Lactococcal plasmid pNP40 encodes a novel, temperature-sensitive restriction-modification system

Abstract

A novel restriction-modification system, designated LlaJI, was identified on pNP40, a naturally occurring 65-kb plasmid from Lactococcus lactis. The system comprises four adjacent similarly oriented genes that are predicted to encode two m(5)C methylases and two restriction endonucleases. The LlaJI system, when cloned into a low-copy-number vector, was shown to confer resistance against representatives of the three most common lactococcal phage species. This phage resistance phenotype was found to be strongly temperature dependent, being most effective at 19 degrees C. A functional analysis confirmed that the predicted methylase-encoding genes, llaJIM1 and llaJIM2, were both required to mediate complete methylation, while the assumed restriction enzymes, specified by llaJIR1 and llaJIR2, were both necessary for the complete restriction phenotype. A Northern blot analysis revealed that the four LlaJI genes are part of a 6-kb operon and that the relative abundance of the LlaJI-specific mRNA in the cells does not appear to contribute to the observed temperature-sensitive profile. This was substantiated by use of a LlaJI promoter-lacZ fusion, which further revealed that the LlaJI operon appears to be subject to transcriptional regulation by an as yet unidentified element(s) encoded by pNP40.

FIG. 1.

Physical maps of the promoter probe vector pPTPL (A) and the cloning vector pPTPi (B). Polylinkers are indicated.

FIG. 2.

(A) Genetic organization of 6.2-kb LlaJI gene cluster. The ISLL6 insertion site, approximately 280 bp downstream of the ATG start codon of llaJIR2 on plasmid pNP10, is indicated. (B) Nucleotide sequences of the promoter region and the 5′ end of llaJIM1 of the LlaJI operon. The ribosome binding site, preceding llaJIM1, is doubly underlined. The extended −10 and −35 sequences of the promoter region are highlighted. A 24-bp perfect inverted repeat structure is indicated by opposing arrows, and a 14-bp direct repeat sequence is shown by broken arrows. The determined transcriptional start site is denoted in lowercase italics.

FIG. 3.

Temperature-dependent phage resistance profile exhibited by LlaJI system. The EOP of φsk1 was determined as a function of the temperature for MG1614 containing the LlaJI system (in plasmid pJO-J).

FIG. 4.

(A) Northern blot analysis of total RNAs isolated from MG1614 and MG1614/pJO-J at three different temperatures and hybridized to an llaJIM1-specific probe. Lanes 1 and 2, RNAs isolated from MG1614 and MG1614/pJO-J, respectively, at 25°C; lanes 3 and 4, RNAs isolated from MG1614 and MG1614/pJO-J, respectively, at 30°C; lanes 5 and 6, RNAs isolated from MG1614 and MG1614/pJO-J, respectively, at 37°C. The positions of the 16S and 23S rRNAs as well as the RNA markers are indicated. (B) Primer extension analyses of total RNAs isolated from MG1614/pNP40 (lane 1), MG1614 (lane 2), and MG1614/pJO-J (lane 3) at 30°C identified the transcriptional start site as a guanine residue (see Fig. 2B).

FIG. 5.

β-Galactosidase assays of LlaJI promoter-lacZ fusion in MG1614 and MG1614/pNP40 backgrounds conducted at 25, 30, and 37°C. 1A, MG1614/pNP40/pJPL at 25°C; 1B, MG1614/pJPL at 25°C; 2A, MG1614/pNP40/pJPL at 30°C; 2B, MG1614/pJPL at 30°C; 3A, MG1614/pNP40/pJPL at 37°C; 3B, MG1614/pJPL at 37°C.