A Protein Thermometer Controls Temperature-Dependent Transcription of Flagellar Motility Genes in Listeria monocytogenes

Facultative bacterial pathogens must adapt to multiple stimuli to persist in the environment or establish infection within a host. Temperature is often utilized as a signal to control expression of virulence genes necessary for infection or genes required for persistence in the environment. However, very little is known about the molecular mechanisms that allow bacteria to adapt and respond to temperature fluctuations. Listeria monocytogenes (Lm) is a food-borne, facultative intracellular pathogen that uses flagellar motility to survive in the extracellular environment and to enhance initial invasion of host cells during infection. Upon entering the host, Lm represses transcription of flagellar motility genes in response to mammalian physiological temperature (37°C) with a concomitant temperature-dependent up-regulation of virulence genes. We previously determined that down-regulation of flagellar motility is required for virulence and is governed by the reciprocal activities of the MogR transcriptional repressor and the bifunctional flagellar anti-repressor/glycosyltransferase, GmaR. In this study, we determined that GmaR is also a protein thermometer that controls temperature-dependent transcription of flagellar motility genes. Two-hybrid and gel mobility shift analyses indicated that the interaction between MogR and GmaR is temperature sensitive. Using circular dichroism and limited proteolysis, we determined that GmaR undergoes a temperature-dependent conformational change as temperature is elevated. Quantitative analysis of GmaR in Lm revealed that GmaR is degraded in the absence of MogR and at 37°C (when the MogR:GmaR complex is less stable). Since MogR represses transcription of all flagellar motility genes, including transcription of gmaR, changes in the stability of the MogR:GmaR anti-repression complex, due to conformational changes in GmaR, mediates repression or de-repression of flagellar motility genes in Lm. Thus, GmaR functions as a thermo-sensing anti-repressor that incorporates temperature signals into transcriptional control of flagellar motility. To our knowledge, this is the first example of a protein thermometer that functions as an anti-repressor to control a developmental process in bacteria

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing

International audienceCurrent sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the world's oceans

Gradable nouns as concepts without prototypes

Non-copy-edited preprint. To appear in L. McNally, E. Castroviejo-Miró, and G. Sassoon (eds). The Semantics of Gradability, Vagueness and Scale Structure. Language, Cognition and Mind series. Springer

The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

A bifunctional O-GlcNAc transferase governs flagellar motility through anti-repression

Flagellar motility is an essential mechanism by which bacteria adapt to and survive in diverse environments. Although flagella confer an advantage to many bacterial pathogens for colonization during infection, bacterial flagellins also stimulate host innate immune responses. Consequently, many bacterial pathogens down-regulate flagella production following initial infection. Listeria monocytogenes is a facultative intracellular pathogen that represses transcription of flagellar motility genes at physiological temperatures (37°C and above). Temperature-dependent expression of flagellar motility genes is mediated by the opposing activities of MogR, a DNA-binding transcriptional repressor, and DegU, a response regulator that functions as an indirect antagonist of MogR. In this study, we identify an additional component of the molecular circuitry governing temperature-dependent flagellar gene expression. At low temperatures (30°C and below), MogR repression activity is specifically inhibited by an anti-repressor, GmaR. We demonstrate that GmaR forms a stable complex with MogR, preventing MogR from binding its DNA target sites. GmaR anti-repression activity is temperature dependent due to DegU-dependent transcriptional activation of gmaR at low temperatures. Thus, GmaR production represents the first committed step for flagella production in L. monocytogenes. Interestingly, GmaR also functions as a glycosyltransferase exhibiting O-linked N-acetylglucosamine transferase (OGT) activity for flagellin (FlaA). GmaR is the first OGT to be identified and characterized in prokaryotes that specifically β-O-GlcNAcylates a prokaryotic protein. Unlike the well-characterized, highly conserved OGT regulatory protein in eukaryotes, the catalytic activity of GmaR is functionally separable from its anti-repression function. These results establish GmaR as the first known example of a bifunctional protein that transcriptionally regulates expression of its enzymatic substrate

Validation of Tn-seq results by gene deletion.

<p>(<b>A</b>) Schematic of FRT scar use for high throughput sequencing. In-frame gene deletions were made using FRT/FLP recombination. Nested PCR using reverse primers 1 and 2 and a forward primer (not shown) was used to amplify the junction between the deletion scar and the gDNA. The sequencing primer is shown. (<b>B</b>) Correlation plot of fitness values of deletions compared to original Tn-seq screen. Each dot represents the fitness value and standard error (SE) for one gene in both screens shown on the x- and y-axes. Shown are paired fitness values from 35 gene deletions tested in 4 different conditions: LB, Cecal fluid, Small Intestine and Pond. A linear regression analysis was used to determine a correlation. (<b>C</b>) Validation of Frt-marked deletions in a 1∶1 competition with a Δ<i>lacZ</i> wild-type for 48 hr in pond water. Median values are indicated. Using a one sample t-test all CI values were significantly different than 1.0 (P≤0.001).</p

Flow diagram of experimental design.

<p>A library of mTn<i>5</i> transposon mutants of <i>V. cholerae</i> was used to infect infant rabbits. After 12 hrs of infection, the host-passaged <i>V. cholerae</i> were collected, pelleted, and resuspended in pond water for 48 hrs at 30°C. In addition, the library was also passaged in LB broth overnight and put directly into pond water for 48 hr at 30°C. After each selective condition the following steps were done: 1. Bacteria were minimally outgrown to a high titer in LB. 2. gDNA was prepared and sheared. 3. Poly-C tails were added to the 3′ ends using Terminal deoxynucleotidyl Transferase. 4. The transposon-genome junctions were amplified by PCR. 4. Barcodes were added to each sample by a second PCR. 5. Multiplexed samples were subjected to massively parallel sequencing.</p

A correlation plot of fitness values from host-passaged <i>V. cholerae</i>.

<p>Each dot represents the fitness values for one gene in the two host compartments shown on the x- and y-axes. Only genes that had calculated fitness values in both conditions were plotted, which represents 93% of all non-essential genes in <i>V. cholerae</i>. A linear regression analysis was used to determine the correlation.</p

Growth-phase dependent survival of <i>V. cholerae</i> in pond water.

<p>(<b>A</b>) Competition assays in pond water. Either a wild type or a Δ<i>lacZ</i> strain were grown overnight to stationary phase and competed 1∶1 with the test strain in pond water for 2 or 8 days as indicated at 30°C. The first column is a control competition between the wild type and Δ<i>lacZ</i> stationary phase cells. Exponential-phase cultures were collected at OD₆₀₀ = 0.3. Host passaged wild type was collected from the cecal fluid of infant rabbits 12 hrs post-infection. Each dot represents a biological replicate, and open squares indicate the limit of detection. Both the exponential phase (P value = <0.0001) and host-passaged (P value = 0.01) at 8 days are statistically different than 1.0. (<b>B</b>) Survival comparison in pond water. Stationary phase, exponential phase and host-passaged <i>V. cholerae</i> were pelleted, washed and resuspended in pond water. The pond cultures were placed standing at 30°C for 6 days and the CFU/ml was plated each day. At least 6 biological replicates were used for each time point. The input CFU at time zero was used to determine 100% survival. The dotted line indicates the limit of detection.</p