Spontaneous mutations in the flhD operon generate motility heterogeneity in Escherichia coli biofilm

Sequence analysis and PCR primers. (DOCX 75 kb

Pleiotropic phenotypes of a Yersinia enterocolitica flhD mutant include reduced lethality in a chicken embryo model

<p>Abstract</p> <p>Background</p> <p>The <it>Yersinia enterocolitica </it>flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. This study investigates the phenotypes of <it>flhD </it>and <it>fliA </it>mutants that result from the complex gene regulation.</p> <p>Results</p> <p>Phenotypes relating to central metabolism were investigated with Phenotype MicroArrays. Compared to the wild-type strain, isogenic <it>flhD </it>and <it>fliA </it>mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source. Both mutants grew more poorly on pyrimidines and L-histidine as sole nitrogen source. Several intermediates of the tricarboxylic acid and the urea cycle, as well as several dipeptides, provided differential growth conditions for the two mutants. Gene expression was determined for selected genes and correlated with the observed phenotypes. Phenotypes relating to virulence were determined with the chicken embryo lethality assay. The assay that was previously established for <it>Escherichia coli </it>strains was modified for <it>Y. enterocolitica</it>. The <it>flhD </it>mutant caused reduced chicken embryo lethality when compared to wild-type bacteria. In contrast, the <it>fliA </it>mutant caused wild-type lethality. This indicates that the virulence phenotype of the <it>flhD </it>mutant might be due to genes that are regulated by FlhD/FlhC but not FliA, such as those that encode the flagellar type III secretion system.</p> <p>Conclusion</p> <p>Phenotypes of <it>flhD </it>and <it>fliA </it>mutants are related to central metabolism and virulence and correlate with gene regulation.</p

Evidence that acetyl phosphate functions as a global signal during biofilm development

We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüß and Wolfe, 1994, Mol Microbiol 12: 973-984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild-type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co-ordinate the expression of surface structures and cellular processes involved in the initial stages of wild-type biofilm development

Relating gene expression data on two-component systems to functional annotations in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Obtaining physiological insights from microarray experiments requires computational techniques that relate gene expression data to functional information. Traditionally, this has been done in two consecutive steps. The first step identifies important genes through clustering or statistical techniques, while the second step assigns biological functions to the identified groups. Recently, techniques have been developed that identify such relationships in a single step.</p> <p>Results</p> <p>We have developed an algorithm that relates patterns of gene expression in a set of microarray experiments to functional groups in one step. Our only assumption is that patterns co-occur frequently. The effectiveness of the algorithm is demonstrated as part of a study of regulation by two-component systems in <it>Escherichia coli</it>. The significance of the relationships between expression data and functional annotations is evaluated based on density histograms that are constructed using product similarity among expression vectors. We present a biological analysis of three of the resulting functional groups of proteins, develop hypotheses for further biological studies, and test one of these hypotheses experimentally. A comparison with other algorithms and a different data set is presented.</p> <p>Conclusion</p> <p>Our new algorithm is able to find interesting and biologically meaningful relationships, not found by other algorithms, in previously analyzed data sets. Scaling of the algorithm to large data sets can be achieved based on a theoretical model.</p

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

A Wash of Ethyl Acetoacetate Reduces Externally Added Salmonella enterica on Tomatoes

The continuously high numbers of food-borne disease outbreaks document that current intervention techniques are not yet satisfactory. This study describes a novel wash for tomatoes that can be used as part of the food processing chain and is designed to prevent contamination with serovars of Salmonella enterica. The wash contains ethyl acetoacetate (EAA) at a concentration of 8% in H2O. This wash reduced live bacterial counts (on Salmonella Shigella agar) of externally added S. Newport MDD14 by 2.3 log, counts of S. Typhimurium ATCC19585 by 1.5 log, and counts of S. Typhimurium FSL R6-0020 by 3.4 log. The naturally occurring background flora of the tomatoes was determined on plate count agar. The log reduction by EAA was 2.1. To mimic organic matter in the wash, we added 1% tomato homogenate to the 8% EAA solution. Prior to using the wash, the tomato homogenate was incubated with the EAA for 2 h. In the presence of the tomato homogenate, the log reductions were 2.4 log for S. Newport MDD14 and 3 log for S. Typhimurium FSL R6-0020. It seems like tomato homogenate did not reduce the efficacy of the EAA wash in the two S. enterica serovars tested. We propose the use of EAA as a wash for tomatoes to reduce bacterial counts of S. enterica well as naturally occurring background flora