Improved cloning vectors and transformation procedure for <i>Lactococcus lactis</i>

Wells, Jeremy M.; Wilson, Peter W.F.; Page, R.

doi:10.1111/j.1365-2672.1993.tb05195.x

Cited by 186 publications

(135 citation statements)

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“…The latter promoter has also been employed for the production of other heterologous proteins in L. lactis: a B. subtilis protease (39), hen egg white lysozyme (40), and colicin V (24). In contrast to several other lactococcal promoters (13,35,44,45), P32 is a constitutive promoter, with no need for specific induction of expression. Because of these advantageous properties, we have used P32 for all constructs.…”

Section: Discussionmentioning

confidence: 99%

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Gaeng

Scherer

Neve³

et al. 2000

Appl Environ Microbiol

131

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Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa L-alanoyl-D-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-L-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the SP slpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SP slpA-ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3 -end coding sequence of SP slpA-ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511⌬(S262-K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent SP SlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L. monocytogenes cells. Moreover, transformation of pSL-PL511⌬C into L. lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.

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Section: Discussionmentioning

confidence: 99%

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Gaeng

Scherer

Neve³

et al. 2000

Appl Environ Microbiol

131

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“…Sequence analyses were performed with the BLAST programmes from the NCBI database. Recombinant plasmids were first selected after transformation into E. coli DH5a, and pGKK1 or pMSP3535 was then transformed into Wells et al (1993).…”

Section: Methodsmentioning

confidence: 99%

Heterologous expression of pneumococcal virulence factor PspC on the surface of Lactococcus lactis confers adhesive properties

et al. 2012

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Lactococcus lactis is a non-pathogenic bacterium that is used in the food industry but is also used as a heterologous host to reveal protein functions of pathogenic bacteria. The adhesin PspC from Streptococcus pneumoniae is a choline-binding protein that is non-covalently anchored to the bacterial cell wall. To assess the exclusive impact of pneumococcal surface protein C (PspC) on the interplay with its host we generated recombinant L. lactis producing a nisin-inducible and covalently anchored variant of PspC on the lactococcal cell surface. A translational fusion of the 59-end of pspC3.4 with the 39-end of hic (pspC11.4) was designed to decorate the surface of L. lactis with a chimeric PspC. The PspC3.4 part comprises the first 281 aa residues of PspC3.4, while the Hic sequence consists of the proline-rich and sortase-anchored domain. The results demonstrated that PspC is sufficient for adhesion and subsequent invasion of host epithelial cells expressing the human polymeric Ig receptor (hpIgR). Moreover, invasion via hpIgR was even more pronounced when the chimeric PspC was produced by lactococci compared with pneumococci. This study shows also for the first time that PspC plays no significant role during phagocytosis by macrophages. In contrast, recruitment of Factor H via the PspC chimer has a dramatic effect on phagocytosis of recombinant but not wild-type lactococci, as Factor H interacts specifically with the amino-terminal part of PspC and mediates the contact with phagocytes. Furthermore, L. lactis expressing PspC increased intracellular calcium levels in pIgR-expressing epithelial cells, thus resembling the effect of pneumococci, which induced release of Ca 2+ from intracellular stores via the PspC-pIgR mechanism. In conclusion, expression of the chimeric PspC confers adhesive properties to L. lactis and indicates the potential of L. lactis as a suitable host to study the impact of individual bacterial factors on their capacity to interfere with the host and manipulate eukaryotic epithelial cells. INTRODUCTIONStreptococcus pneumoniae (pneumococci) is the aetiological agent of community-acquired pneumonia, septicaemia and bacterial meningitis, all associated with high morbidity and mortality (Cartwright, 2002). Pneumococcal infections commence at the nasopharynx, where the bacteria reside as commensals without harming the host niche. Colonization of the respiratory mucosal epithelium is a prerequisite for invasive infections, which are most likely accompanied by dramatic changes in the nasopharyngeal physical barriers and the expression of pneumococcal virulence factors (Orihuela et al., 2004). The repertoire of uptake systems for essential nutrients enables pneumococci to adapt to and survive in different physiological conditions. More importantly, pneumococci have a variety of virulence factors facilitating adherence to host cells and evasion from the host immune system. Pneumococcal adherence to host cells is, similar to other Gram-positive pathogens including Staphylococcus aureus and Streptococcus...

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“…Plasmid DNA was isolated with a Micro plasmid prep kit (Amersham/Pharmacia, Uppsala, Sweden), and total DNA was isolated with an AquaPure genomic DNA isolation kit (Bio-Rad, Veenendaal, The Neth-erlands) used according to the manufacturer's protocols. L. lactis cells were transformed by electroporation as described previously (41). Agarose gel electrophoresis was performed as described by Sambrook et al (30).…”

Section: Methodsmentioning

confidence: 99%

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

Smit

Vlieg

Engels

et al. 2005

Appl Environ Microbiol

119

139

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The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain ␣-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3 terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branchedchain ␣-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.Flavor formation in cheeses is mainly the result of catabolism of milk proteins, sugar, and lipids. Degradation of proteins into amino acids followed by conversion of these amino acids by Lactococcus spp. is very important for flavor formation in semihard types of cheese, like Gouda (for reviews see references 12, 25, 38, and 43). One of the predominant amino acid conversion routes is initiated by transaminases. Transamination of amino acids yields the corresponding ␣-keto acids, which do not have strong flavor properties (12, 13) but are the precursors of various aldehydes, alcohols, acids, and (thio-)esters. The latter compounds generally have much lower odor thresholds and strongly contribute to cheese flavor. This is exemplified by the production of flavor compounds from leucine. Transamination of leucine results in the production of ␣-isocaproic acid (KICA). Interestingly, only a limited number of Lactococcus lactis strains are capable of decarboxylating this ␣-keto acid to 3-methylbutanal (36, 37). The decarboxylating activity is observed mainly in L. lactis strains with nondairy origins, whereas the industrial strains tested hardly showed this activity (36). Subsequently, (de)hydrogenation of 3-methylbutanal results in 3-methylbutanol or 3-methylbutyric acid, which also contributes to the flavor of cheese, but the odor thresholds for these compounds are higher than the odor threshold for 3-methylbutanal (24, 29). The rate-determining step in this route is the decarboxylation of KICA, and therefore, control of this enzyme offers good prospects for the design of starter cultures with tailored flavor production (37, 43).The enzyme performing this reaction has not been identified. A number of thiamine pyrophosphate (TPP)-dependent

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Improved cloning vectors and transformation procedure for Lactococcus lactis

Cited by 186 publications

References 20 publications

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Heterologous expression of pneumococcal virulence factor PspC on the surface of Lactococcus lactis confers adhesive properties

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

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