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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.

Bacteria adopt social behavior to expand into new territory, led by specialized swarmers, before forming a biofilm. Such mass migration of Bacillus subtilis on a synthetic medium produces hyperbranching dendrites that transiently (equivalent to 4 to 5 generations of growth) maintain a cellular monolayer over long distances, greatly facilitating single-cell gene expression analysis. Paradoxically, while cells in the dendrites (nonswarmers) might be expected to grow exponentially, the rate of swarm expansion is constant, suggesting that some cells are not multiplying. Little attention has been paid to which cells in a swarm are actually multiplying and contributing to the overall biomass. Here, we show in situ that DNA replication, protein translation and peptidoglycan synthesis are primarily restricted to the swarmer cells at dendrite tips. Thus, these specialized cells not only lead the population forward but are apparently the source of all cells in the stems of early dendrites. We developed a simple mathematical model that supports this conclusion.

A very large type I polypeptide begins to reel out from a ribosome; minutes later, the still unidentifiable polypeptide, largely lacking secondary structure, is now in some cases a thousand or more residues longer. Synthesis of the final hundred C-terminal residues commences. This includes the identity code, the secretion signal within the last 50 amino acids, designed to dock with a waiting ATP binding cassette (ABC) transporter. What happens next is the subject of this review, with the main, but not the only focus on hemolysin HlyA, an RTX protein toxin secreted by the type I system. Transport substrates range from small peptides to giant proteins produced by many pathogens. These molecules, without detectable cellular chaperones, overcome enormous barriers, crossing two membranes before final folding on the cell surface, involving a unique autocatalytic process. Unfolded HlyA is extruded posttranslationally, C-terminal first. The transenvelope “tunnel” is formed by HlyB (ABC transporter), HlyD (membrane fusion protein) straddling the inner membrane and periplasm and TolC (outer membrane). We present a new evaluation of the C-terminal secretion code, and the structure function of HlyD and HlyB at the heart of this nanomachine. Surprisingly, key details of the secretion mechanism are remarkably variable in the many type I secretion system subtypes. These include alternative folding processes, an apparently distinctive secretion code for each type I subfamily, and alternative forms of the ABC transporter; most remarkably, the ABC protein probably transports peptides or polypeptides by quite different mechanisms. Finally, we suggest a putative structure for the Hly-translocon, HlyB, the multijointed HlyD, and the TolC exit.

This chapter concentrates mainly on structural and mechanistic aspects of ABC (ATP-binding cassette) transporters and, as an example of the physiological significance of these proteins, on lipid transport, vitally important for human health. The chapter considers those aspects of ABC transporter function that appear reasonably well established, those that remain controversial and what appear to be emerging themes. Although we have seen dramatic progress in ABC protein studies in the last 20 years, we are still far from a detailed molecular understanding of function. Nevertheless two critical steps – capture and release of allocrites (transport substrates) involving a binding cavity in the membrane domain, and hydrolysis of ATP by the NBD (nucleotide-binding domain) dimer – are now described by persuasive and testable models: alternating access, and sequential firing of catalysis sites respectively. However, these need to be tested rigorously by more structural and biochemical studies. Other aspects considered include the level at which ATP binding and dimer activation are controlled, the nature of the power stroke delivering mechanical energy for transport, and some unexpected and intriguing differences between importers and exporters. The chapter also emphasizes that some ABC transporters, although important for elimination of toxic compounds (xenobiotics), are also increasingly seen to play crucial roles in homoeostatic regulation of membrane biogenesis and function through translocation of endogenous allocrites such as cholesterol. Another emerging theme is the identification of accessory domains and partners for ABC proteins, resulting in a corresponding widening of the range of activities. Finally, what are the prospects for translational research and ABC transporters?

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.

International audience; Escherichia coli regulates cytosolic free Ca(2+) in the micromolar range through influx and efflux. Herein, we show for the first time that ATP is essential for Ca(2+) efflux and that ATP levels also affect generation time. A transcriptome analysis identified 110 genes whose expression responded to an increase in cytosolic Ca(2+) (41 elevated, 69 depressed). Of these, 3 transport proteins and 4 membrane proteins were identified as potential Ca(2+) transport pathways. Expression of a further 943 genes was modified after 1 h in growth medium containing Ca(2+) relative to time zero. Based on the microarray results and other predicted possible Ca(2+) transporters, the level of cytosolic free Ca(2+) was measured in selected mutants from the Keio knockout collection using intracellular aequorin. In this way, we identified a knockout of atpD, coding for a component of the F(o)F(1) ATPase, as defective in Ca(2+) efflux. Seven other putative Ca(2+) transport proteins exhibited normal Ca(2+) handling. The defect in the DeltaatpD knockout cells could be explained by a 70% reduction in ATP. One millimolar glucose or 1 mM methylglyoxal raised ATP in the DeltaatpD knockout cells to that of the wild type and restored Ca(2+) efflux. One millimolar 2,4-dinitrophenol lowered the ATP in wild type to that in the DeltaatpD cells. Under these conditions, a similar defect in Ca(2+) efflux in wild type was observed in DeltaatpD cells. Ten millimolar concentration of Ca(2+) resulted in a 30% elevation in ATP in wild type and was accompanied by a 10% reduction in generation time under these conditions. Knockouts of pitB, a potential Ca(2+) transporter, atoA, the beta subunit of acetate CoA-transferase likely to be involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate kinase, all indicated no defect in Ca(2+) efflux. We therefore propose that ATP is most likely to regulate Ca(2+) efflux in E. coli through an ATPase.

The results here are the first demonstration of a physiological agonist opening Ca2+ channels in bacteria. Bacteria in the gut ferment glucose and other substrates, producing alcohols, diols, ketones and acids, that play a key role in lactose intolerance, through the activation of Ca2+ and other ion channels in host cells and neighbouring bacteria. Here we show butane 2,3-diol (5–200 mM; half maximum 25 mM) activates Ca2+ transients in E. coli, monitored by aequorin. Ca2+-transient magnitude depended on external Ca2+ (0.1–10 mM). meso-Butane 2,3-diol was approximately twice as potent as 2R,3R (?) and 2S,3S (+) butane 2,3-diol. There were no detectable effects on cytosolic free Ca2+ of butane 1,3-diol, butane 1,4-diol and ethylene glycol. The glycerol fermentation product propane 1,3-diol only induced significant Ca2+ transients in 10 mM external Ca2. Ca2+ butane 2,3-diol Ca2+ transients were due to activation of Ca2+ influx, followed by activation of Ca2+ efflux. The effect of butane 2,3-diol was abolished by La3+, and markedly reduced as a function of growth phase. These results were consistent with butane 2,3-diol activating a novel La3+-sensitive Ca2+ channel. They have important implications for the role of butane 2,3-diol and Ca2+ in bacterial-host cell signalling.

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.

The ABC-transporter haemolysin B is a central component of the secretion machinery that translocates the toxin, haemolysin A, in a Sec-independent fashion across both membranes of E. coli . Here, we report the X-ray crystal structure of the nucleotide-binding domain (NBD) of HlyB. The molecule shares the common overall architecture of ABC-transporter NBDs. However, the last three residues of the Walker A motif adopt a 3 10 helical conformation, stabilized by a bound anion. In consequence, this results in an unusual interaction between the Walker A lysine residue and the Walker B glutamate residue. As these residues are normally required to be available for ATP binding, for catalysis and for dimer formation of ABC domains, we suggest that this conformation may represent a latent monomeric form of the NBD. Surprisingly, comparison of available NBD structures revealed a structurally diverse region (SDR) of about 30 residues within the helical arm II domain, unique to each of the eight NBDs analyzed. As this region interacts with the transmembrane part of ABC-transporters, the SDR helps to explain the selectivity and/or targeting of different NBDs to their cognate transmembrane domains.

A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB-NBD) and a C-terminal 23kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence. Employing surface plasmon resonance, we were able to demonstrate that the HlyB-NBD and HlyA1 interact with a K(D) of approximately 4 microM. No interaction was detected between the HlyA fragment and unrelated NBDs, OpuAA, involved in import of osmoprotectants, and human TAP1-NBD, involved in the export of antigenic peptides. Moreover, a truncated version of HlyA1, lacking the secretion signal, failed to interact with the HlyB-NBD. In addition, we showed that ATP accelerated the dissociation of the HlyB-NBD/HlyA1 complex. Taking these results together, we propose a model for an early stage of initiation of secretion in vivo, in which the NBD of HlyB, specifically recognizes the C terminus of the transport substrate, HlyA, and where secretion is initiated by subsequent displacement of HlyA from HlyB by ATP.

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+.

Haemolysin B (HlyB) is a transmembrane protein which belongs to the superfamily of ABC transporters. In vivo, it mediates the non-classical translocation of the 107 kDa toxin HlyA across both membranes of Escherichia coli together with haemolysin D and the outer membrane protein TolC. The cytosolic ATP-binding domain of HlyB has been overexpressed and purified as an N-terminal His-tag fusion protein. Here, the crystallization of the ATPase domain of HlyB in the presence of ATP is described. A native data set has been obtained at a resolution of 2.8 A. Crystals belong to the primitive tetragonal space group P4(x)2(1)2, where x is very likely to be 1 or 3, with unit-cell parameters a = b = 104.6, c = 125.8 A, alpha = beta = gamma = 90 degrees.

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.

I have tried to cover the minimal properties of the prolific number of protein secretion systems identified presently, particularly in Gram negative bacteria. New systems, however, are being reported almost by the month and certainly I have missed some. With the accumulating evidence one remains in awe of the complexity of some pathways, with the Type III, IV and VI especially fearsome and impressive. These systems illustrate that protein secretion from bacteria is not only about passage of large polypeptides across a bilayer but also through long tunnels, raising quite different questions concerning mechanisms. The mechanism of transport via the Sec-translocase-translocon is well on the way to full understanding, although a structure of a stuck intermediate would be very helpful. The understanding of the precise details of the mechanism of targeting specificity, and actual polypeptide translocation in other systems is, however, far behind. Groups willing to do the difficult (and risky) work to understand mechanism should therefore be more actively encouraged, perhaps to pursue multidisciplinary, collaborative studies. In writing this review I have become fascinated by the cellular regulatory mechanisms that must be necessary to orchestrate the complex flow of so many polypeptides, targeted by different signals to such a wide variety of transporters. I have tried to raise questions about how this might be managed but much more needs to be done in this area. Clearly, this field is very much alive and the future will be full of revealing and surprising twists in the story.

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.

Haemolysin B (HlyB) is essential for secretion of the 107 x 10(3) Mr haemolysin A protein from Escherichia coli and is a member of a family of highly conserved, apparently ATP-dependent surface proteins in many organisms. We have shown in this study that both HlyB and HlyD fractionate primarily with the cytoplasmic membrane of E. coli and are accessible to proteases after removal of the outer membrane. We have measured experimentally the topological organization of HlyB within the membrane by construction of fusions to beta-lactamase as a reporter. The predicted folding of HlyB, with a minimum of six transmembrane segments, does not always coincide with regions of highest average hydrophobicity. This suggests that HlyB may have a novel organization within the bilayer. From our data and comparative sequence analysis, we have been able to predict very similar topological models for the other members of the HlyB family.

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 results here are the first demonstration of a family of carbohydrate fermentation products opening Ca2+ channels in bacteria. Methylglyoxal, acetoin (acetyl methyl carbinol), diacetyl (2,3 butane dione), and butane 2,3 diol induced Ca2+ transients in Escherichia coli, monitored by aequorin, apparently by opening Ca2+ channels. Methylglyoxal was most potent (K(1/2) = 1 mM, 50 mM for butane 2,3 diol). Ca2+ transients depended on external Ca2+ (0.1-10 mM), and were blocked by La3+ (5 mM). The metabolites affected growth, methylglyoxal being most potent, blocking growth completely up to 5 h without killing the cells. But there was no affect on the number of viable cells after 24 h. These results were consistent with carbohydrate products activating a La3+-sensitive Ca2+ channel, rises in cytosolic Ca2+ possibly protecting against certain toxins. They have important implications in bacterial-host cell signalling, and where numbers of different bacteria compete for the same substrates, e.g., the gut in lactose and food intolerance.

The results here are the first clear demonstration of a physiological role for cytosolic Ca(2+) in Escherichia coli by releasing a Ca(2+) binding protein, apoaequorin, from inclusion bodies. In growth medium LB the cytosolic free Ca(2+) was 0.1-0.3 microM. Addition of EGTA reduced this to

International audience; TNP-modified nucleotides have been used extensively to study protein-nucleotide interactions. In the case of ABC-ATPases, application of these powerful tools has been greatly restricted due to the significantly higher affinity of the TNP-nucleotide for the corresponding ABC-ATPase in comparison to the non-modified nucleotides. To understand the molecular changes occurring upon binding of the TNP-nucleotide to an ABC-ATPase, we have determined the crystal structure of the TNP-ADP/HlyB-NBD complex at 1.6A resolution. Despite the higher affinity of TNP-ADP, no direct fluorophore-protein interactions were observed. Unexpectedly, only water-mediated interactions were detected between the TNP moiety and Tyr(477), that is engaged in pi-pi stacking with the adenine ring, as well as with two serine residues (Ser(504) and Ser(509)) of the Walker A motif. Interestingly, the side chains of these two serine residues adopt novel conformations that are not observed in the corresponding ADP structure. However, in the crystal structure of the S504A mutant, which binds TNP-ADP with similar affinity to the wild type enzyme, a novel TNP-water interaction compensates for the missing serine side chain. Since this water molecule is not present in the wild type enzyme, these results suggest that only water-mediated interactions provide a structural explanation for the increased affinity of TNP-nucleotides towards ABC-ATPases. However, our results also imply that in silico approaches such as docking or modeling cannot directly be applied to generate 'affinity-adopted' ADP- or ATP-analogs for ABC-ATPases.

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.

YloQ, from Bacillus subtilis, was identified previously as an essential nucleotide-binding protein of unknown function. YloQ was successfully over-expressed in Escherichia coli in soluble form. The purified protein displayed a low GTPase activity similar to that of other small bacterial GTPases such as Bex/Era. Based on the demonstrated GTPase activity and the unusual order of the yloQ G motifs, we now designate this protein as CpgA (circularly permuted GTPase). An unexpected property of this low abundance GTPase was the demonstration, using gel filtration and ultracentrifugation analysis, that the protein formed stable dimers, dependent upon the concentration of YloQ(CpgA), but independent of GTP. In order to investigate function, cpgA was placed under the control of the pspac promotor in the B. subtilis chromosome. When grown in E or Spizizen medium in the absence of IPTG, the rate of growth was significantly reduced. A large proportion of the cells exhibited a markedly perturbed morphology, with the formation of swollen, bent or 'curly' shapes. To confirm that this was specifically due to depleted CpgA a plasmid-borne cpgA under pxyl control was introduced. This restored normal cell shape and growth rate, even in the absence of IPTG, provided xylose was present. The crystal structure of CpgA(YloQ) suggests a role as a translation initiation factor and we discuss the possibility that CpgA is involved in the translation of a subset of proteins, including some required for shape maintenance.

The relatively simple type 1 secretion system in gram-negative bacteria is nevertheless capable of transporting polypeptides of up to 800 kDa across the cell envelope in a few seconds. The translocator is composed of an ABC-transporter, providing energy through ATP hydrolysis (and perhaps the initial channel across the inner membrane), linked to a multimeric Membrane Fusion Protein (MFP) spanning the initial part of the periplasm and forming a continuous channel to the surface with an outer membrane trimeric protein. Proteins targeted to the translocator carry an (uncleaved), poorly conserved secretion signal of approximately 50 residues. In E. coli the HlyA toxin interacts with both the MFP (HlyD) and the ABC protein HlyB, (a half transporter) triggering, via a conformational change in HlyD, recruitment of the third component, TolC, into the transenvelope complex. In vitro, HlyA, through its secretion signal, binds to the nucleotide binding domain (NBD or ABC-ATPase) of HlyB in a reaction reversible by ATP that may mimic initial movement of HlyA into the translocation channel. HlyA is then transported rapidly, apparently in an unfolded form, to the cell surface, where folding and release takes place. Whilst recent structural studies of TolC and MFP-like proteins are providing atomic detail of much of the transport path, structural analysis of the HlyB NBD and other ABC ATPases, have revealed details of the catalytic cycle within an NBD dimer and a glimpse of how the action of HlyB is coupled to the translocation of HlyA.

Nucleotide-binding domains (NBD) are highly conserved constituents of ATP-binding cassette (ABC) transporters. Members of this family couple ATP hydrolysis to the transfer of various molecules across cell membranes. The NBD of the HlyB transporter, HlyB-NBD, was characterized with respect to its uncoupled ATPase activity, oligomeric state, and stability in solution. Experimental data showed that both the nature and pH of an assay buffer influenced the level of protein activity. Comparative analysis of protein stability and ATPase activity in various buffers suggests an inverse relationship between the two. The highest ATPase activity was detected in HEPES, pH 7.0. A kinetic analysis of the ATPase activity in this buffer revealed an enzyme concentration dependence and ATP-induced protein oligomerization. Assuming that the dimer is the active form of enzyme, at least half of the purified HlyB-NBD was estimated to be a dimer at 1.2 microM under the most optimal conditions for ATP hydrolysis. This is about 2 orders of magnitude lower than reported for other canonical ABC-ATPases. The maximum reaction velocity of 0.6 micromol/mg x min at 22 degrees C and the apparent kinetic constant K(app)(0.5) of 0.26 mM for ATP were determined for the dimerized HlyB-NBD. Gel filtration experiments with the wild-type protein and HlyB-NBD mutated in a key catalytic residue, H662A, provided further evidence for ATP-induced protein dimerization. ATPase activity experiments with protein mixtures composed of wild-type and the ATPase-deficient H662A mutant demonstrated that one intact NBD within a dimer is sufficient for ATP hydrolysis. This single site turnover might suggest a sequential mechanism of ATP hydrolysis in the intact HlyB transporter.

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 general properties of ABC transporters, from bacteria to humans, including a brief history of their initial discovery, are considered. ABC transporters, one of the largest protein super families and vital for human health, are in toto responsible for the transport of an enormous range of molecules from ions (CFTR) or anti-tumour drugs (Pgp/MDR) to large polypeptides. Nevertheless, all ABC transporters are powered by a conserved ATPase the ABC or NBD domain, using in all probability the same basic mechanism of action for the hydrolysis of ATP and its coupling to the transport process. Based on recent high resolution structures of several NBDs and an intact transporter, a model of how dimers of these important proteins function will be discussed, with particular attention to HlyB, the ABC transporter from E. coli.

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.

Many ABC transporters, composed of appropriate membrane and ABC components, are concerned with import or export of relatively small molecules. In contrast, ABC transporters in bacteria required for secretion of RTX toxins and related proteins have been the exception, seemingly embracing a number of different concepts in order to account for translocation of protein substrates, in some cases with sizes over 400 kDa. In all probability, such substrates, secreted by the socalled type 1 pathway, directly access the interior of the transporter from the cytoplasm, by-passing the bilayer. This chapter reconciles the implications of such mammoth transport substrates, or allocrite with a transport mechanism, which still probably shares many of the same features fundamental to other ABC proteins. It reviews the best-studied examples of the type 1 system, which are in Gram-negative bacteria, where two membranes have to be negotiated. In this case, at least one further auxiliary protein in the outer membrane is required to provide the exit to the external medium. This chapter considers the three major examples of this kind of ABC transporter which have been studied in the most detail, HlyB (HlyA toxin transport), PrtD (protease transport), and HasD (transport of a hemebinding protein, HasA).

Those who studied ABC proteins, in the form of HisP and MalK, were fascinated by the mechanism of histidine and maltose uptake in Gram-negative bacteria, certainly an esoteric subject. The reason for the fascination of ABC proteins and their associated partners, however, does not stop at the sheer size of this superfamily but is compelling because of the enormous breadth of biological processes that they embrace. Everything changed dramatically in the mid-1980s with the realization that P-glycoprotein (Pgp), responsible for multidrug resistance and a serious obstacle to effective antitumor chemotherapy, was also an ABC transporter. This was followed quickly by the identification of the CFTR protein as a novel ABC transporter, and the subject has never looked back, with now thousands of ABC genes in the database and the avalanche continues as new genome sequences accumulate.

The purpose of this chapter is to highlight some of the major characteristics of bacterial ABC systems and the breadth of their functions. Prokaryote ABC-dependent transport systems, whether exporters or importers, all adhere to the usual formula of a basic four-unit structure, two membrane components and two units of ABC-ATPases. The term ABC transporter describes the ABC-ATPase plus its associated integral membrane domains, whether fused to the ABC or separately encoded. This core transporter or translocation complex may be further supplemented with essential accessory or auxiliary subunits (usually encoded separately): the external ligand-binding protein in the case of ABC importers, or the MFP (membrane fusion protein) and the OMP-F (outer membrane protein factor) or OMA (outer membrane auxiliary) integral to the inner membrane and outer membrane, respectively. In the case of ABC transporters, the entire complex may sometimes be referred to as the translocon, while for the importers the term permease is also used to describe the entire complex.

Since the initial characterization of the hemolysin secretion system (Hly) from the Gram-negative bacterium, E. coli, by W. Goebel’s group in the late 1970s, there has been a surprising proliferation of discoveries of distinct pro¬tein secretion mechanisms in many Gram-negative bacteria. Both for the Hly system and for other secretion pathways, developments have been the most dramatic over the last 5 years and, therefore, in this review we have placed most detailed emphasis upon this period up to approximately mid-1996. Previous reviews have generally agreed on the classification of secretion Types I, II, III, indicated in this review. Here (see also reference 5), we have proposed a classification for additional pathways: Type IV (auto-transporter systems like the IgA pathway); Type V for surface pilins (E. coli Pap system), the functionally related Type Va, although not homologous with the pilin system, are transported from the periplasm via a single outer membrane “translocator” protein; Type VI for the special case of the filamentous phage (nucleo-protein) secretory pathway. The secretion of flagellar proteins on to the cell surface, although not discussed in this review, could be considered yet another pathway (for review see reference 5a). However, the biogenesis of the flagellum involves at least eight proteins with homology to proteins of the Type III pathway. We emphasize that Types IV–VI, as defined here, are useful working classifications but a generally agreed classification for these secretion pathways in the literature has not yet emerged. What can be generally agreed upon is that Types I, III and VI are one step processes with translocation from cytoplasm directly on to the cell surface or to the medium, while other pathways employ a two-step mechanism involving initial export to the periplasm targeted by an N-terminal signal via the Sec-machinery (general export pathway). The second stage then involves translocation across the outer membrane by various mechanisms. Importantly, the proteins of the latter group are apparently translocated across the outer membrane in a fully folded form, while proteins translocated directly to the outside of the cell (Types I, III) and perhaps IV may be required to re-fold on the surface.

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.