The catabolite repressor protein-cyclic AMP complex regulates csgD and biofilm formation in uropathogenic Escherichia coli

The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD. The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD. IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874–5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406–3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD. Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation

“Good Mothers Work”: How Maternal Employment Shapes Women’s Expectation of Work and Family in Contemporary Urban China

Drawing on 70 in‐depth interviews, I investigated how maternal employment shapes urban young Chinese women’s work–family expectation in a context of rapid social change. These interviews indicated that respondents attached strong moral meaning to mothers’ wage work, regarding it as integral to a “good” mother and an “ideal” woman. This moralization of maternal employment, in turn, led contemporary young Chinese women to view wage work as a taken‐for‐granted choice. Yet different from their own mothers, these young women were confronted with profound transformation across various domains of the postreform Chinese society. The normative expectation of women’s wage work, coupled with slow‐to‐change expectations about women’s roles at home and in a changing labor market, intensified young women’s burden of “doing it all.” This research highlights the importance of bringing the macro‐level context back into the mother–daughter dyad to understand the intergenerational transmission of gender beliefs and behavior.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162814/2/josi12389_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162814/1/josi12389.pd

Gene Activation by Dissociation of an Inhibitor from a Transcriptional Activation Domain▿

Gal4 is a prototypical eukaryotic transcriptional activator whose recruitment function is inhibited in the absence of galactose by the Gal80 protein through masking of its transcriptional activation domain (AD). A long-standing nondissociation model posits that galactose-activated Gal3 interacts with Gal4-bound Gal80 at the promoter, yielding a tripartite Gal3-Gal80-Gal4 complex with altered Gal80-Gal4 conformation to enable Gal4 AD activity. Some recent data challenge this model, whereas other recent data support the model. To address this controversy, we imaged fluorescent-protein-tagged Gal80, Gal4, and Gal3 in live cells containing a novel GAL gene array. We find that Gal80 rapidly dissociates from Gal4 in response to galactose. Importantly, this dissociation is Gal3 dependent and concurrent with Gal4-activated GAL gene expression. When galactose-triggered dissociation is followed by galactose depletion, preexisting Gal80 reassociates with Gal4, indicating that sequestration of Gal80 by Gal3 contributes to the observed Gal80-Gal4 dissociation. Moreover, the ratio of nuclear Gal80 to cytoplasmic Gal80 decreases in response to Gal80-Gal3 interaction. Taken together, these and other results provide strong support for a GAL gene switch model wherein Gal80 rapidly dissociates from Gal4 through a mechanism that involves sequestration of Gal80 by galactose-activated Gal3

Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another

The bacterial curli system possesses a potent and selective inhibitor of amyloid formation.

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids

Solution NMR of CsgC/CsgE interactions with ¹⁵N labeled α-synuclein.

<p>¹H-¹⁵N HSQC spectra at 10°C for 100 μM α-synuclein alone (red data in all panels) and upon addition of a 1-to-1 molar ratio of CsgC (<b>A</b>, blue) and CsgE (<b>B</b>, blue), and for a 1-to-5 molar ratio sample of CsgC and α-synuclein that had been shaken at 37°C for 48 h (<b>C,</b> blue). The data shown in <b>A</b> and <b>B</b> demonstrate that blue and red signals overlap except for His50 (<b>Insets</b> in <b>A</b> and <b>B</b>) and these spectra did not change over the course of three days. The visible chemical shifts in <b>C</b> were analyzed by NMR diffusion experiments to obtain an estimate of the molecular size. <b>D.</b> Analysis of perturbed residues in α-synuclein in the incubated CsgC-synuclein sample, based on the ¹H-¹⁵N HSQC peak intensities in <b>C</b> and reported assignments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140194#pone.0140194.ref033" target="_blank">33</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140194#pone.0140194.ref034" target="_blank">34</a>]. Boxed residues represent chemical shifts assigned to specific residues (78 of the 140 residues were identified and used for the analysis). After shaking, 38 residues disappeared or broadened severely in the new species as judged from the peak intensities. Residues that broadened beyond detection are shown in red, and residues that lost > 90% of the original intensity are shown in yellow. Marked in bold are residues that show no apparent chemical shift change (Δω < 0.02 ppm, calculated as Δω = |0.2Δ¹⁵N+Δ¹H|).</p

Aggregation of α-synuclein in the presence of CsgC and CsgE.

<p><b>A.</b> ThT assay for α-synuclein aggregation with and without 1-to-10 molar ratio of CsgE:synuclein (red) or 1-to-10 molar ratio of CsgC:synuclein (blue). <b>B.</b> Bar graph showing the lag time for α-synuclein aggregation at 3 different ratios of CsgE and CsgC (1-to-3, 1-to-10, 1-to-100; 70 μM α-synuclein in all cases). ‘*’ Denotes no rise in ThT emission after 85 hrs. The error bars represent three experimental replicates. <b>C.</b> Fluorescence microscopy of end products of ThT assay for α-synuclein alone and for 1-to-3, 1-to-10, and 1-to-100 molar ratio of CsgE/CsgC-to-synuclein mixtures. Scale bar 100 μm. <b>D-F.</b> AFM images of end products after ThT experiments for α-synuclein alone (<b>D</b>), and in the presence of CsgE (<b>E</b>) and CsgC (<b>F</b>), as indicated. Scale bar 1 μm.</p