Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon

To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB–A or lacZ–A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn 5 , but, unlike another similar cassette, it lacks IS 1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non-selective conditions at 42°C, and then in the presence of sucrose at 30°C, allowing positive selection for both plasmid excision and curing. Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E. coli chromosome.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72324/1/j.1365-2958.1991.tb00791.x.pd

RfaH Suppresses Small RNA MicA Inhibition of fimB Expression in Escherichia coli K-12

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE , and hence MicA, in the absence of RfaH. Additional work con- firms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression

The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

<p>Abstract</p> <p>Background</p> <p>GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria.</p> <p>Methods</p> <p>An <it>in vitro </it>binding assay was used to assay the binding of recombinant GP2 to defined strains of <it>E. coli </it>that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding.</p> <p>Results</p> <p>GP2 binds <it>E. coli </it>that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues.</p> <p>Conclusion</p> <p>GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the <it>Enterobacteriacae </it>family.</p

Recurrent RNA motifs as probes for studying RNA-protein interactions in the ribosome

To understand how the nucleotide sequence of ribosomal RNA determines its tertiary structure, we developed a new approach for identification of those features of rRNA sequence that are responsible for formation of different short- and long-range interactions. The approach is based on the co-analysis of several examples of a particular recurrent RNA motif. For different cases of the motif, we design combinatorial gene libraries in which equivalent nucleotide positions are randomized. Through in vivo expression of the designed libraries we select those variants that provide for functional ribosomes. Then, analysis of the nucleotide sequences of the selected clones would allow us to determine the sequence constraints imposed on each case of the motif. The constraints shared by all cases are interpreted as providing for the integrity of the motif, while those ones specific for individual cases would enable the motif to fit into the particular structural context. Here we demonstrate the validity of this approach for three examples of the so-called along-groove packing motif found in different parts of ribosomal RNA

Voice, autonomy and utopian desire in participatory film-making with young refugees

This article is a reflection on what reflexive documentary scholars call the ‘moral dimension’ (Nash 2012: 318) of a participatory filmmaking project with refugee young people, who wanted to make a film to support other new young arrivals in the process of making home in Scotland. In the first part, we highlight some of the challenges of collaborating with refugee young people, in light of the often de-humanising representations of refugees in mainstream media and the danger of the triple conflation of authenticity-voice-pain in academic narratives about refugees. In the second part, we show how honouring young people’s desire to convey the hopeful aspects of making home, emerged as a key pedagogical strategy to affirm their expert position and encourage their participation in the project. Revisiting key moments of learning and interaction, we demonstrate how young people’s process of ‘finding a voice’ in moment-by-moment filmmaking practice was not a linear, developmental process towards ‘pure’ individual empowerment and singular artistic expression. Their participation in shaping their visual (self-)representation in the final film, was embedded in the dialogical process and pragmatic requirements of a collaborative film production, in which voice, autonomy and teacher authority were negotiated on a moment-by-moment basis. We conclude that it is vital for a reflexive practice and research to not gloss over the moral dilemmas in the name of progressive ideals, for example, when representations are co-created by project filmmakers/educators, but embrace these deliberations as part of the ‘fascinating collaborative matrix’ (Chambers 2019: 29) of participatory filmmaking

Elevated Incidence of Dental Caries in a Mouse Model of Cystic Fibrosis

Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model.). are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse

Temperature Control of Fimbriation Circuit Switch in Uropathogenic Escherichia coli: Quantitative Analysis via Automated Model Abstraction

Uropathogenic Escherichia coli (UPEC) represent the predominant cause of urinary tract infections (UTIs). A key UPEC molecular virulence mechanism is type 1 fimbriae, whose expression is controlled by the orientation of an invertible chromosomal DNA element—the fim switch. Temperature has been shown to act as a major regulator of fim switching behavior and is overall an important indicator as well as functional feature of many urologic diseases, including UPEC host-pathogen interaction dynamics. Given this panoptic physiological role of temperature during UTI progression and notable empirical challenges to its direct in vivo studies, in silico modeling of corresponding biochemical and biophysical mechanisms essential to UPEC pathogenicity may significantly aid our understanding of the underlying disease processes. However, rigorous computational analysis of biological systems, such as fim switch temperature control circuit, has hereto presented a notoriously demanding problem due to both the substantial complexity of the gene regulatory networks involved as well as their often characteristically discrete and stochastic dynamics. To address these issues, we have developed an approach that enables automated multiscale abstraction of biological system descriptions based on reaction kinetics. Implemented as a computational tool, this method has allowed us to efficiently analyze the modular organization and behavior of the E. coli fimbriation switch circuit at different temperature settings, thus facilitating new insights into this mode of UPEC molecular virulence regulation. In particular, our results suggest that, with respect to its role in shutting down fimbriae expression, the primary function of FimB recombinase may be to effect a controlled down-regulation (rather than increase) of the ON-to-OFF fim switching rate via temperature-dependent suppression of competing dynamics mediated by recombinase FimE. Our computational analysis further implies that this down-regulation mechanism could be particularly significant inside the host environment, thus potentially contributing further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs

A Review of Dietary Prevention of Human Papillomavirus-Related Infection of the Cervix and Cervical Intraepithelial Neoplasia

The natural history of cervical cancer suggests that prevention can be achieved by modification of the host's immune system through a nutrient-mediated program. This study reviews the preventive role of dietary intake on cervical intraepithelial neoplasia (CIN) induced by human papillomavirus (HPV). Electronic databases were searched using relevant keywords such as, but not limited to, human papillomavirus infection, cervical intraepithelial neoplasia, lifestyle factors, nutrients intake, and diet. High consumption of fruit and vegetables appears to be protective against CIN. The findings also highlight the possibility of consuming high levels of specific nutrients, vitamins, and minerals, and retaining sufficient level of these elements in the body, especially those with high antioxidants and antiviral properties, to prevent progression of transient and persistent HPV infections to high-grade CIN 2 and 3 (including in situ cervical cancer). The protective effect is not significant for high-risk HPV persistent infections and invasive cervical cancer. Although it appears that intake of specific nutrients, vitamins, and minerals may be good in CIN prevention, there is lack of evidence from controlled trial to confirm this. Health professionals shall focus on implementation of a balanced-diet prevention strategy at an early stage for cervical cancer prevention

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins

AD51B in Familial Breast Cancer

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C&gt;T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk