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1.
Gene ; 183(1-2): 129-36, 1996 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-8996097

RESUMO

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.


Assuntos
Vetores Genéticos/genética , Micobacteriófagos/genética , Mycobacterium/isolamento & purificação , Clonagem Molecular/métodos , Cosmídeos/genética , Expressão Gênica , Genes Reporter/genética , Cinética , Luciferases/biossíntese , Luciferases/genética , Lisogenia , Testes de Sensibilidade Microbiana/métodos , Micobacteriófagos/crescimento & desenvolvimento , Mycobacterium/virologia , Regiões Promotoras Genéticas/genética , Mapeamento por Restrição , Superinfecção
2.
Proc Natl Acad Sci U S A ; 93(7): 3132-7, 1996 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-8610181

RESUMO

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.


Assuntos
Cromossomos Bacterianos , Genoma Bacteriano , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Mapeamento Cromossômico , Cosmídeos , Elementos de DNA Transponíveis , Desoxirribonucleases de Sítio Específico do Tipo II , Biblioteca Gênica , Marcadores Genéticos , Mapeamento por Restrição , Especificidade da Espécie
4.
Mol Microbiol ; 11(4): 629-39, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7910936

RESUMO

Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall. The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic. The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems. Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria. In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated. The M. smegmatis asd gene was isolated by complementation in Escherichia coli. This gene was then used to isolate the asd genes from other mycobacteria. The asd-complementing fragments from BCG and M. smegmatis were sequenced. An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask). Primer extension analysis revealed that the only transcriptional start in this region is found 5' of the ask gene. This observation indicates that the mycobacterial ask and asd genes are in an operon.


Assuntos
Aspartato Quinase/genética , Aspartato-Semialdeído Desidrogenase/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Mycobacterium/genética , Óperon , Sequência de Aminoácidos , Sequência de Bases , Sequência Consenso , Ácido Diaminopimélico/metabolismo , Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mycobacterium/enzimologia , Fases de Leitura Aberta , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
5.
Proc Natl Acad Sci U S A ; 88(12): 5433-7, 1991 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-2052623

RESUMO

Mycobacteria, particularly Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium, are major pathogens of man. Although insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been possible to apply it to the mycobacteria. To overcome intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M. tuberculosis and bacille Calmette-Guérin (BCG) vaccine strains, we developed a system for random shuttle mutagenesis. A genomic library of Mycobacterium smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in Escherichia coli and these transposon-containing recombinant plasmids were reintroduced into mycobacterial chromosomes by homologous recombination. This system has allowed us to isolate several random auxotrophic mutants of M. smegmatis. To extend this strategy to M. tuberculosis and BCG, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E. coli and reintroduced into the mycobacteria. Surprisingly for prokaryotes, both BCG and M. tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes at a frequency of 10(-5) to 10(-4) relative to the number of transformants obtained with autonomously replicating vectors. Thus the efficient illegitimate recombination of linear DNA fragments provides the basis for an insertional mutagenesis system for M. tuberculosis and BCG.


Assuntos
Mutagênese , Mycobacterium/genética , Recombinação Genética , Southern Blotting , DNA Circular/genética , Genes Bacterianos , Mutação , Plasmídeos
6.
Res Microbiol ; 141(7-8): 931-9, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2101484

RESUMO

Bacille Calmette-Guerin (BCG), currently the most widely used vaccine in the world, was originally administered for many years as an oral vaccine. The low frequency of serious complications, inexpensive production, and adjuvanticity make BCG an ideal candidate for a recombinant vaccine vehicle. Although mycobacteria are slow growing and not yet well characterized genetically, we have recently developed technology for the genetic manipulation of BCG and other mycobacteria. Phage and plasmid systems based on a shuttle strategy to manipulate DNA in Escherichia coli and transfer it to mycobacteria have been developed. We have established that the aminoglycoside phosphotransferase gene can be used as an effective selectable marker in the mycobacteria and that a foreign antigen from Mycobacterium leprae can be expressed in BCG. Furthermore, a thorough analysis of mycobacterial expression sequences has been undertaken to optimize the expression of foreign antigens in BCG. We constructed an expression probe shuttle plasmid with beta-galactosidase as reporter gene, and have used it successfully to identify multiple mycobacteriophage DNA sequences with varying levels of constitutive or regulable promoter activity. Further genetic advances required for development of recombinant BCG into an effective recombinant vaccine vehicle, including possibilities for oral administration, are adumbrated.


Assuntos
Vacina BCG/genética , Mycobacterium bovis/imunologia , Administração Oral , Animais , Vacina BCG/administração & dosagem , Genes Bacterianos , Vetores Genéticos , Humanos , Mycobacterium bovis/genética , Plasmídeos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética
7.
Infect Immun ; 57(5): 1535-41, 1989 May.
Artigo em Inglês | MEDLINE | ID: mdl-2565292

RESUMO

Comparative DNA hybridization studies of genomic DNA indicated that, while different isolates of armadillo-derived Mycobacterium leprae have a high degree of homology, binding of M. leprae genomic DNA to DNA of other species of mycobacteria or to corynebacteria was low, establishing that M. leprae is only remotely genetically related to any of the species examined. Several candidate leprosy vaccine mycobacterial strains were similarly found to have little genetic similarity to M. leprae. In contrast, the DNAs of the slow-growing mycobacteria M. tuberculosis, M. africanum, M. bovis, and M. microti were found to be very closely related. In the course of these studies, an M. leprae-specific repetitive sequence, greater than 15-fold per genome equivalent, was identified that might be useful for diagnostic and epidemiological studies.


Assuntos
Antígenos de Bactérias/genética , Vacinas Bacterianas , DNA Bacteriano/genética , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Southern Blotting , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico
9.
Acta Leprol ; 7 Suppl 1: 203-7, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2503991

RESUMO

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system which allows for the transfer, mutation, and expression of specific genes. Genetic analysis of mycobacteria has been exceedingly difficult since the mycobacteria grow slowly and no natural efficient method of gene transfer within the pathogenic has thus far been found. Using a molecular genetic approach, we have developed both the vectors and the methodology for efficient gene transfer in the mycobacteria. Initially, a novel of type of mycobacteriophage vector was developed, termed a shuttle phasmid. This hybrid shuttle vector replicates in Escherichia coli as a plasmid and in mycobacteria as a phage, capable of introducing foreign DNA into a wide variety of mycobacterial species. A set of shuttle phasmids, constructed from a temperate mycobacteriophage, retained their ability to lysogenize their mycobacterial hosts and could thus introduce foreign DNA stably into mycobacterial cells. An E. coli gene conferring kanamycin-resistance was cloned into these vectors and shown to express in the mycobacteria, thus providing the first selectable marker gene for subsequent genetic studies. Using kanamycin-resistance gene as a selection, the M. fortuitum plasmid pAL5000 replicon, and electroporation; a plasmid transformation system has been developed for both M. smegmatis and BCG. We now plan to use these phage and plasmid systems to analyze, genetically, the virulence attributes of the pathogenic mycobacteria. In addition, by introducing and expressing foreign antigens in BCG, we hope to develop a novel recombinant multi-vaccine vehicle capable of conferring immunity to a variety of bacterial, viral, and parasitic pathogens.


Assuntos
Mycobacterium/genética , Vacina BCG/isolamento & purificação , Vacinas Bacterianas/isolamento & purificação , Genes Bacterianos , Vetores Genéticos , Mycobacterium/imunologia , Mycobacterium/patogenicidade , Plasmídeos , Transformação Genética , Virulência
10.
Proc Natl Acad Sci U S A ; 85(18): 6987-91, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2842799

RESUMO

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes. Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers. First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E. coli and phage capable of lysogenizing the mycobacterial host. These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome. When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E. coli was introduced into M. smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained. Second, to develop a plasmid transformation system in mycobacteria, M. fortuitum/E. coli hybrid plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed. When introduced into M. smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var. Bacille-Calmette-Guérin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants. These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines.


Assuntos
Regulação da Expressão Gênica , Lisogenia , Mycobacterium/genética , Transformação Bacteriana , Clonagem Molecular , Escherichia coli/genética , Canamicina Quinase , Hanseníase/microbiologia , Métodos , Mycobacterium/enzimologia , Fosfotransferases/genética , Plasmídeos , Tuberculose/microbiologia
11.
Nature ; 327(6122): 532-5, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-3473289

RESUMO

Mycobacteria are major pathogens of man and animals. There are approximately 10 million cases of tuberculosis world wide with an annual mortality of three million people. Leprosy, caused by Mycobacterium leprae, afflicts over ten million people, primarily in developing countries. M. tuberculosis and mycobacteria of the M. avium-intracellulare-scrofulaceum (MAIS) group are major opportunistic pathogens of patients with acquired immune deficiency syndrome (AIDS). M. paratuberculosis is the cause of Jöhne's disease in cattle. Yet, BCG (Bacille Calmette-Guerin), an avirulent strain of M. bovis, is the most widely used human vaccine in the world, having been administered to about 2.5 X 10(9) people since 1948 (ref. 4). BCG was highly protective against tuberculosis in England, but has been found not to be effective in preventing pulmonary tuberculosis in adults in Southern India. We have initiated studies to develop the methodology for efficient gene transfer in mycobacteria. We have constructed recombinant shuttle phasmids which are chimaeras containing mycobacteriophage DNA into which an E. coli cosmid is inserted. They can replicate in E. coli as plasmids and in mycobacteria as phages, and transfer DNA across both genera. These shuttle vectors permit for the first time the introduction of foreign DNA by infection into M. smegmatis and BCG. By introducing and ultimately expressing genes for protective antigens for a variety of pathogens, it may be possible to develop cultivatable mycobacteria into useful multivaccine vehicles.


Assuntos
DNA Bacteriano/genética , DNA Recombinante/metabolismo , Mycobacterium/genética , Plasmídeos , Bacteriófagos/genética , Clonagem Molecular , Replicação do DNA , Engenharia Genética/métodos , Vetores Genéticos , Transfecção
12.
Infect Immun ; 52(1): 101-9, 1986 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2937735

RESUMO

Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts.


Assuntos
DNA Bacteriano/genética , Vetores Genéticos , Proteínas de Choque Térmico , Mycobacterium/genética , Salmonella typhimurium/genética , Serina Endopeptidases , Streptococcus mutans/genética , Proteases Dependentes de ATP , DNA Recombinante , Dextranase/genética , Endopeptidases/genética , Regulação da Expressão Gênica , Teste de Complementação Genética , Glucosiltransferases/genética , Mutação , Peptídeo Hidrolases/genética , Recombinases Rec A/genética
13.
Proc Natl Acad Sci U S A ; 83(6): 1926-30, 1986 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2869492

RESUMO

Genomic libraries of Mycobacterium leprae DNA partially digested with Pst I were constructed in the expression vector pYA626, which contains the promoter region from the Streptococcus mutans gene encoding aspartate beta-semialdehyde dehydrogenase, which is very efficiently expressed in Escherichia coli. We have detected several clones that complement a mutation in the citrate synthase gene of E. coli. Southern blot analysis demonstrated that the complementing DNA was M. leprae DNA. Sodium dodecyl sulfate/polyacrylamide gel analysis of polypeptides produced by minicells containing the citrate synthase-complementing recombinant molecules demonstrated the production of a 46-kDa polypeptide. When the citrate synthase-complementing fragment was cloned in pYA626 in the reverse orientation, the recombinant molecule was no longer able to complement the mutation in the citrate synthase gene and no longer produced the 46-kDa polypeptide. When the DNA fragment was cloned in the Pst I site of pHC79, so as to allow expression from the beta-lactamase promoter, the resulting recombinant failed to complement the mutation in the E. coli citrate synthase gene yet still produced the 46-kDa polypeptide, but in one-fourth the amount than when expressed from the S. mutans asd promoters. This demonstrates that M. leprae translational sequences can be recognized by E. coli translational machinery. Promoter expression vectors can be used to obtain expression of protein antigens to be used for early diagnosis of leprosy or components of a vaccine and proteins that are targets of potential antileprosy drugs.


Assuntos
Regulação da Expressão Gênica , Genes Bacterianos , Mycobacterium leprae/genética , Regiões Promotoras Genéticas , Streptococcus mutans/genética , Aspartato-Semialdeído Desidrogenase/biossíntese , Aspartato-Semialdeído Desidrogenase/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Citrato (si)-Sintase/biossíntese , Citrato (si)-Sintase/genética , Clonagem Molecular , DNA Bacteriano/genética , DNA Recombinante , Escherichia coli/genética , Teste de Complementação Genética , Vetores Genéticos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
14.
J Bacteriol ; 161(3): 1093-102, 1985 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3882664

RESUMO

Molecular analysis of DNA from Mycobacterium leprae, "Mycobacterium lufu," and Mycobacterium vaccae has demonstrated that the G + C (guanine plus cytosine) contents of the DNAs are 56, 61, and 65%, respectively, and that the genome sizes are 2.2 X 10(9), 3.1 X 10(9), and 3.1 X 10(9) daltons, respectively. Because of the significant differences in both G + C content and genome size among M. leprae, "M. lufu," and M. vaccae DNAs, these species are not related, although hybridization experiments under nonstringent conditions, with two separate cloned M. leprae DNA inserts as probes, indicate that there are some conserved sequences among the DNAs. The G + C content of Dasypus novemcinctus (armadillo, the animal of choice for cultivating M. leprae) DNA was determined to be 36%. Genomic libraries potentially representing more than 99.99% of each genome were prepared by cloning into the cosmid vector, pHC79, in Escherichia coli K-12. A genomic library representing approximately 95% of the genome of M. vaccae was prepared in pBR322. M. leprae DNA was subcloned from the pHC79::M. leprae library into an expression vector, pYA626. This vector is a 3.8-kilobase derivative of pBR322 in which the promoter region of the asd (aspartate semialdehyde dehydrogenase) gene from Streptococcus mutans has been inserted in place of the EcoRI-to-PstI fragment of pBR322. Several (44% of those tested) pYA626::M. leprae recombinants and one pBR322::M. vaccae recombinant synthesized new polypeptides in minicells of E. coli, indicating that mycobacterial DNA can be expressed in E. coli K-12, although expression is probably dependent upon use of nonmycobacterial promoters recognized by the E. coli transcription-translation apparatus.


Assuntos
DNA Bacteriano/genética , Mycobacterium leprae/genética , Mycobacterium/genética , Proteínas de Bactérias/genética , Clonagem Molecular , DNA Recombinante , Desoxirribonucleotídeos/análise , Escherichia coli/genética , Regulação da Expressão Gênica , Vetores Genéticos , Peso Molecular , Hibridização de Ácido Nucleico
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