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PubMed1980

[Temperature-sensitive mutant of bacteriophage D3 and its use for the purpose of demonstrating the location of prophage in Pseudomonas aeruginosa cells].

For the first time a thermoinducible mutant, known as D3ct, has been obtained from Pseudomonas aeruginosa phage and used for lysogenizing culture PAO2604 Met--Ilv--. After a temperature shock phage D3ctwas induced from lysogenic strain PAO2604 (D3ct), and its development went along the lytic line. From the population of PAO2604 (D3ct) cells only 0.07% survived; in these cells the faulty excision of the prophage probably occurred at a high temperature. After thermoinduction 2.8% of the colonies formed by the survivors were auxotrophic in leucine. Such frequency of the appearance of the additional nutritional requirement for leucine suggests that the prophage was incorporated into the chromosome adjacent to some gene responsible for biosynthesis of leucine. In the process of faulty excision phage D3 retained a part of the host chromosome.

Bacteriophages↗

PubMed1995

Cure of prophage in Staphylococcus aureus by furocoumarin photoadditions.

In a previous report curing of prophage in Staphylococcus aureus by treatment with 8-methoxypsoralen plus near-UV (365 nm) radiation was described. In the present work the use of angelicin and 4,5',8-trimethylpsoralen and repeated irradiation show that monoadducts and DNA interstrand cross-links formed by furocoumarin photoaddition are involved in the curing of the prophage responsible for the suppression of the lipolytic activity in a putative repair proficient strain of Staphylococcus aureus.

DNA Adducts↗

PubMed1995

[Origin and properties of Pseudomonas aeruginosa PAO1 clones surviving after induction of prophages-transposons].

Various mutations cancelling the lethal effect of phage lytic development and simultaneous phenotypic modifications were found in rare clones surviving after incubation at 42 degrees C of Pseudomonas aeruginosa (D3112 cts 15), lysogenic for thermoinducible mutant cts 15 of the transposable prophage (TP) D3112. All mutations arose prior to thermal induction. Temperature induction of other bacteriophages (nontransposable) did not lead to selection of bacterial morphological mutants. Therefore, it was concluded that mutagenesis occurred upon the partial (reversible) TP derepression accompanied by coupled replication-transposition of TP, the latter being the direct cause of the mutator effect. Isolation of the P. aeruginosa PAO1 mutant R10 (this mutant is resistant to infection with TP at 42 degrees C) allowed the proper selection and examination of numerous survivors. Comparison of their types derived from lysogens with different prophage location indicated that the number of secondary sites where TP integration is possible without the loss of cell viability is limited. Several transposition events occurred in the history of some survivors (during a repeated or single derepression event). Type D clones, which produce small colonies, are of special interest, because mechanisms underlying the survival of such clones are extremely diverse, and their phenotypes indicate the possibility of stable chromosomal rearrangements in the genome of P. aeruginosa.

Cloning, Molecular↗

PubMed1995

[Mutagenic effect of oxidizing agents on the thermally-induced prophage lambda cI857: effect of a system of oxidative stress].

The lethal and mutagenic effects of hydrogen peroxide H2O (2-20 mM), cumene hydroperoxide (0.2-2.0 mM), and potassium permanganate KMnO4 (0.25-1.0 mM) on the heat-inducible lambda cI857 prophage were studied under conditions of heat-induction immediately after the mutagenic treatment of lysogenic cells of Escherichia coli oxyR+ or oxyR delta 3. Within the range of the doses used, these agents decreased prophage survival by 3-5 orders of magnitude and increased mutation frequency by up to 0.2% under the action of hydrogen peroxide and cumene hydroperoxide, and up to 1.5% in the case of KMnO4 (in oxyR+ cells). In the absence of the inducible OxyR system of oxidative stress, both lethal and mutagenic effects of H2O were enhanced. The oxyR delta 3 mutation increased lethal and mutagenic effects of cumene hydroperoxide and KMnO4 only at the highest concentrations used. Apparently, the OxyR system does not repair lesions induced by oxidative agents, but only prevents their formation.

Bacteriophage lambda↗

PubMed1976

Studies on megacinogeny in Bacillus cereus. II. Bacillus cereus isolates characterized by prophage-controlled production of megacin A (phospholipase A).

Five out of a number of Bacillus cereus strains isolated from soil produced high titre specific bacteriocin (megacin A) in mitomycin C-induced cultures. In the course of cultivation with ethidium bromide, the strains gave off segregants not producing bacteriocin (cin-). The lysate of two wild strains formed plaques on the corresponding cin- bacteria. The two phages (wx23 and wx26) were identical in antigenic structure with phage wx was present in the lysate of B. cereus strain W, and converted cin- derivatives into cultures producing megacin A (phospholipase A). The phages produced plaques at 26 degrees C but not at 37 degrees C. In the lysates of the remaining three strains phages were not detected with biological and morphological methods; these cultures have been assumed to carry defective prophage genome. As the corresponding prophages are responsible for the determination of inducible phospholipase A production, phages named wx seem to form a separate group of B. cereus phages.

Bacillus cereus↗

PubMed1995

[Cloning and analysis of prophage PBSX repressor gene from Bacillus subtilis].

The repressor gene and its allele--a temperature sensitive mutant have been cloned from the defective prophage PBSX of Bacillus subtilis by means of PCR technique. The characterization of these sequences suggest that wild-type and mutant gene have the same open reading frame, which probably encode a repressor protein of 113 amino acids, and the putative promoter region and ribosome binding sites within the sequences have also been identified. The results from complementation experiments demonstrate that repressor encoded by wild-type gene is capable of blocking the temperature-shift to induce PBSX prophage in Bacillus subtilis.

Amino Acid Sequence↗

PubMed2003

Sunscreen products increase virus production through prophage induction in marine bacterioplankton.

Classical pollutants (e.g., hydrocarbon, pesticides) have been recently recognized to induce lytic cycle in lysogenic bacteria, but information on micro-pollutants is almost completely lacking. We investigated the effects of cosmetic sun products (sunscreen and solar oil) on viral abundance and bacterial activity. We found that both sunscreen and solar oil acted as pollutants, inducing viral development and controlling bacterial abundance and production, thus leading to an increase of the virus to bacterium ratio. Short-term experiments revealed that sunscreen supplementation induced the lytic cycle in a large fraction of total bacterial abundance (13-24% of bacteria, at low and high concentrations, respectively), whereas solar oil had a lower impact (6-9%). A synchronized development of the phage-host system was observed only after sunscreen addition. The addition of sunscreen, even at low concentrations, had a significant impact on all enzymatic activities (aminopeptidase, glucosidase, and phosphatase), which increased significantly. However, when enzymatic activities were normalized per cell, a selective enhancement was observed for certain enzymes (e.g., aminopeptidase) and inhibition for others (e.g., glucosidase). These results indicate that sunscreen products can modify C, N, and P biogeochemical cycling in seawater and increase virus abundance through prophage induction in marine bacterioplankton.

Aminopeptidases↗

PubMed2004

Identification of a P2-related prophage remnant locus of Photorhabdus luminescens encoding an R-type phage tail-like particle.

Analysis of the Photorhabdus luminescens genome sequence revealed that the pts region is related to the tail synthesis gene core of the P2 phage. The pts locus encodes a DNA invertase homologue. PCR-RFLP analysis showed the two potential tail fiber regions of the pts locus present DNA inversions. Electron microscopy revealed a phage tail-like particle, related to the R-type family and named R-photorhabdicin, in the culture supernatant of P. luminescens. Mass spectrometry analysis of two sub-units of R-photorhabdicin revealed that they are encoded by the pts locus. The role of this P2-related prophage remnant in the Photorhabdus genome is discussed.

Bacteriophage P2↗

PubMed2005

Molecular properties of the two-component cell lysis system encoded by prophage phigaY of Lactobacillus gasseri JCM 1131T: cloning, sequencing, and expression in Escherichia coli.

Shotgun cloning of the Lactobacillus gasseri JCM 1131T whole DNA yielded two recombinant plasmids, p118gaY1 and p118gaY2, which directed cell lysis activity. Sequencing analysis revealed that the two plasmids carried almost identical inserted genes in following orders (truncated genes, in parentheses): in p118gaY1, (orf149)-orf92-holgaY-lysgaY-orf35-attL-(mnaAgaY1); in p118gaY2, (orfXgaY1)-orf169-orf149-orf92-holgaY-lysgaY-orf35-attP-(intgaY). The lysgaY-encoded protein (designated as LysgaY, 33.7 kDa) showed significant homology with putative muramidases (peptidoglycan-degrading enzyme) of the Lactobacillus phage phiadh, Lj965, Lj928, LL-H, mv4, and mv1. By zymogram analysis, LysgaY overproduced in Escherichia coli exhibited lytic activity towards 17 Gram-positive bacterial strains, including lactobacilli, lactococci, and staphylococci. The holgaY-encoded protein (15.7 kDa) contained three potential transmembrane helices, resembling putative holins (cytoplasmic membrane-disrupting protein) of Lj928 and Lj965. On the other hand, another clone p118gaYR obtained by EcoRI-shotgun cloning carried the (ptsCgaY1)-attR-(intgaY) genes. Three sequences, attL, attP, and attR, had a 47-bp common (core) sequence, and the core of attR was located in 3' region of a potential tRNA(Arg) gene. These results suggested that (i) attL and attR are phage-host junctions, left- and right-arms, respectively, (ii) attP is a phage attachment site, and (iii) intgaY is an integrase gene for phage integration and/or excision. After mitomycin C-induction, phage particles were demonstrated by electron microscopy. The prophage (phigaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY-LysgaY), closely resembling that of Lj928 as well as Lj965.

Amino Acid Sequence↗

PubMed2005

Functional characterization of the repA replication gene of linear plasmid prophage N15.

The prophage of coliphage N15 is not integrated into the chromosome, but exists as a linear plasmid molecule with covalently closed ends. The only phage gene required for replication of circular N15 miniplasmids is repA (gene 37). Here we show that RepA-driven replication of the N15-based circular and linear miniplasmids is independent of host DnaB helicase protein, but requires the host DnaG primase. Replication of phage N15 DNA during lytic growth following infection does not depend on either DnaG or DnaB, but DnaG is required for lytic development after induction of the N15 lysogen. Finally, protein sequence analysis and replication data using different mutant strains suggest that RepA protein combines helicase and primase functions.

Adenosine Triphosphatases↗

PubMed2006

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes.

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.

Bacillus Phages↗

PubMed2002

Integration and excision of the Mycobacterium tuberculosis prophage-like element, phiRv1.

The genomes of Mycobacterium tuberculosis H37Rv and CDC1551 each contain two prophage-like elements, phiRv1 and phiRv2. The phiRv1 element is not only absent from Mycobacterium bovis BCG but is in different locations within the two sequenced M. tuberculosis genomes; in both cases phiRv1 is inserted into a REP13E12 repeated sequence, which presumably contains the bacterial attachment site, attB, for phiRv1. Although phiRv1 is probably too small to encode infectious phage particles, it may nevertheless have an active integration/excision system and be capable of moving from one chromosomal position to another. We show here that the M. tuberculosis H37Rv phiRv1 element does indeed encode an active site-specific recombination system in which an integrase of the serine recombinase family (Rv1586c) catalyses integration and excision and a small, basic phiRv1-encoded protein (Rv1584c) controls the directionality of re-combination. Integration-proficient plasmid vectors derived from phiRv1 efficiently transform BCG, can utilize four of the seven REP13E12 sites present in BCG as attachment sites, and can occupy more than one site simultaneously.

Amino Acid Sequence↗

PubMed2004

Analysis of a novel prophage-encoded group A Streptococcus extracellular phospholipase A(2).

Group A Streptococcus (GAS) is an important human pathogen that causes many types of infections, including pharyngitis and severe invasive diseases. We recently sequenced the genome of a serotype M3 strain and identified a prophage-encoded secreted phospholipase A(2) designated SlaA. To study SlaA structure-activity relationships, 20 site-specific mutants were constructed by alanine-replacement mutagenesis and purified to apparent homogeneity. Enzymatic activity was greatly reduced by alanine replacement of amino acid residues previously described as crucial in the catalytic mechanism of secreted phospholipase A(2). Similarly, substitution of five residues in an inferred Ca(2+)-binding loop and three residues in the inferred active site region resulted in loss of activity of 76.5% or greater relative to the wild-type enzyme. Analysis of enzyme substrate specificity confirmed SlaA as a phospholipase A(2), with activity against multiple phospholipid head groups and acyl chains located at the sn-2 position. PCR analysis of 1,189 GAS strains representing 48 M protein serotypes commonly causing human infections identified the slaA gene in 129 strains of nine serotypes (M1, M2, M3, M4, M6, M22, M28, M75, and st3757). Expression of SlaA by strains of these serotypes was confirmed by Western immunoblot. SlaA production increased rapidly and substantially on co-culture with Detroit 562 human pharyngeal epithelial cells. Together, these data provide new information about a novel extracellular enzyme that participates in GAS-human interactions.

Alleles↗

PubMed2005

Structure and lytic activity of a Bacillus anthracis prophage endolysin.

We report a structural and functional analysis of the lambda prophage Ba02 endolysin (PlyL) encoded by the Bacillus anthracis genome. We show that PlyL comprises two autonomously folded domains, an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the crystal structure of the catalytic domain; its three-dimensional fold is related to that of the cell wall amidase, T7 lysozyme, and contains a conserved zinc coordination site and other components of the catalytic machinery. We demonstrate that PlyL is an N-acetylmuramoyl-L-alanine amidase that cleaves the cell wall of several Bacillus species when applied exogenously. We show, unexpectedly, that the catalytic domain of PlyL cleaves more efficiently than the full-length protein, except in the case of Bacillus cereus, and using GFP-tagged cell wall-binding domain, we detected strong binding of the cell wall-binding domain to B. cereus but not to other species tested. We further show that a related endolysin (Ply21) from the B. cereus phage, TP21, shows a similar pattern of behavior. To explain these data, and the species specificity of PlyL, we propose that the C-terminal domain inhibits the activity of the catalytic domain through intramolecular interactions that are relieved upon binding of the C-terminal domain to the cell wall. Furthermore, our data show that (when applied exogenously) targeting of the enzyme to the cell wall is not a prerequisite of its lytic activity, which is inherently high. These results may have broad implications for the design of endolysins as therapeutic agents.

Amidohydrolases↗

PubMed2005

Structural and genetic analyses reveal a key role in prophage excision for the TorI response regulator inhibitor.

TorI (Tor inhibition protein) has been identified in Escherichia coli as a protein inhibitor acting through protein-protein interaction with the TorR response regulator. This interaction, which does not interfere with TorR DNA binding activity, probably prevents the recruitment of RNA polymerase to the torC promoter. In this study we have solved the solution structure of TorI, which adopts a prokaryotic winged-helix arrangement. Despite no primary sequence similarity, the three-dimensional structure of TorI is highly homologous to the (lambda)Xis, Mu bacteriophage repressor (MuR-DBD), and transposase (MuA-DBD) structures. We propose that the TorI protein is the structural missing link between the (lambda)Xis and MuR proteins. Moreover, in vivo assays demonstrated that TorI plays an essential role in prophage excision. Heteronuclear NMR experiments and site-directed mutagenesis studies have pinpointed out key residues involved in the DNA binding activity of TorI. Our findings suggest that TorI-related proteins identified in various pathogenic bacterial genomes define a new family of atypical excisionases.

Amino Acid Sequence↗

PubMed2005

Effect of nutrient addition and environmental factors on prophage induction in natural populations of marine synechococcus species.

A series of experiments were conducted with samples collected in both Tampa Bay and the Gulf of Mexico to assess the impact of nutrient addition on cyanophage induction in natural populations of Synechococcus sp. The samples were virus reduced to decrease the background level of cyanophage and then either left untreated or amended with nitrate, ammonium, urea, or phosphate. Replicate samples were treated with mitomycin C to stimulate cyanophage induction. In five of the nine total experiments performed, cyanophage induction was present in the non-nutrient-amended control samples. Stimulation of cyanophage induction in response to nutrient addition (phosphate) occurred in only one Tampa Bay sample. Nutrient additions caused a decrease in lytic (or control) phage production in three of three offshore stations, in one of three estuarine experiments, and in a lysogenic marine Synechococcus in culture. These results suggest that the process of cyanophage induction as an assay of Synechococcus lysogeny was not inorganically nutrient limited, at least in the samples examined. More importantly, it was observed that the level of cyanophage induction (cyanophage milliliter(-1)) was inversely correlated to Synechococcus and cyanophage abundance. Thus, the intensity of the prophage induction response is defined by ambient population size and cyanophage abundance. This corroborates prior observations that lysogeny in Synechococcus is favored during times of low host abundance.

Bacteriophages↗

PubMed2005

Identification of novel Salmonella enterica serovar Typhimurium DT104-specific prophage and nonprophage chromosomal sequences among serovar Typhimurium isolates by genomic subtractive hybridization.

Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments-irsA, the HldD homologue, and three fragments with sequence similarity to prophages-were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.

Bacteriophage Typing↗

PubMed2005

EspJ is a prophage-carried type III effector protein of attaching and effacing pathogens that modulates infection dynamics.

Enterohemorrhagic Escherichia coli, enteropathogenic E. coli, and Citrobacter rodentium are highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attaching and effacing (A/E) lesions. These pathogens utilize a type III secretion system (TTSS) apparatus, encoded by the locus of enterocyte effacement, to translocate bacterial effector proteins into epithelial cells. Here, we report the identification of EspJ (E. coli-secreted protein J), a translocated TTSS effector that is carried on the 5' end of the cryptic prophage CP-933U. Infection of epithelial cells in culture revealed that EspJ is not required for A/E lesion activity in vivo and ex vivo. However, in vivo studies performed with mice demonstrated that EspJ possesses properties that influence the dynamics of clearance of the pathogen from the host's intestinal tract, suggesting a role in host survival and pathogen transmission.

Animals↗