Two loci in the genome of the transposable phage B39 of Pseudomonas aeruginosa affecting the process of integration. I. Study of the properties of phages with the Pde- phenotype and mapping of the rms site

Mutants and recombinants of transposable Pseudomonas aeruginosa bacteriophage B39 with a specific phenotype Pde- (pleiotropic developmental effect) were studied. Pde- phages produce clear minute plaques on lawns of P. aeruginosa PAO1 and fail to grow in cells of PAO1 harbouring Rms 163 (Inc P5) plasmid. Pde+ character is under control of the two loci in phage genome which were designated pdeX and pdeY. In hybrid phages the pdeX and pdeY loci originating from different transposable phages (pdeX from B39 and pdeY from PH132) do not accomplish their function and, as a result, the hybrid phages have the Pde- phenotype. The frequency of integration (f.o.i.) of Pde- phages into bacterial chromosome is lower than f.o.i. for Pde+ phages, as well as the frequency of stable lysogenization of infected bacteria; lytic development of the Pde- phages is also limited. The great difference among the transposable phages in their reaction to the presence of Rms163 plasmid is caused by some differences in the specific rms site in the phage genome. The site is located inside the interval 1.1-3.9 kb of the physical genome map, being closely linked to cI gene of phage B39. The growth of Pde- phages in cells with Rms163 can be restored, due to additional mutations in phage genes affecting lysogenization.     

Restriction alleviation by bacteriophages lambda and lambda reverse.

Deletion analysis indicated that the phage lambda restriction alleviation gene(s) ral resides between the cIII and N genes. The Ral+ phenotype was expressed only when lambda ral+ carried a modification such that it was resistant to restriction by the host specificity system. Under these conditions, Ral function protected superinfecting unmodified phages from restriction by EcoK or EcoB but not from restriction by EcoP1. Ral-protected phage DNA was not concomitantly K and B modified, but rather received only the modification specified by the system of the restricting host. Possible mechanisms for Ral action are discussed. Of the other lambdoid phages tested, the hybrid phage lambda rev had Ral activity, whereas phi 80vir and one lambda-P22 hybrid did not. The restriction alleviation activity of lambda rev called Lar, may be the same as the activity expressed in sbcA- strains of Escherichia coli, but it was functionally separable from exonuclease VIII activity (the product of the recE gene), which is also expressed in sbcA- strains.     

Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. The establishment of a lysogenic state and the effectiveness of lytic development

Expression of transposable phages (TP) of Pseudomonas aeruginosa in the cells of P. putida was studied. The high efficiency of phage lytic development was shown both as a consequence of zygotic induction after transfer of the RP4::TPc+ plasmid into nonlysogenic recipients, and as a result of heat induction of lysogens PpG1 (D3112cts15). The high phage yield (20-25 particles of D3112cts phage per one cell of P. putida) is an evidence for a high level of transposition in the cells of this bacterial species. Plasmids RP4::TP are transferred into cells of PpG1 and PAO1 with similar frequency. However, the efficiency of establishment of the lysogenic state is lower in PpG1. Transposable phages of P. aeruginosa can integrate into the chromosome of PpG1 producing stable inducible lysogens. The presence of RP4 in the P. putida cells is not necessary for expression of transposable phages. The transposable phage D3112cts15 can be used in experiments of interspecies transduction of plasmids and chromosomal genes.     

Identification of a replication protein and repeats essential for DNA replication of the temperate lactococcal bacteriophage TP901-1

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.     

Structural characterization and assembly of the distal tail structure of the temperate lactococcal bacteriophage TP901-1

The tail structures of bacteriophages infecting gram-positive bacteria are largely unexplored, although the phage tail mediates the initial interaction with the host cell. The temperate Lactococcus lactis phage TP901-1 of the Siphoviridae family has a long noncontractile tail with a distal baseplate. In the present study, we investigated the distal tail structures and tail assembly of phage TP901-1 by introducing nonsense mutations into the late transcribed genes dit (orf46), tal(TP901-1) (orf47), bppU (orf48), bppL (orf49), and orf50. Transmission electron microscopy examination of mutant and wild-type TP901-1 phages showed that the baseplate consisted of two different disks and that a central tail fiber is protruding below the baseplate. Evaluation of the mutant tail morphologies with protein profiles and Western blots revealed that the upper and lower baseplate disks consist of the proteins BppU and BppL, respectively. Likewise, Dit and Tal(TP901-1) were shown to be structural tail proteins essential for tail formation, and Tal(TP901-1) was furthermore identified as the tail fiber protein by immunogold labeling experiments. Determination of infection efficiencies of the mutant phages showed that the baseplate is fundamental for host infection and the lower disk protein, BppL, is suggested to interact with the host receptor. In contrast, ORF50 was found to be nonessential for tail assembly and host infection. A model for TP901-1 tail assembly, in which the function of eight specific proteins is considered, is presented.     

Identification of the lower baseplate protein as the antireceptor of the temperate lactococcal bacteriophages TP901-1 and Tuc2009

The first step in the infection process of tailed phages is recognition and binding to the host receptor. This interaction is mediated by the phage antireceptor located in the distal tail structure. The temperate Lactococcus lactis phage TP901-1 belongs to the P335 species of the Siphoviridae family, which also includes the related phage Tuc2009. The distal tail structure of TP901-1 is well characterized and contains a double-disk baseplate and a central tail fiber. The structural tail proteins of TP901-1 and Tuc2009 are highly similar, but the phages have different host ranges and must therefore encode different antireceptors. In order to identify the antireceptors of TP901-1 and Tuc2009, a chimeric phage was generated in which the gene encoding the TP901-1 lower baseplate protein (bppL(TP901-1)) was exchanged with the analogous gene (orf53(2009)) of phage Tuc2009. The chimeric phage (TP901-1C) infected the Tuc2009 host strain efficiently and thus displayed an altered host range compared to TP901-1. Genomic analysis and sequencing verified that TP901-1C is a TP901-1 derivative containing the orf53(2009) gene in exchange for bppL(TP901-1); however, a new sequence in the late promoter region was also discovered. Protein analysis confirmed that TP901-1C contains ORF53(2009) and not the lower baseplate protein BppL(TP901-1), and it was concluded that BppL(TP901-1) and ORF53(2009) constitute antireceptor proteins of TP901-1 and Tuc2009, respectively. Electron micrographs revealed altered baseplate morphology of TP901-1C compared to that of the parental phage.     

Temperate phages TP901-1 and phiLC3, belonging to the P335 species, apparently use different pathways for DNA injection in Lactococcus lactis subsp. cremoris 3107

Five mutants of Lactococcus lactis subsp. cremoris 3107 resistant to phage TP901-1 were obtained after treatment with ethyl methanesulfonate. Two of the mutants were also resistant to phage phiLC3. The remaining three mutants were as sensitive as 3107. Mutants E46 and E100 did not adsorb the two phages. Mutants E119, E121 and E126 adsorbed phage phiLC3 as well as 3107 but phage TP901-1 with significantly reduced efficiency. All, except E46, could be lysogenized with phage TP901-BC1034, a derivative of TP901-1 harboring an erythromycin-resistance marker. However, the lysogenization frequency was 10(3)-10(4) fold higher for 3107 than for the mutants. Mitomycin C induction of lysogenized mutants 3107 indicated that phage propagation was not affected in these four mutants. Electron microscopy and analysis of total DNA of infected cells showed that DNA was liberated from the phage particle during infection of strain 3107 with TP901-1 and that intracellular phage DNA replication occurred. This was not the case for mutants E121 and E126. This strongly suggests that some step starting with triggering DNA release and ending with DNA injection is impaired during infection with TP901-1. As such impairment was not seen when infecting E119, E121 and E126 with phiLC3, we conclude that TP901-1 and phiLC3 either are differently triggered by their receptor or utilize different pathways of injection.     

Identification of a Replication Protein and Repeats Essential for DNA Replication of the Temperate Lactococcal Bacteriophage TP901-1

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.     

Evaluation of the usefulness of three collections of bacteriophages for typing methicillin-resistant Staphylococcus aureus,isolated in Moscow hospitals

The typing of S. aureus methicillin-resistant strains, isolated in different hospitals of Moscow; was carried out with the use of three collections of phages: the International Set of Phages; the set of phages of the International Center of S. aureus phage typing in London (L); and the experimental collection of phages of the Gamaleya Institute of Epidemiology and Microbiology in Moscow (M). In this study made with the use of both the phages of the International Diagnostic Set and phages L in the standard typing dose of 1 TP about 6% of the cultures under study proved to be sensitive. When the typing dose was increased to 100 TP the phages of the international diagnostic set lyzed 75.5% of the cultures. The typed strains were found to belong to phage types 77 (71.7%), 77/84/85 (19.6%) and 94/96 (6.5%). At a concentration of 100 TP phages L lyzed 83.7% of the cultures, but the dominating phage types could not be determined due to a great variety of phage markers. In contrast to the two preceding collections, the third phage collection M was composed in such a way that in the study of the investigated culture the specificity of its restriction modification was primarily evaluated and only then the presence of antiphage immunity was determined. This latter collection was used in the evaluation of 93.1% of the cultures. By the specificity of their restriction specification system the majority of them were classified with two new groups, heretofore not described. Only this collection M made it possible to differentiate epidemic and sporadic strains and to evaluate the epidemic situation in all 6 hospitals.     

Characteristics of phages-transposons of Pseudomonas aeruginosa belonging to 2 groups distinguished by DNA-DNA homology

14 new transposable phages (TP) were isolated from approx. 200 clinical isolates of Pseudomonas aeruginosa. The frequent occurrence of TP of P. aeruginosa has been confirmed. There are at least two different groups of TP, namely, the group of D3112 and that of B3. The distinctive features of phages belonging to the groups are as follows: 1) low level of DNA-DNA homology (less than 10%), the whole region of homology in phage genomes of different groups being located on right genome end (29-38 kb); only one of phages of the B3 group shows an additional homology with D3112 DNA outside the above mentioned region; 2) a variable DNA is observed on the left end of the B3 group phage genomes and no such DNA is revealed on the left end of genomes of the D3112 group phages; 3) all phages of the B3 group have specific type of interaction with RPL11 plasmid, which distinguish them from phages of the D3112 group; 4) phages belonging to the two groups differ greatly in their growth in cells harbouring pMG7 plasmid which mediates production of PaeR7 endonuclease and in the number of DNA sites sensitive to SalGI, PstI, BglII endonucleases. Since some of the B3 group phage genomes possess BamH1 sites, resistance to this enzyme cannot be regarded as a general characteristics for all TP of P. aeruginosa, as it was earlier proposed. Some aspects of modular hypothesis of bacteriophage evolution concerning, in particular, the ways of module formation are discussed.     

Comparative FISH-mapping of six expressed gene loci to river buffalo and sheep chromosomes.

Six expressed gene loci (NF1, CRYB1, CHRNB1, TP53, P4HB and GH1), recently assigned to cattle chromosome 19 by both radiation hybrid analysis and FISH-mapping, were comparatively FISH-mapped to river buffalo chromosome (BBU) 3p and sheep chromosome (OAR) 11, extending the physical map in these two important bovids. The six loci mapped to the same homoeologous chromosome bands of BBU 3p and OAR 11, and their gene order was centromere-NF1-CRYB1-CHNRB1-TP53-(GH1, P4HB).     

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.     

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively. Correspondence to: R. A. Ledford     

Effect of umuC mutations on targeted and untargeted ultraviolet mutagenesis in bacteriophage lambda

Mutagenesis of phage lambda towards clear-plaque phenotype (c+----c) results in two classes of mutants that can be distinguished genetically and morphologically. Indirect mutagenesis, i.e. mutagenesis of unirradiated phage lambda c+ stimulated by the ultraviolet irradiation of the Escherichia coli host, results in mixed bursts (c/c+) of turbid wild-type and clear-plaque mutant phages. Pure bursts of lambda c mutants are induced by irradiation of the phage genome. Irradiation of both phages and host bacteria stimulates the production of the two classes of mutant clones. We show that three different mutant alleles of the E. coli umuC gene only prevent the appearance of pure bursts of clear-plaque mutants, while mixed bursts are produced at least as frequently in umuC mutants as in the umuC+ parent.     

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.     

Nonsense and insertion mutants in the relA gene of E. coli: cloning relA.

We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.     

Phage response to CRISPR-encoded resistance in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.