Identification of four loci isolated from two Streptococcus thermophilus phage genomes responsible for mediating bacteriophage resistance

Sequence data derived from the Streptococcus thermophilus phages phiO1205 and phi7201 indicated that each of these phages contains a distinct DNA region dedicated to replication. Southern blotting experiments showed that phages infecting S. thermophilus may be divided into at least two groups, each containing the presumptive replication functions of either φO1205 (group I) or φ7201 (group II). Specific regions from the putative replication module of each of the two phages were examined for their ability to provide phage resistance.     

Improved generalized transducing bacteriophage for Caulobacter crescentus.

Caulobacter phage phi Cr30T, a temperature-sensitive derivative of the lytic, generalized transducing phage phi Cr30, was isolated as a double temperature-sensitive recombinant in a cross between phi Cr30ts1 and phi Cr30ts2, phi Cr30T mediated generalized transduction of Caulobacter crescentus at frequencies comparable to those of phi Cr30 and eliminated the requirement for irradiation of transducing lysates to prevent killing of transductants on the plate.     

Genetic characterization of a phi80 transducing bacteriophage carrying the histidine operon of Salmonella typhimurium.

A phi 80 transducing phage, phi 80imm lambdadhis, carrying the Salmonella his-gnd region, was characterized by immunity studies, tonB deletion analysis, and marker rescue analysis. Phi 80imm lambdadhis retains the phage immunity region of the phi 80-lambda hybrid phage from which it was derived. Bacterial genes replace most late phage genes. Deletion analysis shows the prophage gene order to be immlambda-his-gnd and indicates the orientation of the his operon to be hisOGDCBHAFIE-gnd. The structure of phi 80imm lambdadhis is remarkably similar to two independently isolated phi 80 phages that carry the his-gnd region of Escherichia coli and that, like phi80imm lambdahis, were derived by directed gene transposition to the tonB locus. A derivative of phi 80imm lambdadhis that is phi 80 immune is also reported.     

Characterization of generalized transducing phage phi w39 heteroimmune to phage P1 in Escherichia coli W39

Generalized transducing phage similar to phage P1 in Escherichia coli was isolated from E. coli W39, an antigenic test strain of the O121 group. This phage, designated phi w39, was reciprocally heteroimmune to phages P1 and P7, but nonreciprocally heteroimmune to phage D6. Transduction experiments using various R plasmids with different molecular weights suggested that phage phi w39 could transduce at least 65 megadaltons DNA. As in the case of P1 prophage, phi w39 prophage existed as a plasmid belonging to incompatibility group Y and carried a dnaB-like function. The molecular weight of phi w39 plasmid was nearly the same as that of plasmid, i.e., 58.6 megadaltons. Despite the pronounced structural and functional similarity of phages phi w39 and P1, restriction cleavage patterns of their genomes differed considerably.     

Characterization of Serratia entomophila Bacteriophages and the Phage-Resistant Mutant Strain BC4B

Successful large-scale fermentations of the bacterium Serratia entomophila for use in biological control of the soil-dwelling insect Costelytra zealandica has required the development of a phage-resistant mutant, BC4B. We report our investigations into S. entomophila phages and the nature of the phage resistance mechanism of strain BC4B. The parental strain of BC4B, A1MO2, was found to contain two previously unidentified prophages, (phi)9A and (phi)9B, which were UV inducible and also released spontaneously in large numbers. BC4B was shown to be completely cured of (phi)9A. Single lysogens of (phi)9A and (phi)9B were not homoimmune to any other S. entomophila phages. However, on the basis of DNA-DNA homology, all S. entomophila phages except (phi)CW3 were shown to have significant regions of homology and also packaged their DNA via pac-like mechanisms. The failure of phage particles to adsorb was identified as the basis of phage resistance in BC4B. In addition, it was demonstrated that all known S. entomophila phages are naturally temperature sensitive.     

Purification and characterization of repressor of temperate S. aureus phage phi11

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.     

Mur-LH, the Broad-Spectrum Endolysin of Lactobacillus helveticus Temperate Bacteriophage φ-0303

-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of -0303 in Escherichia coli DH5α. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of -0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCR amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages. Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium.     

Effects of the Recipient Strain and Ultraviolet Irradiation on Transduction Kinetics of the Penicillinase Plasmid of Staphylococcus aureus

When the penicillinase plasmid of Staphylococcus aureus PS 81(P(81))(T(81)) was transferred to its cured derivative of PS 81(N(P))(T(81)), there was a fivefold increase in the transduction frequency of penicillinase plasmid markers after ultraviolet (UV) irradiation of the phage instead of the expected decrease typical for plasmid-borne markers. These results were independent of the transducing phage, the donor, and the method of curing the recipient and were also obtained with a cured derivative of PS 80(PI(80)). With PS 52, a naturally occurring penicillin-sensitive strain, and a cured transductant of PS 52 as the recipients, typical plasmid kinetics were observed. The plasmid location of penicillinase plasmid markers in transductants was confirmed by their instability in ethidium bromide (EB). In a cross between isogenic plasmids (PI(258)penZ cad x PI(258)penI asa ero), transductants were doubly selected for cadmium and erythromycin resistances. There was a twofold increase in transduction frequency after UV irradiation of the transducing phage and an increase in the proportion of recombinant type transductants. CsCl-EB density centrifugation revealed that plasmid deoxyribonucleic acid (DNA) was present in PS 81(P(81))(N(T)) and its cured derivative [PS 81(N(P))(N(T))], but not in PS 52. Sucrose gradient analysis of plasmid DNA showed that the penicillinase plasmid of PS 81(P(81))(N(T)) was larger than the plasmid in its cured derivative. Thus, the cured derivative contains plasmid DNA which appears to recombine with the incoming plasmid, causing the rise in transduction frequency noted after UV irradiation of transducing phage.     

General transduction in Rhizobium meliloti

General transduction by phage phi M12 in Rhizobium meliloti SU47 and its derivatives is described. Cotransduction and selection for Tn5 insertions which are closely linked to specific loci were demonstrated. A derivative of SU47 carrying the recA::Tn5 allele of R. meliloti 102F34 could be transduced for plasmid R68.45 but not for chromosomally located alleles. Phage phi M12 is morphologically similar to Escherichia coli phage T4, and restriction endonuclease analysis indicated that the phage DNA was ca. 160 kilobases in size.     

VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi

We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.     

DNA sequence analysis, expression, distribution, and physiological role of the Xaa-prolyldipeptidyl aminopeptidase gene fromLactobacillus helveticus CNRZ32

Lactobacillus helveticus CNRZ32 possesses and Xaa-prolyldipeptidyl aminopeptidase (PepX), which releases amino-terminal dipeptides from peptides containing proline residues in the penultimate position. The PepX gene, designatedpepX, fromLb. helveticus CNRZ32 was sequenced. Analysis of the sequence identified a putative 2379-bppepX open-reading frame, which encodes a polypeptide of 793 amino acid residues with a deduced molecular mass of 88 111 Da, The gene shows significant sequence identity with sequencedpepX genes from lactic acid bacteria. The product of the gene contains a motif that is almost identical with the active-site motif of the serine-dependent PepX from lactococci. The introduction ofpepX intoLactococcus lactis LM0230 on either pGK12 (a low-copy-number plasmid vector) did not result in a significant increase in PepX activity, while the introduction ofpepX into CNRZ32 on pGK12 resulted in a four-fold increase in PepX activity. Southern hybridization experiments revealed that thepepX gene from CNRZ32 is well conserved in lactobacilli, pediococci and streptococci. The physiological role of PepX during growth in lactobacillus MRS (a rich medium containing protein hydrolysates along with other ingredients) and milk was examined by comparing growth of CNRZ32 and a CNRZ32 PepX-negative derivative in MRS. However, the CRNZ32 PepX-negative derivative grew in milk at a reduced specific growth rate when compared to wild-type CNRZ32. Introduction of the cloned PepX determinant into the CNRZ32 PepX-negative derivative resulted in a construct with a specific growth rate similar to that of wild-type CNRZ32.     

Morphological and genetic characterization of a bacteriophage-resistant Bacillus subtilis macrofiber-producing strain.

Bacillus subtilis C6 phi R4 is an SPO1-resistant derivative of strain C6D, a left-hand macrofiber-producing strain described previously (N. H. Mendelson, Proc. Natl. Acad. Sci. U.S.A. 75:2478-2482, 1978). In addition to the phage resistance property, strain C6 phi R4 differs from its parent in macrofiber organization and formation of aggregates in liquid shake cultures. The phage resistance mutation was located in the gtaC gene. The macrofiber organization and aggregation phenotypes also appear to be controlled by the gtaC locus. Strains constructed by introduction of the gtaC mutation into C6D appear to be identical to the original C6 phi R4 strain in all phenotypic properties. In contrast, other constructs carrying either gtaA or gtaB that are resistant to SPO1 do not display the characteristic C6 phi R4 morphological phenotypes.     

The expression of Streptomyces and Escherichia coli drug-resistance determinants cloned into the Streptomyces phage phi C31

Lysogens obtained by infecting Streptomyces albus G with a phi C31-pBR322 chimaeric prophage or its delta W12 deletion derivative had increased tetracycline resistance. The ability of the delta W12 derivative to transduce tetracycline resistance was inactivated by inserting a viomycin resistance determinant (vph) into the BamHI site of the pBR322 tet gene, and restored by excising the vph gene. Another deletion mutant (delta W17) of the chimaera, carrying an intact tet gene, was normally unable to transduce tetracycline resistance. This inability was correlated with the finding, by Southern hybridisation analysis, that the att site required for insertion of phi C31 prophage into the host chromosome was located within the delta W17 deletion. Use of phi C31 lysogenic recipient permitted the integration of the att-deleted phage, presumably by homologous recombination, giving tetracycline-resistant double lysogens. This technique was extended to S. coelicolor A3(2) in the detection of derivatives of the att-deleted phage into which a thiostrepton-resistance determinant (tsr) had been inserted in vitro. Phage released from double lysogens were mainly recombinants. One such recombinant is a PstI vector for DNA cloning, able to accommodate up to 6 kb of introduced DNA.     

Isolation of Specialized Transducing Bacteriophages for Gluconate 6-Phosphate Dehydrogenase (gnd) of Escherichia coli

Specialized transducing phages for gluconate 6-phosphate dehydrogenase (gnd), a constitutive enzyme in Escherichia coli, have been isolated using a method previously described for other genes. The gnd-his region, carried on an F' episome, was first transposed to tonB. Rare phages carrying gnd were selected, by transduction, from phi80 lysogens of these strains; one phage also carried his (phi80gndhis). From the transductants, high-frequency transducing lysates were obtained; low multiplicity of infection then yielded defective lysogens. tonB deletion analysis of the phi80dgndhis lysogen shows the order of genes in the prophage to be imm80...hisOGD...gnd; according to a marker rescue experiment most phage late genes have been replaced by bacterial deoxyribonucleic acid. A heat-inducible, lysis-defective lambda-phi80 hybrid derivative of phi80dgndhis has been prepared.     

Isolation and Characterization of Specialized φ80 Transducing Phages Carrying Regions of the Salmonella typhimurium trp Operon

We have isolated a series of nondefective phi80 specialized transducing phage which carry segments of the Salmonella typhimurium trp operon. These phage were obtained from a lysogenic derivative of a merozygote constructed by transferring an S. typhimurium trp episome into an Escherichia coli strain which lacks the normal phi80 attachment site. The deoxyribonucleic acid (DNA) from one such phage was purified and employed in DNA-ribonucleic acid (RNA) hybridization studies. The results obtained show that, under our hybridization conditions, heterologous hybridization is less efficient than homologous hybridization. It was also observed that not all S. typhimurium trp messenger RNA can readily anneal to E. coli trp operon DNA. Heterologous hybrids consisting of S. typhimurium trp messenger RNA and E. coli trp operon DNA were estimated to have a dissociation constant 10-fold larger than that of homologous hybrids.     

Evidence for a Plasmid-Linked Restriction-Modification System in Lactobacillus helveticus

The presence of a restriction-modification (R/M) system against two bacteriophages, 328-B1 and hv, was demonstrated in three Lactobacillus helveticus strains, CNRZ 1094, CNRZ 1095, and CNRZ 1096. In addition, the burst size of phage 328-B1 in the three restrictive strains CNRZ 1094, CNRZ 1095, and CNRZ 1096 was reduced with respect to the values obtained in its propagating strain, CNRZ 328. Heating at 60°C did not inactivate the R/M system. Nonrestrictive variants from CNRZ 1094 were easily obtained under several culture conditions, but treatment with novobiocin at 42°C followed by storage at −20°C resulted in drastic elimination of the R⁺/M⁺ phenotype from all clones tested. Electrophoretic analysis of CNRZ 1094 nonrestrictive variants revealed the concomitant loss of a 34-kb plasmid. Four EcoRI fragments from the 34-kb plasmid were cloned in the Escherichia coli vector pACYC184. The use of one or several of these fragments as probes confirmed the plasmidic location of the genes responsible for the R/M system. These probes also showed the presence of R/M plasmids in the two other restrictive strains, CNRZ 1095 and CNRZ 1096. Lactose-fermenting ability and/or proteolytic capacity was not linked to the 34-kb plasmid.     

Two groups of bacteriophages infecting Streptococcus thermophilus can be distinguished on the basis of mode of packaging and genetic determinants for major structural proteins.

A comparative study of 30 phages of Streptococcus thermophilus was performed based on DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and host range data. All phages exhibited distinct DNA restriction profiles, with some phages displaying similarly sized restriction fragments. DNA homology was shown to be present among all 30 phages. The phages could be divided into two groups on the basis of their packaging mechanism as was derived from the appearance of submolar DNA fragments in restriction enzyme digests and the presence (cos-containing phages) or absence (pac-containing phages) of cohesive genomic extremities. Interestingly, the 19 identified cos-containing phages possessed two major structural proteins (32 and 26 kDa) in contrast to the remaining 11 pac-containing phages, which possessed three major structural proteins (41, 25, and 13 kDa). Southern hybridization demonstrated that all pac-containing phages tested contain homologs of the genes encoding the three major structural proteins of the pac-containing phage O1205, whereas all cos-containing phages tested exhibit homology to the gene specifying one of the structural components of the cos-containing phage phi 7201. Fifty-seven percent of the phages (both cos and pac containing) possessed the previously identified 2.2-kb EcoRI fragment of the temperate S. thermophilus phage Sfi18 (H. Brüssow, A. Probst, M. Frémont, and J. Sidoti, Virology 200:854-857, 1994). No obvious correlation was detected between grouping based on packaging mechanism and host range data obtained with 39 industrial S. thermophilus strains.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.