Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module.     

A conserved C-terminal region in Gp71 of the small isometric-head phage LL-H and ORF474 of the prolate-head phage JCL1032 is implicated in specificity of adsorption of phage to its host, Lactobacillus delbrueckii

Thirty-five phage-resistant mutants of Lactobacillus delbrueckii subsp. lactis ATCC 15808 were selected. Thirty-three of these mutants were assigned to the Bes group, while the remaining two were grouped under the Ads designation. Bes group mutants adsorbed phage LL-H but did not allow efficient phage development. Preliminary evidence suggests that these strains exhibit a mutation that changes the DNA specificity of a restriction-modification system. The Ads group mutants did not adsorb the small isometric-head phage LL-H. The results suggest that there are at least three different types of phage receptors in L. delbrueckii: two that are specific for small isometric-head phages and one that is specific for prolate-head phage JCL1032. Five LL-H host-range mutants which could overcome the adsorption block (a-type mutants) were selected and investigated by sequencing the genes g71 and g17, which encode minor and major tail proteins, respectively. Each of the a-type mutants carried a nucleotide change at the 3' end of gene g71. No mutations were observed in gene g17. Comparison of the gene product of g71 of phage LL-H with its homolog in JCL1032 (ORF474) showed that these proteins had very similar C-terminal regions. No similarities were found at the N-terminal part of the proteins. We conclude that the C-terminal portion of the protein encoded by g71 of phage LL-H and its homolog in phage JCL1032 determines the adsorption specificities of these phages on L. delbrueckii.     

Defective site-specific integration elements are present in the genome of virulent bacteriophage LL-H of Lactobacillus delbrueckii.

The phage attachment site, attP, and the integrase-encoding gene, int, are sufficient to promote site-specific integration of the temperate phage mv4 genome into the chromosome of the Lactobacillus delbrueckii host (L. Dupont, B. Boizet-Bonhoure, M. Coddeville, F. Auvray, and P. Ritzenthaler, J. Bacteriol. 177:586--595, 1995). The mv4 genome region containing these elements was compared at the nucleotide and amino acid levels with that of the closely related virulent phage LL-H. Complex DNA rearrangements were identified; a truncated integrase gene and two sites homologous to the mv4 attP site were detected in the genome of the virulent phage LL-H. These observations suggest that the two phages derive from a common temperate ancestor.     

Molecular interaction between lipoteichoic acids and Lactobacillus delbrueckii phages depends on D-alanyl and alpha-glucose substitution of poly(glycerophosphate) backbones

Lipoteichoic acids (LTAs) have been shown to act as bacterial counterparts to the receptor binding proteins of LL-H, LL-H host range mutant LL-H-a21, and JCL1032. Here we have used LTAs purified by hydrophobic interaction chromatography from different phage-resistant and -sensitive strains of Lactobacillus delbrueckii subsp. lactis. Nuclear magnetic resonance analyses revealed variation in the degree of alpha-glucosyl and D-alanyl substitution of the 1,3-linked poly(glycerophosphate) LTAs between the phage-sensitive and phage-resistant strains. Inactivation of phages was less effective if there was a high level of D-alanine residues in the LTA backbones. Prior incubation of the LTAs with alpha-glucose-specific lectin inhibited the LL-H phage inactivation. The overall level of decoration or the specific spatial combination of alpha-glucosyl-substituted, D-alanyl-substituted, and nonsubstituted glycerol residues may also affect phage adsorption.     

Effective plasmid pX3 transduction in Lactobacillus delbrueckii by bacteriophage LL-H

High-frequency plasmid transductions in Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus strains mediated by pac-type bacteriophages were observed and further investigated. The frequency of plasmid transduction by phages LL-H and LL-S attained levels of from 0.10 to about 1 with plasmid pX3, but only about 2 × 10⁻² with plasmid pJK650. Infection of L. delbrueckii subsp. lactis strain LKT(pX3) or ATCC 15808(pX3) with phage LL-H resulted in intensive concatemerization of plasmid pX3, and most progeny phage particles contained concatemers of plasmid DNA instead of phage LL-H DNA. The synthesis of phage LL-H DNA was depressed. No evident homology or recombination was observed between phage LL-H DNA and plasmid pX3. The unusually high frequency of plasmid pX3 transduction by phage LL-H could be considered to result from specific interaction(s) between a particular phage and plasmid. These interactions may include pX3-mediated blockage of phage LL-H DNA replication and effective use of a particular pac-like site located about 1 kb from BglII in the smaller NdeI-BglII fragment of plasmid pX3. Phage LL-H together with plasmid vector pX3 could be used as effective plasmid transduction tools for genetic engineering of L. delbrueckii subsp. lactis and subsp. bulgaricus strains.     

Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends.

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.     

Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends.

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.     

Selection of human chromosome 21-specific DNA probes for genetic analysis in Alzheimer's dementia and Down syndrome

Summary We used a mouse-human somatic cell hybrid to construct a chromosome 21-enriched library in phage vector EMBL4. In all, 35 phage clones containing human inserts were identified by differential screening with total human and mouse DNA. Whole recombinant phages were regionally mapped on chromosome 21 by Southern blot analysis using competitive hybridisation conditions to block repetitive sequences. Ten phage clones mapped proximal to a translocation breakpoint in band 21q21.2, while 25 mapped distal to this point. Three of the phage clones identify restriction fragment length polymorphisms. Polymorphic chromosome 21 markers may be useful in the genetic analysis of Alzheimer's dementia and Down syndrome.     

Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin.

LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N-terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.     

Transduction of Low-Copy Number Plasmids by Bacteriophage P22

The generalized transducing bacteriophage of Salmonella typhimurium, P22, can transduce plasmids in addition to chromosomal markers. Previous studies have concentrated on transduction of pBR322 by P22 and P22HT, the high transducing mutant of P22. This study investigates the mechanism of P22HT transduction of low-copy number plasmids, namely pSC101 derivatives. We show that P22HT transduces pSC101 derivatives that share homology with the chromosome by two distinct mechanisms. In the first mechanism, the plasmid integrates into the chromosome of the donor by homologous recombination. This chromosomal fragment is then packaged in the transducing particle. The second mechanism is a size-dependent mechanism involving a putative plasmid multimer. We propose that this multimer is formed by interplasmidic recombination. In contrast, P22HT can efficiently transduce pBR322 by a third mechanism, which is independent of plasmid homology with the chromosome. It has been proposed that the phage packages a linear concatemer created during rolling circle replication of pBR322, similar in fashion to phage genome packaging. This study investigates the role of RecA, RecD, and RecF recombination proteins in plasmid/plasmid and plasmid/chromosome interactions that form packageable substrates in the donor. We also examine the resolution of various transduced plasmid species in the recipient and the roles of RecA and RecD in these processes.     

Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox.

Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.     

Biodiversity of Lactococcus lactis bacteriophages in Polish dairy environment

We present here the results of an exploration of the bacteriophage content of dairy wheys collected from milk plants localized in various regions of Poland. Thirty-three whey samples from 17 regions were analyzed and found to contain phages active against L. lactis strains. High phage titer in all whey samples suggested phage-induced lysis to be the main cause of fermentation failures. In total, over 220 isolated phages were examined for their restriction patterns, genome sizes, genetic groups of DNA homology, and host ranges. Based on DNA digestions the identified phages were classified into 34 distinct DNA restriction groups. Phage genome sizes were estimated at 14-35 kb. Multiplex PCR analysis established that the studied phages belong to two out of the three main lactococcal phage types--c2 and 936, while P335-type phages were not detected. Yet, analyses of bacterial starter strains revealed that the majority of them are lysogenic and carry prophages of P335-type in their chromosome. Phage geographical distribution and host range are additionally discussed.     

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.     

Bacteriophages of lactobacilli

Lactobacilli are members of the bacterial flora of lactic starter cultures used to generate lactic acid fermentation in a number of animal or plant products used as human or animals foods. They can be affected by phage outbreaks, which can result in faulty and depreciated products. Two groups of phages specific of Lactobacillus casei have been thoroughly studied. 1. The first group is represented by phage PL-1. This phage behaves as lytic in its usual host L. casei ATCC 27092, but can lysogenize another strain, L. casei ATCC 334. Bacterial receptors of this phage are located in a cell-wall polysaccharide and rhamnose is the main component of the receptors. Ca2+ and adenosine triphosphate (ATP) are indispensable to ensure the injection of the phage DNA into the bacterial cell. The phage DNA is double-stranded, mostly linear, but with cohesive ends which enables it to be circularized. The vegetative growth of PL-1 proceeds according to the classical mode. Cell lysis is produced by an N-acetyl-muramidase at the end of vegetative growth. 2. The second group is represented by the temperate phage phi FSW of L. casei ATCC27139. It has been shown how virulent phages originate from this temperate phage in Japanese dairy plants. The lysogenic state of phi FSW can be altered either by point mutations or by the insertion of a mobile genetic element called ISL 1, which comes from the bacterial chromosome. This is the first transposable element that has been described in lactobacilli. Lysogeny appears to be widespread among lactobacilli since one study showed that 27% of 148 strains studied, representing 15 species, produced phage particles after induction by mitomycin C. Similarly, 23 out of 30 strains of Lactobacillus salivarius are lysogenic and produce, after induction by mitomycin C, temperate phages, killer particles, or defective phages. Temperate phages have also been found in 10 out of 105 strains of Lactobacillus bulgaricus or Lactobacillus lactis after induction by mitomycin C. Phages so far studied of the latter 2 and closely related lactobacilli, either temperate or isolated as lytic, may be divided into 4 unrelated groups called a, b, c and d. Most of these phages are found in group a and an unquestionable relationship has already been shown between lytic phages and temperate phages that belong to this group. Lytic phage LL-H of L. lactis LL 23, isolated in Finland, is one of the most representative of those of group a and has been extensively studied on the molecular level.     

Excision process of integrated plasmid pBD12 from the Bacillus subtilis chromosome

We studied the behavior of pBD12 plasmid integrated into Bacillus subtilis chromosome via homologous recombination. One copy of the plasmid was integrated into the chromosome, it conferred resistance to low concentrations of antibiotics. Clones with enhanced resistance bearing autonomous plasmid DNAs appeared with a frequency 10(-6) in rec+ but not in recE strain with the integrated plasmid. By restriction and hybridization analysis of some excised plasmids, the sites of excision were determined, chromosomal location of pBD12 plasmid was found to be at the terminal fragment of prophage DNA, so that the att site of phi 105 phage is supposed to be situated on the EcoRI fragment of phage DNA.     

Posttranslational processing of Uukuniemi virus glycoproteins G1 and G2

A total of 26 polypeptides have been resolved by gel electrophoresis of purified phage PBSX, 3 of which belong to the head and the remainder to the tail. After mitomycin C treatment, synthesis of 11 additional proteins which are not found in the assembled phage particle was demonstrated, all but 4 being under the control of the phage repressor. Existence of a prehead and of a precursor of the main capsid protein (molecular weight, 35,000) suggested phage head maturation which is accompanied by cleavage of the precursor (molecular weight, 36,500). The role of induced proteins related and unrelated to PBSX is discussed. Finally, the estimated phage genome mass of 4 X 10(7) daltons exceeded by more than four times its head capacity, which could explain the defectiveness of the phage.     

HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli genome, and location of the tonB gene.

The HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli chromosome are presented. Analysis of phage DNAs carrying tonB mutations has allowed identification of a 1,730-base pair HindII fragment containing at least part of the tonB gene. This fragment is 4,020 base pairs from the end of trpA, with the total distance from attphi80 to trpA being 6,550 +/- 800 base pairs. Properties of hybrid plasmids containing insertions of various tonB+ restriction fragments suggest that tonB lies completely within the 1,730-base pair fragment. In addition, apparent fusions of beta-galactoside to proteins within the tonB region suggest that the entire region codes for more than one polypeptide.     

A new insertion sequence, IS121, is found on the Mu dI1 (Ap lac) bacteriophage and the Escherichia coli K-12 chromosome

We have discovered a new insertion sequence, now designated IS121, as a component of the Mu dI1 (Ap lac) phage. This sequence is 1.2 kilobases long and contains single recognition sites for the HincII, Bg1II, and HindIII restriction endonucleases. IS121 is present in at least three copies in the chromosome of several Escherichia coli K-12 strains. When present in the nonconjugative plasmid pBR322, IS121 can mediate cointegrate formation with an F' lac plasmid and transfer of pBR322 sequences to suitable recipients. IS121 is also capable of precise or nearly precise excision. As part of the study of IS121, we have determined the physical structure of the Mu dI1 (Ap lac) phage and established an extensive restriction endonuclease map of this phage. A revised schema for the formation of the Mu dI1 (Ap lac) phage is presented.     

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.