Bacterial neural networksJ. P. Armitage et al.

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 0950-382X Blackwell Publishing Ltd, 200347Meeting Report

Molecular Microbiology (2003) 47(2), 583593

MicroMeeting Thinking and decision making, bacterial style: Bacterial Neural Networks, Obernai, France, 7th12th June 2002
Judith P. Armitage,1* Charles J. Dorman,2 Klaas Hellingwerf,3 Rudiger Schmitt,4 David Summers5 and Barry Holland6 1 Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. 2 Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Trinity College, Dublin 2, Ireland. 3 Laboratory for Microbiology, Swammerdam Institute for Life Science, University of Amsterdam, 1018 WV Amsterdam, Netherlands. 4 Department of Genetics, University of Regensburg, D-93040 Regensburg, Germany. 5 Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK. 6 Institut de Genetique et Microbiologie, UMR-CNRS 8621, Universite Paris XI, 91405 Orsay, France. Summary Bacteria exhibit a bewildering range of behavioural responses and permutations of metabolic pathways for maximum exploitation of their environment. These are based on sensory perception of external and internal signals through batteries of surface and cytoplasmic receptors, evaluation of complex information ows and rapid decision making. Appreciation of the diversity of bacterial behaviour and adaptation capacities requires the study of a broad range of organisms and at this meeting we sampled more than 30 species with new ndings which included the nature of gaseous receptors, advances in chemotaxis, subversion of host defences by pathogens, adaptation to high salt, community life and its obvious benets, cell to cell communications and even the nature of bacterial circadian rhythms. With around 80 bacterial genomes now completed, and many more almost there, it was appropriate to complete the meeting with an introduction to Systems Biology and prospects for simulating the virtual cell. The versatility and seemingly intelligent behaviour of bacteria will continue to fascinate, and this meeting on Bacterial Neural Networks fully reected the excitement of this eld.

The third Euroconference carrying the sweeping title Bacterial Neural Networks took place earlier this year in the beautiful Alsatian town of Obernai, France, under the auspices of the European Science Foundation (Euresco) and the European Commissions Human Potential Program. This forum successfully brought together a diverse group of researchers interested in understanding not only the rich repertoire of regulatory networks and sophisticated signalling mechanisms, but also the fascinating behaviour and extraordinary capacity of the different bacteria on the planet to adapt and survive in a wide range of environments, by seemingly intelligent use of sensory perception. Although only a small fraction of the subject could be touched upon in the time available, the meeting was able to convey the excitement and beauty of several different systems in a range of bacteria, allied to major advances in the understanding of some of these processes at the molecular level. The range of approaches and interpretations led to some extremely lively, broad based and enlightening discussions with an audience including a large proportion of graduate students and other young scientists at the outset of their careers in Microbiology. The clear overall impact of the meeting was that we still have a lot more questions than answers, but may now be in a position to use multidisciplinary approaches to address a number of these questions. It also became very apparent that bacteria still have a lot of surprises in store! In general, only the plenary talks are summarized here to illustrate the breadth of the meeting.

Signalling, behaviour and development

Accepted 17 October, 2002, *For correspondence. E-mail judith. armitage@bioch.ox.ac.uk; Tel. (+44) 1865275299; Fax (+44) 1865275297.

Signalling, behaviour and development are interlinked processes enabling bacteria in a wide range of environ-

2003 Blackwell Publishing Ltd

584 J. P. Armitage et al. ments to detect and translate external signals to regulate expression, movement or differentiation, so that survival, dispersal through spores or colonization of new territory, are maximized. Bacterial chemotaxis, regulated by two component signal transduction systems, may well be the rst complete behavioural system to be understood as a sequence of molecular events and this therefore formed a major theme at the beginning of the meeting. This led on to other two component pathways, for sensing hydrogen and oxygen concentration, cell population density, osmotic stress or for the transition to growth on solid surfaces. It will be a goal of future conferences in the Bacterial Neural Network series to learn more about intracellular communications between the diverse systems and about actions of global regulators that link and tune the different pathways in a cell. Aerotaxis is a specialized form of chemotaxis which appears to use different input systems in different species to control a common output into the chemosensory pathway. Barry Taylor (Loma Linda, CA, USA) described how the 300 or so aerotaxis, Aer, homodimer receptors in Escherichia coli sense redox changes in the respiratory electron transport chain. These changes, detected by the N-terminal FAD-binding PAS domain, are transmitted to the C-terminal domain that then transduces signals to the histidine kinase, CheA, of the chemosensory system (Fig. 1). The sensing and signalling domains of Aer are linked by a hydrophobic membrane anchor and a conserved HAMP domain. Both PAS and HAMP domains are found in sensory proteins from bacteria to eukaryotes. Mutational and second-site suppressor analyses identied contacts between the PAS and HAMP domains as the most probable path for the intramolecular transmission of conformational signals, initiated in the PAS domain in response to a change in the redox state of the bound FAD (Taylor et al., 2001). Unlike Aer, other chemotaxis receptors, for example in E. coli, are transmembrane homodimers composed of an external ligand-binding domain, four transmembrane helices, and internal methyl-accepting and signalling domains. Chemoreceptors form higher order aggregates with other signalling proteins at the cell poles, although the signicance of these structures is not yet clear. The precise mechanism by which receptors transmit signals

Fig. 1. Diagramatic representation of the E. coli aerotaxis receptor, Aer, and a classical chemoreceptor (MCP) showing the intracellular signalling pathway controlling switching of the agellar motor. The MCP is a transmembrane receptor controlling the phosphorylation state of the histidine protein kinase, CheA (A) via the linker, CheW (W) and the two response regulators, CheY (Y) (motor binding) and CheB [a methyl esterase involved with the methyl transferase, CheR(R), in controlling methylation of the receptor glutamates and thus adaptation]. Aer lacks the periplasmic domain and the adaptation sites, but has FAD cofactor. ETS-electron transport chain, CM-cytoplasmic membrane. (From B. L.Taylor).

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Bacterial neural networks 585 from outside the cell to the internal transduction chain and the role of receptor aggregation are the subject of a great deal of recent research. Sherry Mowbray (Uppsala, Sweden) analysed the effect of point mutations in the ligand binding site of the E. coli MCP, the aspartate receptor (Tar). Curiously, mutations that changed the afnity of aspartate binding by four orders of magnitude had little, if any, effect on signalling as measured by adaptation-dependent methylation of the receptor and the ability of the bacteria to spread through soft nutrient agar plates. Like other available biological data, these results suggest that the signalling of an individual receptor is binary, that is, it is either on or off. Signalling apparently begins with motions within a subunit in the periplasmic domain. The subsequent changes in periplasmic regions are not yet understood, although it seems likely that these are modulated by the higher order aggregates (Bjrkman et al., 2001). Both Aer and the classical receptors, such as Tar, signal to the response regulator, CheY, a small 14 kDa protein which binds the E. coli agellar motor when phosphorylated. CheY has been studied in great detail at the atomic and biochemical level for many years, it was therefore exciting to hear from Michael Eisenbach (Rehovot, Israel) how, in addition to phosphorylation at a conserved aspartate, the activity of CheY is enhanced by acetylation on up to ve lysine residues (Barak and Eisenbach, 2001). Moreover, the CheY-modifying phosphorylation and acetylation enzymes appear to mutually regulate their activities, suggesting that they link the level of chemotactic signalling to its metabolic state and that the acetylation system may compensate for cellular variations in the levels of the phosphorylating and dephosphorylating enzymes of CheY. Many bacterial species, including the a subgroup members, Rhodobacter sphaeroides and Sinorhizobium meliloti have multiple chemotactic sensors and pathways which signal to the agella motor. Judy Armitage (Oxford, UK) showed how R. sphaeroides has multiple homologues of the E. coli chemosensory proteins, including 4CheW, 6 CheY, 4 CheA, 3CheR, 2 CheB and 12 receptor homologues. Nevertheless, most of these were shown not to be redundant (Porter et al., 2002). The receptors turn out to be spatially separated; some in the membrane, localized to the poles (as in E. coli) with specic CheA, CheR and CheW homologues. Other receptors however, interacting with a different subset of chemosensory proteins, are in the cytoplasm and may sense the intracellular metabolic state of cells (Fig. 2). Remarkably, the cytoplasmic proteins are organized in structures with the appearance of organelles that are actively segregated upon cell division (Wadhams et al., 2002). The different components of the chemosensory networks are differentially expressed under different growth conditions, suggesting
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Fig. 2. Localization of chemosensory proteins in R. sphaeroides. The gfp gene was cloned onto either the 3 or 5 end of the chemosensory gene and the wild-type gene replaced with the fusion, in the genome behind the native promoter, and the recombinant checked for normal behaviour. A. McpG-GFP (one of eight transmembrane receptors) localized to the poles. B. TlpC-GFP localized to a cluster in the cytoplasm. C. A single strain expressing both McpG-GFP and TlpC-GFP. (From G. Wadhams and J. P. Armitage)

that this species can tune its behavioral system to match the prevailing environment. The chemotaxis signalling pathway of Sinorhizobium meliloti has two response regulators, CheY1 and CheY2, and, as is the case with all non-enteric species, lacks the CheZ phosphatase. Rather than switching direction of motor rotation to change swimming direction, S. meliloti slows rotation, with CheY2-P binding the motor and CheY1 acting as a phosphate sink, compensating for the lack of CheZ. Birgit Scharf (Regensburg, Germany) presented data suggesting that the relative proportion of two antagonistic cytoplasmic proteins, MotD (fast) and CheY2-P (slow) compete for sites at the motor base to control the speed of agellar rotation at any given moment. This concept is supported by CheY2 point mutants with either high or low afnity for the motor switch (Schmitt, 2002).

586 J. P. Armitage et al. Among other two component systems discussed, Oliver Lenz (Berlin, Germany) provided an interesting comparison to Aer. The H2-sensing signal transduction chain, Hox, regulates the use of molecular hydrogen (H2) as an energy source in the aerobe Ralstonia eutropha. R. eutropha has both a membrane-bound and a cytoplasmic [NiFe] hydrogenase to feed electrons either directly into the respiratory chain or indirectly reduce NAD. When sufcient H2 is present in the environment, the multimeric hydrogenases are synthesised as metal-free precursors and subsequently assembled in a complex process to reach the catalytic site. Transcription of the two sets of hydrogenase enzymes is positively controlled by a histidine kinase two component system which is, interestingly, a third [NiFe] hydrogenase, the regulatory hydrogenase or RH, which does not participate in energy conversion. The formation of a tight complex between RH and the cytoplasmic sensor kinase, HoxJ, permits the latter to monitor redox changes through a PAS domain and hence to control transcription through a downstream response regulator, HoxA. Increased H2 alters the kinase/phosphatase activity of HoxJ such that HoxA becomes unphosphorylated, which is the active form that triggers hydrogenase gene expression. This unusually inverse activation behaviour is in contrast to the mechanisms of orthodox two-component systems (Lenz et al., 2002). In yet another variation on signal sensing, evidence was presented by Bert Poolman (Groningen, Netherlands) that osmosensors in the membrane of Lactococcus lactis are activated by ionic changes in the cytoplasm on osmotic up- or downshifts. There has been a great deal of speculation about the mechanisms that allow osmotic change to be sensed. Many possible signalling mechanisms were experimentally tested and a picture emerged in which it seems likely that osmotic upshifts are sensed through the intracellular ionic strength. For the osmosensing ABC transporter of L. lactis there is strong evidence that the ionic signal is transduced to the protein complex via changes in proteinlipid interactions rather than direct sensing of ion concentration by the proteins (van der Heide et al., 2001; Poolman et al., 2002). More complex signalling systems More complex signalling systems were also an outstanding feature of the meeting. For example, purple bacteria such as R. capsulatus display dramatic metabolic diversity, allowing them to exploit, as available, light, hydrogen and organic compounds as energy sources. Carl Bauer (Indiana, USA) explained how the shift from aerobic to anaerobic conditions causes induction of not only of bacteriochlorophyll and carotenoid genes, but also of nitrogenase and rubisco genes. Expression is regulated by a global two component pathway, RegB/A, homologues of which are present in a wide range of related species. The membrane spanning histidine kinase, RegB, responds to oxygen dependent electron transport through a high afnity terminal oxidase, controlling the activity of the transcriptional activator, RegA (Swem et al., 2001). In addition, however, there is a repressor, CtrJ which, although lacking a metal centre, still responds to the redox state. CtrJ appears to interact directly with molecular oxygen, which induces an intramolecular S-S bond in the DNA binding domain of the molecule. This oxidized form of CtrJ binds to promoters of genes encoding, for example, certain cytochromes of the photosynthetic apparatus repressing their expression (Masuda et al., 2002). The validity of the neural net concept in phospho-relay control systems was addressed by Klaas Hellingwerf (Amsterdam, Netherlands). He listed the essential features of a neural network: multiple parallel paths, logical operators, auto-amplication, feed-back loops and crosstalk (Fig. 3). The rst four are apparent in many systems, but what about cross-talk? This has long been a subject of debate, not least because of the large number of two component systems identied in many species. In vitro transphosphorylation experiments have demonstrated that non-cognate phosphoryl transfer can readily be observed, but reports of cross-talk in vivo have been rarer. In experiments with the Arc, Uhp, Pho and Ntr phosphorelay systems, Hellingwerf found no convincing evidence of cross-talk. It appeared that the effects observed in vitro were obscured in vivo by the phosphatase activity of the unphosphorylated sensory kinases (Verhamme et al., 2002). The existence of cross-talk has been discussed for over a decade but it is clear that much remains to be done before proof of its biological signicance can be claimed. Differentiation/developmental systems Differentiation/developmental systems require a response to a series of cues, leading to co-ordinated changes in gene expression. In Streptomyces, differentiation of the vegetative mycelium into sporulating aerial hyphae and antibiotic-producing hyphae is co-ordinated by both extracellular and intracellular signalling. Keith Chater (John Innes Centre, Norwich, UK) took as his starting point the antibiotic production and aerial mycelium deciencies of Streptomyces coelicolor mutants in bldA, the gene for the only tRNA in this GC-rich organism capable of translating the rare codon UUA. He showed that for each of three different antibiotic pathways, bldA-dependence was due to the presence of TTA codons in pathway-specic regulatory genes, and therefore that there was no pleiotropically acting, TTA-containing regulatory gene. This implied that a distinct bldA target gene (or genes) controls morphological differentiation. This target turned out to be the S. coelicolor version of adpA, a gene which in Streptomy 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 583 593

Bacterial neural networks 587

Fig. 3. Diagrammatic representation of a virtual microbial cell with several sensory systems working in parallel (2 component, quorum sensing, cell-cell contact). Because logical operations are being carried out (e.g. two kinases may phosphorylate a single regulator) and some may show auto-amplication or feedback control (intracellular curved black arrows), all prerequisites that would make this system classify as a neural network would be fullled if, in addition, there is (extensive) cross-talk (i.e. broken arrows). Additional phosphoryl groups may be provided to this system through compounds such as Acetyl-phosphate (Ac~P), Carbamoyl-phosphate (Carb~P) and/or ATP (represented through DGp, the phosphorylation potential). Abbreviations: Dp-electrochemical proton gradient. Input pathways in green/blue, sensory pathways blue, output pathways red/blue. (With thanks to Mark Roberts)

ces griseus is needed to activate both morphological differentiation and antibiotic production. However, in S. coelicolor adpA appears unnecessary for antibiotic production, and its own regulation also differs in the two species in S. griseus it is expressed only in the presence of an extracellular quorum indicator, the gammabutyrolactone A-factor, whereas in S. coelicolor the gamma-butyrolactone SCB1 is not needed for expression of adpA (Chater, 2001). At a more complex level Lotte Sogaard-Andersen (Odense, Denmark) introduced the topic of intercellular signalling in Myxococcus xanthus populations. Individual cells can migrate over a surface using gliding motility (with a speed that is only 10- to 100-fold faster than continental drift). In response to starvation cells aggregate to form spore-lled fruiting bodies. This entire process of aggregation and sporulation is dependent on ve consecutive molecular signals (A to E), exchanged between the cells in a specic sequential order (i.e. B, A, D, E and C).
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Sogaard-Andersen focused on the C-signal, which induces aggregation as well as sporulation. The C-signal is cell surface associated rather than diffusible and Csignal transmission involves direct contact between cells. An ordered increase in the level of C-signalling during development together with the contact dependent C-signal transmission mechanism is essential for the spatial coordination of aggregation and sporulation. Surprisingly C-signal turns out to be a 17 kDa membrane associated protein (p17), with homology to part of an NAD binding alcohol dehydrogenase. p17 is formed by proteolytic cleavage of a 25 kDa precursor protein (p25) encoded by the csgA gene. p25 binds NAD+, however, p17 lacks most of the NAD+ binding pocket and does not bind NAD+, therefore must have a structural rather than catalytic role. At the molecular level C-signal induces phosphorylation of the key DNA binding response regulator FruA, which in turn induces methylation of FrzCD and transcription of developmental genes (Fig. 4) (Kruse et al., 2001).

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Fig. 4. Model of the C-signal transduction pathway. The diagram illustrates contactdependent C-signal transmission between two cells. For simplicity, the components in the pathway are only shown in the cell to the right. These components are also present in the cell to the left. In that cell, only csgA is shown. The ordered increase in C-signalling levels is caused by the two signal amplication loops labelled 1 and 2. -(indicates the, still, hypothetical C-signal sensor. HPK indicates the hypothetical FruA histidine protein kinase activated in response to the reception of the C-signal. (From L. Sgaard-Andersen)

Bacteria evidently enjoy and prot from community life and Jim Shapiro (Chicago, USA) presented a visual and cellular analysis of circularly expanding colonies of Proteus mirabilis which swarm on agar surfaces as alternating populations of long multiagellate swarmers and short rods. Swarming motility is a collective process. Isolated swarmer cells do not migrate over the agar, only groups or rafts of aligned swarmer cells, encased in acidic exopolysaccharide. This co-ordinated multicellular alternation of swarming and consolidation phases leads to formation of symmetrical colonies with periodically spaced terraces. This pattern formation is dependent on the presence of a mixture of amino acids in the swarm substrate, but not on factors such as the rate of swarming or the amount of carbon or energy source available, nor on cycles of nutrient exhaustion and chemotactic migration. A dened lifespan of swarmer cells plus a cell-density threshold for collective migration is therefore thought to be involved in forming swarm patterns (Esipov and Shapiro, 1998). Continuing the theme of community life, a biolm is a functional consortium of bacteria (usually in nature composed of multiple species), enclosed in a matrix of extracellular biopolymers. Ian Sutherland (Edinburgh, UK) introduced their formation and general properties. Biolms that develop on solid surfaces exposed to a continuous ow of nutrients form thick layers consisting of differentiated mushroom and pillar-like structures separated by water-lled spaces. The structures are composed primarily of an extracellular polysaccharide matrix in which the bacteria are embedded. He highlighted some of the differences between bacteria living planktonically and those in biolms. Whereas in planktonic environments one bacteriocin producing strain will always outcompete another, stable biolms can form with bacteriocin producing and sensitive strains present. However, dual species biolms formed by bacteriocin producing strains tend to be smaller and show increased sensitivity to antibiotics and disinfec-

tants such as triclosan. Bacteriocins may therefore have a major role in biolm formation, competition within biolms and reducing colonization of existing biolms rather than in planktonic environments. Sutherland also suggested that the sensitivity to the common disinfectant triclosan may be because it can inhibit the synthesis of precursors of homoserine lactones and thus disrupt quorum sensing that may be important in biolm formation (Tait and Sutherland, 2002). In recent years, it has become apparent that under appropriate conditions, even E. coli can produce biolms. David Clarke (Bath, UK) studied changes in the physiology and gene expression of E. coli switching from a planktonic (free-living) to a sessile (surface-attached) life-style. The Rcs two-component pathway was found to respond immediately but transiently to contacts with a solid surface. In a working model, the hybrid membrane sensorkinase, RcsC, senses the presence of a solid surface and, by means of a complex phosphorelay, phosphorylates the response regulator, RcsB. This, in turn, promotes the transcription of genes for the production and secretion of capsular polysaccharides, colonic acid, required for the formation and differentiation of biolms. In addition, new evidence indicates that the Rcs-pathway positively regulates the expression of genes for periplasmic, as well as inner and outer membrane proteins of yet unknown function (Clarke et al., 2002). In other studies of biolms, Elizabeth Lopez-Tenorio (Kobe, Japan) suggested that while quorum sensing may be required to initiate biolm formation in E. coli, type 1 mbriae and genes involved in controlling cell division, such as ftsZ, minE and minD are needed to produce mature, thick biolms. In contrast increased agellar activity, for example, led to their dissolution. As might be expected therefore, biolm formation is almost certainly the result of a large number of interactive positive and negative signals. Biolms in nature rarely involve a single monoculture and the surface of lungs in CF patients can contain many
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Bacterial neural networks 589 resources and recreational lakes. Cyanobacterial blooms are thought to have killed many Pleistocene mammals whose skeletons are found in the sediments of an ancient lake near Halle, Germany. Elke Dittmann (Berlin, Germany) presented evidence that microcystin, although considered an endotoxin, functions in cell-cell communication within M. aeruginosa populations. The control of the export of this peptide depends not only on population density, but also light intensity which may activate its dedicated ABC transporter (Dittmann et al., 2001). In Gram-positive organisms, cellcell signalling is often modulated by peptides, either unmodied or processed after translation, that are sensed by the producing cells by a typical two-component signal transduction system. Michiel Kleerebezem (Wageningen, Netherlands) described the regulation of production of (modied) antimicrobial peptides (AMP). The type I AMP, nisin, controlled expression (NICE) system of Lactococcus lactis is similar to the subtilin regulated expression (SURE) system of Bacillus subtilis. The construction of functional chimeric promoters between the spa and nis promoters showed that both subtilin and nisin responsive promoter elements contain pentanucleotide direct repeats responsible for the AMP mediated response. Similarly, chimeras between the sensors, NisK and SpaK, of these two systems were found to be functional and led to engineered cross-talk between these two AMP-regulatory systems. The nisin and subtilin regulatory systems may be examples of a common quorum-sensing signalling system in many Gram-positive bacteria. Nevertheless, in Streptomyces, secondary metabolite production can be controlled by lactone-based pheromones, a class more normally associated with the Gram-negative bacteria (Kleerebezem and Quadri 2001). These ndings illustrate that our understanding of the quorum sensing eld is still far from complete and that there is still room for surprises in this area of molecular biology.

Fig. 5. Identication of Pseudomonas genes differentially expressed in coculture with Streptococci using a random promoter library screening approach. Expression of Pseudomonas reporters in (A) monoculture versus (B) co-culture. (Expression was normalized and grouped using CLUSTER, Eisen et al., 1998). Only differentially expressed clones are shown (approximately 5% of all reporters). Red to green gradient indicates levels of expression from high to low. The screen identied genes which were (I) downregulated and (II) upregulated in co-culture. (K. Duan and M. G. Surette, unpublished data).

different bacterial species, both Gram-positive and Gramnegative. Michael Surette (Calgary, Canada) described how he has left behind the relative simplicity of quorum sensing in systems involving a single bacterial species, and has begun to explore intercellular signalling in mixed biolms. His talk demonstrated that despite their complexity, these systems are nevertheless amenable to study. The changing community of microbes in the lungs of cystic brosis (CF) patients was described. These included chronic infections, the rise and fall of normal lung ora, and occasional infections by secondary pathogens. Strong evidence for the importance of interspecies communication in the pathology of the CF lung came from a demonstration that most normal ora isolated from CF sputum produce chemical signals that increase the pathogenicity of Pseudomonas aeruginosa in animal models. A random promoter library was developed to analyse the response of pseudomonads in mixed cultures, and the complexity of species interplay was demonstrated by the observation that different members of the respiratory tract ora could induce the activation of different gene sets in their pseudomonad neighbours (Fig. 5). Keeping abreast of the number of cells in your population by quorum sensing may control a multiplicity of density-dependent processes including pathogenicity, plasmid transfer by conjugation and of course biolm formation. Certain planktonic freshwater cyanobacteria, like Microcystis aeruginosa, produce the hepatotoxic microcystin that is a potential drinking hazard in water
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Hostbacterial interactions and signalling Hostbacterial interactions and signalling is an obvious area where complex interactions and signalling occurs. Some spectacular advances were described concerning infection of mammalian cells and the extraordinary mechanisms employed by bacteria to subvert the host cells regulatory circuits to ensure a successful invasion. Type 1 mbriae are used by many Gram-negative bacterial species for initial attachment to host cells, and they also contribute to biolm formation. In Escherichia coli, phase-variable expression of mbriae is the result of an invertible genetic switch whose default setting is the OFF phase. Charles Dorman (Dublin, Ireland) described new data showing that the mE gene that encodes the recom-

590 J. P. Armitage et al. binase responsible for maintaining the switch in the OFF phase uses the same switch to tag its mRNA product for rapid or delayed turnover. The switch, located at the 3 end of the mE gene, contains a Rho-dependent transcription terminator that operates only when the switch is in the OFF phase. In the ON phase, mE transcription reads through the switch and the result is a more stable message that is translated to give greatly enhanced levels of the FimE recombinase. This in turn inverts the switch back into the OFF phase (Joyce and Dorman, 2002). The signals that modulate the performance of this molecular mechanism will be of great future interest. Having attached successfully to the host surface, many pathogens move to a more intimate level of association. In several cases so-called type III secretion systems are crucial for this next phase, with Yersinia, for example, injecting proteins (Yops), including a host-like phosphatase, into macrophages. Global regulation plays an essential role in the co-ordination of the assault launched by Yersinia on its unfortunate host. Guy Cornelis (Basel, Switzerland) used new time lapse studies to show that the process of contacting the surface of the cell and injecting through the type III secretion apparatus, the injectisome made up of 25 Ysc proteins, takes only about one minute, suggesting that all the Yops are preformed (Cornelis, 2002). Also on the theme of bacterial redirection of host cellular events, Brenden Kenny (Bristol, UK) discussed some recent developments in enteropathogenic E. coli (EPEC) and the enterocyte effacement (LEE) pathogenicity island. He concentrated on the effector proteins Tir and Map, which are also injected into host cells through a type III secretion system. Tir, the translocated intimin receptor, has been studied for several years and is of special interest as it becomes the receptor for intimate host-pathogen contact, following modication of Tir by the host. These modications involve phosphorylation of specic residues within Tir, such as at Ser-434 by the cAMP-dependent protein kinase A and at Tyr-474 by an unidentied tyrosine protein kinase. Less well characterized is Map, a mitochondrial associated protein, which is targeted to host cell mitochondria where it alters mitochondrial ability to maintain a membrane potential. The rst 44 amino acids of this 203-residue polypeptide are believed to act as a motif for N-terminal cleavage and mitochondrial targeting, but the precise role of Map in the mitochondrion remains elusive. Suggested roles include the regulation of apoptotic events or interference with respiratory functions. A surprising revelation was that Map also possesses a mitochondrialindependent function, activating the host small GTPase protein, Cdc42, leading to lopodia formation at the site of infection (Fig. 6). Moreover, this activity is inhibitory to Tir-mediated cytoskeletal rearrangements and is down regulated in a Tir and intimin-dependent mechanism (Kenny et al., 2002). It is clear that we are at an early stage in our understanding of the multiple and complex contributions of Map to EPEC pathogenesis. Plant pathogens, like their mammalian counterparts, employ a battery of secreted degradative enzymes, regulated by quorum sensing and specic 2-component regulatory systems. Tapio Palva (Helsinki) described the virulence factors employed by the plant pathogen Erwinia carotovora, among them a type III secretion system encoded by the hrp locus and a number of plant cell wall degrading enzymes. One of the central virulence regulators in E. carotovora, the PehR-PehS 2-component system responds to external calcium and magnesium. It resembles the PhoP-PhoQ system of Salmonella spp., which also plays a key role in virulence, especially in interactions with the macrophage where responses to Mg2+ levels are crucial. Interestingly, E. carotovora also possesses a counterpart to the PmrA-PmrB twocomponent system of Salmonella spp. In the animal pathogen the PmrA-PmrB system is concerned with responses to iron and is partly under the control of PhoPPhoQ, forming a regulatory cascade. At present it is unclear if a similar relationship exists between PmrAPmrB of E. carotovora and the PehR-PehS system. A regulatory cascade does link the ExpS-ExpA (or GacSGacA) two-component system and the RsmA-rsmB posttranscriptional regulator. The rsmB gene codes for a small regulatory RNA whose half-life is governed by the RsmA RNA binding protein. This cascade plays a central role in

Fig. 6. Map-induced lopodia formation. Scanning electron micrograph in pseudo-colour showing lododia formation at site of infection of host cell by enteropathogenic E. coli (EPEC) [reproduced from Kenny et al. (2002) with permission]. 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 583 593

Bacterial neural networks 591 controlling extracellular virulence protein production by E. carotovora (Hyytiainen et al., 2001). A signicant difference between the animal and plant pathogens is the necessity of the latter to develop methods for dealing with a thick protective host cell wall. Consequently, E. carotovora expresses an impressive arsenal of cell wall degrading enzymes. Nevertheless, as with the animal pathogens, the key to a successful infection is the appropriate expression of virulence determinants in response to sensing the correct cues provided by the host. Quorum sensing is also very important in the initiation of an infection by E. carotovora suggesting that a minimum inoculum is needed to ensure a successful assault on the plant without triggering the plant defense systems. Much progress has been made in elucidating the details of the defense pathways, and surprisingly a key role for the tryptophan biosynthetic pathway in the plant has been determined. Particularly important are the Trp-pathwayderived secondary metabolites called indole glucosinolates which have now been shown to be induced in response to attack by E. carotovora (Brader et al., 2001). The Trp pathway is also crucial in the biosynthesis of auxin plant hormones. Jos Vanderleyden (Leuven) described the presence of auxin biosynthesis pathways in bacteria that form associative relationships with plants. Azospirillum brasilense possesses at least three pathways for auxin biosynthesis, at least two of which use tryptophan as a precursor. The ipdC gene is involved in auxin biosynthesis and is induced by low pH, exogenous auxin and entry into stationary phase (Lambrecht et al., 1999). Using a promoter probe transposon library, plantresponsive genes in symbiotic bacteria were detected. Among these was the casA gene from Rhizobium etli coding for a calmodulin-like protein (calsymin) which is essential for normal formation of bacteroids and the ability to x nitrogen. This protein is expressed by the bacterium exclusively during interaction with the plant and the casA gene is repressed by a TetR-like repressor called CasR. Rhizobium etli also possesses a sophisticated array of quorum-sensing systems. The best characterized of these involves the saturated long chain 3-hydroxy-acylhomoserine lactone produced by CinI, which is the ligand for the putative transcription regulator CinR. Mutants decient in this signalling system are impaired in symbiosis (Daniels et al., 2002). The second system, encoded by the raiI and raiR genes, is involved in the production and activities of at least six other autoinducer molecules. Mutants decient in CinI or RaiR also have abnormal growth rates. Therefore, these signalling pathways appear to be crucial for successful bacterial growth and bacteria plant interactions. The most tyrannical and unavoidable global regulator must be time itself. Susan Golden (Texas A and M, USA)
2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 583 593

reported progress in the analysis of mechanisms underpinning the circadian rhythms found in the cyanobacterium Synechococcus elongatus. Such is the prevalence of clock-watching in this organism that its effect is apparent in the periodic transcription of genes throughout the entire genome. Evidence for the sophistication of the clockwork is the demonstration now that separate timing circuits with different periods can co-exist. Previous studies had identied a number of clock genes, kai (for cycle), and new studies indicate that a component of the Kai oscillator, KaiC, autophosphorylates on several thr/ser residues. KaiC also interacts with a typical histidine sensor kinase (Iwasaki et al., 2002). The interplay between these phospho-proteins and presumed phosphatases, promises to be a key element in the cascade, which ultimately controls RNA polymerase action on a 24-h clock basis. A central, if not yet understood, role for sigma factors in the mechanism of periodic transcription was revealed by the effects of mutations in the genes for four group 2 sigma factors (rpoD2, rpoD3, rpoD4 and sigC) (Nair et al., 2002). Each single or pair-wise inactivation of these genes altered the period, amplitude, phase or waveform of circadian expression from the psbA1 promoter. However, only a subset of alleles affected expression from the kaiB promoter. Thus inactivation of sigC lengthened the circadian period of activity of the psbA1 promoter by 2 h but had no effect on kaiB. Systems biology Systems biology is dening new approaches to studying the totality of enzyme reactions, protein interactions, and their overarching metabolic and regulatory circuits, constituting the dening characteristics of a living cell. At the Obernai meeting we were presented with new data from transcriptome and proteome analysis in bacteria and new bioinformatics approaches to the handling of the avalanche of data now accumulating from Systems biology. Analysis of competence as a model system in Streptococcus pneumoniae by Alessandra Polissi (GlaxoWellcome, Italy) using high-density microarrays, revealed two waves of induced gene expression, at least 20 new genes associated with competence and the identication of consensus promoter sequences. Her talk highlighted not only the strengths, but possible problems of mass microarray screening, such as identifying the correct cut-off level. Whereas such gene proling has so far lagged behind the analysis of mammalian systems, these studies conrm its value for analysis of complex circuits in bacteria. Proteomics, combining 2D gel separation and high throughput mass spectrometry analysis to analyse gene expression at the protein level under different metabolic and environmental conditions, can be used to diagnose the function of proteins guilt by association with the assignment of

592 J. P. Armitage et al. proteins to stimulons and regulons (Rimini et al., 2000). Ruth VanBogelen (Pzer, Ann Arbor, USA) described efforts using compilations of 2D gel patterns for many different stress and metabolic conditions to establish a data base of proteomic signatures, allowing pattern or prole matching, to identify common signals or switch points that control regulons. The resulting examples so far, include the ribosome as the candidate target point for heat and cold shock regulons and ppGpp in the regulation of fatty acid and amino acid biosynthesis. Together these approaches are creating the discipline of molecular physiology. Another new discipline is now emerging from Biomathematics, not only to cope with informational overload but also to simulate the regulation of large complex interacting networks and metabolic pathways. Igor Goryanin (GSK, UK) showed that such approaches are yielding predictive characteristics and therefore generating hypotheses. Many of the models under development and the related software work on the principal of progressively increasing the level of complexity of the analysis. These models tend to be based on E. coli at the moment because of the mass of existing data and its easy manipulation (Covert et al., 2001). Interestingly, the contributors in this section of the meeting were all from industry, underlining the need for more academics in Europe to become more deeply involved in the Systems Biology, Biomathematics approach. Encouragingly, we were left with the idea of the need for a worldwide effort to co-ordinate studies in this eld, involving collaborations between Industry and Academia, in which some industrial companies at least are willing to provide a transparent, co-coordinating role. If anyone has any suggestions for topics they feel would t into this forum or would like to express an interest in the next meeting in the series, to be held in 2004, they are encouraged to contact any of the authors. References
Barak, R., and Eisenbach, M. (2001) Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis. Mol Microbiol 40: 731743. Bjrkman, A.M., Dunten, P., Sandgren, M.O.J., Dwarakanath, V.N., and Mowbray, S.L. (2001) Mutations that affect ligand binding to the E. coli aspartate receptor: implications for transmembrane signaling. J Biol Chem 276: 28082815. Brader, G., Tas, E., and Palva, E.T. (2001) Jasmonatedependent induction of indole glucosinolates in Arabidopsis by culture ltrates of the non-specic pathogen Erwinia carotovora. Plant Physiol 126: 849860. Chater, K.F. (2001) Regulation of sporulation in Streptomyces coelicolor: a checkpoint multiplex? Curr Opin Microbiol 4: 667673. Clarke, D.J., Joyce, S.A., Toutain, C.M., Jacq, A., and Holland, I.B. (2002) Genetic analysis of the RcsC sensor kinase from Escherichia coli K-12. J Bacteriol 184: 1204 1208. Cornelis, G.R. (2002) Yersinia type III secretion: send in the effectors. J Cell Biol 58: 401408. Covert, M.W., Schilling, C.S., Famili, I., Edwards, J.S., Goryanin, I.I., Selkov, E., and Palsson, B.O. (2001) Metabolic modeling of microbial strains in silico. TIBS 26: 179 186. Daniels, R., De Vos, D., Desair, J., Raedschelders, G., Luyten, E., Rosemeyer, V., et al. (2002) The cin quorum sensing locus of Rhizobium etli CNPAF512 affects growth and symbiotic nitrogen xation. J Biol Chem 277: 462 468. Dittmann, E., Erhard, M., Kaebernick, M., Scheler, C., Neilan, B.A., von Dohren, H., and Borner, T. (2001) Altered expression of two light-dependent genes in a microcystinlacking mutant of Microcystis aeruginosa PCC 7806. Microbiology 147: 31133119. Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95: 14863 14868. Esipov, S., and Shapiro, J.A. (1998) Kinetic model of Proteus mirabilis swarm colony development. J Math Biol 36: 249 268. van der Heide, T., Stuart, M.C.A., and Poolman, B. (2001) On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine. EMBO J 20: 70227032. Hyytiainen, H., Montesano, M., and Palva, E.T. (2001) Global regulators ExpA (GacA) and KdgR modulates extracellular enzyme gene expression through the RsmA-rsmB system in Erwinia carotovora subsp. carotovora. Mol Plant Microbe Interact 14: 931938. Iwasaki, H., Nishiwaki, T., Kitayama, Y., Nakajima, M., and Kondo. T. (2002) KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria. Proc Natl Acad Sci USA 99: 1578815793. Joyce, S.A., and Dorman, C.J. (2002) A Rho-dependent phase-variable transcription terminator controls expression of the FimE recombinase in Escherichia coli. Mol Microbiol 45: 11071117. Kenny, B., Ellis, S., Leard, A., Warawa, J., Mellor, H., and Jepson, M.A. (2002) Co-ordinate regulation of distinct host signalling pathways by multifunctional enteropathogenic E.coli (EPEC) effector molecules. Mol Microbiol 44: 1095 1107. Kleerebezem, M., and Quadri, L.E. (2001) Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior. Peptides 22: 15791596. Kruse, T., Lobedanz, S., Berthelsen, N.M., and SgaardAndersen, L. (2001) C-signal: a cell surface-associated morphogen that induces and co-ordinates multicellular fruiting body morphogenesis and sporulation in Myxococcus xanthus. Mol Microbiol 40: 156168. Lambrecht, M., Vande Broek, A., Dosselare, F., and Vanderleyden, J. (1999) The ipdC promoter auxinresponsive element of Azospirillum brasilense, a prokaryotic ancestral form of the plant AuxRE? Mol Microbiol 32: 889891.
2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 583 593

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Lenz, O., Bernhard, M., Buhrke, T., Schwartz, E., and Friedrich, B. (2002) The hydrogen-sensing apparatus in Ralstonia eutropha. J Mol Microbiol Biotechnol 4: 255 262. Masuda, S., Dong, C., Swem, D., Setterdahl, A.T., Knaff, D.B., and Bauer, C.E. (2002) Aerobic repression of photosystem synthesis through formation of an intramolecular disulde bond in CrtJ. Proc Natl Acad Sci USA 99: 7078 7083. Nair, U., Ditty, J.L., Min, H., and Golden, S.S. (2002) Roles for sigma factors in global circadian regulation of the cyanobacterial genome. J Bacteriol 184: 35303538. Poolman, B., Blount, P., Folgering, J., Friesen, R.H.E., Moe, P.C., and van der Heide, T. (2002) How do membrane proteins sense water stress? Mol Microbiol 44: 889 902. Porter, S.L., Warren, A.V., Martin, A.C., and Armitage, J.P. (2002) The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis. Mol Microbiol 46: 10811094. Rimini, R., Jansson, B., Feger, G., Roberts, T.C., de Francesco, M., Domenici, E., Frandsen, N., and Polissi, A. (2000) Global analysis of transcription kinetics during competence development in Streptococcus pneumoniae using high density DNA arrays. Mol Microbiol 36: 1279 1289. Schmitt, R. (2002) Sinorhizobial chemotaxis: a departure from the enterobacterial paradigm. Microbiology 148: 627 631. Swem, L., Elsen, S., Bird, T.H., Myllykallio, H., Daldal, F., and Bauer, C.E. (2001) The RegB/RegA two-component regulatory system controls synthesis of photosynthesis and respiratory electron transfer components in Rhodobacter capsulatus. J Mol Biol 309: 121138. Tait, K., and Sutherland, I.W. (2002) Antagonistic interactions amongst bacteriocin-producing enteric bacteria in dual species biolms. J Appl Microbiol 93: 345352. Taylor, B.L., Rebbapragada, A., and Johnson, M.S. (2001) The FAD-PAS domain as a sensor for behavioral responses in Escherichia coli. Antioxid Redox Signal 3: 867879. Verhamme, D.T., Arents, J.C., Crielaard, W., Postma, P.W., and Hellingwerf, K.J. (2002) Investigation of in vivo crosstalk between key two-component systems of Escherichia coli. Microbiology 148: 6978. Wadhams, G.H., Martin, A.C., Porter, S.L., Maddock, J.R., Mantotta, J.C., King, H.M., and Armitage, J.P. (2002) TlpC, a novel chemotaxis signalling protein in Rhodobacter sphaeroides, localises to a discrete region in the cytoplasm Mol Microbiol 46: 12111221.

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