Clear-plaque Mutants Research Articles

Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage

BackgroundWhole genome sequencing promises to revolutionize our ability to link genotypic and phenotypic variation in a wide range of model and non-model species.ResultsHere we describe the isolation and characterization of a novel mycobacteriophage named BGlluviae that grows on Mycobacterium smegmatis mc2155. BGlluviae normally produces turbid plaques but a spontaneous clear plaque was also recovered. The genomic DNA from pure populations of the BGlluviae phage and the clear plaque mutant were sequenced. A single substitution, at amino acid 54 (I to T), in the immunity repressor protein resulted in a clear plaque phenotype.ConclusionsThis substitution is predicted to be located at the subunit interaction interface of the repressor protein, and thus prevents the establishment of lysogeny.

Clear Plaque Mutants of Lactococcal Phage TP901-1.

We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1.

SOS repair of 8-methoxypsoralene monoadducts in DNA of lambda bacteriophage and plasmids is mediated by MucA 2 ′ B, but not UmuD 2 ′ C (PolV) polymerase

The light-induced action of 8-methoxypsoralen (8-MOP) on λ phage and plasmids yields monoadducts and interstrand crosslinks. The survival and clear plaque mutation frequency in the phage photosensitized with 8-MOP and irradiated with UV at wavelength > 320 nm are increased whenthe wild-type host (Escherichia coli uvr+) is subjected to UV irradiation (wavelength = 254 nm) prior to phage inoculation. These phenomena are known as "W reactivation" and "W mutagenesis." It is shown that 8-MOP monoadducts in λ DNA in- duce clear mutations in the phage inoculated to UV-irradiated excision repair mutants of E. coli only when the error-prone repair is performed by MucA B, but not PolV (UmuD'2C) polymerase. The efficiency of the SOS repair (W reactivation) of 8-MOP monoadducts in plasmid and λ phage DNA also only increases with the presence of pKM101 plasmid muc+ in E. coli uvr-.

Phage-Borne Factors and Host LexA Regulate the Lytic Switch in Phage GIL01

The Bacillus thuringiensis temperate phage GIL01 does not integrate into the host chromosome but exists stably as an independent linear replicon within the cell. Similar to that of the lambdoid prophages, the lytic cycle of GIL01 is induced as part of the cellular SOS response to DNA damage. However, no CI-like maintenance repressor has been detected in the phage genome, suggesting that GIL01 uses a novel mechanism to maintain lysogeny. To gain insights into the GIL01 regulatory circuit, we isolated and characterized a set of 17 clear plaque (cp) mutants that are unable to lysogenize. Two phage-encoded proteins, gp1 and gp7, are required for stable lysogen formation. Analysis of cp mutants also identified a 14-bp palindromic dinBox1 sequence within the P1-P2 promoter region that resembles the known LexA-binding site of Gram-positive bacteria. Mutations at conserved positions in dinBox1 result in a cp phenotype. Genomic analysis identified a total of three dinBox sites within GIL01 promoter regions. To investigate the possibility that the host LexA regulates GIL01, phage induction was measured in a host carrying a noncleavable lexA (Ind(-)) mutation. GIL01 formed stable lysogens in this host, but lytic growth could not be induced by treatment with mitomycin C. Also, mitomycin C induced β-galactosidase expression from GIL01-lacZ promoter fusions, and induction was similarly blocked in the lexA (Ind(-)) mutant host. These data support a model in which host LexA binds to dinBox sequences in GIL01, repressing phage gene expression during lysogeny and providing the switch necessary to enter lytic development.

Serendipity

Serendipity

Molecular Characterization of a Variant of Bacillus anthracis -Specific Phage AP50 with Improved Bacteriolytic Activity

The genome sequence of a Bacillus anthracis-specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.

Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages

HK620 is a temperate lambdoid bacteriophage that adsorbs to the O-antigen of its host, Escherichia coli H. The genome of a temperature-sensitive clear-plaque mutant consists of 38,297 nucleotides in which we recognize 60 open reading frames (orfs). Eighteen of these lie in a region of the genome that we call the virion structure domain. The other 42 orfs lie in what we call the metabolic domain. Virions of HK620 resemble those of phage P22. The virion structural orfs encode three kinds of putative proteins relative to the virion proteins of P22: (1) those that are nearly (about 90 %) identical; (2) those that are weakly (about 30 %) identical; and (3) those composed of nearly and weakly identical segments. We hypothesize that these composite proteins form bridges between the virion proteins of the other two kinds. Three of the putative virion proteins that are only weakly identical to P22 proteins are 71, 60 and 79 % identical to proteins encoded by the phage APSE-1, whose virions also resemble those of P22. Because the hosts of APSE-1 and HK620 have been separated from each other by an estimated 200 My, we propose using the amino acid differences that have accumulated in these proteins to estimate a biological clock for temperate lambdoid phages. The putative transcriptional regulatory gene circuitry of HK620 seems to resemble that of phage lambda. Integration, on the other hand, resembles that of satellite phage P4 in that the attP sequence lies between the leftward promoter and int rather than downstream of int. Comparing the metabolic domains of several lambdoid phage genomes reveals seven short conserved sequences roughly defining boundaries of functional modules. We propose that these boundary sequences are foci of genetic recombination that serve to assort the modules and make the metabolic domain highly mosaic genetically.

The anti-immunity system of phage-plasmid N15: identification of the antirepressor gene and its control by a small processed RNA.

N15 is a temperate virus of Escherichia coli related to lambdoid phages. However, unlike all other known phages, the N15 prophage is maintained as a low copy number linear DNA molecule with covalently closed ends. The primary immunity system at the immB locus is structurally and functionally comparable to that of lambdoid phages, and encodes the immunity repressor CB. We have characterized a second locus, immA, in which clear plaque mutations were mapped, and found that it encodes an anti-immunity system involved in the choice between the lytic and the lysogenic cycle. Three open reading frames at the immA locus encode an inhibitor of cell division (icd ), an antirepressor (antA) and a gene that may play an ancillary role in anti-immunity (antB ). These genes may be transcribed from two promoters: the upstream promoter Pa is repressed by the immunity repressor CB, whereas the downstream promoter Pb is constitutive. Full repression of the anti-immunity system is achieved by premature transcription termination elicited by a small RNA (CA RNA) produced by processing of the leader transcript of the anti-immunity operon. The N15 anti-immunity system is structurally and functionally similar to the anti-immunity system of bacteriophage P1 and to the immunity system of satellite phage P4.

Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice.

Big Blue mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor-intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073-9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C --> A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3' flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G --> A and G --> T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G --> A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations.

General transducing phages like Salmonella phage P22 isolated using a smooth strain of Escherichia coli as host.

A smooth colony strain, resistant to phages lambda and P22, was isolated from sewage and identified as Escherichia coli (strain H). Four temperate phages plaquing on strain H were isolated from sewage. The archetype, HK620, does not plaque on strains C and K12 of E. coli nor on the LT2 strain of Salmonella enterica. Bacterial mutants resistant to a clear plaque mutant of HK620 produce rough colonies. Some are also galactose-negative, a few are histidine auxotrophs, and most show sensitivity to lambda. HK620 can transduce a wide variety of auxotrophic mutants of E. coli H to prototrophy. It can recombine with lambda but its virions resemble those of P22.

General transducing phages like Salmonella phage P22 isolated using a smooth strain of Escherichia coli as host

A smooth colony strain, resistant to phages λ and P22, was isolated from sewage and identified as Escherichia coli (strain H). Four temperate phages plaquing on strain H were isolated from sewage. The archetype, HK620, does not plaque on strains C and K12 of E. coli nor on the LT2 strain of Salmonella enterica. Bacterial mutants resistant to a clear plaque mutant of HK620 produce rough colonies. Some are also galactose-negative, a few are histidine auxotrophs, and most show sensitivity to λ. HK620 can transduce a wide variety of auxotrophic mutants of E. coli H to prototrophy. It can recombine with λ but its virions resemble those of P22.

Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii.

The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1. is a member of the Siphoviridae family with a B1 morphology. It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 x 10(7) daltons. BCJA1 was stable over the pH range of 6-11. A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40. The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36500 and 28000, respectively. The genome size was estimated to be between 32.1 and 34.8 kb. The percent G + C content of purified bacteriophage DNA was 45.6. The wildtype bacteriophage is temperate but a clear plaque mutant was isolated.

Characterization of the primary immunity region of the Escherichia coli linear plasmid prophage N15

N15 is the only bacteriophage of Escherichia coli known to lysogenize as a linear plasmid. Clear-plaque mutations lie in at least two regions of the 46-kb genome. We have cloned, sequenced, and characterized the primary immunity region, immB. This region contains a gene, cB, whose product shows homology to lambdoid phage repressors. The cB3 mutation confers thermoinducibility on N15 lysogens, consistent with CB being the primary repressor of N15. Downstream of cB lies the locus of N15 plasmid replication. Upstream of cB lies an operon predicted to encode two products: one homologous to the late repressor of P22 (Cro), the other homologous to the late antiterminator of phi 82 (Q). The Q-like protein is essential for phage development. We show that CB protein regulates the expression of genes that flank the cB gene by binding to DNA at symmetric 16-bp sites. Three sites are clustered upstream of cB and overlap a predicted promoter of the cro and Q-like genes as well as two predicted promoters of cB itself. Two sites downstream of cB overlap a predicted promoter of a plasmid replication gene, repA, consistent with the higher copy number of the mutant, N15cB3. The leader region of repA contains terminators in both orientations and a putative promoter. The organization of these regulatory elements suggests that N15 plasmid replication is controlled not only by CB but also by an antisense RNA and by a balance between termination and antitermination.

Cloning of the early promoters of Pseudomonas aeruginosa bacteriophage D3: sequence of the immunity region of D3.

The early promoters of bacteriophage D3 of Pseudomonas aeruginosa were cloned and physically mapped to the right 25% of the phage genome. The promoters were cloned into promoter selection vector pQF26, and their relative strengths, the direction of transcription, and whether they were directly regulated by repressor were determined. A 3.3-kb fragment of the genome containing the immunity region was sequenced and analyzed (GenBank accession number: L22692). The promoter activity associated with this region was determined to be bidirectional and repressible, indicating that this region contains operator-promoter complexes. Sequence and functional analyses suggest that this region is analogous to the immunity region of coliphage lambda. Two strong promoters, one of which was repressible, were found to be located adjacent to the immunity region. Clear-plaque mutant phage D3c contains insertion element IS222, which causes it to behave as a repressor-negative (c1) variant. The site of insertion of IS222 was sequenced and determined to lie within the c1 gene open reading frame. This phage shows remarkable similarity in genomic organization to coliphage lambda and its relatives.

Genes for the establishment and maintenance of lysogeny by the temperate coliphage 186

To identify the genes in coliphage 186 that are required for lysogeny, we isolated clear-plaque mutants. Complementation studies and DNA sequencing identified two genes, the cI gene for the immunity maintenance repressor and the cII gene, which is required only for the establishment of lysogeny. One mutant carried a change in the LexA-binding site controlling expression of the antirepression protein Tum.

Bacteriophage phi 105clz induces the GroEL-homologue protein in Bacillus subtilis.

Using two-dimensional polyacrylamide gel electrophoresis, the GroEL homologue of Bacillus subtilis was shown to be induced upon infection with phi 105clz, a clear plaque mutant of the temperate bacteriophage phi 105. Western blotting of one dimensional polyacrylamide gels also showed the induction of the GroEL homologue when cells were infected with phi 105clz.

Identification and nucleotide sequence analysis of an open reading frame involved in high-frequency conversion of turbid to clear plaque mutants of filamentous phage Cf1t

Clear plaque mutants (Cf1c) isolated from the temperate filamentous phage Cf1t occurred at a frequency of approximately 10 −3. The phage yield from Cf1c-infected cells was higher than that from Cf1t-infected cells. Results of spot complementation tests implied that the turbid plaque phenotype is dominant. DNA fragment substitution studies indicated that a Ncol/ Kpnl fragment of 591 by was responsible for the determination of plaque turbidity. Sequence data from four Cf1c isolates revealed base pair alterations and a deletion located in the upstream region of an open reading frame (ORFII) which might encode a 18.2-kDa protein. When the ORFII in Cfi t was disrupted by a frameshift mutation, this recombinant phage formed clear plaques. These observations suggest that ORFII may participate in the formation of turbid plaques. ORFII does not show significant homology with the sequence of f1 gpII, gpV, or other known phage proteins.

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.

X-ray inactivation, Weigle reactivation, and Weigle mutagenesis of the lysogenic Vibrio κ phage

Vibrio cholerae lysogenic kappa phage was inactivated by X-ray (60 kV) in a dose-dependent manner, the inactivation dose leading to 37% survival (D37) in phosphate-buffered saline (PBS), pH 7.4, being 0.36 kGy. The phages were significantly protected against X-ray irradiation when histidine or cysteine or both were present in PBS or when phages were irradiated in nutrient broth. Maximum protection was offered when both histidine (10.0 mM) and cysteine (10.0 mM) were present in PBS (dose enhancement factor being 4.17). The X-irradiated kappa phages also underwent a small but significant Weigle reactivation and also Weigle mutagenesis in the UV-irradiated V. cholerae host H218Smr. The Weigle factor or the frequency of clear-plaque mutants increased with increasing UV dose, attained a maximum at a UV dose of 2.4 J m-2, and thereafter decreased gradually with a further increase of the UV dose. The X-ray dose (D)--survival (S) curves could be empirically described by the equation S = exp[-(aD + bD2)], where a and b are constants depending on the irradiation conditions, and a good agreement between the theoretical curves and experimental data was obtained.

Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences

Twenty-nine clear-plaque mutants of bacteriophage lambda were isolated from a Shigella dysenteriae lysogen. Three were associated with insertions in the cI gene: two were due to insertion of IS600, and the third resulted from insertion of a new element, IS911. IS911 is 1,250 base pairs (bp) long, carries 27-bp imperfect terminal inverted repeats, and generates 3-bp duplications of the target DNA on insertion. It was found in various copy numbers in all four species of Shigella tested and in Escherichia coli K-12 but not in E. coli W. Analysis of IS911-mediated cointegrate molecules indicated that the majority were generated without duplication of IS911. They appeared to result from direct insertion via one end of the element and the neighboring region of DNA, which resembles a terminal inverted repeat of IS911. Nucleotide sequence analysis revealed that IS911 carries two consecutive open reading frames which code for potential proteins showing similarities to those of the IS3 group of elements.