The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract

BACKGROUND: The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model. RESULTS: Cystitis was generated in Macaque fascularis monkeys by infecting the bladder with a 1:1 mixture of the uropathogenic Escherichia coli isolate DS17 and a derivative where the uvrY gene had been disrupted with a kanamycin resistance gene. Urine was collected through bladder punctuation at subsequent time intervals and the relative amount of uvrY mutant was determined. This showed that inactivation of the UvrY response regulator leads to a reduced fitness. In similar competitions in culture flasks with Luria Broth (LB) the uvrY mutant rather had a higher fitness than the wild type. When the competitions were done in flasks with human urine the uvrY mutant initially had a lower fitness. This was followed by a fluctuation in the level of mutant in the long-term culture, with a pattern that was specific for the individual urines that were tested. Addition of LB to the different urine competition cultures however clearly led to a consistently higher fitness of the uvrY mutant. CONCLUSION: This paper demonstrates that the BarA-UvrY two-component system is a determinant for virulence in a monkey cystitis model. The observed competition profiles strengthen our previous hypothesis that disruption of the BarA-UvrY two-component system impairs the ability of the bacteria to switch between different carbon sources. The urine in the bladder contains several different carbon sources and its composition changes over time. Inability to efficiently switch between the carbon sources may thus provide an explanation to the reduced fitness of the uvrY mutant in the cystitis model

Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy

<p>Abstract</p> <p>Background</p> <p>Curli, cellulose and the cell surface protein BapA are matrix components in <it>Salmonella </it>biofilms. In this study we have investigated the roles of these components for the morphology of bacteria grown as colonies on agar plates and within a biofilm on submerged mica surfaces by applying atomic force microscopy (AFM) and light microscopy.</p> <p>Results</p> <p>AFM imaging was performed on colonies of <it>Salmonella </it>Typhimurium grown on agar plates for 24 h and on biofilms grown for 4, 8, 16 or 24 h on mica slides submerged in standing cultures. Our data show that in the wild type curli were visible as extracellular material on and between the cells and as fimbrial structures at the edges of biofilms grown for 16 h and 24 h. In contrast to the wild type, which formed a three-dimensional biofilm within 24 h, a curli mutant and a strain mutated in the global regulator CsgD were severely impaired in biofilm formation. A mutant in cellulose production retained some capability to form cell aggregates, but not a confluent biofilm. Extracellular matrix was observed in this mutant to almost the same extent as in the wild type. Overexpression of CsgD led to a much thicker and a more rapidly growing biofilm. Disruption of BapA altered neither colony and biofilm morphology nor the ability to form a biofilm within 24 h on the submerged surfaces. Besides curli, the expression of flagella and pili as well as changes in cell shape and cell size could be monitored in the growing biofilms.</p> <p>Conclusion</p> <p>Our work demonstrates that atomic force microscopy can efficiently be used as a tool to monitor the morphology of bacteria grown as colonies on agar plates or within biofilms formed in a liquid at high resolution.</p

Titration-free massively parallel pyrosequencing using trace amounts of starting material

Continuous efforts have been made to improve next-generation sequencing methods for increased robustness and for applications on low amounts of starting material. We applied double-stranded library protocols for the Roche 454 platform to avoid the yield-reducing steps associated with single-stranded library preparation, and applied a highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) method. The MGB-probe qPCR, which can detect as low as 100 copies, was used to quantify the amount of effective library, i.e. molecules that form functional clones in emulsion PCR. We also demonstrate that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labour-intensive and costly titration can be eliminated and trace amounts of starting material such as precious clinical samples, transcriptomes of small tissue samples and metagenomics on low biomass environments is applicable

Antisense RNA protects mRNA from RNase E degradation by RNA–RNA duplex formation during phage infection

The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg2+, pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA–mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression

Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway

MicA is a trans-encoded small non-coding RNA, which downregulates porin-expression in stationary-phase. In this work, we focus on the role of endoribonucleases III and E on Salmonella typhimurium sRNA MicA regulation. RNase III is shown to regulate MicA in a target-coupled way, while RNase E is responsible for the control of free MicA levels in the cell. We purified both Salmonella enzymes and demonstrated that in vitro RNase III is only active over MicA when in complex with its targets (whether ompA or lamB mRNAs). In vivo, MicA is demonstrated to be cleaved by RNase III in a coupled way with ompA mRNA. On the other hand, RNase E is able to cleave unpaired MicA and does not show a marked dependence on its 5′ phosphorylation state. The main conclusion of this work is the existence of two independent pathways for MicA turnover. Each pathway involves a distinct endoribonuclease, having a different role in the context of the fine-tuned regulation of porin levels. Cleavage of MicA by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the eukaryotic RNAi system, where RNase III-like enzymes play a pivotal role

Characterization of the role of ribonucleases in Salmonella small RNA decay

In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. To better understand the turnover of regulatory sRNAs in the model pathogen, Salmonella typhimurium, we have constructed mutants for several ribonucleases (RNase E, RNase G, RNase III, PNPase) and Poly(A) Polymerase I. The expression profiles of four sRNAs conserved among many enterobacteria, CsrB, CsrC, MicA and SraL, were analysed and the processing and stability of these sRNAs was studied in the constructed strains. The degradosome was a common feature involved in the turnover of these four sRNAs. PAPI-mediated polyadenylation was the major factor governing SraL degradation. RNase III was revealed to strongly affect MicA decay. PNPase was shown to be important in the decay of these four sRNAs. The stability of CsrB and CsrC seemed to be independent of the RNA chaperone, Hfq, whereas the decay of SraL and MicA was Hfq-dependent. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs

Multiple Novel Nesprin-1 and Nesprin-2 Variants Act as Versatile Tissue-Specific Intracellular Scaffolds

<div><h3>Background</h3><p>Nesprins (<u>N</u>uclear <u>e</u>nvelope <u>s</u>pectrin-<u>r</u>epeat <u>p</u>roteins) are a novel family of giant spectrin-repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of ∼1mDa and ∼800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms.</p> <h3>Results</h3><p>In this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types.</p> <h3>Conclusions</h3><p>These studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account for the diverse range of disease phenotypes observed when these genes are mutated.</p> </div

Heme-Oxygenases during Erythropoiesis in K562 and Human Bone Marrow Cells

In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity

Children’s perspective in planning : A case study on the impact of densification on children’s outdoor environments in preschool and school

Barn som samhällsgrupp tenderar att vara tämligen osynliga inom ramen för den fysiska planeringen. Därmed är syftet med denna studie att bidra med en ökad förståelse för hur barnperspektivet implementeras i den fysiska planeringen, men även undersöka hur förtätning påverkar barns utemiljöer, specifikt förskole- och skolgårdar. Detta eftersom det i täta stadsdelar föreligger en problematik gällande att allmänna ytor tas i anspråk för bebyggelse. Den empiri som ligger till grund för studien har samlats in genom kvalitativa intervjuer samt granskning av dokument, vilket sedan analyserats med hjälp av en tematisk analys. Genom den tematiska analysen har två huvudteman identifierats, integreringen av barnperspektivet i planprocessen och barns plats i den kompakta staden, som belyser det rådande klimatet i den kommunala fysiska planeringen utifrån de studerade fallen. Utifrån denna tematik, med stöd av tidigare forskning, har det kunnat påvisats att barnperspektivet inte implementeras i tillräckligt stor utsträckning. Detta eftersom det finns flertalet problematiska aspekter i arbetet med stadsutveckling, varav två är resursfrågan och intressekonflikter.To begin with children can be seen as a rather invisible group within the work of spatial planning. Thus, the aim of this thesis is to contribute with an increased understanding regarding how the child perspective is implemented in spatial planning, but also investigate how outdoor environments for children are affected by densification, specifically preschool yards, and schoolyards. Densified areas tend to be problematic regarding the fact that land often tends to be limited, which results in public areas being claimed for buildings. The empirical basis that the study consists of is a thematic analysis, which is based on qualitative interviews and reviews of documents. Two main themes have been identified through the thematic analysis, these are integration of child perspective and children’s place in the compact city. Furthermore, this illustrates the current climate regarding municipal spatial planning based on the cases that have been studied. The findings in this thesis show that the child perspective is not being implemented to a sufficient extent. Reasons for this are that there are problematic aspects, such as resources and conflicts of interests, these aspects need to be dealt with regarding the implementation of child perspective in spatial planning

Children’s perspective in planning : A case study on the impact of densification on children’s outdoor environments in preschool and school

Barn som samhällsgrupp tenderar att vara tämligen osynliga inom ramen för den fysiska planeringen. Därmed är syftet med denna studie att bidra med en ökad förståelse för hur barnperspektivet implementeras i den fysiska planeringen, men även undersöka hur förtätning påverkar barns utemiljöer, specifikt förskole- och skolgårdar. Detta eftersom det i täta stadsdelar föreligger en problematik gällande att allmänna ytor tas i anspråk för bebyggelse. Den empiri som ligger till grund för studien har samlats in genom kvalitativa intervjuer samt granskning av dokument, vilket sedan analyserats med hjälp av en tematisk analys. Genom den tematiska analysen har två huvudteman identifierats, integreringen av barnperspektivet i planprocessen och barns plats i den kompakta staden, som belyser det rådande klimatet i den kommunala fysiska planeringen utifrån de studerade fallen. Utifrån denna tematik, med stöd av tidigare forskning, har det kunnat påvisats att barnperspektivet inte implementeras i tillräckligt stor utsträckning. Detta eftersom det finns flertalet problematiska aspekter i arbetet med stadsutveckling, varav två är resursfrågan och intressekonflikter.To begin with children can be seen as a rather invisible group within the work of spatial planning. Thus, the aim of this thesis is to contribute with an increased understanding regarding how the child perspective is implemented in spatial planning, but also investigate how outdoor environments for children are affected by densification, specifically preschool yards, and schoolyards. Densified areas tend to be problematic regarding the fact that land often tends to be limited, which results in public areas being claimed for buildings. The empirical basis that the study consists of is a thematic analysis, which is based on qualitative interviews and reviews of documents. Two main themes have been identified through the thematic analysis, these are integration of child perspective and children’s place in the compact city. Furthermore, this illustrates the current climate regarding municipal spatial planning based on the cases that have been studied. The findings in this thesis show that the child perspective is not being implemented to a sufficient extent. Reasons for this are that there are problematic aspects, such as resources and conflicts of interests, these aspects need to be dealt with regarding the implementation of child perspective in spatial planning