High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals

Savijoki, Kirsi; Kahala, Minna; Palva, Airi

doi:10.1016/s0378-1119(96)00717-2

Cited by 65 publications

(60 citation statements)

References 24 publications

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“…These results agree well with the predicted mass of Ply118 Staphylococcal nuclease as a reporter for SP SlpA-mediated secretion. The S. aureus nuclease (SNase, Nuc) is a useful reporter for the protein secretion ability of gram-positive bacterial cells (30,36). We have used a truncated Nuc protein (lacking its own signal peptide) as a reporter for SP SlpA-mediated secretion from L. lactis cells.…”

Section: Cloning and Expression Of Functional Phage Endolysins In Lmentioning

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

127

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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: Cloning and Expression Of Functional Phage Endolysins In Lmentioning

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

127

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confidence: 99%

“…Furthermore, we have demonstrated the applicability of the slpA expression and secretion signals for heterologous protein production (14,27) and have shown that a nonadhesive lactic acid bacterium can be provided with the capacity for adhesion to human intestinal cell lines by providing it with the receptor-binding region of L. brevis ATCC 8287 SlpA (S. Å vall-Jääskeläinen et al, submitted for publication).…”

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confidence: 99%

“…We have previously characterized the S-layer protein gene (slpA) of L. brevis ATCC 8287 (40), studied its in vivo expression, which is directed by two active adjacent slpA promoters (13), and found that, in this L. brevis strain, only a single-copy S-layer protein gene can be found (40; A. Palva et al, unpublished results). Furthermore, we have demonstrated the applicability of the slpA expression and secretion signals for heterologous protein production (14,27) and have shown that a nonadhesive lactic acid bacterium can be provided with the capacity for adhesion to human intestinal cell lines by providing it with the receptor-binding region of L. brevis ATCC 8287 SlpA (S. Å vall-Jääskeläinen et al, submitted for publication).Our adhesion studies further indicate that the neotype strain of L. brevis, ATCC 14869, adheres more strongly to human enterocytes in vitro than L. brevis ATCC 8287, and even the binding of ATCC 14869 to pig intestinal epithelial cells was found (M. Jakava-Viljanen and A. Palva, unpublished data). To study whether the S-layer protein of ATCC 14869, like that of L. brevis ATCC 8287, mediates adhesion and whether the L. brevis S-layer proteins have structural similarity in their epithelial cell binding domains, we have started to investigate the S-layer protein of ATCC 14869 in more detail.…”

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Isolation of Three New Surface Layer Protein Genes ( slp ) from Lactobacillus brevis ATCC 14869 and Characterization of the Change in Their Expression under Aerated and Anaerobic Conditions

Jakava-Viljanen

Åvall-Jääskeläinen

Messner³

et al. 2002

J Bacteriol

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Two new surface layer (S-layer) proteins (SlpB and SlpD) were characterized, and three slp genes (slpB, slpC, and slpD) were isolated, sequenced, and studied for their expression in Lactobacillus brevis neotype strain ATCC 14869. Under different growth conditions, L. brevis strain 14869 was found to form two colony types, smooth (S) and rough (R), and to express the S-layer proteins differently. Under aerobic conditions R-colony type cells produced SlpB and SlpD proteins, whereas under anaerobic conditions S-colony type cells synthesized essentially only SlpB. Anaerobic and aerated cultivations of ATCC 14869 cells in rich medium also resulted in S-layer protein patterns similar to those of the S-and R-colony type cells, respectively. Electron microscopy suggested the presence of only a single S-layer with an oblique structure on the cells of both colony forms. The slpB and slpC genes were located adjacent to each other, whereas the slpD gene was not closely linked to the slpB-slpC gene region. Northern analyses confirmed that both slpB and slpD formed a monocistronic transcription unit and were effectively expressed, but slpD expression was induced under aerated conditions. slpC was a silent gene under the growth conditions tested. The amino acid contents of all the L. brevis ATCC 14869 S-layer proteins were typical of S-layer proteins, whereas their sequence similarities with other S-layer proteins were negligible. The interspecies identity of the L. brevis S-layer proteins was mainly restricted to the Nterminal regions of those proteins. Furthermore, Northern analyses, expression of a PepI reporter protein under the control of the slpD promoter, and quantitative real-time PCR analysis of slpD expression under aerated and anaerobic conditions suggested that, in L. brevis ATCC 14869, the variation of S-layer protein content involves activation of transcription by a soluble factor rather than DNA rearrangements that are typical for most of the S-layer phase variation mechanisms known.Surface layers (S layers) are monomolecular crystalline arrays of proteinaceous subunits present in almost all archaea and all major phylogenetic groups of bacteria (26,33). Most of the S layers are composed of subunits of a single protein or glycoprotein species capable of forming symmetrical arrays and covering the cell surface during all stages of growth. Slayer proteins commonly contain high numbers of acidic and hydrophobic amino acids but lack overall amino acid sequence homology with corresponding proteins from unrelated species. Moreover, a low pI is typical for S-layer proteins (31) except for those from different lactobacilli (5, 40) and Methanothermus fervidus (6). Diverse functions have been proposed for S layers, such as acting as molecular sieves, protective coats, molecular and ion traps, cell shape determinants, and promoters for cell adhesion and surface recognition (26). There is also increasing evidence that S-layer-carrying bacteria may use Slayer variation, by expressing alternative S-layer protein genes, for ada...

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“…In addition, following the production of proteins by LAB using a specific expression vector they should be properly folded, targeted and sometimes recovered. Several vectors included secretion cassettes, such as those based on the secretion signal of the lactococcal Usp45 protein , the expression and secretion signals of S-layer proteins (Savijoki et al, 1997), the PrtP signal sequence (Kajikawa et al, 2010) or the M6 carboxy-terminal domain to anchor proteins to the cell wall (Reveneau et al, 2002). The nisin-controlled expression (NICE) system, based on the autoregulation mechanism of the bacteriocin nisin, is a very effective expression vector for production of heterologous proteins in LAB (Mierau & Kleerebezem, 2005).…”

Section: Functional Properties Of Lactobacillimentioning

confidence: 99%

Genetically Engineered Lactobacilli for Technological and Functional Food Applications

Yebra¹,

Monedero²,

Pérez-Martı́nez³

et al. 2012

Food Industrial Processes - Methods and Equipment

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High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals

Cited by 65 publications

References 24 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

Isolation of Three New Surface Layer Protein Genes ( slp ) from Lactobacillus brevis ATCC 14869 and Characterization of the Change in Their Expression under Aerated and Anaerobic Conditions

Genetically Engineered Lactobacilli for Technological and Functional Food Applications

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