Sequence analysis of the lactococcal plasmid pNP40: a mobile replicon for coping with environmental hazards

Abstract

The conjugative lactococcal plasmid pNP40, identified in Lactococcus lactis subsp. diacetylactis DRC3, possesses a potent complement of bacteriophage resistance systems, which has stimulated its application as a fitness-improving, food-grade genetic element for industrial starter cultures. The complete sequence of this plasmid allowed the mapping of previously known functions including replication, conjugation, bacteriocin resistance, heavy metal tolerance, and bacteriophage resistance. In addition, functions for cold shock adaptation and DNA damage repair were identified, further confirming pNP40's contribution to environmental stress protection. A plasmid cointegration event appears to have been part of the evolution of pNP40, resulting in a "stockpiling" of bacteriophage resistance systems.

FIG. 1.

Genetic map of the lactococcal plasmid pNP40. Block arrows and lines represent identified ORFs. A number of functional gene clusters are indicated.

FIG. 2.

(A) Genetic organization of the replication region of pNP40. B. Sequence of repA and upstream and downstream regions. Translational start sites of repA and repB are in boldface, presumed ribosome-binding sites are underlined, and predicted consensus −10 and −35 sequences of the repB promoter are shaded. Inverted repeat structures are represented by opposing arrows, and the 40-bp directly repeated putative iteron sequences are indicated by the dashed arrows.

FIG. 3.

(A) Genetic organization to the pNP40 conjugation region (Top). Checkered arrows represent ORFs encoding predicted membrane-spanning proteins. Conserved gene clusters (and percent identities) similar to segments of the conjugal transfer region of the enterococcal plasmids pCF10 and pAM373 are indicated. (B) Sequence of the region downstream of the mobD gene, predicted to contain the oriT. The mobD translational stop codon is in boldface, and the putative transcriptional terminator for the conjugation operon is indicated by opposing arrows (IR1). A perfect 22-bp inverted repeat (IR2) is indicated; this repeat was also noted to constitute two perfect 22-bp tandem repeats.

FIG.4.

(A) β-Galactosidase assays of the cspC promoter transcriptional fusion (present on pAKC) after 2 and 4 h of cold shock at 10°C. The graph depicts the fold increase in promoter activity after cold shock treatment relative to the promoter activity under non-cold-shock conditions (i.e., 30°C). Bars: 1, MG1614/pAKC (2 h at 10°C); 2, MG1614/pAKC (4 h at 10°C); 3, MG1614/pNP40/pAKC (2 h at 10°C); 4, MG1614/pNP40/pAKC (4 h at 10°C). Absolute values are listed beneath the graph. (B) β-Galactosidase assays of the cspD promoter transcriptional fusion (present on pAKD) after 2 and 4 h of cold shock at 10°C. The graph depicts the fold increase in promoter activity after cold shock treatment relative to the promoter activity under non-cold-shock conditions (i.e., 30°C). Bars: 1, MG1614/pAKD (2 h at 10°C); 2, MG1614/pAKD (4 h at 10°C); 3, MG1614/pNP40/pAKD (2 h at 10°C); 4, MG1614/pNP40/pAKD (4 h at 10°C). Absolute values are listed beneath the graph. (C) Survival of MG1614 and MG1614/pNP40 frozen at −20°C after successive freeze-thaw cycles after exposure to a cold shock at 10°C for 0, 2, and 4 h as indicated.

FIG. 4—

Continued.

FIG. 5.

(A) Growth of MG1614 and MG1614/pNP40 in response to MMC. The arrow on the graph indicates when MMC was added to the culture. (B) β-Galactosidase assay of the orfU promoter transcriptional fusion (pAK17) in the MG1614 host background at 15, 30, 60, and 90 min after exposure to MMC. The graph depicts the fold increase in promoter activity (and the P value) in the presence of MMC relative to the promoter activity at each time point in the absence of MMC. (C) Sequence of orfU, umuC (IL1403), and hdiR upstream regions. The −10 and −35 sequences are shaded, and the orfU start codon is in boface. Sequences corresponding to the core HdiR binding motif (ATCAGW₅CTGAT) are underlined.

FIG. 6.

Intracellular sk1 DNA replication in MG1614 (A) and MG1614/pNP40 (B). (A) Lane 1 contains purified sk1 control DNA, whereas lanes 2 to 5 represent total DNA samples isolated at 0, 20, 40, and 60 min postinfection, respectively, as indicated above each lane. Lysis had initiated with this host after 40 min. (B) Lane 1 contains purified sk1 control DNA, whereas lanes 2 to 8 represent total DNA isolated at 0, 20, 40, 60, 80, 100, and 120 min postinfection, respectively, as indicated above each lane. No lysis was evident with this host during the course of the experiment.