Isolation and characterization of an acyl -lipid desaturase mutant of Bacillus cereus ATCC 14579

CHAZARRETA-CIFRE, LORENA; DIEGO DE MENDOZA; ALTABE, SILVIA

Rosario

Congreso; IX-SAMIGE; 2013

Sociedad Argentina de Microbiologia General

Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrheal type of food-associated illness that is becoming increasingly important in the industrialized world. B. cereus Group are Gram-positive spore-forming, facultative anaerobic bacteria that have the ability to grow at temperatures between 4ºC and 50°C depending on the strain. Temperature is one of the most important environmental factor to which microorganism have to respond. Cold adaptation requires several changes in bacterial cells, in particular, membrane modifications, mainly in the fatty acyl moieties. These modifications are known to decrease the melting point of fatty acids and to improve bacterial adaptation to lower growth temperatures. The best characterized of these adjustments is the biosynthesis of unsaturated fatty acids (UFAs) that is carried out by fatty acid desaturases. A fatty acid desaturase is a special type of oxygenase that can remove two hydrogens from a fatty acyl chain, catalyzing the formation of a double bond in the substrate. Desaturases use activated molecular oxygen and two reducing equivalents for catalysis. In previous work we have described that B. cereus ATCC14579 has two acyl lipid desaturases, BC2983 and BC0400 that are involved in the synthesis of UFAs with double bonds in D5 and D10 positions, respectively. In this work we report the isolation and characterization of B. cereus D5-Des, D10-Des and D5,10-Des mutants, named LSC2983, LSC0400 and LSC2904, respectively. The construction of these mutant strains was carried out by a simple and efficient method that uses a temperature-sensitive vector that carries a selectable marker. The impact of these mutations on FA composition analyzed by GC-MS showed that mutant strain LSC2983 did not synthesize D5-UFAs when grown in minimal medium. This mutant grows as wild type strain in minimal medium at low temperature showing that D5-UFAs synthesis is not essential for B. cereus growth indicating that an additional mechanism is also involved in cold adaptation. On the other hand, B. cereus LSC0400 did not synthesize D10-UFAs and strain LSC2904 did not have detectable levels of UFAs as determined by GC-MS. Furthermore, these two mutant strains were unable to growth in minimal medium supplemented with amino acids and the defect could be overcome by adding oleic acid to the media. Therefore our experiments demonstrate that D10-Des is essential for B. cereus growth in minimal medium and provide the basis for developing new antibacterial strategies. Código de Resumen: FM-004 Sección: Fisiología Microbiana