic fowl. We have identified a DNA region which is present in Salmonella serotypes commonly isolated from livestock and domestic fowl (S. enterica subspecies I) but absent from reptile-associated Salmonella serotypes (S. bongori and S. enterica subspecies II to VII). This DNA region was cloned from Salmonella serotype Typhimurium and sequence analysis revealed the presence of a 6,105-bp open reading frame, designated shdA, whose product's deduced amino acid sequence displayed homology to that of AIDA-I from diarrheagenic Escherichia coli, MisL of serotype Typhimurium, and IcsA of Shigella flexneri. The shdA gene was located adjacent to xseA at 52 min, in a 30-kb DNA region which is not present in Escherichia coli K-12. A serotype Typhimurium shdA mutant was shed with the feces in reduced numbers and for a shorter period of time compared to its isogenic parent. A possible role for the shdA gene during the expansion in host range of S. enterica subspecies I to include warm-blooded vertebrates is discussed

In vivo characterization of connective tissue remodeling using infrared photoacoustic spectra

Premature cervical remodeling is a critical precursor of spontaneous preterm birth, and the remodeling process is characterized by an increase in tissue hydration. Nevertheless, current clinical measurements of cervical remodeling are subjective and detect only late events, such as cervical effacement and dilation. Here, we present a photoacoustic endoscope that can quantify tissue hydration by measuring near-infrared cervical spectra. We quantify the water contents of tissue-mimicking hydrogel phantoms as an analog of cervical connective tissue. Applying this method to pregnant women in vivo, we observed an increase in the water content of the cervix throughout pregnancy. The application of this technique in maternal healthcare may advance our understanding of cervical remodeling and provide a sensitive method for predicting preterm birth

CYP2A6 metabolism in the development of smoking behaviors in young adults

Cytochrome P450 2A6 (CYP2A6) encodes the enzyme responsible for the majority of nicotine metabolism. Previous studies support that slow metabolizers smoke fewer cigarettes once nicotine dependent but provide conflicting results on the role of CYP2A6 in the development of dependence. By focusing on the critical period of young adulthood, this study examines the relationship of CYP2A6 variation and smoking milestones. A total of 1209 European American young adults enrolled in the Collaborative Study on the Genetics of Alcoholism were genotyped for CYP2A6 variants to calculate a previously well-validated metric that estimates nicotine metabolism. This metric was not associated with the transition from never smoking to smoking initiation nor with the transition from initiation to daily smoking (P > 0.4). But among young adults who had become daily smokers (n = 506), decreased metabolism was associated with increased risk of nicotine dependence (P = 0.03) (defined as Fagerström Test for Nicotine Dependence score ≥4). This finding was replicated in the Collaborative Genetic Study of Nicotine Dependence with 335 young adult daily smokers (P = 0.02). Secondary meta-analysis indicated that slow metabolizers had a 53 percent increased odds (OR = 1.53, 95 percent CI 1.11-2.11, P = 0.009) of developing nicotine dependence compared with normal metabolizers. Furthermore, secondary analyses examining four-level response of time to first cigarette after waking (>60, 31-60, 6-30, ≤5 minutes) demonstrated a robust effect of the metabolism metric in Collaborative Study on the Genetics of Alcoholism (P = 0.03) and Collaborative Genetic Study of Nicotine Dependence (P = 0.004), illustrating the important role of this measure of dependence. These findings highlight the complex role of CYP2A6 variation across different developmental stages of smoking behaviors