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Surface faulting earthquakes are known to cluster in time from historical and palaeoseismic studies, but the mechanism(s) responsible for clustering, such as fault interaction, strain-storage, and evolving dynamic topography, are poorly quantified, and hence not well understood. We present a quantified replication of observed earthquake clustering in central Italy. Six active normal faults are studied using 36 Cl cosmogenic dating, revealing out-of-phase periods of high or low surface slip-rate on neighboring structures that we interpret as earthquake clusters and anticlusters. Our calculations link stress transfer caused by slip averaged over clusters and anti-clusters on coupled fault/shear-zone structures to viscous flow laws. We show that (1) differential stress fluctuates during fault/shear-zone interactions, and (2) these fluctuations are of sufficient magnitude to produce changes in strain-rate on viscous shear zones that explain slip-rate changes on their overlying brittle faults. These results suggest that fault/shear-zone interactions are a plausible explanation for clustering, opening the path towards process-led seismic hazard assessments., (© 2022. The Author(s).)

Routine immunoassays for insulin and C-peptide have the potential to cross-react with partially processed proinsulin products, although in healthy patients these are present at such low levels that the interference is insignificant. Elevated concentrations of proinsulin and des-31,32 proinsulin arising from pathological conditions, or injected insulin analogues, however can cause significant assay interferences, complicating interpretation. Clinical diagnosis and management therefore sometimes require methods that can distinguish true insulin and C-peptide from partially processed proinsulin or injected insulin analogues. In this scenario, the high specificity of mass spectrometric analysis offers potential benefit for patient care. A high throughput targeted LC-MS/MS method was developed as a fit for purpose investigation of insulin, insulin analogues, C-peptide and proinsulin processing intermediates in plasma samples from different patient groups. Using calibration standards and bovine insulin as an internal standard, absolute concentrations of insulin and C-peptide were quantified across a nominal human plasma postprandial range and correlated strongly with immunoassay-based measurements. The ability to distinguish between insulin, insulin analogues and proinsulin intermediates in a single extraction is an improvement over existing immunological based techniques, offering the advantage of exact identification of the species being measured. The method promises to aid in the detection of circulating peptides which have previously been overlooked but may interfere with standard insulin and C-peptide immunoassays., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To analyse the peptidomics of mouse enteroendocrine cells (EECs) and human gastrointestinal (GI) tissue and identify novel gut derived peptides., Methods: High resolution nano-flow liquid chromatography mass spectrometry (LC-MS/MS) was performed on (i) flow-cytometry purified NeuroD1 positive cells from mouse and homogenised human intestinal biopsies, (ii) supernatants from primary murine intestinal cultures, (iii) intestinal homogenates from mice fed high fat diet. Candidate bioactive peptides were selected on the basis of species conservation, high expression/biosynthesis in EECs and evidence of regulated secretionin vitro. Candidate novel gut-derived peptides were chronically administered to mice to assess effects on food intake and glucose tolerance., Results: A large number of peptide fragments were identified from human and mouse, including known full-length gut hormones and enzymatic degradation products. EEC-specific peptides were largely from vesicular proteins, particularly prohormones, granins and processing enzymes, of which several exhibited regulated secretion in vitro. No regulated peptides were identified from previously unknown genes. High fat feeding particularly affected the distal colon, resulting in reduced peptide levels from GCG, PYY and INSL5. Of the two candidate novel peptides tested in vivo, a peptide from Chromogranin A (ChgA 435-462a) had no measurable effect, but a progastrin-derived peptide (Gast p59-79), modestly improved glucose tolerance in lean mice., Conclusion: LC-MS/MS peptidomic analysis of murine EECs and human GI tissue identified the spectrum of peptides produced by EECs, including a potential novel gut hormone, Gast p59-79, with minor effects on glucose tolerance., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Meal ingestion triggers secretion of a variety of gut and endocrine peptides important in diabetes research which are routinely measured by immunoassays. However, similarities between some peptides (glucagon, oxyntomodulin and glicentin) can cause specificity issues with immunoassays. We used a liquid chromatography/tandem mass spectrometry (LC/MS/MS) methodology to unambiguously monitor multiple gut peptides in human plasma., Methods: A simple acetonitrile-based protein precipitation step, followed by evaporation and solid-phase extraction, removed high-abundance proteins from samples prior to nano-LC/MS/MS analysis on an Orbitrap Q-Exactive Plus mass spectrometer using a data-dependent methodology. Database searching using PEAKS identified multiple gut-derived peptides, including peptides in the mid-pg/mL range. The relative levels of these and previously characterised peptides were assessed in plasma samples from gastrectomised and control subjects during an oral glucose tolerance test., Results: Analysis of plasma extracts revealed significantly elevated levels of a number of peptides following glucose ingestion in subjects who had undergone gastrectomy compared with controls. These included GLP-1(7-36), GLP-1(9-36), glicentin, oxyntomodulin, GIP(1-42), GIP(3-42), PYY(1-36), PYY(3-36), neurotensin, insulin and C-peptide. Motilin levels decreased following glucose ingestion. Results showed good correlation with immunoassay-derived concentrations of some peptides in the same samples. The gastrectomy group also had higher, but non-glucose-dependent, circulating levels of peptides from PIGR and DMBT1., Conclusions: Overall, the approach showed that a fast, generic and reproducible LC/MS/MS methodology requiring only a small volume of plasma was capable of the multiplexed detection of a variety of diabetes-related peptides., (© 2020 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Whether the stress-loading of faults to failure in earthquakes appears to be random or to an extent explainable, given constraints on fault/shear-zone interaction and the build-up and release of stress over many earthquake cycles, is a key question for seismic hazard assessment. Here we investigate earthquake recurrence for a system of 25 active normal faults arranged predominantly along strike from each other, allowing us to isolate the effects of stress-loading due to regional strain versus across- and along-strike fault interaction. We calculate stress changes over 6 centuries due to interseismic loading and 25 > Mw 5.5 earthquakes. Where only one fault exists across strike, stress-loading is dominated by the regional tectonics through slip on underlying shear zones and fault planes have spatially smooth stress with predominantly time-dependent stress increase. Conversely, where faults are stress-loaded by across-strike fault interactions, fault planes have more irregular stress patterns and interaction-influenced stress loading histories. Stress-loading to failure in earthquakes is not the same for all faults and is dependent on the geometry of the fault/shear-zone system.

Guanylin, a peptide implicated in regulation of intestinal fluid secretion, is expressed in the mucosa, but the exact cellular origin remains controversial. In a new transgenic mouse model fluorescent reporter protein expression driven by the proguanylin promoter was observed throughout the small intestine and colon in goblet and Paneth(-like) cells and, except in duodenum, in mature enterocytes. In Ussing chamber experiments employing both human and mouse intestinal tissue, proguanylin was released predominantly in the luminal direction. Measurements of proguanylin expression and secretion in cell lines and organoids indicated that secretion is largely constitutive and requires ER to Golgi transport but was not acutely regulated by salt or other stimuli. Using a newly-developed proguanylin assay, we found plasma levels to be raised in humans after total gastrectomy or intestinal transplantation, but largely unresponsive to nutrient ingestion. By LC-MS/MS we identified processed forms in tissue and luminal extracts, but in plasma we only detected full-length proguanylin. Our transgenic approach provides information about the cellular origins of proguanylin, complementing previous immunohistochemical and in-situ hybridisation results. The identification of processed forms of proguanylin in the intestinal lumen but not in plasma supports the notion that the primary site of action is the gut itself.

Successive locations of individual large earthquakes (M w  > 5.5) over years to centuries can be difficult to explain with simple Coulomb stress transfer (CST) because it is common for seismicity to circumvent nearest-neighbour along-strike faults where coseismic CST is greatest. We demonstrate that Coulomb pre-stress (the cumulative CST from multiple earthquakes and interseismic loading on non-planar faults) may explain this, evidenced by study of a 667-year historical record of earthquakes in central Italy. Heterogeneity in Coulomb pre-stresses across the fault system is >±50 bars, whereas coseismic CST is <±2 bars, so the latter will rarely overwhelm the former, explaining why historical earthquakes rarely rupture nearest neighbor faults. However, earthquakes do tend to occur where the cumulative coseismic and interseismic CST is positive, although there are notable examples where earthquake propagate across negatively stressed portions of faults. Hence Coulomb pre-stress calculated for non-planar faults is an ignored yet vital factor for earthquake triggering.

Enteroendocrine cells (EECs) produce hormones such as glucagon-like peptide 1 and peptide YY that regulate food absorption, insulin secretion, and appetite. Based on the success of glucagon-like peptide 1-based therapies for type 2 diabetes and obesity, EECs are themselves the focus of drug discovery programs to enhance gut hormone secretion. The aim of this study was to identify the transcriptome and peptidome of human EECs and to provide a cross-species comparison between humans and mice. By RNA sequencing of human EECs purified by flow cytometry after cell fixation and staining, we present a first transcriptomic analysis of human EEC populations and demonstrate a strong correlation with murine counterparts. RNA sequencing was deep enough to enable identification of low-abundance transcripts such as G-protein-coupled receptors and ion channels, revealing expression in human EECs of G-protein-coupled receptors previously found to play roles in postprandial nutrient detection. With liquid chromatography-tandem mass spectrometry, we profiled the gradients of peptide hormones along the human and mouse gut, including their sequences and posttranslational modifications. The transcriptomic and peptidomic profiles of human and mouse EECs and cross-species comparison will be valuable tools for drug discovery programs and for understanding human metabolism and the endocrine impacts of bariatric surgery., (© 2019 by the American Diabetes Association.)

The 28th December 1908 Messina earthquake (Mw 7.1), Italy, caused >80,000 deaths and transformed earthquake science by triggering the study of earthquake environmental effects worldwide, yet its source is still a matter of debate. To constrain the geometry and kinematics of the earthquake we use elastic half-space modelling on non-planar faults, constrained by the geology and geomorphology of the Messina Strait, to replicate levelling data from 1907-1909. The novelty of our approach is that we (a) recognise the similarity between the pattern of vertical motions and that of other normal faulting earthquakes, and (b) for the first time model the levelling data using the location and geometry of a well-known offshore capable fault. Our results indicate slip on the capable fault with a dip to the east of 70° and 5 m dip-slip at depth, with slip propagating to the surface on the sea bed. Our work emphasises that geological and geomorphological observations supporting maps of capable non-planar faults should not be ignored when attempting to identify the sources of major earthquakes.

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression. Circulating GDF15 levels manifest very modest changes in response to moderate caloric surpluses or deficits in mice or humans, differentiating it from classical intestinally derived satiety hormones and leptin. However, GDF15 levels do increase following sustained high-fat feeding or dietary amino acid imbalance in mice. We demonstrate that GDF15 expression is regulated by the integrated stress response and is induced in selected tissues in mice in these settings. Finally, we show that pharmacological GDF15 administration to mice can trigger conditioned taste aversion, suggesting that GDF15 may induce an aversive response to nutritional stress., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Bariatric surgery is widely used to treat obesity and improves type 2 diabetes beyond expectations from the degree of weight loss. Elevated post-prandial concentrations of glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and insulin are widely reported, but the importance of GLP-1 in post-bariatric physiology remains debated. Here, we show that GLP-1 is a major driver of insulin secretion after bariatric surgery, as demonstrated by blocking GLP-1 receptors (GLP1Rs) post-gastrectomy in lean humans using Exendin-9 or in mice using an anti-GLP1R antibody. Transcriptomics and peptidomics analyses revealed that human and mouse enteroendocrine cells were unaltered post-surgery; instead, we found that elevated plasma GLP-1 and PYY correlated with increased nutrient delivery to the distal gut in mice. We conclude that increased GLP-1 secretion after bariatric surgery arises from rapid nutrient delivery to the distal gut and is a key driver of enhanced insulin secretion., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Diagnosis of pancreatic neuroendocrine tumours requires the study of patient plasma with multiple immunoassays, using multiple aliquots of plasma. The application of mass spectrometry based techniques could reduce the cost and amount of plasma required for diagnosis., Methods: Plasma samples from two patients with pancreatic neuroendocrine tumours were extracted using an established acetonitrile-based plasma peptide enrichment strategy. The circulating peptidome was characterised using nano and high flow rate liquid chromatography/mass spectrometry (LC/MS) analyses. To assess the diagnostic potential of the analytical approach, a large sample batch (68 plasmas) from control subjects, and aliquots from subjects harbouring two different types of pancreatic neuroendocrine tumour (insulinoma and glucagonoma), were analysed using a 10-min LC/MS peptide screen., Results: The untargeted plasma peptidomics approach identified peptides derived from the glucagon prohormone, chromogranin A, chromogranin B and other peptide hormones and proteins related to control of peptide secretion. The glucagon prohormone derived peptides that were detected were compared against putative peptides that were identified using multiple antibody pairs against glucagon peptides. Comparison of the plasma samples for relative levels of selected peptides showed clear separation between the glucagonoma and the insulinoma and control samples., Conclusions: The combination of the organic solvent extraction methodology with high flow rate analysis could potentially be used to aid diagnosis and monitor treatment of patients with functioning pancreatic neuroendocrine tumours. However, significant validation will be required before this approach can be clinically applied., (© 2018 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.)

Background: Altered enteroendocrine hormone responses are widely believed to underlie the beneficial effects of bariatric surgery in type 2 diabetes. While elevated postprandial glucagon-like peptide-1 (GLP-1) is considered one of the mediators, increased postprandial glucagon levels have recently been implicated., Objectives: We investigated hormonal responses in lean patients after prophylactic total gastrectomy (PTG), as a model of Roux-en-Y gastric bypass without the confounding effects of obesity or massive weight loss., Setting: University hospital, United Kingdom., Methods: Ten participants after PTG and 9 healthy volunteers were recruited for oral glucose tolerance tests. Plasma glucose, insulin, GLP-1, peptide YY, glucose-dependent insulinotropic-polypeptide, glucagon, oxyntomodulin, glucagon (1-61) , and glicentin levels were assessed using immunoassays and/or mass spectrometry., Results: PTG participants exhibited accelerated plasma glucose appearance, followed, in 3 of 10 cases, by hypoglycemia (<3 mM glucose). Plasma GLP-1, peptide YY, glucose-dependent insulinotropic-polypeptide, glicentin, and oxyntomodulin responses were elevated, and glucagon appeared to rise in PTG participants when measured with a glucagon-specific enzyme-linked immunosorbent assay. We revisited the specificity of this assay, and demonstrated significant cross-reactivity with glicentin and oxyntomodulin at concentrations observed in PTG plasma. Reassessment of glucagon with the same assay using a modified protocol, and by liquid chromatography-mass spectrometry, demonstrated suppression of glucagon secretion after oral glucose tolerance tests in both PTG and control cohorts., Conclusions: Care should be taken when assessing glucagon levels in the presence of elevated plasma levels of other proglucagon products. Substantial elevation of GLP-1 and insulin responses after PTG likely contribute to the observed hypoglycemia, and mirror similar hormone levels and complications observed in bariatric weight loss patients., (Copyright © 2018 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

The temporal evolution of slip on surface ruptures during an earthquake is important for assessing fault displacement, defining seismic hazard and for predicting ground motion. However, measurements of near-field surface displacement at high temporal resolution are elusive. We present a novel record of near-field co-seismic displacement, measured with 1-second temporal resolution during the 30 th October 2016 M w 6.6 Vettore earthquake (Central Italy), using low-cost Global Navigation Satellite System (GNSS) receivers located in the footwall and hangingwall of the Mt. Vettore - Mt. Bove fault system, close to new surface ruptures. We observe a clear temporal and spatial link between our near-field record and InSAR, far-field GPS data, regional measurements from the Italian Strong Motion and National Seismic networks, and field measurements of surface ruptures. Comparison of these datasets illustrates that the observed surface ruptures are the propagation of slip from depth on a surface rupturing (i.e. capable) fault array, as a direct and immediate response to the 30 th October earthquake. Large near-field displacement ceased within 6-8 seconds of the origin time, implying that shaking induced gravitational processes were not the primary driving mechanism. We demonstrate that low-cost GNSS is an accurate monitoring tool when installed as custom-made, short-baseline networks.

Many areas of the Earth's crust deform by distributed extensional faulting and complex fault interactions are often observed. Geodetic data generally indicate a simpler picture of continuum deformation over decades but relating this behaviour to earthquake occurrence over centuries, given numerous potentially active faults, remains a global problem in hazard assessment. We address this challenge for an array of seismogenic faults in the central Italian Apennines, where crustal extension and devastating earthquakes occur in response to regional surface uplift. We constrain fault slip-rates since ~18 ka using variations in cosmogenic 36 Cl measured on bedrock scarps, mapped using LiDAR and ground penetrating radar, and compare these rates to those inferred from geodesy. The 36 Cl data reveal that individual faults typically accumulate meters of displacement relatively rapidly over several thousand years, separated by similar length time intervals when slip-rates are much lower, and activity shifts between faults across strike. Our rates agree with continuum deformation rates when averaged over long spatial or temporal scales (10 4  yr; 10 2  km) but over shorter timescales most of the deformation may be accommodated by <30% of the across-strike fault array. We attribute the shifts in activity to temporal variations in the mechanical work of faulting.

The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal "LytTR" DNA-binding domain. Here the RcoM(Bx)-1 protein from Burkholderia xenovorans LB400 was heterologously expressed and then purified in a form with minimal bound CO (~10%) and was found to stably bind this effector with a nanomolar affinity. DNase I protection assays demonstrated that the CO-associated form binds with a micromolar affinity to two ~60-bp DNA regions, each comprised of a novel set of three direct-repeat binding sites spaced 21 bp apart on center. Binding to each region was independent, while binding to the triplet binding sites within a region was cooperative, depended upon spacing and sequence, and was marked by phased DNase I hyperactivity and protection patterns consistent with considerable changes in the DNA conformation of the nucleoprotein complex. Each protected binding site spanned a conserved motif (5'-TTnnnG-3') that was present, in triplicate, in putative RcoM-binding regions of more than a dozen organisms. In vivo screens confirmed the functional importance of the conserved "TTnnnG" motif residues and their triplet arrangement and were also used to determine an improved binding motif [5'-CnnC(C/A)(G/A)TTCAnG-3'] that more closely corresponds to canonical LytTR domain/DNA-binding sites. A low-affinity but CO-dependent binding of RcoM(Bx)-1 to a variety of DNA probes was demonstrated in vitro. We posit that for the RcoM(Bx)-1 protein, the high CO affinity combined with multiple low-affinity DNA-binding events constitutes a transcriptional "accumulating switch" that senses low but persistent CO levels.

The CO-responsive transcriptional regulator RcoM from Burkholderia xenovorans (BxRcoM) was recently identified as a Cys(thiolate)-ligated heme protein that undergoes a redox-mediated ligand switch; however, the Cys bound to the Fe(III) heme was not identified. To that end, we generated and purified three Cys-to-Ser variants of BxRcoM-2--C94S, C127S, and C130S--and examined their spectroscopic properties in order to identify the native Cys(thiolate) ligand. Electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies demonstrate that the C127S and C130S variants, like wild-type BxRcoM-2, bind a six-coordinate low-spin Fe(III) heme using a Cys/His ligation motif. In contrast, electronic absorption and resonance Raman spectra of the C94S variant are most consistent with a mixture of five-coordinate high-spin and six-coordinate low-spin Fe(III) heme, neither of which are ligated by a Cys(thiolate) ligand. The EPR spectrum of C94S is dominated by a large, axial high-spin Fe(III) signal, confirming that the native ligation motif is not maintained in this variant. Together, these data reveal that Cys(94) is the distal Fe(III) heme ligand in BxRcoM-2; by sequence alignment, Cys(94) is also implicated as the distal Fe(III) heme ligand in BxRcoM-1, another homologue found in the same organism.

Vfr, a transcription factor homologous to the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP), regulates many aspects of virulence in Pseudomonas aeruginosa. Vfr, like CRP, binds to cAMP and then recognizes its target DNA and activates transcription. Here we report that Vfr has important functional differences from CRP in terms of ligand sensing and response. First, Vfr has a significantly higher cAMP affinity than does CRP, which might explain the mysteriously unidirectional functional complementation between the two proteins (S. E. H. West et al., J. Bacteriol. 176:7532-7542, 1994). Second, Vfr is activated by both cAMP and cGMP, while CRP is specific to cAMP. Mutagenic analyses show that Thr133 (analogous to Ser128 of CRP) is the key residue for both of these distinct Vfr properties. On the other hand, substitutions that cause cAMP-independent activity in Vfr are similar to those seen in CRP, suggesting that a common cAMP activation mechanism is present. In the course of these analyses, we found a remarkable class of Vfr variants that have completely reversed the regulatory logic of the protein: they are active in DNA binding without cAMP and are strongly inhibited by cAMP. The physiological impact of Vfr's ligand sensing and response is discussed, as is a plausible basis for the fundamental change in protein allostery in the novel group of Vfr variants., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first step of CO(2) fixation in the Calvin-Benson-Bassham (CBB) cycle. Besides its function in fixing CO(2) to support photoautotrophic growth, the CBB cycle is also important under photoheterotrophic growth conditions in purple nonsulfur photosynthetic bacteria. It has been assumed that the poor photoheterotrophic growth of RubisCO-deficient strains was due to the accumulation of excess intracellular reductant, which implied that the CBB cycle is important for maintaining the redox balance under these conditions. However, we present analyses of cbbM mutants in Rhodospirillum rubrum that indicate that toxicity is the result of an elevated intracellular pool of ribulose-1,5-bisphosphate (RuBP). There is a redox effect on growth, but it is apparently an indirect effect on the accumulation of RuBP, perhaps by the regulation of the activities of enzymes involved in RuBP regeneration. Our studies also show that the CBB cycle is not essential for R. rubrum to grow under photoheterotrophic conditions and that its role in controlling the redox balance needs to be further elucidated. Finally, we also show that CbbR is a positive transcriptional regulator of the cbb operon (cbbEFPT) in R. rubrum, as seen with related organisms, and define the transcriptional organization of the cbb genes.

Rhodospirillum rubrum (Esmarch 1887) Molisch 1907 is the type species of the genus Rhodospirillum, which is the type genus of the family Rhodospirillaceae in the class Alphaproteobacteria. The species is of special interest because it is an anoxygenic phototroph that produces extracellular elemental sulfur (instead of oxygen) while harvesting light. It contains one of the most simple photosynthetic systems currently known, lacking light harvesting complex 2. Strain S1(T) can grow on carbon monoxide as sole energy source. With currently over 1,750 PubMed entries, R. rubrum is one of the most intensively studied microbial species, in particular for physiological and genetic studies. Next to R. centenum strain SW, the genome sequence of strain S1(T) is only the second genome of a member of the genus Rhodospirillum to be published, but the first type strain genome from the genus. The 4,352,825 bp long chromosome and 53,732 bp plasmid with a total of 3,850 protein-coding and 83 RNA genes were sequenced as part of the DOE Joint Genome Institute Program DOEM 2002.

Low levels of carbon monoxide inhibit the N(2)-dependent growth of Rhodospirillum rubrum unless the ∼100-residue CowN protein is expressed. Expression requires the CO-responsive regulator RcoM and is maximal in cells grown in the presence of CO and a poor nitrogen source, consistent with the role of CowN in N(2) fixation.

Nitrogenase not only reduces atmospheric nitrogen to ammonia, but also reduces protons to hydrogen (H(2)). The nitrogenase system is the primary means of H(2) production under photosynthetic and nitrogen-limiting conditions in many photosynthetic bacteria, including Rhodospirillum rubrum. The efficiency of this biological H(2) production largely depends on the nitrogenase enzyme and the availability of ATP and electrons in the cell. Previous studies showed that blockage of the CO(2) fixation pathway in R. rubrum induced nitrogenase activity even in the presence of ammonium, presumably to remove excess reductant in the cell. We report here the re-characterization of cbbM mutants in R. rubrum to study the effect of Rubisco on H(2) production. Our newly constructed cbbM mutants grew poorly in malate medium under anaerobic conditions. However, the introduction of constitutively active NifA (NifA*), the transcriptional activator of the nitrogen fixation (nif) genes, allows cbbM mutants to dissipate the excess reductant through the nitrogenase system and improves their growth. Interestingly, we found that the deletion of cbbM alters the posttranslational regulation of nitrogenase activity, resulting in partially active nitrogenase in the presence of ammonium. The combination of mutations in nifA, draT and cbbM greatly increased H(2) production of R. rubrum, especially in the presence of excess of ammonium. Furthermore, these mutants are able to produce H(2) over a much longer time frame than the wild type, increasing the potential of these recombinant strains for the biological production of H(2).

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) protein; P(II) in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.

The protein Clp from Xanthomonas axonopodis pv. citri regulates pathogenesis and is a member of the CRP (cyclic AMP receptor protein) superfamily. We show that unlike the DNA-binding activity of other members of this family, the DNA-binding activity of Clp is allosterically inhibited by its effector and that cyclic di-GMP serves as that effector at physiological concentrations.

Nitrogenase activity in Rhodospirillum rubrum and in some other photosynthetic bacteria is regulated in part by the availability of light. This regulation is through a posttranslational modification system that is itself regulated by P(II) homologs in the cell. P(II) is one of the most broadly distributed regulatory proteins in nature and directly or indirectly senses nitrogen and carbon signals in the cell. However, its possible role in responding to light availability remains unclear. Because P(II) binds ATP, we tested the hypothesis that removal of light would affect P(II) by changing intracellular ATP levels, and this in turn would affect the regulation of nitrogenase activity. This in vivo test involved a variety of different methods for the measurement of ATP, as well as the deliberate perturbation of intracellular ATP levels by chemical and genetic means. To our surprise, we found fairly normal levels of nitrogenase activity and posttranslational regulation of nitrogenase even under conditions of drastically reduced ATP levels. This indicates that low ATP levels have no more than a modest impact on the P(II)-mediated regulation of NifA activity and on the posttranslational regulation of nitrogenase activity. The relatively high nitrogenase activity also shows that the ATP-dependent electron flux from dinitrogenase reductase to dinitrogenase is also surprisingly insensitive to a depleted ATP level. These in vivo results disprove the simple model of ATP as the key energy signal to P(II) under these conditions. We currently suppose that the ratio of ADP/ATP might be the relevant signal, as suggested by a number of recent in vitro analyses.

The activity of NifA, the transcriptional activator of the nitrogen fixation (nif) gene, is tightly regulated in response to ammonium and oxygen. However, the mechanisms for the regulation of NifA activity are quite different among various nitrogen-fixing bacteria. Unlike the well-studied NifL-NifA regulatory systems in Klebsiella pneumoniae and Azotobacter vinelandii, in Rhodospirillum rubrum NifA is activated by a direct protein-protein interaction with the uridylylated form of GlnB, which in turn causes a conformational change in NifA. We report the identification of several substitutions in the N-terminal GAF domain of R. rubrum NifA that allow NifA to be activated in the absence of GlnB. Presumably these substitutions cause conformational changes in NifA necessary for activation, without interaction with GlnB. We also found that wild-type NifA can be activated in a GlnB-independent manner under certain growth conditions, suggesting that some other effector(s) can also activate NifA. An attempt to use Tn5 mutagenesis to obtain mutants that altered the pool of these presumptive effector(s) failed, though much rarer spontaneous mutations in nifA were detected. This suggests that the necessary alteration of the pool of effector(s) for NifA activation cannot be obtained by knockout mutations.

Spectroscopic characterization of the newly discovered heme-PAS domain sensor protein BxRcoM-2 reveals that this protein undergoes redox-dependent ligand switching and CO- and NO-induced ligand displacement. The aerobic bacterium Burkholderia xenovorans expresses two homologous heme-containing proteins that promote CO-dependent transcription in vivo. These regulators of CO metabolism, BxRcoM-1 and BxRcoM-2, are gas-responsive heme-PAS domain proteins like mammalian neuronal PAS domain protein 2 (NPAS2) and the direct oxygen sensor from Escherichia coli ( EcDos). BxRcoM-2 was studied using electronic absorption, MCD, resonance Raman, and EPR spectroscopies. In the Fe(III) oxidation state, the heme is low-spin and six-coordinate with a cysteine(thiolate) as one of the two ligands. The sixth ligand is a histidine (His (74)), which is present in all states of the protein that were studied. Reduction to the Fe(II) oxidation state results in replacement of the cysteine(thiolate) with a neutral thioether ligand, Met (104). CO and NO bind to the Fe(II) BxRcoM-2 heme opposite the histidine ligand. Thus, BxRcoM-2 employs coordination state changes similar to those known for CO-sensing CooA, with redox-dependent loss of a cysteine(thiolate) ligand and displacement of a relatively weakly bound axial ligand by the effector gas molecule. Like EcDos, the weakly bound axial ligand that is displaced is methionine.

Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 A) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.

Genomic analysis suggested the existence of a CO-sensing bacterial transcriptional regulator that couples an N-terminal PAS fold domain to a C-terminal DNA-binding LytTR domain. UV/visible-light spectral analyses of heterologously expressed, purified full-length proteins indicated that they contained a hexacoordinated b-type heme moiety that avidly binds CO and NO. Studies of protein variants strongly suggested that the PAS domain residues His74 and Met104 serve as the heme Fe(II) axial ligands, with displacement of Met104 upon binding of the gaseous effectors. Two RcoM (regulator of CO metabolism) homologs were shown to function in vivo as CO sensors capable of regulating an aerobic CO oxidation (cox) regulon. The genetic linkage of rcoM with both aerobic (cox) and anaerobic (coo) CO oxidation systems suggests that in different organisms RcoM proteins may control either regulon type.

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions.

The nitrogen regulatory protein P(II) and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P(II) homolog, GlnJ, has higher affinity for an AmtB(1)-containing membrane than the other two P(II) homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB(1) complex in the presence of ATP and 2-KG, supporting a role for P(II) as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K(d) of 2-KG for GlnJ bound to AmtB(1) is 340 microM, which is higher than that required for uridylylation of GlnJ in vitro, about 5 microM. This supports a model where multiple 2-KG and ATP molecules must bind a P(II) trimer to stimulate release of P(II) from AmtB(1), in contrast to the lower 2-KG requirement for productive uridylylation of P(II) by GlnD.

The CooA family of proteins are prokaryotic CO-sensing transcription factors that regulate the expression of genes involved in the utilization of CO as an energy source. They are homodimeric proteins that contain two hemes. Each monomer contains an N-terminal heme-binding domain and a C-terminal DNA-binding domain. Binding of CO to the heme leads to activation by a large reorientation of the DNA-binding domain such that the DNA-binding domain is in position for specific DNA recognition. The crystal structure of CooA from Rhodospirillum rubrum [RrCooA; Lanzilotta et al. (2000), Nature Struct. Biol. 7, 876-880] in the inactive CO-free off-state shows that the N-terminal Pro residue of monomer A coordinates the heme of monomer B and vice versa. It now appears that the CO replaces the Pro ligand and that this change is coupled to the activation process. However, precisely how the replacement of the Pro ligand by CO results in structural changes some 25 A from the CO-binding site remains unknown. Here, the structure of a CooA variant from the thermophilic bacterium Carboxydothermus hydrogenoformans (ChCooA) is reported in which one monomer is fully in the on-state. The N-terminal region that is displaced by CO binding is now positioned between the heme-binding and DNA-binding domains, which requires movement of the N-terminus by approximately 20 A and thus serves as a bridge between the two domains that helps to orient the DNA-binding domain in position for DNA binding.

The cAMP receptor protein (CRP) of Escherichia coli exists in an equilibrium between active and inactive forms, and the effector, cAMP, shifts that equilibrium to the active form, thereby allowing DNA binding. For this equilibrium shift, a C-helix repositioning around the C-helix residues Thr-127 and Ser-128 has been reported as a critical local event along with proper beta4/beta5 positioning. Here we show that another C-helix residue, Arg-123, has a unique role in cAMP-dependent CRP activation in two different ways. First, Arg-123 is important for proper cAMP affinity, although it is not critical for the conformational change with saturating amounts of cAMP. Second, Arg-123 is optimal for stabilizing the inactive conformation of CRP when cAMP is absent, thereby allowing a maximal range of regulation by cAMP. However, Arg-123 does not appear to be critical for a functional response to cAMP, as has been proposed previously (Berman, H. M., Ten Eyck, L. F., Goodsell, D. S., Haste, N. M., Korney, A., and Taylor, S. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 45-50). Based on mutagenic evidence, we also propose the basis for the stabilization of the inactive form to be through a salt interaction between Asp-68 and Arg-123.

CooA is a transcription factor from Rhodospirillum rubrum that is regulated by the binding of the small molecule effector, CO, to a heme moiety in the protein. The heme in CooA is axially ligated by two endogenous donors in the Fe(III) and Fe(II) states of the protein, and CO binding to the Fe(II) state results in replacement of the distal ligand. Reduction of the heme in the absence of CO results in a ligand switch on the proximal side, in which a cysteine thiolate in the Fe(III) state is replaced by a histidine in the Fe(II) state. Recently, a variant, termed RW CooA, was designed to respond to a new effector; Fe(II) RW CooA shows high specificity and induced DNA-binding activity in the presence of imidazole. Spectroscopic characterization of the imidazole adducts of RW CooA revealed that, unlike CO, imidazole binds to both Fe(III) RW CooA and Fe(II) RW CooA. The spectral characteristics are consistent with normal function of the redox-mediated ligand switch; Fe(III)-imidazole RW CooA bears a thiolate ligand and Fe(II)-imidazole RW CooA bears a neutral donor ligand. Since the effector binds to both redox states, RW CooA was used to probe the role of the redox-mediated ligand switch in the CooA activation mechanism. Functional studies of Fe(III)-imidazole and Fe(II)-imidazole ligated RW CooA demonstrate that only the Fe(II)-imidazole form is active for DNA binding. Thus, the ligand switch is essential for the activating conformational change and may prevent aberrant activation of CooA by other neutral diatomic molecules.

The heme-containing protein CooA of Rhodospirillum rubrum regulates the expression of genes involved in CO oxidation. CooA binds its target DNA sequence in response to CO binding to its heme. Activity measurements and resonance Raman (RR) spectra are reported for CooA variants that bind DNA even in the absence of CO, those in which the wild-type residues at the 121-126 positions, TSCMRT, are replaced by the residues AYLLRL or RYLLRL, and also for variants that bind DNA poorly in the presence of CO, such as L120S and L120F. The Fe-C and C-O stretching resonance Raman (RR) frequencies of all CooAs examined deviate from the expected back-bonding correlation in a manner indicating weakening of the Fe-His-77 proximal ligand bond, and the extent of weakening correlates positively with DNA binding activity. The (A/R) YLLRL variants have detectable populations of a 5-coordinate heme resulting from partial dissociation of the endogenous distal ligand, Pro-2. Selective excitation of this population reveals downshifted Fe-His-77-stretching RR bands, confirming the proximal bond weakening. These results support our previous hypothesis that the conformational change required for DNA binding is initiated by displacement of the heme into an adjacent hydrophobic cavity once CO displaces the Pro-2 ligand. Examination of the crystal structure reveals a physical basis for these results, and a mechanism is proposed to link heme displacement to conformational change.

CooA of Rhodospirillum rubrum is a CO-sensing, heme-containing transcriptional activator that regulates the expression of the genes responsible for CO oxidation. We randomized the codons for residues 75-77 of CooA which include two proximal heme ligands, screened for both CO-dependent and CO-independent variants, and characterized in vivo and in vitro properties of selected CooA variants. The analysis showed that small residues at position 75 are critical and that, as previously suspected, His77 is absolutely necessary for CO responsiveness of CooA. Many hemeless variants altered at those residues were found to be constitutively active. We propose that proximal heme pocket residues of wild-type CooA have important role in stabilizing both active and inactive heme positions for its CO-sensing function.

The P(II) family of proteins is found in all three domains of life and serves as a central regulator of the function of proteins involved in nitrogen metabolism, reflecting the nitrogen and carbon balance in the cell. The genetic elimination of the genes encoding these proteins typically leads to severe growth problems, but the basis of this effect has been unknown except with Escherichia coli. We have analysed a number of the suppressor mutations that correct such growth problems in Rhodospirillum rubrum mutants lacking P(II) proteins. These suppressors map to nifR3, ntrB, ntrC, amtB(1) and the glnA region and all have the common property of decreasing total activity of glutamine synthetase (GS). We also show that GS activity is very high in the poorly growing parental strains lacking P(II) proteins. Consistent with this, overexpression of GS in glnE mutants (lacking adenylyltransferase activity) also causes poor growth. All of these results strongly imply that elevated GS activity is the causative basis for the poor growth seen in R. rubrum mutants lacking P(II) and presumably in mutants of some other organisms with similar genotypes. The result underscores the importance of proper regulation of GS activity for cell growth.

The AmtB protein transports uncharged NH(3) into the cell, but it also interacts with the nitrogen regulatory protein P(II), which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P(II) homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB(1) and amtB(2), have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB(1) are defective in their response to both addition and darkness, while mutants lacking AmtB(2) show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB(1) is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB(1)-P(II) regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P(II) is proposed. Other results demonstrate a degree of specificity in interaction of AmtB(1) with the different P(II) homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P(II) proteins regulate processes involved in nitrogen acquisition and utilization.

In Rhodospirillum rubrum, NifA, the transcriptional activator for the nif genes, is posttranslationally activated only by the uridylylated form of GlnB, one of three P(II) homologs in the organism. We have used the yeast two-hybrid system to detect variants of GlnB that interact better with NifA than does wild-type GlnB. When examined for physiological effects in R. rubrum, these GlnB* variants activated NifA in the presence of NH(4)(+), which normally blocks NifA activation completely, and in the absence of GlnD, whose uridylylation of GlnB is also normally essential for NifA activation. When these variants were tested in the two-hybrid system for their interaction with NtrB, a receptor that should interact with the nonuridylylated form of GlnB, they were uniformly weaker than wild-type GlnB in that interaction. When expressed in R. rubrum either as single-copy integrants or on multiple-copy plasmids, these variants were also dramatically altered in terms of their ability to regulate several other receptors involved in nitrogen metabolism, including GlnE, NtrB/NtrC, and DRAT (dinitrogenase reductase ADP-ribosyl transferase)-DRAG (dinitrogenase reductase-activating glycohydrolase). The consistent pattern throughout is that these GlnB variants partially mimic the uridylylated form of wild-type GlnB, even under nitrogen-excess conditions and in strains lacking GlnD. The results suggest that the role of uridylylation of GlnB is primarily to shift the equilibrium of GlnB from a "nitrogen-sufficient" form to a "nitrogen-deficient" form, each of which interacts with different but overlapping receptor proteins in the cell. These GlnB variants apparently shift that equilibrium through direct structural changes.

CooA, the CO-sensing heme protein from Rhodospirillum rubrum, regulates the expression of genes that encode a CO-oxidation system, allowing R. rubrum to use CO as a sole energy source. To better understand the gas-sensing regulation mechanism used by R. rubrum CooA and its homologs in other organisms, we characterized spectroscopically and functionally the Fe(II), Fe(II)-NO, and Fe(II)-CO forms of CooA from Carboxydothermus hydrogenoformans. Surprisingly, and unlike R. rubrum CooA, C. hydrogenoformans CooA binds NO to form a six-coordinate Fe(II)-NO heme that is active for DNA binding in vitro and in vivo. In contrast, R. rubrum CooA, which is exquisitely specific for CO, forms a five-coordinate Fe(II)-NO adduct that is inactive for DNA binding. Based on analyses of protein variants and temperature studies, NO-dependent DNA binding by C. hydrogenoformans CooA is proposed to result from a greater apparent stability of the six-coordinate Fe(II)-NO adduct at room temperature. Results from the present study strengthen the proposal that CO specificity in the CooA activation mechanism is based on the requirement for a small, neutral distal ligand, which in turn affects the relative positioning of the ligand-bound heme.