Systems analysis of bioenergetics and growth of the extreme halophile Halobacterium salinarum

Halobacterium salinarum is a bioenergetically flexible, halophilic microorganism that can generate energy by respiration, photosynthesis, and the fermentation of arginine. In a previous study, using a genome-scale metabolic model, we have shown that the archaeon unexpectedly degrades essential amino acids under aerobic conditions, a behavior that can lead to the termination of growth earlier than necessary. Here, we further integratively investigate energy generation, nutrient utilization, and biomass production using an extended methodology that accounts for dynamically changing transport patterns, including those that arise from interactions among the supplied metabolites. Moreover, we widen the scope of our analysis to include phototrophic conditions to explore the interplay between different bioenergetic modes. Surprisingly, we found that cells also degrade essential amino acids even during phototropy, when energy should already be abundant. We also found that under both conditions considerable amounts of nutrients that were taken up were neither incorporated into the biomass nor used as respiratory substrates, implying the considerable production and accumulation of several metabolites in the medium. Some of these are likely the products of forms of overflow metabolism. In addition, our results also show that arginine fermentation, contrary to what is typically assumed, occurs simultaneously with respiration and photosynthesis and can contribute energy in levels that are comparable to the primary bioenergetic modes, if not more. These findings portray a picture that the organism takes an approach toward growth that favors the here and now, even at the cost of longer-term concerns. We believe that the seemingly "greedy" behavior exhibited actually consists of adaptations by the organism to its natural environments, where nutrients are not only irregularly available but may altogether be absent for extended periods that may span several years. Such a setting probably predisposed the cells to grow as much as possible when the conditions become favorable

Active Membrane Fluctuations Studied by Micropipet Aspiration

We present a detailed analysis of the micropipet experiments recently reported in J-B. Manneville et al., Phys. Rev. Lett. 82, 4356--4359 (1999), including a derivation of the expected behaviour of the membrane tension as a function of the areal strain in the case of an active membrane, i.e., containing a nonequilibrium noise source. We give a general expression, which takes into account the effect of active centers both directly on the membrane, and on the embedding fluid dynamics, keeping track of the coupling between the density of active centers and the membrane curvature. The data of the micropipet experiments are well reproduced by the new expressions. In particular, we show that a natural choice of the parameters quantifying the strength of the active noise explains both the large amplitude of the observed effects and its remarkable insensitivity to the active-center density in the investigated range. [Submitted to Phys Rev E, 22 March 2001]Comment: 14 pages, 5 encapsulated Postscript figure

Attractant and Repellent Signaling Conformers of Sensory Rhodopsin−Transducer Complexes†

ABSTRACT: 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 pKa of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by ∼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 onephoton 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 connecte

Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses

Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structures reported for the same intermediate cannot be reconciled in terms of differing extents of change on a single conformational trajectory. New measurements of image phases obtained by cryo-electron microscopy of the D96G/F171C/F219L triple mutant provide independent validation for the description of the large protein conformational change derived at 3.2 Å resolution by electron crystallography of 2D crystals, but do not support atomic models for light-driven conformational changes derived using X-ray crystallography of 3D crystals. Our findings suggest that independent determination of phase information from 2D crystals can be an important tool for testing the accuracy of atomic models for membrane protein conformational changes

Intracellular protein determination using droplet-based immunoassays

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from 50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells

Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein

Replacement of aspartate 85 (D85) in bacteriorhodopsin (BR) by threonine but not be asparagine creates at pH<7 an anion-binding site in the molecular similar to that in chloride pump halorhodopsin. Binding of various anions to BR-D85T causes a blue shift of the absorption maximum by maximally 57 nm. Connected to this color change is a change in the absorption difference spectrum of the initial state and the longest living photo intermediate from a positive difference maximum at 460 nm in the absence of transported anions to one at 630 nm in their presence. Increasing anion concentration cause decreasing decay times of this intermediate. At physiological pH, BR-D85T but not BR-D85N transports chloride ions inward in green light, protons outward in blue or green light and protons inward in white light (directions refer to the intact cell). The proton movements are observable also in BR-D85N. Thus, creation of an anion-binding site in BR is responsible for chloride transport and introduction of anion-dependent spectroscopic properties at physiological pH. The different transport modes are explained with the help of the recently proposed IST model, which states that after light-induced isomerization of the retinal an ion transfer step and an accessibility change of the active site follow. The latter two steps occur independently. In order to complete the cyclic event, the accessibility change, ion transfer and isomerization state have to be reversed. The relative rates of accessibility changes and ion transfer steps define ultimately the vectoriality of ion transfers. All transport modes described here for the same molecule can satisfactorily be described in the framework of this general concept