Phosphorylation of rhodopsin as a possible mechanism of adaptation.

Light-induced phosphorylation of rhodopsin has been extensively studied by a number of investigators from a biochemical point of view. However, little is known about the physiological function of this reaction. The slow rates measured for phosphorylation and dephosphorylation suggest that it may be involved in visual adaptation rather than in excitation. This paper presents biochemical data obtained from phosphorylation experiments in isolated photoreceptor membranes as well as in the physiological system of whole retinas and living animals. An attempt is made to compare the phosphorylation reaction with visual adaptation hypotheses taken from the electrophysiological literature. Finally, effects of cyclic nucleotide metabolism on the sensitivity of photoreceptors are presented and discussed.     

Pseudomonas syringae lipopolysaccharides: Immunochemical characteristics and structure as a basis for strain classification

Lipopolysaccharide (LPS) preparations of 34 Pseudomonas syringae strains of 19 pathovars were prepared by saline extraction from wet cells and purified by repeated ultracentrifugation. The preparations reacted with homologous O-antisera, obtained by rabbit immunization with heat-killed bacterial cells. Through inhibition of homologous reactions between LPS preparations of heterologous strains (enzyme immunoassay, EIA), it was established for the first time that high serological affinity between strains is observed only if their LPS contains O-specific polysacc haride chains (OPS) comprised of completely identical rather than partially similar units. The central linear part of the OPS was found to be serologically inert when shielded with side groups. Data on immunochemical characteristics of the LPS and OPS structure are analyzed in relation to the design of P. syringae classification scheme.     

Comparative analysis of the lipopolysaccharides of a rough and a smooth strain of Pseudomonas syringae pv. phaseolicola

The lipopolysaccharides (LPS) of a rough (R) and a smooth (S) strain of Pseudomonas syringae pv. phaseolicola were analysed. The S-LPS revealed markedly more rhamnose and fucose, but less glucose, than the R-LPS. The presence of 3-O-methyl-rhamnose (acofriose) in the S-LPS was confirmed by cochromatography with authentic acofriose. SDS polyacrylamide gel electrophoresis of the S-LPS demonstrated a cluster of regularly spaced high molecular weight fractions, which was almost lacking in the R-LPS. The main fatty acids of the lipid A of both LPS species were 3-OH-10:0,3-OH-12:0,2-OH-12:0, and 12:0. Two N-linked diesters were demonstrated: 3-O(12:0)-12:0 and 3-O(2-OH-12:0)-12:0. S-LPS was subjected to mild hydrolysis and the degraded polysaccharide separated into three fractions by gel permeation chromatography on a Fractogel TSK HW-50 column. Fraction I, representing nearly only the O-specific side chain, consisted of rhamnose and fucose in a molar ratio of 4:1, with 4% of the rhamnose being 3-O-methylated (acofriose). Fraction II, representing mostly core material, was composed of glucose, rhamnose, heptose, glucosamine, galactosamine, alanine, and a still unidentified amino compound, in an approximate molar ratio of 3:1:1:1:1:1:1, and KDO. Fraction III consisted of released monomers and salts. The LPS was highly phosphorylated (3.28% phosphorus in the core fraction). The thus characterized composition of the LPS O-chain seems to be unique for the pathovar phaseolicola of P. syringae, although many similarities exist to other pathovars as well as to other bacterial species.Abbreviations LPS lipopolysacchairdes - GC/MS combined gas liquid chromatography-mass spectrometry - HVE high voltage electrophoresis - KDO 2-keto-3-deoxyoctonic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate P.s. pv. phaseolicola is termed P. phaseolicola in the text     

Structural heterogeneity in the lipopolysaccharides of Pseudomonas syringae with O-polysaccharide chains having different repeating units.

Studies by sugar and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy revealed a structural heterogeneity in the O-polysaccharides of Pseudomonas syringae pvs. coronafaciens IMV 9030 and atrofaciens IMV 8281 owing to the presence of different types of repeating units. In strain IMV 9030, the major repeating units are a linear alpha-L-rhamnose trisaccharide and a tetrasaccharide (A, n=0 or 1). A minor repeating unit is a branched pentasaccharide with an alpha-L-rhamnose main chain and a lateral 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) residue (B, X=2, n=1). In strain IMV 8281, all repeating units are branched and differ in size and position of substitution of one of the alpha-L-rhamnose residues (tetrasaccharide, B, X=3, n=0; pentasaccharides, B, X=2 or 3, n=1). [structure--see text] Reinvestigation of the structure of the branched O-polysaccharide of P. syringae pv. tomato IPGR 140 showed that, together with the major tetrasaccharide repeating unit (B, X=3, n=0) [Knirel, Y. A., et al. Carbohydr. Res. 1993, 243, 199-204], it has a minor pentasaccharide repeating unit (B, X=3, n=1).     

Phosphorylation of rhodopsin as a possible mechanism of adaptation

Light-induced phosphorylation of rhodopsin has been extensively studied by a number of investigators from a biochemical point of view. However, little is known about the physiological function of this reaction. The slow rates measured for phosphorylation and dephosphorylation suggest that it may be involved in visual adaptation rather than in excitation. This paper presents biochemical data obtained from phosphorylation experiments in isolated photoreceptor membranes as well as in the more physiological system of whole retinas and living animals. An attempt is made to compare the phosphorylation reaction with visual adaptation hypotheses taken from the electrophysiological literature. Finally, effects of cyclic nucleotide metabolism on the sensitivity of photoreceptors are presented and discussed.Abbreviations used ATP adenosine 5-triphosphate - GTP guanosine 5-triphosphate - ROS rod outer segments - IBMX isobutylmethylxanthine - cyclic GMP guanosine 3, 5-monophosphate Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

Low-temperature-induced changes in composition and fluidity of lipopolysaccharides in the antarctic psychrotrophic bacterium Pseudomonas syringae

The Antarctic psychrotrophic bacterium Pseudomonas syringae was more sensitive to polymyxin B at a lower (4 degrees C) temperature of growth than at a higher (22 degrees C) temperature. The amount of hydroxy fatty acids in the lipopolysaccharides (LPS) also increased at the lower temperature. These changes correlated with the increase in fluidity of the hydrophobic phase of lipopolysaccharide aggregates in vitro.     

Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae.

One of the chromosomal regions of Pseudomonas syringae pv. syringae encoding pathogenicity factors had been mapped into a 3.9-kilobase-pair fragment in previous studies. Promoter probe analysis indicated the existence of a promoter near one end of the fragment. DNA sequencing of this fragment revealed the existence of a consensus promoter sequence in the region of the promoter activity and two open reading frames (ORFs) downstream. These ORFs, ORF1 and ORF2, encoded putative polypeptides of 40 and 83 kilodaltons, respectively. All ORF1::Tn5 as well as ORF2::Tn5 mutant strains were nonpathogenic on susceptible host bean plants and were unable to elicit hypersensitive reactions on nonhost tobacco plants. The deduced amino acid sequence of the 83-kilodalton polypeptide contained features characteristic of known integral membrane proteins. Fusion of the lacZ gene to ORF2 led to the expression of a hybrid protein inducible in Escherichia coli. The functions of the putative proteins encoded by ORF1 and ORF2 are unknown at present.     

ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature

RecD is essential for growth at low temperature in the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro, with the ability to unwind short DNA duplexes containing only 5' overhangs or forked ends. Five of the mutant proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in motif III) and R660A (in motif VI) completely lost both ATPase and helicase activities. Three other mutants, T259A in motif Ia, R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity, but had no helicase activity. While all RecD proteins had DNA-binding activity, the mutants of motifs IV and V displayed reduced binding, and the motif II mutant showed a higher degree of binding to ssDNA. Significantly, only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4 degrees C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.     

Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.     

Aspartate aminotransferase is involved in cold adaptation in psychrophilic Pseudomonas syringae

CSM2, a cold-sensitive mutant of psychrophilic Pseudomonas syringae, grows like wild-type cells when cultured at 22 and 28°C; but at 4°C, the growth is retarded. In CSM2, AAT (coding for aspartate aminotransferase) is identified as the mutated gene. The expression of AAT in Pseudomonas syringae was transiently enhanced when cells were shifted from 22 to 4°C indicating that AAT is cold-inducible. Complementation of the mutated AAT transformed CSM2 from a cold-sensitive phenotype to a cold-resistant phenotype like the wild-type cells, thus providing evidence for the first time that AAT is required for low-temperature growth.     

The possible functional role of neuronal gangliosides in temperature adaptation of vertebrates

Comparative studies on brain gangliosides of more than 60 vertebrate species show correlations between concentration and the level of evolutionary organization: poikilothermic lower vertebrates (fish, amphibs, reptiles) contain about 110 to 700 μg ganglioside bound NeuAc/g. fresh wt., homeothermic birds and mammals, on the other side, 500 to 1000 μg. The composition of brain gangliosides in poikilotherms is much more complex and variable (more multisialogangliosides) as compared with homeotherms (domination of less polar fractions). There are distinct correlations between brain ganglioside composition and state of thermal adaptation: Fishes being adapted to habitates with extreme temperatures (antarctic icefish — tropic fish) are characterized by quite opposite ganglioside patterns (domination of high versus less polar fractions). During seasonal acclimatization and experimental acclimation of fish to cold or during hibernation and early postnatal development of mammals poly-sialylations of brain gangliosides occur. With regard to this the individual brain structures react differently.

The results are taken for evidence that variations in the composion of synaptic membrane-bound gangliosides may induce long-term alterations in viscosity and permeability of the neuronal membrane by which the neuronal transmission might be kept on a constant level during the process of temperature adaptation.     

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae.

Plant-associated pseudomonads are commonly exposed to copper bactericides, which are applied to reduce the disease incidence caused by these bacteria. Consequently, many of these bacteria have acquired resistance or tolerance to copper salts. We recently conducted a survey of 37 copper-resistant (Cur) Pseudomonas spp., including P. cepacia, P. fluorescens, P. syringae, and P. viridiflava, and found that a subset of the P. syringae strains showed a dramatic increase in exopolysaccharide (EPS) production on mannitol-glutamate medium containing CuSO4 at 250 micrograms/ml. A modified carbazole assay indicated that the EPS produced on copper-amended media contained high levels of uronic acids, suggesting that the EPS was primarily alginic acid. Uronic acids extracted from selected strains were further confirmed to be alginate by demonstrating their sensitivity to alginate lyase and by descending paper chromatography following acid hydrolysis. Subinhibitory levels of arsenate, cobalt, lithium, rubidium, molybdenum, and mercury did not induce EPS production, indicating that alginate biosynthesis is not induced in P. syringae cells exposed to these heavy metals. A 200-kb plasmid designated pPSR12 conferred a stably mucoid phenotype to several P. syringae recipients and also increased their resistance to cobalt and arsenate. A cosmid clone constructed from pPSR12 which conferred a stably mucoid phenotype to several P. syringae strains but not to Pseudomonas aeruginosa was obtained. Results obtained in this study indicate that some of the signals and regulatory genes for alginate production in P. syringae differ from those described for alginate production in P. aeruginosa.     

Phosphorylation of paramyosin and its possible role in the catch mechanism.

Paramyosin isolated from the adductor muscle of Mercenaria mercenaria was shown to contain three to five phosphate groups/molecule; the actual number varied depending on the method used to extract the protein. Dephosphorylation resulted in an increase in the solubility of paramyosin near pH 7 and near physiological ionic strength. This behavior suggests that the number of phosphates/molecule may be a determining factor in the aggregation behavior of paramyosin-containing myofilaments. Thus, phosphorylation may be involved in catch contractions since correlations have been demonstrated earlier between catch contraction of molluscan muscles and aggregation properties of paramyosin (Ruegg, J. C. (1971) Physiol. Rev. 51, 201-249).     

Gene amplification and cold adaptation of pepsin in Antarctic fish. A possible strategy for food digestion at low temperature

Cold-adapted organisms have developed a number of adjustments at the molecular level to maintain metabolic functions at low temperatures. Among other features, they can produce enzymes characterized by a high turnover number or a high catalytic efficiency. The present work is aimed at investigating the process of food digestion at low temperature through the study of pepsins in Antarctic notothenioids. For such a purpose, we have cloned and sequenced three forms of pepsin A and a single form of gastricsin from the gastric mucosa of Trematomus bernacchii (rock cod). Phylogenetic analysis has suggested that the three pepsin A isotypes arose from two gene duplication events leading to the most ancestral pepsin A3 and to the most recent forms represented by pepsin A1 and pepsin A2. Molecular modeling has unraveled significant structural differences in these enzymes with respect to their mesophilic counterparts. Hydropathy and flexibility determined on the substrate-binding subsites of Antarctic and mesophilic pepsins have shown for pepsin A2 reduced hydropathy and increased flexibility at the level of the substrate cleft, features typical of cold-adapted enzymes. Northern blot analysis of RNA from rock cod gastric mucosa hybridized with molecular probes designed on specific regions of different pepsin forms has shown that rock cod pepsin genes are expressed at comparable levels. The present results suggest that the Antarctic rock cod adopted two different strategies to accomplish efficient protein digestion at low temperature. One mechanism is the gene duplication that increases enzyme production to compensate for the reduced kinetic efficiency, the other is the expression of a new enzyme provided with features typical of cold-adapted enzymes.