Molecular Characterization of Three Small Isometric-Headed Bacteriophages Which Vary in Their Sensitivity to the Lactococcal Phage Resistance Plasmid pTR2030

Abstract

Lactococcus lactis LMA12-4 is a pTR2030 transconjugant that has been used as an industrial starter culture because of its resistance to phages predominant in cheese plants. Plasmid pTR2030 interferes with susceptible phages in this host strain via two mechanisms, restriction and modification (R/M) and abortive infection (Hsp). After prolonged use of LMA12-4 transconjugants in the industry, two different bacteriophages, designated nck202.+48 (4)48) and nck202.+50 (+50), were isolated which could produce plaques on LMA12-4 containing pTR2030. In this study, these two phages were characterized and compared with a third phage, nck2O2.+31 (+31), which is susceptible to both the R/M and Hsp activities encoded by pTR2030. Phage +48 was not susceptible to inhibition by Hsp, whereas +50 was unaffected by either the R/M or Hsp mechanisms. All three were small isometric-headed phages, but small differences were noted between the phages in the structural details of the tail base plate, susceptibility to chloroform treatment, and requirements for calcium infectivity. The phage genomes were all between 29.9 and 31.9 kb in length. Phages +31 and +48 harbored cohesive ends, whereas the phage +50 genome was circularly permuted, terminally redundant, and carried a putative packaging initiation site. DNA-DNA hybridization experiments conducted between the phages revealed a common region in +48 and +50 that may correlate with the resistance of the two phages to the Hsp-abortive infection induced by pTR2030. Phage +50 also harbored DNA sequences that shared homology to pTR2030 in the region where R/M activities have been localized on the plasmid. Molecular characterization of the three phages localized regions within the genomes of the pTR2030-resistant phages that may be responsible for circumventing plasmid-encoded Hsp and R/M defense mechanisms in lactococci.

Isolation and characterization of phages infecting Bacillus cereus

“…Chloroform had no effect on phage viability for 30 min at 22°C. Titres before and after CHCl 3 treatment were the same for both FWLBc1 and FWLBc2 (10 6 and 10 7 PFU ml )1 , respectively), indicating chloroform resistance (Alatossava and Claenhammer 1991;Oh et al 1999).…”

Sequence analysis of the Lactococcus lactis temperate bacteriophage BK5-T and demonstration that the phage DNA has cohesive ends

“…At present, only two lactococcal phage species (type phages P335 and BK5-T) contain temperate phage members (20). Phage species P335 contains temperate phages which possess DNA with either cohesive or noncohesive ends (1,28). However, members of this group show DNA-DNA homology with phage R1-T which has been shown previously (19) to share little DNA-DNA homology with BK5-T.…”

Bacteriophage and bacteriophage resistance in lactic acid bacteria

“…Phage 4,50 is an industrial isolate initially identified in a cheese plant using a phage-resistant starter strain [36]. The industrial strain is a derivative of L. lactis subsp, lactis NCK202 harbouring the phage resistance plasmid pTR2030 (the mechanisms of resistance encoded by pTR2030 are discussed below).…”

“…The industrial phage q~50 is unaffected by the LlaI R/M system encoded by the resistance plasmid pTR2030 [36]. However, it is sensitive to other R/M systems, suggesting a specific resistance against Llal.…”