Yoram Gerchman

A synthetic multicellular system for programmed pattern formation.

Pattern formation is a hallmark of coordinated cell behaviour in both single and multicellular organisms. It typically involves cell–cell communication and intracellular signal processing. Here we show a synthetic multicellular system in which genetically engineered ‘receiver’ cells are programmed to form ring-like patterns of differentiation based on chemical gradients of an acyl-homoserine lactone (AHL) signal that is synthesized by ‘sender’ cells. In receiver cells, ‘band-detect’ gene networks respond to user-defined ranges of AHL concentrations. By fusing different fluorescent proteins as outputs of network variants, an initially undifferentiated ‘lawn’ of receivers is engineered to form a bullseye pattern around a sender colony. Other patterns, such as ellipses and clovers, are achieved by placing senders in different configurations. Experimental and theoretical analyses reveal which kinetic parameters most significantly affect ring development over time. Construction and study of such synthetic multicellular systems can improve our quantitative understanding of naturally occurring developmental processes and may foster applications in tissue engineering, biomaterial fabrication and biosensing.

Na+/H+ antiporters

Na(+)/H(+) antiporters are membrane proteins that play a major role in pH and Na(+) homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants. The emerging genomic sequence projects already have started to reveal that the Na(+)/H(+) antiporters cluster in several families. Structure and function studies of a purified antiporter protein have as yet been conducted mainly with NhaA, the key Na(+)/H(+) antiporter of Escherichia coli. This antiporter has been overexpressed, purified and reconstituted in a functional form in proteoliposomes. It has recently been crystallized in both 3D as well as 2D crystals. The NhaA 2D crystals were analyzed by cryoelectron microscopy and a density map at 4 A resolution was obtained and a 3D map was reconstructed. NhaA is shown to exist in the 2D crystals as a dimer of monomers each composed of 12 transmembrane segments with an asymmetric helix packing. This is the first insight into the structure of a polytopic membrane protein. Many Na(+)/H(+) antiporters are characterized by very dramatic sensitivity to pH, a property that corroborates their role in pH homeostasis. The molecular mechanism underlying this pH sensitivity has been studied in NhaA. Amino acid residues involved in the pH response have been identified. Conformational changes transducing the pH change into a change in activity were found in loop VIII-IX and at the N-terminus by probing trypsin digestion or binding of a specific monoclonal antibody respectively. Regulation by pH of the eukaryotic Na(+)/H(+) antiporters involves an intricate signal transduction pathway (recently reviewed by Yun et al., Am. J. Physiol. 269 (1995) G1-G11). The transcription of NhaA has been shown to be regulated by a novel Na(+)-specific regulatory network. It is envisaged that interdisciplinary approaches combining structure, molecular and cell biology as well as genomics should be applied in the future to the study of this important group of transporters.

Histidine 225, a Residue of the NhaA-Na+/H+ Antiporter of Escherichia coli Is Exposed and Faces the Cell Exterior

Cysteine residues were found nonessential in the mechanism of the NhaA antiporter activity of Escherichia coli. The functional C-less NhaA has provided the groundwork to study further histidine 225 of NhaA which has previously been suggested to play an important role in the activation of NhaA at alkaline pH (Rimon, A., Gerchman, Y., Olami, Y., Schuldiner, S. and Padan, E. (1995) J. Biol. Chem. 270, 26813-26817). C-less H225C was constructed and shown to possess an antiporter activity 60% of that of C-less antiporter and a pH profile similar to that of both the C-less or wild-type antiporters. Remarkably, whereas neither the wild-type nor the C-less antiporters were affected by N-ethylmaleimide, C-less H225C was inhibited by this reagent. To determine the degree of alkylation of the antiporter protein by N-ethylmaleimide, antiporter derivatives tagged at their C termini with six histidines residues were constructed. Alkylation of C-less H225C was measured by labeling of everted membrane vesicles with [14C]N-ethylmaleimide, affinity purification of the His-tagged antiporter, and determination of the radioactivity of the purified protein. This assay showed that H225C is alkylated to a much higher level than any of the native cysteinyl residues of NhaA reaching saturation at alkyl/NhaA stoichiometry of 1. The wild-type derivative showed at least 10-fold less alkylation even at higher concentrations, suggesting that H225C resides in a domain that is much more exposed to N-ethylmaleimide than the native cysteinyl residues of NhaA. Since H225C residues both in right-side out and inside-out membrane vesicles were quantitatively alkylated by N-ethylmaleimide, this assay was used to determine the accessibility of H225C to other SH reagents by titrating the H225C left free to react with N-ethylmaleimide, following exposure of the membranes to the reagents. Furthermore, since membrane-impermeant probes can react with residues in membrane-embedded protein only if accessible to the medium containing the reagent, the assay was used to determine the membrane topology of H225C. As expected for a membrane-permeant probe, p-chloromercuribenzoate reacted with H225C as efficiently as N-ethylmaleimide in both membrane orientations. Similar results were obtained with methanethiosulfonate ethylammonium supporting the recent observations that this probe is membrane-permeant. On the other hand, both membrane-impermeant reagents p-chloromercuribenzosulfonate and methanethiosulfonate ethyl-trimethyl ammonium bromide reacted with H225C 10-fold more in right-side out than in inside-out vesicles, and p-chloromercuribenzosulfonate also blocked completely the H225C in intact cells. These results strongly suggest that H225C is exposed at the periplasmic face of the membrane.

Bacterial communities in floral nectar.

Floral nectar is regarded as the most important reward available to animal-pollinated plants to attract pollinators. Despite the vast amount of publications on nectar properties, the role of nectar as a natural bacterial habitat is yet unexplored. To gain a better understanding of bacterial communities inhabiting floral nectar, culture-dependent and -independent (454-pyrosequencing) methods were used. Our findings demonstrate that bacterial communities in nectar are abundant and diverse. Using culture-dependent method we showed that bacterial communities of nectar displayed significant variation among three plant species: Amygdalus communis, Citrus paradisi and Nicotiana glauca. The dominant class in the nectar bacterial communities was Gammaproteobacteria. About half of the isolates were novel species (< 97% similarities of the 16S rRNA gene with known species). Using 454-pyrosequencing we demonstrated that nectar microbial community are distinct for each of the plant species while there are no significant differences between nectar microbial communities within nectars taken from different plants of the same species. Primary selection of the nectar bacteria is unclear; it may be affected by variations in the chemical composition of the nectar in each plant. The role of the rich and diverse nectar microflora in the attraction-repulsion relationships between the plant and its nectar consumers has yet to be explored.

Melatonin as an antioxidant and its semi-lunar rhythm in green macroalga Ulva sp.

The presence and role of melatonin in plants are still under debate owing to difficulties of identification and quantification. Accordingly, although it has been frequently proposed that melatonin acts as an antioxidant in phototrophic organisms, experimental data on its physiological role are scarce. This study describes the use of a rapid and simple new method for quantification of melatonin in the marine macroalga Ulva sp., organisms routinely exposed to tide-related environmental stresses and known for their high tolerance to abiotic conditions. The method was used here to show that exposure to oxidative stress-inducing environmental conditions (elevated temperature and heavy metals) induced a rise in melatonin level in the algae. Addition of exogenous melatonin alleviated the algae from cadmium-induced stress. Interestingly, although the algae were taken from a culture growing free floating and kept under constant photoperiod and water level, they exhibited a semi-lunar rhythm of melatonin levels that correlated with predicted spring tides. The correlation can probably be interpreted as reflecting preparation for predicted low tides, when the algae are exposed to increasing temperature, desiccation, and salinity, all known to induce oxidative stress. Given the simplicity of the described method it can easily be adapted for the study of melatonin in many other phototrophic organisms. These results provide, for the first time, experimental data that support both an antioxidant role for melatonin and its semi-lunar rhythm in macroalgae.

Light-dependent oxygen consumption in nitrogen-fixing cyanobacteria plays a key role in nitrogenase protection1

All colonial diazotrophic cyanobacteria are capable of simultaneously evolving O2 through oxygenic photosynthesis and fixing nitrogen via nitrogenase. Since nitrogenase is irreversibly inactivated by O2, accommodation of the two metabolic pathways has led to biochemical and/or structural adaptations that protect the enzyme from O2. In some species, differentiated cells (heterocysts) are produced within the filaments. PSII is absent in the heterocysts, while PSI activity is maintained. In other, nonheterocystous species, however, a “division of labor” occurs whereby individual cells within a colony appear to ephemerally fix nitrogen while others evolve oxygen. Using membrane inlet mass spectrometry (MIMS) in conjunction with tracer 18O2 and inhibitors of photosynthetic and respiratory electron transport, we examined the light dependence of O2 consumption in Trichodesmium sp. IMS 101, a nonheterocystous, colonial cyanobacterium, and Anabaena flos‐aquae (Lyngb.) Bréb. ex Bornet et Flahault, a heterocystous species. Our results indicate that in both species, intracellular O2 concentrations are maintained at low levels by the light‐dependent reduction of oxygen via the Mehler reaction. In N2‐fixing Trichodesmium colonies, Mehler activity can consume ∼75% of gross O2 production, while in Trichodesmium utilizing nitrate, Mehler activity declines and consumes ∼10% of gross O2 production. Moreover, evidence for the coupling between N2 fixation and Mehler activity was observed in purified heterocysts of Anabaena, where light accelerated O2 consumption by 3‐fold. Our results suggest that a major role for PSI in N2‐fixing cyanobacteria is to effectively act as a photon‐catalyzed oxidase, consuming O2 through pseudocyclic electron transport while simultaneously supplying ATP in both heterocystous and nonheterocystous taxa.

A pH-dependent conformational change of NhaA Na(+)/H(+) antiporter of Escherichia coli involves loop VIII-IX, plays a role in the pH response of the protein, and is maintained by the pure protein in dodecyl maltoside.

Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII–IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni2+-NTA column. Loop VIII–IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na+/H+ antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII–IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH.

A Point Mutation (G338S) and Its Suppressor Mutations Affect Both the pH Response of the NhaA-Na+/H+ Antiporter as Well as the Growth Phenotype of Escherichia coli

pH controls the activity of the NhaA Na+/H+ antiporter of Escherichia coli. In the present work we show that replacement of glycine 338 of NhaA with serine (G338S) alleviates the pH control of the antiporter. Monitoring Na+-dependent collapse of ΔpH, to assess antiporter activity in isolated membrane vesicles, shows that the mutant protein is practically independent of pH, between pH 7 and 9, and even at pH 6 is 70% active. Similarly the purified reconstituted mutant protein catalyzes pH-independent passive efflux of22Na from proteoliposomes as well as ΔpH-driven influx. Whereas the native NhaA in isolated membrane vesicles is exposed to digestion by trypsin only above pH 7, the mutated protein is degraded already at pH 6.5. ΔnhaAΔnhaB cells transformed with a plasmid encoding the pH-independent antiporter are sensitive to Na+ but not to K+ at alkaline pH, while growing in the presence of both ions at neutral pH. Several possibilities that could explain the Na+ sensitivity of the mutant at alkaline pH were excluded; Western analysis and measurement of Na+/H+ antiporter activity in membrane vesicles, isolated from cells shifted to the non-permissive growth conditions, showed neither reduced expression of G338S-NhaA nor defective activity. The finding that the mutated protein is electrogenic led to the retraction of the idea that the protein is active in vitro but not in vivo at alkaline pH, when only Δψ exists in the cells. The Na+ concentration needed for half-maximal activity of G338S in isolated everted membrane vesicles is similar to that of the wild type. Therefore an increase in intracellular Na+ due to a reduced antiporter affinity could not explain the results. It is suggested that the loss of growth at alkaline pH in the presence of Na+ is due to the loss of the pH control of the mutated NhaA. Indeed, in the four mutations suppressing G338S phenotype, growth at alkaline pH was restored together with the pH regulation of NhaA. Three of the four suppressor mutations cluster in helix IV, whereas the original mutation is in helix XI, suggesting that the two helixes interact.

Why do many galls have conspicuous colors? A new hypothesis

Galls are abnormal plant growth induced by various parasitic organisms, mainly insects. They serve as “incubators” for the developing insects in which they gain nutrition and protection from both abiotic factors and natural enemies. Galls are typically armed with high levels of defensive secondary metabolites. Conspicuousness by color, size and shape is a common gall trait. Many galls are colorful (red, yellow etc.) and therefore can be clearly distinguished from the surrounding host plant organs. Here we outlined a new hypothesis, suggesting that chemically protected galls which are also conspicuous are aposematic. We discuss predictions, alternative hypotheses and experimental tests of this hypothesis.

Replacements of Histidine 226 of NhaA-Na+/H+ Antiporter of Escherichia coli CYSTEINE (H226C) OR SERINE (H226S) RETAIN BOTH NORMAL ACTIVITY AND pH SENSITIVITY, ASPARTATE (H226D) SHIFTS THE pH PROFILE TOWARD BASIC pH, AND ALANINE (H226A) INACTIVATES THE CARRIER AT ALL pH VALUES

We have previously shown that replacement of His-226 in the NhaA Na+/H+ antiporter of Escherichia coli to Arg (H226R) shifts the pH profile of the antiporter toward acidic pH and as a result a ΔnhaAΔnhaB strain bearing this mutation is Na+ sensitive at alkaline pH (Gerchman, Y., Olami, Y., Rimon, A., Taglicht, D., Schuldiner, S. and Padan, E.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1212-1216). In the present work the role of His-226 in the response of NhaA to pH has been studied in detail. The Na+ sensitivity of the ΔnhaAΔnhaB mutant bearing the H226R-NhaA plasmid at alkaline pH provided a very powerful tool to isolate revertants and suppressants of H226R growing on high Na+ at alkaline pH. With this approach cysteine (H226C) and serine (H226S) replacements were found to efficiently replace His-226 and yield an antiporter, which like the wild-type protein, is activated by pH between pH 7 and 8. These results imply that polarity and/or hydrogen bonding, the common properties shared by these amino acid residues, are essential at position 226 for pH regulation of NhaA. This suggestion was substantiated by site-directed mutagenesis of His-226 either to alanine (H226A) or aspartate (H226D). Whereas H226A-NhaA shows very low activity which is not activated by pH, H226D-NhaA is active and regulated by pH. The pH profile of H226D is shifted by half a pH unit toward alkaline pH, as opposed to the previously isolated mutant H226R which has a pH profile shift, to the same extent, but toward acidic pH. It is suggested that charge modifies the pH profile but is not essential for the pH regulation of NhaA.