BACKGROUND
the streptococcal  <dig> kda mcra-like proteins represent a family of proteins which are present in a wide range of bacteria.
it has been reported that some members of the mcra-like proteins have fatty acid hydratase activity  <cit> .
recently, the mcra protein from streptococcus pyogenes m <dig> was reported to be a fad-containing enzyme, which acts as hydratase on cis- <dig>  - and trans- <dig> -double bonds of c- <dig>  c- <dig> non-esterified fatty acids producing 10-hydroxy and  <dig> -dihydroxy fatty acids  <cit> .
the expression of the mcra gene was found to be up-regulated by the stress responsive alternative transcription factor σb in staphylococcus aureus  <cit> .
it could be speculated that in vivo conditions such as high temperature at the site of infection, leading to stress in s. aureus may induce the stress responsive σb transcription factor  <cit> .
it is a general belief that the mcra protein is involved in virulence in staphylococcus and streptococcus  <cit> .
a  <dig> kda streptococcal mcra protein was up-regulated in group a streptococcus during experimental mouse soft tissue infection  <cit>  and in the asymptomatic phase of infection in cynomolgus macaques monkeys infected with group a streptococcus  <cit> .
however, a mcra deletion mutant in streptococcus pyogenes showed only mild alteration in virulence properties, such as blood survival, adherence and internalization to human keratinocytes  <cit> .
dunman et al.
found the mcra gene was down-regulated by agr, one of the most studied virulence response regulators in staphylococcus aureus, along with other cell surface virulence factors  <cit> .
growth-phase associated regulation of the mcra 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.
the transcriptional regulatory protein rgg coordinates amino acid catabolism and virulence factor expression in streptococcus pyogenes  <cit> .
also, the mcra gene was up-regulated by the mgra regulon in stapylococcus aureus.
this regulon has been shown to affect multiple staphylococcus aureus genes involved in virulence and antibiotic resistance, but also genes involved in osmotic stress response , prop  and gbsa )  <cit> .
the mcra protein shows over 50% homology with linoleate isomerases from lactobacillus acidophilus  and lb.
reuteri pyr <dig> linoleate isomerase  <cit> , a protein that catalyses the formation of -cla from linoleic acid.
cla refers to several positional  and geometric  isomers of linoleic acid c18: <dig> cis- <dig> , cis- <dig>  octadecadienoic acid] with conjugated double bonds, and the  and  isomers in particular, have been shown to exert beneficial effects on human metabolism, including anti- carcinogenic, anti-diabetogenic and anti-atherogenic activities and body composition alteration effects.
cla is formed via biotransformation of linoleic acid, as a direct result of the action of the ruminal microbiota, of which butyrivibrio fibrisolvens is the foremost  <cit> .
the mechanism of microbial production of cla was characterised using washed cells of the strain lb.
acidophilus aku  <dig>  <cit>  and involves the production of hydroxy fatty acids as precursor to formation of cla.
when isolated and introduced to the washed cells, these hydroxy fatty acids were rapidly transformed to their respective cla isomers.
thus, cla formation by lb.
acidophilus was found to consist of two distinct steps, step one: the hydration of linoleic acid to 10-hydroxy-18: <dig> and step two: the subsequent dehydration and isomerisation of these hydroxy fatty acids to the c <dig> t <dig> cla and t <dig> t <dig> cla isomers.
we have previously reported the ability of a range of bifidobacterium spp.
to produce the c <dig> t <dig> cla isomer from free linoleic acid  <cit>  and have shown that administration of cla producing b. breve ncimb  <dig> to mice and pigs in combination with linoleic acid alters host tissue fatty acid composition  <cit> , including elevated liver c <dig> t <dig> cla, coupled with reductions in the proinflammatory cytokines tumour necrosis factor-α  and interferon-γ .
the enzyme responsible for cla production in bifidobacterium has not been reported.
the aims of this study were 1) to investigate if the mcra protein from b. breve ncimb  <dig> 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 thus we report a role for mcra in stress protection in b. breve.
methods
cultures and media
b. breve ncimb  <dig> was cultured in mrs media  supplemented with  <dig> %  l-cysteine hydrochloride  under anaerobic conditions using anaerobic jars with 'anaerocult a' gas packs  at 37°c.
lactococcus lactis nz <dig>  was cultured at 30°c in m <dig>  broth and/or agar containing glucose .
l. lactis carrying the plasmid pnz <dig> was routinely cultured in the presence of chloramphenicol  as a selective marker.
corynebacterium glutamicum atcc  <dig> was cultured aerobically in bhi media  at 37°c.
c. glutamicum atcc  <dig> carrying the shuttle vector pclik 5a mcs pddh  was routinely cultured in the presence of kanamycin  as a selective marker.
escherichia coli jm <dig> carrying the plasmid poxo <dig> was routinely cultured aerobically in bhi at 37°c in the presence of chloramphenicol  as a selective marker.
dna sequence analysis and primer design
the sequence encoding the  <dig> kda mcra  protein from lb.
reuteri pyr <dig>  <cit>  was compared to sequence databases , using the blast suite of programs  <cit> .
proteins exhibiting significant similarity were aligned using dnastar software  and conserved motifs were identified.
degenerate oligonucleotide primers and primers specific for bifidobacteria  were designed to these motifs  and used in pcr reactions to amplify a ~ <dig> kb internal region of the sequence as follows: primer ev1a forward and ev2a reverse general, degenerate): 5'- ta,tgg,iii,aci,atg,tt,gci,tt,ga-3' and 5'- ga,ga,tc,igt,igt,aa,iac,igt,tc 3', ev1b forward and ev2b reverse : 5'-tac,tgg,iii,acc,atg,ttc,gci,ttc,gaa-3' and 5'- ga,gta,ttc,gt,gt,gaa,gac,gt,tc 3'.
pcr reactions were performed in a hybaid pcr express unit  in  <dig> μl with an annealing temperature of 45°c.
each reaction contained  <dig> μl of each primer ,  <dig> μl of template in  <dig> μl mgcl <dig> ,  <dig> μl dntp master mix ,  <dig> μl 10x nh <dig> reaction buffer and  <dig>  μl biotaq dna polymerase  .
the resulting ~ <dig> kb fragment was cloned into pcr  <dig> - topo and sequenced .
chromosome walking by inverse pcr
to obtain flanking chromosomal sequence, two primers were designed to terminal regions of the known chromosomal dna sequence .
the genomic dna from b. breve ncimb  <dig> was digested with a range of restriction enzymes followed by ligation with dna ligase as described by the supplier .
the circularized fragments were subsequently used as templates in the inverse pcr reactions with the terminal primers.
the reactions were performed in the same way as the standard pcr reactions but with an annealing temperature of 50°c and the resulting fragments  were cloned into the pcr <dig> -topovector and sequenced.
the two terminal primers used were primer a : 3'-cgttctcgaccttggtgttgtatcggaatt 5' and primer b : 5' -gtaccgaccgacaagatcgagtcgcttgcc-3'.
from the approach described above, it was confirmed by sequencing that the 3' end of the gene was obtained from re-circularized xbai digests; however the 5' end was not yet acquired.
a second round of inverse pcr was performed with primers a   and pcs <dig>  <dig>   targeted to re-circularized kpni digests of genomic dna, which provided another  <dig> bp of the upstream region of the gene.
the last round of inverse pcr to obtain the missing  <dig> bp from the 5' end of the gene was carried out with primers er <dig>  and er <dig>  targeted to circularized haeii digests.
dna manipulations and plasmid construction
genomic dna was isolated from b. breve ncimb  <dig> by a modified method described by hoffmann et al.
<cit> .
the  <dig> mcra gene from b. breve ncimb  <dig>  was amplified from genomic dna with primers emy <dig> and emy <dig> .
pcr was performed with high fidelity expand polymerase as described by the supplier  using  <dig> ng genomic dna as a template.
pcr conditions were as follows:  <dig> cycles of  <dig> min,  <dig> s denaturation ,  <dig> s annealing ,  <dig> min elongation  followed by  <dig> cycles of  <dig> s ,  <dig> s ,  <dig> min +  <dig> s/cycle  and finally, one  <dig> min cycle at 72°c.
the mcra gene was amplified with primers emy <dig> and emy <dig>  for cloning in pclik, resulting in the construct pemyc <dig> which was transformed into c. glutamicum.
recombinant plasmids were double digested with the same enzymes to verify the correct clone and electroporated into l. lactis nz <dig>  <dig> and c. glutamicum atcc  <dig>  electrocompetent l. lactis were prepared and transformed according to the method described by de ruyter et al.
<cit> , while electrocompetent c. glutamicum cells were prepared as described by luchansky et al.
<cit> .
for enzymatic assays, the recombinant b. breve mcra protein was produced in e. coli as n-terminal 6x his-tag fusion.
the complete mcra gene was amplified using primers bbi_fw <dig> and bbi_rv <dig> .
ligation with pet <dig> vector  resulted in a construct pbbi <dig> containing n-terminal fusion of 6x his tag and b. breve mcra protein.
the authenticity of the clones was verified by sequencing .
investigation of the stress tolerance of mcra protein-overproducing l. lactis and c. glutamicum
the mcra protein was expressed in lactococci using the nice system  <cit> , which allows maximal protein expression at sub-lethal concentrations of nisin, and is tightly controlled, such that there is negligible expression in the absence of nisin  <cit> .
the mcra protein was also expressed in c. glutamicum using the shuttle vector pclik 5a mcs pddh.
this vector replicates in both e. coli and c. glutamicum and contains a c. glutamicum promoter  and a kanamycin resistance gene which function in both e. coli and c. glutamicum.
l. lactis pnz <dig> and pemyl <dig> cells were induced with nisin  at od <dig> nm ~  <dig>  and cultures were left to grow for an additional  <dig>  h prior to heat treatment.
c. glutamicum pclik and pemyc <dig> were grown to od <dig> nm ~  <dig>  -  <dig> , prior to stress.
thermotolerance of l. lactis and c. glutamicum was monitored at 51°c in broth over 30- <dig> min.
aliquots were removed at  <dig>   <dig>   <dig>   <dig> and  <dig> min, plated on agar and incubated at 30°c  and 37°c  for  <dig> h. salt tolerance was determined following resuspension in broth containing  <dig> m nacl  and  <dig>  m nacl  and culture viability was monitored over 60- <dig> min with aliquots taken at  <dig> min intervals.
solvent tolerance was determined on addition of butanol 1-5% , and survival was monitored over  <dig> min with aliquots taken at  <dig> min intervals.
heterologous expression, protein extraction and sds-page
the putative linoleic acid isomerase was expressed in poxo <dig> in e. coli using the t <dig> system .
e. coli poxo <dig>  and e. coli pemye <dig> cultures were aerated vigorously until the od600nm reached approximately  <dig> , at which point isopropyl-β-d-thiogalactopyranoside  was added to final concentration of  <dig>  mm to induce expression of the t <dig> rna polymerase.
aliquots were taken at intervals after iptg induction at  <dig>   <dig>   <dig> and  <dig> h for whole cell protein extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis .
similarly, l. lactis pemyl <dig> and pnz <dig>  were cultured to od600nm ~ <dig>  and induced with nisin  and allowed to grow for another  <dig> h prior to protein extraction.
c. glutamicum pemyc <dig> and pclik  were cultured as described previously for  <dig> h prior to protein extraction.
to extract proteins, cells  were harvested and washed in  <dig> mm tris hcl, ph  <dig>  and resuspended in  <dig>  ml extraction solution ) and bead-beaten for  <dig> ×  <dig> sec with  <dig> min rest on ice.
the cell lysate was then centrifuged to remove beads and cell debris and the protein extracts analysed by sds-page using a mini protean ii cell unit  by the method of laemmli  <cit>  with a 10% acrylamide resolving gel and 4% stacking gel.
a low molecular weight standard ranging from  <dig>  to  <dig>  da  was used as a molecular weight marker.
production and purification of recombinant b. breve mcra protein from e. coli
for protein production, e. coli bl <dig> star strain  harboring pbbi <dig> plasmid was used.
bacteria were cultivated in 2xyt medium , yeast extract , nacl ) supplied with kanamycin  at 37°c until od <dig> reached  <dig> .
at that point, iptg was added to final concentration of  <dig>  mm, the cells were changed to 16°c and harvested by centrifugation  after  <dig> h induction time.
cells were resuspended in buffer a  in 1: <dig> v/v ratio, frozen in liquid nitrogen and stored at -20°c.
for protein purification, cells were lysed by adding lysozyme  and dnasei  in buffer a and  <dig> mm mgcl <dig> and centrifuged for  <dig> min at  <dig>  g. the supernatant was loaded on histrap column , the column was washed with  <dig> column volumes of buffer a, and the protein was eluted with  <dig> column volumes of buffer a and  <dig>  m imidazol.
after elution, the buffer was exchanged to buffer b  by ultrafiltration and protein concentration was estimated spectrophotometrically using calculated absorption coefficient ε <dig> =  <dig>
immunoblotting
<dig> μg of protein were subjected to sds page followed by protein transfer to nitrocellulose membrane at  <dig> v,  <dig> ma/cm <dig> gel surface for  <dig> h. the membrane was washed with buffer b and blocked for one hour in buffer b containing 5%  milk powder.
his-tagged proteins were detected using anti-his tag antibody  in combination with a secondary antibody coupled with alkaline phosphatase and nitrotetrazolium blue/5-bromo-4-chloro-3-indolylphosphate staining.
spectroscopic assays
solution of purified b. breve mcra protein had yellow colour, therefore for initial characterization uv-vis spectrum of purified protein in the range 300- <dig> nm was recorded with a single beam absorption spectrometer .
for the reference, spectra of fad and fmn in buffer b were recorded in the same way.
for these measurements, b. breve mcra protein, fad and fmn were brought within the concentration range 10- <dig> pm.
the absence of covalent bond between cofactor and b. breve mcra protein was confirmed in the following way;  <dig> pl of the protein  was boiled for  <dig> min at 100°c and centrifuged at  <dig>  g for  <dig> min.
the spectrum of the supernatant was then recorded as described above.
activity assay, lipid extraction and derivatization
to demonstrate the enzymatic activity of b. breve mcra protein, a panel of free fatty acids was tested in the reactions with the purified protein.
typically, 10- <dig> pg of substrate was mixed with  <dig> pg of purified protein in  <dig> ml of buffer b and incubated for  <dig> h at 25°c.
fatty acids were extracted from the reaction mixture with  <dig> ml chloroform:methanol  according to bligh and dyer  <cit> .
free fatty acids were dissolved in methanol and converted to corresponding methyl esters with -diazomethane.
for gc- ms analysis, fatty acid methyl esters were dissolved in acetonitrile and hydroxy groups were modified by n,o-bis--trifluoroacetamide , resulting in fatty acid trimethylsilylethers.
gc-ms analysis was performed as previously described  <cit> .
phylogenetic analysis
homologs to the previously identified linoleate isomerase from lb.
acidophilus  and oleate hydratase, elizabethkingia meningoseptica  were selected from blast analysis.
these homologs, along with mcra from b. breve ncimb  <dig> discussed in this manuscript were aligned using t-coffee  <cit>  and protein trees were built using the phylip package  <cit> .
the cladogram was then visualised using dendroscope  <cit> .
RESULTS
previous studies by rosson et al.
<cit>  reported on the isolation and purification of a putative linoleic acid isomerase from lb.
reuteri.
in these studies, protein sequencing had identified a  <dig> kda protein with homology to mcra.
based on this finding, we designed primers to conserved regions shared by a range of mcra proteins in the database  and found that we could pcr the gene from a range of different bifidobacterium strains and propionibacterium freudenreichii shermanii .
given that b. breve ncimb  <dig> is known to be capable of producing c <dig> t <dig> cla from linoleic acid  <cit> , we decided to study this strain in terms of cla production.
using primers ev1a forward and ev2a reverse, we amplified a  <dig> kb internal fragment of the putative isomerase gene from b. breve.
sequencing of this revealed up to 70% homology to mcra proteins from various genera.
following this, we primer walked in both 5'and 3' directions, which resulted in the full sequence of b. breve putative isomerase .
sequence analysis
the complete  <dig> bp nucleotide sequence from b. breve ncimb  <dig> encodes a protein of  <dig> amino acids with homology to other " <dig> kilodalton mcra" proteins.
the calculated molecular weight of the protein is  <dig>  da.
comparison with sequences in the database revealed that the cloned mcra protein showed 71% homology with putative  <dig> kda mcra from streptococcus mutans ua <dig>  and 51% homology with lb.
acidophilus linoleate isomerase .
a non-canonical flavin binding motif 21e) is present at the n-terminus of b. breve mcra protein, similar to streptococcus and lactobacillus mcra proteins  <cit> .
the protein is cytoplasmic, but may have one trans-membrane helix as predicted by psortb .
upstream of the orf is a putative dehydrogenase or reductase protein with ~50% homology with b. longum ncc <dig>  <dig> -diketo-d-gluconic acid reductase .
downstream of the tga stop codon is a region of dyad symmetry, capable of forming a stable stem-and-loop structure.
the potential hairpin consists of 13-bp stem separated by a 3-bp mis-priming and a 5- bp loop .
over-expression of b. breve mcra protein in e. coli
since no over-expression of the putative linoleic acid isomerase could be confirmed following sds-page of the recombinant lactococci and corynebacteria, possibly due to masking of the protein by other proteins of similar molecular weight , the putative lai  gene was expressed in poxo <dig> in e. coli using the t <dig> system .
following growth and induction at od600nm  <dig>  with  <dig>  mm iptg, aliquots of e. coli poxo <dig>  and e. coli pemye <dig> were taken at  <dig>   <dig>   <dig> and  <dig> h after induction for whole cell protein extraction and sds-page.
a protein band of approximately  <dig> kda was visualized, corresponding to the expected molecular weight of the putative isomerase protein, which was absent in the iptg induced control culture e. coli poxo <dig> .
following incubation for 48- <dig> h with free linoleic acid , the substrate for bacterial conversion to c <dig> t <dig> cla, no isomerase activity could be confirmed among the heterologous clones carrying the lai gene in the bacterial supernatant by gc , or by measuring od <dig> nm  <cit> .
production of b. breve mcra in e. coli.
and enzymatic assays
b. breve mcra protein was overproduced in e. coli and purified on ni2+-nta column .
the major band with apparent molecular weight of  <dig> kda was visualized on sds page after the elution.
immunoblotting with anti-his tag antibodies confirmed that this band contained 6x his tag .
purified b. breve mcra protein has flavin-like uv-vis absorption spectrum with maxima around  <dig> and  <dig> nm  and the flavin cofactor is released in the solution upon protein denaturation by heat.
the spectral properties of b. breve mcra are consistent with a recombinant mcra protein from s. pyogenes, which was found to contain a non-covalently bound fad and exhibited double bond hydratase activity producing 10- and 13-hydroxy fatty acids  <cit> .
these data prompted us to examine the enzymatic activity of b. breve mcra using common c <dig> and c <dig> fatty acids as substrates.
to this end, purified protein was incubated with free fatty acids and the reaction products were analyzed by gc-ms. indeed, we observed conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids , but myristoleic acid or shorter monounsaturated fatty acids were not substrates.
the presence of trans-double bonds as well as the double bonds beyond the position c <dig> abolished hydratase activity.
no conversion of esterified fatty acids such as methyl linoleate or cla isomers was observed.
phylogenetic analysis
stress tolerance of mcra-overproducing lactococcus and corynebacterium
to determine if heterologous lactococcus and corynebacterium containing the mcra gene exhibited improved stress tolerance, the heterologous cultures along with vector controls of lactococcus  and corynebacterium  were assayed for hydratase activity by measuring 9-hydroxy fatty acids without and with linoleic acid supplementation of the medium.
indeed, those cultures that expressed mcra/hydratase accumulated increasing amounts of 10-hoa and 10-hoe in the culture medium .
next, the heterologous cultures were exposed to heat , salt  and solvent  butanol × 45- <dig> min) stresses.
l. lactis pnz <dig> reduced in viability by  <dig> log cfu/ml following heat stress at 51°c for  <dig> min, whereas l. lactis pemyl <dig> only reduced in viability by  <dig> log cfu/ml .
the difference in survivability between c. glutamicum pclik and pemyc <dig> was ~ <dig>  log cfu/ml when heat stressed at 51°c for  <dig> min, a decline in survivability by < <dig>  log cfu/ml for l. lactis pemyl <dig> and < <dig> log cfu/ml for c. glutamicum pclik .
similarly, when induced l. lactis pnz <dig> was exposed to solvent stress  butanol), a decrease in viability of > <dig>  log cfu/ml was observed, whereas induced l. lactis pemyl <dig> only decreased ~ <dig>  log cfu/ml following  <dig> min in 3%  butanol, i.e.
a difference of nearly  <dig> log cfu/ml between the cultures under identical conditions.
c. glutamicum pemyc <dig> exposed to 3%  butanol stress for  <dig> min survived ~  <dig>  log cfu/ml better than the vector control  .
upon confirming that the clones containing the mcra protein were less sensitive to heat and solvent stresses, we examined the hypothesis that the protein also protected the cultures from salt stress.
following growth and induction by nisin  and additional growth for  <dig>  h, l. lactis pnz <dig>  and pemyl <dig> cultures were exposed to  <dig> m nacl for  <dig> min.
however, no difference in salt tolerance was observed at concentrations of  <dig> m nacl.
a similar observation was made for c. glutamicum pclik and pemyc <dig> which were grown in  <dig>  m nacl for  <dig> min.
both c. glutamicum control  and pemyc <dig> cultures survived in  <dig>  m nacl for  <dig> min with no apparent decline in viable numbers .
discussion
several recent reports have linked mcra and oleic acid hydratase activity  <cit> , which was first observed more than forty years ago in pseudomonas sp.
<cit> .
however, the significance of this hydratase activity for the bacterial host is not yet clearly established.
our study identified mcra from b. breve as a fad-containing hydratase with an additional role in stress protection .
interestingly, a recent study by o'flaherty and klaenhammer  indicated a role for mcra in stress tolerance, cell wall division and adherence to caco- <dig> cells  <cit> .
furthermore, all three characterized enzymes from this family are highly specific for free fatty acids as the previously characterized linoleic acid isomerase from p. acnes  <cit> .
thus, both enzyme activities seem to form a new class of fad-dependent fatty acid modifying enzymes.
this finding may be even further supported by phylogenetic groupings .
given the wide distribution of the mcra gene between human pathogenic and commensal bacteria, it could be speculated that it is not directly linked to virulence but to a general stress protection, which could also be useful for a pathogen i.e.
increased heat at the site of infection.
our study confirmed the increased viability of heterologous lactococcus and corynebacterium transformed with the mcra gene during heat and solvent stresses as a difference in viability of  <dig> log cfu/ml following heat stress at 51°c for  <dig> min and 3%  butanol stress for  <dig> min was observed between l. lactis pemyl <dig> and pnz <dig> .
likewise, the introduced mcra improved the survivability of c. glutamicum by ~ <dig>  log cfu/ml difference between c. glutamicum pclik and pemyl <dig> following heat stress at 51°c for  <dig> min, and ~ <dig>  log cfu/ml following 3%  butanol stress for  <dig> min .
the same host, l. lactis nz <dig> has previously been used for comparative studies of stress performance following transformation with the well known heat shock protein groesl in plasmid pnz <dig>  desmond et al.
<cit>  compared the viability of induced  and heat treated cells at 54°c for  <dig> min of the same host l. lactis nz <dig> transformed with either pnz <dig> or pnz8048-groesl.
at that temperature, both l. lactis pnz <dig> and un-induced l. lactis pnz8048-groesl  reduced in viability by  <dig>  log cfu/ml, whereas the induced l. lactis  declined by  <dig>  log cfu/ml during the heat stress.
furthermore, induced groesl-overproducing l. lactis  was able to grow and increase by  <dig>  log cfu/ml during  <dig> h exposure to  <dig> %  butanol, compared with l. lactis  and un-induced l. lactis  which declined in viability after  <dig> h , a trend that continued over the  <dig> h period of solvent challenge.
it has been reported that the response of cells to sublethal heat and ethanol exposure induce essentially identical stress responses because of the alteration in the membrane fluidity by these stresses  <cit> .
heat and ethanol stresses cause similar changes to plasma membrane protein composition, reducing the levels of plasma membrane h+- atpase protein and inducing the plasma membrane-associated hsp <dig>  both stresses also stimulate the activity of the fraction of h+-atpase remaining in the plasma membrane.
the resulting enhancement to catalysed proton efflux from the cell represents a considerable energy demand, yet may help to counteract the adverse effects for homeostasis of the increased membrane permeability that results from stress.
CONCLUSIONS
this study has shown that the mcra protein in bifidobacteria is a fad containing hydratase enzyme, which may be catalysing the first step in cla production in this species, and has an additional function in bacterial stress protection.
abbreviations
<dig> -dihoa:  <dig> -dihydroxy octadecanoic acid; fad: flavin adenine dinucleotide; fmn: flavin adenine mononucleotide; hepes: 4--  <dig> -piperazine ethane-sulfonic acid; 10-hoa: 10-hydroxy octadecanoic acid; 10-hoe: -10- hydroxy-12-octadecenoic acid; hplc: high pressure liquid chromatography; mcra: myosin cross-reactive antigen; ms: mass-spectroscopy; pai: propionibacterium acnes isomerase; tris: 2-amino-2-propane-  <dig>  3-diol,
authors' contributions
er-c: collection and analysis of data, writing of manuscript, al: collection and analysis of data, cg: collection and analysis of data rpr: design of experiments, editing of manuscript, oos: collection and analysis of data, sfff: design of experiments, gf: design of experiments if: design of experiments, analysis of data and writing of manuscript, and cs: design of experiments, data analysis and editing of manuscript.
all authors have read and approved the final manuscript.
supplementary material
additional file 1
map of putative isomerase gene.
map of putative isomerase gene and chromosome walking strategy  used to obtain the full sequence.
click here for file
additional file 2
mcra gene sequence data from bifidobacterium strains and propionibacterium freudenreichii shermanii.
<dig> kb pcr fragments following pcr of genomic dna from a range of bifidobacterial strains and propionibacterium freudenreichii shermanii  <dig> using oligonucleotide primers ev1a forward and ev2a reverse.
lanes 1- <dig> in the following order; b. adolescentis; b. breve 2257; b. breve 702258; b. breve 8815; b. dentium 2243; b. infantis 2205; b. lactis bb 12; b. longum 2259; b. longum bb 536; bifidobacterium sp.
35612; bifidobacterium sp.
35687; propionibacterium freudenreichii shermanii  <dig>
click here for file
additional file 3
putative linoleic acid isomerase protein.
panel a. e. coli jm <dig> poxo <dig>  and pemye <dig>  induced with  <dig>  mm iptg.
samples taken after  <dig>   <dig>   <dig> and  <dig> h growth for protein extraction and sds-page.
panel b. c. glutamicum pclik  and pmyc <dig>  following  <dig> h growth prior to protein extraction.
panel c. l. lactis pnz <dig>  and pemyl <dig>  following induction with nisin  <dig> and  <dig> ng/ml, respectively, and further growth for  <dig> h prior to protein extraction.
click here for file
additional file 4
cla forming activity in bacterial strains expressing mcra from b. breve.
gc chromatogram of supernatant from b. breve ncimb  <dig> , l. lactis pemyl <dig> , and l. lactis pnz <dig>  following growth in linoleic acid   and  for 48- <dig> h. similar results were obtained for recombinant c. glutamicum and e. coli.
click here for file
additional file 5
hydratase activity in bacterial strains expressing mcra from b. breve.
amounts of 10-hoa  and 10-hoe  in supernatant from l. lactis pemyl <dig> , and l. lactis pnz <dig>  following growth in linoleic acid   for 2- <dig> h. data represent the mean values of  <dig> measurements.
similar results were obtained for recombinant c. glutamicum.
click here for file
acknowledgements
this study was funded by science foundation ireland, through the alimentary pharmabiotic centre, which is a research centre funded by sfi , the irish government's national development plan and the european union .
al was founded by a georg-christoph-lichtenberg stipend of the ph.
d. program "molecular biology".

 the streptococcal kda mcra-like proteins represent a family of proteins which are present in a wide range of bacteria. it has been reported that some members of the mcra-like proteins have fatty acid hydratase activity. recently, the mcra protein from streptococcus pyogenes m was reported to be a fad-containing enzyme, which acts as hydratase on cis- - and trans- -double bonds of c- c- non-esterified fatty acids producing 10-hydroxy and -dihydroxy fatty acids. the expression of the mcra gene was found to be up-regulated by the stress responsive alternative transcription factor σb in staphylococcus aureus. it could be speculated that in vivo conditions such as high temperature at the site of infection, leading to stress in s. aureus may induce the stress responsive σb transcription factor. it is a general belief that the mcra protein is involved in virulence in staphylococcus and streptococcus. a kda streptococcal mcra protein was up-regulated in group a streptococcus during experimental mouse soft tissue infection and in the asymptomatic phase of infection in cynomolgus macaques monkeys infected with group a streptococcus. however, a mcra deletion mutant in streptococcus pyogenes showed only mild alteration in virulence properties, such as blood survival, adherence and internalization to human keratinocytes. dunman et al. found the mcra gene was down-regulated by agr, one of the most studied virulence response regulators in staphylococcus aureus, along with other cell surface virulence factors. growth-phase associated regulation of the mcra 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. the transcriptional regulatory protein rgg coordinates amino acid catabolism and virulence factor expression in streptococcus pyogenes. also, the mcra gene was up-regulated by the mgra regulon in stapylococcus aureus. this regulon has been shown to affect multiple staphylococcus aureus genes involved in virulence and antibiotic resistance, but also genes involved in osmotic stress response, prop and gbsa ). the mcra protein shows over 50% homology with linoleate isomerases from lactobacillus acidophilus and lb. reuteri pyr linoleate isomerase, a protein that catalyses the formation of -cla from linoleic acid. cla refers to several positional and geometric isomers of linoleic acid c18: cis-, cis- octadecadienoic acid] with conjugated double bonds, and the and isomers in particular, have been shown to exert beneficial effects on human metabolism, including anti- carcinogenic, anti-diabetogenic and anti-atherogenic activities and body composition alteration effects. cla is formed via biotransformation of linoleic acid, as a direct result of the action of the ruminal microbiota, of which butyrivibrio fibrisolvens is the foremost. the mechanism of microbial production of cla was characterised using washed cells of the strain lb. acidophilus aku and involves the production of hydroxy fatty acids as precursor to formation of cla. when isolated and introduced to the washed cells, these hydroxy fatty acids were rapidly transformed to their respective cla isomers. thus, cla formation by lb. acidophilus was found to consist of two distinct steps, step one: the hydration of linoleic acid to 10-hydroxy-18: and step two: the subsequent dehydration and isomerisation of these hydroxy fatty acids to the c t cla and t t cla isomers. we have previously reported the ability of a range of bifidobacterium spp. to produce the c t cla isomer from free linoleic acid and have shown that administration of cla producing b. breve ncimb to mice and pigs in combination with linoleic acid alters host tissue fatty acid composition, including elevated liver c t cla, coupled with reductions in the proinflammatory cytokines tumour necrosis factor-α and interferon-γ. the enzyme responsible for cla production in bifidobacterium has not been reported. the aims of this study were 1) to investigate if the mcra protein from b. breve ncimb 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 thus we report a role for mcra in stress protection in b. breve. methods cultures and media b. breve ncimb was cultured in mrs media supplemented with % l-cysteine hydrochloride under anaerobic conditions using anaerobic jars with'anaerocult a' gas packs at 37°c. lactococcus lactis nz was cultured at 30°c in m broth and/or agar containing glucose. l. lactis carrying the plasmid pnz was routinely cultured in the presence of chloramphenicol as a selective marker. corynebacterium glutamicum atcc was cultured aerobically in bhi media at 37°c. c. glutamicum atcc carrying the shuttle vector pclik 5a mcs pddh was routinely cultured in the presence of kanamycin as a selective marker. escherichia coli jm carrying the plasmid poxo was routinely cultured aerobically in bhi at 37°c in the presence of chloramphenicol as a selective marker. dna sequence analysis and primer design the sequence encoding the kda mcra protein from lb. reuteri pyr was compared to sequence databases, using the blast suite of programs. proteins exhibiting significant similarity were aligned using dnastar software and conserved motifs were identified. degenerate oligonucleotide primers and primers specific for bifidobacteria were designed to these motifs and used in pcr reactions to amplify a ~ kb internal region of the sequence as follows: primer ev1a forward and ev2a reverse general, degenerate): 5'- ta,tgg,iii,aci,atg,tt,gci,tt,ga-3' and 5'- ga,ga,tc,igt,igt,aa,iac,igt,tc 3', ev1b forward and ev2b reverse: 5'-tac,tgg,iii,acc,atg,ttc,gci,ttc,gaa-3' and 5'- ga,gta,ttc,gt,gt,gaa,gac,gt,tc 3'. pcr reactions were performed in a hybaid pcr express unit in μl with an annealing temperature of 45°c. each reaction contained μl of each primer, μl of template in μl mgcl, μl dntp master mix, μl 10x nh reaction buffer and μl biotaq dna polymerase. the resulting ~ kb fragment was cloned into pcr - topo and sequenced. chromosome walking by inverse pcr to obtain flanking chromosomal sequence, two primers were designed to terminal regions of the known chromosomal dna sequence. the genomic dna from b. breve ncimb was digested with a range of restriction enzymes followed by ligation with dna ligase as described by the supplier. the circularized fragments were subsequently used as templates in the inverse pcr reactions with the terminal primers. the reactions were performed in the same way as the standard pcr reactions but with an annealing temperature of 50°c and the resulting fragments were cloned into the pcr -topovector and sequenced. the two terminal primers used were primer a: 3'-cgttctcgaccttggtgttgtatcggaatt 5' and primer b: 5' -gtaccgaccgacaagatcgagtcgcttgcc-3'. from the approach described above, it was confirmed by sequencing that the 3' end of the gene was obtained from re-circularized xbai digests; however the 5' end was not yet acquired. a second round of inverse pcr was performed with primers a and pcs targeted to re-circularized kpni digests of genomic dna, which provided another bp of the upstream region of the gene. the last round of inverse pcr to obtain the missing bp from the 5' end of the gene was carried out with primers er and er targeted to circularized haeii digests. dna manipulations and plasmid construction genomic dna was isolated from b. breve ncimb by a modified method described by hoffmann et al.. the mcra gene from b. breve ncimb was amplified from genomic dna with primers emy and emy. pcr was performed with high fidelity expand polymerase as described by the supplier using ng genomic dna as a template. pcr conditions were as follows: cycles of min, s denaturation, s annealing, min elongation followed by cycles of s, s, min + s/cycle and finally, one min cycle at 72°c. the mcra gene was amplified with primers emy and emy for cloning in pclik, resulting in the construct pemyc which was transformed into c. glutamicum. recombinant plasmids were double digested with the same enzymes to verify the correct clone and electroporated into l. lactis nz and c. glutamicum atcc electrocompetent l. lactis were prepared and transformed according to the method described by de ruyter et al., while electrocompetent c. glutamicum cells were prepared as described by luchansky et al.. for enzymatic assays, the recombinant b. breve mcra protein was produced in e. coli as n-terminal 6x his-tag fusion. the complete mcra gene was amplified using primers bbi_fw and bbi_rv. ligation with pet vector resulted in a construct pbbi containing n-terminal fusion of 6x his tag and b. breve mcra protein. the authenticity of the clones was verified by sequencing. investigation of the stress tolerance of mcra protein-overproducing l. lactis and c. glutamicum the mcra protein was expressed in lactococci using the nice system, which allows maximal protein expression at sub-lethal concentrations of nisin, and is tightly controlled, such that there is negligible expression in the absence of nisin. the mcra protein was also expressed in c. glutamicum using the shuttle vector pclik 5a mcs pddh. this vector replicates in both e. coli and c. glutamicum and contains a c. glutamicum promoter and a kanamycin resistance gene which function in both e. coli and c. glutamicum. l. lactis pnz and pemyl cells were induced with nisin at od nm ~ and cultures were left to grow for an additional h prior to heat treatment. c. glutamicum pclik and pemyc were grown to od nm ~ -, prior to stress. thermotolerance of l. lactis and c. glutamicum was monitored at 51°c in broth over 30- min. aliquots were removed at and min, plated on agar and incubated at 30°c and 37°c for h. salt tolerance was determined following resuspension in broth containing m nacl and m nacl and culture viability was monitored over 60- min with aliquots taken at min intervals. solvent tolerance was determined on addition of butanol 1-5%, and survival was monitored over min with aliquots taken at min intervals. heterologous expression, protein extraction and sds-page the putative linoleic acid isomerase was expressed in poxo in e. coli using the t system. e. coli poxo and e. coli pemye cultures were aerated vigorously until the od600nm reached approximately, at which point isopropyl-β-d-thiogalactopyranoside was added to final concentration of mm to induce expression of the t rna polymerase. aliquots were taken at intervals after iptg induction at and h for whole cell protein extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. similarly, l. lactis pemyl and pnz were cultured to od600nm ~ and induced with nisin and allowed to grow for another h prior to protein extraction. c. glutamicum pemyc and pclik were cultured as described previously for h prior to protein extraction. to extract proteins, cells were harvested and washed in mm tris hcl, ph and resuspended in ml extraction solution ) and bead-beaten for × sec with min rest on ice. the cell lysate was then centrifuged to remove beads and cell debris and the protein extracts analysed by sds-page using a mini protean ii cell unit by the method of laemmli with a 10% acrylamide resolving gel and 4% stacking gel. a low molecular weight standard ranging from to da was used as a molecular weight marker. production and purification of recombinant b. breve mcra protein from e. coli for protein production, e. coli bl star strain harboring pbbi plasmid was used. bacteria were cultivated in 2xyt medium, yeast extract, nacl ) supplied with kanamycin at 37°c until od reached. at that point, iptg was added to final concentration of mm, the cells were changed to 16°c and harvested by centrifugation after h induction time. cells were resuspended in buffer a in 1: v/v ratio, frozen in liquid nitrogen and stored at -20°c. for protein purification, cells were lysed by adding lysozyme and dnasei in buffer a and mm mgcl and centrifuged for min at g. the supernatant was loaded on histrap column, the column was washed with column volumes of buffer a, and the protein was eluted with column volumes of buffer a and m imidazol. after elution, the buffer was exchanged to buffer b by ultrafiltration and protein concentration was estimated spectrophotometrically using calculated absorption coefficient ε = immunoblotting μg of protein were subjected to sds page followed by protein transfer to nitrocellulose membrane at v, ma/cm gel surface for h. the membrane was washed with buffer b and blocked for one hour in buffer b containing 5% milk powder. his-tagged proteins were detected using anti-his tag antibody in combination with a secondary antibody coupled with alkaline phosphatase and nitrotetrazolium blue/5-bromo-4-chloro-3-indolylphosphate staining. spectroscopic assays solution of purified b. breve mcra protein had yellow colour, therefore for initial characterization uv-vis spectrum of purified protein in the range 300- nm was recorded with a single beam absorption spectrometer. for the reference, spectra of fad and fmn in buffer b were recorded in the same way. for these measurements, b. breve mcra protein, fad and fmn were brought within the concentration range 10- pm. the absence of covalent bond between cofactor and b. breve mcra protein was confirmed in the following way; pl of the protein was boiled for min at 100°c and centrifuged at g for min. the spectrum of the supernatant was then recorded as described above. activity assay, lipid extraction and derivatization to demonstrate the enzymatic activity of b. breve mcra protein, a panel of free fatty acids was tested in the reactions with the purified protein. typically, 10- pg of substrate was mixed with pg of purified protein in ml of buffer b and incubated for h at 25°c. fatty acids were extracted from the reaction mixture with ml chloroform:methanol according to bligh and dyer. free fatty acids were dissolved in methanol and converted to corresponding methyl esters with -diazomethane. for gc- ms analysis, fatty acid methyl esters were dissolved in acetonitrile and hydroxy groups were modified by n,o-bis--trifluoroacetamide, resulting in fatty acid trimethylsilylethers. gc-ms analysis was performed as previously described. phylogenetic analysis homologs to the previously identified linoleate isomerase from lb. acidophilus and oleate hydratase, elizabethkingia meningoseptica were selected from blast analysis. these homologs, along with mcra from b. breve ncimb discussed in this manuscript were aligned using t-coffee and protein trees were built using the phylip package. the cladogram was then visualised using dendroscope.  previous studies by rosson et al. reported on the isolation and purification of a putative linoleic acid isomerase from lb. reuteri. in these studies, protein sequencing had identified a kda protein with homology to mcra. based on this finding, we designed primers to conserved regions shared by a range of mcra proteins in the database and found that we could pcr the gene from a range of different bifidobacterium strains and propionibacterium freudenreichii shermanii. given that b. breve ncimb is known to be capable of producing c t cla from linoleic acid, we decided to study this strain in terms of cla production. using primers ev1a forward and ev2a reverse, we amplified a kb internal fragment of the putative isomerase gene from b. breve. sequencing of this revealed up to 70% homology to mcra proteins from various genera. following this, we primer walked in both 5'and 3' directions, which resulted in the full sequence of b. breve putative isomerase. sequence analysis the complete bp nucleotide sequence from b. breve ncimb encodes a protein of amino acids with homology to other " kilodalton mcra" proteins. the calculated molecular weight of the protein is da. comparison with sequences in the database revealed that the cloned mcra protein showed 71% homology with putative kda mcra from streptococcus mutans ua and 51% homology with lb. acidophilus linoleate isomerase. a non-canonical flavin binding motif 21e) is present at the n-terminus of b. breve mcra protein, similar to streptococcus and lactobacillus mcra proteins. the protein is cytoplasmic, but may have one trans-membrane helix as predicted by psortb. upstream of the orf is a putative dehydrogenase or reductase protein with ~50% homology with b. longum ncc -diketo-d-gluconic acid reductase. downstream of the tga stop codon is a region of dyad symmetry, capable of forming a stable stem-and-loop structure. the potential hairpin consists of 13-bp stem separated by a 3-bp mis-priming and a 5- bp loop. over-expression of b. breve mcra protein in e. coli since no over-expression of the putative linoleic acid isomerase could be confirmed following sds-page of the recombinant lactococci and corynebacteria, possibly due to masking of the protein by other proteins of similar molecular weight, the putative lai gene was expressed in poxo in e. coli using the t system. following growth and induction at od600nm with mm iptg, aliquots of e. coli poxo and e. coli pemye were taken at and h after induction for whole cell protein extraction and sds-page. a protein band of approximately kda was visualized, corresponding to the expected molecular weight of the putative isomerase protein, which was absent in the iptg induced control culture e. coli poxo. following incubation for 48- h with free linoleic acid, the substrate for bacterial conversion to c t cla, no isomerase activity could be confirmed among the heterologous clones carrying the lai gene in the bacterial supernatant by gc, or by measuring od nm. production of b. breve mcra in e. coli. and enzymatic assays b. breve mcra protein was overproduced in e. coli and purified on ni2+-nta column. the major band with apparent molecular weight of kda was visualized on sds page after the elution. immunoblotting with anti-his tag antibodies confirmed that this band contained 6x his tag. purified b. breve mcra protein has flavin-like uv-vis absorption spectrum with maxima around and nm and the flavin cofactor is released in the solution upon protein denaturation by heat. the spectral properties of b. breve mcra are consistent with a recombinant mcra protein from s. pyogenes, which was found to contain a non-covalently bound fad and exhibited double bond hydratase activity producing 10- and 13-hydroxy fatty acids. these data prompted us to examine the enzymatic activity of b. breve mcra using common c and c fatty acids as substrates. to this end, purified protein was incubated with free fatty acids and the reaction products were analyzed by gc-ms. indeed, we observed conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but myristoleic acid or shorter monounsaturated fatty acids were not substrates. the presence of trans-double bonds as well as the double bonds beyond the position c abolished hydratase activity. no conversion of esterified fatty acids such as methyl linoleate or cla isomers was observed. phylogenetic analysis stress tolerance of mcra-overproducing lactococcus and corynebacterium to determine if heterologous lactococcus and corynebacterium containing the mcra gene exhibited improved stress tolerance, the heterologous cultures along with vector controls of lactococcus and corynebacterium were assayed for hydratase activity by measuring 9-hydroxy fatty acids without and with linoleic acid supplementation of the medium. indeed, those cultures that expressed mcra/hydratase accumulated increasing amounts of 10-hoa and 10-hoe in the culture medium. next, the heterologous cultures were exposed to heat, salt and solvent butanol × 45- min) stresses. l. lactis pnz reduced in viability by log cfu/ml following heat stress at 51°c for min, whereas l. lactis pemyl only reduced in viability by log cfu/ml. the difference in survivability between c. glutamicum pclik and pemyc was ~ log cfu/ml when heat stressed at 51°c for min, a decline in survivability by < log cfu/ml for l. lactis pemyl and < log cfu/ml for c. glutamicum pclik. similarly, when induced l. lactis pnz was exposed to solvent stress butanol), a decrease in viability of > log cfu/ml was observed, whereas induced l. lactis pemyl only decreased ~ log cfu/ml following min in 3% butanol, i.e. a difference of nearly log cfu/ml between the cultures under identical conditions. c. glutamicum pemyc exposed to 3% butanol stress for min survived ~ log cfu/ml better than the vector control. upon confirming that the clones containing the mcra protein were less sensitive to heat and solvent stresses, we examined the hypothesis that the protein also protected the cultures from salt stress. following growth and induction by nisin and additional growth for h, l. lactis pnz and pemyl cultures were exposed to m nacl for min. however, no difference in salt tolerance was observed at concentrations of m nacl. a similar observation was made for c. glutamicum pclik and pemyc which were grown in m nacl for min. both c. glutamicum control and pemyc cultures survived in m nacl for min with no apparent decline in viable numbers. discussion several recent reports have linked mcra and oleic acid hydratase activity, which was first observed more than forty years ago in pseudomonas sp.. however, the significance of this hydratase activity for the bacterial host is not yet clearly established. our study identified mcra from b. breve as a fad-containing hydratase with an additional role in stress protection. interestingly, a recent study by o'flaherty and klaenhammer indicated a role for mcra in stress tolerance, cell wall division and adherence to caco- cells. furthermore, all three characterized enzymes from this family are highly specific for free fatty acids as the previously characterized linoleic acid isomerase from p. acnes. thus, both enzyme activities seem to form a new class of fad-dependent fatty acid modifying enzymes. this finding may be even further supported by phylogenetic groupings. given the wide distribution of the mcra gene between human pathogenic and commensal bacteria, it could be speculated that it is not directly linked to virulence but to a general stress protection, which could also be useful for a pathogen i.e. increased heat at the site of infection. our study confirmed the increased viability of heterologous lactococcus and corynebacterium transformed with the mcra gene during heat and solvent stresses as a difference in viability of log cfu/ml following heat stress at 51°c for min and 3% butanol stress for min was observed between l. lactis pemyl and pnz. likewise, the introduced mcra improved the survivability of c. glutamicum by ~ log cfu/ml difference between c. glutamicum pclik and pemyl following heat stress at 51°c for min, and ~ log cfu/ml following 3% butanol stress for min. the same host, l. lactis nz has previously been used for comparative studies of stress performance following transformation with the well known heat shock protein groesl in plasmid pnz desmond et al. compared the viability of induced and heat treated cells at 54°c for min of the same host l. lactis nz transformed with either pnz or pnz8048-groesl. at that temperature, both l. lactis pnz and un-induced l. lactis pnz8048-groesl reduced in viability by log cfu/ml, whereas the induced l. lactis declined by log cfu/ml during the heat stress. furthermore, induced groesl-overproducing l. lactis was able to grow and increase by log cfu/ml during h exposure to % butanol, compared with l. lactis and un-induced l. lactis which declined in viability after h, a trend that continued over the h period of solvent challenge. it has been reported that the response of cells to sublethal heat and ethanol exposure induce essentially identical stress responses because of the alteration in the membrane fluidity by these stresses. heat and ethanol stresses cause similar changes to plasma membrane protein composition, reducing the levels of plasma membrane h+- atpase protein and inducing the plasma membrane-associated hsp both stresses also stimulate the activity of the fraction of h+-atpase remaining in the plasma membrane. the resulting enhancement to catalysed proton efflux from the cell represents a considerable energy demand, yet may help to counteract the adverse effects for homeostasis of the increased membrane permeability that results from stress.  this study has shown that the mcra protein in bifidobacteria is a fad containing hydratase enzyme, which may be catalysing the first step in cla production in this species, and has an additional function in bacterial stress protection. abbreviations -dihoa: -dihydroxy octadecanoic acid; fad: flavin adenine dinucleotide; fmn: flavin adenine mononucleotide; hepes: 4-- -piperazine ethane-sulfonic acid; 10-hoa: 10-hydroxy octadecanoic acid; 10-hoe: -10- hydroxy-12-octadecenoic acid; hplc: high pressure liquid chromatography; mcra: myosin cross-reactive antigen; ms: mass-spectroscopy; pai: propionibacterium acnes isomerase; tris: 2-amino-2-propane- 3-diol, authors' contributions er-c: collection and analysis of data, writing of manuscript, al: collection and analysis of data, cg: collection and analysis of data rpr: design of experiments, editing of manuscript, oos: collection and analysis of data, sfff: design of experiments, gf: design of experiments if: design of experiments, analysis of data and writing of manuscript, and cs: design of experiments, data analysis and editing of manuscript. all authors have read and approved the final manuscript. supplementary material additional file 1 map of putative isomerase gene. map of putative isomerase gene and chromosome walking strategy used to obtain the full sequence. click here for file additional file 2 mcra gene sequence data from bifidobacterium strains and propionibacterium freudenreichii shermanii. kb pcr fragments following pcr of genomic dna from a range of bifidobacterial strains and propionibacterium freudenreichii shermanii using oligonucleotide primers ev1a forward and ev2a reverse. lanes 1- in the following order; b. adolescentis; b. breve 2257; b. breve 702258; b. breve 8815; b. dentium 2243; b. infantis 2205; b. lactis bb 12; b. longum 2259; b. longum bb 536; bifidobacterium sp. 35612; bifidobacterium sp. 35687; propionibacterium freudenreichii shermanii click here for file additional file 3 putative linoleic acid isomerase protein. panel a. e. coli jm poxo and pemye induced with mm iptg. samples taken after and h growth for protein extraction and sds-page. panel b. c. glutamicum pclik and pmyc following h growth prior to protein extraction. panel c. l. lactis pnz and pemyl following induction with nisin and ng/ml, respectively, and further growth for h prior to protein extraction. click here for file additional file 4 cla forming activity in bacterial strains expressing mcra from b. breve. gc chromatogram of supernatant from b. breve ncimb, l. lactis pemyl, and l. lactis pnz following growth in linoleic acid and for 48- h. similar results were obtained for recombinant c. glutamicum and e. coli. click here for file additional file 5 hydratase activity in bacterial strains expressing mcra from b. breve. amounts of 10-hoa and 10-hoe in supernatant from l. lactis pemyl, and l. lactis pnz following growth in linoleic acid for 2- h. data represent the mean values of measurements. similar results were obtained for recombinant c. glutamicum. click here for file acknowledgements this study was funded by science foundation ireland, through the alimentary pharmabiotic centre, which is a research centre funded by sfi, the irish government's national development plan and the european union. al was founded by a georg-christoph-lichtenberg stipend of the ph. d. program "molecular biology".
furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls. mcra 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 production, and this protein has an additional function in bacterial stress protection. mcra 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 c abolished hydratase activity. we also found that heterologous lactococcus and corynebacterium expressing mcra accumulated increasing amounts of 10-hoa and 10-hoe in the culture medium. the aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen from bifidobacterium breve ncimb mcra from b. breve ncimb was cloned, sequenced and expressed in heterologous hosts and the recombinant proteins assessed for enzymatic activity against fatty acid substrates. the hydroxy fatty acids produced were not metabolised further.
 the aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen from bifidobacterium breve ncimb mcra from b. breve ncimb was cloned, sequenced and expressed in heterologous hosts and the recombinant proteins assessed for enzymatic activity against fatty acid substrates.  mcra 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 c 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.  mcra 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 production, and this protein has an additional function in bacterial stress protection. 