Mutations specifically affecting ligand interaction of the Trg chemosensory transducer.

The Trg transducer mediates chemotactic response to galactose and ribose by interacting, respectively, with sugar-occupied galactose- and ribose-binding proteins. Adaptation is linked to methylation of specific glutamyl residues of the Trg protein. This study characterized two trg mutations that affect interaction with binding protein ligands but do not affect methylation or adaptation. The mutant phenotypes indicated that the steady-state activity of methyl-accepting sites is independent of ligand-binding activity. The mutation trg-8 changed arginine 85 to histidine, and trg-19 changed glycine 151 to aspartate. The locations of the mutational changes provided direct evidence for functioning of the amino-terminal domain of Trg in ligand recognition. Cross-inhibition of tactic sensitivity by the two Trg-linked attractants implies competition for a common site on Trg. However, the single amino acid substitution caused by trg-19 greatly reduced the response to galactose but left unperturbed the response to ribose. Thus Trg must recognize the two sugar-binding proteins at nonidentical sites, and the complementary sites on the respective binding proteins should differ. trg-8 mutants were substantially defective in the response to both galactose and ribose. An increase in cellular content of Trg-8 protein improved the response to galactose but not to ribose. It appears that Trg-8 protein is defective in the generation of the putative conformational change induced by ligand interaction. The asymmetry of the mutational defect implies that functional separation of interaction sites could persist beyond the initial stage of ligand binding.     

Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae

The differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses. One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament. The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK. FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay. Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non-motile, non-flagellated phenotype. The flagellins of the mutant had larger molecular weights than their wild-type counterparts, as expected if they retained their 11- to 12-amino-acid leader peptide. Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay. Site-directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin. As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.     

Purification of receptor protein Trg by exploiting a property common to chemotactic transducers of Escherichia coli

The methyl-accepting chemotactic transducers of Escherichia coli were found to bind strongly to Cibacron blue-Sepharose. Among potential elutants tested, only S-adenosylmethionine at moderate concentrations and NaCl at concentrations greater than 1.5 M caused dissociation of these detergent-solubilized transmembrane proteins from the dye. Release by S-adenosylmethionine may be a generalized effect rather than the result of a specific binding site for that compound on transducers. A truncated trg gene was created that coded for the carboxyl-terminal three-fifths of the transducer, which constitutes the cytoplasmic domain common to all four transducers in E. coli. This domain bound to Cibacron blue-Sepharose and was eluted in a pattern similar to that exhibited by intact Trg, indicating that interaction with the dye occurred in this conserved domain. Adherence to Cibacron blue and elution by high salt formed the core of an efficient purification scheme, developed for Trg but applicable to all transducers in E. coli and perhaps to methyl-accepting chemotaxis proteins in other species. Determination of the amino acid sequence at the beginning of purified Trg confirmed that it contained a longer hydrophilic segment at its amino terminus than other transducers of E. coli. The initial methionine of Trg is neither cleaved nor modified, in contrast to the Tar transducer in which the amino terminus was found previously to be blocked. Circular dichroic measurements of purified Trg indicated that the secondary structural organization of the protein is predominantly alpha-helix.     

Limited homology between trg and the other transducer proteins of Escherichia coli.

Transducers are transmembrane proteins that are central to the chemotactic system of Escherichia coli. The proteins transduce ligand recognition into an excitatory signal and function in adaptation as methyl-accepting proteins. The transducer genes tsr, tar, and tap have extensive homology with each other. However, previous studies revealed little indication of homology between those three transducer genes and a fourth gene, trg. We investigated the relationship between trg and the other genes by blot-hybridization experiments and the relationship between Trg and the other transducer proteins by immune precipitation and experiments with an antiserum raised to purified Trg protein. In experiments in which 35% mismatch would be tolerated, weak hybridization of trg was detected to a DNA fragment containing tar and tap but not to a fragment containing tsr. In experiments in which only 30% mismatch would be tolerated, no trg hybridization was apparent either to total chromosomal DNA or to DNA from hybrid plasmids carrying the other transducer genes. An anti-Trg serum formed immune precipitates with the Tsr and Tar proteins as well as with the Trg protein to which it was raised. We conclude that there is homology between Trg and the other transducer, but the homology is more limited than that shared among the other transducers. Furthermore, we found no indication of additional transducer genes closely related to trg. Thus, the trg gene is a somewhat distant cousin within a single transducer gene family of E. coli.     

Effects of glutamines and glutamates at sites of covalent modification of a methyl-accepting transducer.

Chemotactic transducer proteins of Escherichia coli contain four or five methyl-accepting glutamates that are crucial for sensory adaptation and gradient sensing. Two residues arise from posttranslational deamidation of glutamines to yield methyl-accepting glutamates. We addressed the significance of this arrangement by creating two mutated trg genes: trg(5E), coding for a transducer in which all five modification sites were synthesized as glutamates, and trg(5Q), in which all five were glutamines. We found that the normal (3E,2Q) configuration was not an absolute requirement for synthesis, assembly, or stable maintenance of transducers. Both mutant proteins were methylated, although Trg(5Q) had a reduced number of methyl-accepting sites because two glutamines at adjacent residues were blocked for deamidation and thus could not become methyl-accepting glutamates. The glutamine-glutamate balance had striking effects on signaling state. Trg(5E) was in a strong counterclockwise signaling configuration, and Trg(5Q) was in a strong clockwise signaling induced by ligand binding, and alanines substituted at modification sites had an intermediate effect. Chemotactic migration by growing cells containing trg(5E) or trg(5Q) exhibited reduced effectiveness, probably reflecting perturbations of the counterclockwise/clockwise ratio caused by newly synthesized transducers not modified rapidly enough to produce a balanced signaling state during growth. These defects were evident for cells in which other transducers were not available to contribute to balanced signaling or were present at lower levels than the mutant proteins.     

Conversion of mono-polar to peritrichous flagellation in Vibrio alginolyticus

Precise regulation of the number and positioning of flagella are critical in order for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. It has been shown that, in V. alginolyticus cells, the putative GTPase FlhF determines the polar location and production of flagella, while the putative ATPase FlhG interacts with FlhF, preventing it from localizing at the pole, and thus negatively regulating the flagellar number. In fact, no ΔflhF cells have flagella, while a very small fraction of ΔflhFG cells possess peritrichous flagella. In this study, the mutants that suppress inhibition of the swarming ability of ΔflhFG cells were isolated. The mutation induced an increase in the flagellar number and, furthermore, most Vibrio cells appeared to have peritrichous flagella. The sequence of the flagella related genes was successfully determined, however, the location of the suppressor mutation could not been found. When the flhF gene was introduced into the suppressor mutant, multiple polar flagella were generated in addition to peritrichous flagella. On the other hand, introduction of the flhG gene resulted in the loss of most flagella. These results suggest that the role of FlhF is bypassed through a suppressor mutation which is not related to the flagellar genes.     

Isolation and characterization of Chlamydomonas reinhardtii mutants with defects in the induction of flagellar protein synthesis after deflagellation

Amputating the flagella of Chlamydomonas reinhardtii stimulates increased synthesis of many flagellar proteins within 30 min. We have isolated a series of mutants which are defective in this stimulation, taking advantage of the fact that cells which cannot stimulate flagellar protein synthesis cannot regenerate flagella. More than a dozen mutants which have flagella, but cannot regenerate them after amputation, were isolated and studied by in vivo labeling to identify those non-regenerator mutants which were specifically defective in the induction of flagellar protein synthesis. Ten such mutants have been identified, and in each of them flagellar amputation does not stimulate the synthesis of any of the major flagellar proteins. At least four of the mutants display an interesting conditional phenotype. The synthesis of flagellar proteins after deflagellation is defective only in gametic cells; vegetative cells of these mutants are capable of flagellar protein synthesis after flagellar amputation.     

Isolation and Characterization of Chlamydomonas reinhardtii Mutants with Defects in the Induction of Flagellar Protein Synthesis after Deflagellation1,2

Amputating the flagella of Chlamydomonas reinhardtii stimulates increased synthesis of many flagellar proteins within 30 min. We have isolated a series of mutants which are defective in this stimulation, taking advantage of the fact that cells which cannot stimulate flagellar protein synthesis cannot regenerate flagella. More than a dozen mutants which have flagella, but cannot regenerate them after amputation, were isolated and studied by in vivo labeling to identify those non-regenerator mutants which were specifically defective in the induction of flagellar protein synthesis. Ten such mutants have been identified, and in each of them flagellar amputation does not stimulate the synthesis of any of the major flagellar proteins. At least four of the mutants display an interesting conditional phenotype. The synthesis of flagellar proteins after deflagellation is defective only in gametic cells; vegetative cells of these mutants are capable of flagellar protein synthesis after flagellar amputation.     

Evolution of chemotactic-signal transducers in enteric bacteria.

The methyl-accepting chemotactic-signal transducers of the enteric bacteria are transmembrane proteins that consist of a periplasmic receptor domain and a cytoplasmic signaling domain. To study their evolution, transducer genes from Enterobacter aerogenes and Klebsiella pneumoniae were compared with transducer genes from Escherichia coli and Salmonella typhimurium. There are at least two functional transducer genes in the nonmotile species K. pneumoniae, one of which complements the defect in serine taxis of an E. coli tsr mutant. The tse (taxis to serine) gene of E. aerogenes also complements an E. coli tsr mutant; the tas (taxis to aspartate) gene of E. aerogenes complements the defect in aspartate taxis, but not the defect in maltose taxis, of an E. coli tar mutant. The sequence was determined for 5 kilobases of E. aerogenes DNA containing a 3' fragment of the cheA gene, cheW, tse, tas, and a 5' fragment of the cheR gene. The tse and tas genes are in one operon, unlike tsr and tar. The cytoplasmic domains of Tse and Tas are very similar to those of E. coli and S. typhimurium transducers. The periplasmic domain of Tse is homologous to that of Tsr, but Tas and Tar are much less similar in this region. However, several short sequences are conserved in the periplasmic domains of Tsr, Tar, Tse, and Tas but not of Tap and Trg, transducers that do not bind amino acids. These conserved regions include residues implicated in amino-acid binding.     

Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas

Ciliary membranes have a large repertoire of receptors and ion channels that act to transduce information from the environment to the cell. Chlamydomonas offers a tractable system for dissecting the transport and function of ciliary and flagellar membrane proteins. Isolation of ergosterol and sphingolipid-enriched Chlamydomonas flagellar membrane domains identified potential signaling molecules by mass spectroscopy. These include a membrane protein and a matrix flavodoxin protein that are encoded by the AGG2 and AGG3 genes, respectively. Agg2p localizes to the proximal flagellar membrane near the basal bodies. Agg3p is distributed throughout the flagellar matrix, with an increased concentration in the proximal regions where Agg2p is located. Chlamydomonas cells sense light by using a microbial-type rhodopsin , transduce a signal from the cell body to the flagella, and alter the waveform of the flagella to turn a cell toward the light. Protein depletion by RNA interference reveals that both AGG gene products play roles in the orientation of cells to a directional light source. The depleted strains mimic the phenotype of the previously identified agg1 mutant, which swims away from light. We propose that the localization of Agg2p and Agg3p to the proximal region of the flagella may be important for interpreting light signals.     

The LF1 gene of Chlamydomonas reinhardtii encodes a novel protein required for flagellar length control

Flagellar length is tightly regulated in the biflagellate alga Chlamydomonas reinhardtii. Several genes required for control of flagellar length have been identified, including LF1, a gene required to assemble normal-length flagella. The lf1 mutation causes cells to assemble extra-long flagella and to regenerate flagella very slowly after amputation. Here we describe the positional cloning and molecular characterization of the LF1 gene using a bacterial artificial chromosome (BAC) library. LF1 encodes a protein of 804 amino acids with no obvious sequence homologs in other organisms. The single LF1 mutant allele is caused by a transversion that produces an amber stop at codon 87. Rescue of the lf1 phenotype upon transformation was obtained with clones containing the complete LF1 gene as well as clones that lack the last two exons of the gene, indicating that only the amino-terminal portion of the LF1 gene product (LF1p) is required for function. Although LF1 helps regulate flagellar length, the LF1p localizes almost exclusively in the cell body, with <1% of total cellular LF1p localizing to the flagella.     

The 2 microm plasmid causes cell death in Saccharomyces cerevisiae with a mutation in Ulp1 protease

The 2 microm circle plasmid confers no phenotype in wild-type Saccharomyces cerevisiae but in a nib1 mutant, an elevated plasmid copy number is associated with cell death. Complementation was used to identify nib1 as a mutant allele of the ULP1 gene that encodes a protease required for removal of a ubiquitin-like protein, Smt3/SUMO, from protein substrates. The nib1 mutation replaces conserved tryptophan 490 with leucine in the protease domain of Ulp1. Complete deletion of ULP1 is lethal, even in a strain that lacks the 2 microm circle. Partial deletion of ULP1, like the nib1 mutation, results in clonal variations in plasmid copy number. In addition, a subset of these mutant cells produces lineages in which all cells have reduced proliferative capacity, and this phenotype is dependent upon the presence of the 2 microm circle. Segregation of the 2 microm circle requires two plasmid-encoded proteins, Rep1 and Rep2, which were found to colocalize with Ulp1 protein in the nucleus and interact with Smt3 in a two-hybrid assay. These associations and the observation of missegregation of a fluorescently tagged 2 microm circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification plays a role in both plasmid copy number control and segregation.     

A dynein light intermediate chain, D1bLIC, is required for retrograde intraflagellar transport

Intraflagellar transport (IFT), the bidirectional movement of particles along flagella, is essential for flagellar assembly. The motor for retrograde IFT in Chlamydomonas is cytoplasmic dynein 1b, which contains the dynein heavy chain DHC1b and the light intermediate chain (LIC) D1bLIC. To investigate a possible role for the LIC in IFT, we identified a d1blic mutant. DHC1b is reduced in the mutant, indicating that D1bLIC is important for stabilizing dynein 1b. The mutant has variable length flagella that accumulate IFT-particle proteins, indicative of a defect in retrograde IFT. Interestingly, the remaining DHC1b is normally distributed in the mutant flagella, strongly suggesting that the defect is in binding of cargo to the retrograde motor rather than in motor activity per se. Cell growth and Golgi apparatus localization and morphology are normal in the mutant, indicating that D1bLIC is involved mainly in retrograde IFT. Like mammalian LICs, D1bLIC has a phosphate-binding domain (P-loop) at its N-terminus. To investigate the function of this conserved domain, d1blic mutant cells were transformed with constructs designed to express D1bLIC proteins with mutated P-loops. The constructs rescued the mutant cells to a wild-type phenotype, indicating that the function of D1bLIC in IFT is independent of its P-loop.     

Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number

Wild-type Chlamydomonas reinhardtii carry two flagella per cell that are used for both motility and mating. We describe a mutant, vfl-1, in which the biflagellate state is disrupted such that the number of flagella per cell ranges from 0 to as many as 10. vfl-1 cells possess the novel ability to assemble new flagella throughout the G1 portion of the cell cycle, resulting in an average increase of about 0.05 flagella per cell per hour. Such uncoupling of the flagellar assembly cycle from the cell cycle is not observed in other mutants with abnormal flagellar number. Rather than being located in an exclusively apical position characteristic of the wild type, vfl-1 flagella can be at virtually any location on the cell surface. vfl-1 cells display abnormally wide variations in cell size, probably owing to extremely unequal cell divisions. Various ultrastructural abnormalities in the flagellar apparatus are also present, including missing or defective striated fibers and reduced numbers of rootlet microtubules. The pleiotropic defects observed in vfl-1 result from a recessive Mendelian mutation mapped to Chromosome VIII.     

Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina

Mitani, Michiko (National Institute of Genetics, Mishima, Japan), and Tetsuo Iino. Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina. J. Bacteriol. 90:1096-1101. 1965.-The arrangement of flagella was observed by dark-field and electron microscopy in three strains of Salmonella abortivoequina, namely, normal flagellar, curly flagellar, and paralyzed curly flagellar strains. With dark-field microscopy, bundled flagella could be seen in 5 to 10% of actively moving normal or curly mutant cells. Under the electron microscope, a great many bundled flagella were observed in the curly mutant strain, but in the normal strain most of the flagella were dissociated or the bundles were rather loose and irregular. Normal flagella seem to separate easily during the process of preparation, but not the curly ones. Single flagella were found to run parallel with each other and to form a bundle consisting of five or more flagella; the bundle was spirally gyrating, with the characteristic flagellar wave. It is thought that the bundle observed with the electron microscope corresponds to that observed under the dark-field microscope. Further, the marked decrease of bundle formation in the paralyzed curly mutant cells suggests that bundle formation is not caused by curly flagellar structure per se, but corresponds to the mode of locomotion of peritrichously flagellated bacteria.     

Cloning of trg, a gene for a sensory transducer in Escherichia coli.

Clones of trg, a gene which codes for a chemotactic transducer, were isolated linked to ColE1 and pBR322 vectors. Studies with the hybrid plasmids demonstrated unequivocally that trg is the structural gene for methyl-accepting chemotaxis protein III. The Trg protein was found to be structurally complex, electrophoresing as a series of seven bands on high-resolution sodium dodecyl sulfate-polyacrylamide gels. The multiplicity of bands is a function of the activity of cheR, which codes for a methyltransferase, and of cheB, which codes for a demethylase. It appears that Trg, a quantitatively minor transducer, resembles the two major transducer proteins, Tsr and Tar, in that all three are multiply methylated and also multiply modified in a second way which requires an active cheB gene. However, preliminary analysis of the Trg protein indicated that it is significantly less related structurally to the Tsr or Tar protein than those two transducers are to each other. This implies that the features of multiple methylation and cheB-dependent modification are likely to be critical for the common physiological functions in chemotactic excitation and adaptation performed by all three transducers.