Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae

Clark‐Curtiss, Josephine E.; Jacobs, William R.; Docherty, Martin; Ritchie, Leigh; Curtiss, rd R

doi:10.1128/jb.161.3.1093-1102.1985

Cited by 170 publications

(74 citation statements)

References 26 publications

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“…The sensitivity achieved was such as to detect as low as 20 fg of purified M. leprae DNA. Estimations of the number of equivalent bacilli were based on the fact that c. 5 fg of DNA corresponds to one bacterium (Clark-Curtiss et al, 1985). By this estimation, the minimal number of M. leprae that this assay could detect is four bacilli.…”

Section: Discussionmentioning

confidence: 99%

LightCycler™ real-time PCR for rapid detection and quantitation ofMycobacterium lepraein skin specimens

Rudeeaneksin

Srisungngam

Sawanpanyalert

et al. 2008

FEMS Immunol Med Microbiol

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Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and bacterial enumeration by microscopic examination is required for leprosy classification, choice in choosing and monitoring chemotherapy regimens, and diagnosis of relapse. However, detection and quantification using standard microscopy yields results of limited specificity and sensitivity. We describe an extremely sensitive and specific assay for the detection and quantification of M. leprae in skin biopsy specimens. Primers that amplified a specific 171-bp fragment of M. leprae 16S rRNA gene were chosen and specificity was verified by amplicon melting temperature. The method is sensitive enough to detect as low as 20 fg of M. leprae DNA, equivalent to four bacilli. The assay showed 100% concordance with clinical diagnosis in cases of multibacillary patients, and 50% of paucibacillary leprosy. The entire procedure of DNA extraction and PCR could be performed in c. 3 h. According to normalized quantitative real-time PCR, the patients in this study had bacilli numbers in the range of 1.07 x 10(2) -1.65 x 10(8) per 6-mm3 skin biopsy specimen. This simple real-time PCR assay is a facile tool with possible applications for rapid detection and simultaneous quantification of leprosy bacilli in clinical samples.

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Section: Discussionmentioning

confidence: 99%

LightCycler™ real-time PCR for rapid detection and quantitation ofMycobacterium lepraein skin specimens

Rudeeaneksin

Srisungngam

Sawanpanyalert

et al. 2008

FEMS Immunol Med Microbiol

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“…Chromosomal DNA was isolated from Z. mobilis CP4 as described previously [12]. The DNA was partially digested with Sau3A, and fragments of 5-7 kb were isolated using a sucrose gradient [13]. Libraries of these fragments were constructed by ligation into the dephosphorylated BamHI site of pUC18 [14].…”

Section: Isolation Of 2 Mobilis Chromosomal Dna and Construction Of mentioning

confidence: 99%

An allosterically insensitive class of cyclohexadienyl dehydrogenase from Zymomonas mobilis

Zhao

Xia

Ingram

et al. 1993

European Journal of Biochemistry

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The key enzyme of tyrosine biosynthesis in many Gram-negative prokaryotes is cyclohexadienyl dehydrogenase. The Zymomonas mobilis gene ( t y r o coding for this enzyme was cloned in Escherichia coli by camplementation of a tyrosine auxotroph. The tyrC gene was 882 bp long, encoding a protein with a calculated molecular mass of 32086 Da. The Z. mobilis cyclohexadienyl dehydrogenase expressed in E. coli was purified to electrophoretic homogeneity. The subunit molecular mass of the purified enzyme was 32kDa as determined by SDSPAGE. The ratio of the activity of arogenate dehydrogenase to that of prephenate dehydrogenase (approximately 3 : 1) remained constant throughout purification, and the two activities were therefore inseparable. The genetic and biochemical data obtained demonstrated a single enzyme protein capable of catalyzing either of two reactions. K, values of 0.25 mM and 0.18 mM were obtained for prephenate and L-arogenate, respectively. The K, value obtained for NAD+ (0.09 mM) was the same regardless of whether the enzyme was assayed as arogenate dehydrogenase or as prephenate dehydrogenase. Unlike the corresponding enzyme of Pseudomonas aemginosa or E. coli, the cyclohexadienyl dehydrogenase of Z. mobilis lacks sensitivity to feedback inhibition by L-tyrosine. A typical NAD+-binding domain was found to be located at the N-terminus of the protein.Although the deduced amino-acid sequence of the Z. mobilis cyclohexadienyl dehydrogenase showed relatively low identity (19-32 %) with the prephenate dehydrogenases of Bacillus subtilis and Saccharomyces cerevisiae, as well as with the cyclohexadienyl dehydrogenase components of the bifunctional T-proteins of E. coli and Erwinia herbicola, a presumptive motif was identified which may correspond to critical residues of the binding site for cyclohexadienyl substrate molecules.Immediately upstream of tyrC a portion of a gene was sequenced and found to exhibit clearcut homology of the deduced amino-acid sequence with the B. subtilis hzsH gene product. Thus, the Zymomonas gene organization is reminiscent of the linkage of genes encoding a tyrosine-pathway dehydrogenase and a histidine-pathway aminotransferase in B. subtilis.

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“…In addition, the failure to grow M. leprae in vitro has limited the data that are available for its taxonomic classification. Moreover such data that are available are often ambiguous; for example the guanine plus cytosine content of M. leprae purified from experimentally infected armadillos was found to be 56%, which is lower than the 64-67% found for cultivable mycobacteria [1].…”

Section: Introductionmentioning

confidence: 89%

“…Previously, we isolated rRNA from M. leprae recovered from armadillo tissue and identified the nucleotide sequence near to the 3'-end [2] by the method of primer extension [3]. We have used the same procedure to obtain the nucleotide sequence (nucleotide positions 1305-1508) near to the 3'-end of 16SrRNA of M. tuberculosis (63.7% G + C [4]), Mycobacterium vaccae (63% G + C [1]) and Mycobacterium smegmatis (66.7% G + C [5]). The results are of interest because of their taxonomic and evolutionary significance for M. leprae.…”

Section: Introductionmentioning

confidence: 99%

Determination and evolutionary significance of nucleotide sequences near to the 3′-end of 16S ribosomal RNA of mycobacteria

Estrada-G¹

1989

FEMS Microbiology Letters

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The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae. No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria. The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera. For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence. The observations support the assignment of M. leprae to the genus Mycobacterium.

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Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae

Cited by 170 publications

References 26 publications

LightCycler™ real-time PCR for rapid detection and quantitation ofMycobacterium lepraein skin specimens

LightCycler™ real-time PCR for rapid detection and quantitation ofMycobacterium lepraein skin specimens

An allosterically insensitive class of cyclohexadienyl dehydrogenase from Zymomonas mobilis

Determination and evolutionary significance of nucleotide sequences near to the 3′-end of 16S ribosomal RNA of mycobacteria

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