[Show abstract][Hide abstract] ABSTRACT: Seventy-five aerobic heterotrophs have been isolated from a packed-column bioreactor inoculated with soil from Antarctica.
The column was maintained at 10°C and continuously fed with a casein-containing medium to enrich protease producers. Twenty-eight
isolates were selected for further characterization on the basis of morphology and production of clearing zones on skim milk
plates. Phenotypic tests indicated that the strains were mainly psychrotrophs and presented a high morphological and metabolical
diversity. The extracellular protease activities tested were optimal at neutral pH and between 30 and 45°C. 16S ribosomal
DNA sequence analyses showed that the bioreactor was colonized by a wide variety of taxons, belonging to various bacterial
divisions: α-, β-, and γ-Proteobacteria; the Flexibacter-Cytophaga-Bacteroides group; and high G+C gram-positive bacteria and low G+C gram-positive bacteria. Some strains represent candidates for new
species of the genera Chryseobacterium and Massilia. This diversity demonstrates that the bioreactor is an efficient enrichment tool compared to traditional isolation strategies.
[Show abstract][Hide abstract] ABSTRACT: A comparison of the crystal structure of the dimeric enzyme citrate synthase from the psychrophilic Arthrobacter strain DS2-3R with that of the structurally homologous enzyme from the hyperthermophilic Pyrococcus furiosus reveals a significant difference in the accessibility of their active sites to substrates. In this work, we investigated
the possible role in cold activity of the greater accessibility of the Arthrobacter citrate synthase. By site-directed mutagenesis, we replaced two alanine residues at the entrance to the active site with
an arginine and glutamate residue, respectively, as found in the equivalent positions of the Pyrococcus enzyme Also, we introduced a loop into the active site of the psychrophilic citrate synthase, again mimicking the situation
in the hyperthermophilic enzyme. Analysis of the thermoactivity and thermostability of the mutant enzymes reveals that cold
activity is not significantly compromised by the mutations, but rather the affinity for one of the substrates, acetyl-CoA,
is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R) has an increased thermostability but a reduced temperature optimum for catalytic activity. This unexpected relationship between stability and activity is discussed with
respect to the nature of the dependence of catalytic activity on temperature.
Full-text · Article · Oct 2001 · Protein engineering
[Show abstract][Hide abstract] ABSTRACT: Recombinant citrate synthase from a psychrotolerant bacterium, DS2-3R, recently isolated in Antarctica, has been crystallized. The crystals belong to space group P6122 or P6522, with cell dimensions a = b = 70.8, c = 307.8 A. Diffraction data collected on a synchrotron from a cryoprotected crystal extends to at least 2.0 A. Knowledge of the structure of this enzyme will add to the understanding of cold activity and thermolability, and will be of biotechnological interest. Previously, the structure of citrate synthase from Archaea inhabiting environments at 328 and 373 K, has been reported. This present study will extend our understanding of the structural integrity and activity of proteins at the temperature extremes of life.
No preview · Article · Oct 1998 · Acta Crystallographica Section D Biological Crystallography
[Show abstract][Hide abstract] ABSTRACT: Following the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second citrate synthase of this organism, recently described by the authors, is in fact a 2-methylcitrate synthase that possesses citrate synthase activity as a minor component. Whereas the hexameric citrate synthase is constitutively produced, the 2-methylcitrate synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylcitrate is proposed. The citrate synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus, are approximately 40% identical in sequence to the Escherichia coli 2-methylcitrate synthase and also possess 2-methylcitrate synthase activity. The data are discussed with respect to the structure, function and evolution of citrate synthase and 2-methylcitrate synthase.
[Show abstract][Hide abstract] ABSTRACT: Background: The structural basis of adaptation of enzymes to low temperature is poorly understood. Dimeric citrate synthase has been used as a model enzyme to study the structural basis of thermostability, the structure of the enzyme from organisms living in habitats at 55°C and 100°C having previously been determined. Here the study is extended to include a citrate synthase from an Antarctic bacterium, allowing us to explore the structural basis of cold activity and thermostability across the whole temperature range over which life is known to exist.
[Show abstract][Hide abstract] ABSTRACT: The gene encoding citrate synthase from a novel bacterial isolate (DS2-3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, purified from DS2-3R, are cold-active, with a temperature optimum of 31 degrees C. In addition the enzymes are rapidly inactivated at 45 degrees C, and show significant activity at 10 degrees C and below. Comparison of amino acid sequences indicates that DS2-3R citrate synthase is most closely related to the enzyme from gram-positive bacteria. The amino acid sequence of the DS2-3R enzyme shows several features previously recognised in other cold-active enzymes, including an extended surface loop, an increase in the occurrence of charged residues and a decrease in the number of proline residues in loops. Other changes observed in some psychrophilic enzymes, such as a decrease in isoleucine content and in arginine/(arginine+lysine) content, were not seen in this case.
Full-text · Article · Sep 1997 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: The conserved glutamate residue at position 65 of the Propionigenium modestum c subunit is directly involved in binding and translocation of Na+ across the membrane. The site-specific introduction of the cQ32I and cS66A substitutions in the putative vicinity to cE65 inhibited growth of the single-site mutants on succinate minimal agar, indicating that both amino acid residues are important for proper function of the oxidative phosphorylation system. This growth inhibition was abolished, however, if the cF84L/cL87V double mutation was additionally present in the P. modestum c subunit. The newly constructed Escherichia coli strain MPC848732I, harboring the cQ32I/cF84L/cL87V triple mutation, revealed a change in the coupling ion specificity from Na+ to H+. ATP hydrolysis by this enzyme was therefore not activated by NaCl, and ATP-driven H+ transport was not affected by this alkali salt. Both activities were influenced, however, by LiCl. These data demonstrate the loss of the Na+ binding site and retention of Li+ and H+ binding sites within this mutant ATPase. In the E. coli strain MPC848766A (cS66A/cF84L/cL87V), the specificity of the ATPase was further restricted to H+ as the exclusive coupling ion. Therefore, neither Na+ nor Li+ stimulated the ATPase activity, and no ATP-driven Li+ transport was observed. The ATPase of the E. coli mutant MPC32N (cQ32N) was activated by NaCl and LiCl. The mutant ATPase exhibited a 5-fold higher Km for NaCl but no change in the Km for LiCl in comparison to that of the parent strain. These results demonstrate that the binding of Na+ to the c subunit of P. modestum requires liganding groups provided by Q32, E65, and S66. For the coordination of Li+, two liganding partners, E65 and S66, are sufficient, and H+ translocation was mediated by E65 alone.
[Show abstract][Hide abstract] ABSTRACT: Expression studies of Propionigenium modestum ATPase genes in various combinations with Escherichia coli ATPase genes were performed in the unc deletion mutant strain E. coli DK8. Plasmids containing the whole unc operon from P. modestum were unable to complement the E. coli unc deletion mutant. Although all ATPase subunits were expressed from the plasmids, there was no detectable ATP hydrolysing activity, indicating that the F1 part was not functional. Transformants expressing an E. coli F1-P. modestum F0 hybrid exhibited considerable ATPase activities. Binding of the F1 part to the membrane was very weak, however, and the coupling between ATP hydrolysis and Na+ transport was impaired. After combining the genes for E. coli ATPase subunits alpha, beta, gamma, delta and epsilon and the hydrophilic part of subunit b with P. modestum ATPase subunits a and c and the hydrophobic part of subunit b on a plasmid, a non-functional hybrid ATPase was expressed in E. coli. The ATPase was only loosely bound to the membrane, from which it was solubilized with Triton X-100 and purified. Subunit b and a proteolytic degradation product were the only F0 subunits detectable in the purified enzyme. A stable F0 complex is thus not formed with the hybrid b subunit. The absence of a functional F0 complex was in accord with proton-conduction measurements with bacterial vesicles. The only functional Na(+)-translocating ATPase expressed in E. coli thus far consists of E. coli subunits alpha, beta, gamma and epsilon, and P. modestum subunits delta, a, b and c [Kaim, G. & Dimroth, P. (1993) Eur. J. Biochem. 218, 937-944]. During the cloning conducted in our present study, errors in the sequence entry into the EMBL data bank (accession no. X58461) for the P. modestum ATPase alpha and beta subunits became evident, which are corrected in this paper.
Preview · Article · Sep 1995 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to construct functional hybrid ATPases consisting of all Escherichia coli ATPase subunits excepts the F0 subunits a or c which were replaced by the respective subunits of the Propionigenium modestum ATPase. This would give valuable information on the subunit(s) conferring the coupling ion specificity. Plasmids were constructed that carried the gene for subunit c (uncE) or subunit a (uncB) behind a tac promoter. These plasmids were transformed into E. coli strains which differed with respect to the unc operon and the expression of the P. modestum genes was verified biochemically. Enhanced expression of the P. modestum genes led to strong growth inhibition of all E. coli strains tested. However, the expressed P. modestum proteins could not functionally complement E. coli strains that lacked the homologous subunit.
No preview · Article · Feb 1994 · Archives of Microbiology
[Show abstract][Hide abstract] ABSTRACT: Expression of the iap gene of Listeria monocytogenes in the L. monocytogenes rough mutant RIII and in Bacillus subtilis DB104 caused the disruption of the cell chains which these two strains normally form under exponential growth conditions. The p60 protein produced by L. monocytogenes and B. subtilis DB104 also exhibited bacteriolytic activity detected in denaturing polyacrylamide gels containing heat-killed Micrococcus lysodeikticus. Purification of the p60 protein led to aggregation of p60 and loss of the cell chain disruption and bacteriolytic activities. A cysteine residue in the C-terminal part of p60 which is conserved in all p60-like proteins from the other Listeria species seems to be essential for both activities. The iap gene could not be inactivated without a loss of cell viability, indicating that p60 is an essential housekeeping protein for L. monocytogenes and probably also for other Listeria species. These data suggest that p60 possesses a murein hydrolase activity required for a late step in cell division.
Full-text · Article · Jul 1993 · Journal of Bacteriology
[Show abstract][Hide abstract] ABSTRACT: We report here the N-terminal protein sequences of the subunits of the ATPase from Propionigenium modestum. Subunits c, b, delta, alpha and beta start with an N-terminal methionine residue, the gamma and epsilon subunits have an alanine N-terminus, from which N-formylmethionine was hydrolyzed by posttranslational modification, and subunit a contains a blocked N-terminus. Each of the N-terminal sequences exactly matches a portion of the DNA sequence in the gene encoding the respective subunit protein on the unc operon. Thus, the exact translational start for each subunit protein can be identified and the primary structures of the protein transcripts can be clearly defined. Based on these data the putative size of the open reading frame that was envisaged from the DNA sequence had to be revised for the alpha and delta subunits.