A model of excitation and adaptation in bacterial chemotaxis

Bacterial chemotaxis is widely studied because of its accessibility and because it incorporates processes that are important in a number of sensory systems: signal transduction, excitation, adaptation, and a change in behavior, all in response to stimuli. Quantitative data on the change in behavior are available for this system, and the major biochemical steps in the signal transduction/processing pathway have been identified. We have incorporated recent biochemical data into a mathematical model that can reproduce many of the major features of the intracellular response, including the change in the level of chemotactic proteins to step and ramp stimuli such as those used in experimental protocols. The interaction of the chemotactic proteins with the motor is not modeled, but we can estimate the degree of cooperativity needed to produce the observed gain under the assumption that the chemotactic proteins interact directly with the motor proteins.

“Switch I” mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by σ54-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of σ54-holoenzyme—residues 206–220—is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrCA216V and NtrCG219K, have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrCA216C and NtrCG219C, are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix–turn–helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with σ54-holoenzyme. The conserved region in which these amino acid substitutions occur (206–220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21ras.

Controlled unzipping of a bacterial surface layer with atomic force microscopy

We have combined high-resolution atomic force microscopy (AFM) imaging and force spectroscopy to gain insight into the interaction forces between the individual protomers of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans. After imaging the HPI layer, the AFM stylus was attached to individual protomers by enforced stylus-sample contact to allow force spectroscopy experiments. Imaging of the HPI layer after recording force-extension curves allowed adhesion forces to be correlated with structural alterations. By using this approach, individual protomers of the HPI layer were found to be removed at pulling forces of ≈300 pN. Furthermore, it was possible to sequentially unzip entire bacterial pores formed by six HPI protomers. The combination of high-resolution AFM imaging of individual proteins with the determination of their intramolecular forces is a method of studying the mechanical stability of supramolecular structures at the level of single molecules.

Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein

In the current model for bacterial cell division, FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other proteins such as FtsA. This putative protein complex ultimately generates the division septum. Herein we report that FtsZ and FtsA proteins tagged with green fluorescent protein (GFP) colocalize to division-site ring-like structures in living bacterial cells in a visible space between the segregated nucleoids. Cells with higher levels of FtsZ–GFP or with FtsA–GFP plus excess wild-type FtsZ were inhibited for cell division and often exhibited bright fluorescent spiral tubules that spanned the length of the filamentous cells. This suggests that FtsZ may switch from a septation-competent localized ring to an unlocalized spiral under some conditions and that FtsA can bind to FtsZ in both conformations. FtsZ–GFP also formed nonproductive but localized aggregates at a higher concentration that could represent FtsZ nucleation sites. The general domain structure of FtsZ–GFP resembles that of tubulin, since the C terminus of FtsZ is not required for polymerization but may regulate polymerization state. The N-terminal portion of Rhizobium FtsZ polymerized in Escherichia coli and appeared to copolymerize with E. coli FtsZ, suggesting a degree of interspecies functional conservation. Analysis of several deletions of FtsA–GFP suggests that multiple segments of FtsA are important for its localization to the FtsZ ring.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

Proton uptake by bacterial reaction centers: The protein complex responds in a similar manner to the reduction of either quinone acceptor

In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone QA− and the secondary acceptor QB− after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near QB; some of them carry additional mutations distant from the quinone sites (M231Arg → Leu, M43Asn → Asp, M5Asn → Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala → Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either QA− or QB−, suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to QB is evident from examination of the pattern of H+/QA− proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q− species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.

Chloroplast small heat shock proteins: Evidence for atypical evolution of an organelle-localized protein

Knowledge of the origin and evolution of gene families is critical to our understanding of the evolution of protein function. To gain a detailed understanding of the evolution of the small heat shock proteins (sHSPs) in plants, we have examined the evolutionary history of the chloroplast (CP)-localized sHSPs. Previously, these nuclear-encoded CP proteins had been identified only from angiosperms. This study reveals the presence of the CP sHSPs in a moss, Funaria hygrometrica. Two clones for CP sHSPs were isolated from a F. hygrometrica heat shock cDNA library that represent two distinct CP sHSP genes. Our analysis of the CP sHSPs reveals unexpected evolutionary relationships and patterns of sequence conservation. Phylogenetic analysis of the CP sHSPs with other plant CP sHSPs and eukaryotic, archaeal, and bacterial sHSPs shows that the CP sHSPs are not closely related to the cyanobacterial sHSPs. Thus, they most likely evolved via gene duplication from a nuclear-encoded cytosolic sHSP and not via gene transfer from the CP endosymbiont. Previous sequence analysis had shown that all angiosperm CP sHSPs possess a methionine-rich region in the N-terminal domain. The primary sequence of this region is not highly conserved in the F. hygrometrica CP sHSPs. This lack of sequence conservation indicates that sometime in land plant evolution, after the divergence of mosses from the common ancestor of angiosperms but before the monocot–dicot divergence, there was a change in the selective constraints acting on the CP sHSPs.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections

Many Gram-positive bacteria covalently tether their surface adhesins to the cell wall peptidoglycan. We find that surface proteins of Staphylococcus aureus are linked to the cell wall by sortase, an enzyme that cleaves polypeptides at a conserved LPXTG motif. S. aureus mutants lacking sortase fail to process and display surface proteins and are defective in the establishment of infections. Thus, the cell wall envelope of Gram-positive bacteria represents a surface organelle responsible for interactions with the host environment during the pathogenesis of bacterial infections.

Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions

Amphipols are a new class of surfactants that make it possible to handle membrane proteins in detergent-free aqueous solution as though they were soluble proteins. The strongly hydrophilic backbone of these polymers is grafted with hydrophobic chains, making them amphiphilic. Amphipols are able to stabilize in aqueous solution under their native state four well-characterized integral membrane proteins: (i) bacteriorhodopsin, (ii) a bacterial photosynthetic reaction center, (iii) cytochrome b6f, and (iv) matrix porin.

Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold

We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded β-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (i) that the molecular recognition of fluorescein is specific and (ii) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins “anticalins.” Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice.

Electrostatic interactions between rotor and stator in the bacterial flagellar motor

Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor–stator interface.

Diversity and phylogeny of gephyrin: Tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins

Gephyrin is essential for both the postsynaptic localization of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (Moco) in different peripheral organs. Several alternatively spliced gephyrin transcripts have been identified in rat brain that differ in their 5′ coding regions. Here, we describe gephyrin splice variants that are differentially expressed in non-neuronal tissues and different regions of the adult mouse brain. Analysis of the murine gephyrin gene indicates a highly mosaic organization, with eight of its 29 exons corresponding to the alternatively spliced regions identified by cDNA sequencing. The N- and C-terminal domains of gephyrin encoded by exons 3–7 and 16–29, respectively, display sequence similarities to bacterial, invertebrate, and plant proteins involved in Moco biosynthesis, whereas the central exons 8, 13, and 14 encode motifs that may mediate oligomerization and tubulin binding. Our data are consistent with gephyrin having evolved from a Moco biosynthetic protein by insertion of protein interaction sequences.

A 1.2-Å snapshot of the final step of bacterial cell wall biosynthesis

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of β-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-Å resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type d-alanyl-d-alanine carboxypeptidase/transpeptidase (EC 3.4.16.4) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

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.