Mechanism of Transcription Activation at the comG Promoter by the Competence Transcription Factor ComK of Bacillus subtilis

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type -subunits and with C-terminal deletion mutants of the -subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.Peer reviewe

Biochemical characterization of a molecular switch involving the heat shock protein ClpC, which controls the activity of ComK, the competence transcription factor of Bacillus subtilis

Development of genetic competence in Bacillus subtilis is controlled by the competence-specific transcription factor ComK. ComK activates transcription of itself and several other genes required for competence. The activity of ComK is controlled by other genes including mecA, clpC, and comS. We have used purified ComK, MecA, ClpC, and synthetic ComS to study their interactions and have demonstrated the following mechanism for ComK regulation. ClpC, in the presence of ATP, forms a ternary complex with MecA and ComK, which prevents ComK from binding to its specific DNA target. This complex dissociates when ComS is added, liberating active ComK. ClpC and MecA function as a molecular switch, in which MecA confers molecular recognition, connecting ClpC to ComK and to ComS

The bdbDC operon of Bacillus subtilis encodes thiol-disulfide oxidoreductases required for competence development

The development of genetic competence in the Gram-positive eubacterium Bacillus subtilis is a complex postexponential process. Here we describe a new bicistronic operon, bdbDC, required for competence development, which was identified by the B. subtilis Systematic Gene Function Analysis program. Inactivation of either the bdbC or bdbD genes of this operon results in the loss of transformability without affecting recombination or the synthesis of ComK, the competence transcription factor. BdbC and BdbD are orthologs of enzymes known to be involved in extracytoplasmic disulfide bond formation. Consistent with this, BdbC and BdbD are needed for the secretion of theEscherichia coli disulfide bond-containing alkaline phosphatase, PhoA, by B. subtilis. Similarly, the amount of the disulfide bond-containing competence protein ComGC is severely reduced in bdbC or bdbD mutants. In contrast, the amounts of the competence proteins ComGA and ComEA remain unaffected by bdbDC mutations. Taken together, these observations imply that in the absence of either BdbC or BdbD, ComGC is unstable and that BdbC and BdbD catalyze the formation of disulfide bonds that are essential for the DNA binding and uptake machinery

Criterion Validity and Applicability of Motor Screening Instruments in Children Aged 5-6 Years:A Systematic Review

The detection of motor developmental problems, especially developmental coordination disorder, at age 5–6 contributes to early interventions. Here, we summarize evidence on (1) criterion validity of screening instruments for motor developmental problems at age 5–6, and (2) their applicability. We systematically searched seven databases for studies assessing criterion validity of these screening instruments using the M-ABC as reference standard. We applied COSMIN criteria for systematic reviews of screening instruments to describe the correlation between the tests and the M-ABC. We extracted information on correlation coefficients or area under the receiver operating curve, sensitivity and specificity, and applicability in practice. We included eleven studies, assessing eight instruments: three performance-based tests (MAND, MOT 4–6, BFMT) and five questionnaires (DCD-Q, PQ, ASQ-3, MOQ-T-FI, M-ABC-2-C). The quality of seven studies was fair, one was good, and three were excellent. Seven studies reported low correlation coefficients or AUC (<0.70), four did not report these. Sensitivities ranged from 21–87% and specificities from 50–96%, with the MOT4–6 having the highest sensitivity and specificity. The DCD-Q, PQ, ASQ-3, MOQ-T-FI, and M-ABC-2-C scored highest on applicability. In conclusion, none of the instruments were sufficiently valid for motor screening at age 5–6. More research is needed on screening instruments of motor delay at age 5–6

Childhood prediction models for hypertension later in life:A systematic review

BACKGROUND: Hypertension, even during childhood, increases the risk of developing atherosclerosis and cardiovascular disease. Therefore, starting prevention of hypertension early in the life course could be beneficial. Prediction models might be useful for identifying children at increased risk of developing hypertension, which may enable targeted primordial prevention of cardiovascular disease. OBJECTIVE: To provide an overview of childhood prediction models for future hypertension. METHODS: Embase and Medline were systematically searched. Studies were included that were performed in the general population, and that reported on development or validation of a multivariable model for children to predict future high blood pressure, prehypertension or hypertension. Data were extracted using the CHARMS checklist for prediction modelling studies. RESULTS: Out of 12 780 reviewed records, six studies were included in which 18 models were presented. Five studies predicted adulthood hypertension, and one predicted adolescent prehypertension/hypertension. BMI and current blood pressure were most commonly included as predictors in the final models. Considerable heterogeneity existed in timing of prediction (from early childhood to late adolescence) and outcome measurement. Important methodological information was often missing, and in four studies information to apply the model in new individuals was insufficient. Reported area under the ROC curves ranged from 0.51 to 0.74. As none of the models were validated, generalizability could not be confirmed. CONCLUSION: Several childhood prediction models for future hypertension were identified, but their value for practice remains unclear because of suboptimal methods, limited information on performance, or the lack of external validation. Further validation studies are indicated.This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0

Analysis of Stochastic Strategies in Bacterial Competence: A Master Equation Approach

Competence is a transiently differentiated state that certain bacterial cells reach when faced with a stressful environment. Entrance into competence can be attributed to the excitability of the dynamics governing the genetic circuit that regulates this cellular behavior. Like many biological behaviors, entrance into competence is a stochastic event. In this case cellular noise is responsible for driving the cell from a vegetative state into competence and back. In this work we present a novel numerical method for the analysis of stochastic biochemical events and use it to study the excitable dynamics responsible for competence in Bacillus subtilis. Starting with a Finite State Projection (FSP) solution of the chemical master equation (CME), we develop efficient numerical tools for accurately computing competence probability. Additionally, we propose a new approach for the sensitivity analysis of stochastic events and utilize it to elucidate the robustness properties of the competence regulatory genetic circuit. We also propose and implement a numerical method to calculate the expected time it takes a cell to return from competence. Although this study is focused on an example of cell-differentiation in Bacillus subtilis, our approach can be applied to a wide range of stochastic phenomena in biological systems

Evaluation of a nationwide Dutch guideline to detect Lynch syndrome in patients with endometrial cancer

Objective: In the Netherlands a nationwide guideline was introduced in 2016, which recommended routine Lynch syndrome screening (LSS) for all women with endometrial cancer (EC) <70 years of age. LSS consists of immunohistochemical (IHC) staining for loss of mismatch repair (MMR) protein expression, supplemented with MLH1 methylation analysis if indicated. Test results are evaluated by the treating gynaecologist, who refers eligible patients to a clinical geneticist. We evaluated the implementation of this guideline. Methods: From the nation-wide pathology database we selected all women diagnosed with EC < 70 years of age, treated from 1.6.2016–1.6.2017 in 14 hospitals. We collected data on the results of LSS and follow up of cases with suspected LS. Results: In 183 out of 204 tumours (90%) LSS was performed. In 41 cases (22%) MMR protein expression was lost, in 25 cases due to hypermethylation of the MLH1 promotor. One patient was known with a pathogenic MLH1 variant. The option of genetic counselling was discussed with 12 of the 15 remaining patients, of whom three declined. After counselling by the genetic counsellor nine patients underwent germline testing. In two no pathogenic germline variant was detected, two were diagnosed with a pathogenic PMS2 variant, and five with a pathogenic MSH6 variant, in concordance with the IHC profiles. Conclusion: Coverage of LSS was high (90%), though referral for genetic counselling could be improved. Gynaecologists ought to be aware of the benefits and possible drawbacks of knowing mutational status, and require training in discussing this with their patients

Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops