Dataset Information

Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303.

ABSTRACT: phi-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of phi-0303 in Escherichia coli DH5alpha. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of phi-0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCR amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages. Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium.

SUBMITTER: Deutsch SM

PROVIDER: S-EPMC321252 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303.

Deutsch Stéphanie-Marie SM Guezenec Stéphane S Piot Michel M Foster Simon S Lortal Sylvie S

Applied and environmental microbiology 20040101 1

phi-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of phi-0303 in Escherichia coli DH5alpha. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of phi-0303 containing six open reading frames (ORFs) a ...[more]

PMID: 14711630

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Project description:Peptides in the 3-kDa ultrafiltrate of fermented whey protein isolate (WPI) medium could be responsible for the antivirulence activity of Lactobacillus helveticus LH-2 and Lactobacillus acidophilus La-5 against Salmonella Typhimurium. Non-fermented and fermented media containing 5.6% WPI were fractionated at a 3 kDa cut-off and the filtrate was analyzed by mass spectrometry. The non-fermented WPI medium contained 109 milk derived peptides, which originated from ?-casein (52), ?s1-casein (22), ?s2-casein (10), ?-casein (8), and ?-lactoglobulin (17). Most of these peptides were not found in the fermented media, except for 14 peptides from ?-casein and one peptide from ?s2-casein. Database searches confirmed that 39 out of the 109 peptides had established physiological functions, including angiotensin-converting-enzyme (ACE) inhibitory, antioxidant, antimicrobial, or immunomodulating activity. A total of 75 peptides were found in the LH-2 cell free spent medium (CFSM): 54 from ?-casein, 14 from k-casein, 4 from ?-lactoglobulin and 3 from ?s2-casein. From these peptides, 19 have previously been associated with several categories of bioactivity. For La-5 CFSM, a total of 15 peptides were sequenced: 8 from ?-casein, 5 from ?s1-casein, 2 from ?-lactoglobulin. Only 5 of these have previously been reported as having bioactivity. Many of the peptides remaining in the fermented medium would contain low-affinity residues for oligopeptide binding proteins and higher resistance to peptidase hydrolysis. These properties of the sequenced peptides could explain their accumulation after fermentation despite the active proteolytic enzymes of LH-2 and La-5 strains. Down-regulated expression of hilA and ssrB genes in S. Typhimurium was observed in the presence of La-5 and LH-2 CFSM. Downregulation was not observed for the Salmonella oppA mutant strain exposed to the same CFSM used to treat the S. Typhimurium DT104 wild-type strain. This result suggests the importance of peptide transport by S. Typhimurium for down regulation of virulence genes in Salmonella.

Dataset Information

Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303.

Publications

Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303.

Similar Datasets

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