Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris

Abstract

The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. Plasmid replication and stability have been extensively studied for several plasmids found in gram-negative species. These studies have led to identification of several plasmidencoded elements which mediate plasmid replication, control plasmid copy number, and stabilize plasmid inheritance (18,24,26,30 the reasons for the differing abilities of L. lactis and L. cremoris strains to maintain pSK11L, we attempted to identify the pSK11L replication origin and regions involved in plasmid maintenance since these regions would be most likely to interact with host plasmid replication and maintenance functions.We report here that a 14.8-kbp PvuII fragment of pSK11L confers the replication ability and the temperature-sensitive pSK11L maintenance phenotype of L. lactis LM0230. We also report identification of several regions on this fragment which appear to affect pSK11L maintenance. Several of these regions affect pSK11L maintenance differently in L. lactis LM0230 and L. cremoris EB5. MATERIALS AND METHODSBacterial strains and plasmids. The L. lactis strains used in this study included LM0230, a plasmid-free, prophage-free derivative of C2 (9), and JF3216, an LM0230 transformant containing pSK11L, a lactose plasmid from L. cremoris SK11 (10). The L. cremoris strain used was EB5, a plasmidfree derivative of KR1 (3). Strains LM0230 and EB5 were grown at 32°C in M17 broth (33) supplemented with 0.5% glucose (M17-G). Transformants derived from these strains were grown at 25°C in M17-G containing erythromycin (Em) (10 jig/ml). Strain JF3216 was grown at 25°C in M17 broth supplemented with 0.5% lactose. The Escherichia coli plasmid used to clone the replication region of pSK11L was pVA891, a derivative of pACYC184 (4) containing the pAM,1 Emr-encoding gene (21). pVA891 and its derivatives were maintained in E. coli XL1-Blue

A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts

“…This is most likely due to the theta-type mode of replication, which gives structural and segregational stability (19). Furthermore, L. lactis vectors of the theta family have narrow host ranges (15,25,39), preventing or at least reducing horizontal plasmid transfer to other microorganisms.…”

Genetic and biochemical characterization of the oligopeptide transport system of Lactococcus lactis

“…These authors characterized a 1.65-kb XbaI-XhoI fragment from the L. lactis subsp. cremoris lactose plasmid pSK11L, which facilitated the Campbell-like integration of the thermosensitive replicating plasmid pSK11L into the lactococcal chromosome (7,16). It appears that this entire XbaI-XhoI fragment is present in the lactococcal piece of DNA cloned in pVS8 ( Fig.…”

Complete Sequences of Four Plasmids ofLactococcus lactissubsp.cremorisSK11 Reveal Extensive Adaptation to the Dairy Environment

“…cremoris SK11 (previously described as Streptococcus cremoris SK11) is a phage-resistant strain used in cheese making. Several studies of this strain and its derivatives have shown that proteinase activity and phage resistance are plasmid-encoded functions that can be cured, while the plasmid-encoded lactose utilization ability is extremely stable (17,18,29,38). Detailed studies demonstrated that SK11 cultures are heterogeneous with respect to plasmid content, and isolates were found to contain different combinations of at least eight different plasmids (17,18).…”

“…Curing experiments suggest that phage resistance is encoded on the next largest plasmid, pSK112, but no sequence information is available (17,63). The most extensively studied plasmid is pSK113, better known as pSK11L or the lactose (Lac) plasmid, which is 47 to 48 kb long (17,29,38,74,75). This plasmid encodes a module with the lactose operon, flanked by ISS1 elements, which is essential for its lactose-fermenting ability (38).…”