A triggered-suicide system designed as a defense against bacteriophages.

A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.     

Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage phi31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage phi31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage phi31 was inhibited to an efficiency of plaquing (EOP) of 10(-6) in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the phi31 EOP to <10(-7). Phage phi31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the phi31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.     

Lysogenic conversion caused by phage phi 80. III. The mapping of the conversion gene and additional characterization of the phenomenon

The cor gene specifies lysogenic conversion caused by the lambdoid phage phi 80. The cor gene product inhibits tonA function in infected and lysogenic cells. The cells harboring pBR322 plasmid with the cloned cor gene of phi 80 became resistant to the phages T1 and phi 80 (TonA phenotype). The cor gene was mapped between 24 and 13 genes on the phi 80 phage genetic map. It is not essential for phage lytic growth. Its presence in lysogens leads up to accumulation of tonA mutants in a cell population.     

The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage phi X174

Most bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell. The ssDNA phage phi X174 uses a single lysis gene, E, encoding a 91-amino-acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis. Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E-mediated lysis and phi X174 plaque formation. The slyD gene encodes a FKBP-type peptidyl-prolyl cis-trans isomerase (PPIase). To investigate the molecular basis of this unique FKBP-dependence, spontaneous plaque-forming mutants of phi X174 were isolated on a slyD lawn. All of these Epos ('plates on slyD') suppressors encode proteins with either a R3H or L19F change. The double mutant was also isolated and generated the largest plaques on the slyD lawn. A c-myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function. Western blots and pulse-chase labelling experiments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis-sufficient level of Epos to accumulate. Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect. These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD- dependence phenotype for identifying elements of PPIase selectivity.     

Mutation in the Xanthomonas campestris xanA gene required for synthesis of xanthan and lipopolysaccharide drastically reduces the efficiency of bacteriophage (phi)L7 adsorption

(Phi)L7 is a lytic phage infecting the gram-negative Xanthomonas campestis pv. campestris, a plant pathogen. To study phage-host interaction, a (phi)L7-resistant mutant was isolated from strain Xc17 by mini-Tn5 transposition and designated CH7LR. CH7LR could not plate (phi)L7 in double-layered assay and formed turbid clearing zones when the cell lawn was dropped with a high titer of (phi)L7. Sequence analysis showed that the mutated gene is xanA coding for phosphoglucomutase/phosphomannomutase, required for the synthesis of lipopolysaccharide and exopolysaccharide (xanthan). The involvement of xanA was confirmed by isolating another mutant with interrupted xanA and complementing with the cloned wild-type gene. Nonmucoid mutants are still sensitive to (phi)L7, indicating that xanthan is not involved in (phi)L7 adsorption. Since the mutants still exhibited low efficiencies of phage adsorption, we predict, by analogy with the cases in other bacteriophages of gram-negative bacteria, that other outer membrane components such as a protein are required for the formation of a complex receptor.