The conjugative plasmid pTR2030 encodes two bacteriophage defense mechanisms in lactococci, restriction modification (R+/M+) and abortive infection (Hsp+).

pTR2030 is a conjugative plasmid which encodes resistance to bacteriophage in lactococci by a mechanism that aborts the phage infection (Hsp+). Subcloning and in vivo deletion events showed that two independent mechanisms of resistance are located on a 13.6-kilobase Bg/II fragment cloned in pSA3; one mechanism is responsible for the abortive infection, and the other incodes a restriction modification system. The introduction of pTR2030 or the recombinant plasmid pTK6 resulted in the loss of a resident restriction modification plasmid in Lactococcus lactis NCK202 which was not previously identified.     

The conjugative plasmid pTR2030 encodes two bacteriophage defense mechanisms in lactococci, restriction modification (R+/M+) and abortive infection (Hsp+)

pTR2030 is a conjugative plasmid which encodes resistance to bacteriophage in lactococci by a mechanism that aborts the phage infection (Hsp+). Subcloning and in vivo deletion events showed that two independent mechanisms of resistance are located on a 13.6-kilobase Bg/II fragment cloned in pSA3; one mechanism is responsible for the abortive infection, and the other incodes a restriction modification system. The introduction of pTR2030 or the recombinant plasmid pTK6 resulted in the loss of a resident restriction modification plasmid in Lactococcus lactis NCK202 which was not previously identified.     

Abortive phage resistance mechanism AbiZ speeds the lysis clock to cause premature lysis of phage-infected Lactococcus lactis

The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10(-6), without affecting phage adsorption. AbiZ causes phage phi31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of phi31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage phi31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis.     

Conjugal transfer from Streptococcus lactis ME2 of plasmids encoding phage resistance, nisin resistance and lactose-fermenting ability: evidence for a high-frequency conjugative plasmid responsible for abortive infection of virulent bacteriophage

Streptococcus lactis ME2 exhibits at least three mechanisms which confer resistance to virulent bacteriophage. These include plasmid-induced interference with phage adsorption, host-controlled restriction and modification activities, and a heat-sensitive mechanism which suppresses development of virulent phage. Conjugal mating experiments were done with S. lactis ME2 to determine if phage-defence mechanisms present in this strain could be mobilized, associated with plasmid DNA elements and phenotypically characterized in transconjugants. Agar-surface matings of S. lactis ME2 with S. lactis LM0230 demonstrated that lactose-fermenting ability (Lac+) was transferred in a conjugation-like process at frequencies of 10(-6) per donor cell and was associated with a 40 MDal plasmid designated pTR1040. Resistance to nisin (Nisr) was acquired or lost simultaneously with Lac+, indicating that pTR1040 carried determinants for both phenotypes. Lac+ Nisr transconjugants that carried a 30 MDal plasmid (pTR2030) exhibited a heat-sensitive phage-defence mechanism (Hsp+) which limited the burst size and plaque size of phage c2 without altering the efficiency of plaquing (e.o.p.) or the level of adsorption. The ability of phage c2 to initiate plaquing at an e.o.p. of 1.0 indicated that DNA injection and early viral gene expression are not affected in the Hsp+ transconjugants. We suggest, therefore, that the Hsp+ phenotype may result from plasmid-induced abortive infection of phage dependent on the presence of pTR2030. Hsp+ transconjugants carrying pTR2030 also promoted high-frequency conjugal transfer of Lac+ Nisr associated with pTR1040 (greater than 10(-1) per donor cell). It was concluded that Hsp+ and determinants for conjugal transfer ability (Tra+) are located on pTR2030.     

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.     

Nucleotide sequence and distribution of the pTR2030 resistance determinant (hsp) which aborts bacteriophage infection in lactococci

The lactococcal plasmid pTR2030 encodes resistance to bacteriophage attack via two mechanisms, an abortive-infection mechanism, designated Hsp, and a restriction and modification system. We present the complete sequence of the hsp structural gene. The gene is 1,887 base pairs in length and encodes a protein with a predicted molecular mass of 73.8 kilodaltons. The upstream region was cloned in a promoter-screening vector and shown to direct the constitutive expression of the cat-86 gene. An internal probe was used to determine the distribution of the hsp sequence in industrially significant lactococcal strains and to evaluate its relatedness to another lactococcal plasmid implicated in an abortive-infection-type mechanism, pNP40. No homology was detected, suggesting that this gene is not widely distributed in lactococci. Therefore, there are at least two independent abortive-infection genotypes in lactococci.     

Nucleotide sequence and distribution of the pTR2030 resistance determinant (hsp) which aborts bacteriophage infection in lactococci.

The lactococcal plasmid pTR2030 encodes resistance to bacteriophage attack via two mechanisms, an abortive-infection mechanism, designated Hsp, and a restriction and modification system. We present the complete sequence of the hsp structural gene. The gene is 1,887 base pairs in length and encodes a protein with a predicted molecular mass of 73.8 kilodaltons. The upstream region was cloned in a promoter-screening vector and shown to direct the constitutive expression of the cat-86 gene. An internal probe was used to determine the distribution of the hsp sequence in industrially significant lactococcal strains and to evaluate its relatedness to another lactococcal plasmid implicated in an abortive-infection-type mechanism, pNP40. No homology was detected, suggesting that this gene is not widely distributed in lactococci. Therefore, there are at least two independent abortive-infection genotypes in lactococci.     

The bacteriophage resistance plasmid pTR2030 forms high-molecular-weight multimers in lactococci.

Lactococcus lactis ME2 can transfer a 46-kb plasmid, pTR2030, which encodes abortive phage infection (Hsp) and restriction/modification (R/M) activities. pTR2030 can be detected as a monomeric plasmid in transconjugants at low copy number, but not in ME2. pTR2030-specific probes were cloned and used to determine the location of the element in ME2. No homology was observed between these pTR2030-specific probes and the CsCl-purified plasmid content of ME2. However, probes specific for pTR2030 hybridized strongly to a high-molecular-weight moiety, and not to chromosomal DNA, in total DNA isolated by a gentle lysis procedure. The absence of junction fragments indicates that pTR2030 forms high-molecular-weight multimers in lactococci. A phage-sensitive derivative of ME2, L. lactis N1, is cured of pTR2030 and no longer possesses the high-molecular-weight species. When pTR2030 was reintroduced to N1 via conjugation, an ME2-like phage-insensitive phenotype was restored. pTR2030 could remain as a detectable monomeric plasmid in the N1 transconjugants or could revert to the high-molecular-weight structure.     

Localization, cloning, and expression of genetic determinants for bacteriophage resistance (Hsp) from the conjugative plasmid pTR2030

Genetic determinants for a bacteriophage resistance mechanism (Hsp+) encoded by plasmid pTR2030 (46.2 kilobases [kb]) were localized by mapping an 11.5-kb deletion that accompanied the transition of Lactococcus lactis LMA12-4 transconjugants (M. E. Sanders, P. J. Leonard, W. D. Sing, and T. R. Klaenhammer, Appl. Environ. Microbiol. 52:1001-1007, 1986) from phage resistance to phage sensitivity. The deleted 34.7-kb replicon (pTR2023, Hsp-) retained its conjugative ability, demonstrating that the phage resistance and conjugal transfer determinants were genetically distinct. The Hsp region of pTT2030, which was contained within a 13.6-kb BglII fragment, was cloned into the BamHI site of bacteriophage lambda EMBL3, and Hsp was subcloned into the Escherichia coli-Streptococcus shuttle vector pSA3. The recombinant plasmids pTK6 and pTK9 were recovered in E. coli HB101 and contained a 13.6-kb insert in opposite orientations. L. Lactis MG1363 transformants carrying pTK6 or pTK9 exhibited a significant reduction in plaque size, in addition to a slight reduction in the efficiency of plaquing for both prolate and small isometric phages. Phenotypic reactions observed for the recombinant plasmids suggest that pTR2030-encoded Hsp acts similarly against both prolate and small isometric phages. Tn5 mutagenesis was used to define the region essential for the expression of the Hsp+ phenotype. Any of four insertions within a 3-kb region resulted in the loss of phage resistance, whereas a further 26 insertions outside this locus had no effect on Hsp expression. In vitro deletion analysis confirmed that the 3-kb region contained all the information necessary for the observed resistance.     

Characterization of insertion sequence IS946, an Iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030.

The self-transmissible plasmid pTR2030 mobilized nonconjugative heterologous cloning vectors pGK12 (Cmr Emr) and pSA3 (Emr) at frequencies of 10(-5) to 10(-6) per input donor. Transconjugants harbored a 51- or 58-kilobase (kb) plasmid not found in the parental strains that cotransferred at high frequency with Cmr Emr and pTR2030-encoded phage resistance (Hsp+) in second-round matings (10(-1) per input donor). Restriction endonuclease mapping and DNA-DNA hybridization identified the 51- to 58-kb plasmids as pTR2030::vector cointegrates. Examination of four cointegrates indicated that pGK12 and pSA3 had inserted within two locations on pTR2030. Resolution of the cointegrates generated vector derivatives containing a 0.8-kb insert of pTR2030 DNA. Restriction analyses of several resolution plasmids indicated that the 0.8-kb element had inserted into various positions within pGK12 and pSA3 and in certain cases had inactivated the Cmr or Emr marker of pGK12. A conjugative mobilization assay demonstrated that the 0.8-kb element, designated IS946, mediated transpositional recombination. Nucleotide sequence determination identified IS946 as an 808-base-pair (bp) insertion sequence sharing ca. 96% homology with lactococcal insertion sequence ISS1. IS946 differed by 27 and 31 bp from ISS1S and ISS1T, respectively, and in 2 of 226 amino acids in the deduced sequence of the putative transposase. IS946 has perfect 18-bp terminal inverted repeats, identical to ISS1, and similarly generated 8-bp direct repeats of the target site upon insertion.     

Localization, cloning, and expression of genetic determinants for bacteriophage resistance (Hsp) from the conjugative plasmid pTR2030.

Genetic determinants for a bacteriophage resistance mechanism (Hsp+) encoded by plasmid pTR2030 (46.2 kilobases [kb]) were localized by mapping an 11.5-kb deletion that accompanied the transition of Lactococcus lactis LMA12-4 transconjugants (M. E. Sanders, P. J. Leonard, W. D. Sing, and T. R. Klaenhammer, Appl. Environ. Microbiol. 52:1001-1007, 1986) from phage resistance to phage sensitivity. The deleted 34.7-kb replicon (pTR2023, Hsp-) retained its conjugative ability, demonstrating that the phage resistance and conjugal transfer determinants were genetically distinct. The Hsp region of pTT2030, which was contained within a 13.6-kb BglII fragment, was cloned into the BamHI site of bacteriophage lambda EMBL3, and Hsp was subcloned into the Escherichia coli-Streptococcus shuttle vector pSA3. The recombinant plasmids pTK6 and pTK9 were recovered in E. coli HB101 and contained a 13.6-kb insert in opposite orientations. L. Lactis MG1363 transformants carrying pTK6 or pTK9 exhibited a significant reduction in plaque size, in addition to a slight reduction in the efficiency of plaquing for both prolate and small isometric phages. Phenotypic reactions observed for the recombinant plasmids suggest that pTR2030-encoded Hsp acts similarly against both prolate and small isometric phages. Tn5 mutagenesis was used to define the region essential for the expression of the Hsp+ phenotype. Any of four insertions within a 3-kb region resulted in the loss of phage resistance, whereas a further 26 insertions outside this locus had no effect on Hsp expression. In vitro deletion analysis confirmed that the 3-kb region contained all the information necessary for the observed resistance.     

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.     

Naturally occurring lactococcal plasmid pAH90 links bacteriophage resistance and mobility functions to a food-grade selectable marker

The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866-876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.     

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.     

Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r1t Temperate Bacteriophage but Not Induction of r1t 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 r₁t, 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 r₁t. Strain R1Cs and a derivative of this strain that was relysogenized with r₁t, designated R1Cs(r₁t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r₁t) were evaluated for sensitivity to r₁t phage and induction of r₁t prophage, respectively. The temperate phage r₁t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r₁t lysogen [T-R1Cs(r₁t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r₁t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r₁t 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.     

Naturally Occurring Lactococcal Plasmid pAH90 Links Bacteriophage Resistance and Mobility Functions to a Food-Grade Selectable Marker

The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866–876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.     

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.     

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.     

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.