Rise and rise of the ABC transporter families.

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.

Type I Secretion Systems-One Mechanism for All?

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 ions in the extracellular space, which triggers folding of the entire protein.

Bacillus subtilis Swarmer Cells Lead the Swarm, Multiply, and Generate a Trail of Quiescent Descendants.

Unlabelled: 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.

Type I Protein Secretion-Deceptively Simple yet with a Wide Range of Mechanistic Variability across the Family.

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.

The Type 1 secretion pathway - the hemolysin system and beyond.

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 mimic chain reaction.

It is sometimes speculated that the equivalent of the polymerase chain reaction might be developed for identification of peptides, proteins or other molecules. In general, though, it is believed that there can be no way to amplify targets such as proteins. Natural amplification systems do, however, exist as in the case of certain autoinducer systems in bacteria.

ABC transporters, mechanisms and biology: an overview.

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.

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers.

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.

Crystallization and preliminary X-ray crystallographic studies of an oligomeric species of a refolded C39 peptidase-like domain of the Escherichia coli ABC transporter haemolysin B.

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.

The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system.

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.

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin.

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.

The extraordinary diversity of bacterial protein secretion mechanisms.

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.

ATP regulates calcium efflux and growth in E. coli.

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).

CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis.

The conserved prpC, prkC, cpgA locus in Bacillus subtilis encodes 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 PrpC in vitro.

Identification of genes required for different stages of dendritic swarming in Bacillus subtilis, with a novel role for phrC.

Highly branched dendritic swarming of B. subtilis on 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 situ localisation and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry.

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.

The GTPase CpgA is implicated in the deposition of the peptidoglycan sacculus in Bacillus subtilis.

Depletion of the Bacillus subtilis GTPase CpgA produces abnormal cell shapes, nonuniform deposition of cell wall, and five- to sixfold accumulation of peptidoglycan precursors. Nevertheless, the inherent structure of the cell wall appeared mostly unchanged. The results are consistent with CpgA being involved in coordinating normal peptidoglycan deposition.

pH and monovalent cations regulate cytosolic free Ca(2+) in E. coli.

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.

Water-mediated protein-fluorophore interactions modulate the affinity of an ABC-ATPase/TNP-ADP complex.

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.

Methylglyoxal and other carbohydrate metabolites induce lanthanum-sensitive Ca2+ transients and inhibit growth in E. coli.

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).

Cytosolic Ca2+ regulates protein expression in E. coli through release from inclusion bodies.

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.

A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer.

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.

Fermentation product butane 2,3-diol induces Ca2+ transients in E. coli through activation of lanthanum-sensitive Ca2+ channels.

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 (5-200mM; half maximum 25mM) activates Ca2+ transients in E.

The motor domains of ABC-transporters. What can structures tell us?

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.