Campylobacter spp. are among the most commonly reported bacterial causes of acute diarrheal disease in humans worldwide. Potential virulence factors include motility, chemotaxis, colonization ability, adhesion to intestinal cells, invasion and epithelial translocation, intracellular survival, and formation of toxins. Probiotic Lactobacillus and Bifidobacterium strains are known to have an inhibitory effect against the growth of various foodborne pathogens. The objective of this study was to investigate the effect of Lactobacillus acidophilus strain La-5 and Bifidobacterium longum strain NCC2705 cell-free spent media (CFSM) on the expression of virulence genes (cadF, cdtB, flaA, and ciaB) of Campylobacter jejuni strain 81-176 and a luxS mutant, using real-time PCR. Our results demonstrated that the CFSM of both probiotic strains were able to down-regulate the expression of ciaB (ratio of -2.80 and -5.51, respectively) and flaA (ratio of -7.00 and -5.13, respectively) in the wild-type Campylobacter strain. In the luxS mutant, where the activated methyl cycle is disrupted, only the ciaB gene (ratio -7.21) was repressed in the presence of La-5 CFSM. A supplementation of homocysteine to restore the disrupted cycle was able to partially reestablish the probiotic effect of both strains. luxS and the activated methyl cycle might play an active role in the modulation of virulence of C. jejuni by probiotic extracts.

Aim:  To isolate eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) genes from Shewanella baltica MAC1 and to examine recombinant production of EPA and DHA in E. coli to investigate cost‐effective, sustainable and convenient alternative sources for fish oils. Methods and Results:  A fosmid library was prepared from the genomic DNA of S. baltica MAC1 and was screened for EPA and DHA genes by colony hybridization using a partial fragment of the S. baltica MAC1 pfaA and pfaD genes as probes. Analysis of total fatty acids isolated from transgenic E. coli positive for pfaA and pfaD genes by gas chromatography and gas chromatography‐mass spectrometry indicated recombinant production of both EPA and DHA. Analysis of the complete nucleotide sequence for the isolated gene cluster showed 16 putative open reading frames (ORFs). Among those, four ORFs showed homology with pfaA, pfaB, pfaC and pfaD genes of the EPA and/or DHA biosynthesis gene clusters; however, the protein domains of these genes were different from other EPA/DHA biosynthesis genes. Conclusions:  The EPA and DHA gene cluster was cloned successfully. The transgenic E. coli strain carrying the omega‐3 gene cluster was able to produce both EPA and DHA. The isolated gene cluster contained all the genes required for the recombinant production of both EPA and DHA in E. coli. Significance and Impact of the Study:  These findings have implications for any future use of the EPA and DHA gene cluster in other micro‐organisms, notably those being used for fermentation. Recombinant production of both EPA and DHA by E. coli or any other micro‐organism has great potential to add economic value to a variety of industrial and agricultural products.

Transposon Tn5 mutagenesis was used to generate random mutations in Shewanella baltica MAC1, a polyunsaturated fatty acid (PUFA)-producing bacterium. Three mutants produced 3-5 times more eicosapentaenoic acid (EPA 20:5 n-3) compared to the wild type at 10 degrees C. One of the mutants produced 0.3 mg EPA g(-1) when grown at high temperature (30 degrees C). Moreover, 2 mg docosahexaenoic acid (DHA 22:6 n-3) g(-1) was produced by S. baltica mutants at 4 degrees C. Sequencing of insertion mutation(s) showed 96% homology to trimethylamine N-oxide (TMAO) reductase gene and 85% homology to rRNA operons of E. coli. Tn5 transposon mutagenesis therefore is a suitable technique to increase PUFA formation in bacteria.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35 ± 0.5 mg g(-1) cell dry weight) and EPA (0.12 ± 0.04 mg g(-1) cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.

Omega-3 fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on human health. The probiotic bacterium Escherichia coli Nissle is unable to produce either EPA or DHA. Escherichia coli Nissle was transformed with the pfBS-PS plasmid carrying the EPA/DHA gene cluster, previously isolated from a marine bacterium. The transgenic E. coli Nissle produced EPA when grown at 10ºC (16.52 ± 1.4 mg g(-1) cell dry weight), 15ºC (31.36 ± 0.25 mg g(-1) cell dry weight), 20ºC (13.71 ± 2.8 mg g(-1) cell dry weight), 25ºC (11.33 ± 0.44 mg g(-1) cell dry weight) or 30ºC (0.668 ± 0.073 mg g(-1) cell dry weight). Although DHA was also produced at all these temperatures, it comprised less than 0.2% of total extracted fatty acids. Transcriptomic analysis using Reverse Transcription qPCR showed upregulation of the entire gene cluster in E. coli Nissle. Among EPA/DHA genes, pfaB, pfaC and pfaD were overexpressed (expression ratio of 181.9, 39.86 and 131.61, respectively) as compared to pfaA (expression ratio of 3.40) and pfaE (expression ratio of 4.05). The EPA/DHA-producing probiotic E. coli Nissle may be used as a safe, alternative and economic source for the industrial and pharmaceutical production of EPA and DHA.