X-ray structure analysis of bacteriorhodopsin at 1.3 Å resolution.

Bacteriorhodopsin (bR) of Halobacterium salinarum is a membrane protein that acts as a light-driven proton pump. bR and its homologues have recently been utilized in optogenetics and other applications. Although the structures of those have been reported so far, the resolutions are not sufficient for elucidation of the intrinsic structural features critical to the color tuning and ion pumping properties. Here we report the accurate crystallographic analysis of bR in the ground state. The influence of X-rays was suppressed by collecting the data under a low irradiation dose at 15 K. Consequently, individual atoms could be separately observed in the electron density map at better than 1.3 Å resolution. Residues from Thr5 to Ala233 were continuously constructed in the model. The twist of the retinal polyene was determined to be different from those in the previous models. Two conformations were observed for the proton release region. We discuss the meaning of these fine structural features.

Light-independent phospholipid scramblase activity of bacteriorhodopsin from Halobacterium salinarum.

The retinylidene protein bacteriorhodopsin (BR) is a heptahelical light-dependent proton pump found in the purple membrane of the archaeon Halobacterium salinarum. We now show that when reconstituted into large unilamellar vesicles, purified BR trimers exhibit light-independent lipid scramblase activity, thereby facilitating transbilayer exchange of phospholipids between the leaflets of the vesicle membrane at a rate >10,000 per trimer per second. This activity is comparable to that of recently described scramblases including bovine rhodopsin and fungal TMEM16 proteins. Specificity tests reveal that BR scrambles fluorescent analogues of common phospholipids but does not transport a glycosylated diphosphate isoprenoid lipid. In silico analyses suggest that membrane-exposed polar residues in transmembrane helices 1 and 2 of BR may provide the molecular basis for lipid translocation by coordinating the polar head-groups of transiting phospholipids. Consistent with this possibility, extensive coarse-grained molecular dynamics simulations of a BR trimer in an explicit phospholipid membrane revealed water penetration along transmembrane helix 1 with the cooperation of a polar residue (Y147 in transmembrane helix 5) in the adjacent protomer. These results suggest that the lipid translocation pathway may lie at or near the interface of the protomers of a BR trimer.

The Effects of HZE Particles, γ and X-ray Radiation on the Survival and Genetic Integrity of Halobacterium salinarum NRC-1, Halococcus hamelinensis, and Halococcus morrhuae.

Three halophilic archaea, Halobacterium salinarum NRC-1, Halococcus hamelinensis, and Halococcus morrhuae, have been exposed to different regimes of simulated outer space ionizing radiation. Strains were exposed to high-energy heavy ion (HZE) particles, namely iron and argon ions, as well as to γ radiation (60Co) and X-rays, and the survival and the genetic integrity of the 16S rRNA gene were evaluated. Exposure to 1 kGy of argon or iron ions at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute for Radiological Sciences (NIRS) in Japan did not lead to a detectable loss in viability; only after exposure to 2 kGy of iron ions a decline in survival was observed. Furthermore, a delay in growth was manifested following exposure to 2 kGy iron ions. DNA integrity of the 16S rRNA was not compromised up to 1 kGy, with the exception of Hcc. hamelinensis following exposure to argon particles. All three strains showed a high resistance toward X-rays (exposed at the DLR in Cologne, Germany), where Hcc. hamelinensis and Hcc. morrhuae displayed better survival compared to Hbt. salinarum NRC-1. In all three organisms the DNA damage increased in a dose-dependent manner. To determine a biological endpoint for survival following exposure to γ radiation, strains were exposed to up to 112 kGy at the Beta-Gamma-Service GmbH (BGS) in Germany. Although all strains were incubated for up to 4 months, only Hcc. hamelinensis and Hcc. morrhuae recovered from 6 kGy of γ radiation. In comparison, Hbt. salinarum NRC-1 did not recover. The 16S rRNA gene integrity stayed remarkably well preserved up to 48 kGy for both halococci. This research presents novel data on the survival and genetic stability of three halophilic archaea following exposure to simulated outer space radiation. Key Words: Halophilic archaea-Radiation-Survival. Astrobiology 17, 110-117.

Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H(+)-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (∼ 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.

The Survival and Resistance of Halobacterium salinarum NRC-1, Halococcus hamelinensis, and Halococcus morrhuae to Simulated Outer Space Solar Radiation.

UNLABELLED: Solar radiation is among the most prominent stress factors organisms face during space travel and possibly on other planets. Our analysis of three different halophilic archaea, namely Halobacterium salinarum NRC-1, Halococcus morrhuae, and Halococcus hamelinensis, which were exposed to simulated solar radiation in either dried or liquid state, showed tremendous differences in tolerance and survivability. We found that Hcc. hamelinensis is not able to withstand high fluences of simulated solar radiation compared to the other tested organisms. These results can be correlated to significant differences in genomic integrity following exposure, as visualized by random amplified polymorphic DNA (RAPD)-PCR. In contrast to the other two tested strains, Hcc. hamelinensis accumulates compatible solutes such as trehalose for osmoprotection. The addition of 100 mM trehalose to the growth medium of Hcc. hamelinensis improved its survivability following exposure. Exposure of cells in liquid at different temperatures suggests that Hbt. salinarum NRC-1 is actively repairing cellular and DNA damage during exposure, whereas Hcc. morrhuae exhibits no difference in survival. For Hcc. morrhuae, the high resistance against simulated solar radiation may be explained with the formation of cell clusters. Our experiments showed that these clusters shield cells on the inside against simulated solar radiation, which results in better survival rates at higher fluences when compared to Hbt. salinarum NRC-1 and Hcc. hamelinensis. Overall, this study shows that some halophilic archaea are highly resistant to simulated solar radiation and that they are of high astrobiological significance. KEY WORDS: Halophiles-Solar radiation-Stress resistance-Survival.

The rad2 gene of haloarchaeum Halobacterium salinarum is functional in the repair of ultraviolet light induced DNA photoproducts.

There are a lot of bacterial and eukaryotic DNA repair gene homologs among sequenced archaeal genomes but there is little information about DNA repair mechanisms and the interaction of involved repair proteins. In order to study DNA repair mechanisms in the third domain of life, we studied these processes in the model archaeon, Halobacterium salinarum. H. salinarum has homologs of eukaryotic nucleotide excision repair genes such as rad2 gene. A functional analysis of rad2 was performed by knocking down of this gene. We introduced an antisense RNA expression vector into the cells and the sensitivity of transformants against ultraviolet light exposure was measured to determine whether rad2 gene performs any role in the repair of the DNA lesions induced by UV light or not. Our data suggests that rad2 is functional in this pathway and knocked down strains were unable to completely repair the UV induced DNA damages. In this study, for the first time antisense RNA is used for functional analysis of a gene in H. salinarum and it is shown that antisense RNA could be used as a reliable genetic tool for understanding of the archaeal genetics.

Preliminary ultrasonication affects the rate of the bacteriorhodopsin bleaching and the effectiveness of the reconstitution process in bacterioopsin.

The formation process of polymer films based on bacteriorhodopsin (BR) analogs requests a high amount of BR samples one time only. The common technique for apomembrane formation (preparation of bacterioopsin, BO) is not designed to be operated with high concentrations and high volumes of BR, so the use of this technique results in a low rate of BR bleaching. To accelerate the process of BR bleaching preliminary sonication was used. It was used just as preliminary sonication before bleaching of BR samples, so also sonication was used before reconstitution of resulted BO samples. These modifications of the common technique lead to an acceleration of BR bleaching and an increase in effectiveness of reconstitution of BO in comparison with the nonmodified technique. The quantitative results of sonication's effect on the bleaching acceleration and the effectiveness of reconstitution are different depending on the BR strains.

Insight into a single halobacterium using a dual-bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination.

The light-driven outward proton transporter assists energy production via an ATP synthase system best exemplified by the bacteriorhodopsin (BR) from Halobacterium salinarum, HsBR. As the only archaea able to survive in the resource-limited ecosystem of the Dead Sea, Haloarcula marismortui has been reported to have a unique dual-BR system, consisting of HmBRI and HmBRII, instead of only a single BR in a cell (solo-BR). The contribution of this dual-BR system to survival was investigated. First, native H. marismortui and H. salinarum cells were tested in water that had been adjusted to mimic the conditions of Dead Sea water. These archaea were shown to accumulate protons and reduce pH in their periplasmic regions, which disabled further proton transportation functionality in H. salinarum but not in H. marismortui. Then, pH-dependent photocurrent measurements using purified BR proteins demonstrated that HsBR and HmBRI were functional at pH > 5.0 and that HmBRII was functional at pH > 4.0. Our results indicate that the dual-HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light-driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH of the Dead Sea.

Effects of intracellular Mn on the radiation resistance of the halophilic archaeon Halobacterium salinarum.

Ionizing radiation (IR) is of particular interest in biology because its exposure results in severe oxidative stress to the cell's macromolecules. Our recent work with extremophiles supports the idea that IR resistance is most likely achieved by a metabolic route, effected by manganese (Mn) antioxidants. Biochemical analysis of "super-IR resistant" mutants of H. salinarum, evolved over multiple cycles of exposure to high doses of IR, confirmed the key role for Mn antioxidants in the IR resistance of this organism. Analysis of the proteome of H. salinarum "super-IR resistant" mutants revealed increased expression for proteins involved in energy metabolism, replenishing the cell with reducing equivalents depleted by the oxidative stress inflicted by IR. Maintenance of redox homeostasis was also activated by the over-expression of coenzyme biosynthesis pathways involved in redox reactions. We propose that in H. salinarum, increased tolerance to IR is a combination of metabolic regulatory adjustments and the accumulation of Mn-antioxidant complexes.

Potential applications of bacteriorhodopsin mutants.

Bacteriorhodopsin (BR), a model system in biotechnology, is a G-protein dependent trans membrane protein which serves as a light driven proton pump in the cell membrane of Halobacterium salinarum. Due to the linkage of retinal to the protein, it seems colored and has numbers of versatile properties. As in vitro culture of the Halobacteria is very difficult, and isolation is time consuming and usually inefficient, production of genetically modified constructs of the protein is essential. There are three important characteristics based on protein catalytic cycle and molecular functions of photo-electric, photochromic and proton transporting, which makes this protein as a strategic molecule with potential applications in biotechnology. Such applications include protein films, used in artificial retinal implants, light modulators, three-dimensional optical memories, color photochromic sensors, photochromic and electrochromic papers and ink, biological camouflage and photo detectors for biodefense and non-defense purposes.

Photo-induced bleaching of sensory rhodopsin II (phoborhodopsin) from Halobacterium salinarum by hydroxylamine: identification of the responsible intermediates.

Sensory rhodopsin II from Halobacterium salinarum (HsSRII) is a retinal protein in which retinal binds to a specific lysine residue through a Schiff base. Here, we investigated the photobleaching of HsSRII in the presence of hydroxylamine. For identification of intermediate(s) attacked by hydroxylamine, we employed the flash-induced bleaching method. In order to change the concentration of intermediates, such as M- and O-intermediates, experiments were performed under varying flashlight intensities and concentrations of azide that accelerated only the M-decay. We found the proportional relationship between the bleaching rate and area under the concentration-time curve of M, indicating a preferential attack of hydroxylamine on M. Since hydroxylamine is a water-soluble reagent, we hypothesize that for M, hydrophilicity or water-accessibility increases specifically in the moiety of Schiff base. Thus, hydroxylamine bleaching rates may be an indication of conformational changes near the Schiff base. We also considered the possibility that azide may induce a small conformational change around the Schiff base. We compared the hydroxylamine susceptibility between HsSRII and NpSRII (SRII from Natronomonas pharaonis) and found that the M of HsSRII is about three times more susceptible than that of the stable NpSRII. In addition, long illumination to HsSRII easily produced M-like photoproduct, P370. We thus infer that the instability of HsSRII under illumination may be related to this increase of hydrophilicity at M and P370.

Sensory rhodopsin-I as a bidirectional switch: opposite conformational changes from the same photoisomerization.

The phototaxis receptor sensory rhodopsin I (SRI) exists in two protein conformations, each of which is converted to the other by light absorption by the protein's retinylidene chromophore. One conformer inhibits a histidine-kinase attached to its bound transducer HtrI and its formation induces attractant motility responses, whereas the other conformer activates the kinase and its formation induces repellent responses. We performed Fourier transform infrared spectroscopy with temperature, pH, and mutation-induced shifts in the conformer equilibrium, and found that both conformers when present in the unphotolyzed dark state contain an all-trans retinal configuration that is photoisomerized to 13-cis, i.e., the same photoisomerization causes the opposite conformational change in the photointerconvertible pair of conformers depending on which conformer is present in the dark. Therefore, switching between the protein global conformations that define the two conformers is independent of the direction of isomerization. Insights into this phenomenon are gained from analysis of the evolution of the receptor from light-driven proton pumps, which use similar conformers for transport. The versatility of the conformational changes of microbial rhodopsins, including conformer interexchangeability in the photocycle as shown here, is likely a significant factor in the evolution of the diverse functionality of this protein family.

Coordinating the structural rearrangements associated with unidirectional proton transfer in the bacteriorhodopsin photocycle induced by deprotonation of the proton-release group: a time-resolved difference FTIR spectroscopic study.

In the photocycle of bacteriorhodopsin at pH 7, proton release from the proton releasing group (PRG) to the extracellular medium occurs during formation of the M intermediate. This proton release is inhibited at acidic pH, below the pK(a) of the PRG, approximately 6 in M, and instead occurs later in the cycle as the initial state is restored from the O intermediate. Here, structural changes related to deprotonation of the PRG have been investigated by time-resolved FTIR spectroscopy at 25 degrees C. The vibrational features at 2100-1790, 1730-1685, 1661, and 1130-1045 cm(-1) have greater negative intensity in the pure M-minus-BR spectrum and even in the M-minus-BR spectrum, that is present earlier together with the L-minus-BR spectrum, at pH 7, than in the corresponding M-minus-BR spectra at pH 5 or 4. The D212N mutation abolishes the decreases in the intensities of the broad feature between 1730 and 1685 cm(-1) and the band at 1661 cm(-1). The 1730-1685 cm(-1) feature may arise from transition dipole coupling of the backbone carbonyl groups of Glu204, Phe208, Asp212, and Lys216 interacting with Tyr57 and C(15)-H of the chromophore. The 1661 cm(-1) band, which is insensitive to D(2)O substitution, may arise by interaction of the backbone carbonyl of Asp212 with C(15)-H. The 2100-1790 cm(-1) feature with a trough at 1885 cm(-1) could be due to a water cluster. Depletion of these bands upon deprotonation of the PRG is attributable to disruption of a coordinated structure, held in place by interactions of Asp212. Deprotonation of the PRG is also accompanied by disruption of the interaction of the water molecule near Arg82. The liberated Asp212 may stabilize the protonated state of Asp85 and thus confer unidirectionality to the transport.

Prokaryotic phototaxis.

Microorganisms have various mechanisms at their disposal to react to (changes in) their ambient light climate (i.e., intensity, color, direction, and degree of polarization). Of these, one of the best studied mechanisms is the process of phototaxis. This process can be described as a behavioral migration-response of an organism toward a change in illumination regime. In this chapter we discuss three of these migration responses, based on swimming, swarming, and twitching motility, respectively. Swimming motility has been studied using a wide range of techniques, usually microscopy based. We present a detailed description of the assays used to study phototaxis in liquid cultures of the phototrophic organisms Halobacterium salinarum, Halorhodospira halophila, and Rhodobacter sphaeroides and briefly describe the molecular basis of these responses. Swarming and twitching motility are processes taking place at the interface between a solid phase and a liquid or gas phase. Although assays to study these processes are relatively straightforward, they are accompanied by technical complications, which we describe. Furthermore, we discuss the molecular processes underlying these forms of motility in Rhodocista centenaria and Synechocystis PCC6803. Recently, it has become clear that also chemotrophic organisms contain photoreceptor proteins that allow them to respond to their ambient light climate. Surprisingly, light-modulated motility responses can also be observed in the chemotrophic organisms Escherichia coli and Acinetobacter calcoaceticus. In the light-modulated surface migration not only "che-like" signal transduction reactions may play a role, but in addition processes as modulation of gene expression and even intermediary metabolism.

Salt shield: intracellular salts provide cellular protection against ionizing radiation in the halophilic archaeon, Halobacterium salinarum NRC-1.

The halophilic archaeon Halobacterium salinarum NRC-1 was used as a model system to investigate cellular damage induced by exposure to high doses of ionizing radiation (IR). Oxidative damages are the main lesions from IR and result from free radicals production via radiolysis of water. This is the first study to quantify DNA base modification in a prokaryote, revealing a direct relationship between yield of DNA lesions and IR dose. Most importantly, our data demonstrate the significance of DNA radiation damage other than strand breaks on cell survival. We also report the first in vivo evidence of reactive oxygen species scavenging by intracellular halides in H. salinarum NRC-1, resulting in increased protection against nucleotide modification and carbonylation of protein residues. Bromide ions, which are highly reactive with hydroxyl radicals, provided the greatest protection to cellular macromolecules. Modified DNA bases were repaired in 2 h post irradiation, indicating effective DNA repair systems. In addition, measurements of H. salinarum NRC-1 cell interior revealed a high Mn/Fe ratio similar to that of Deinococcus radiodurans and other radiation-resistant microorganisms, which has been shown to provide a measure of protection for proteins against oxidative damage. The work presented here supports previous studies showing that radiation resistance is the product of mechanisms for cellular protection and detoxification, as well as for the repair of oxidative damage to cellular macromolecules. The finding that not only Mn/Fe but also the presence of halides can decrease the oxidative damage to DNA and proteins emphasizes the significance of the intracellular milieu in determining microbial radiation resistance.

Coupling between the retinal thermal isomerization and the Glu194 residue of bacteriorhodopsin.

Glu194 is a residue located at the end of F helix on the extracellular side of the light-induced proton pump bacteriorhodopsin (BR). Currently, it is well recognized that Glu194 and Glu204 residues, along with water clusters, constitute the proton release group of BR. Here we report that the replacement of Glu194 for Gln affects not only the photocycle of the protein but also has tremendous effect on the all-trans to 13-cis thermal isomerization. We studied the pH dependence of the dark adaptation of the E194Q mutant and performed HPLC analysis of the isomer compositions of the light- and partially dark-adapted states of the mutant at several pH values. Our data confirmed that E194Q exhibits extremely slow dark adaptation over a wide range of pH. HPLC data showed that a significantly larger concentration of all-trans isomer was present in the samples of the E194Q mutant even after prolonged dark adaptation. After 14 days in the dark the 13-cis to all-trans ratio was 1:3 in the mutant, compared to 2:1 in the wild type. These data clearly indicate the involvement of Glu194 in control of the rate of all-trans to 13-cis thermal isomerization.

Excitation of the M intermediates of bacteriorhodopsin.

Protein electric response signals (PERS) of the M intermediates of wild-type bacteriorhodopsin (bR) were recorded. Contrary to earlier findings reporting on a single-phase response upon excitation of the M intermediates, a kinetic analysis of the signals revealed the existence of three components, the fastest and the slowest ones of negative, while the middle one of positive sign with respect to the normal direction of proton pumping. Based on proton motion indicator experiments and molecular dipole calculations, the components were assigned to proton transfer steps and conformational changes driving the bR molecule back from the M to the ground state upon blue light excitation. The fastest, negative pump component was assigned to the proton transfer from D85 to the Schiff base. The subsequent positive component was attributed to rearrangements in the protein core (in the vicinity of the retinal molecule), triggered by the primary proton transfer process. The slowest component was established to reflect charge rearrangements associated with proton uptake by the protein from the bulk.

A tin oxide transparent electrode provides the means for rapid time-resolved pH measurements: application to photoinduced proton transfer of bacteriorhodopsin and proteorhodopsin.

An electrochemical cell was previously reported in which bacteriorhodopsin (BR, purple membrane) was adsorbed on the surface of a transparent SnO(2) electrode, and illumination resulted in potential or current changes (Koyama et al., Science 265:762-765, 1994; Robertson and Lukashev, Biophys. J. 68:1507-1517, 1995; Koyama et al., Photochem. Photobiol. 68:400-406, 1998). In this paper, we concluded that pH changes caused by proton transfer by the deposited BR or proteorhodopsin (PR) films lead to the flash-induced potential change in the SnO(2) electrode. Thus, the signals originate from BR and PR acting as light-driven proton pumps. This conclusion was drawn from the following observations. (1) The relation between the potential of a bare electrode and pH is linear for a wide pH range. (2) The flash-induced potential changes decrease with an increase in the buffer concentration. (3) The action spectrum of PR agrees well with the absorption spectrum. (4) The present electrode can monitor the pH change in the time range from 10 ms to several hundred milliseconds, as deduced by comparing the SnO(2) signal with the signals of pH-sensitive dyes. Using this electrode system, flash-induced proton transfer by BR was measured for a wide pH range from 2 to 10. From these data, we reconfirmed various pK(a) values reported previously, indicating that the present method can give the correct pK(a) values. This is the first report to estimate these pK(a) values directly from the proton transfer. We then applied this method to flash-induced proton transfer of PR. We observed proton uptake followed by release for the pH range from 4 to 9.5, and in other pH ranges, proton release followed by uptake was observed.

Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy.

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.

Automatic reconstruction of molecular and genetic networks from discrete time series data.

We apply a mathematical algorithm which processes discrete time series data to generate a complete list of Petri net structures containing the minimal number of nodes required to reproduce the data set. The completeness of the list as guaranteed by a mathematical proof allows to define a minimal set of experiments required to discriminate between alternative network structures. This in principle allows to prove all possible minimal network structures by disproving all alternative candidate structures. The dynamic behaviour of the networks in terms of a switching rule for the transitions of the Petri net is part of the result. In addition to network reconstruction, the algorithm can be used to determine how many yet undetected components at least must be involved in a certain process. The algorithm also reveals all alternative structural modifications of a network that are required to generate a predefined behaviour.