Holins kill without warning.

Holins comprise the most diverse functional group of proteins known. They are small bacteriophage-encoded proteins that accumulate during the period of late-protein synthesis after infection and cause lysis of the host cell at a precise genetically programmed time. It is unknown how holins achieve temporal precision, but a conserved feature of their function is that energy poisons subvert the normal scheduling mechanism and instantly trigger membrane disruption. On this basis, timing has been proposed to involve a progressive decrease in the energized state of the membrane until a critical triggering level is reached. Here, we report that membrane integrity is not compromised after the induction of holin synthesis until seconds before lysis. The proton motive force was monitored by the rotation of individual cells tethered by a single flagellum. The results suggest an alternative explanation for the lysis "clock," in which holin concentrations build to a critical level that leads to formation of an oligomeric complex that disrupts the membrane.

Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene.

The 308 residue MotB protein anchors the stator complex of the Escherichia coli flagellar motor to the peptidoglycan of the cell wall. Together with MotA, it comprises the transmembrane channel that delivers protons to the motor. At the outset of the mutational analysis of MotB described here, we found that the non-motile phenotype of a DeltamotAB strain was rescued better by a pmotA(+)B(+) plasmid than the non-motile phenotype of a DeltamotB strain was rescued by a pmotB(+) plasmid. Transcription in each case was from the inducible tac promoter but relied on the native ribosome-binding site (RBS). This result confirms that translational coupling to motA is important for normal translation of the motB mRNA, since overproduction of MotA in trans did not improve complementation by pmotB. However, introduction of an optimized RBS into pmotB (to generate pmotB(o)) did. To dissect the function of the periplasmic domain of MotB, site-directed mutagenesis was used to replace Gln, Ser, and Tyr codons scattered throughout motB with amber (UAG) codons. Plasmid-borne motB(am) genes were introduced into sup(o), supE, and supF strains to see what motility defects were imposed by particular amber mutations and whether the defects could be suppressed by amber-suppressor tRNAs inserting the native or heterologous amino acids. Amber mutations at codon 268 or earlier in pmotB, and at codon 261 or earlier in pmotB(o) or pmotAB, eliminated motility. Thus, in agreement with the deletion analysis of motB by another laboratory, we conclude that the portion of MotB carboxyl-terminal to its peptidoglycan-binding motif (residues 161 to 264) is not essential. In strains containing supE or supF alleles, motility defects associated with motB(am) mutations were suppressed weakly, if at all, in pmotB. In contrast, motility defects conferred by most motB(am) mutations in pmotB(o) or pmotAB could be suppressed to a significant extent. However, the S18(am), Q100(am), Q112(am), Q124(am), Y201(am), and Y208(am) mutations were still suppressed extremely poorly. Full-length MotB was present at very low levels in suppressor strains containing the first four mutations, but Y201(am) and Y208(am) were suppressed efficiently at the translational level. We suggest that a translational pause by suppressor tRNAs reading UAG at these two positions may divert the nascent polypeptide into an alternative folding pathway that traps MotB in a non-functional conformation. We further propose that MotA and MotB form a stable pre-assembly complex in the membrane. In this complex, MotB exists in a form that cannot associate with peptidoglycan and blocks the proton-conducting channel. Opening of the channel and attachment to the cell wall may occur when the complex collides with a flagellar basal body and MotA makes specific contacts with the C ring and/or the MS ring.

Allele-specific suppression as a tool to study protein-protein interactions in bacteria.

Suppression analysis is well suited to study the interactions of gene products. It offers the advantage of simplicity for any organism for which a convenient genetic system has been developed, which holds for a wide spectrum of bacteria and an ever-increasing number of unicellular as well as complex eukaryotes. No other method provides as much information about the functional relationships of biological macromolecules. The intrinsic value of suppression analysis is enhanced by advances in genomics and in biophysical techniques for investigating the properties of nucleic acids and proteins, such as X-ray crystallography, liquid and solid-state nuclear magnetic resonance, electron spin labeling, and isothermal calorimetry. These approaches confirm and complement whatever is revealed by genetics. Despite these sterling qualities, suppression analysis has its dangers, less in execution than in conceptualization of experiments and interpretation of data. A consistent nomenclature is essential for a uniform and widespread understanding of the results. Familiarity with the genetic background and idiosyncracies of the organism studied is critical in avoiding extraneous phenomena that can affect the outcome. Finally, it is imperative not to underestimate potentially bizarre and improbable consequences that can transpire when rigorous genetic selection is maintained for an appreciable length of time. The article begins with a somewhat pedagogical discussion of genetic terminology. It then moves on to the necessary precautions to observe while planning and conducting suppression analysis. The remainder of the article considers different manifestations of suppression: bypass suppression; gradients of suppression; suppression by relaxed specificity; allele-specific "suppression at a distance"; and true conformational suppression. The treatment is not exhaustive, but representative examples have been gleaned from the recent bacterial literature.

Model of maltose-binding protein/chemoreceptor complex supports intrasubunit signaling mechanism.

The Tar protein of Escherichia coli is unique among known bacterial chemoreceptors in that it generates additive responses to two very disparate ligands, aspartate and maltose. Aspartate binds directly to the periplasmic (extracytoplasmic) domain of Tar. Maltose first binds to maltose-binding protein (MBP). MBP then assumes a closed conformation in which it can interact with the periplasmic domain of Tar. MBP residues critical for binding Tar were identified in a screen of mutations that cause specific defects in maltose chemotaxis. Mutations were introduced into a plasmid-borne malE gene that encodes a mutant form of MBP in which two engineered Cys residues spontaneously generate a disulfide bond in the oxidizing environment of the periplasmic space. This disulfide covalently crosslinks the NH3-terminal and COOH-terminal domains of MBP and locks the protein into a closed conformation. Double-Cys MBP confers a dominant-negative phenotype for maltose taxis, and we reasoned that third mutations that relieve this negative dominance probably alter residues that are important for the initial interaction of MBP with Tar. The published three-dimensional structures of MBP and the periplasmic domain of E. coli Tar were docked in a computer simulation that juxtaposed the residues in MBP identified in this way with residues in Tar that have been implicated in maltose taxis. The resulting model of the MBP-Tar complex exhibits good complementarity between the surfaces of the two proteins and supports the idea that aspartate and MBP may each initiate an attractant signal through Tar by inducing similar conformational changes in the chemoreceptor.

A mechanism for simultaneous sensing of aspartate and maltose by the Tar chemoreceptor of Escherichia coli.

The Tar chemoreceptor of Escherichia coli exhibits partial sensory additivity. Tar can mediate simultaneous responses to two disparate ligands, aspartate and substrate-loaded maltose-binding protein (MBP). To investigate how one receptor generates concurrent signals to two stimuli, ligand-binding asymmetry was imposed on the rotationally symmetric Tar homodimer. Mutations causing specific defects in aspartate or maltose chemotaxis were introduced pairwise into plasmid-borne tar genes. The doubly mutated tar genes did not restore aspartate or maltose chemotaxis in a strain containing a chromosomal deletion of tar (delta tar). However, when Tar proteins with complementing sets of mutations were co-expressed from compatible plasmids, the resulting heterodimeric receptors enabled delta tar cells to respond to aspartate or maltose. The effect of one attractant on the response to the other depended on the relative orientations of the functional binding sites for aspartate and MBP. When the sites were in the 'same' orientation, saturating levels of one attractant strongly inhibited chemotaxis to the other. In the 'opposite' orientation, such inhibitory effects were negligible. These data demonstrate that opposing subunits of Tar can transmit signals to aspartate and maltose independently if the ligands are restricted to the 'opposite' binding orientation. When aspartate and MBP bind in the 'same' orientation, they compete for signalling through one subunit. In the wild-type Tar dimer, aspartate and MBP can bind in either the 'same' or the 'opposite' orientation, a freedom that can explain the partial additivity of the aspartate and maltose responses that is seen with tar+ cells.

Chimeric chemoreceptors in Escherichia coli: signaling properties of Tar-Tap and Tap-Tar hybrids.

The Tap (taxis toward peptides) receptor and the periplasmic dipeptide-binding protein (DBP) of Escherichia coli together mediate chemotactic responses to dipeptides. Tap is a low-abundance receptor. It is present in 5- to 10-fold-fewer copies than high-abundance receptors like Tar and Tsr. Cells expressing Tap as the sole receptor, even from a multicopy plasmid at 5- to 10-fold-overexpressed levels, do not generate sufficient clockwise (CW) signal to tumble and thus swim exclusively smoothly (run). To study the signaling properties of Tap in detail, we constructed reciprocal hybrids between Tap and Tar fused in the linker region between the periplasmic and cytoplasmic domains. The Tapr hybrid senses dipeptides and is a good CW-signal generator, whereas the Tarp hybrid senses aspartate but is a poor CW-signal generator. Thus, the poor CW signaling of Tap is a property of its cytoplasmic domain. Eighteen residues at the carboxyl terminus of high-abundance receptors, including the NWETF sequence that binds the CheR methylesterase, are missing in Tap. The Tart protein, created by removing these 18 residues from Tar, has diminished CW-signaling ability. The Tapl protein, made by adding the last 18 residues of Tar to the carboxyl terminus of Tap, also does not support CW flagellar rotation. However, Tart and Tapl cross-react well with antibody directed against the conserved cytoplasmic region of Tsr, whereas Tap does not cross-react with this antibody. Tap does cross-react, however, with antibody directed against the low-abundance chemoreceptor Trg. The hybrid, truncated, and extended receptors exhibit various levels of methylation. However, Tar and Tapl, which contain a consensus CheR-binding motif (NWETF) at their carboxyl termini, exhibit the highest basal levels of methylation, as expected. We conclude that no simple correlation exists between the abundance of a receptor, its methylation level, and its CW-signaling ability.

Maltose-binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor.

The Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L-aspartate and to maltose. Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer. Maltose, in contrast, first binds to the periplasmic maltose-binding protein (MBP), which in its ligand-stabilized closed form then interacts with Tar. Intragenic complementation was used to determine the MBP-binding site on the Tar dimer. Mutations causing certain substitutions at residues Tyr-143, Asn-145, Gly-147, Tyr-149, and Phe-150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg-73, Met-76, Asp-77, and Ser-83. These two sets of mutations defined two complementation groups when the defective proteins were co-expressed at equal levels from compatible plasmids. We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically. Mutations affecting Met-75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer. When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane-distal apex of the domain and also extended onto one shoulder of the apical region.

Extragenic suppression of motA missense mutations of Escherichia coli.

The MotA and MotB proteins are thought to comprise elements of the stator component of the flagellar motor of Escherichia coli. In an effort to understand interactions among proteins within the motor, we attempted to identify extragenic suppressors of 31 dominant, plasmid-borne alleles of motA. Strains containing these mutations were either nonmotile or had severely impaired motility. Four of the mutants yielded extragenic suppressors mapping to the FlaII or FlaIIIB regions of the chromosome. Two types of suppression were observed. Suppression of one type (class I) probably results from increased expression of the chromosomal motB gene due to relief of polarity. Class I suppressors were partial deletions of Mu insertion sequences in the disrupted chromosomal motA gene. Class I suppression was mimicked by expressing the wild-type MotB protein from a second, compatible plasmid. Suppression of the other type (class II) was weaker, and it was not mimicked by overproduction of wild-type MotB protein. Class II suppressors were point mutations in the chromosomal motB or fliG genes. Among 14 independent class II suppressors characterized by DNA sequencing, we identified six different amino acid substitutions in MotB and one substitution in FliG. A number of the strongest class II suppressors had alterations of residues 136 to 138 of MotB. This particular region within the large, C-terminal periplasmic domain of MotB has previously not been associated with a specific function. We suggest that residues 136 to 138 of MotB may interact directly with the periplasmic face of MotA or help position the N-terminal membrane-spanning helix of MotB properly to interact with the membrane-spanning helices of the MotA proton channel.

Attractant signaling by an aspartate chemoreceptor dimer with a single cytoplasmic domain.

Signal transduction across cell membranes often involves interactions among identical receptor subunits, but the contribution of individual subunits is not well understood. The chemoreceptors of enteric bacteria mediate attractant responses by interrupting a phosphotransfer circuit initiated at receptor complexes with the protein kinase CheA. The aspartate receptor (Tar) is a homodimer, and oligomerized cytoplasmic domains stimulate CheA activity much more than monomers do in vitro. Intragenic complementation was used to show in Escherichia coli that heterodimers containing one full-length and one truncated Tar subunit mediated responses to aspartate in the presence of full-length Tar homodimers that could not bind aspartate. Thus, a Tar dimer containing only one cytoplasmic domain can initiate an attractant (inhibitory) signal, although it may not be able to stimulate kinase activity of CheA.

Maltose-binding protein containing an interdomain disulfide bridge confers a dominant-negative phenotype for transport and chemotaxis.

Bacterial substrate-binding proteins exist in an equilibrium among four forms: open/substrate-free, open/substrate-bound, closed/substrate-free, and closed/substrate-bound. Ligands stabilize the closed conformation, whereas the open conformation predominates in the substrate-free species. In its closed form, the NH2-terminal and COOH-terminal domains of maltose-binding protein (MBP) are proposed to be aligned to allow residues in both domains to interact simultaneously with complementary sites on the MalF and MalG proteins of the maltodextrin uptake system or with the Tar chemotactic signal transducer. However, the initial interaction might occur with an open/substrate-bound form of the binding protein, which would then close in contact with MalFG or Tar. Ligand would help stabilize this complex. We introduced cysteines (G69C and S337C) by site-directed mutagenesis into each domain of MBP and found that they formed an interdomain disulfide cross-link that should hold the protein in a closed conformation. This mutant MBP confers a dominant-negative phenotype for growth on maltose, for maltose transport, and for maltose chemotaxis. The growth and transport defects are partially reversed when the cells are exposed to the reducing agent dithiothreitol. We conclude that the cross-linked form of MBP competes with wild-type MBP in vivo for interaction with MalFG and Tar.

Mutations in motB suppressible by changes in stator or rotor components of the bacterial flagellar motor.

Five proteins (MotA, MotB, FliG, FliM and FliN) may be involved in energizing flagellar rotation in Escherichia coli. To study interactions between the Mot proteins, and between them and the three Fli proteins of the switch-motor complex, we have isolated extragenic suppressors of dominant and partially dominant motB missense mutations. Four of the 13 motB mutations yielded partially allele-specific suppressors. Of the suppressing mutations, 57 are in the motA gene, eight are in fliG, and one is in fliM; no suppressor was identified in fliN. The prevalence of suppressors in fliG suggests that FliG interacts rather directly with the Mot proteins. The behaviour of cells in tethering and swarm assays indicates that the motA suppressors are more efficient than the fliG or fliM suppressors. Some of the suppressing mutations themselves confer distinctive phenotypes in motB+ cells. We propose a model in which mutations affecting residues in or near the putative peptidoglucan-binding region of MotB misalign the stator relative to the rotor. We suggest that most of the suppressors restore motility by introducing compensatory realignments in MotA or FliG.

Motility protein interactions in the bacterial flagellar motor.

Five proteins (MotA, MotB, FliG, FliM, and FliN) have been implicated in energizing flagellar rotation in Escherichia coli and Salmonella typhimurium. One model for flagellar function envisions that MotA and MotB comprise the stator of a rotary motor and that FliG, FliM, and FliN are part of the rotor. MotA probably functions as a transmembrane proton channel, and MotB has been proposed to anchor MotA to the peptidoglycan of the cell wall. To study interactions between the Mot proteins themselves and between them and other components of the flagellar motor, we attempted to isolate extragenic suppressors of 13 dominant or partially dominant motB missense mutations. Four of these yielded suppressors, which exhibited widely varying efficiencies of suppression. The pattern of suppression was partially alleles-specific, but no suppressor seriously impaired motility in a motB+ strain. Of 20 suppressors from the original selection, 15 were characterized by DNA sequencing. Fourteen of these cause single amino acid changes in MotA. Thirteen alter residues in, or directly adjacent to, the putative periplasmic loops of MotA, and the remaining one alters a residue in the middle of the fourth predicted transmembrane helix of MotA. We conclude that the MotA and MotB proteins form a complex and that their interaction directly involves or is strongly influenced by the periplasmic loops of MotA. The 15th suppressor from the original selection and 2 motB suppressors identified during a subsequent search cause single amino acid substitutions in FliG. This finding suggests that the postulated Mot-protein complex may be in close proximity to FliG at the stator-rotor interface of the flagellar motor.

The dipeptide permease of Escherichia coli closely resembles other bacterial transport systems and shows growth-phase-dependent expression.

The dipeptide permease (Dpp) of Escherichia coli transports peptides consisting of two or three L-amino acids. The periplasmic dipeptide-binding protein (DBP), encoded by the dppA gene, also serves as a chemoreceptor. We sequenced the dpp locus, which comprises an operon of five genes, dppABCDE. Its organization is the same as the oligopeptide permease (opp) operon of Salmonella typhimurium and the spo0K operon of Bacillus subtilis. The dpp genes are also closely related to the hbpA gene, which encodes a haem-binding lipoprotein, and four other genes in an unlinked operon of unknown function in Haemophilus influenzae. Each Dpp protein has an Opp, Spo0K and H. influenzae homologue. Transcription of the dpp operon initiates 165 bases upstream of the predicted dppA start codon. The start site for transcription is preceded by potential -35 and -10 regions of a sigma 70 promoter. During exponential growth in Luria-Bertani (LB) broth, the level of dpp mRNA increases in two steps, one between A590 0.2 and 0.4 and one between A590 0.7 and 1.0. The 310 nucleotides between dppA and dppB include a RIP (repetitive IHF-binding palindromic) element, whose deletion from a multi-copy plasmid causes fivefold and 10-fold reductions in the levels of upstream and downstream dpp mRNA, respectively.

Maltose chemotaxis involves residues in the N-terminal and C-terminal domains on the same face of maltose-binding protein.

The periplasmic maltose-binding protein (MBP) of Escherichia coli is the recognition component of the maltose chemoreceptor and of the active transport system for maltose. It interacts with the Tar chemotactic signal transducer and the integral cytoplasmic-membrane components (the MalF and MalG proteins) of the maltose transport system. Maltose binds in a cleft between the globular N-terminal and C-terminal domains of MBP, which are connected by a moveable hinge. The two domains undergo a large motion relative to one another as the protein moves from the open, unbound state to the closed, ligand-bound state. We generated, by doped-primer mutagenesis, amino acid substitutions that specifically disrupt the chemotactic function of MBP. These substitutions cluster in two well-defined regions that are nearly contiguous on the surface of MBP in its closed conformation. One region is in the N-terminal domain and one is in the C-terminal domain. The distance between the two regions is expected to change substantially as the protein goes from the open to the closed form. These results support a model in which ligand binding brings two recognition sites on MBP into the proper spatial relationship to interact with complementary sites on Tar. Mutations in MBP that appear to cause defects in interaction with MalF and MalG are distributed differently from mutations that primarily affect maltose taxis. We conclude that the regions of MBP that contact Tar and those that contact MalF and MalG are adjacent on the face of the protein opposite the hinge connecting the two domains and that those regions are largely, although perhaps not entirely, distinct.

Introduction to bacterial motility and chemotaxis.

Bacteria swim by rotating semirigid, left-handed helical flagellar filaments; counterclockwise (CCW) rotation produces straight swims, known as "runs," and clockwise (CW) rotation generates abrupt changes in direction, known as "tumbles." As a cell moves through its environment, alternately running and tumbling, it detects spatial gradients of attractants and repellents by making temporal comparisons of their concentration. These chemicals bind to receptors in the cell envelope to modulate the activity of the chemotactic signal transducers, proteins that span the cytoplasmic membrane. Signals generated by the transducers control the motion of the flagella to promote migration up attractant gradients and down repellent gradients. Chemotactic adaptation, accomplished by methylation-demethylation of the transducers, cancels out these signals. Adaptation is an essential component of the "memory" that allows bacteria to use a temporal mechanism to detect spatial gradients. Both signaling and adaptation are mediated by changes in the level of phosphorylation of several cytoplasmic chemotaxis (Che) proteins. The activity of the transducers regulates the rate of autophosphorylation of the CheA protein, which then passes the phosphate on to other proteins. In particular, phosphorylated CheY protein controls the frequency of tumbling because it promotes CW flagellar rotation, and the CheB esterase modulates adaptation because its nonphosphorylated form removes methyl groups from the transducers much more slowly than its phosphorylated form.

Comparison of sequences from the malB regions of Salmonella typhimurium and Enterobacter aerogenes with Escherichia coli K12: a potential new regulatory site in the interoperonic region.

The malE and malK genes from Salmonella typhimurium, and the malEFG operon and a portion of malK from Enterobacter aerogenes were cloned and sequenced. Plasmid-borne malE genes from both species and the malF and malG genes from E. aerogenes were expressed normally in Escherichia coli, and their products function in maltose transport. This shows that the malB products from the three species are interchangeable, at least in the combinations tested. The general genetic organization of the malB region is conserved. Potential binding sites and distances between them are highly conserved in the regulatory intervals. An unexpected conserved region was detected, which we call the U box, and which could be another target for a regulatory protein. This hypothesis is supported by the presence of the U box in the regulatory region of the pulA-malX operon in Klebsiella pneumoniae. The intergenic region between malE and malF from S. typhimurium and E. aerogenes, contains inverted repeats similar to the palindromic units (PU or REP) found at the same location in E. coli. The predicted amino acid sequence of the encoded proteins showed 90% or more identity in every pairwise comparison of species.