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Mycoplasma hyopneumoniae p65 Surface Lipoprotein Is a Lipolytic Enzyme with a Preference for Shorter-Chain Fatty Acids. Jono A. Schmidt, 2004.Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine . p65 is an immunodominant surface lipoprotein of M . hyopneumoniae that is specifically recognized during disease . Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes . To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons . After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein . The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP) . Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester . Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39°C . Calcium ions did not increase the activity of recombinant GST-p65 . Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M . hyopneumoniae in vitro . Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC . The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases . This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma . Expression of the Pseudomonas putida OCT Plasmid Alkane Degradation Pathway Is Modulated by Two Different Global Control Signals: Evidence from Continuous Cultures. M. Alejandro Dinamarca, 2003.Expression of the genes of the alkane degradation pathway encoded in the Pseudomonas putida OCT plasmid are subject to negative and dominant global control depending on the carbon source used and on the physiological status of the cell . We investigated the signals responsible for this control in chemostat cultures under conditions of nutrient or oxygen limitation . Our results show that this global control is not related to the growth rate and responds to two different signals . One signal is the concentration of the carbon source that generates the repressing effect (true catabolite repression control) . The second signal is influenced by the level of expression of the cytochome o ubiquinol oxidase, which in turn depends on factors such as oxygen availability or the carbon source used . Since under carbon limitation conditions the first signal is relieved but the second signal is not, we propose that modulation mediated by the cytochrome o ubiquinol oxidase is not classical catabolite repression control but rather a more general physiological control mechanism . The two signals have an additive, but independent, effect, inhibiting induction of the alkane degradation pathway . Linkage of High Rates of Sulfate Reduction in Yellowstone Hot Springs to Unique Sequence Types in the Dissimilatory Sulfate Respiration Pathway. Susan Fishbain, 2003.Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment . Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89°C . Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture .
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