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.     

Ecological role of energy taxis in microorganisms

Motile microorganisms rapidly respond to changes in various physico-chemical gradients by directing their motility to more favorable surroundings. Energy generation is one of the most important parameters for the survival of microorganisms in their environment. Therefore it is not surprising that microorganisms are able to monitor changes in the cellular energy generating processes. The signal for this behavioral response, which is called energy taxis, originates within the electron transport system. By coupling energy metabolism and behavior, energy taxis is fine-tuned to the environment a cell finds itself in and allows efficient adaptation to changing conditions that affect cellular energy levels. Thus, energy taxis provides cells with a versatile sensory system that enables them to navigate to niches where energy generation is optimized. This behavior is likely to govern vertical species stratification and the active migration of motile cells in response to shifting gradients of electron donors and/or acceptors which are observed within microbial mats, sediments and soil pores. Energy taxis has been characterized in several species and might be widespread in the microbial world. Genome sequencing revealed that many microorganisms from aquatic and soil environments possess large numbers of chemoreceptors and are likely to be capable of energy taxis. In contrast, species that have a fewer number of chemoreceptors are often found in specific, confined environments, where relatively constant environmental conditions are expected. Future studies focusing on characterizing behavioral responses in species that are adapted to diverse environmental conditions should unravel the molecular mechanisms underlying sensory behavior in general and energy taxis in particular. Such knowledge is critical to a better understanding of the ecological role of energy taxis.     

Methyl-accepting protein associated with bacterial sensory rhodopsin I.

In vivo radiolabeling of Halobacterium halobium phototaxis mutants and revertants with L-[methyl-3H] methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in phototaxis. The lability of the radiolabeled bands to mild base treatment indicated that the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing phototaxis receptors in H. halobium. It was absent in a strain that contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based on the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By [3H]retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced [3H]retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the signal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I.     

Independence and interdependence of Dif and Frz chemosensory pathways in Myxococcus xanthus chemotaxis

Dif and Frz, two Myxococcus xanthus chemosensory pathways, are required in phosphatidylethanolamine (PE) chemotaxis for excitation and adaptation respectively. DifA and FrzCD, the homologues of methyl-accepting chemoreceptors in the two pathways, were examined for methylation in the context of chemotaxis and inter-pathway interactions. Evidence indicates that DifA may not undergo methylation, but signals transmitting through DifA do modulate FrzCD methylation. Results also revealed that M. xanthus possesses Dif-dependent and Dif-independent PE-sensing mechanisms. Previous studies showed that FrzCD methylation is decreased by negative chemostimuli but increased by attractants such as PE. Results here demonstrate that the Dif-dependent sensory mechanism suppresses the increase in FrzCD methylation in attractant response and elevates FrzCD methylation upon negative stimulation. In other words, FrzCD methylation is governed by opposing forces from Dif-dependent and Dif-independent sensing mechanisms. We propose that the Dif-independent but Frz-dependent PE sensing leads to increases in FrzCD methylation and subsequent adaptation, while the Dif-dependent PE signalling suppresses or diminishes the increase in FrzCD methylation to decelerate or delay adaptation. We contend that these antagonistic interactions are crucial for effective chemotaxis in this gliding bacterium to ensure that adaptation does not occur too quickly relative to the slow speed of M. xanthus movement.     

Immunospecific retention of oligonucleotides possessing N6-methyladenosine and 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were immobilized on Sepharose and the resulting immunoadsorbents tested for their ability to retain specific oligonucleotides possessing the corresponding antigenic haptens (i.e. m6A and m7G). Results obtained with oligonucleotides derived from ribonuclease T1 digests of Escherichia coli tRNA (previously labeled with [methyl-3H]methionine) indicated that each immunoadsorbent quantitatively and exclusively retained those methyl-3H-labeled oligonucleotides possessing [methyl-3H]m6A and [methyl-3H]m7G. Elution and subsequent characterization of the retained methyl-3H-labeled oligonucleotides via DEAE-cellulose chromatography revealed the presence of several small oligonucleotides containing m7G and a single, larger oligonucleotide containing m6A. These findings are in accord with previously sequenced structures which indicate that numerous bacterial tRNA species possess m7G while only tRNAVal contains m6A.     

Color-sensitive motility and methanol release responses in Rhodobacter sphaeroides

Blue-light-induced repellent and demethylation responses, characteristic of behavioral adaptation, were observed in Rhodobacter sphaeroides. They were analyzed by computer-assisted motion analysis and through the release of volatile tritiated compounds from [methyl-(3)H]methionine-labeled cells, respectively. Increases in the stop frequency and the rate of methanol release were induced by exposure of cells to repellent light signals, such as an increase in blue- and a decrease in infrared-light intensity. At a lambda of >500 nm the amplitude of the methanol release response followed the absorbance spectrum of the photosynthetic pigments, suggesting that they function as photosensors for this response. In contrast to the previously reported motility response to a decrease in infrared light, the blue-light response reported here does not depend on the number of photosynthetic pigments per cell, suggesting that it is mediated by a separate sensor. Therefore, color discrimination in taxis responses in R. sphaeroides involves two photosensing systems: the photosynthetic pigments and an additional photosensor, responding to blue light. The signal generated by the former system could result in the migration of cells to a light climate beneficial for photosynthesis, while the blue-light system could allow cells to avoid too-high intensities of (harmful) blue light.     

Transmethylation of Sterols in Aerobically Adapting Saccharomyces cerevisiae

The transmethylation of methyl-(14)C-methionine and methyl-(14)C-adenosylmethionine into the nonsaponifiable lipids of anaerobically grown yeast during adaptation to aerobic conditions was investigated. The rate and extent of methyl transfer increased with aeration time and was dependent upon the presence of a fermentable carbon source and O(2). Methionine and adenosylmethionine uptake rates increased in adaptation buffer but did not seem to be the rate-limiting factor for transmethylation under the conditions studied. Thinlayer chromatography of the nonsaponifiable fraction after exposure to label showed the labeled product to be ergosterol. Samples taken after short-term exposure to label were composed of two labeled steroidal products, one with kinetics of an ergosterol precursor.     

Primary structure and functional analysis of the soluble transducer protein HtrXI in the archaeon Halobacterium salinarium.

Signal transduction in the archaeon Halobacterium salinarium is mediated by a family of 13 soluble and membrane-bound transducers. Here, we report the primary structure and functional analysis of one of the smallest halobacterial putative transducers, HtrXI. Hydropathy plot analysis of the primary structure predicts no membrane-spanning segments in HtrXI. The fractionation of the H. salinarium proteins confirmed that HtrXI is a soluble protein. Capillary assay with an HtrXI deletion mutant and a complemented strain revealed that this soluble transducer is involved in Asp and Glu taxis. In vivo analysis of the methylesterase activity of the htrXI-1 deletion mutant suggests that HtrXI plays an important role in the adaptation of the chemotactic responses to His, Asp, and Glu, which are attractants for halobacteria. Stimulation by Asp and Glu causes demethylation of HtrXI and of another putative transducer, HtrVII. But addition of His to halobacterial cells increases HtrXI methylation together with that of other putative transducers. In the absence of HtrXI, stimulation by either Glu or His does not decrease or increase the methylation of any putative transducers. Therefire, the HtrXI transducer appears to have a complex role in chemotaxis signal transduction.     

Differential turnover of methyl groups on methyl-accepting membrane proteins of irreversibly sickled erythrocytes

Methyl group turnover rates for specific methyl-accepting membrane proteins in intact irreversibly sickled cells (ISCs) have been determined. The turnover of methyl groups on all methyl acceptor membrane proteins carboxylmethylated in ISCs is not concerted but proceeds in an ordered sequence which is markedly different from that exhibited by unfractionated normal erythrocytes (AA). In ISCs methyl group turnover based on initial demethylation rate constants is most rapid for membrane polypeptides migrating in sodium dodecyl sulfate at 30,000-39,000 Da, 40,000-55,000 Da, polypeptides comigrating with cytoskeletal component band 4.1, and band 4.5. In contrast, initial methyl group turnover rates obtained after less than 20% of the total methyl groups are turned over in (AA) cells show most rapid demethylation rates for membrane polypeptides migrating at 40,000-55,000 Da, polypeptides comigrating with band 4.5, cytoskeletal components bands 2.1 and 4.1. Results also show significant differences between ISCs and most dense fractions from normal (AA) and nonsickle hemolytic anemias in the demethylation of cytoskeletal proteins, bands 2.1 and 4.1. These findings indicate qualitative differences in accessibility of methyl acceptor substrates to the methylating-demethylating enzyme activity in the cytosol of irreversibly sickled cells compared to discocytic control erythrocytes.     

Evaluation of Genetic and Epigenetic Modification in Rapeseed （Brassica napus） Induced by Salt Stress

Salinity is an important limiting environmental factor for rapeseed production worldwide. In this study, we assessed the extent and pattern of DNA damages caused by salt stress in rapeseed plants. Amplified fragment length polymorphism （AFLP） analysis revealed dose-related increases in sequence alterations in plantlets exposed to 10-1000 mmol/L sodium chloride. In addition, individual plantlets exposed to the same salt concentration showed different AFLP and selected region amplified polymorphism banding patterns. These observations suggested that DNA mutation in response to salt stress was random in the genome and the effect was dose-dependant. DNA methylation changes in response to salt stress were also evaluated by methylation sensitive amplified polymorphism （MSAP）. Three types of MSAP bands were recovered. Type Ⅰ bands were observed with both isoschizomers Hpa Ⅱ and Msp Ⅰ, while type Ⅱ and type Ⅲ bands were observed only with Hpa Ⅱ and Msp Ⅰ, respectively. Extensive changes in types of MSAP bands after NaCI treatments were observed, including appearance and disappearance of type Ⅰ, Ⅱ and Ⅲ bands, as well as exchanges between either type Ⅰand type Ⅱ or type Ⅰ and type Ⅲ bands. An increase of 0.2-17.6% cytosine methylated CCGG sites were detected in plantlets exposed to 10- 200 mmol/L salt compared to the control, and these changes included both de novo methylation and demethylation events. Nine methylation related fragments were also recovered and sequenced, and one sharing a high sequence homology with the ethylene responsive element binding factor was identified. These results demonstrated clear DNA genetic and epigenetic alterations in planUets as a response to salt stress, and these changes may suggest a mechanism for plants adaptation under salt stress.     

Effects of long-range taxis and population pressure on the range expansion of invasive species in heterogeneous environments

We consider a new model for biological invasions in periodic patchy environments, in which long-range taxis and population pressure are incorporated in the framework of reaction-diffusion-advection equations. We assume that long-range taxis is induced by a weighted integral of stimuli within a certain sensing range. Population pressure is incorporated in the diffusion coefficient that linearly increases with population density. We first analyze the model in the absence of population pressure and demonstrate how the sensing length of long-range taxis influences the range expansion pattern of invasive species and its rate of spread. The effects of population pressure are examined for both homogeneous and periodic patchy environments. For the homogeneous environment, an exact and explicit traveling wave solution and the spreading speed are obtained. For the periodic patchy environment, we find numerically that a population starting from any localized distribution evolves to a traveling periodic wave if the null solution of the RDA equation is locally unstable, and that the traveling wave speed significantly increases with increasing population pressure. Furthermore, the population pressure and taxis intensity synergistically enhance the spreading speed when they are increased together.     

Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets

The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.     

The accumulation of the light-harvesting 2 complex during remodeling of the Rhodobacter sphaeroides intracytoplasmic membrane results in a slowing of the electron transfer turnover rate of photochemical reaction centers

A functional proteomic analysis of the intracytoplasmic membrane (ICM) development process was performed in Rhodobacter sphaeroides during adaptation from high-intensity illumination to indirect diffuse light. This initiated an accelerated synthesis of the peripheral light-harvesting 2 (LH2) complex relative to that of LH1-reaction center (RC) core particles. After 11 days, ICM vesicles (chromatophores) and membrane invagination sites were isolated by rate-zone sedimentation and subjected to clear native gel electrophoresis. Proteomic analysis of gel bands containing the RC-LH1 and -LH2 complexes from digitonin-solubilized chromatophores revealed high levels of comigrating electron transfer enzymes, transport proteins, and membrane assembly factors relative to their equivalent gel bands from cells undergoing adaptation to direct low-level illumination. The GroEL chaperonin accounted for >65% of the spectral counts in the RC-LH1 band from membrane invagination sites, which together with the appearance of a universal stress protein suggested that the viability of these cells was challenged by light limitation. Functional aspects of the photosynthetic unit assembly process were monitored by near-IR fast repetition rate analysis of variable fluorescence arising from LH-bacteriochlorophyll a components. The quantum yield of the primary charge separation during the early stages of adaptation showed a gradual increase (variable/maximal fluorescence = 0.78-0.83 between 0 and 4 h), while the initial value of ~70 for the functional absorption cross section (σ) gradually increased to 130 over 4 days. These dramatic σ increases showed a direct relation to gradual slowing of the RC electron transport turnover rate (τ(QA)) from ~1.6 to 6.4 ms and an ~3-fold slowing of the rate of reoxidation of the ubiquinone pool. These slowed rates are not due to changes in UQ pool size, suggesting that the relation between increasing σ and τ(QA) reflects the imposition of constraints upon free diffusion of ubiquinone redox species between the RC and cytochrome bc(1) complex as the membrane bilayer becomes densely packed with LH2 rings.     

Demethylation of methyl-accepting chemotaxis proteins in Escherichia coli induced by the repellents glycerol and ethylene glycol.

The addition of glycerol or ethylene glycol caused not only severe tumbling but also a drastic decrease in the methylation level of methyl-accepting chemotaxis proteins (MCPs) in Escherichia coli. Experiments with various mutants having defects in their MCPs showed that the demethylation occurred in all three kinds of MCPs, MCPI, II, and III. The addition of an attractant to the glycerol- or ethylene glycol-treated cells resulted in a distinct increase in the methylation level of the relevant MCP, indicating that glycerol and ethylene glycol do not directly damage the methylation-demethylation system in the cell. The time courses of adaptation and MCP demethylation upon addition of these repellents were consistent with each other. Furthermore, both the response time and the extent of MCP demethylation were increased in parallel with increasing concentrations of glycerol or ethylene glycol. These results indicate that the adaptation to these repellents is performed by the demethylation of MCPs. Thus, glycerol and ethylene glycol are novel repellents, which utilize not just one but all three kinds of MCPs for both information processing and adaptation.     

Multiple Chemosensory Defects in Daf-11 and Daf-21 Mutants of Caenorhabditis Elegans

Phenotypic analysis of the daf-11 and daf-21 mutants of Caenorhabditis elegans suggests that they have defects in components shared by processes analogous to vertebrate taste and olfaction. daf-11 and daf-21 mutations were previously shown to cause inappropriate response to the dauer-inducing pheromone. By mutational analysis and by disabling specific chemosensory sensilla with a laser, we show that neurons in the amphid sensilla are required for this pheromone response. Using behavioral assays, we find that daf-11 and daf-21 mutants are not defective in avoidance of certain non-volatile repellents, but are defective in taxis to non-volatile attractants. In addition, both mutants are defective in taxis to volatile attractants detected primarily by the amphid neuron AWC, but respond normally to volatile attractants detected primarily by AWA. We propose that daf-11 and daf-21 mediate sensory transduction for both volatile and non-volatile compounds in specific amphid neurons.     

Methylation and demethylation of solubilized chemoreceptors from thermophilic bacterium PS-3

Methyl-accepting chemotaxis proteins (MCPs) were solubilized from the membrane of thermophilic bacterium PS-3 in the presence of Triton X-100. The solubilized MCPs could be methylated and demethylated. Methylation of the solubilized MCPs reached a steady state, at which the methylation and demethylation rates were equal. The solubilized MCPs were purified by anti-MCPs Sepharose 4B column chromatography. The purified MCPs could also be methylated and demethylated without reconstituting them into liposomes. As suggested by the results of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified MCPs, ion-exchange chromatography showed that MCPs consisted of at least two components. Each component appeared on SDS gel electrophoresis as multiple bands in the 64K to 70K dalton range or in the 70K to 84K dalton range. The initial rate and level of methylation of the solubilized MCPs were increased by the addition of attractants: glutamate, L-serine, L-aspartate, D-glucose, etc. The threshold of the glutamate concentration for this increase was about 10(-7) M. The rate of demethylation was also increased by attractants.     

Change of sludge consortium in response to sequential adaptation to benzene, toluene, and o-xylene

Activated sludge was sequentially adapted to benzene, toluene, and o-xylene (BTX) to study the effects on the change of microbial community. Sludge adapted to BTX separately degraded each by various rates in the following order; toluene>o-xylene>benzene. Degradation rates were increased after exposure to repeated spikes of substrates. Eleven different kinds of sludge were prepared by the combination of BTX sequential adaptations. Clustering analyses (Jaccard, Dice, Pearson, and cosine product coefficient and dimensional analysis of MDS and PCA for DGGE patterns) revealed that acclimated sludge had different features from nonacclimated sludge and could be grouped together according to their prior treatment. Benzene- and xylene-adapted sludge communities showed similar profiles. The sludge profile was affected from the point of the final adaptation substrate regardless of the adaptation sequence followed. In the sludge adapted to 50 ppm toluene, Nitrosomonas sp. and bacterium were dominant, but these bands were not dominant in benzene and benzene after toluene adaptations. Instead, Flexibacter sp. was dominant in these cultures. Dechloromonas sp. was dominant in the culture adapted to 50 ppm benzene. Thauera sp. was the main band in the sludge adapted to 50 ppm xylene, but became vaguer as the xylene concentration was increased. Rather, Flexibacter sp. dominated in the sludge adapted to 100 ppm xylene, although not in the culture adapted to 250 ppm xylene. Two bacterial species dominated in the sludge adapted to 250 ppm xylene, and they also existed in the sludge adapted to 250 ppm xylene after toluene and benzene.     

Divergent adaptation to Qinghai-Tibetan Plateau implicated from transciptome study of Gymnocypris dobula and Schizothorax nukiangensis

The Schizothoracine fishes are widely distributed in the Qinghai-Tibetan Plateau (QTP) area and its peripheral regions, which provide a prime example of adaptation in highland aquatic environments. Recent progresses have revealed various genetic adaptations of these fishes by comparing to distantly related lowerland species, however, comparative studies on closely-related species of different altitudes are still lacking. In this study, we sequenced and annotated a primitive Schizothoracine fish Schizothorax nukiangensis Tsao and a highly specialized one Gymnocypris dobula. We performed evolutionary analyses to investigate the candidate genes and signaling pathways involved QTP highland adaptation in both Schizothoracine fishes. Analysis of the 11,007 one-copy orthologs to the primitive cyprinid species, Danio rerio, revealed that both G. dobula and S. nukiangensis showed elevated evolutionary rates. A large number of genes related to hypoxia, including genes involved metabolic processes and cardiovascular system development, exhibited signatures of positive selection in both Schizothoracine fishes, but very few positively selected genes were found overlapping among these Schizothoracines. Our results indicated divergent genetic adaptation to highland environment for aquatic species living in QTP.