Martin Kessel

Enzymatic and Structural Similarities between the Escherichia coli ATP-dependent Proteases, ClpXP and ClpAP*

Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (M r46,000) associate to form a six-membered ring (M r ∼ 280,000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (∼2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min−1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500‐Å‐long secreted protein that is rich in β‐structure and contains two regions, R1 and R2, of tandem 19‐residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel β‐sheets; or (ii) a β‐helix in which the polypeptide chain is coiled to form three long parallel β‐sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional ‘covert’ copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the β‐helix model that the shaft domain – a 350 Å rod – should consist of a continuous run of these motifs, apart from loop inserts. The N‐terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin‐like structure. Drawing on crystal structures of known β‐helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins – all large secreted proteins of bacterial provenance – that have similar repeats and probably also similar structures.

Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli.

Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage λ CII transcriptional activator plays a key role in the lysis‐lysogeny decision by phage λ. Here we demonstrate that the E. coli ATP‐dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat‐shock transcription factor σ32. Proteolysis of σ32 requires, in vivo, the presence of the DnaK‐DnaJ‐GrpE chaperone machine. Neither DnaK‐DnaJ‐GrpE nor GroEL‐GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP‐dependent proteolysis of CII in vitro. The degradation of CII is at least 10‐fold faster than that of σ32. Electron microscopy revealed that purified FtsH forms ring‐shaped structures with a diameter of 6–7 nm.

V. Functions of S‐layers

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Translocation pathway of protein substrates in ClpAP protease.

Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATPγS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through ≈150 Å into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.

Six‐fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP‐dependent activator, ClpY

ClpQ (HsIV) is a homolog of the β‐subunits of the 20S proteasome. In E. coli, it is expressed from an operon that also encodes ClpY (HsIU), an ATPase homologous to the protease chaperone, ClpX. ClpQ (subunit M r 19 000) and ClpY (subunit M r 49 000) were purified separately as oligomeric proteins with molecular weights of ∼220 000 and ∼350 000, respectively, estimated by gel filtration. Mixtures of ClpY and ClpQ displayed ATP‐dependent proteolytic activity against casein, and a complex of the two proteins was isolated by gel filtration in the presence of ATP. Image processing of negatively stained electron micrographs revealed strong six‐fold rotational symmetry for both ClpY and ClpQ, suggesting that the subunits of both proteins are arranged in hexagonal rings. The molecular weight of ClpQ combined with its symmetry is consistent with a double hexameric ring, whereas the data on ClpY suggest only one such ring. The symmetry mismatch previously observed between hexameric ClpA and heptameric ClpP in the related ClpAP protease is apparently not reproduced in the symmetry‐matched ClpYQ system.

Halobacteroides halobius gen. nov., sp. nov., a Moderately Halophic Anaerobic Bacterium from the Bottom Sediments of the Dead Sea

Summary An anaerobic, long rod-shaped, motile, Gram-negative, non-sporulating bacterium wasisolated from Dead Sea sediment. It required NaCl concentrations between 1.4 and 2.8 mol/l, and optimal growth with doubling times of about 1 hour was found in 1.5-2.5 mol NaCl/l and 37-42 °C. Fermentation products on glucose were ethanol, acetate, hydrogen and carbon dioxide. Fermentable substrates included several mono- and oligosaccharides, starch and pyruvate. The Guanine plus Cytosine content of its DNA was 30.7 mol . Analysis of the oligonucleotides in a digest of its 16S rRNA showed that the isolate was an eubacterium, but was not a member of any of the major eubacterial subgroups defined to date. We propose to name it Halobacteroides halobius gen. nov., sp. nov., belonging to a new family, the Haloanaerobiaceae. The type strain is strain MD-1 (ATCC 35273).

E. coli Transports Aggregated Proteins to the Poles by a Specific and Energy-Dependent Process

Aggregation of proteins due to failure of quality control mechanisms is deleterious to both eukaryotes and prokaryotes. We found that in Escherichia coli, protein aggregates are delivered to the pole and form a large polar aggregate (LPA). The formation of LPAs involves two steps: the formation of multiple small aggregates and the delivery of these aggregates to the pole to form an LPA. Formation of randomly distributed aggregates, their delivery to the poles, and LPA formation are all energy-dependent processes. The latter steps require the proton motive force, activities of the DnaK and DnaJ chaperones, and MreB. About 90 min after their formation, the LPAs are dissolved in a process that is dependent upon ClpB, DnaK, and energy. Our results confirm and substantiate the notion that the formation of LPAs allows asymmetric inheritance of the aggregated proteins to a small number of daughter cells, enabling their rapid elimination from most of the bacterial population. Moreover, the results show that the processing of aggregated proteins by the protein quality control system is a multi-step process with distinct spatial and temporal controls.

Halogeometricum borinquense gen. nov., sp. nov., a novel halophilic archaeon from Puerto Rico

A novel extremely halophilic archaeon was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism is very pleomorphic, motile and requires at least 8% (w/v) NaCl to grow. Polar lipid composition revealed the presence of a novel non-sulfate-containing glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. The G + C content of the DNA is 59 mol%. On the basis of 16S rRNA sequence data, the new isolate cannot be classified in one of the recognized genera, but occupies a position that is distantly related to the genus Haloferax. All these features justify the creation of a new genus and a new species for the family Halobacteriaceae, order Halobacteriales. The name Halogeometricum borinquense gen. nov., sp. nov. is proposed. The type strain is ATCC 700274T.

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at −150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.