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G F Hatfull

Publications and source records attributed to G F Hatfull.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mycobacteriophage TM4: genome structure and gene expression.

Mycobacteriophage TM4 is a dsDNA-tailed phage that infects both fast-growing and slow-growing strains of mycobacteria. While TM4 has been used extensively for the construction of mycobacterial shuttle phasmids and for the delivery of reporter genes and transposons into mycobacterial cells, little is known about its genetics or molecular biology. We describe here the complete 52,797 bp genome sequence of TM4 and a map of its genome organization. While not a close relative of other mycobacteriophages, TM4 encodes several proteins with sequence similarity to those of other bacteriophages--including L5 and D29--indicating that they have common ancestry. In addition, TM4 encodes proteins with similarity to haloperoxidases, glutaredoxins and the WhiB family of transcriptional regulators. Following infection, TM4 genes are expressed in a defined temporal pattern, with the virion structural proteins expressed late in the phage growth cycle. Understanding the genetics of TM4 will greatly facilitate its use as a tool for the genetic manipulation of the mycobacteria.

Bacteriophage Typing↗
PubMed1998

Characterization of the dnaG locus in Mycobacterium smegmatis reveals linkage of DNA replication and cell division.

We have isolated a UV-induced temperature-sensitive mutant of Mycobacterium smegmatis that fails to grow at 42 degrees C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division. Complementation of this mutant with an M. smegmatis genomic library and subsequent subcloning reveal that the defect lies within the M. smegmatis dnaG gene encoding DNA primase. Sequence analysis of the mutant dnaG allele reveals a substitution of proline for alanine at position 496. Thus, dnaG is an essential gene in M. smegmatis, and DNA replication and cell division are coupled processes in this species. Characterization of the sequences flanking the M. smegmatis dnaG gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a dgt gene encoding dGTPase. The organization of this operon is conserved in Mycobacterium tuberculosis and Mycobacterium leprae, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.

Alleles↗
PubMed1998

Characterization of the mIHF gene of Mycobacterium smegmatis.

Integration of mycobacteriophage L5 requires the mycobacterial integration host factor (mIHF) in vitro. mIHF is a 105-residue heat-stable polypeptide that is not obviously related to HU or any other small DNA-binding proteins. mIHF is most abundant just prior to entry into stationary phase and is essential for the viability of Mycobacterium smegmatis.

Alleles↗
PubMed1998

Mycobacteriophages.

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Bacteriological Techniques↗
PubMed1997

Transcriptional silencing by the mycobacteriophage L5 repressor.

The success of a temperate bacteriophage is dependent upon its ability to completely shut down expression of its lytic genes during lysogenic growth. Mycobacteriophage L5 accomplishes this by an atypical phage repressor, gp71, which binds to multiple asymmetric DNA sites. L5 gp71 regulates transcription initiation at an early lytic promoter, Pleft, but also affects downstream gene expression at 'stoperator' sites in the phage genome. The L5 genome is replete with stoperator sites located within short intergenic spaces in both the early and late lytic operons and oriented specifically with respect to transcription. Binding of gp71 to these sites results in a strong orientation-dependent polar effect on downstream gene expression and global silencing of prophage gene expression.

Base Sequence↗
PubMed1997

Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis.

Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30 degrees C but not at 37 degrees C (TM4) or 38.5 degrees C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn10(kan) occurred in a site-specific fashion in M. smegmatis whereas Tn5367 transposed apparently randomly in M. phlei, Bacille Calmette-Guérin (BCG), and M. tuberculosis. Sequence analysis of the M. tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M. tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M. phlei, BCG, and M. tuberculosis.

DNA Transposable Elements↗
PubMed1997

Characterization of the mycobacteriophage L5 attachment site, attP.

Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.

Bacteriophages↗
PubMed1997

A putative ABC-transport operon of Mycobacterium smegmatis.

We have recently described the mpr gene of Mycobacterium smegmatis whose product confers resistance to mycobacteriophages L5 and D29 when overproduced (Barsom and Hatfull (1996) Mol. Microbiol. 21, 159-170). We have determined the nt sequence of approximately 3.5 kb immediately adjacent to mpr which appears to encode components of an ATP-binding cassette (ABC) transport system. Four closely-spaced open reading frames (ORF) were identified although two of these may cooperate to produce an integral membrane component of the transport system via a programmed translational frameshift. Another putative protein is also predicted to be an integral membrane protein, while the third is an ABC-transporter protein. We propose that these three putative proteins form a mycobacterial membrane-bound complex involved in protein-dependent transport. This is the first ABC-transport system to be described in mycobacteria.

ATP-Binding Cassette Transporters↗
PubMed1997

Mycobacteriophage L5 infection of Mycobacterium bovis BCG: implications for phage genetics in the slow-growing mycobacteria.

Mycobacteriophage L5 is a well-characterized temperate phage that forms stable lysogens in Mycobacterium smegmatis. The host range of L5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as Mycobacterium tuberculosis and bacille Calmette-Guerin (BCG). Moreover, luciferase reporter phage derivatives of L5 failed to produce light from BCG, suggesting that infection is blocked at or before the stage of DNA injection. In this study, we demonstrate that L5 infection of slow growing mycobacteria specifically requires a high concentration of Ca2+, conditions that differs from those required for infection of M. smegmatis by L5 and for infection of BCG by the closely related phage D29. In addition, we show that there are specific genetic determinants of L5 that confer the ability to infect slow growing mycobacteria, without altering infection of M. smegmatis. These observations extend the use of phage L5 for the diagnosis and analysis of tuberculosis and other mycobacterial diseases.

Calcium↗
PubMed1996

A novel host factor for integration of mycobacteriophage L5.

Bacterial integration host factors (IHFs) play central roles in the cellular processes of recombination, DNA replication, transcription, and bacterial pathogenesis. We describe here a novel mycobacterial IHF (mIHF) of Mycobacterium smegmatis and Mycobacterium tuberculosis that stimulates integration of mycobacteriophage L5. mIHF is the product of a single gene and is unrelated at the sequence level to other integration host factors. By itself, mIHF does not bind preferentially to attP DNA, although it significantly alters the pattern of integrase (Int) binding, promoting the formation of specific integrase-mIHF-attP intasome complexes.

Amino Acid Sequence↗
PubMed1996

Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria.

Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.

Cloning, Molecular↗
PubMed1996

Characterization of Mycobacterium smegmatis gene that confers resistance to phages L5 and D29 when overexpressed.

Bacteriophage infection requires a specific interaction with the outer surface of a bacterial host followed by interaction with the cell membrane and phage DNA injection. Phages of the mycobacteria encounter a cell wall that is rich in unusual lipid- and sugar-containing components which form a formidable barrier that must be passed to gain access to the membrane. We describe here a gene of Mycobacterium smegmatis that confers resistance to mycobacteriophages L5 and D29. The phage-resistance phenotype results not from mutation but from elevated expression of a wild-type gene. It appears that the product of this multicopy phage-resistance (mpr) gene may alter the structure of the host cell wall or membrane, thereby inhibiting productive phage DNA injection.

Amino Acid Sequence↗
PubMed1996

Positions of strand exchange in mycobacteriophage L5 integration and characterization of the attB site.

Mycobacteriophage L5 integrates into the genome of Mycobacterium smegmatis via site-specific recombination between the phage attP site and the bacterial attB site. These two sites have a 43-bp common core sequence within which strand exchange occurs and which overlaps a tRNAGly gene at attB. We show here that a 29-bp segment of DNA is necessary and sufficient for attB function and identify the positions of strand exchange.

Anticodon↗
PubMed1995

Transcriptional regulation of repressor synthesis in mycobacteriophage L5.

Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. Lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. We show here that there are three promoters located upstream of gene 71 which are active in an L5 lysogen but which do not require any phage-encoded proteins. In early lytic growth, gene 71 is also transcribed from a promoter, Pleft, located at the right end of the genome and which appears to be a target of gp71 regulation. A model is given for the regulation of L5 life cycles.

Amino Acid Sequence↗
PubMed1995

L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria.

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.

Bacterial Typing Techniques↗
PubMed1994

Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence.

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.

Animals↗
PubMed1994

Bacteriophages as tools for vaccine development.

The construction of live recombinant bacterial vaccines requires a reasonably sophisticated genetic system for the introduction, stabilization and expression of foreign antigen genes. Bacteriophages offer a rich collection of tools that can be used for vaccine construction, including site-specific integration-proficient vectors, non-antibiotic selectable markers and signals for efficient transcription and translation of foreign genes. We describe the characterization of a temperate phage of the mycobacteria, mycobacteriophage L5, and application of these phage studies for the construction of recombinant BCG vaccines.

BCG Vaccine↗