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.Lactococcus lactis, a gram-positive lactic acid bacterium, has been extensively exploited for the production of a variety of fermented dairy products. L. lactis strains exhibit biotechnologically important activities, which contribute to the character of the final food product, e.g., lactose utilization and protease production, and in addition encode properties that specifically provide a selective advantage to the bacterium itself, e.g., heavy metal resistance, bacteriocin production and/or immunity, and bacteriophage resistance (47). Many of these industrially significant traits have been found to be encoded by plasmids, which are omnipresent among this species, with most isolates containing multiple plasmids ranging in size from 2 to 80 kb (11).In recent decades, extensive research has established the molecular mechanisms governing many of these activities, in particular with respect to bacteriophage resistance (47). Lactococcal strains used for many food fermentations are known to be persistently challenged by phages and probably as a consequence have evolved numerous bacteriophage resistance strategies (48, 67).Presently, there are 30 completely sequenced lactococcal plasmids, the largest being pSK11P, a 75.8-kb plasmid isolated from L. lactis subsp. cremoris SK11 (47, 62). This plasmid encodes a variety of functions, including copper resistance, proteolytic activity, cold shock proteins, and cation transport activities, and displays clear "markings" of multiple recombination events that may have contributed to its evolution (62).Previous studies of a similarly sized plasmid, pNP40, originally identified in L. lactis subsp. diacetylactis DRC3 (45), revealed that this molecule, besides its encoded nisin and cadmium resistance determinants, is responsible for an impressive bacteriophage resistance profile (16,19,20,50,65). Two such systems, AbiE and AbiF, were found to provide significant resistance that correlates to an abortive infection phenotype (19).In addition, on the basis of phenotypic evidence, the presence of a third mechanism active at the stage of phage DNA injection was proposed (20). Most recently, a fourth resistance system, the LlaJI restriction-modification system, was identified (50).In the present stud...

Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis

“…One target gene of HdiR was a umuC-like gene, present in L. lactis IL1403 but absent in another L. lactis strain, MG1363 (58). O'Driscoll and coworkers (44) recently reported the presence of a umuC ortholog in the Lactococcus plasmid pNP40 preceded by a repeat structure highly similar to the HdiR-binding sequence. EMSA revealed that the target sequence of S. uberis HdiR binding is located within an IR sequence that differs from the target of L. lactis HdiR.…”

Diversity of Tn 1546 and Its Role in the Dissemination of Vancomycin-Resistant Enterococci in Portugal

“…AF408195). The diversity found among Tn1546 variants containing this IS might be explained by genetic exchange between sequences containing IS1216, as IS6 family members are involved in mechanisms associated with cointegrative processes and replicative transposition (6,33). Interestingly, Tn1546 types containing IS1216 (D, X, PP-1, PP-2, PP-11, PP-13, PP15, PP-16, PP-17, PP-20, PP-23, PP-25, PP-28, and PP-29) were isolated from different sources, which might mirror wide dissemination of vancomycin resistance plasmids which have evolved by different recombinatorial events throughout different hosts.…”

Lantibiotic Resistance

“…Since then, several groups have isolated nisin-resistant lactococcal strains containing nsr on a plasmid (181)(182)(183). The mechanism by which NSR confers resistance is proposed to involve the proteolytic degradation of nisin, mediated by a C-terminally conserved, tail-specific protease (TSPc) domain (184,185). TSPc-containing enzymes efficiently cleave substrates that have apolar residues and a free alpha-carboxylate at the C terminus and have been identified and characterized in a range of species, including E. coli (186).…”