Aromatic acids are chemoattractants for Pseudomonas putida

A quantitative capillary assay was used to show that aromatic acids, compounds that are chemorepellents for Escherichia coli and Salmonella sp., are chemoattractants for Pseudomonas putida PRS2000. The most effective attractants were benzoate; p-hydroxybenzoate; the methylbenzoates; m-, p-, and o-toluate; salicylate; DL-mandelate; beta-phenylpyruvate; and benzoylformate. The chemotactic responses to these compounds were inducible. Taxis to benzoate and m-toluate was induced by beta-ketoadipate, a metabolic intermediate formed when benzoate is dissimilated via enzymes specified by chromosomal genes. Benzoylformate taxis was induced by benzoylformate and L(+)-mandelate. Taxis to mandelate, benzoylformate, and beta-phenylpyruvate was exhibited by cells grown on mandelate, but not by cells grown on benzoate. Cells grown on benzoate were chemotactic to benzoate, the toluates, p-hydroxybenzoate, and salicylate. These results show that P. putida synthesizes at least two distinct chemoreceptors for aromatic acids. Although DL-mandelate was an effective attractant in capillary assays, additional experiments indicated that the cells were actually responding to benzoylformate, a metabolite formed from mandelate. With the exception of mandelate taxis, chemotaxis to aromatic acids was not dependent on the expression of pathways for aromatic degradation. Therefore, the tactic responses exhibited by cells cannot be attributed to an effect of the oxidation of aromatic acids on the energy metabolism of cells.     

TOL plasmid can prevent induction of chemotactic responses to aromatic acids

Growth conditions that elicited positive chemotaxis to benzoate and m-toluate in TOL- Pseudomonas putida cells failed to elicit taxis to these compounds in TOL+ cells. The inability of TOL+ cells to respond to these aromatic acids appears to be due to the preferential expression of TOL-encoded genes for aromatic degradation over chromosomally encoded genes. Expression of chromosomal genes for aromatic degradation is required for cells to form beta-ketoadipate, the inducer of benzoate and m-toluate taxis.     

Effect of outer membrane permeability on chemotaxis in Escherichia coli.

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.     

Chemoattractants elicit methylation of specific polypeptides in Spirochaeta aurantia

On the basis of this investigation, chemotaxis in Spirochaeta aurantia correlates with methylation of specific polypeptides which are presumed to be analogous to the methyl-accepting chemotaxis proteins (MCPs) in bacteria such as Escherichia coli. The polypeptides exhibited apparent molecular weights in the range of 55,000 to 65,000. Generally, two major presumptive MCP bands and three minor bands were observed on sodium dodecyl sulfate-polyacrylamide gels. Upon addition of D-glucose to S. aurantia cells, methylation of the presumptive MCPs increased for 10 to 12 min to a level greater than 4 times the level of methylation in the absence of D-glucose. Removal of D-glucose resulted in a decrease in methylation of the presumptive MCPs to a level similar to that in unstimulated cells. All attractants tested, including a non-metabolizable attractant (alpha-methyl-D-glucoside) stimulated methylation of the presumptive MCPs (from 1.7 to 4.3 times the level of methylation in unstimulated cells). D-Mannitol, a metabolizable sugar which is not an attractant for S. aurantia, did not stimulate methylation. Stimulation of methylation by D-galactose occurred in cells induced for D-galactose taxis but not in uninduced cells. These data are indicative of an evolutionary relationship between the chemotaxis systems of spirochetes and of flagellated bacteria.     

Chemotaxis of Pseudomonas aeruginosa: involvement of methylation.

The involvement of a protein methyl transfer system in the chemotaxis of Pseudomonas aeruginosa was investigated. When a methionine auxotroph of P. aeruginosa was starved for methionine, chemotaxis toward serine, measured by a quantitative capillary assay, was reduced 80%, whereas background motility was unaffected or increased. When unstarved bacteria were labeled with L-[methyl-3H]methionine, a labeled species of 73,000 molecular weight which was methylated in response to stimulation by L-serine was identified. Under appropriate electrophoretic conditions, the 73,000 molecular weight species was resolved into two bands, both of which responded to stimulation by L-serine, L-arginine, and alpha-aminoisobutyrate (AIB) with an increased incorporation of methyl label. Arginine, which elicited the strongest chemotactic response in the capillary assay, also stimulated the greatest methylation response. Methylation of the 73,000 molecular weight species reached a maximum 10 min after stimulation by AIB and returned to the unstimulated level upon removal of the AIB. In vitro labeling of cell extracts with S-adenosyl[methyl-3H]methionine indicated that the 73,000 molecular weight species are methylated by an S-adenosylmethionine-mediated reaction. These results indicate that chemotaxis of P. aeruginosa toward amino acids is mediated by dynamic methylation and demethylation of methyl-accepting chemotaxis proteins analogous to those of the enteric bacteria.     

Methyl transfer in chemotaxis toward sugars by Bacillus subtilis.

Like amino acids, the sugars glucose and the nonmetabolizable 2-deoxyglucose caused a turnover of methyl groups on the methyl-accepting chemotaxis proteins. These sugars also caused methanol formation on addition. Thus, in contrast to chemotaxis in Escherichia coli, taxis to phosphotransferase sugars by Bacillus subtilis utilizes the methyl-accepting chemotaxis proteins.     

Nitrobenzoates and aminobenzoates are chemoattractants for Pseudomonas strains

Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the beta-ketoadipate pathway, has a well-characterized beta-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (beta-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the beta-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (beta-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a beta-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.     

Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.     

Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa.

A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N'-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. The pctA gene was inactivated by insertion of a kanamycin resistance gene cassette into the wild-type gene, resulting in the same observed deficiency in taxis toward L-amino acids as PCT1. In vivo methyl labeling experiments with L-[methyl-3H]methionine showed that this knockout mutant lacked an MCP with a molecular weight of approximately 68,000.     

Metabolism-dependent taxis towards (methyl)phenols is coupled through the most abundant of three polar localized Aer-like proteins of Pseudomonas putida

Comparatively little is known about directed motility of environmental bacteria to common aromatic pollutants. Here, by expressing different parts of a (methyl)phenol-degradative pathway and the use of specific mutants, we show that taxis of Pseudomonas putida towards (methyl)phenols is dictated by its ability to catabolize the aromatic compound. Thus, in contrast to previously described chemoreceptor-mediated chemotaxis mechanisms towards benzoate, naphthalene and toluene, taxis in response to (methyl)phenols is mediated by metabolism-dependent behaviour. Here we show that P. putida differentially expresses three Aer-like receptors that are all polar-localized through interactions with CheA, and that inactivation of the most abundant Aer2 protein significantly decreases taxis towards phenolics. In addition, the participation of a sensory signal transduction protein composed of a PAS, a GGDEF and an EAL domain in motility towards these compounds is demonstrated. The results are discussed in the context of the versatility of metabolism-dependent coupling and the necessity for P. putida to integrate diverse metabolic signals from its native heterogeneous soil and water environments.     

Cytoplasmic pH mediates pH taxis and weak-acid repellent taxis of bacteria.

Bacteria migrate away from an acid pH and from a number of chemicals, including organic acids such as acetate; the basis for detection of these environmental cues has not been demonstrated. Membrane-permeant weak acids caused prolonged tumbling when added to Salmonella sp. or Escherichia coli cells at pH 5.5. Tethered Salmonella cells went from a prestimulus behavior of 14% clockwise rotation to 80% clockwise rotation when 40 mM acetate was added and remained this way for more than 30 min. A low external pH in the absence of weak acid did not markedly affect steady-state tumbling frequency. Among the weak acids tested, the rank for acidity (salicylate greater than benzoate greater than acetate greater than 5,5-dimethyl-2,4-oxazolidinedione) was the same as the rank for the ability to collapse the transmembrane pH gradient and to cause tumbling. At pH 7.0, the tumbling responses caused by the weak acids were much briefer. Indole, a non-weak-acid repellent, did not cause prolonged tumbling at low pH. Two chemotaxis mutants (a Salmonella mutant defective in the chemotaxis methylesterase and an E. coli mutant defective in the methyl-accepting protein in MCP I) showed inverse responses of enhanced counterclockwise rotation in the first 1 min after acetate addition. The latter mutant had been found previously to be defective in the sensing of gradients of extracellular pH and (at neutral pH) of acetate. We conclude (i) that taxes away from acid pH and membrane-permeant weak acids are both mediated by a pH-sensitive component located either in the cytoplasm or on the cytoplasmic side of the membrane, rather than by an external receptor (as in the case of the attractants), and (ii) that both of these taxes involve components of the chemotaxis methylation system, at least in the early phase of the response.     

Control of the chemotactic behavior of Bacillus subtilis cells

The effects of nigericin, valinomycin and some lipophilic cations on the motile behavior of non-starved and methionine-starved Bacillus subtilis cells were studied. For valinomycin and nigericin a quantitative relationship between the flux in the proton-motive force and the duration of the twiddle response was found. Lipophilic cations bind to the ion gate controlling the twiddle frequency and thereby cause the cells to swim smoothly. To explain the transmission of the chemotactic signal a model is given in which receptors, a hyperpolarizing wave, an ion gate and two methylation sites, viz. methyl-accepting chemotaxis proteins and a further methylation site (MT), play a role. For the transmission of the signal caused by an attractant both the hyperpolarizing wave and an interaction between receptor and methylation site (MT) are needed. The methyl-accepting chemotaxis proteins are involved in the adaptation/deadaptation to altered levels of attractant. Artificial changes in the proton-motive force act directly on the ion gate, which finally controlls the twiddle frequency of the cells.Abbreviations KT medium potassium taxis medium - NAT medium sodium taxis medium - HT medium acidic taxis medium - OHT medium alkaline taxis medium - ImT medium imidazole taxis medium - GT medium glycylgycine taxis medium - Di-S-C₃(5) 3,3-dipropyl-2,2-thiacarbocyanine iodide - TPAs⁺ tetraphenylarsonium ion - TPMP⁺ triphenylmethylphosphonium ion - DDA⁺ dibenzyldimethylammonium ion - TPB^- tetraphenylboron ion - pmf proton-motive force - MCP methyl-accepting chemotaxis protein - MT methylation site - membrane potential     

Role of methylation in aerotaxis in Bacillus subtilis.

Taxis to oxygen (aerotaxis) in Bacillus subtilis was characterized in a capillary assay and in a temporal assay in which the concentration of oxygen in a flow chamber was changed abruptly. A strong aerophilic response was present, but there was no aerophobic response to high concentrations of oxygen. Adaptation to a step increase in oxygen concentration was impaired when B. subtilis cells were depleted of methionine to prevent methylation of the methyl-accepting chemotaxis proteins. There was a transient increase in methanol release when wild-type B. subtilis, but not a cheR mutant that was deficient in methyltransferase activity, was stimulated by a step increase or a step decrease in oxygen concentration. The methanol released was quantitatively correlated with demethylation of methyl-accepting chemotaxis proteins. This indicated that methylation is involved in aerotaxis in B. subtilis in contrast to aerotaxis in Escherichia coli and Salmonella typhimurium, which is methylation independent.     

Identification of methylation sites in Thermotoga maritima chemotaxis receptors

Adaptation in bacterial chemotaxis involves reversible methylation of specific glutamate residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins. The specific sites of methylation in Salmonella enterica and Escherichia coli chemoreceptors, identified 2 decades ago, established a consensus sequence for methylation by methyltransferase CheR. Here we report the in vitro methylation of chemoreceptors from Thermotoga maritima, a hyperthermophile that has served as a useful source of chemotaxis proteins for structural analysis. Sites of methylation have been identified by liquid chromatography-mass spectrometry/mass spectrometry. Fifteen sites of methylation were identified within the cytoplasmic domains of four different T. maritima chemoreceptors. The results establish a consensus sequence for chemoreceptor methylation sites in T. maritima that is distinct from the previously identified consensus sequence for E. coli and S. enterica. These findings suggest that consensus sequences for posttranslational modifications in one organism may not be directly extrapolated to analogous modifications in other bacteria.     

Methylation of FrzCD, a methyl-accepting taxis protein of Myxococcus xanthus, is correlated with factors affecting cell behavior.

Myxococcus xanthus, a nonflagellated gliding bacterium, exhibits multicellular behavior during vegetative growth and fruiting body formation. The frizzy (frz) genes are required to control directed motility for these interactions. The frz genes encode proteins that are homologous to all of the major enteric chemotaxis proteins, with the exception of CheZ. In this study, we characterized FrzCD, a protein which is homologous to the methyl-accepting chemotaxis proteins from the enteric bacteria. FrzCD, unlike the other methyl-accepting chemotaxis proteins, was found to be localized primarily in the cytoplasmic fraction of cells. FrzCD migrates as a ladder of bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reflecting heterogeneity due to methylation or demethylation and to deamidation. FrzCD was shown to be methylated in vivo when cells were exposed to yeast extract or Casitone and demethylated when starved in buffer. We used the methylation state of FrzCD as revealed by Western blot (immunoblot) analyses to search for stimuli that are recognized by the frz signal transduction system. Common amino acids, nucleotides, vitamins, and sugars were not recognized, but certain lipids and alcohols were recognized. For example, the saturated fatty acids capric acid and lauric acid stimulated FrzCD methylation, whereas a variety of other saturated fatty acids did not. Lauryl alcohol and lipoic acid also stimulated methylation, as did phospholipids containing lauric acid. In contrast, several short-chain alcohols, such as isoamyl alcohol, and some other solvents caused demethylation. The relatively high concentrations of the chemicals required for a response may indicate that these chemicals are not the relevant signals recognized by M. xanthus in nature. Isoamyl alcohol and isopropanol also had profound effects on the behavior of wild-type cells, causing them to reverse continuously. Cells of frzB, frzF, and frzG mutants also reversed continuously in the presence of isoamyl alcohol, whereas cells of frzA, frzCD, or frzE mutants did not. On the basis of the data presented, we propose a model for the frz signal transduction pathway in M. xanthus.     

Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate.

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.     

Adaptation in bacterial chemotaxis: CheB-dependent modification permits additional methylations of sensory transducer proteins

Sensory transduction in E. coli consists of two phases, excitation and adaptation, both of which involve the methyl-accepting chemotaxis proteins (MCPs). These molecules relay transmembrane signals and are reversibly methylated during adaptation of E. coli to environmental stimuli. Each MCP contains multiple sites of methylation, and we identified six of these sites in MCPI. Recently, a second covalent modification of MCPs has been identified, which is not methylation. This modification, designated CheB-dependent modification, is stimulated by repellents and causes a net increase in the negative charge of MCPI and MCPII by one or two charges. We demonstrate that one CheB modification occurs on the methyl-accepting methionine-and lysine-containing tryptic peptide in MCPI and MCPII, and the second CheB modification is on an arginine-containing tryptic peptide. The CheB modification allows three additional methyl groups to be incorporated into the methyl-accepting methionine-lysine peptide, while not actually creating all of these methylation sites. The two CheB modifications occur sequentially. A possible mechanism by which CheB modification permits additional methylations and the role of CheB modification in bacterial chemotaxis are discussed.     

Structural features of methyl-accepting taxis proteins conserved between archaebacteria and eubacteria revealed by antigenic cross-reaction.

A number of eubacterial species contain methyl-accepting taxis proteins that are antigenically and thus structurally related to the well-characterized methyl-accepting chemotaxis proteins of Escherichia coli. Recent studies of the archaebacterium Halobacterium halobium have characterized methyl-accepting taxis proteins that in some ways resemble and in other ways differ from the analogous eubacterial proteins. We used immunoblotting with antisera raised to E. coli transducers to probe shared structural features of methyl-accepting proteins from archaebacteria and eubacteria and found substantial antigenic relationships. This implies that the genes for the contemporary methyl-accepting proteins are related through an ancestral gene that existed before the divergence of arachaebacteria and eubacteria. Analysis by immunoblot of mutants of H. halobium defective in taxis revealed that some strains were deficient in covalent modification of methyl-accepting proteins although the proteins themselves were present, while other strains appeared to be missing specific methyl-accepting proteins.     

BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum

Halophilic archaea, such as eubacteria, use methyl-accepting chemotaxis proteins (MCPs) to sense their environment. We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids. The C-terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation. Hydropathy analysis predicts an enterobacterial-type transducer protein topology for BasT, with an extracellular putative ligand-binding domain flanked by two transmembrane helices and a cytoplasmic domain. BasT-inactivated mutant cells are missing a membrane protein radiolabelled with L-[methyl-3H]-methionine in wild-type cells, confirming that BasT is methylatable and membrane bound. Behavioural analysis of the basT mutant cells by capillary and chemical-in-plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine. The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover. Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine. Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H. salinarum.     

Characterization of Halobacterium halobium mutants defective in taxis.

Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.