Reversible ADP-ribosylation as a mechanism of enzyme regulation in procaryotes

Several cases of ADP-ribosylation of endogenous proteins in procaryotes have been discovered and investigated. The most thoroughly studied example is the reversible ADP-ribosylation of the dinitrogenase reductase from the photosynthetic bacteriumRhodospirillum rubrum and related bacteria. A dinitrogenase reductase ADP-ribosyltransferase (DRAT) and a dinitrogenase reductase ADP-ribose glycohydrolase (DRAG) fromR. rubrum have been isolated and characterized. The genes for these proteins have been isolated and sequences and show little similarity to the ADP-ribosylating toxins. Other targets for endogenous ADP-ribosylation by procaryotes include glutamine synthetase inR. rubrum andRhizobium meliloti and undefined proteins inStreptomyces griseus andPseudomonas maltophila.     

Phosphorylation of glucose by a guanosine-5′-triphosphate (GTP)-dependent glucokinase in Fibrobacter succinogenes subsp. succinogenes S85

Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg⁺² and K⁺ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-d-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pl of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent K_m values of 55 M for glucose and 72 M for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.Abbreviations CHES cyclohexylaminoethanesulfonic acid - GK glucokinase - PEP phosphoenolpyruvate Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article no. 2186     

Role of sodium ions for sulfate transport and energy metabolism in Desulfovibrio salexigens

The sodium ion gradient and the membrane potential were found to be the driving forces of sulfate accumulation in the marine sulfate reducer Desulfovibrio salexigens. The protonmotive force of –158 mV, determined by means of radiolabelled membrane-permeant probes, consisted of a membrane potential of –140 mV and a pH gradient (inside alkaline) of 0.3 at neutral pH_out. The sodium ion gradient, as measured with silicone oil centrifugation and atomic absorption spectroscopy, was eightfold ([Na⁺]_out/[Na⁺]_in) at an external Na⁺ concentration of 320 mM. The resulting sodium ionmotive force was –194 mV and enabled D. salexigens to accumulate sulfate 20000-fold at low external sulfate concentrations (<0.1 M). Under these conditions high sulfate accumulation occurred electrogenically in symport with three sodium ions (assuming equilibrium with the sodium ion-motive force). With increasing external sulfate concentrations sulfate accumulation decreased sharply, and a second, low-accumulating system symported sulfate electroneutrally with two sodium ions. The sodium-ion gradient was built up by electrogenic Na⁺/H⁺ antiport. This was demonstrated by (i) measuring proton translocation upon sodium ion pulses, (ii) studying uptake of sodium salts in the presence or absence of the electrical membrane potential, and (iii) the inhibitory effect of the Na⁺/H⁺ antiport inhibitor propylbenzilylcholin-mustard HCl (PrBCM). With resting cells ATP synthesis was found after proton pulses (changing the pH by three units), but neither after pulses of 500 mM sodium ions, nor in the presence of the uncoupler tetrachorosalicylanilide (TCS). It is concluded that the energy metabolism of the marine strain D. salexigens is based primarily on the protonmotive force and a protontranslocating ATPase.Abbreviations MOPS morpholinopropanesulfonic acid - TCS tetrachlorosalicylanilide - PrBCM propylbenzilylcholin-mustard HCl - Tris tris(hydroxymethyl)aminomethane - TPP⁺ bromide tetraphenylphosphonium bromide     

Characterisation and pathogenicity of bacteria from shoot tips of the globe artichoke (Cynara scolymus L.)

Bacteria have been isolated from shoot tips of symptomless globe artichoke plants. These were identified as Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas spp., Serratia liquefaciens, Enterobacter agglomerans/Erwinia, Agrobacterium radiobacter, an unidentified member of Rhizobiaceae and another classified in the “corynebacteria” group. The most frequently isolated species was P. fluorescens, biovars II and III. The endogenous character of these bacteria was studied in plants growing in vitro and in the open field. P. fluorescens, P. marginalis, S. liquefaciens and E. agglomerans/Erwinia caused symptoms in plants growing in vitro, but only P. fluorescens biovar II and P. marginalis produced symptoms in plants growing in open fields. Differences in pathogenicity were observed on inoculated plants growing in vitro or in the open field. This suggests that several endophytic bacterial species may be responsible for the high levels of contaminants found during the micropropagation of globe artichoke.     

Terminal electron transport system (ETS)-activity in the sediment of Lake Balaton,Hungary

Terminal electron transport system (ETS)-activity of the sediment and plankton of Lake Balaton, the largest shallow lake of Central Europe was measured by tetrazolium-reduction biweekly during 1989–1990 and in the spring of 1991. Sediment proved to be enzymatically active to 30-35 cm down in the hypertrophic Keszthely Bay and to 15–20 cm down in the meso-eutrophic Siófok Basin. Sediment ETS-activity exceeded planktonic activity 15 to 24 fold.The total activity m^–2 showed one or two order of magnitude higher respiratory potential in Lake Balaton than needed for complete oxidation of the planktonic primary production; most of this potential was detected in the upper 3–5 cm sediment layer in springs. Incubations of cell-free homogenates of sediment bacteria showed that ETS remains active days after death of organisms at low temperature. Accumulated postmortem ETS-activity derived from the benthic diatoms, bacteria, plankton deposit and dead summer macrophytes seems to be responsible for the high ETS-activity of the sediment in the warming periods in springs. These enzyme fractions may contribute to the rapid oxidation of the alkaline, well-aerated lake.     

Mass transfer limitation of sulfate in methanogenic aggregates

The role of mass transfer limitation of sulfate as a factor governing the competition between sulfate reducing and methane producing bacteria in methanogenic aggregates was theoretically evaluated by the calculation of steady-state sulfate microprofiles using a reference set of parameters obtained from the literature. The shooting method was used as a numerical technique for solving the mathematical model. The effect of the parameters on mass transport limitation was tested by varying each reference value of the parameters with a factor of 3. Sulfate limitation within granules prevailed at moderate (0.1 kg m(-3)) and low sulfate concentrations in the bulk liquid, at high maximum sulfate utilization rates (3.73 x 10(-5) kg SO(4) (2-) kg(-1) VSS S(-1) or biomass concentrations (40 KG VSS m(-3)), and in large aggregates (radius of 7.5 10(-4) m). The effective diffusion coefficient of sulfate and the affinity constant were less determinative for the penetration depth of sulfate within a granule. (c) 1994 John Wiley & Sons, Inc.     

Chloroflexus-like organisms from marine and hypersaline environments: Distribution and diversity

We report the presence of a diverse number ofChloroflexus-like organisms in intertidal marine and submerged hypersaline microbial mats using light, infrared fluorescence, and electron microscopy. The intertidal organisms appear morphologically very similar to thermophilicC. aurantiacus while the 2 hypersaline strains are larger and have a more complex ultrastructure composed of chlorosome-bearing internal membranes that appear to arise as invaginations of the cell membrane. By comparing spectroradiometry of microbial mat layers with microscopic observations, we have confirmed that theChloroflexus-like organisms are major constituents of the hypersaline microbial mat communities. In situ studies on mat layers dominated byChloroflexus-like organisms showed that sulfide-dependent photoautotrophic activity sustained by near infrared radiation prevailed. Autoradiographic analyses revealed that autotrophy was sustained in the filaments by 750 nm radiation. Three morphologically distinct strains are now maintained in mixed culture. One of these appears to be growing photoautotrophically.     

Comparison of phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities among dissimilatory sulfate-reducing bacteria

Abstract Twenty-five isolates of dissimilatory sulfate-reducing bacteria were clustered based on similarity analysis of their phospholipid ester-linked fatty acids (PLFA). Of these, 22 showed that phylogenetic relationships based on the sequence similarity of their 16S rRNA directly paralleled the PLFA relationships. Desulfobacter latus and Desulfobacter curvatus grouped with the other Desulfobacter spp. by 16S rRNA comparison but not with the PLFA analysis as they contained significantly more monoenoic PLFA than the others. Similarly, Desulfovibrio africanus clustered with the Desulfovibrio spp. by 16S rRNA but not with them when analyzed by PLFA patterns because of higher monoenoic PLFA content. Otherwise, clustering obtained with either analysis was essentially congruent. The relationships defined by PLFA patterns appeared robust to shifts in nutrients and terminal electron acceptors. Additional analyses utilizing the lipopolysaccharide-lipid A hydroxy fatty acid patterns appeared not to shift the relationships based on PLFA significantly except when completely absent, as in Gram-positive bacteria. Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns. Determination of bacterial relationships based on different selective pressures for various cellular components provides more clues to evolutionary history leading to a more rational nomenclature.     

Detection of Antibody-Coated Bacteria in Expectorated Sputum for Diagnosis of Lower Respiratory Infections

We evaluated antibody-coated bacteria (ACB) in expectorated sputum to discriminate contaminating or colonizing organisms from true pathogens. We examined 60 expectorated sputum samples from 51 patients with lower respiratory infections (chronic obstructive pulmonary disease 25, pneumonia 20, purulent tracheobronchitis 6). All samples were examined with quantitative culture and immunofluorescent demonstration of ACB. From the results of quantitative culture, we divided specimens into pathogen-isolated and pathogen-free samples. Among pathogen-isolated samples, in which we isolated accepted pathogenic organisms at ≥ 10⁷ colony-forming units per ml, 16 of 23 samples were ACB-positive (69.5%). In contrast, among pathogen-free samples, in which we isolated accepted pathogens at < 10⁷ colony forming units per ml or only upper respiratory flora, only 3 of 37 samples were ACB-positive (8.1%). The ACB-positive rate was significantly higher in pathogen-isolated than in pathogen-free samples (P < 0.001). Consequently, detecting ACB in expectorated sputum shows good potential as another criterion for distinguishing contaminating or colonizing organisms from true pathogens.