Characterization of AbiR, a Novel Multicomponent Abortive Infection Mechanism Encoded by Plasmid pKR223 of Lactococcus lactis subsp. lactis KR2

The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, “early”-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.     

Involvement of the LlaKR2I methylase in expression of the AbiR bacteriophage defense system in Lactococcus lactis subsp. lactis biovar diacetylactis KR2

The native lactococcal plasmid, pKR223, from Lactococcus lactis subsp. lactis biovar diacetylactis KR2 encodes two distinct bacteriophage-resistant mechanisms, the LlaKR2I restriction and modification (R/M) system and the abortive infection (Abi) mechanism, AbiR, that impedes bacteriophage DNA replication. This study completed the characterization of AbiR, revealing that it is the first Abi system to be encoded by three genes, abiRa, abiRb, and abiRc, arranged in an operon and that it requires the methylase gene from the LlaKR2I R/M system. An analysis of deletion and insertion clones demonstrated that the AbiR operon was toxic in L. lactis without the presence of the LlaKR2I methylase, which is required to protect L. lactis from AbiR toxicity. The novelty of the AbiR system resides in its original gene organization and the unusual protective role of the LlaKR2I methylase. Interestingly, the AbiR genetic determinants are flanked by two IS982 elements generating a likely transposable AbiR composite. This observation not only substantiated the novel function of the LlaKR2I methylase in the AbiR system but also illustrated the evolution of the LlaKR2I methylase toward a new and separate cellular function. This unique structure of both the LlaKR2I R/M system and the AbiR system may have contributed to the evolution of the LlaKR2I methylase toward a novel role comparable to that of the cell cycle-regulated methylases that include Dam and CcrM methylases. This new role for the LlaKR2I methylase offers a unique snapshot into the evolution of the cell cycle-regulated methylases from an existing R/M system.     

Localization of Separate Genetic Loci for Reduced Sensitivity towards Small Isometric-Headed Bacteriophage sk1 and Prolate-Headed Bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2

The mechanism of reduced sensitivity to the small isometric-headed bacteriophage sk1 encoded on a 19-kilobase (kb) HpaII fragment subcloned from pKR223 of Lactococcus lactis subsp. lactis KR2 was examined. The reduced sensitivity to phage sk1 was due to a modest restriction/modification (R/M) system that was not active against prolate-headed phage c2. The genetic loci for the R/M system against sk1 and the abortive phage infection (Abi) mechanism effective against phage c2 were then localized by restriction mapping, subcloning, and deletion analysis. The restriction gene was localized to a region of a 2.7-kb EcoRV fragment and included an EcoRI site within that fragment. The modification gene was found to be physically separable from the restriction gene and was present on a 1.75-kb BstEII-XbaI fragment. The genetic locus for the Abi phenotype against phage c2 was localized to a region containing a 1.3-kb EcoRI fragment. Attempts to clone the c2 Abi mechanism independent of the sk1 R/M system were unsuccessful, suggesting that expression of the abi genes required sequences upstream of the modification gene. Some pGBK17 (vector pGB301 plus a 19-kb HpaII insert fragment) transformants exhibited the R/M system against phage sk1 but lost the Abi mechanism against phage c2. These transformants contained a 1.2- to 1.3-kb insertion in the Abi region. The data identified genetic loci on a cloned 19-kb HpaII fragment responsible for restriction activity and for modification activity against a small isometric-headed phage and for Abi activity against prolate-headed phage c2. A putative insertion element was also found to inactivate the abi gene(s).     

A Starter Culture Rotation Strategy Incorporating Paired Restriction/ Modification and Abortive Infection Bacteriophage Defenses in a Single Lactococcus lactis Strain

Three derivatives of Lactococcus lactis subsp. lactis NCK203, each with a different pair of restriction/ modification (R/M) and abortive infection (Abi) phage defense systems, were constructed and then rotated in repeated cycles of a milk starter culture activity test (SAT). The rotation proceeded successfully through nine successive SATs in the presence of phage and whey containing phage from previous cycles. Lactococcus cultures were challenged with 2 small isometric-headed phages, (phi)31 and ul36, in one rotation series and with a composite of 10 industrial phages in another series. Two native lactococcal R(sup+)/M(sup+) plasmids, pTRK68 and pTRK11, and one recombinant plasmid, pTRK308, harboring a third distinct R/M system were incorporated into three NCK203 derivatives constructed separately for the rotation. The R(sup+)/M(sup+) NCK203 derivatives were transformed with high-copy-number plasmids encoding four Abi genes, abiA, abiC, per31, and per50. Various Abi and R/M combinations constructed in NCK203 were evaluated for their effects on cell growth, level of phage resistance, and retardation of phage development during repeated cycles of the SAT. The three NCK203 derivatives chosen for use in the SAT exhibited additive effects of the R/M and Abi phenotypes against sensitive phages. In such combinations, phage escaping restriction are prevented from completing their infective cycle by an abortive response that kills the host cell. The rotation series successfully controlled modified, recombinant, and mutant phages which were resistant to any one of the individual defense systems by presenting a different set of R/M and Abi defenses in the next test of the rotation.     

Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis.

The natural plasmid pSRQ800 isolated from Lactococcus lactis subsp. lactis W1 conferred strong phage resistance against small isometric phages of the 936 and P335 species when introduced into phage-sensitive L. lactis strains. It had very limited effect on prolate phages of the c2 species. The phage resistance mechanism encoded on pSRQ800 is a temperature-sensitive abortive infection system (Abi). Plasmid pSRQ800 was mapped, and the Abi genetic determinant was localized on a 4.5-kb EcoRI fragment. Cloning and sequencing of the 4.5-kb fragment allowed the identification of two large open reading frames. Deletion mutants showed that only orf1 was needed to produce the Abi phenotype. orf1 (renamed abiK) coded for a predicted protein of 599 amino acids (AbiK) with an estimated molecular size of 71.4 kDa and a pI of 7.98. DNA and protein sequence alignment programs found no significant homology with databases. However, a database query based on amino acid composition suggested that AbiK might be in the same protein family as AbiA. No phage DNA replication nor phage structural protein production was detected in infected AbiK+ L. lactis cells. This system is believed to act at or prior to phage DNA replication. WHen cloned into a high-copy vector, AbiK efficiency increased 100-fold. AbiK provides another powerful tool that can be useful in controlling phages during lactococcal fermentations.     

Characterization of a novel type II restriction-modification system, Sth368I, encoded by the integrative element ICESt1 of Streptococcus thermophilus CNRZ368

A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to phiST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5'-GATC-3'. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5'-GATC-3' was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5'-GATC-3'. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5'-GATC-3' and is related to the Sau3AI and LlaKR2I restriction systems.     

Identification of a recA homolog (recALP) on the conjugative lactococcal phage resistance plasmid pNP40: evidence of a role for chromosomally encoded recAL in abortive infection.

The determinants for two bacteriophage resistance mechanisms, AbiE and AbiF, are separated by approximately 3,300 nucleotides on the lactococcal plasmid pNP40 (P. Garvey, G.F. Fitzgerald, and C. Hill, Appl. Environ. Microbiol. 61:4321-4328, 1995). DNA sequence analysis of the intervening region led to the identification of two open reading frames (ORFs) which are transcribed in the opposite direction to the Abi determinants. One of these ORFs encodes a recA homolog (designated recALP). This is the first report of a recA-like determinant located to a plasmid. The second ORF (orfU) shares homology with the umuC gene of the SOS response. Analysis of a number of lactococcal strains confirmed the presence of recALP-like sequences in at least two other lactococcal strains. The proximity of the recA and umuC homologs suggested a possible role in the phase resistance encoded by the Abi determinants. However, no evidence was obtained to demonstrate a function for either ORF in the expression of either AbiE or AbiF. Nor could the recALP gene restore resistance to mitomycin in a recA-deficient lactococcal strain, VEL1122. Interestingly, it was shown that the chromosomally encoded recA is necessary for complete expression of the AbiF phenotype, confirming a role for RecA in this abortive infection system.     

Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.

The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by Tn5 mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. lactis subsp. lactis ME2) and abi416 (Abi+, from L. lactis subsp. lactis IL416). Unlike abiA, the action of abiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.     

AbiG, a genotypically novel abortive infection mechanism encoded by plasmid pCI750 of Lactococcus lactis subsp. cremoris UC653.

AbiG is an abortive infection (Abi) mechanism encoded by the conjugative plasmid pCI750 originally isolated from Lactococcus lactis subsp. cremoris UC653. Insensitivity conferred by this Abi manifested itself as complete resistance to phi 712 (936 phage species) with only partial resistance to phi c2 (c2 species). The mechanism did not inhibit phage DNA replication. The smallest subclone of pCI750 which expressed the Abi phenotype contained a 3.5-kb insert which encoded two potential open reading frames. abiGi (750 bp) and abiGii (1,194 bp) were separated by 2 bp and appeared to share a single promoter upstream of abiGi. These open reading frames showed no significant homology to sequences of either the DNA or protein databases; however, they did exhibit the typical low G+C content (29 and 27%, respectively) characteristic of lactococcal abi genes. In fact, the G+C content of a 7.0-kb fragment incorporating the abiG locus was 30%, which may suggest horizontal gene transfer from a species of low G+C content. In this context, it is notable that remnants of IS elements were observed throughout this 7.0-kb region.     

Phage abortive infection mechanism from Lactococcus lactis subsp. lactis, expression of which is mediated by an Iso-ISS1 element.

A 5-kb DNA fragment conferring a phage abortive infection phenotype (Abi+) has been cloned from Lactococcus lactis subsp. lactis IL416. The Abi+ determinant was subcloned on a 2-kb fragment which carried an Iso-ISS1 element and an open reading frame of 753 bp designated ORFX. Deletion within ORFX entailed the loss of the Abi+ phenotype, establishing that ORFX is the structural abi-416 gene. The expression of abi-416 was shown to be mediated by the Iso-ISS1 element, which contains a sequence fitting the consensus sequence for gram-positive promoters.     

Phage abortive infection mechanism from Lactococcus lactis subsp. lactis, expression of which is mediated by an Iso-ISS1 element.

A 5-kb DNA fragment conferring a phage abortive infection phenotype (Abi+) has been cloned from Lactococcus lactis subsp. lactis IL416. The Abi+ determinant was subcloned on a 2-kb fragment which carried an Iso-ISS1 element and an open reading frame of 753 bp designated ORFX. Deletion within ORFX entailed the loss of the Abi+ phenotype, establishing that ORFX is the structural abi-416 gene. The expression of abi-416 was shown to be mediated by the Iso-ISS1 element, which contains a sequence fitting the consensus sequence for gram-positive promoters.     

Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.     

Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.     

Identification of three different plasmid-encoded restriction/modification systems in Streptococcus lactis subsp. cremoris W56

Abstract Streptococcus lactis subsp. cremoris W56 ( S. cremoris W56) is a strain partially resistant to phage attack. Derivatives which had lost either plasmid pJW563 or pJW566 no longer expressed the restriction and modification systems encoded by these plasmids. Genetic evidence for the correlation between the plasmids and the R/M systems was obtained by transformation. In addition, a third R/M system was discovered among the transformants and was shown to be encoded by pJW565. Thus, genetic evidence for at least 3 distinct R/M systems encoded by plasmids in S. cremoris W56 is presented. One of the R/M-systems showed stronger restriction of the isometric phage p2 than of the prolate phage c2. The other two systems restricted both classes of phages with equal efficiencies.     

The ability of the plasmid-encoded restriction and modification system LlaBIII to protect Lactococcus lactis against bacteriophages

AIMS: To investigate the potential of the plasmid-encoded restriction and modification (R/M) system LlaBIII to protect Lactococcua lactis against bacteriophages during milk fermentations. METHODS AND RESULTS: The R/M system LlaBIII on plasmid pJW566 was cloned with a chloramphenicol cassette, resulting in plasmid pJK1. When introduced into L. lactis strains, pJK1 conferred increased phage resistance against the three most common lactococcal phage species 936, c2, and P335 and three unclassified industrial phages. The growth of the strains in RSM was not affected by the presence of plasmid pJK1. CONCLUSIONS: The plasmid-encoded R/M system LlaBIII has great ability to protect L. lactis strains against bacteriophages in milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluates the ability of the LlaBIII R/M system to function as a phage defence mechanism which is an essential step prior to considering utilizing it for improving starter cultures.     

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.     

Cloning and sequencing of LlaDCHI [corrected] restriction/modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system.

The natural 7.8-kb plasmid pSRQ700 was isolated from Lactococcus lactis subsp. cremoris DCH-4. It encodes a restriction/modification system named LlaDCHI [corrected]. When introduced into a phage-sensitive L. lactis strain, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species, namely, 936, c2, and P335. The LlaDCHI [corrected] endonuclease was purified and found to cleave the palindromic sequence 5'-GATC-3'. It is an isoschizomer of Streptococcus pneumoniae DpnII. The plasmid pSRQ700 was mapped, and the genetic organization of LlaDCHI [corrected] was localized. Cloning and sequencing of the entire LlaDCHI [corrected] system allowed the identification of three open reading frames. The three genes (llaIIA, llaIIB, and llaIIC) overlapped and are under one putative promoter. A putative terminator was found at the end of llaIIC. The genes llaIIA and llaIIB coded for m6A methyltransferases, and llaIIC coded for an endonuclease. The LlaDCHI [corrected] system shares strong genetic similarities with the DpnII system. The deduced amino acid sequence of M.LlaIIA was 75% identical with that of M.DpnII, whereas M.LlaIIB was 88% identical with M.DpnA. However, R.LlalII shared only 31% identity with R.DpnII.     

Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi(+)) with phage phi31. HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.