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Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution, the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that currently comprises seven different types based on structural homology in the TMDs.

This review will inevitably be influenced by my personal experience and personal view of the progression of this amazing family of proteins. This has generated a huge literature in over nearly five decades, some ideas have bloomed and faded while others have persisted, other contributions simply become redundant, overtaken by better techniques. At the outset, the pioneers had no idea of the magnitude of the topic they were working on, then a very rough idea of the significance emerged and, progressively, the picture becomes sharper and finally extraordinary. I have tried to produce at least an outline of that progression. My apologies for the also inevitable omissions, especially relating to the mass of biochemical and spectroscopy and genetical studies. I decided to prioritise structural biology because structures when successful are definitive and of course provide a 'visual' image. However, I tried to limit the structural aspects to the proteins that reflected the main advances.

Type I secretion systems (T1SS) are widespread in Gram-negative bacteria, especially in pathogenic bacteria, and they secrete adhesins, iron-scavenger proteins, lipases, proteases, or pore-forming toxins in the unfolded state in one step across two membranes without any periplasmic intermediate into the extracellular space. The substrates of T1SS are in general characterized by a C-terminal secretion sequence and nonapeptide repeats, so-called GG repeats, located N terminal to the secretion sequence. These GG repeats bind Ca 2+ ions in the extracellular space, which triggers folding of the entire protein. Here we summarize our current knowledge of how Gram-negative bacteria secrete these substrates, which can possess a molecular mass of up to 1,500 kDa. We also describe recent findings that demonstrate that the absence of periplasmic intermediates, the “classic” mode of action, does not hold true for all T1SS and that we are beginning to realize modifications of a common theme.

International audience; Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.

Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of β-lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the β-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.

The ATP-binding cassette (ABC)-transporter haemolysin (Hly)B, a central element of a Type I secretion machinery, acts in concert with two additional proteins in Escherichia coli to translocate the toxin HlyA directly from the cytoplasm to the exterior. The basic set of crystal structures necessary to describe the catalytic cycle of the isolated HlyB-NBD (nucleotide-binding domain) has now been completed. This allowed a detailed analysis with respect to hinge regions, functionally important key residues and potential energy storage devices that revealed many novel features. These include a structural asymmetry within the ATP dimer that was significantly enhanced in the presence of Mg2+, indicating a possible functional asymmetry in the form of one open and one closed phosphate exit tunnel. Guided by the structural analysis, we identified two amino acids, closing one tunnel by an apparent salt bridge. Mutation of these residues abolished ATP-dependent cooperativity of the NBDs. The implications of these new findings for the coupling of ATP binding and hydrolysis to functional activity are discussed.

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC50 of 16 μM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.

Type 1 secretion systems (T1SS) are wide-spread among Gram-negative bacteria. An important example is the secretion of the hemolytic toxin HlyA from uropathogenic strains. Secretion is achieved in a single step directly from the cytosol to the extracellular space. The translocation machinery is composed of three indispensable membrane proteins, two in the inner membrane, and the third in the outer membrane. The inner membrane proteins belong to the ABC transporter and membrane fusion protein families (MFPs), respectively, while the outer membrane component is a porin-like protein. Assembly of the three proteins is triggered by accumulation of the transport substrate (HlyA) in the cytoplasm, to form a continuous channel from the inner membrane, bridging the periplasm and finally to the exterior. Interestingly, the majority of substrates of T1SS contain all the information necessary for targeting the polypeptide to the translocation channel — a specific sequence at the extreme C-terminus. Here, we summarize our current knowledge of regulation, channel assembly, translocation of substrates, and in the case of the HlyA toxin, its interaction with host membranes. We try to provide a complete picture of structure function of the components of the translocation channel and their interaction with the substrate. Although we will place the emphasis on the paradigm of Type 1 secretion systems, the hemolysin A secretion machinery from E. coli, we also cover as completely as possible current knowledge of other examples of these fascinating translocation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

The export of proteins to the Escherichia coli periplasm is a well established system for heterologous protein production. With a better understanding of the protein export (SecA, Y-dependent) process and a greater awareness of the conditions necessary for correct folding of proteins in the periplasm, serious efforts are now being made to manipulate this system to achieve substantial increases in the yield of authentically folded proteins. Further advances in the development of methods for the recovery of recombinant proteins from the culture medium have made the use of fusion proteins secreted by the protein A or haemolysin pathways a more attractive option. Recent studies of the haemolysin system indicate its ability to secrete a wide range of polypeptides, including normally cytoplasmic proteins. As their features and potential applications become much clearer, a rapidly expanding number of protein-secretion mechanisms in Gram-negative bacteria are becoming available for heterologous protein expression. Most, if not all, of these systems can be successfully transplanted into E. coli, providing a wider choice of systems for the future.

Summary Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1-D and 2-D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat- or SDS-treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross-reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat-resistant Ca2+-binding protein from Saccharo-polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+.

Structure and Mechanism of ABC Transporters ABCA1-Mediated HDL Formation Bile Formation, the Enterohepatic Circulation and Cholestasis Deciphering the Role of Human Efflux Proteins in Cellular and Tissue Permeability The Photoreceptor ABC Transporter and Its Role in Inherited Retinal Degenerative Diseases ABCs in Immunity - The Antigen Translocation Machinery The ABCs of Mitochondrial Metabolic Disorders The Role of ABCC Family Members for the Disposition of Endogenous Compounds and Drugs ABC Transporter Mutations in Heritable Skin Diseases CFTR, Elucidating the Basis and Influencing the Outcome of a Major Genetic Disease Physiology and Pathology of ABCC8 Getting into the Peroxisome with the ABCD Family ABC Transporters and Sterol Homeostasis Hot Topics, including: Multidrug Transporters in Stem Cells and Cancer Stem Cells Targeting Regulation of ABC Transporters Nuclear Receptors involved in Regulating Bile Flow Gene and Small Molecule Therapy for Cystic Fibrosis.

International audience; The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.

The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyper-branched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp + (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30–40 %), it is dispensable at high humidity. Dendritic migration (flagella- and surfactin-dependent) of strains 168 sfp + swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (single-cell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication.

While the role of calcium binding proteins (CaBPs) in cell signaling pathways and homeostasis is well established in eukaryotic cells, the physiological function of CaBPs in prokaryotes is unknown. Although several CaBPs have been identified and sequences predicted in a variety of prokaryotic genomes, biochemical and functional characterization is lacking. We hypothesize that CaBPs play an important role in Ca2+ homeostasis and that Ca2+ ions regulate several processes in bacterial cells. The purpose of this work was to study the effects of Ca2+ in the B. subtilis proteome, to identify CaBPs altered (increased or decreased) by the addition of Ca2+ -chelators (EGTA, BAPTA) or CaCl2, and to examine Ca2+ homeostasis in B. subtilis cells utilizing various analytical techniques. 45Ca-autoradiography and antibody-crossreactivity were used to detect CaBPs. These proteins were identified by LC-MS/MS. Intracellular calcium levels [Ca2+]i were measured using the photoprotein aequorin. Our results show that remarkable global changes in protein abundance occurred in the B. subtilis proteome as a result of CaCl2 or chelator 58 treatments compared to control cells. Six proteins appeared to be modulated by high levels of extracellular Ca2+. These proteins were increased after Ca2+ -chelator treatments and reduced upon Ca2+ addition. Moreover, these proteins bound radioactive 45Ca2+, and showed a shift in molecular weight in the presence of Ca2+ /EGTA. B. subtilis cells thightly regulate cytosolic Ca2+ levels. Taken together, these results suggest an important role of Ca2+ ions in B. subtilis.

The conservedprpC,prkC,cpgAlocus inBacillus subtilisencodes respectively a Ser/Thr phosphatase, the cognate sensor kinase (containing an external PASTA domain suggested to bind peptidoglycan precursors) and CpgA, a small ribosome-associated GTPase that we have shown previously is implicated in shape determination and peptidoglycan deposition. In this study, in a search for targets of PrkC and PrpC, we showed that,in vitro, CpgA itself is phosphorylated on serine and threonine, and another GTPase, the translation factor EF-Tu, is also phosphorylated by the kinase on the conserved T384 residue. Both substrates are dephosphorylated by PrpCin vitro. In addition, we identified YezB, a 10.3 kDa polypeptide, and a component of the stressosome, as a substrate for both enzymesin vitroand apparentlyin vivo. We propose that the PrpC/PrkC/CpgA system constitutes an important element of a regulatory network involved in the coordination of cell wall expansion and growth inB. subtilis.

Highly branched dendritic swarming ofB. subtilison synthetic B-medium involves a developmental-like process that is absolutely dependent on flagella and surfactin secretion. In order to identify new swarming genes, we targeted the two-component ComPA signalling pathway and associated global regulators. In liquid cultures, the histidine kinase ComP, and the response regulator ComA, respond to secreted pheromones ComX and CSF (encoded byphrC) in order to control production of surfactin synthases and ComS (competence regulator). In this study, for what is believed to be the first time, we established that distinct early stages of dendritic swarming can be clearly defined, and that they are amenable to genetic analysis. In a mutational analysis producing several mutants with distinctive phenotypes, we were able to assign the genessfp(activation of surfactin synthases),comA, abrBandcodY(global regulators),hag(flagellin),mecAandyvzB(hag-like),and swrB(motility), to the different swarming stages. Surprisingly, mutations in genescomPX, comQ, comS,rapCandoppD, which are normally indispensable for import of CSF, had only modest effects, if any, on swarming and surfactin production. Therefore, during dendritic swarming, surfactin synthesis is apparently subject to novel regulation that is largely independent of the ComXP pathway; we discuss possible alternative mechanisms for drivingsrfABCDtranscription. We showed that thephrCmutant, largely independent of any effect on surfactin production, was also, nevertheless, blocked early in swarming, forming stunted dendrites, with abnormal dendrite initiation morphology. In a mixed swarm co-inoculated withphrC sfp+andphrC+sfp(GFP), an apparently normal swarm was produced. In fact, while initiation of all dendrites was of the abnormalphrCtype, these were predominantly populated bysfpcells, which migrated faster than thephrCcells. This and other results indicated a specific migration defect in thephrCmutant that could not betrans-complemented by CSF in a mixed swarm. CSF is the C-terminal pentapeptide of the surface-exposed PhrC pre-peptide and we propose that the residual PhrC 35 aa residue peptide anchored in the exterior of the cytoplasmic membrane has an apparently novel extracellular role in swarming.

International audience; Surfactins are a family of heptacyclopeptides in which the C-terminal carbonyl is linked with the beta-hydroxy group of a fatty acid acylating the N-terminal function of a glutamic acid residue. The fatty acyl chain is 12-16 carbon atoms long. These compounds, which are secreted by the Gram-positive bacterium Bacillus subtilis in stationary phase in liquid cultures, play an important role in swarming communities on the surface of agar media in the formation of dendritic patterns. TOF secondary ion MS (TOF-SIMS) imaging was used to map surfactins within 16-17 h swarming patterns, with a 2 mum spatial resolution. Surfactins were mainly located in the central mother colony (the site of initial inoculation), in a 'ring' surrounding the pattern and along the edges of the dendrites. In the mother colony and the interior of the dendrites, surfactins with shorter chain lengths are present, whereas in the ring surrounding the swarm community and between dendrites, surfactins with longer fatty acyl chain lengths were found. A quantitative analysis by MALDI-TOF MS showed a concentration gradient of surfactin from the mother colony to the periphery. The concentration of surfactin was approximately 400 pmol/mL in the mother colony and approximately 10 pmol/mL at the base of the dendrites, decreasing to 2 pmol/mL at their tips.

International audience; The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca(2+) in E. coli through Ca(2+) influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca(2+) was 0.2-0.5 microM. In the presence of external Ca(2+) (1 mM) at alkaline pH this rose to 4 microM, being reduced to 0.9 microM at acid pH. Removal of external Ca(2+) caused an immediate decrease in cytosolic free Ca(2+) at 50-100 nM s(-1). Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca(2+)/H(+)exchanger, appeared not to be a major Ca(2+)-efflux pathway. In the absence of added Na(+), but with 1 mM external Ca(2+), cytosolic free Ca(2+) rose to approximately 10 microM. The addition of Na(+)(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca(2+) efflux. However, this effect was not specific, since K(+) also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca(2+) efflux. The results were consistent with H(+) and monovalent cations competing with Ca(2+) for a non-selective ion influx channel. Ca(2+) entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca(2+) in Escherichia coli. The number of Ca(2+) ions calculated to move per cell per second ranged from 6 million ions per second. This raises fundamental questions about the nature and regulation of Ca(2+) transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.

The transport of substrates across a cellular membrane is a vitally important biological function essential for cell survival. ATP-binding cassette (ABC) transporters constitute one of the largest subfamilies of membrane proteins, accomplishing this task. Mutations in genes encoding for ABC transporters cause different diseases, for example, Adrenoleukodystrophy, Stargardt disease or Cystic Fibrosis. Furthermore, some ABC transporters are responsible for multidrug resistance, presenting a major obstacle in modern cancer chemotherapy. In order to translocate the enormous variety of substrates, ranging from ions, nutrients, small peptides to large toxins, different ABC-transporters utilize the energy gained from ATP binding and hydrolysis. The ATP binding cassette, also called the motor domain of ABC transporters, is highly conserved among all ABC transporters. The ability to purify this domain rather easily presents a perfect possibility to investigate the mechanism of ATP hydrolysis, thus providing us with a detailed picture of this process. Recently, many crystal structures of the ATP-binding domain and the full-length structures of two ABC transporters have been solved. Combining these structural data, we have now the opportunity to analyze the hydrolysis event on a molecular level. This review provides an overview of the structural investigations of the ATP-binding domains, highlighting molecular changes upon ATP binding and hydrolysis.

The ABC transporter HlyB is a central element of the HlyA secretion machinery, a paradigm of Type I secretion. Here, we describe the crystal structure of the HlyB‐NBD (nucleotide‐binding domain) with H662 replaced by Ala in complex with ATP/Mg 2+ . The dimer shows a composite architecture, in which two intact ATP molecules are bound at the interface of the Walker A motif and the C‐loop, provided by the two monomers. ATPase measurements confirm that H662 is essential for activity. Based on these data, we propose a model in which E631 and H662, highly conserved among ABC transporters, form a catalytic dyad. Here, H662 acts as a ‘linchpin’, holding together all required parts of a complicated network of interactions between ATP, water molecules, Mg 2+ , and amino acids both in cis and trans , necessary for intermonomer communication. Based on biochemical experiments, we discuss the hypothesis that substrate‐assisted catalysis, rather than general base catalysis might operate in ABC‐ATPases.

The natural wild-type Bacillus subtilis strain 3610 swarms rapidly on the synthetic B medium in symmetrical concentric waves of branched dendritic patterns. In a comparison of the behavior of the laboratory strain 168 ( trp ) on different media with that of 3610, strain 168 ( trp ), which does not produce surfactin, displayed less swarming activity, both qualitatively (pattern formation) and in speed of colonization. On E and B media, 168 failed to swarm; however, with the latter, swarming was arrested at an early stage of development, with filamentous cells and rafts of cells (characteristic of dendrites of 3610) associated with bud-like structures surrounding the central inoculum. In contrast, strain 168 apparently swarmed efficiently on Luria-Bertani (LB) agar, colonizing the entire plate in 24 h. However, analysis of the intermediate stages of development of swarms on LB medium demonstrated that, in comparison with strain 3610, initiation of swarming of 168 ( trp ) was delayed and the greatly reduced rate of expansion of the swarm was uncoordinated, with some regions advancing faster than others. Moreover, while early stages of swarming in 3610 are accompanied by the formation of large numbers of dendrites whose rapid advance involves packs of cells at the tips, strain 168 advanced more slowly as a continuous front. When sfp + was inserted into the chromosome of 168 ( trp ) to reestablish surfactin production, many features observed with 3610 on LB medium were now visible with 168. However, swarming of 168 ( sfp + ) still showed some reduced speed and a distinctive pattern compared to swarming of 3610. The results are discussed in terms of the possible role of surfactin in the swarming process and the different modes of swarming on LB medium.

This series of conferences beautifully illustrates and reflects the dramatic pace of development within the field of bacteriology. In the initial meeting the talks tended to be about the molecular details of single systems. Only 5 years on the talks centered on the complex integrative nature of signaling networks, their sensitivity to the changing external environment, and, in the case of pathogens and symbionts, their dialogue with their hosts. What is becoming very clear from the rapid increase in data on sensory networks in increasing numbers of bacterial species is that no two species are alike. Indeed, no cell within a population is identical to its neighbor, and each is influenced by its stage in the cell cycle, its recent and historical past, and its local environment. We need to make sense of this increasing volume of information in order to develop a deeper understanding of particular species and to create generic models that will allow some insight into how individual cells, dispersed populations, and communities of bacteria (single and mixed species) will respond to change. This is the challenge of the next series of meetings. Taking on this challenge requires the true integration of different disciplines, combining the skills of the microbial physiologist and biochemist, the physicist, the computational and mathematical modelers, the engineers, and the technologists. Each needs to understand the other's language and then mobilize their different skills to address a common biologically driven problem: the design and operation of bacterial sensory networks. We must not however neglect past experience, the roots of bacterial physiology, but combine this with new knowledge of the molecular details of the component parts of these complex circuits to develop (or revive?) a broad holistic approach. This will involve combining the accumulated wealth of biochemical and metabolic data with the current flood of data from genomics and proteomics, brought into a coherent whole through modelling, in order to produce useful guides on the way to the "total" understanding of the complex nature that makes up a bacterium.

After optimizing the conditions, including nutrients and temperature, swarming of Bacillus subtilis 3610 was obtained on a synthetic, fully defined medium. The swarms formed highly branched (dendritic) patterns, generated by successive waves of moving cells. A detailed microscopic in situ analysis of swarms 1 and 2 revealed varied cell morphologies and a remarkable series of events, with cells assembling into different ‘structures’, as the architecture of the swarm developed. Long filamentous cells begin to form before the onset of the first swarming (11 h) and are again observed at later stages in the interior of individual mature dendrites. Swarm 2, detected at 18–22 h, is accompanied by the rapid movement of a wave of dispersed (non-filamentous) cells. Subsequently at the forward edge of this swarm, individual cells begin to cluster together, gradually forming de novo the shape of a dendrite tip with progressive lengthening of this new structure ‘backwards' towards the swarm centre. In both swarms 1 and 2, after the initial clustering of cells, there is the progressive appearance of a spreading monolayer of rafts (4–5 non-filamented cells, neatly aligned). The alternative possible roles of the rafts in the development of the swarm are discussed.

We previously described a mutation feeB1 conferring a temperature-sensitive filamentation phenotype and resistance to the calmodulin inhibitor 48/80 in Escherichia coli, which constitutes a single base change in the acceptor stem of the rare tRNA3Leu recognizing CUA codons. We now describe a second mutant, feeA1, unlinked to feeB, but displaying a similar phenotype, 48/80 resistance and a reduced growth rate at the permissive temperature, 30 degrees C, and temperature-sensitive, forming short filaments at 42 degrees C. In the feeA mutant, tRNA3Leu expression (but not that of tRNA1Leu) was reduced approximately fivefold relative to the wild type. We previously showed that the synthesis of beta-galactosidase, which unusually requires the translation of 6-CUA codons, was substantially reduced, particularly at 42 degrees C, in feeB mutants. The feeA mutant also shows drastically reduced synthesis of beta-galactosidase at the non-permissive temperature and reduced levels even at the permissive temperature. We also show that increased copy numbers of the abundant tRNA1Leu, which can also read CUA codons at low efficiency, suppressed the effects of feeA1 under some conditions, providing further evidence that the mutant was deficient in CUA translation. This, and the previous study, demonstrates that mutations which either reduce the activity of tRNA3Leu or the cellular amount of tRNA3Leu confer resistance to the drug 48/80, with concomitant inhibition of cell division at 42 degrees C.

Shigella flexneri kills macrophages through apoptosis, involving the induction of host cell DNA fragmentation and characteristic morphological changes. Shigella can only cause damage if it escapes from the phagolysosome into the cytoplasm. The S. flexneri cytotoxic genes have been localized to the ipa operon of shigella's virulence plasmid. ipaB, C and D deletion mutants are not invasive and therefore not cytotoxic. In order to distinguish genes involved in the escape from the phagolysosome as distinct from cytotoxicity, we constructed Shigella strains that secrete low amounts of Escherichia coli haemolysin (hly(low)). These strains can escape into the cytoplasm of the macrophage even in the absence of the invasion plasmid as verified by electron microscopy and resistance to chloroquine. Macrophages were infected with different ipa mutants expressing hly(low). Both delta ipaC hly(low) and delta ipaD hly(low) were cytotoxic whilst delta ipaB hly(low) and a hly(low) strain cured of shigella's pathogenicity plasmid were not. Furthermore, both delta ipaC hly(low) and delta ipaD hly(low) killed through apoptosis as shown by both changes in ultrastructural morphology and fragmentation of the host cell DNA. These results demonstrate that ipaB is essential for S. flexneri to induce apoptosis in macrophages.

Summary The release of haemolysin from Escherichia coli involves direct secretion across both the inner and outer membranes. Secretion of HlyA is dependent upon a specific membrane export complex composed of HlyB, -D and possibly TolC. HlyA is targeted to the medium via the membrane translocation complex, by a novel C-terminal secretion signal. Previous studies involving deletion and fusion analyses have given contradictory results for the minimal length (20–60 residues) of this HlyA signal region and little is known of the nature of the specific residues and structural features required for function. In this study we have analysed, quantitatively, the effect upon secretion of many point mutations introduced into the HlyA C-terminus. The results indicate the presence of a minimal secretion signal domain whose proximal boundary extends to at least residue –46 and which contains at least four Individual residues essential for maximal secretion levels. We propose that such residues act co-operatively, forming multiple contact points with the translocator proteins, with the ‘best fit’ promoting maximal levels of secretion.

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Secretion of foreign proteins from microorganisms in general and from Escherichia coli and Bacillus subtilis in particular is still a favoured option for the subsequent recovery of such proteins on a commercial scale. The variety of natural bacterial systems now being studied and the likely limiting factors in their adaptation for the export of heterologous proteins are reviewed. One major limiting factor is the lack of basic knowledge of the secretion mechanism. The details of one such system now being intensively studied, the secretion of haemolysin from E. coli, are described.

Preparation of an in vitro coupled transcription-translation system from E. coli strains lacking exonuclease V has greatly improved the system for use with added linear DNA fragments. In fact, in extracts of these mutants linear fragments are stable for several hours. However the cell extracts show a high level of endogenous background. To avoid this complication extracts were prepared at 30 degrees C from a mutant carrying a temperature-sensitive exonuclease V. Polypeptides coded by a specific DNA region, e.g., delineated by restriction endonuclease sites, can now be easily identified.

DNA restriction enzyme fragments have been used to programme the synthesis of polypeptides in an in vitro system without apparent loss in fidelity compared with supercoiled templates. The system is extremely sensitive, less than 1 microgram of DNA can be used to direct the synthesis of 35S-labelled polypeptides of sufficiently high specific activity such that products can be identified by SDS-PAGE after a few hours autoradiography. The ability to analyse fragments can be used to readily assign specific proteins to small regions of the coding template, to identify cloned gene products distinct from those of the vector, and to identify cloned genes expressed from their own promoters. The in vitro system can be used successfully with bacterial DNA from other species and efficient extracts can be prepared from any E. coli K-12 strain, which should greatly facilitate the purification of factors controlling the expression of specific genes by complementation assay.

Most secreted proteins of both eukaryotes and prokaryotes are synthesized as preproteins with NH2-terminal hydrophobic signal sequences which are removed during transfer of the proteins across the membrane1,2. The role of cleavable signal sequences in the insertion of proteins into the cytoplasmic membrane is not clear. Rothman and Lenard have proposed that the assembly of those integral membrane proteins that have a substantial extracytoplasmic domain (ectoproteins) occurs by a similar mechanism to that of secreted proteins, involving a cleavable signal sequence, but with complete secretion being prevented by a hydrophobic COOH–terminal region3. In eukaryotes, several ectoproteins seem to be synthesized in this way4–6 but in prokaryotes, the coat protein of M13 phage is the only reported example of a cytoplasmic membrane protein that is made as a preprotein2,7, although a non-cleavable signal-like sequence has been shown for the E. coli lactose permease8. We show here that penicillin-binding proteins 5 and 6 of Escheri-chia coli are the first examples of cytoplasmic membrane proteins, encoded by bacteria, that are processed.

Many biological processes are coupled to ATP hydrolysis. We describe here a class of closely related ATP-binding proteins, from several bacterial species, which are associated with a variety of cellular functions including membrane transport, cell division, nodulation in Rhizobium and haemolysin export. These proteins comprise a family of structurally and functionally related subunits which share a common evolutionary origin, bind ATP and probably serve to couple ATP hydrolysis to each of these biological processes. This finding suggests a specific role for ATP in cell division, nodulation during nitrogen fixation and protein export, and allows us to assign a probable function to one of the protein components from each of these systems.

The synthesis of a membrane-bound MalE ,B-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%o. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.