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Abstract
BackgroundThe aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates.ResultsMCRA catalysed the conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but shorter chain fatty acids were not used as substrates, while the presence of trans-double bonds and double bonds beyond the position C12 abolished hydratase activity. The hydroxy fatty acids produced were not metabolised further. We also found that heterologous Lactococcus and Corynebacterium expressing MCRA accumulated increasing amounts of 10-HOA and 10-HOE in the culture medium. Furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls.ConclusionsMCRA protein in B. breve can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, which may be catalysing the first step in conjugated linoleic acid (CLA) production, and this protein has an additional function in bacterial stress protection.
Highlights
The aim of this study was to determine the catalytic activity and physiological role of myosin-crossreactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258
We have previously reported the ability of a range of Bifidobacterium spp. to produce the c9,t11 conjugated linoleic acid (CLA) isomer from free linoleic acid [15,16,17] and have shown that administration of CLA producing B. breve NCIMB 702258 to mice and pigs in combination with linoleic acid alters host tissue fatty acid composition [18], including elevated liver c9,t11 CLA, coupled with reductions in the proinflammatory cytokines tumour necrosis factor-a (TNF-a) and interferon-g (IFN-g)
The aims of this study were 1) to investigate if the MCRA protein from B. breve NCIMB 702258 was active as a fatty acid hydratase, by introducing the gene encoding MCRA into Corynebacterium glutamicum and Lactococcus lactis and analysis of the enzymatic activity of the expressed protein in the heterologous hosts and 2) to investigate whether this led to increased stress tolerance in the new hosts, and we report a role for MCRA in stress protection in B. breve
Summary
The aim of this study was to determine the catalytic activity and physiological role of myosin-crossreactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. It has been reported that some members of the MCRA-like proteins have fatty acid hydratase activity [1,2]. The expression of the mcra gene was found to be upregulated by the stress responsive alternative transcription factor sB in Staphylococcus aureus [3]. It could be gene was abrogated in a rgg mutant strain of Streptococcus pyogenes causing the mutant strain to synthesize the protein during both the exponential and stationary phases of growth, in contrast to the wild-type strain which only synthesized the protein in the exponential phase of growth. This regulon has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance, and genes involved in osmotic stress response (opuD (N315SA1987, encoding the glycine betaine transporter), proP (N315-SA0531, encoding a proline/betaine transport homolog) and gbsA (SA2406, encoding glycine betaine aldehyde dehydrogenase)) [9]
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