Abstract: The present invention provides a purified and isolated nucleic acid encoding mycobacterial isocitrate lyase, as well as mutated forms of the nucleic acid. Further provided are purified and isolated isocitrate lyase proteins and mutated isocitrate lyase proteins. Additionally, the present invention provides vectors which comprises nucleic acid sequences encoding mycobacterial isocitrate lyase and mutated forms of this nucleic acid, as well as host cells containing these vectors. Also provided is a mycobacterium containing one or more mutations in its isocitrate lyase gene. Further provided by the present invention are agents that inhibit the activity or expression of a mycobacterial lyase protein, a method of identifying these, and a method of producing them. Finally, the present invention also provides a method of identifying genes required for persistence of mycobacteria.

Abstract: A mutated mycobacterium selected from the class consisting of mutated M.bovis-BCG, mutated M.tuberculosis, and mutated M. leprae. The mutation of M.bovis-BCG, M.tuberculosis, or M. leprae is preferably effected through an insertional mutation of a mycobacterial gene. The insertional mutagenesis may be effected, for example, through illegitimate recombination or by a mycobacterial transposon. Such mutated mycobacteria may then be transformed with an expression vector(s) containing a complement gene to the gene which is mutated, and preferably also including a heterologous gene.

Abstract: The present invention provides a recombinant M. tuberculosis mycobacterium that is auxotrophic for leucine. The present invention also provides a vaccine comprising a recombinant M. tuberculosis mycobacterium that is auxotrophic for leucine, as well as a method for treating or preventing tuberculosis in a subject comprising administering to the subject a recombinant M. tuberculosis mycobacterium that is auxotrophic for leucine in an amount effective to treat or prevent tuberculosis in the subject.

Abstract: Disclosed is a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation, where an “unmarked mutation” is a mutated nucleotide sequence introduced into a mycobacterium where the introduced mutated nucleotide sequence does not contain a selectable marker, such as a gene conferring antibiotic resistance to the recombinant mycobacterium incorporating the mutated nucleotide sequence. Also disclosed is a method for preparing a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation, as well as a vaccine comprising a recombinant slow-growing mycobacterium having at least one mycobacterial gene containing an unmarked mutation dispersed in a physiologically acceptable carrier. Further disclosed is a method of treating or preventing tuberculosis in a subject comprising administering the vaccine of the present invention in an amount effective to treat or prevent tuberculosis in the subject.

Abstract: The present invention provides a purified and isolated nucleic acid encoding mycobacterial isocitrate lyase, as well as mutated forms of the nucleic acid. Further provided are purified and isolated isocitrate lyase proteins and mutated isocitrate lyase proteins. Additionally, the present invention provides vectors which comprises nucleic acid sequences encoding mycobacterial isocitrate lyase and mutated forms of this nucleic acid, as well as host cells containing these vectors. Also provided is a mycobacterium containing one or more mutations in its isocitrate lyase gene. Further provided by the present invention are agents that inhibit the activity or expression of a mycobacterial lyase protein, a method of identifying these, and a method of producing them. Finally, the present invention also provides a method of identifying genes required for persistence of mycobacteria.

Abstract: Recombinant mycobacterial vaccine vehicles capable of expressing DNA of interest which encodes at least one protein antigen for at least one pathogen against which an immune response is desired and which can be incorporated into the mycobacteria or stably integrated into the mycobacterial genome. The vaccine vehicles are useful for administration to mammalian hosts for purposes of immunization. A recombinant vector which replicates in E. coli but not in mycobacteria is also disclosed. The recombinant vector includes 1) a mycobacterial gene or portions thereof, necessary for recombination with homologous sequences in the genome of mycobacteria transformed with the recombinant plasmid; 2) all or a portion of a gene which encodes a polypeptide or protein whose expression is desired in mycobacteria transformed with the recombinant plasmid; 3) DNA sequences necessary for replication and selection in E coli; and 4) DNA sequences necessary for selection in mycobacteria (e.g., drug resistance).

Abstract: This invention relates to mycobacterial species-specific reporter mycobacteriophages (reporter mycobacteriophages), methods of producing said reporter mycobacteriophages and the use of said reporter mycobacteriophages for the rapid diagnosis of mycobacterial infection and the assessment of drug susceptibilities of mycobacterial strains in clinical samples. In particular, this invention is directed to the production and use of luciferase reporter mycobacteriophages to diagnose tuberculosis. The mycobacterial species-specific reporter mycobacteriophages comprise mycobacterial species-specific mycobacteriophages which contain reporter genes and transcriptional promoters therein. When the reporter mycobacteriophages are incubated with clinical samples which may contain the mycobacteria of interest, the gene product of the reporter genes will be expressed if the sample contains the mycobacteria of interest, thereby diagnosing mycobacterial infection.

Abstract: Disclosed are novel recombinant mutant strains of mycobacteria that are deficient for the synthesis or transport of dimycoserosalphthiocerol (“DIM”). The present invention also provides a method of producing a recombinant mutant mycobacterium that is deficient for the synthesis or transport of DIM, comprising mutating a nucleic acid responsible for the synthesis or transport of dimycoserosalphthiocerol, including a nucleic acid comprising the promoter for the pps operon, fadD28 or mmpL7. The present invention also provides a vaccine comprising a DIM mutant mycobacterium of the present invention, as well as a method for the treatment or prevention of tuberculosis in a subject using the vaccine.

Abstract: Recombinant mycobacterial vaccine vehicles capable of expressing DNA of interest which encodes at least one protein antigen for at least one pathogen against which an immune response is desired and which can be incorporated into the mycobacteria or stably integrated into the mycobacterial genome. The vaccine vehicles are useful for administration to mammalian hosts for purposes of immunization. A recombinant vector which replicates in E. coli but not in mycobacteria is also disclosed. The recombinant vector includes 1) a mycobacterial gene or portions thereof, necessary for recombination with homologous sequences in the genome of mycobacteria transformed with the recombinant plasmid; 2) all or a portion of a gene which encodes a polypeptide or protein whose expression is desired in mycobacteria transformed with the recombinant plasmid; 3) DNA sequences necessary for replication and selection in E. coli; and 4) DNA sequences necessary for selection in mycobacteria (e.g., drug resistance).

Abstract: The present invention provides a method for high frequency of allelic exchange in the slow-growing mycobacteria using in vitro generated specialized transducing mycobacteriophages, as well as the recombinant slow-growing mycobacteria generated using the disclosed method. A transducing mycobacteriophage of the present invention comprises a conditional mycobacteriophage containing an E. coli bacteriophage lambda cosmid inserted into a non-essential region of the mycobacteriophage, said cosmid containing a mutated DNA substrate which is homologous to a wildtype nucleic acid sequence of a slow-growing mycobacterium. When slow-growing mycobacteria infected with the conditional transducing phage are cultured under conditions wherein the conditional transducing phage does not replicate, the mutated DNA substrate is incorporated into the chromosomal DNA of the slow-growing mycobacteria by homologous recombination, thereby generating the recombinant slow-growing mycobacteria of the present invention.

Abstract: This invention relates to the identification, cloning, sequencing and characterization of the iniB, iniA and iniC genes of mycobacteria which are induced by a broad class of antibiotics that act by inhibiting cell wall biosynthesis, including the first line antituberculosis agents, isoniazid and ethambutol. The present invention provides purified and isolated iniB, iniA, iniC and iniB promoter nucleic acids which may comprise the iniBAC operon, as well as mutated forms of these nucleic acids. The present invention also provides one or more single-stranded nucleic acid probes which specifically hybridize to the iniB, iniA, iniC and iniB promoter nucleic acids, and mixtures thereof, which may be formulated in kits, and used in the diagnosis of drug-resistant mycobacterial strain. The present invention also provides methods for the screening and identification of drugs effective against Mycobacterium tuberculosis using induction of the iniB promoter.

Abstract: This invention relates to mycobacterial species-specific reporter mycobacteriophages (reporter mycobacteriophages), methods of producing said reporter mycobacteriophages and the use of said reporter mycobacteriophages for the rapid diagnosis of mycobacterial infection and the assessment of drug susceptibilities of mycobacterial strains in clinical samples. In particular, this invention is directed to the production and use of luciferase reporter mycobacteriophages to diagnose tuberculosis. The mycobacterial species-specific reporter mycobacteriophages comprise mycobacterial species-specific mycobacteriophages which contain reporter genes and transcriptional promoters therein. When the reporter mycobacteriophages are incubated with clinical samples which may contain the mycobacteria of interest, the gene product of the reporter genes will be expressed if the sample contains the mycobacteria of interest, thereby diagnosing mycobacterial infection.

Abstract: The present invention refers in general to novel recombinant mycobacteria that are auxotrophic for diaminopimelate. In particular, this invention relates to novel auxotrophic recombinant mycobacteria, to methods of making the mycobacteria, and to uses of the mycobacteria to deliver vaccines. This invention also provides for uses of the mycobacteria in drug screening processes.

Abstract: This invention relates to the identification, cloning, sequencing and characterization of the embCAB operon which determines mycobacterial resistance to the antimycobacterial drug ethambutol. The embCAB operon encodes the proteins which are the target of action of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium leprae for ethambutol. The present invention provides purified and isolated embC, embA, and embB nucleic acids which comprise the embCAB operon, as well as mutated forms of these nucleic acids. The present invention also provides one or more single-stranded nucleic acid probes which specifically hybridize to the wild type embCAB operon or the mutated embCAB operon, and mixtures thereof, which may be formulated in kits, and used in the diagnosis of drug-resistant mycobacterial strain. The present invention also provides methods for the treatment and prevention of mycobacterial infections.

Abstract: The present invention provides for novel vector constructs comprising an origin of replication; a nucleotide sequence encoding an intein, the nucleotide sequence having a unique restriction enzyme site, critical amino acid residues located at the splice junctions of the intein, the intein inserted into a nucleotide sequence encoding a selectable marker; and a nucleotide sequence encoding suitable regulatory elements so as to effect expression of the vector construct in a suitable host cell. The vector constructs of the present invention may contain a DNA of interest cloned into a unique restriction site of the intein, and may be used as a vaccine alone or transformed into a vaccine vector. The vector constructs of this invention may further be used in methods of selecting translated open reading frames or genes, leading to the identification of potentially protective antigens of pathogenic organisms.

Abstract: The present invention provides a conditional shuttle phasmid constructed by inserting a cosmid into a non-essential region of the TM4 mycobacteriophage that introduces DNA of interest into mycobacteria, especially M. tuberculosis complex organisms and other slow growing mycobacteria. The present invention provides a recombinant mycobacterium which expresses a DNA of interest incorporated into its chromosome by a TM4 conditional shuttle phasmid containing the DNA of interest. The present invention further provides a mycobacterial auxotrophic mutant and a method of generating auxotrophic mutants.

Abstract: Recombinant mycobacterial vaccine vehicles capable of expressing DNA of interest which encodes at least one protein antigen for at least one pathogen against which an immune response is desired and which can be incorporated into the mycobacteria or stably integrated into the mycobacterial genome. The vaccine vehicles are useful for administration to mammalian hosts for purposes of immunization. A recombinant vector which replicates in E. coli but not in mycobacteria is also disclosed. The recombinant vector includes 1) a mycobacterial gene or portions thereof, necessary for recombination with homologous sequences in the genome of mycobacteria transformed with the recombinant plasmid; 2) all or a portion of a gene which encodes a polypeptide or protein whose expression is desired in mycobacteria transformed with the recombinant plasmid; 3) DNA sequences necessary for replication and selection in E. coli; and 4) DNA sequences necessary for selection in mycobacteria (e.g., drug resistance).

Abstract: Recombinant mycobacterial vaccine vehicles capable of expressing DNA of interest which encodes at least one protein antigen for at least one pathogen against which an immune response is desired and which can be incorporated into the mycobacteria or stably integrated into the mycobacterial genome. The vaccine vehicles are useful for administration to mammalian hosts for purposes of immunization. A recombinant vector which replicates in E. coli but not in mycobacteria is also disclosed. The recombinant vector includes 1) a mycobacterial gene or portions thereof, necessary for recombination with homologous sequences in the genome of mycobacteria transformed with the recombinant plasmid; 2) all or a portion of a gene which encodes a polypeptide or protein whose expression is desired in mycobacteria transformed with the recombinant plasmid; 3) DNA sequences necessary for replication and selection in E. coli; and 4) DNA sequences necessary for selection in mycobacteria (e.g., drug resistance).

Abstract: This invention relates to InhA enzyme crystals and to methods of growing said crystals. This invention is further directed to the utilization of said crystals to determine the three dimensional structure of InhA enzyme utilizing heavy atom derivatives of said crystals, and to the identification and development of compounds which inhibit the biochemical activity of InhA enzyme in bacteria and plants.

Abstract: Polynucleotides associated with virulence in mycobacteria, and particularly a fragment of DNA isolated from M. bovis that contains a region encoding a putative sigma factor. Also provided are methods for a DNA sequence or sequences associated with virulence determinants in mycobacteria, and particularly in M. tuberculosis and M. bovis. The invention also provides corresponding polynucleotides associated with avirulence in mycobacteria. In addition, the invention provides a method for producing strains with altered virulence or other properties which can themselves be used to identify and manipulate individual genes.