Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases

Abstract

At low temperatures, Bacillus cereus synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions ⌬5 and ⌬10, as well as ⌬5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative ⌬5 desaturase and a fatty acid acyl-lipid desaturase in the B. cereus ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in Bacillus subtilis confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have ⌬5 and ⌬10 desaturase activities. Thus, these ORFs were correspondingly named desA (⌬5 desaturase) and desB (⌬10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when B. subtilis expressing B. cereus desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from B. cereus. Bacillus cereus is widespread in nature, being frequently isolated from soil and growing plants. This microorganism is also well adapted to growth in the intestinal tract of insects and mammals. From these habitats, it easily spreads to food, where it may cause an emetic or a diarrheal type of food-associated illness that is becoming increasingly important in the industrialized world. The B. cereus group is constituted by Gram-positive, spore-forming, facultative anaerobic bacteria that have the ability to grow at temperatures between 4°C and 50°C, depending on the strain (1). Temperature is one of the most important environmental factors to which microorganisms have to respond. Cold adaptation in bacteria requires several changes in cellular components, in particular, membrane modifications, mainly in the fatty acyl moieties (2). These modifications are known to decrease the melting point of fatty acids (FAs) and to improve bacterial adaptation to lower growth temperatures (3). Of these adjustments, the best characterized is the biosynthesis of unsaturated fatty acids (UFAs), which is carried out by FA desaturases, 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 (4-6).The biosynthesis of UFAs in bacilli was extensively investigated by Fulco (7), who described that most species have a negligible amount of UFAs in their membrane lipids. In some Bacillus species that synthesize ⌬5 UFAs, such as Bacillus megaterium and Bacillus subtilis, the proportion of UFAs has been observed to dramatically increase when the bacteria are grown at low temperatures (7). ...

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth

“…The genes encoding the two desaturases DesA and DesB were also downregulated in the ΔcasK/R strain compared to the WT strain. These B. cereus desaturases are responsible for UFA biosynthesis (Chazarreta Cifre et al, 2013).…”

“…Basically, the proportion of Δ5-UFAs, which are specifically unsaturated by DesA, was higher in the WT strain than in the ΔcasK/R strain. B. cereus DesA is a Δ5 desaturase homologous to B. subtilis Des protein (Chazarreta Cifre et al, 2013) that catalyzes the fixation of double bonds in the Δ5 position on FA. An extensive investigation of the biosynthesis of UFAs in Bacilli (Fulco, 1969) showed that for some Bacillus species that synthesize Δ5 UFAs, such as B. megaterium and B. subtilis, proportion of Δ5-UFAs increases when the bacteria are grown at low temperatures.…”

“…In B. subtilis, the Des protein was found to be responsible for maintaining membrane fluidity during low-temperature growth (Cybulski et al, 2002). In B. cereus, two desaturases DesA and DesB were described (Chazarreta Cifre et al, 2013), but no mechanism similar to B. subtilis DesK/R has yet been described for members of the B. cereus group.…”

Fatty acid profiles and desaturase-encoding genes are different in thermo- and psychrotolerant strains of the Bacillus cereus Group

“…The number of desaturases varies depending on species, e.g. two desaturases have been identified in Bacillus cereus stricto or ss [ 10 ] whereas B. subtilis counts only one known desaturase [ 11 ]. The FA composition of bacterial cells is also known to vary according to species, especially in Bacillus and related genera [ 12 , 13 ], and has been included among important features for describing new taxa of the aerobic endospore-forming bacteria [ 14 ].…”

Xanthomonas campestris FabH is required for branched-chain fatty acid and DSF-family quorum sensing signal biosynthesis

“…Using similar pathways1212, many Gram-positive organisms, such as the bacillus, staphylococci, and streptomycetes, are able to produce BCFAs with odd- and even-numbered carbon chains13141516. However, the substrate specificity of 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) in Gram-positive bacteria is distinct from that in E. coli .…”