Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening

Ruyter, Pascalle G.G.A. de; Kuipers, Oscar P.; Meijer, W.C.; Vos, Willem M. de

doi:10.1038/nbt1097-976

Cited by 134 publications

(74 citation statements)

References 19 publications

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“…For the construction of pTal 2009 mut-2⌬N, suitable primers were designed and site-directed mutagenesis was carried out using the PCR-based SOEing technique (22). To check expression of tal 2009 in L. lactis, the DNA fragment corresponding to tal 2009 (15). Electrotransformation of plasmid DNA into E. coli was performed as described by Sambrook et al (47), while that of L. lactis NZ9800 was performed as described by Wells et al (54).…”

Section: Methodsmentioning

confidence: 99%

Bacteriophage Tuc2009 Encodes a Tail-Associated Cell Wall-Degrading Activity

Kenny¹,

McGrath²,

Fitzgerald

et al. 2004

J Bacteriol

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Tuc2009 is a P335-type member of the tailed-phage supergroupLactic acid bacteria are economically important bacteria used in the production of fermented foods such as cheeses, yogurts, and sausages. Tuc2009 is a 38,347-bp lysogenic member of the P335 type of the Siphoviridae supergroup of noncontractile-tailed bacteriophages (GenBank accession no. NC_002703) and was originally identified in Lactococcus lactis subsp. cremoris UC509, a strain used in Cheddar cheese production, following mitomycin C induction (2, 42).Muralytic enzymes or lysins degrade the peptidoglycan (PG) matrix and play essential roles for both phages and bacteria. "Autolysins" is the term used for lysins which are produced by bacteria and involved in cell division, while the term "endolysins" refers to lytic enzymes involved in phage release. Some bacteria also produce lysins which act as class III bacteriocins. Lysins fall into three categories, glycosidases, amidases, and endopeptidases, depending on the type of chemical bond they cleave within the PG. Glycosidases can be further subdivided into the muramidases, glucosaminidases, and transglycosylases (55). Progeny release for many double-stranded-DNA-tailed phages has been shown to employ a lysis system involving one or more holins in conjunction with an endolysin. The holins function by forming pores in the cytoplasmic membrane of the host, thereby abolishing membrane potential and allowing the endolysin to access the PG layer.Lysins exhibit a modular design (16). While a portion (usually the N-terminal part in the case of endolysins) encodes bond cleavage, a second segment is involved in substrate binding. This is believed to help the enzymatic efficiency and specificity of such muralytic enzymes by locating the active motif directly at the site of the substrate and causing endolysins to lyse only those bacteria possessing both the specifically recognized binding region and the target bond of the cleaving domain. It is this specificity of target recognition that could make lysins attractive therapeutic agents. Indeed, studies have demonstrated the usefulness of lysins by specifically lysing streptococci which had colonized mice (38). The lysin is thus said to demonstrate independently functioning domains, as shown for the choline-binding motif of the majority of lysins of Streptococcus pneumoniae and its phages (16) and the endolysin of Tuc2009 (50). Furthermore, the level of homology between these modules from endolysins and autolysins is supportive of the modular theory of phage evolution, as it indicates that the genes encoding such enzymes have arisen as a result of genomic exchange and rearrangement (16).While the cellular PG layer gives structural support to the bacterium, it also represents a formidable barrier across which the phage must transport its DNA during the infection process. Several proteins used by phages infecting gram-negative bacteria to perform this task of "hole punching" have been characterized (45). Phages T4, T7, PRD1, and 6, all of which infect gram-negative ho...

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Section: Methodsmentioning

confidence: 99%

Bacteriophage Tuc2009 Encodes a Tail-Associated Cell Wall-Degrading Activity

Kenny¹,

McGrath²,

Fitzgerald

et al. 2004

J Bacteriol

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“…This ability makes holins the regulators of both the timing of host lysis and the yield of the phage progeny. Despite the increasing number of holin family members described by genetic analysis and their promising use in biotechnology, their structure and molecular mechanism of action are still unknown (2)(3)(4)(5)(6).…”

mentioning

confidence: 99%

Reconstitution of Holin Activity with a Synthetic Peptide Containing the 1–32 Sequence Region of EJh, the EJ-1 Phage Holin

Haro

Vélez

Goormaghtigh

et al. 2003

Journal of Biological Chemistry

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Pneumococcal EJ-1 phage holin (EJh) is a hydrophobic polypeptide of 85 amino acid residues displaying lethal inner membrane disruption activity. To get an insight into holin structure and function, several peptides representing the different topological regions predicted by sequence analysis have been synthesized. Peptides were structurally characterized in both aqueous buffer and membrane environments, and their potential to induce membrane perturbation was determined. Among them, only the N-terminal predicted transmembrane helix increased the membrane permeability. This segment, only when flanked by the positive charged residues on its N-terminal side, which are present in the sequence of the full-length protein, folds into a major ␣-helix structure with a transmembrane preferential orientation. Fluorescein quenching experiments of Nterminal-labeled peptide evidenced the formation of oligomers of variable size depending on the peptideto-lipid molar ratio. The self-assembling tendency correlated with the formation of transmembrane pores that permit the release of encapsulated dextrans of various sizes. When analyzed by atomic force microscopy, peptide-induced membrane lesions are visualized as transbilayer holes. These findings are the first evidence for a lytic domain in holins and for the nature of membrane lesions caused by them.

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“…The importance of these genes in the induction and control of L. lactis cell lysis during cheese ripening was previously demonstrated and discussed (de Ruyter et al 1997;Sanders et al 1997). In all cases, the insertion of these genes within Ll.LtrB completely abolished mobility in both induced and uninduced conditions (Fig.…”

Section: Insertion Of Long Foreign Sequences Within Llltrb Significamentioning

confidence: 99%

Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron

Plante¹,

Cousineau²

2006

RNA

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The Ll.LtrB intron, from the low G+C gram-positive bacterium Lactococcus lactis, was the first bacterial group II intron shown to splice and mobilize in vivo. The detailed retrohoming and retrotransposition pathways of Ll.LtrB were studied in both L. lactis and Escherichia coli. This bacterial retroelement has many features that would make it a good gene delivery vector. Here we report that the mobility efficiency of Ll.LtrB expressing LtrA in trans is only slightly affected by the insertion of fragments <100 nucleotides within the loop region of domain IV. In contrast, Ll.LtrB mobility efficiency is drastically decreased by the insertion of foreign sequences >1 kb. We demonstrate that the inhibitory effect caused by the addition of expression cassettes on Ll.LtrB mobility efficiency is not sequence specific, and not due to the expression, or the toxicity, of the cargo genes. Using genetic screens, we demonstrate that in order to maintain intron mobility, the loop region of domain IV, more specifically domain IVb, is by far the best region to insert foreign sequences within Ll.LtrB. Poisoned primer extension and Northern blot analyses reveal that Ll.LtrB constructs harboring cargo sequences splice less efficiently, and show a significant reduction in lariat accumulation in L. lactis. This suggests that cargo-containing Ll.LtrB variants are less stable. These results reveal the potential, yet limitations, of the Ll.LtrB group II intron to be used as a gene delivery vector, and validate the random insertion approach described in this study to create cargo-containing Ll.LtrB variants that are mobile.

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Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening

Cited by 134 publications

References 19 publications

Bacteriophage Tuc2009 Encodes a Tail-Associated Cell Wall-Degrading Activity

Bacteriophage Tuc2009 Encodes a Tail-Associated Cell Wall-Degrading Activity

Reconstitution of Holin Activity with a Synthetic Peptide Containing the 1–32 Sequence Region of EJh, the EJ-1 Phage Holin

Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron

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