Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in Escherichia coli K-12

The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote E. coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening in vitro, we identified the whole set of ‘constitutive’ promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ70), RpoS (σ38), RpoH (σ32), RpoF (σ28) or RpoE (σ24), in the absence of other supporting regulatory factors. In contrast, RpoN sigma (σ54), involved in expression of nitrogen-related genes and also other cellular functions, requires an enhancer (or activator) protein, such as NtrC, for transcription initiation. In this study, a series of gSELEX screenings were performed to search for promoters recognized by the RpoN RNAP holoenzyme in the presence and absence of the major nitrogen response enhancer NtrC, the best-characterized enhancer. Based on the RpoN holoenzyme-binding sites, a total of 44 to 61 putative promoters were identified, which were recognized by the RpoN holoenzyme alone. In the presence of the enhancer NtrC, the recognition target increased to 61–81 promoters. Consensus sequences of promoters recognized by RpoN holoenzyme in the absence and presence of NtrC were determined. The promoter activity of a set of NtrC-dependent and -independent RpoN promoters was verified in vivo under nitrogen starvation, in the presence and absence of RpoN and/or NtrC. The promoter activity of some RpoN-recognized promoters increased in the absence of RpoN or NtrC, supporting the concept that the promoter-bound NtrC-enhanced RpoN holoenzyme functions as a repressor against RpoD holoenzyme. Based on our findings, we propose a model in which the RpoN holoenzyme fulfils the dual role of repressor and transcriptase for the same set of genes. We also propose that the promoter recognized by RpoN holoenzyme in the absence of enhancers is the ‘repressive’ promoter. The presence of high-level RpoN sigma in growing E. coli K-12 in rich medium may be related to the repression role of a set of genes needed for the utilization of ammonia as a nitrogen source in poor media. The list of newly identified regulatory targets of RpoN provides insight into E. coli survival under nitrogen-depleted conditions in nature.

Misleading localization by 18F-fluorocholine PET/CT in familial hypocalciuric hypercalcemia type-3: a case report

Background Familial hypocalciuric hypercalcemia (FHH) is a heterogeneous autosomal-dominant disorder of calcium hemostasis that may be difficult to distinguish clinically from mild primary hyperparathyroidism. Loss-of-function mutations mainly involving Arg15 residue of the adaptor-related protein complex 2, sigma subunit 1 (AP2S1) cause a rarer, more recently recognized form of FHH, FFH type-3. Recently, 18F-fluorocholine positron emission tomography/computed tomography (FCH-PET/CT) showed superior sensitivity to conventional imaging in localizing parathyroid adenomas. We report a new FFH type-3 patient who underwent unnecessary parathyroidectomy in association with misleading FCH-PET/CT imaging. Case presentation A 29-year old woman was initially evaluated for parathyroid hormone (PTH)-dependent hypercalcemia in 2013. Medical history was positive only for chronic constipation and malaise with no personal or family history of hypercalcemia, kidney stones, or neck surgery. Over seven years, serum calcium level was 2.51–2.89 mmol/L with concomitant PTH level of 58.7–94.8 mmol/L. Serum phosphate levels were in the low/low normal range. Serum creatinine and magnesium levels were normal. 25-hydroxy vitamin D level was 13 nmol/L. 24-hour urine calcium level was 1.92 mmol/day but increased to 6.99 mmol/day after treatment with cholecalciferol 1000 IU daily. Bone mineral density and renal ultrasound were normal. Parathyroid ultrasound showed two hypoechoic nodules inferior to the left and right thyroid lobes; however, 99mtechnitium-sestamibi scans (2013, 2016, 2018) were negative. FCH-PET/CT (2019) showed focal uptake co-localizing with the nodule inferior to the left thyroid lobe. The patient underwent left inferior parathyroidectomy and pathology was consistent with parathyroid hyperplasia. However, postoperatively, serum calcium and PTH levels remained elevated and FCH-PET/CT and ultrasound showed persistence of the uptake/nodule. Whole exome sequencing showed Arg15Cys mutation in the AP2S1 gene characteristic of FHH type-3. Conclusions In this new case of FHH type-3, FCH-PET/CT failed to localize to the hyperplastic parathyroid glands and localized instead to apparently a lymph node. This, together with increased urinary calcium after vitamin D supplementation, led to unnecessary parathyroidectomy. Given the increasingly lower cost of genetic testing and the cost of follow up and unnecessary surgery, it may prudent to include genetic testing for FHH early on in patients with mild PTH-dependent hypercalcemia.

Ap2s1 mutation in mice causes familial hypocalciuric hypercalcemia type 3

Mutations of the adaptor protein-2 sigma subunit (AP2S1) gene which encodes AP2σ2, a component of the ubiquitous AP2 heterotetrameric complex involved in endosomal trafficking of the calcium-sensing receptor (CaSR), cause familial hypocalciuric hypercalcemia type 3 (FHH3). FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with marked hypercalcemia and occasional hypophosphatemia and osteomalacia. To further characterise the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcemia, hypermagnesemia, hypophosphatemia, and increased plasma concentrations of parathyroid hormone, fibroblast growth factor 23 and alkaline phosphatase activity, but normal pro-collagen type 1 N-terminal pro-peptide and 1,25 dihydroxyvitamin D. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. The AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2σ2 and the other AP2 subunits, and the CaSR. Cinacalcet, a CaSR allosteric activator, ameliorated the hypercalcemia and elevated PTH concentrations, but not the diminished AP2σ2-CaSR interaction. Thus, our studies have established a mouse model with a germline loss-of-function AP2S1 mutation that is representative for FHH3 in humans, and demonstrated that cinacalcet corrects the abnormalities of plasma calcium and PTH.

Coordinated Hibernation of Transcriptional and Translational Apparatus during Growth Transition ofEscherichia colito Stationary Phase

ABSTRACTIn the process ofEscherichia coliK-12 growth from exponential phase to stationary, marked alteration takes place in the pattern of overall genome expression through modulation of both parts of the transcriptional and translational apparatus. In transcription, the sigma subunit with promoter recognition properties is replaced from the growth-related factor RpoD by the stationary-phase-specific factor RpoS. The unused RpoD is stored by binding with the anti-sigma factor Rsd. In translation, the functional 70S ribosome is converted to inactive 100S dimers through binding with the ribosome modulation factor (RMF). Up to the present time, the regulatory mechanisms of expression of these two critical proteins, Rsd and RMF, have remained totally unsolved. In this study, attempts were made to identify the whole set of transcription factors involved in transcription regulation of thersdandrmfgenes using the newly developed promoter-specific transcription factor (PS-TF) screening system. In the first screening, 74 candidate TFs with binding activity to both of thersdandrmfpromoters were selected from a total of 194 purified TFs. After 6 cycles of screening, we selected 5 stress response TFs, ArcA, McbR, RcdA, SdiA, and SlyA, for detailed analysisin vitroandin vivoof their regulatory roles. Results indicated that bothrsdandrmfpromoters are repressed by ArcA and activated by McbR, RcdA, SdiA, and SlyA. We propose the involvement of a number of TFs in simultaneous and coordinated regulation of the transcriptional and translational apparatus. By using genomic SELEX (gSELEX) screening, each of the five TFs was found to regulate not only thersdandrmfgenes but also a variety of genes for growth and survival.IMPORTANCEDuring the growth transition ofE. colifrom exponential phase to stationary, the genome expression pattern is altered markedly. For this alteration, the transcription apparatus is altered by binding of anti-sigma factor Rsd to the RpoD sigma factor for sigma factor replacement, while the translation machinery is modulated by binding of RMF to 70S ribosome to form inactive ribosome dimer. Using the PS-TF screening system, a number of TFs were found to bind to both thersdandrmfpromoters, of which the regulatory roles of 5 representative TFs (one repressor ArcA and the four activators McbR, RcdA, SdiA, and SlyA) were analyzed in detail. The results altogether indicated the involvement of a common set of TFs, each sensing a specific environmental condition, in coordinated hibernation of the transcriptional and translational apparatus for adaptation and survival under stress conditions.

Characterization of Chilean Pseudomonas syringae pv actinidiae strains isolated from infected orchards

Pseudomonas syringae pv. actinidiae (PSA) strain is a major problem for the kiwifruit industry worldwide. So far, 5 biovars of PSA have been identified, of which the most virulent form is biovar 3. This is the only biovar that has been detected in Chile, which is the third kiwifruit exporter country and is currently suffering from canker disease produced by PSA. Single nucleotide polymorphisms (SNPs) analyses have classified the biovar 3 strain into three groups: the European, the New Zealander and the Chilean groups, which have evolved from a common Chinese PSA ancestor. Although Chilean strains have been used in phylogenetic analysis, there is no information about genomic diversity within this group or whether they present microbiological characteristics that could affect its virulence. In this work we studied 15 Chilean bacterial isolates collected from orchards with canker disease symptoms, and classified them as PSA using a different PCR techniques. To gain more information on the relationship between the isolates we sequenced part of three conserved genes widely used to classify bacterial strains: gtl (Citrate Synthase), rpoD (Sigma subunit of RNA polymerase II) and gyrB (Gyrase B). Using these sequences we performed a phylogenetic analysis that included some PSA reference sequences. Fourteen PSA Chilean isolates were grouped with PSA reference strains and three of them formed a subgroup within the PSA clade, suggesting clear differences at the genomic level among the isolates. We evaluated three microbiological traits in all the isolates: motility (swimming and swarming), and ability to induce a hypersensitive response in tobacco plants. All the isolates were able to induce the hypersensitive response in tobacco plants and were also able to perform both types of movements in appropriated growing conditions.

Characterization of Chilean Pseudomonas syringae pv actinidiae strains isolated from infected orchards

Pseudomonas syringae pv. actinidiae (PSA) strain is a major problem for the kiwifruit industry worldwide. So far, 5 biovars of PSA have been identified, of which the most virulent form is biovar 3. This is the only biovar that has been detected in Chile, which is the third kiwifruit exporter country and is currently suffering from canker disease produced by PSA. Single nucleotide polymorphisms (SNPs) analyses have classified the biovar 3 strain into three groups: the European, the New Zealander and the Chilean groups, which have evolved from a common Chinese PSA ancestor. Although Chilean strains have been used in phylogenetic analysis, there is no information about genomic diversity within this group or whether they present microbiological characteristics that could affect its virulence. In this work we studied 15 Chilean bacterial isolates collected from orchards with canker disease symptoms, and classified them as PSA using a different PCR techniques. To gain more information on the relationship between the isolates we sequenced part of three conserved genes widely used to classify bacterial strains: gtl (Citrate Synthase), rpoD (Sigma subunit of RNA polymerase II) and gyrB (Gyrase B). Using these sequences we performed a phylogenetic analysis that included some PSA reference sequences. Fourteen PSA Chilean isolates were grouped with PSA reference strains and three of them formed a subgroup within the PSA clade, suggesting clear differences at the genomic level among the isolates. We evaluated three microbiological traits in all the isolates: motility (swimming and swarming), and ability to induce a hypersensitive response in tobacco plants. All the isolates were able to induce the hypersensitive response in tobacco plants and were also able to perform both types of movements in appropriated growing conditions.

Structural basis for the sequestration of the anti-σ70factor Rsd from σ70by the histidine-containing phosphocarrier protein HPr

Histidine-containing phosphocarrier protein (HPr) is a general component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) involved in the phosphorylation-coupled transport of numerous sugars called PTS sugars. HPr mainly exists in a dephosphorylated form in the presence of PTS sugars in the medium, while its phosphorylation increases in the absence of PTS sugars. A recent study revealed that the dephosphorylated form of HPr binds and antagonizes the function of the antisigma factor Rsd. This anti-sigma factor sequesters the housekeeping sigma factor σ70to facilitate switching of the sigma subunit on RNA polymerase from σ70to the stress-responsive sigma factor σSin stationary-phase cells. In this study, the structure of the complex of Rsd and HPr was determined at 2.1 Å resolution and revealed that the binding site for HPr on the surface of Rsd partly overlaps with that for σ70. The localization of the phosphorylation site on HPr at the binding interface for Rsd explains why phosphorylation of HPr abolishes its binding to Rsd. The mutation of crucial residues involved in the HPr–Rsd interaction significantly influenced the competition between HPr and σ70for binding to Rsd bothin vitroandin vivo. The results provide a structural basis for the linkage of global gene regulation to nutrient availability in the external environment.