Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40.

The lactococcal plasmid pNP40, from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp. lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity. Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs). Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity. DNA sequence analysis of this region revealed the presence of a single complete ORF. The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity. The mechanisms encoded by pPG01 and pCG1 in L. lactis subsp. lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively. Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition. The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.     

The Lactococcal Plasmid pNP40 Encodes a Third Bacteriophage Resistance Mechanism, One Which Affects Phage DNA Penetration

The lactococcal plasmid pNP40 mediates insensitivity to (phi)c2 by an early-acting phage resistance mechanism in addition to the previously identified abortive infection system, AbiF, in the Lactococcus lactis subsp. lactis MG1614 background. A second abortive infection determinant on pNP40, AbiE, does not confer resistance to (phi)c2. The early-acting mechanism on pNP40 does not prevent phage adsorption nor does it appear to operate by restriction/modification. Phage DNA was not detected in pNP40-containing cells until 30 min following exposure to (phi)c2 compared with 5 min in a sensitive host; however, electroporation of phage DNA into resistant hosts resulted in the release of phage progeny from a dramatically elevated number of cells compared with conventionally infected hosts. It appears therefore that pNP40 encodes a novel phage resistance mechanism which blocks DNA penetration specifically for (phi)c2.     

Construction of a Bacteriophage-Resistant Derivative of Lactococcus lactis subsp. lactis 425A by Using the Conjugal Plasmid pNP40

Lactococcus lactis subsp. lactis 425A is an atypical strain which excretes a high concentration of α-acetolactate when grown in milk. The conjugative lactococcal plasmid pNP40, which encodes phage and nisin resistance, was introduced to strain 425A by conjugation, using resistance to phage and nisin as a selection. No phage-nisin resistance mutants were encountered. Transconjugants display complete resistance at both 21 and 39°C to those phage previously identified as lytic for 425A. Transconjugants lose their resistance characteristics when spontaneously cured of pNP40. The commercially important property of 425A—production of high levels of α-acetolactic acid—is unaffected by the presence of pNP40.     

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage.

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.     

Restriction enzyme analysis of lactose and bacteriocin plasmids from Streptococcus lactis subsp. diacetylactis WM4 and cloning of BclI fragments coding for bacteriocin production.

The 131.1-kilobase (kb) bacteriocin production (Bac) plasmid pNP2 and the 63.6-kb lactose metabolism (Lac) plasmid pCS26, from Streptococcus lactis subsp. diacetylactis WM4, as well as pWN8, a 116.7-kb recombinant plasmid from a Lac+ transconjugant, were analyzed with restriction enzymes to determine the origin of pWN8. Plasmid pWN8 conferred a Lac+ Bac- phenotype, contained DNA derived from pCS26 and pNP2, and, like pNP2, exhibited self-transmissibility (Tra+). In cloning attempts, Bac+ transformant S. lactis KSH1 was isolated. The recombinant plasmid, pKSH1, contained three BclI fragments from pNP2. Bac- transformants which individually contained each of the three fragments were also identified. Comparison of restriction maps of pKSH1 and pNP2 revealed an 18.4-kb region common to both plasmids, involving two of the three BclI fragments. S. lactis KSH1 also exhibited greater inhibitory activity against the indicator strain S. diacetylactis 18-16 than did a strain containing the 131.1-kb Bac plasmid.     

Restriction enzyme analysis of lactose and bacteriocin plasmids from Streptococcus lactis subsp. diacetylactis WM4 and cloning of BclI fragments coding for bacteriocin production

The 131.1-kilobase (kb) bacteriocin production (Bac) plasmid pNP2 and the 63.6-kb lactose metabolism (Lac) plasmid pCS26, from Streptococcus lactis subsp. diacetylactis WM4, as well as pWN8, a 116.7-kb recombinant plasmid from a Lac+ transconjugant, were analyzed with restriction enzymes to determine the origin of pWN8. Plasmid pWN8 conferred a Lac+ Bac- phenotype, contained DNA derived from pCS26 and pNP2, and, like pNP2, exhibited self-transmissibility (Tra+). In cloning attempts, Bac+ transformant S. lactis KSH1 was isolated. The recombinant plasmid, pKSH1, contained three BclI fragments from pNP2. Bac- transformants which individually contained each of the three fragments were also identified. Comparison of restriction maps of pKSH1 and pNP2 revealed an 18.4-kb region common to both plasmids, involving two of the three BclI fragments. S. lactis KSH1 also exhibited greater inhibitory activity against the indicator strain S. diacetylactis 18-16 than did a strain containing the 131.1-kb Bac plasmid.     

A genetic dissection of the LlaJI restriction cassette reveals insights on a novel bacteriophage resistance system

Background  

Restriction/modification systems provide the dual function of protecting host DNA against restriction by methylation of appropriate bases within their recognition sequences, and restriction of foreign invading un-methylated DNA, such as promiscuous plasmids or infecting bacteriphage. The plasmid-encoded LlaJI restriction/modification system from Lactococcus lactis recognizes an asymmetric, complementary DNA sequence, consisting of 5'GACGC'3 in one strand and 5'GCGTC'3 in the other and provides a prodigious barrier to bacteriophage infection. LlaJI is comprised of four similarly oriented genes, encoding two 5mC-MTases (M1.LlaJI and M2.LlaJI) and two subunits responsible for restriction activity (R1.LlaJI and R2.LlaJI). Here we employ a detailed genetic analysis of the LlaJI restriction determinants in an attempt to characterize mechanistic features of this unusual hetero-oligomeric endonuclease.     

Use of M13mp phages to study gene regulation, structure and function: cloning and recombinational analysis of genes of the Salmonella typhimurium histidine operon

A restriction map was determined for a phi 80 lambda dhis transducing phage DNA carrying the Salmonella typhimurium histidine operon. DNA fragments containing the promoter/regulatory region and the first two structural genes of the histidine operon (hisOGD) were identified by their ability to direct regulated synthesis of histidinol dehydrogenase (product of hisD) in a coupled in vitro protein synthesizing system. A 3.1-kb SalI-EcoRI restriction fragment containing the hisOGD region, was subcloned into phage M13mp8 and M13mp9 RF DNAs. Methods are described for shuttling mutant and wild-type bacterial DNA sequences between the M13mp::his phage and host bacterial genomes. Of novel importance is the use of the phage M13 gene II amber mutation to obtain integration of the M13mp::his phage genome into the homologous his region of the bacterial chromosome following transduction of recipients lacking an amber suppressor. This method can be used to facilitate allele replacement with genes carried on M13 transducing phages.     

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.     

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).     

Increasing phage resistance of cheese starters : a case study using Lactococcus lactis DPC4268

This study serves as an example of strategies used to increase the phage resistance of an important Irish Cheddar cheese starter, Lactococcus lactis DPC4268. It describes the emergence and persistence of a lytic bacteriophage, 4268, that has a relatively large burst size and exhibits no homology to the most common phage types encountered in Irish cheese plants. Inherent difficulties were encountered that prevented the effective introduction of conjugative phage-resistance plasmids pNP40 and pMRC01 to strain DPC4268. In fact, pNP40-associated Abi systems were naturally present in six of 19 starters. Control of phage 4268 was eventually achieved by generating a mutant of DPC4268, which was subsequently used for cheese manufacture.     

Two genes involved in the phase-variable phi C31 resistance mechanism of Streptomyces coelicolor A3(2).

The phage growth limitation (Pgl) system of Streptomyces coelicolor confers resistance to phi C31 and its homoimmune phages. The positions of the pgl genes within a 16-kb clone of S. coelicolor DNA were defined by subcloning, insertional inactivation, and deletion mapping. Nucleotide sequencing and functional analysis identified two genes, pglY and pglZ, required for the Pgl+ (phage-resistant) phenotype. pglY and pglZ, which may be translationally coupled, are predicted to encode proteins with M(r)S of 141,000 and 104,000, respectively. Neither protein shows significant similarity to other known proteins, but PglY has a putative ATP/GTP binding motif. The pglY and pglZ genes are cotranscribed from a single promoter which appears to be constitutive and is not induced by phage infection.     

Characterization of the nuclear ribosomal DNA of Euglena gracilis

A phage lambda recombinant library containing Euglena gracilis genomic DNA was screened for nuclear rDNA sequences. A recombinant phage was isolated that contained an 11.5-kb nuclear rDNA sequence. The 11.5-kb insert was mapped with restriction endonucleases and was shown to represent a complete rDNA repeat unit that carried the genes for the 19S, 25S, 5.8 S and 5 S cytoplasmic rRNAs. The 2000 rDNA repeat units per haploid genome are organized in the form of identical tandem repeats.     

Expression of the uvrB gene of Escherichia coli: in vitro construction of a pMB9 uvrB plasmid.

Bacteriophage lambdab2att2 [lambdab2cI857intam6(deltabioAB)bioFCD+uvrB+phr+] codes for a function(s) that confers UV resistance (Uvr+) and reactivation of irradiated phage (Hcr+) to an Uvr-Hcr-Escherichia coli strain. It was demonstrated that these functions are expressed under the control of bacterial regulatory elements located on lambdab2att2 DNA. The location of the E. coli uvrB gene on the DNA of this transducing phage was established by heteroduplex and restriction-enzyme analyses. Recombinant DNA molecules were constructed in vitro from plasmid pMB9 (Tcr), as the vector, and an EcoRI fragment (Eco-RI-F) of lambdab2att2 DNA. The resulting plasmid, designated pNP5, has a molecular weight of 5.1 X 10(6) and replicates in a relaxed fashion. Transformation of E. coli uvrB with plasmid pNP5 resulted in clones that are Uvr+ Tcr. Irradiation of bacteria transformed with plasmid pNP5 with low UV doses revealed a complete restoratation of the Uvr+ phenotype by the presence of the cloned EcoRI-F DNA, while only a partial restoration was observed after irradiation with high UV doses. Likewise, the Hcr+ character was also partially restored due to the presence of pNP5. No correlation was found between the acquired Uvr+, Hcr+ properties, and the presence of correndonuclease II activity in an extract of bacteria that harbor plasmid pNP5.     

Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2.

A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp. lactis C2 was transformed with a chromosomal library of the wild-type, phage-sensitive strain. Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive. A cause-and-effect relationship between the cloned chromosomal fragments and the phage-sensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment. The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases. The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb. Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb. The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region. Further subcloning indicated that a single EcoRI site within the 4.5-kb region must lie within the gene or its promoter. The required 4.5-kb region was sequenced and found to code for one partial and two complete open reading frames. The gene required for complementation was functionally mapped by Tn5 mutagenesis and localized to one of the two complete open reading frames, which was designated pip (an acronym for phage infection protein). pip is 2,703 bases in length. Potential promoters start 206 and 212 bases upstream of the open reading frame. A ribosome binding site and a seven-base spacer precede the GTG (Val) translation initiation codon. The amino acid sequence deduced from the gene has 901 residues and an M(r) of 99,426. Hydropathy analysis revealed four to six potential membrane-spanning regions, one near the amino terminus and the others at the extreme carboxyl terminus. The amino terminus has characteristics of a signal sequence. The putative protein would have a 650-residue, central polar domain.     

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.     

Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478.

A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, SalI, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb SalI fragment of R478.