Anscriptional regulator (mleR). Only the closely connected species Lactobacillus rhamnosus also harbors an MLE-encoding gene cluster and an ME-encoding gene cluster. The presence of two pathways for L-malate utilization in Lb. casei prompted us to investigate their role in L-malate utilization as a carbon supply, no matter if their expression is concertedly regulated at a transcriptional level and no matter whether both pathways are functionally intertwined.Components AND METHODSStrains and growth circumstances. The strains and plasmids utilised inside the present study are listed in Table 1. Lb. casei was routinely grown in MRS broth (Oxoid) at 37 under static circumstances. Lactococcus lactis MG1363 was grown in M17 medium (Oxoid) supplemented with 27.75 mM glu-Received 11 April 2013 Accepted 27 June 2013 Published ahead of print eight July 2013 Address correspondence to Manuel Z��iga, [email protected]. * Present address: Jos?Mar Landete, Departamento de Tecnolog de Alimentos, INIA, Madrid, Spain. Supplemental material for this article could be discovered at http://dx.doi.org/10.1128 /AEM.01177-13. Copyright ?2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.01177-September 2013 Volume 79 NumberApplied and Environmental Microbiologyp. 5509 ?aem.asm.orgLandete et al.LCABL_30730 maeE maeP maeK maeRBLcorA LCABL_08040 mleR mleS mleT tpx LCABL_30730 maeE maeP maeKBL315 ( maeR) MR (mleR) MT (mleT) MRST ( mleRST) MS (mleS) MPs (maeP)corA LCABL_erypRVmleSmleTtpxcorA LCABL_mleRmleSerypRVtpxcorA LCABL_in-frame deletionmleT?tpxcorA LCABL_mleRstop codonmleSmleTtpxLCABL_30730 maeK maeRmaeEmaePstop codonLCABL_30730 maeK erypRVmaeE corA LCABL_maePmaeR tpxMPT (maeP mleT)mleRmleSFIG 1 Schematic representation of your mae and mle gene clusters in Lb. casei BL23 and derivative strains made use of inside the present study.cose. Escherichia coli DH5 strains were grown in LB medium (BD Difco) at 37 with aeration. The antibiotics made use of had been 100 g ampicillin of ml 1 for E. coli and five g of erythromycin ml 1 for Lb. casei and Lc. lactis. Development assays and gene expression analyses had been carried out at 30 in malic enzyme induction (MEI) medium (tryptone, five g liter 1; yeast extract, 5 g liter 1; K2HPO4, six g liter 1; KH2PO4, 4 g liter 1; MgSO4?H2O, 0.2 g liter 1; MnSO4, 0.05 g liter 1; Tween 80, 1 ml liter 1; cysteine, 0.5 g liter 1) as previously described (three). Medium pH was adjusted to 6.1S,2S-DHAC-Phenyl Trost Ligand site 8, 5.N-Boc-dolaproine custom synthesis 5, or four.PMID:23710097 5 with HCl. At the very least three independent replicatesof every development curve had been obtained. The results have been expressed as averages the common deviations. DNA techniques. Typical solutions have been employed for cloning in E. coli (18). Restriction enzymes and T4 DNA ligase had been purchased from New England BioLabs. Taq DNA polymerase for PCR screening was from Biotools (B M Labs, Madrid, Spain). Platinum Pfx DNA polymerase (Life Technologies S.A., Madrid, Spain) was used for cloning purposes. Plasmids have been isolated with a GFX Micro Plasmid Prep kit (GE Healthcare). DNA from Lb. casei was isolated using the DNA isolation kit for cellsTABLE 1 Strains and plasmids used within this studyStrain or plasmid Strains Escherichia coli DH5 Lactobacillus casei BL23 BL315 MRST MR MT MTc MS MPs MPT Lactococcus lactis MG1363 Plasmids pRV300 pRVmaePstop pRVmle pRVmleR pRVmleS pRVmleT pT1NX pT1mleTaCharacteristics or relevant genotypea F endA1 hsdR17 gyrA96 thi-1 recA1 relA1 supE44 lacU169 ( 80 lacZ M15) Wild-type strain BL23 maeR BL23 mleRST BL23 mleR::pRV300; Eryr BL23 mleT::pRV300; Eryr MRST carrying plasmid pT1mleT BL23 m.