Dataset Information

Lactococcus lactis gene yjgB encodes a gamma-D-glutaminyl-L-lysyl-endopeptidase which hydrolyzes peptidoglycan.

ABSTRACT: YjgB is one of five peptidoglycan hydrolases previously identified in Lactococcus lactis. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithiothreitol. Peptidoglycan-hydrolyzing activity of YjgB was detected in growing cells of an L. lactis strain overexpressing YjgB, as revealed by the presence of disaccharide (DS)-dipeptide in the muropeptide composition of the overexpressing strain. YjgB hydrolyzes the peptide chains of L. lactis muropeptides between gamma-D-Gln and L-Lys residues. Its hydrolytic activity was detected on DSs with tetra- and pentapeptide chains, whereas hydrolytic activity was very low on DS-tripeptides. Thus, we demonstrated that YjgB is an endopeptidase which cleaves gamma-D-Gln-L-Lys bonds in peptide chains of L. lactis peptidoglycan.

SUBMITTER: Redko Y

PROVIDER: S-EPMC2074913 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Lactococcus lactis gene yjgB encodes a gamma-D-glutaminyl-L-lysyl-endopeptidase which hydrolyzes peptidoglycan.

Redko Yulia Y Courtin Pascal P Mézange Christine C Huard Carine C Chapot-Chartier Marie-Pierre MP

Applied and environmental microbiology 20070720 18

YjgB is one of five peptidoglycan hydrolases previously identified in Lactococcus lactis. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithi ...[more]

PMID: 17644633

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Project description:BackgroundLactic acid bacteria are a family of "generally regarded as safe" organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields.ResultsTo expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4-2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding.ConclusionsOverall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

Project description:BACKGROUND:Important industrial traits have been linked to plasmids in Lactococcus lactis. RESULTS:The dairy isolate L. lactis subsp. lactis biovar diacetylactis FM03P was sequenced revealing the biggest plasmidome of all completely sequenced and published L. lactis strains up till now. The 12 plasmids that were identified are: pLd1 (8277 bp), pLd2 (15,218 bp), pLd3 (4242 bp), pLd4 (12,005 bp), pLd5 (7521 bp), pLd6 (3363 bp), pLd7 (30,274 bp), pLd8 (47,015 bp), pLd9 (15,313 bp), pLd10 (39,563 bp), pLd11 (9833 bp) and pLd12 (3321 bp). Structural analysis of the repB promoters and the RepB proteins showed that eleven of the plasmids replicate via the theta-type mechanism, while only plasmid pLd3 replicates via a rolling-circle replication mechanism. Plasmids pLd2, pLd7 and pLd10 contain a highly similar operon involved in mobilisation of the plasmids. Examination of the twelve plasmids of L. lactis FM03P showed that 10 of the plasmids carry putative genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as lactose utilisation (lacR-lacABCDFEGX), citrate uptake (citQRP), peptide degradation (pepO and pepE) and oligopeptide uptake (oppDFBCA), uptake of magnesium and manganese (2 mntH, corA), exopolysaccharides production (eps operon), bacteriophage resistance (1 hsdM, 1 hsdR and 7 different hsdS genes of a type I restriction-modification system, an operon of three genes encoding a putative type II restriction-modification system and an abortive infection gene) and stress resistance (2 uspA, cspC and cadCA). Acquisition of these plasmids most likely facilitated the adaptation of the recipient strain to the dairy environment. Some plasmids were already lost during a single propagation step signifying their instability in the absence of a selective pressure. CONCLUSIONS:Lactococcus lactis FM03P carries 12 plasmids important for its adaptation to the dairy environment. Some of the plasmids were easily lost demonstrating that propagation outside the dairy environment should be minimised when studying dairy isolates of L. lactis.

Dataset Information

Lactococcus lactis gene yjgB encodes a gamma-D-glutaminyl-L-lysyl-endopeptidase which hydrolyzes peptidoglycan.

Publications

Lactococcus lactis gene yjgB encodes a gamma-D-glutaminyl-L-lysyl-endopeptidase which hydrolyzes peptidoglycan.

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