The <i>Lactococcus lactis</i> sex‐factor aggregation gene <i>cluA</i>

Godon, Jean‐Jacques; Jury, Karen; Shearman, Claire; Gasson, Michael J.

doi:10.1111/j.1365-2958.1994.tb01053.x

Cited by 45 publications

(53 citation statements)

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“…M6 (49 kDa) is among the best characterized of the cell wall-anchored proteins and has already been successfully used to drive cell wall anchoring of recombinant fusion proteins to the surface of Streptococcus gordonii (25). More than 60 cell wall-anchored proteins have been identified in gram-positive organisms, and 2 such proteins, a cell wall proteinase and a clumping factor, were characterized in the model LAB, Lactococcus lactis (11,17,22). All these proteins share a rather similar C-terminal anchoring tail of about 35 amino acids, suggesting that the anchoring mechanism is conserved in gram-positive organisms.…”

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Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria

et al. 1997

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The M6 protein from Streptococcus pyogenes is the best-characterized member of a family of cell envelopeassociated proteins. Based on the observation that the C-terminal sorting signals of these proteins can drive cell wall anchoring of heterologous unanchored proteins, we have cloned and expressed the emm6 structural gene for the M6 protein in various lactic acid bacteria (LAB). The emm6 gene was successfully expressed from lactococcal promoters in several Lactococcus lactis strains, an animal-colonizing Lactobacillus fermentum strain, Lactobacillus sake, and Streptococcus salivarius subsp. thermophilus. The M6 protein was efficiently anchored to the cell wall in all strains tested. In lactobacilli, essentially all detectable M6 protein was cell wall associated. These results suggest the feasibility of using the C-terminal anchor moiety of M6 for protein surface display in LAB.Surface presentation of heterologous molecules in grampositive bacteria is of increasing interest for applications in various fields of biotechnology. Major achievements concerning the surface display of heterologous antigens (25, 33), immunoglobulins (19), and enzymes (40) were recently reported. The absence of an outer membrane in gram-positive bacteria makes them particularly attractive for use in the exposure of bioactive molecules to the extracellular compartment.Lactic acid bacteria (LAB) constitute a family of gram-positive bacteria which are extensively used in the fermentation of raw agricultural products and in the manufacture of a wide variety of food products (5). The burst of information concerning LAB genetics and metabolism, as well as the development of expression and secretion tools, has opened the door for new (non)alimentary applications of these bacteria, such as those described above (8,29). The use of LAB as in vivo delivery vectors for biologically active molecules (e.g., antigens, enzymes, or biological peptides) is made attractive by their nonpathogenicity and ability to survive passage along an oral route down to the intestine. The fulfillment of this project necessitates a delivery system comprising a vehicule (in this case, a LAB species) and a system to present molecules at the cell surface. To do this, we examined the cell wall-anchoring potential of the M6 protein of Streptococcus pyogenes. M6 (49 kDa) is among the best characterized of the cell wall-anchored proteins and has already been successfully used to drive cell wall anchoring of recombinant fusion proteins to the surface of Streptococcus gordonii (25). More than 60 cell wall-anchored proteins have been identified in gram-positive organisms, and 2 such proteins, a cell wall proteinase and a clumping factor, were characterized in the model LAB, Lactococcus lactis (11,17,22). All these proteins share a rather similar C-terminal anchoring tail of about 35 amino acids, suggesting that the anchoring mechanism is conserved in gram-positive organisms. The anchoring structure includes an LPXTG motif followed by a stretch of about 23 hydrophobic amino acids a...

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Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria

et al. 1997

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“…To demonstrate that this chromosomal rearrangement was strictly linked to the presence of the sex factor, we performed a similar experiment using the 5Ј end of the cluA gene as a probe. This gene, cloned from the sex factor of strain MG1363 (20), contains an ApaI site and has been precisely located on the MG1363 genome map (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

Genome Plasticity among Related Lactococcus Strains: Identification of Genetic Events Associated with Macrorestriction Polymorphisms

2000

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The genomic diversity of nine strains of the Lactococcus lactis subsp. cremoris (NCDO712, NCDO505, NCDO2031, NCDO763, MMS36, C2, LM0230, LM2301, and MG1363) was studied by macrorestriction enzyme analysis using pulsed-field gel electrophoresis. These strains were considered adequate for the investigation of genomic plasticity because they have been described as belonging to the same genetic lineage. Comparison of ApaI and SmaI genome fingerprints of each strain revealed the presence of several macrorestriction fragment length polymorphisms (RFLPs), despite a high degree of similarity of the generated restriction patterns. The physical map of the MG1363 chromosome was used to establish a genome map of the other strains and allocate the RFLPs to five regions. Southern hybridization analysis correlated the polymorphic regions with genetic events such as chromosomal inversion, integration of prophage DNA, and location of the transposon-like structures carrying conjugative factor or oligopeptide transport system.

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“…All insertions located in the Tra domain, unique to CluA compared to other gram-positive cell surface protein homologues (16), were affected in sex factor transfer. The importance of this region was confirmed by replacement of charged amino acids located along the length of the Tra domain, which showed that these amino acids are essential for optimized DNA transfer.…”

Section: Discussionmentioning

confidence: 99%

“…CluA exhibits significant homology with two groups of cell surface proteins found in other gram-positive bacteria (16): two enterococcal proteins involved in plasmid conjugation, Asa1 (12) and Asc10 (22), and two streptococcal proteins involved in binding to salivary glycoproteins, SpaP (23) and Ssp5 (8). A comparison of the amino acid sequences of CluA with the sequences of these four other proteins led to identification of six distinct domains in CluA (16), including four conserved domains (domains I, II, IV, and V), a variable domain (domain III), and an additional domain (designated domain IVB in this study) that is specific to CluA and is located at the C-terminal end between domains IV and V. In previous work, we investigated the role of CluA in aggregation and conjugation by controlling the level of protein expression using the lactococcal nisA promoter (29). In a sex factor-negative MG1363 derivative, overexpression of CluA resulted in aggregation of the cells, indicating that CluA is the only sex factor component required for aggregation.…”

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confidence: 99%

“…CluA is a 136-kDa surface-bound protein encoded by the chromosomally located sex factor of Lactococcus lactis MG1363 (16); it is associated with cell aggregation linked to high-frequency transfer of the sex factor (13,14). The amino terminus of CluA has a signal sequence typical of secreted proteins, and the carboxy terminus has a cell wall binding motif that includes a conserved LPXTGE motif (11).…”

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The Tra Domain of the Lactococcal CluA Surface Protein Is a Unique Domain That Contributes to Sex Factor DNA Transfer

2006

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CluA is a cell surface-presented protein that causes cell aggregation and is essential for a high-efficiency conjugation process in Lactococcus lactis. We know from previous work that in addition to promoting cell-to-cell contact, CluA is involved in sex factor DNA transfer. To define the CluA domains involved in aggregation and in transfer, we first performed random mutagenesis of the cluA gene using a modified mini-Tn7 element which generated five amino acid insertions located throughout the encoded protein. Thirty independent cluA insertion mutants expressing modified CluA proteins at the cell surface were isolated and characterized further. The level of aggregation of each mutant was determined. The cell binding capacity of CluA was affected strongly when the protein had a mutation in its N-terminal region, which defined an aggregation domain extending from amino acid 153 to amino acid 483. Of the cluA mutants that still exhibited aggregation, eight showed an attenuated ability to conjugate, and six mutations were located in a 300-amino-acid C-terminal region of the protein defining a transfer domain (Tra). This result was confirmed by a phenotypic analysis of an additional five mutants obtained using site-directed mutagenesis in which charged amino acids of the Tra domain were replaced by alanine residues. Two distinct functional domains of the CluA protein were defined in this work; the first domain is involved in cell binding specificity, and the Tra domain is probably involved in the formation of the DNA transport machinery. This is the first report of a protein involved in conjugation that actively contributes to DNA transfer and mediates contact between donor and recipient strains.

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The Lactococcus lactis sex‐factor aggregation gene cluA

Cited by 45 publications

References 36 publications

Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria

Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria

Genome Plasticity among Related Lactococcus Strains: Identification of Genetic Events Associated with Macrorestriction Polymorphisms

The Tra Domain of the Lactococcal CluA Surface Protein Is a Unique Domain That Contributes to Sex Factor DNA Transfer

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