Role of transducer proteins in photoaxis signaling of Halobacterium salinarum

In Halobacterium salinarum phototaxis is mediated by the visual pigment-like photoreceptors sensory rhodopsin I (SRI) and II (SRII). SRI is a receptor for attractant orange and repellent UV-blue light, and SRII is a receptor for repellent blue-green light, and transmit signals through the membrane-bound transducer proteins HtrI and HtrII, respectively. ^ The primary sequences of HtrI and HtrII predict 2 transmembrane helices (TM1 and TM2) followed by a hydrophilic cytoplasmic domain. HtrII shows an additional large periplasmic domain for chemotactic ligand binding. The cytoplasmic regions are homologous to the adaptation and signaling domains of eubacterial chemotaxis receptors and, like their eubacterial homologs, modulate the transfer of phosphate groups from the histidine protein kinase CheA to the response regulator CheY that in turn controls flagellar motor rotation and the cell's swimming behavior. HtrII and Htrl are dimeric proteins which were predicted to contain carboxylmethylation sites in a 4-helix bundle in their cytoplasmic regions, like eubacterial chemotaxis receptors. ^ The phototaxis transducers of H. salinarum have provided a model for studying receptor/tranducer interaction, adaptation in sensory systems, and the role of membrane molecular complexes in signal transduction. ^ Interaction between the transducer HtrI and the photoreceptor SRI was explored by creating six deletion constructs of HtrI, with progressively shorter cytoplasmic domains. This study confirmed a putative chaperone-like function of HtrI, facilitating membrane insertion or stability of the SRI protein, a phenomenon previously observed in the laboratory, and identified the smallest HtrI fragment containing interaction sites for both the chaperone-like function and SRI photocycle control. The active fragment consisted of the N-terminal 147 residues of the 536-residue HtrI protein, a portion of the molecule predicted to contain the two transmembrane helices and the first ∼20% of the cytoplasmic portion of the protein. ^ Phototaxis and chemotaxis sensory systems adapt to stimuli, thereby signaling only in response to changes in environmental conditions. Observations made in our and in other laboratories and homologies between the halobacterial transducers with the chemoreceptors of enteric bacteria anticipated a role for methylation in adaptation to chemo- and photostimuli. By site directed mutagenesis we identified the methylation sites to be the glutamate pairs E265–E266 in HtrI and E513–E514 in HtrII. Cells containing the unmethylatable transducers are still able to perform phototaxis and adapt to light stimuli. By pulse-chase analysis we found that methanol production from carboxylmethyl group hydrolysis occurs upon specific photo stimulation of unmethylatable HtrI and HtrII and is due to turnover of methyl groups on other transducers. We demonstrated that the turnover in wild-type H. salinarum cells that follows a positive stimulus is CheY-dependent. The CheY-feedback pathway does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell. ^ Assembly of signaling molecules into architecturally defined complexes is considered essential in transmission of the signals. The spectroscopic characteristics of SRI were exploited to study the stoichiometric composition in the phototaxis complex SRI-HtrI. A molar ratio of 2.1 HtrI: 1 SRI was obtained, suggesting that only 1 SRI binding site is occupied on the HtrI homodimer. We used gold-immunoelectron microscopy and light fluorescence microscopy to investigate the structural organization and the distribution of other halobacterial transducers. We detected clusters of transducers, usually near the cell's poles, providing a ultrastructural basis for the global effects and intertransducer communication we observe. ^

Immobilizing bacteriorhodopsin on a single electron transistor

As awareness of potential human and environmental impacts from toxins has increased, so has the development of innovative sensors. Bacteriorhodopsin (bR) is a light activated proton pump contained in the purple membrane (PM) of the bacteria Halobacterium salinarum. Bacteriorhodopsin is a robust protein which can function in both wet and dry states and can withstand extreme environmental conditions. A single electron transistor(SET) is a nano-scale device that exploits the quantum mechanical properties of electrons to switch on and off. SETs have tremendous potential in practical applications due to their size, ultra low power requirements, and electrometer-like sensitivity. The main goal of this research was to create a bionanohybrid device by integrating bR with a SET device. This was achieved by a multidisciplinary approach. The SET devices were created by a combination of sputtering, photolithography, and focused ion beam machining. The bionanomaterial bacteriorhodopsin was created through oxidative fermentation and a series of transmembrane purification processes. The bR was then integrated with the SET by electrophoretic deposition, creating a bionanohybrid device. The bionanohybrid device was then characterized using a semiconductor parametric analyzer. Characterization demonstrated that the bR modulated the operational characteristics of the SET when bR was activated with light within its absorbance spectrum. To effectively integrate bacteriorhodopsin with microelectromechanical systems (MEMS) and nanoelectromechanical systems (NEMS), it is critical to know the electrical properties of the material and to understand how it will affect the functionality of the device. Tests were performed on dried films of bR to determine if there is a relationship between inductance, capacitance, and resistance (LCR) measurements and orientation, light-on/off, frequency, and time. The results indicated that the LCR measurements of the bR depended on the thickness and area of the film, but not on the orientation, as with other biological materials such as muscle. However, there was a transient LCR response for both oriented and unoriented bR which depended on light intensity. From the impedance measurements an empirical model was suggested for the bionanohybrid device. The empirical model is based on the dominant electrical characteristics of the bR which were the parallel capacitance and resistance. The empirical model suggests that it is possible to integrate bR with a SET without influencing its functional characteristics.

Bacteriorhodopsin protein hybrids for chemical and biological sensing

Bacteriorhodopsin (bR), an optoelectric protein found in Halobacterium salinarum, has the potential for use in protein hybrid sensing systems. Bacteriorhodopsin has no intrinsic sensing properties, however molecular and chemical tools permit production of bR protein hybrids with transducing and sensing properties. As a proof of concept, a maltose binding protein-bacteriorhodopsin ([MBP]-bR) hybrid was developed. It was proposed that the energy associated with target molecule binding, maltose, to the hybrid sensor protein would provide a means to directly modulate the electrical output from the MBP-bR bio-nanosensor platform. The bR protein hybrid is produced by linkage between bR (principal component of purified purple membrane [PM]) and MBP, which was produced by use of a plasmid expression vector system in Escherichia coli and purified utilizing an amylose affinity column. These proteins were chemically linked using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which facilitates formation of an amide bond between a primary carboxylic acid and a primary amine. The presence of novel protein hybrids after chemical linkage was analyzed by SDSPAGE. Soluble proteins (MBP-only derivatives and unlinked MBP) were separated from insoluble proteins (PM derivatives and unlinked PM) using size exclusion chromatography. The putatively identified MBP-bR protein hybrid, in addition to unlinked bR, was collected. This sample was normalized for bR concentration to native PM and both were deposited onto indium tin oxide (ITO) coated glass slides by electrophoretic sedimentation. The photoresponse of both samples, activated using 100 Watt tungsten lamp at 10 cm distance, were equal at 175 mV. Testing of deposited PM with 1 mM sucrose or 1 mM maltose showed no change in the photoresponse of the xiv material, however addition of 1 mM maltose to the deposited MBP-bR linked hybrid material elicited a 57% decrease in photoresponse indicating a positive response for targeting of maltose. This chemically linked MBP-bR hybrid protein, with bacteriorhodopsin, as a photoresponsive transducing substrate, shows promise for creation of a universal sensing array by attachment of other pertinent sensing materials, in lieu of the maltose binding protein utilized. This strategy would allow significant reduction in sensor size, while increasing responsiveness and sensitivity at nano and picomolar levels.

Attractant and repellent signaling conformers of sensory rhodopsin-transducer complexes.

Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor sensory rhodopsin I and its transducer subunit (SRI-HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pK(a) of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by approximately 1.5 units from that of the inwardly connected conformer. The pK(a) difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the one-photon excitation of the SRI-HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversely, a second near-UV photon drives the complex back to the inwardly connected conformer in the repellent signaling photoreaction. The results suggest a model of the color-discriminating dual-signaling mechanism in which phototaxis responses (his-kinase modulation) result from the photointerconversion of the two oppositely connected SRI-HtrI conformers by one-photon and two-photon activation. Furthermore, we find that the related repellent phototaxis SRII-HtrII receptor complex has an outwardly connected retinylidene Schiff base like the repellent signaling forms of the SRI-HtrI complex, indicating the general applicability of macro conformational changes, which can be detected by the connectivity switch, to phototaxis signaling by sensory rhodopsin-transducer complexes.

A Schiff base connectivity switch in sensory rhodopsin signaling.

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI-HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.

Three strategically placed hydrogen-bonding residues convert a proton pump into a sensory receptor.

In haloarchaea, light-driven ion transporters have been modified by evolution to produce sensory receptors that relay light signals to transducer proteins controlling motility behavior. The proton pump bacteriorhodopsin and the phototaxis receptor sensory rhodopsin II (SRII) differ by 74% of their residues, with nearly all conserved residues within the photoreactive retinal-binding pocket in the membrane-embedded center of the proteins. Here, we show that three residues in bacteriorhodopsin replaced by the corresponding residues in SRII enable bacteriorhodopsin to efficiently relay the retinal photoisomerization signal to the SRII integral membrane transducer (HtrII) and induce robust phototaxis responses. A single replacement (Ala-215-Thr), bridging the retinal and the membrane-embedded surface, confers weak phototaxis signaling activity, and the additional two (surface substitutions Pro-200-Thr and Val-210-Tyr), expected to align bacteriorhodopsin and HtrII in similar juxtaposition as SRII and HtrII, greatly enhance the signaling. In SRII, the three residues form a chain of hydrogen bonds from the retinal's photoisomerized C(13)=C(14) double bond to residues in the membrane-embedded alpha-helices of HtrII. The results suggest a chemical mechanism for signaling that entails initial storage of energy of photoisomerization in SRII's hydrogen bond between Tyr-174, which is in contact with the retinal, and Thr-204, which borders residues on the SRII surface in contact with HtrII, followed by transfer of this chemical energy to drive structural transitions in the transducer helices. The results demonstrate that evolution accomplished an elegant but simple conversion: The essential differences between transport and signaling proteins in the rhodopsin family are far less than previously imagined.

Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Protein-protein interaction between phototaxis receptor sensory Rhodopsin I and its transducer HtrI

The molecular complex containing the seven transmembrane helix photoreceptor S&barbelow;ensory R&barbelow;hodopsin I&barbelow; (SRI) and transducer protein HtrI (H&barbelow;alobacterial Transducer for SRI&barbelow;) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function and the signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, the signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components. ^ Twenty-one mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. ^ One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. ^ We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH. (Abstract shortened by UMI.) ^

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base–Asp-73 interhelical salt bridge

Sensory rhodopsin II (SRII) is a repellent phototaxis receptor in the archaeon Halobacterium salinarum, similar to visual pigments in its seven-helix structure and linkage of retinal to the protein by a protonated Schiff base in helix G. Asp-73 in helix C is shown by spectroscopic analysis to be a counterion to the protonated Schiff base in the unphotolyzed SRII and to be the proton acceptor from the Schiff base during photoconversion to the receptor signaling state. Coexpression of the genes encoding mutated SRII with Asn substituted for Asp-73 (D73N) and the SRII transducer HtrII in H. salinarum cells results in a 3-fold higher swimming reversal frequency accompanied by demethylation of HtrII in the dark, showing that D73N SRII produces repellent signals in its unphotostimulated state. Analogous constitutive signaling has been shown to be produced by the similar neutral residue substitution of the Schiff base counterion and proton acceptor Glu-113 in human rod rhodopsin. The interpretation for both seven-helix receptors is that light activation of the wild-type protein is caused primarily by photoisomerization-induced transfer of the Schiff base proton on helix G to its primary carboxylate counterion on helix C. Therefore receptor activation by helix C–G salt-bridge disruption in the photoactive site is a general mechanism in retinylidene proteins spanning the vast evolutionary distance between archaea and humans.

Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers

The halobacterial phototaxis receptors sensory rhodopsin I and II (SRI, SRII) enable the bacteria to seek optimal light conditions for ion pumping by bacteriorhodopsin and/or halorhodopsin. The incoming signal is transferred across the plasma membrane by means of receptor-specific transducer proteins that bind tightly to their corresponding photoreceptors. To investigate the receptor/transducer interaction, advantage is taken of the observation that both SRI and SRII can function as proton pumps. SRI from Halobacterium salinarum, which triggers the positive phototaxis, the photophobic receptor SRII from Natronobacterium pharaonis (pSRII), as well as the mutant pSRII-F86D were expressed in Xenopus oocytes. Voltage-clamp studies confirm that SRI and pSRII function as light-driven, outwardly directed proton pumps with a much stronger voltage dependence than the ion pumps bacteriorhodopsin and halorhodopsin. Coexpression of SRI and pSRII-F86D with their corresponding transducers suppresses the proton transport, revealing a tight binding and specific interaction of the two proteins. These latter results may be exploited to further analyze the binding interaction of the photoreceptors with their downstream effectors.

The primary structures of the Archaeon Halobacterium salinarium blue light receptor sensory rhodopsin II and its transducer, a methyl-accepting protein.

Recently, a large family of transducer proteins in the Archaeon Halobacterium salinarium was identified. On the basis of the comparison of the predicted structural domains of these transducers, three distinct subfamilies of transducers were proposed. Here we report isolation, complete gene sequences, and analysis of the encoded primary structures of transducer gene htrII, a member of family B, and its blue light receptor gene (sopII) of sensory rhodopsin II (SRII). The start codon ATG of the 714-bp sopII gene is one nucleotide beyond the termination codon TGA of the 2298-bp htrII gene. The deduced protein sequence of HtrII predicts a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm. HtrII has a common feature with HtrI, the sensory rhodopsin I transducer; like HtrI, HtrII possesses a hydrophilic loop structure just after the second transmembrane segment. The C-terminal 299 residues (765 amino acid residues total) of HtrII show strong homology to the signaling and methylation domain of eubacterial transducer Tsr. The hydropathy plot of the primary structure of SRII indicates seven membrane-spanning alpha-helical segments, a characteristic feature of retinylidene proteins ("rhodopsins") from a widespread family of photoactive pigments. SRII shows high identity with SRI (42%), bacteriorhodopsin (BR) (32%), and halorhodopsin (24%). The crucial positions for retinal binding sites in these proteins are nearly identical, with the exception of Met-118 (numbering according to the mature BR sequence), which is replaced by Val in SRII. In BR, residues Asp-85 and Asp-96 are crucial in proton pumping. In SRII, the position corresponding to Asp-85 in BR is conserved, but the corresponding position of Asp-96 is replaced by an aromatic Tyr. Coexpression of the htrII and sopII genes restores SRII phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI and insertion in htrII/sopII regions. This paper describes the first example that both HtrI and HtrII exist in the same halobacterial cell, confirming that different sensory rhodopsins SRI and SRII in the same organism have their own distinct transducers.

Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins.

Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features. We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers. This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI. On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H. salinarium: (i) a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; (ii) a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains; and (iii) a cytoplasmic domain without hydrophobic transmembrane segments. We fractionated the halobacterial cell lysate into soluble and membrane fractions and localized different halobacterial methyl-accepting taxis proteins in both fractions.

Purification and Biochemical Characterization of Thermostable Alkaline Phosphatases Produced by Rhizopus microsporus var. rhizopodiformis

The biochemical properties of the alkaline phosphatases (AIPs) produced by Rhizopus micro-sporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1% wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) Stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K(m) 0.28 and 0.22 mmol/L, with upsilon(lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.

Notes et extraits de Pierre-François CHIFFLET, concernant principalement la Franche-Comté et la Bourgogne.

On remarque : « Ex Glossario pervetusto » (f. 3) ; — « Ex chartis ecclesiae de Caritate » (f. 10) ; — « Ex veteri membrana et ex antiquo circulo depicto propè altare Maurimonasterii in Alsatia » (f. 10) ; — « Ex chartis R. P. Stephani Voyrin » (f. 11) ; — « Episcopi Metenses, ex ms. codice Sancti Arnulfi Metensis » (f. 12 v°) ; — Lettre de Hugues Métel à Gerland (f. 14) ; — « Ex variis catalogis sanctorum » (f. 14) ; — « Ex archivio ecclesiae Beatae Magdalenae [Bisuntinae] » (f. 17) ; — « Ex archivio Sancti Vincentii Bisont[ini] » (f. 18) ; — « Ex archivio monasterii Bellaevallis » [Bisuntinae dioecesis] (f. 20) ; — « Ex veteri necrologio Sancti Stephani [Bisuntini], quod est ad margines Martyrologii Bedae perantiqui, ad ejusdem ecclesiae usum accommodati » [cf. ms. de Besançon 712] (f. 23 v°) ; — « Inter martyrologium et regulam, seu librum primum concilii Aquisgranensis, habetur haec sententia absolutionis » (f. 25) ; — « Ante veterem Ordinem Romanum, qui est Liber Pontificalis, habentur sequentes formulae praestandae obedientiae archiepiscopis et ecclesiae Bisuntinae » (f. 25 v°) ; — « Alia ex archivo archiepiscopi [Bisuntini] » (f. 26 v°) ; — « Ex archivio Balernensi » (f. 27 v°) ; — « Ex variis sanctorum historiis manuscriptis » (f. 30) ; — « Extraits concernant Tournus » (f. 34 v°), — notamment : « Ex codice manuscripto Trenorchiensi, membraneo, quod vocare possis Registrum Trenorchiense, incipiens ab anno 1222 et desinens in 1296 ; inscribitur : Feoda ecclesiae Trenorchiensis » (f. 37) ; — « Ex notis R. P. Voyrini » [cf. supra, f. 11] (f. 49) ; — « Inscriptio vetus Luxoviensis » (f. 50 v°) ; — Notes historiques, concernant principalement des abbayes comtoises (f. 51) ; — « Scriptores rerum Burgundicarum » (f. 52) ; — « Ex parvo chartulari Sancti Benigni [Divionensis] » (f. 52 v°) ; — « Ex libro anniversariorum Sancti Benigni [Divionensis] », 1579-1629 (f. 52 v°) ; — « Ex chartulari Sancti Symphoriani Augustodunensis » (f. 53) ; — « Ex chartulari Sancti Marcelli Cabilonensis » (f. 53 v°) ; — « Ex chartulari S. Sequani » [Lingonensis dioecesis] (f. 54 v°) ; — « Ex chartulari Sanctae Capellae Divionensis » (f. 55) ; — « Ex chartulari cathedralis Augustodunensis » (f. 55 v°) ; — « Ex tomo priore chartularis Sancti Stephani Divionensis » (f. 56) ; — « Ex chartulari Cluniacensi » (f. 57) ; — « Ex apographo chartularis Matisconensis, cujus autographum vetus dicebatur Liber catenatus » (f. 57 v°) ; — « Ex chartulari Patriciaci, qui est prioratus dependens a Floriacensi abbatia, situs in comitatu Kadrellensi, dioecesi Augustodunensi » (f. 58 v°) ; — Extraits concernant Autun et les comtes d'Autun (f. 60 v°) ; — « Ex autographis Sancti Benigni Divionensis » (f. 61) ; — « Iterum ex chartulari Sancti Stephani Divionensis » (f. 61) ; — « Ex autographis monasterii puellarum Tartensis » (f. 62) ; — « Iterum ex tabulario Cluniacensi, adnotatis paginis » (f. 62 v°) ; — « Ex majore tabulario ecclesiae Cabilonensis, quod digessit Johannes Germani, Cabilonensis episcopus » (f. 64 v°) ; — « Ex tabulario abbatiae de Buxeria » (f. 65) ; — « Ex autographis monialium Benedictinarum de Pralon » (f. 66) ; — « Ex autographis prioratus Vallis Beatae Mariae juxta Talant, de Ordine Vallis Scholarium, vulgò Bonvau » (f. 66) ; — « Ex kalendario pervetusto Sancti Benigni Divionensis » (f. 66 v°) ; — « Ex martyrologio proprio Sancti Martini Eduensis » (f. 67) ; — « Ex necrologio Sancti Lazari Aeduensis » (f. 67) ; — « Ex chronico Sancti Petri Vivi Senonensis » (f. 67) ; — « Ex chronico S. Medardi Suessionensis » (f. 68) ; — « Ex brevi chronico Sancti Dionysii ad cyclos paschales » (f. 68) ; — « De Saracenis in Gallia profligatis » (f. 69) ; — « Ex necrologio Sancti Augendi Jurensis » (f. 74) ; — « Ex necrologio Sancti Petri Bisuntini » (f. 74) ; — « Ex necrologio veteri Sancti Stephani [Bisuntini] » (f. 74 v°) ; — « Ex registro Innocentii papae VI, anni IX pontificatus, Christi 1361 » (f. 82) ; — « Ex tabulis capituli Sancti Dionysii de Vergeyo, nunc Nuciacensis » (f. 83) ; — « Ex manuscripto codice donationum 208 Joannis, comitis Burgundiae et domini Salinensis, super puteum Salinarum » (f. 89 v°) ; — « Ex archivo Sancti Anatolii Salinensis ; ex magno necrologio chartaceo eique simili membraneo, composito 1390 » (f. 92) ; — « Ex chartis prioratus de Marthereto prope Vesulium » (f. 94) ; — « Ex archivo Sancti Pauli Bisonticensis » (f. 95) ; — « Ex veteri chartulario Sancti Pauli [Bisonticensis] » (f. 95 v°) ; — « Ex veteri manuscripto codice Sancti Pauli [Bisonticensis] » (f. 96) ; — « Ex antiquioribus libris ecclesiae Sancti Stephani [Bisonticensis], scriptis aut in usum adhibitis tempore Hugonis primi, [archiepiscopi Bisonticensis] » (f. 96 v°) ; — « Ex codice Sancti Stephani [Bisonticensis] pervetusto, qui Bisonticensis ecclesiae sacros ritus continet » (f. 97) ; — « Ex antiquis codicibus Sancti Pauli [Bisonticensis] » (f. 98) ; — « Ex antiquo rituum libro » (f. 98) ; — « Ex antiquis letaniis, tempore Hugonis I, [archiepiscopi Bisuntini], in ecclesia Bisontina cani solitis ; ex manuscriptis Sancti Stephani et Sancti Pauli » (f. 99) ; — « Extraits de plusieurs manuscrits de Saint-Paul de Besançon » (f. 99 v°) ; — « Ex manuscripto codice Sanctae Magdalenae [Bisuntinae] » (f. 101) ; — Inscription trouvée à Saint-Ferjeux, en 1627 (f. 101 v°) ; — « Ex antiquis membranis archivii ecclesiae metropolitanae Bisontinae », extraits de bulles pontificales, etc. (f. 102) ; — « Ex archivio Loci Crescentis seu Trium Regum » (f. 105) ; — Notes empruntées, « ut videtur », à Anselme « de Marenchiis » (f. 106) ; — « Ex actis capituli Bisontini » (f. 106 v°) ; — « Ex obitibus adnotatis ad marginem martyrologii cathedralis Aeduensis » (f. 106 v°) ; — Fragment de chronique, 563-1033, tiré « ex veteri membrana monasterii Sancti Augendi » (f. 107) ; — « Ex alia membrana, post chronicon Engolismense » (f. 107) ; — « Ex archivio abbatiae de Aceyo » (f. 108) ; — « Ex archivio abbatiae de Roseriis » (f. 109) ; — « Extrait d'un tome des recognoissances des fiefs du Charolois » (f. 109 v°) ; — « Ex polypthico Sancti Vincentii Cabilonensis » (f. 114) ; — « Ex Aeneae, Parisiensis episcopi, collectione contra Graecos, Biblioth. Thuan., n. 444 » (f. 114) ; — « Consuetudines Cluniacenses, auctore Wdalrico, monacho Cluniacensi » (f. 115) ; — Inscriptions copiées « in pervetusto manuscripto codice, post Persii satyras », etc. (f. 116) ; — Extraits d'une « Vita metrica sancti Eustachii martyris, cognomine Placidae », contenue « in manuscripto codice Cisterciensi », etc. (f. 116 v°) ; — « De archiepiscopis Bisontinis » (f. 119) ; — « Ex archivio capituli Bisontini » (f. 120 v°) ; — « Notitia nostrorum diplomatum » (f. 124-135 r° , 141 r° , 142 v° et 143 r°) ; — « De comitibus Burgundiae in Sancti Stephani basilica Bisontina sepultis, ex veteri membrana archivi ecclesiae metropolitanae » (f. 135 v°) ; — Lettre autogr. de « Pierre-François Chifflet » [à Baluze], Dijon, 18 mars 1657 (f. 161) ; — Fragment de charte, « extrait du thresor de S. Anatoile de Salins » (f. 162) ; — Lettre autogr. de « [fr.] François Du Chemin » à Chifflet, Cîteaux, 16 mai 1657 (f. 167) ; — « Nova regum Francorum series, a quâ Theodericus II et omnes ejus posteri excluduntur, ex manuscripto codice Fontanetensi » (f. 171) ; — « Vita sancti Theutbaldi, Viennensis archiepiscopi », incomplète [Bibliotheca hagiographica latina, 8044 b] (f. 172 et 172 bis) ; — « Ex chartulario Sancti Germani Autisiodorensis », diplômes carolingiens (f. 174) ; — Fragment d'une lettre [adressée vraisemblablement à Chifflet], Paris, octobre 1670 (f. 177) ; — Extraits « ex vitâ sancti Theutbaldi, Viennensis archiepiscopi..., ex manuscripto codice monasterii Sancti Theuderii, ubi sanctus Theobaldus, Viennensis archiepiscopus, requiescit » [extraits de la vie dont un fragment occupe les feuillets 172 et 172 bis] (f. 178) ; — Lettre autogr. de « P[ierre-] F[rançois] Chifflet » [à Baluze], 10 mars (f. 180) ; — Notes sur saint Théobald, archevêque de Vienne (f. 181). Cf. Ulysse Robert, Catalogue des manuscrits relatifs à la Franche-Comté, qui sont conservés dans les bibliothèques publiques de Paris (1878), p. 161-162.