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At least 19 recordsLinked to original sources
PubMed1998

Mycobacteriophage D29 integrase-mediated recombination: specificity of mycobacteriophage integration.

Mycobacteriophage D29 is a lytic phage that infects both fast- and slow-growing species of the mycobacteria. D29 forms clear plaques on lawns of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guérin (BCG) in which a very high proportion of infected cells are killed. However, genomic analysis of D29 demonstrates that it is a close relative of the temperate mycobacteriophage L5, and is presumably a non-temperate derivative of a temperate parent. The D29 genome encodes a putative integrase protein with a primary amino acid sequence similar to that of the L5 integrase; the corresponding int genes fall in colinear positions within the D29 and L5 genomes, immediately flanking and transcribed away from their associated attP sites. We show here that the D29 integrase is functional and catalyzes integrative recombination between the D29 attP site and the M. smegmatis attB site in vitro in an mIHF-dependent manner. D29 integrase also mediates recombination between the L5 attP site and attB DNA and, reciprocally, L5 integrase catalyzes recombination with D29 attP DNA. However, in both in-vitro and in-vivo assays, the D29-encoded integrase recombines the D29 attP more efficiently than the L5 attP, and vice versa, suggesting that each integration system has evolved a degree of specificity of attP recognition. We also present the sequences of the putative attP site and integrase protein of the cryptic prophage-like element phiRv2, and compare them to those of mycobacteriophages L5 and D29.

Amino Acid Sequence↗
PubMed1997

Mycobacteriophage D29 contains an integration system similar to that of the temperate mycobacteriophage L5.

A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis, was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally hold view of D29 as a virulent phage must be reviewed.

Amino Acid Sequence↗
PubMed1987

Characterization of mycobacteriophage I8 and its unrelatedness to mycobacteriophages I1, I3 and I5.

Homology among the genomes of mycobacteriophages I1, I3, I5 and I8 has been studied. Based on restriction endonuclease cleavage patterns, dot blot hybridization and Southern blot hybridization analysis, the DNAs of phages I1, I3 and I5 have been shown to be homologous and indistinguishable, but entirely different from phage I8. Unlike the others, the I8 genome does not harbour any single-strand interruptions. The DNA is 43 kb in length with limited cyclic permutations and has a G + C content of 54%. The presence of 5-methylcytosine in I8 DNA was indicated from the restriction patterns of MspI and HpaII. The number of sites and fragment sizes for several restriction enzymes on I8 DNA has been determined. Phage I8 has a replication cycle of 300 min, with a latent period of 180 min, a rise period of 120 min and a burst size of 100. The viability of phage I8 is significantly reduced by treatment with organic solvents.

DNA, Viral↗
PubMed2005

[Molecular mechanism of the integration and lysis of mycobacteriophage].

Tuberculosis remains one of the major threats to public health. China is one of the heavy TB burden countries. Novel drugs and vaccines are urgently needed to combat the increasingly multidrug resistant TB. Mycobacteriophage is one of the hot topic in TB novel drugs discovery and drug susceptibility test. Phages can multiply via two alternative mechanisms: the lytic cycle or the lysogenic cycle. The lytic cycle ends with the lysis and death of the host cell, whereas the host cell remains alive in the lysogenic cycle. Lysogenic mycobacteriophages were intensively studied to elucidate the integration and lysis mechanisms of mycobacteriophage. The integration of mycobacteriophage requires for attP of bacteriopahge genome, attB of Mycobacterium genome, integrase and integration host factor. Some lysogenic phage, eg. mycobacteriophage Ms6, employ lytic cycle, form new phage, lysis host by the cooperation of lysin and holin, and release phages. There is no reports as to the mycobacteriophage unique to China clinical or environmental isolates. Studies on the integration and lysis molecular mechanism of mycobacteriophage might facilitate future new anti-TB drugs development.

Bacteriolysis↗
PubMed2005

Comparative analysis of the base composition and codon usages in fourteen mycobacteriophage genomes.

To study the possible codon usage and base composition variation in the bacteriophages, fourteen mycobacteriophages were used as a model system here and both the parameters in all these phages and their plating bacteria, M. smegmatis had been determined and compared. As all the organisms are GC-rich, the GC contents at third codon positions were found in fact higher than the second codon positions as well as the first + second codon positions in all the organisms indicating that directional mutational pressure is strongly operative at the synonymous third codon positions. Nc plot indicates that codon usage variation in all these organisms are governed by the forces other than compositional constraints. Correspondence analysis suggests that: (i) there are codon usage variation among the genes and genomes of the fourteen mycobacteriophages and M. smegmatis, i.e., codon usage patterns in the mycobacteriophages is phage-specific but not the M. smegmatis-specific; (ii) synonymous codon usage patterns of Barnyard, Che8, Che9d, and Omega are more similar than the rest mycobacteriophages and M. smegmatis; (iii) codon usage bias in the mycobacteriophages are mainly determined by mutational pressure; and (iv) the genes of comparatively GC rich genomes are more biased than the GC poor genomes. Translational selection in determining the codon usage variation in highly expressed genes can be invoked from the predominant occurrences of C ending codons in the highly expressed genes. Cluster analysis based on codon usage data also shows that there are two distinct branches for the fourteen mycobacteriophages and there is codon usage variation even among the phages of each branch.

Bacteriophages↗
PubMed2006

Use of a mycobacteriophage-based assay for rapid assessment of susceptibilities of Mycobacterium tuberculosis isolates to isoniazid and influence of resistance level on assay performance.

We standardized and assessed the performance of an in-house microtiter assay for determining the susceptibilities of Mycobacterium tuberculosis clinical isolates to isoniazid based on mycobacteriophage amplification technology. Seventy isolates (43 resistant and 27 sensitive according to the BACTEC 460 radiometric method and MIC determination) were studied. The isoniazid resistance molecular mechanism was previously determined by sequencing the entire katG gene and the mabA-inhA regulatory region. The sensitivity of the mycobacteriophage-based assay in detecting isoniazid resistance was 86.1%, the specificity achieved was 92.6%, and the overall accuracy was 88.6%. In order to assess the possible influence of resistance levels on the mycobacteriophage-based-assay sensitivity, the results were analyzed according to the isoniazid MICs. All the isolates exhibiting high-level resistance (MIC > or = 2 microg/ml) were scored as resistant by the mycobacteriophage-based assay (100% concordance), and 95% showed mutations or deletions in the catalytic domain of the katG gene. In contrast, 26.1% of the low-level-resistance strains (MICs, 0.25 to 1 microg/ml) were misclassified, and 66.7% had alterations in the mabA-inhA regulatory region. The mycobacteriophage-based assay could be used as a rapid method to detect the isoniazid susceptibility pattern, although data from those areas with high rates of low-level-resistance strains should be interpreted with caution. The features of the assay make it suitable for widespread application due to its low technical demand and cost.

Antitubercular Agents↗
PubMed1993

Comparative analysis of the genomes of mycobacteriophages infecting saprophytic and pathogenic mycobacteria.

The genomes of a series of mycobacteriophages have been analyzed to disclose possible relationships between genetic characteristics and host range. The percent guanine-plus-cytosine in the DNA of 14 phages was found to be 34.4 to 47.5, as determined by a double-labelling procedure, which is unaffected by the presence of modified bases. The DNA of few mycobacteriophages yielded discordant values when the G + C content was estimated by buoyant density determination and by the double labelling procedure. This observation suggests the possible presence of modified bases in these genomes. The reduced susceptibility of viral DNAs to several restriction endonucleases is suggestive of the occurrence of both methyladenine and methylcytosine in the genome of all the mycobacteriophages studied. Heterologous annealing among the 14 DNAs analyzed yielded 6 hybridization groups. Within one group, the homology level among viral genomes was estimated by comparing the electrophoretic mobilities of restriction fragments: values of 0.8 to 1.3% base substitution have thus been found. A comparison of the genomic characteristics and host range of the mycobacteriophages analyzed suggests a possible relationship between restriction pattern, G + C content, crosshybridization level and host range.

Base Composition↗
PubMed1966

Further studies of infectious DNA extracted from mycobacteriophages.

Results of the previous investigation in which it was found that DNA extracted from D29 mycobacteriophage was infectious for Mycobacterium smegmatis 607, have been extended. DNA extracted from mycobacteriophage D4 and D32 produced plaques when plated on their respective hosts; D28 DNA, extracted in the same manner and tested under similar conditions, failed to show infectivity. Species barriers were not crossed by mycobacteriophage DNA; bacteria resistant to intact phage were not infected with the phage DNA. The efficiency of plating of the DNA is very much lower than that of intact phage; infection of a given host was not accomplished by DNA when titration for plaque formation by the intact phage was less than 10(9) PFU. The base composition of DNA extracted from the four mycobacteriophages and the three propagating hosts was very similar. The bases were paired, adenine with thymine and guanine with cytosine. A relatively higher per cent of guanine-cytosine than of adenine-thymine, was found. The buoyant density of each DNA in CsCl was linearly related to its guanine-cytosine content whereas with the exception of D28 DNA, thermal denaturation temperatures failed to show this relationship. However, the thermal transition profiles were characteristic of double stranded DNA. Additional evidence that D29 DNA forms complexes with basic proteins was obtained. Binding between calf thymus histone and between RNAase and D29 DNA readily occurs with a resultant loss in DNA infectivity. Trypsin and D29 DNA are only weakly reactive.

Animals↗
PubMed2006

Host range of 14 mycobacteriophages in Mycobacterium ulcerans and seven other mycobacteria including Mycobacterium tuberculosis--application for identification and susceptibility testing.

The host range of well-characterized mycobacteriophages, such as D29 and TM4, has been determined, together with that of more recently isolated mycobacteriophages, in Mycobacterium ulcerans, Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium fortuitum and Mycobacterium chelonae. Here, a set of virulent phages for M. ulcerans, a pathogen with a dramatic increase of incidence over the last decade, is demonstrated. In this work, a mycobacteriophage replication assay was adapted for the identification and rifampicin-susceptibility testing of M. ulcerans. Mycobacteriophages have generated a number of useful tools and enabled insights into mycobacterial genetics. With regard to the neglected pathogen M. ulcerans, the findings presented in this work allow the application of a large range of phage-based vectors and markers. The potential of phage therapy can now be evaluated for this extracellular pathogen.

Animals↗
PubMed2002

Expression of Mycobacteriophage Ms6 lysis genes is driven by two sigma(70)-like promoters and is dependent on a transcription termination signal present in the leader RNA.

A mycobacteriophage Ms6 strong promoter region (P(lys)) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem sigma(70)-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region P(lys) drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that beta-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.

Amino Acid Sequence↗
PubMed2004

Cloning and sequencing analysis of the repressor gene of temperate mycobacteriophage L1.

The wild-type and temperature-sensitive (ts) repressor genes were cloned from the temperate mycobacteriophage L1 and its mutant L1cIts391, respectively. A sequencing analysis revealed that the 131st proline residue of the wild-type repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative N-terminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor.

Amino Acid Sequence↗
PubMed2005

[The in vitro killing of intracellular Mycobacterium smegmatis by Mycobacteriophage].

OBJECTIVE: To study the effect of Mycobacteriophage on the lysis of intracellular Mycobacterium smegmatis. METHODS: Peritoneal macrophages from BALB/C mice were incubated with Mycobacterium smegmatis for 4 h, and the extracellular bacteria were removed. Then the infected macrophages were treated for 2 h with normal saline, or different doses of Mycobacteriophages (2.1 x 10(7) PFU, 2.1 x 10(6) PFU, and 2.1 x 10(5) PFU, respectively), all in a volume of 0.1 ml, and then the extracellular phages and Mycobacterium smegmatis were removed by washing. After incubation for 24 h, the number of viable intracellular bacteria was determined. The intracellular changes after infection of host bacteria by bacteriophages in the macrophages were observed by electron microscopy. RESULTS: The logarithm 10 of viable intracellular bacteria unit was 5.74 +/- 0.18 in the saline group, 4.77 +/- 0.08 in the high dose phage group (P < 0.01), 4.97 +/- 0.17 in the moderate dose phage group (P < 0.01), and 5.33 +/- 0.13 in the low dose phage group (P > 0.05). Electron microscopy confirmed the infection of intracellular bacteria by the bacteriophages and the production of filial bacteriophages. CONCLUSIONS: Mycobacteriophages phagocytosed by macrophages are capable of killing the infected mycobacteria. The result suggests that the use of Mycobacteriophages is a potentially novel strategy in the treatment of intracellular bacterial infection.

Animals↗