Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r(1)t Temperate Bacteriophage but Not Induction of r(1)t Prophage in Streptococcus cremoris R1

The effects of pTR2030 on the replication of four small isometric bacteriophages were examined in Streptococcus cremoris R1. Three lytic phages (652, 720, and 751), which were isolated independently over a 29-year period, were unable to form plaques on a pTR2030 transconjugant of S. cremoris R1. The fourth phage evaluated, phage r(1)t, was a temperate phage induced from S. cremoris R1 by treatment with mitomycin C. A prophage-cured derivative of S. cremoris R1, designated R1Cs, was isolated and served as a lytic indicator for phage r(1)t. Strain R1Cs and a derivative of this strain that was relysogenized with r(1)t, designated R1Cs(r(1)t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r(1)t) were evaluated for sensitivity to r(1)t phage and induction of r(1)t prophage, respectively. The temperate phage r(1)t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r(1)t lysogen [T-R1Cs(r(1)t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r(1)t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r(1)t phage from without but did not retard lytic development after prophage induction within the cell. It was suggested that pTR2030-encoded phage resistance to small isometric phages may, therefore, act at the cell surface or membrane to prevent phage DNA passage into the host cell or inhibit early events required for lytic replication of externally infecting phage.     

Bacteriophage Resistance Conferred on Lactic Streptococci by the Conjugative Plasmid pTR2030: Effects on Small Isometric-, Large Isometric-, and Prolate-Headed Phages

A series of reactions between phages, sensitive hosts, and transconjugants where the sensitivity of small isometric-, large isometric-, and prolate-headed phages to pTR2030-induced phage resistance was evaluated in Streptococcus lactis and Streptococcus cremoris strains. Phage-resistant transconjugants were constructed in the desired host by conjugal transfer of lactose-fermenting ability (Lac⁺, pTR1040) and phage resistance (Hsp⁺, pTR2030) from S. lactis TEK1. S. lactis and S. cremoris transconjugants harboring pTR2030 were resistant to all small isometric-headed phages examined. In contrast, prolate- and large isometric-headed phages were either not inhibited in the pTR2030 transconjugants or exhibited a reduction in plaque size without a reduction in the efficiency of plaquing. Small isometric-headed phages subject to pTR2030 induced inhibition shared no significant DNA homology with pTR2030, suggesting that phage immunity genes are not harbored on the plasmid or responsible for resistance. The general effectiveness of pTR2030 against small isometric-headed phages was highly significant since these are the phages which have been isolated most commonly from dairy fermentation plants.     

Conjugal Transfer of Bacteriophage Resistance Determinants on pTR2030 into Streptococcus cremoris Strains

Agar surface conjugal matings were used to introduce heat-sensitive phage resistance (Hsp⁺) determinants carried on the conjugal plasmid pTR2030 into Streptococcus cremoris KH, HP, 924, and TDM1. Lactose-fermenting (Lac⁺) transconjugants were selected from matings of Lac⁻ variants of S. cremoris KH, HP, 924, and TDM1 with Streptococcus lactis ME2 or a high-frequency donor, S. lactis T-EK1 (pTR1040, Lac⁺; pTR2030, Hsp⁺). For all of the S. cremoris strains examined, select Lac⁺ transconjugants were completely resistant to plaquing by their homologous lytic phages. In all cases the plaquing efficiencies were less than 10⁻⁹. Acquisition of a 30-megadalton plasmid (pTR2030) in the S. cremoris phage-resistant transconjugants was demonstrated by direct plasmid analysis, by hybridization with ³²P-labeled probes, or by conjugal transfer of pTR2030 out of the phage-resistant transconjugants into a plasmid-cured recipient, S. lactis LM2302. Acid production, coagulation ability, and proteolytic activity of phage-resistant transconjugants in milk were comparable to those of their phage-sensitive parents. Further, S. cremoris phage-resistant transconjugants were not attacked by phage in starter culture activity tests, which included a 40°C incubation period. The results demonstrated that phage resistance determinants on pTR2030 could be conjugally transferred to a variety of S. cremoris strains and confer resistance to phage under conditions encountered during cheese manufacture. Phage-resistant transconjugants of S. cremoris M43 and HP were also constructed without the use of antiblotic markers to select conjugal recipients from mating mixtures.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages.

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Conjugal Strategy for Construction of Fast Acid-Producing, Bacteriophage-Resistant Lactic Streptococci for Use in Dairy Fermentations

Bacteriophage-resistant dairy streptococci were obtained following conjugal transfer of pTR2030 from a lactose-negative donor, Streptococcus lactis TEK12, to lactose-positive recipient strains, Streptococcus cremoris LMA13 and 924 and S. lactis LMA12. Fast acid-producing, phage-resistant transconjugants were selected by challenge with homologous phage on fast-slow differential agar or lactose indicator agar. Acquisition of pTR2030 by the transconjugants was confirmed by DNA-DNA hybridization. Resistance of transconjugants to homologous phage was complete. Curing or deletion of pTR2030 in the transconjugants confirmed that phage resistance was due to pTR2030 acquisition and not to coincident background mutation. Phage-sensitive pTR2030 deletion derivatives of LMA12 transconjugants were isolated in vivo. The HindIII fragment B of pTR2030 was subcloned into pBR322 to yield a recombinant plasmid, pMET2, useful as a source of pTR2030 DNA. A specific, chemically synthesized oligomer useful as a pTR2030 probe was derived from the sequence of a small portion of pTR2030. The conjugal strategy presented here was effective in yielding fast acid-producing, phage-resistant S. cremoris and S. lactis strains without the use of antibiotic resistance markers and without interfering with the acid-producing ability of the recipient strain.     

DNA-DNA Homology Between Lactic Streptococci and Their Temperate and Lytic Phages

Temperate phages were induced from Streptococcus cremoris R₁, BK₅, and 134. DNA from the three induced phages was shown to be homologous with prophage DNA in the bacterial chromosomes of their lysogenic hosts by the Southern blot hybridization technique. ³²P-labeled DNA from 11 lytic phages which had been isolated on cheese starters was similarly hybridized with DNA from 36 strains of lactic streptococci. No significant homology was detected between the phage and bacterial DNA. Phages and lactic streptococci used included phages isolated in a recently opened cheese plant and all the starter strains used in the plant since it commenced operation. The three temperate phages were compared by DNA-DNA hybridizations with 25 lytic phages isolated on cheese starters. Little or no homology was found between DNA from the temperate and lytic phages. In contrast, temperate phages showed a partial relationship with one another. Temperate phage DNA also showed partial homology with DNA from a number of strains of lactic streptococci, many of which have been shown to be lysogenic. This suggests that many temperate phages in lactic streptococci may be related to one another and therefore may be homoimmune with one another. These findings indicate that the release of temperate phages from starter cells currently in use is unlikely to be the predominant source of lytic phages in cheese plants.     

In vivo genetic exchange of a functional domain from a type II A methylase between lactococcal plasmid pTR2030 and a virulent bacteriophage.

The conjugative plasmid pTR2030 confers bacteriophage resistance to lactococci by two independent mechanisms, an abortive infection mechanism (Hsp+) and a restriction and modification system (R+/M+). pTR2030 transconjugants of lactococcal strains are used in the dairy industry to prolong the usefulness of mesophilic starter cultures. One bacteriophage which has emerged against a pTR2030 transconjugant is not susceptible to either of the two defense systems encoded by the plasmid. Phage nck202.50 (phi 50) is completely resistant to restriction by pTR2030. A region of homology between pTR2030 and phi 50 was subcloned, physically mapped, and sequenced. A region of 1,273 bp was identical in both plasmid and phage, suggesting that the fragment had recently been transferred between the two genomes. Sequence analysis confirmed that the transferred region encoded greater than 55% of the amino domain of the structural gene for a type II methylase designated LlaI. The LlaI gene is 1,869 bp in length and shows organizational similarities to the type II A methylase FokI. In addition to the amino domain, upstream sequences, possibly containing the expression signals, were present on the phage genome. The phage phi 50 fragment containing the methylase amino domain, designated LlaPI, when cloned onto the shuttle vector pSA3 was capable of modifying another phage genome in trans. This is the first report of the genetic exchange between a bacterium and a phage which confers a selective advantage on the phage. Definition of the LlaI system on pTR2030 provides the first evidence that type II systems contribute to restriction and modification phenotypes during host-dependent replication of phages in lactococci.     

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

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 (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, nck202.31 (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.     

Rapid Method To Characterize Lactococcal Bacteriophage Genomes

We present a rapid method to isolate and analyze bacteriophage DNA. Cells are infected and phage replication is allowed to proceed normally for 30 to 60 min. Prior to DNA packaging and cell bursts, the infected cells (1 ml) are harvested and lysed by using a combination of lysozyme and sodium dodecyl sulfate treatments. The total DNA recovered is enriched for phage genomes, and restriction fragments of the phage DNA can be readily visualized on agarose gels. This method was used to grossly compare the genomes of nine lactococcal phages isolated from different cheese plants at different times. The method was also used to visualize the inhibitory effects of pTR2030-induced abortive infection on the replication of phage nck202.31 in its homologous host, Lactococcus lactis NCK203.     

Restriction and Modification in Group N Streptococci: Effect of Heat on Development of Modified Lytic Bacteriophage

The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 × 10⁻⁷ for tr plaqued on 799 and 2.1 × 10⁻⁷ for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50°C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.     

A Strategy for Rotation of Different Bacteriophage Defenses in a Lactococcal Single-Strain Starter Culture System

A new strategy for starter culture rotations was developed for a series of phage-resistant clones genetically derived from a single strain of Lactococcus lactis subsp. lactis. Phage-resistant derivatives carrying different defense systems were constructed via conjugation with various plasmids encoding abortive infection (Abi/Hsp) and/or restriction and modification (R/M) systems of different specificity. The plasmids included pTR2030 (Hsp⁺ R⁺/M⁺), pTN20 (Abi⁺ R⁺/M⁺), pTRK11 (R⁺/M⁺), and pTRK68 (R⁺/M⁺). Selected phage-resistant transconjugants or transformants were evaluated in different rotation sequences through cycles of the Heap-Lawrence starter culture activity test in milk contaminated with phage and whey from the previous cycle. When used in consecutive sequence, derivative strains carrying the R/M systems encoded by pTN20, pTRK11, and pTRK68 retarded phage development when the initial levels of phage contamination were below 10² PFU/ml but not when levels were increased to 10³ PFU/ml. Use of a derivative bearing pTR2030 (Hsp⁺ R⁺/M⁺) at the beginning of the rotation prevented phage development, even when the initial levels of phage contamination were high (10⁶ PFU/ml). Alternating the type and specificity of R/M and Abi defenses through the rotation prevented phage proliferation and in some cases eliminated contaminating phages. A model rotation sequence for the phage defense rotation strategy was developed and performed successfully over nine cycles of the Heap-Lawrence starter culture activity test in the presence of high-titer commercial phage composites. This phage defense rotation strategy is designed to protect a highly specialized Lactococcus strain from phage attack during continuous and extended use in the dairy industry.     

Interactions of Lactobacillus bulgaricus Temperate Bacteriophage 0448 with Host Strains

Lactobacillus bulgaricus LT4(0448) is a lysogenic strain from which a temperate bacteriophage can be induced by mitomycin C or UV irradiation. Lactobacillus lactis CNRZ 326 is an indicator strain for the temperate phage 0448, but this strain lyses only in the presence of Ca²⁺ ions. A resistant culture developed secondarily after phage lysis and grew normally in MRS broth but again lysed abruptly if Ca²⁺ ions were added after two or three transfers. This behavior of the secondary culture and its subcultures is explained by a heterogeneous and fluctuating bacterial population, including clones identical to L. lactis 326, which were sensitive to 0448 and which formed rough colonies, as does the indicator. The proportion of these clones increased in the course of transfers in MRS, explaining lysis when Ca²⁺ was added. The population also included clones which formed smooth colonies (S clones). SI clones, which could not be induced by mitomycin C, were the major type in the initial culture, although they were sensitive to temperate phage 0448. The SI population then decreased and was gradually replaced by SII clones, inducible by mitomycin C and resistant to 0448. These SII clones were lysogenized clones, 326(0448), whose stability was confirmed by growth in the presence of an antiphage serum. When L. bulgaricus LT4(0448) was treated with mitomycin C, several cured LT4 clones were obtained that were related to the clones of the indicator L. lactis 326; they formed rough colonies. They also became sensitive to lytic phages or temperate phages active against L. lactis 326 and insensitive to lytic phages which lysed L. bulgaricus LT4(0448). This suggests that phage 0448 can lead to a lysogenic conversion of host strain LT4.     

Characterization of Temperate <Emphasis Type="Italic">Lactobacillus gasseri</Emphasis> Phage LgaI and Its Impact as Prophage on Autolysis of Its Lysogenic Host Strains

We show by electron microscopy that Lactobacillus gasseri phage LgaI, a temperate phage residing in the chromosome of Lactobacillus gasseri ATCC33323, belongs to the family of Myoviridae phages. The LgaI DNA is packed by the “head-full” mechanism, as demonstrated by analysis of restriction patterns of heated (74°C) or non-heated DNA. By isolating prophage-cured cells, we were able to demonstrate phage LgaI to be responsible for the strong autolytic phenotype observed for Lactobacillus gasseri ATCC33323. In addition, we show that a copy of the LgaI prophage resides in the chromosome of Lactobacillus gasseri NCK102. The LgaI prophage was not inducible in L. gasseri NCK102-adh by mitomycin C, however, it apparently contributed to the autolytic phenotype of this strain.     

Analysis of the Bacteriolytic Enzymes of the Autolytic Lactococcus lactis subsp. cremoris Strain AM2 by Renaturing Polyacrylamide Gel Electrophoresis: Identification of a Prophage-Encoded Enzyme

Lactococcus lactis subsp. cremoris AM2 was previously shown to lyse early and extensively during cheese ripening (M.-P. Chapot-Chartier, C. Deniel, M. Rousseau, L. Vassal, and J.-C. Gripon, Int. Dairy J. 4:251–269, 1994). We analyzed the bacteriolytic activities of autolytic strain AM2 by using renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed with two different substrates in the gel, Micrococcus lysodeikticus and L. lactis autoclaved cells. Several lytic activities were detected in L. lactis AM2; a major lytic activity, designated A2 (46 kDa), was found only with the L. lactis cell substrate. This activity appears to be different from major peptidoglycan hydrolase AcmA characterized previously (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrickman, J. Bacteriol. 177:1554–1563, 1995), which has a similar molecular mass. The two enzymes differ in substrate specificity as well as in sensitivity to pH and different chemical compounds. L. lactis AM2 is lysogenic and mitomycin C inducible. Enzyme A2 was shown to be inducible by mitomycin C and to be prophage encoded. It was identified as an enzyme similar to the lysin encoded by lactococcal small isometric temperate bacteriophages. A prophage-cured derivative of L. lactis AM2 was obtained, and this isolate exhibited different autolytic properties than AM2. After prolonged incubation in the stationary phase after growth on M17 medium, the extent of lysis of an AM2 culture was 60%, whereas over the same period there was almost no lysis in a prophage-cured derivative strain culture. These results suggest that the prophage lytic system is involved in the strain AM2 lysis observed in liquid medium and that it could also be involved in the lysis observed during cheese ripening.     

Bacteriophage PBC1 and Its Endolysin as an Antimicrobial Agent against Bacillus cereus

Bacillus cereus is an opportunistic human pathogen responsible for food poisoning and other, nongastrointestinal infections. Due to the emergence of multidrug-resistant B. cereus strains, the demand for alternative therapeutic options is increasing. To address these problems, we isolated and characterized a Siphoviridae virulent phage, PBC1, and its lytic enzymes. PBC1 showed a very narrow host range, infecting only 1 of 22 B. cereus strains. Phylogenetic analysis based on the major capsid protein revealed that PBC1 is more closely related to the Bacillus clarkii phage BCJA1c and phages of lactic acid bacteria than to the phages infecting B. cereus. Whole-genome comparison showed that the late-gene region, including the terminase gene, structural genes, and holin gene of PBC1, is similar to that from B. cereus temperate phage 250, whereas their endolysins are different. Compared to the extreme host specificity of PBC1, its endolysin, LysPBC1, showed a much broader lytic spectrum, albeit limited to the genus Bacillus. The catalytic domain of LysPBC1 when expressed alone also showed Bacillus-specific lytic activity, which was lower against the B. cereus group but higher against the Bacillus subtilis group than the full-length protein. Taken together, these results suggest that the virulent phage PBC1 is a useful component of a phage cocktail to control B. cereus, even with its exceptionally narrow host range, as it can kill a strain of B. cereus that is not killed by other phages, and that LysPBC1 is an alternative biocontrol agent against B. cereus.     

Prophage Curing in Lactobacillus casei by Isolation of a Thermoinducible Mutant

To eliminate the occurrence of virulent phage in industrial fermentation, attempts were made to obtain prophage-cured derivatives from Lactobacillus casei lysogenic strain S-1. A thermoinducible mutant lysogen was isolated from mutagenized strain S-1, since S-1 cannot be induced under laboratory conditions. The mutation responsible for thermoinducibility was located on the prophage. Prophage-cured strains were selected after heat induction of the mutant. These cured strains did not produce the virulent phage and should be valuable for industrial fermentation.