Controlled expression of CluA in Lactococcus lactis and its role in conjugation

Abstract

CluA is a 136 kDa surface-bound protein encoded by the chromosomally located sex factor of Lactococcus lactis MG1363 and is associated with cell aggregation linked to high-frequency transfer of the sex factor. To further investigate the involvement of CluA in these phenomena, the cluA gene was cloned on a plasmid, downstream from the lactococcal nisA promoter. In a sex-factor-negative MG1363 derivative, nisin-controlled CluA expression resulted in aggregation, despite the absence of the other genes of the sex factor. Therefore, CluA is the only sex factor component responsible for aggregation. The direct involvement of CluA in the establishment of cell-to-cell contact for aggregate formation was observed by electron microscopy using immunogold-labelled CluA antibodies. Inactivation of cluA in an MG1363 background led to a dramatic decrease in sex factor conjugation frequency compared to the parental strain. Increasing levels of CluA expressed in trans in the cluA-inactivated donor strain facilitated a gradual restoration of conjugation frequency, reaching that of the parental strain. In conclusion, CluA is essential for efficient sex factor transfer in conjugation of L. lactis.

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

“…This strain was used as a cluA background strain to monitor the aggregation of the different CluA mutants. Strains FI9980 and FI9981 (29) are FI9979 derivatives that harbor pFI2209, an expression vector containing the P nisA promoter, and pFI2213, the same expression vector containing the wild-type cluA gene, respectively. These two strains were used as negative and positive controls to assess aggregation.…”

“…Plasmid pFI2453 was used as a target for insertion mutagenesis. This plasmid is a derivative of pFI2213 (expression vector pFI2209 containing the wild-type cluA gene under control of the P nisA promoter) (29) in which the unique BsrGI site was removed by blunt ending the 5Ј overhangs using the T4 DNA polymerase (Promega) under the conditions described by the manufacturer. All the relevant plasmids resulting from cluA insertional mutagenesis are listed in Table 1.…”

“…A lysozyme surface extract of each strain was obtained as previously described (29). Briefly, the pellet from a 10-ml culture in SM17 was resuspended in a solution containing 50 mM Tris-HCl (pH 8), 0.1ϫ protease inhibitor cocktail set II (Calbiochem), 10% sucrose, and 25 mg/ml lysozyme.…”

“…The fragments were sequenced in order to verify that no misincorporation had occurred in the DNA sequence during PCR amplification. The cluA gene containing the point mutations was excised with SmaI and XhoI and cloned into the pFI2209 expression vector (29). The five resulting plasmids are listed in Table 2.…”

“…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.…”