A riboswitch-controlled TerC family transporter Alx tunes intracellular manganese concentration in Escherichia coli at alkaline pH.

Cells use transition metal ions as structural components of biomolecules and cofactors in enzymatic reactions, making transition metal ions integral cellular components. Organisms optimize metal ion concentration to meet cellular needs by regulating the expression of proteins that import and export that metal ion, often in a metal ion concentration-dependent manner. One such regulation mechanism is via riboswitches, which are 5'-untranslated regions of an mRNA that undergo conformational changes to promote or inhibit the expression of the downstream gene, commonly in response to a ligand. The yybP-ykoY family of bacterial riboswitches shares a conserved aptamer domain that binds manganese ions (Mn2+). In Escherichia coli, the yybP-ykoY riboswitch precedes and regulates the expression of two different genes: mntP, which based on genetic evidence encodes an Mn2+ exporter, and alx, which encodes a putative metal ion transporter whose cognate ligand is currently in question. The expression of alx is upregulated by both elevated concentrations of Mn2+ and alkaline pH. With metal ion measurements and gene expression studies, we demonstrate that the alkalinization of media increases the cytoplasmic manganese pool, which, in turn, enhances alx expression. The Alx-mediated Mn2+ export prevents the toxic buildup of the cellular manganese, with the export activity maximal at alkaline pH. We pinpoint a set of acidic residues in the predicted transmembrane segments of Alx that play a critical role in Mn2+ export. We propose that Alx-mediated Mn2+ export serves as a primary protective mechanism that fine tunes the cytoplasmic manganese content, especially during alkaline stress.IMPORTANCEBacteria use clever ways to tune gene expression upon encountering certain environmental stresses, such as alkaline pH in parts of the human gut and high concentration of a transition metal ion manganese. One way by which bacteria regulate the expression of their genes is through the 5'-untranslated regions of messenger RNA called riboswitches that bind ligands to turn expression of genes on/off. In this work, we have investigated the roles and regulation of alx and mntP, the two genes in Escherichia coli regulated by the yybP-ykoY  riboswitches, in alkaline pH and high concentration of Mn2+. This work highlights the intricate ways through which bacteria adapt to their surroundings, utilizing riboregulatory mechanisms to maintain Mn2+ levels amidst varying environmental factors.

Allosteric Determinants in High Temperature Requirement A Enzymes Are Conserved and Regulate the Population of Active Conformations.

High temperature requirement A (HtrA) are allosterically regulated enzymes wherein effector binding to the PDZ domain triggers proteolytic activity. Yet, it remains unclear if the inter-residue network governing allostery is conserved across HtrA enzymes. Here, we investigated and identified the inter-residue interaction networks by molecular dynamics simulations on representative HtrA proteases, Escherichia coli DegS and Mycobacterium tuberculosis PepD, in effector-bound and free forms. This information was used to engineer mutations that could potentially perturb allostery and conformational sampling in a different homologue, M. tuberculosis HtrA. Mutations in HtrA perturbed allosteric regulation─a finding consistent with the hypothesis that the inter-residue interaction network is conserved across HtrA enzymes. Electron density from data collected on cryo-protected HtrA crystals revealed that mutations altered the topology of the active site. Ensemble models fitted into electron density calculated from room-temperature diffraction data showed that only a fraction of these models had a catalytically competent active site conformation alongside a functional oxyanion hole thus providing experimental evidence that these mutations influenced conformational sampling. Mutations at analogous positions in the catalytic domain of DegS perturbed the coupling between effector binding and proteolytic activity, thus confirming the role of these residues in the allosteric response. The finding that a perturbation in the conserved inter-residue network alters conformational sampling and the allosteric response suggests that an ensemble allosteric model best describes regulated proteolysis in HtrA enzymes.

A riboswitch-controlled manganese exporter (Alx) tunes intracellular Mn2+ concentration in E. coli at alkaline pH.

Cells use transition metal ions as structural components of biomolecules and cofactors in enzymatic reactions, making transition metals vital cellular components. The buildup of a particular metal ion in certain stress conditions becomes harmful to the organism due to the misincorporation of the excess ion into biomolecules, resulting in perturbed enzymatic activity or metal-catalyzed formation of reactive oxygen species. Organisms optimize metal concentration by regulating the expression of proteins that import and export that metal, often in a metal concentration-dependent manner. One such regulation mechanism is via riboswitches, which are 5'-untranslated regions (UTR) of an mRNA that undergo conformational changes to promote or inhibit the expression of the downstream gene, commonly in response to a ligand. The yybP-ykoY family of bacterial riboswitches shares a conserved aptamer domain that binds manganese (Mn2+). In E. coli, the yybP-ykoY riboswitch precedes and regulates the expression of two genes: mntP, which based on extensive genetic evidence encodes an Mn2+ exporter, and alx, which encodes a putative metal ion transporter whose cognate ligand is currently in question. Expression of alx is upregulated by both elevated intracellular concentrations of Mn2+ and alkaline pH. With metal ion measurements and gene expression studies, we demonstrate that the alkalinization of media increases cytoplasmic Mn2+ content, which in turn enhances alx expression. Alx then exports excess Mn2+ to prevent toxic buildup of the metal inside the cell, with the export activity maximal at alkaline pH. Using mutational and complementation experiments, we pinpoint a set of acidic residues in the predicted transmembrane segments of Alx that play a crucial role in its Mn2+ export. We propose that Alx-mediated Mn2+ export provides a primary protective layer that fine-tunes the cytoplasmic Mn2+ levels, especially during alkaline stress.

Association between initial complexity, frequency of refinements, treatment duration, and outcome in Invisalign orthodontic treatment.

INTRODUCTION: This study explored possible associations between treatment duration, initial complexity, outcomes in Invisalign therapy, and the number of refinements. METHODS: Three-dimensional models (initial, final, and refinements) of 355 Invisalign patients (114 males and 241 females; 33.8 ± 17.1 years) were analyzed using the Peer Assessment Rating (PAR) index questionnaire tool in the Ortho Analyzer software (version 2.0; 3Shape, Copenhagen, Denmark) to calculate the weighted total and individual PAR index scores for each component of the PAR index. Data related to demographics, treatment duration, and the number of refinements were collected. RESULTS: Treatment duration increased as the number of refinements increased. Percent of improvement was higher in PAR ≥22 group than PAR <22 with an increase in the number of refinements: 83.3% vs 73.8% for 2 refinements; 94.7% vs 91.2% for 3 refinements; and 100% vs 85.7% for ≥4 refinements. Those who achieved great improvement or improvement and those who did not were significantly different in treatment duration (P <0.001 and P = 0.027), number of refinements (≥3 refinements; P <0.001), initial occlusal severity (PAR ≥22; P <0.01 and P = 0.031). Most subjects achieved improvement after the first refinement (64.5% for PAR <22 and 78.5% for PAR ≥22). Few had ≥4 refinements, and if they did, none achieved improvement with additional refinements. CONCLUSIONS: Initial complexity for an Invisalign case is associated with treatment duration, achieved outcomes, and the number of refinements. Treatment duration increased with an increased number of refinements. Great improvement or improvement for the first time dropped to 0 if additional refinements were carried out after 3. Therefore, performing additional refinements does not necessarily mean better occlusal outcomes.

Variability associated with mandibular ramus area thickness and depth in subjects with different growth patterns, gender, and growth status.

INTRODUCTION: The purpose of this study was to quantitatively evaluate the ramus bone parameters (ramus thickness and ramus depth) for miniscrew placement. An additional aim was to compare and contrast the ramus bone parameters in growing and nongrowing male and female subjects with hyperdivergent, normodivergent, hypodivergent facial types. METHODS: Cone-beam computed tomography scans of 690 subjects were evaluated. They were classified in terms of growth status, gender, and facial type. Ramus thickness was measured as the distance from the outer (buccal) to the inner (lingual) aspects of the mandibular ramus. Ramus depth was measured as the distance from the anterior border of the ramus to the inferior alveolar nerve canal. The measurements for ramus thickness and ramus depth were performed at 3 different levels bilaterally: (1) occlusal plane (OP), (2) 5 mm above the occlusal plane (5OP), and (3) 10 mm above the occlusal plane (10OP). RESULTS: Males showed a significantly higher ramus thickness than females (P <0.05). Ramus thickness decreased significantly (P <0.05) as we moved superior from the level of OP to 5OP and 10OP in all 3 facial types in both females (growing and nongrowing) and males (growing and nongrowing). Growing females and growing males had significantly higher ramus thickness than nongrowing females and nongrowing males, respectively. Ramus depth increased as we moved higher from the OP to 10OP. Hyperdivergent facial type showed a significantly reduced ramus depth compared with hypodivergent and normodivergent facial type in growing and nongrowing males and females at all 3 locations, namely OP, 5OP, and 10OP (P <0.05). CONCLUSIONS: Because of adequate ramus depth and ramus thickness, 5OP was considered the optimal insertion site for the placement of miniscrews. Patients with a hyperdivergent facial type showed significantly reduced ramus depth than hypodivergent and normodivergent facial types. Ramus thickness in males was significantly higher than in females in all facial types.

Microbial Colonization on Elastomeric Ligatures during Orthodontic Therapeutics: An Overview.

The current review focuses on the studies conducted on the colonization of microorganisms on orthodontic ligatures during orthodontic treatment. The fixed orthodontic appliances have long been associated with an increase in plaque accumulation, bacterial colonization, and resultant enamel decalcification. Voluminous research has been carried out on the microbial colonization of even newer orthodontic materials such as elastomeric ligatures with an evidence of variably increased microbial counts during orthodontic treatment. However, conclusive material-based data for minimal microbial colonization to establish acceptance criteria for the use of elastomeric ligatures are hardly available. Thus, there is a need for further studies with dual emphasis on exploring microbial associations based on surface chemistries of different elastomers and their requisite modifications for hampering microbial biofilms to evolve efficacious oral health friendly orthodontic ligatures.

Evidence of variable bacterial colonization on coloured elastomeric ligatures during orthodontic treatment: An intermodular comparative study.

BACKGROUND: Besides, other factors, the choice of materials used as orthodontic ligatures could be one of the many tools to counter the effects of microbial adhesion, that culminates into dental ailments. Therefore, we assessed bacterial adhesion on elastomeric ligatures with special reference to coloured elastomeric rings during orthodontic treatment. MATERIAL AND METHODS: A split mouth study, involving 240 samples of different elastomeric ligatures from forty orthodontic patients possessing good oral hygiene was carried out. The archwire was ligated to the brackets on both arches with elastomeric rings (superslick, clear transparent , blue and pink) at predetermined quadrants. After six weeks, ligatures from second premolars were removed and processed for bacterial enumeration using standard techniques. Bacterial counts were also determined for stimulated saliva samples taken at 0 and 6 weeks. RESULTS: A statistically significant difference in bacterial counts was obtained amongst different elastomeric modules used. Maximum bacterial counts were found on conventional pigmented elastomeric modules, followed by Superslick module and clear module. More number of bacteria associated with the conventional pink as compared to the conventional blue pigmented modules, however it was not statistically significant. The three bacterial genera Streptococcus Staphylococcus and Aerobic Lactobacilli adhered to elastomeric modules in following predominant pattern i.e. Conventional pink>Conventional Blue>Superslick>Clear. CONCLUSIONS: The studies evidenced colour and material dependent bacterial colonization on orthodontic modules and could be an indicator of bacterial biofilm forming potential based on surface chemistries and a clinically efficacious tool to redesign conventional and modified elastomeric rings as orthodontic ligation accessories. Key words:Bacterial colonization, biofilm, coloured elastomers, orthodontic ligatures.

Growth Inhibition by External Potassium of Escherichia coli Lacking PtsN (EIIANtr) Is Caused by Potassium Limitation Mediated by YcgO.

UNLABELLED: The absence of PtsN, the terminal phosphoacceptor of the phosphotransferase system comprising PtsP-PtsO-PtsN, in Escherichia coli confers a potassium-sensitive (K(s)) phenotype as the external K(+) concentration ([K(+)]e) is increased above 5 mM. A growth-inhibitory increase in intracellular K(+) content, resulting from hyperactivated Trk-mediated K(+) uptake, is thought to cause this K(s) We provide evidence that the K(s) of the ΔptsN mutant is associated with K(+) limitation. Accordingly, the moderate K(s) displayed by the ΔptsN mutant was exacerbated in the absence of the Trk and Kup K(+) uptake transporters and was associated with reduced cellular K(+) content. Conversely, overproduction of multiple K(+) uptake proteins suppressed the K(s) Expression of PtsN variants bearing the H73A, H73D, and H73E substitutions of the phosphorylation site histidine of PtsN complemented the K(s) Absence of the predicted inner membrane protein YcgO (also called CvrA) suppressed the K(s), which was correlated with elevated cellular K(+) content in the ΔptsN mutant, but the ΔptsN mutation did not alter YcgO levels. Heterologous overexpression of ycgO also led to K(s) that was associated with reduced cellular K(+) content, exacerbated by the absence of Trk and Kup and alleviated by overproduction of Kup. Our findings are compatible with a model that postulates that K(s) in the ΔptsN mutant occurs due to K(+) limitation resulting from activation of K(+) efflux mediated by YcgO, which may be additionally stimulated by [K(+)]e, implicating a role for PtsN (possibly its dephosphorylated form) as an inhibitor of YcgO activity. IMPORTANCE: This study examines the physiological link between the phosphotransferase system comprising PtsP-PtsO-PtsN and K(+) ion metabolism in E. coli Studies on the physiological defect that renders an E. coli mutant lacking PtsN to be growth inhibited by external K(+) indicate that growth impairment results from cellular K(+) limitation that is mediated by YcgO, a predicted inner membrane protein. Additional observations suggest that dephospho-PtsN may inhibit and external K(+) may stimulate K(+) limitation mediated by YcgO. It is speculated that YcgO-mediated K(+) limitation may be an output of a response to certain stresses, which by modulating the phosphotransfer capacity of the PtsP-PtsO-PtsN phosphorelay leads to growth cessation and stress tolerance.