Coulomb pre-stress and fault bends are ignored yet vital factors for earthquake triggering and hazard.

Successive locations of individual large earthquakes (Mw > 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.

Slip on a mapped normal fault for the 28th December 1908 Messina earthquake (Mw 7.1) in Italy.

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.

Orogen-scale uplift in the central Italian Apennines drives episodic behaviour of earthquake faults.

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 36Cl measured on bedrock scarps, mapped using LiDAR and ground penetrating radar, and compare these rates to those inferred from geodesy. The 36Cl 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 (104 yr; 102 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.

Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status.

The GlnB (P(II)) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other P(II) homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of dinitrogenase reductase catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

Effects of specific amino acid substitutions on activities of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum.

Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

The heme pocket afforded by Gly117 is crucial for proper heme ligation and activity of CooA.

CooA, a CO-sensing homodimeric transcription activator from Rhodospirillum rubrum, undergoes a conformational change in response to CO binding to its heme prosthetic group that allows it to bind specific DNA sequences. In a recent structural study (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880), it was suggested that CO binding to CooA results in a modest repositioning of the C-helices that serve as the dimer interface. Gly(117) is one of the residues on the C-helix within 7 A of the heme iron on the Pro(2) side of the heme in CooA. Analysis of a series of Gly(117) variants revealed altered CO-sensing function and heme ligation states dependent on the size of the substituted amino acid at this position; bulky substitutions perturbed CooA both spectrally and functionally. A combination of spectroscopic and mutagenic studies showed that a representative Gly(117) variant, G117I CooA, was specifically perturbed in its Pro(2) ligation in both Fe(III) and Fe(II) forms, but comparison with other CooA variants indicated that perturbation of Pro(2) ligation is not the basis for the lack of CO response in G117I CooA. These results have led to the hypothesis that (i) the heme and the C-helix region move toward each other following CO binding and the interaction of the heme with the C-helix is crucial for CooA activation, and (ii) this event occurs only when a properly sized heme pocket is afforded.

CooA: a heme-containing regulatory protein that serves as a specific sensor of both carbon monoxide and redox state.

CooA, the heme-containing carbon monoxide (CO) sensor from the bacterium Rhodospirillum rubrum, is a transcriptional factor that activates expression of certain genes in response to CO. As with other heme proteins, CooA is unable to bind CO when the Fe heme is oxidized, consistent with the fact that some of the regulated gene products are oxygen-labile. Upon reduction, there is an unusual switch of protein ligands to the six-coordinate heme and the reduced heme is able to bind CO. CO binding stabilizes a conformation of the dimeric protein that allows sequence-specific DNA binding, and transcription is activated through contacts between CooA and RNA polymerase. CooA is therefore a novel redox sensor as well as a specific CO sensor. CooA is a homolog of catabolite responsive protein (CRP), whose transcriptionally active conformation has been known for some time. The recent solution of the crystal structure of the CO-free (transcriptionally inactive) form of CooA has allowed insights into the mechanism by which both proteins respond to their specific small-molecule effectors.

Mapping CooA.RNA polymerase interactions. Identification of activating regions 2 and 3 in CooA, the co-sensing transcriptional activator.

CooA is a CO-sensing protein that activates the transcription of genes encoding the CO-oxidation (coo) regulon, whose polypeptide products are required for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the -35 element of the P(cooF) promoter, similar to the arrangement of class II CRP (cAMP receptor protein)- and FNR (fumarate and nitrate reductase activator protein)-dependent promoters when expressed in Escherichia coli. Gain-of-function CooA variants were isolated in E. coli following mutagenesis of the portion of cooA encoding the effector-binding domain. Some of the mutations affect regions of CooA that are homologous to the activating regions (AR2 and AR3) previously identified in CRP and FNR, whereas others affect residues that lie in a region of CooA between AR2 and AR3. These CooA variants are comparable to wild-type (WT) CooA in DNA binding affinity in response to CO but differ in transcription activation, presumably because of altered interactions with E. coli RNA polymerase. Based on predictions of similarity to CRP and FNR, loss-of-function CooA variants were obtained in the AR2 and AR3 regions that have minimal transcriptional activity, yet have WT-like DNA binding affinities in response to CO. This study demonstrates that WT CooA contains AR2- and AR3-like surfaces that are required for optimal transcription activation.

Redox-mediated transcriptional activation in a CooA variant.

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO at a reduced (Fe(II)) heme moiety with resulting conformational changes that promote DNA binding. In this study, we report a variant of CooA, M124R, that is active in transcriptional activation in a redox-dependent manner. Where wild-type CooA is active only in the Fe(II) + CO form, M124R CooA is active in both Fe(II) + CO and Fe(III) forms. Analysis of the pH dependence of the activity of Fe(III) M124R CooA demonstrated that the activity was also coordination state-dependent with a five-coordinate, high-spin species identified as the active form and Cys(75) as the retained ligand. In contrast, the active Fe(II) + CO forms of both wild-type and M124R CooA are six-coordinate and low-spin with a protein ligand other than Cys(75), so that WT and Fe(III) M124R CooA are apparently achieving an active conformation despite two different heme coordination and ligation states. A hypothesis to explain these results is proposed. This study demonstrates the utility of CooA as a model system for the isolation of functionally interesting heme proteins.

Effect of P(II) and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae.

Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnB mutant, with DRAG being inactivated more slowly in response to NH4+ treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In a glnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH4+ addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation in R. rubrum.

Structure of the CO sensing transcription activator CooA.

CooA is a homodimeric transcription factor that belongs to the catabolite activator protein (CAP) family. Binding of CO to the heme groups of CooA leads to the transcription of genes involved in CO oxidation in Rhodospirillum rubrum. The 2.6 A structure of reduced (Fe2+) CooA reveals that His 77 in both subunits provides one heme ligand while the N-terminal nitrogen of Pro 2 from the opposite subunit provides the other ligand. A structural comparison of CooA in the absence of effector and DNA (off state) with that of CAP in the effector and DNA bound state (on state) leads to a plausible model for the mechanism of allosteric control in this class of proteins as well as the CO dependent activation of CooA.

Characterization of variants altered at the N-terminal proline, a novel heme-axial ligand in CooA, the CO-sensing transcriptional activator.

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro(2) of one subunit (Met(1) is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the resulting CooA variants. While Pro(2) appears to be optimal for CooA function, it is not essential and a variety of studied variants at this position have substantial CO-sensing function. Surprisingly, even alterations that add a residue (where Pro(2) is replaced by Met(1)-Tyr(2), for example) accumulate heme-containing CooA with functional properties that are similar to those of wild-type CooA. Other nearby residues, such as Phe(5) and Asn(6) appear to be important for either the structural integrity or the function of CooA. These results are contrasted with those previously reported for alteration of the His(77) ligand on the opposite side of the heme.

Altering the specificity of CooA, the carbon monoxide-sensing transcriptional activator: characterization of CooA variants that bind cyanide in the Fe(II) form with high affinity.

CooA is a carbon monoxide- (CO-) sensing homodimeric heme protein that activates the transcription of genes required for the anaerobic oxidation of CO to CO(2) in the phototrophic bacterium Rhodospirillum rubrum. In this study, we demonstrate that mutational alteration of the histidine residue (His(77)) that serves as a heme ligand in the Fe(II) form of CooA allows high-affinity binding of cyanide (K(d) approximately 0.4 mM) to the heme. In contrast, neither these same variants in the Fe(III) form nor wild-type CooA in either oxidation state was able to bind cyanide even at high concentrations (50 mM). Examination of the pH dependence of spectral changes upon addition of cyanide suggested that the cyanide anion coordinated the heme iron. In addition, the UV-visible absorption spectrum of H77Y Fe(II) CooA without added effectors is also pH-dependent, suggesting that an ionizable amino acid has become solvent-accessible in the absence of His(77). Finally, we demonstrate that the transcriptional activity of H77Y CooA shows a small (1.4-fold) increase in the presence of cyanide, suggesting that the binding of cyanide to this variant promotes the active conformation of H77Y CooA.

Effects of perturbations of the nitrogenase electron transfer chain on reversible ADP-ribosylation of nitrogenase Fe protein in Klebsiella pneumoniae strains bearing the Rhodospirillum rubrum dra operon.

The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in Klebsiella pneumoniae strains transformed by plasmids carrying dra genes from Rhodospirillum rubrum. The dra operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in R. rubrum. In bacteria containing the dra operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. The dra gene products, expressed at a low level in K. pneumoniae, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in R. rubrum. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion of nifD, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.

Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum.

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments.

Electronic absorption, EPR, and resonance raman spectroscopy of CooA, a CO-sensing transcription activator from R. rubrum, reveals a five-coordinate NO-heme.

Electronic absorption, EPR, and resonance Raman spectroscopies revealed that CooA, the CO-sensing transcriptional regulator from Rhodospirillum rubrum, reacts with NO to form a five-coordinate NO-heme. NO must therefore displace both of the heme ligands from six-coordinate, low-spin Fe(II)CooA in forming five-coordinate Fe(II)CooA(NO). CO, in contrast, displaces a single heme ligand from Fe(II)CooA to form six-coordinate Fe(II)CooA(CO). Of a series of common heme-binding ligands, only CO and NO were able to bind to the heme of wild-type CooA; imidazole, azide anion, and cyanide anion had no effect on the heme absorption spectrum. Although NO binds to the heme and displaces the endogenous ligands, NO was not able to induce CooA to bind to its target DNA. The mechanism of CO-dependent activation of CooA is thus more complex than simple displacement of a ligand from the heme iron since NO does not trigger DNA binding. These observations suggest that the CooA heme site discriminates between NO and the biologically relevant signal, CO.

The non-linear viscoelastic behaviour of human hair at moderate extensions.

We report the results of experiments carried out on the stress relaxation of slightly stretched human hair. The results show that human hair is viscoelastic, and that when quickly extended to a moderate degree (0.5-6.5%) and held, the force generated will fall to half its original value in about 15 h. However, the relaxation process is also dependent on extension, so that iso-chronous (constant time) data is non-Hookean. We show that the iso-chronous stress/strain behaviour departs from linearity at about 1% extension and the modulus progressively decreases thereafter according to a power-law relationship with strain.

Probing the heme axial ligation in the CO-sensing CooA protein with magnetic circular dichroism spectroscopy.

The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-)(1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMCD spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA. The spectra are almost identical to each other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.

Importance of cis determinants and nitrogenase activity in regulated stability of the Klebsiella pneumoniae nitrogenase structural gene mRNA.

The Klebsiella pneumoniae nitrogen fixation (nif) mRNAs are unusually stable, with half-lives of 20 to 30 min under conditions favorable to nitrogen fixation (limiting nitrogen, anaerobiosis, temperatures of 30 degrees C). Addition of O2 or fixed nitrogen or temperature increases to 37 degrees C or more result in the dramatic destabilization of the nif mRNAs, decreasing the half-lives by a factor of 3 to 5. A plasmid expression system, independent of nif transcriptional regulation, was used to define cis determinants required for the regulated stability of the 5.2-kb nifHDKTY mRNA and to test the model suggested by earlier work that NifA is required in trans to stabilize nif mRNA under nif-derepressing conditions. O2 regulation of nifHDKTY mRNA stability is impaired in a plasmid containing a deletion of a 499-bp region of nifH, indicating that a site(s) required for the O2-regulated stability of the mRNA is located within this region. The simple model suggested from earlier work that NifA is required for stabilizing nif mRNA under conditions favorable for nitrogen fixation was disproved, and in its place, a more complicated model involving the sensing of nitrogenase activity as a component of the system regulating mRNA stability is proposed. Analysis of nifY mutants and overexpression suggests a possible involvement of the protein in this sensing process.

Incorporation of molybdenum into the iron-molybdenum cofactor of nitrogenase.

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99Mo to follow the incorporation of Mo into precursors. 99Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis. 99Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99Mo on this protein required nucleotide.