Summary: Substrains of Mycobacterium bovis BCG (BCG) have been divided into two major groups, high and low producers, on the basis of the amount of secretion of the MPB70 protein. The antigen is produced in high concentration by BCG Tokyo, Moreau, Russia and Sweden (high-producer substrains), whereas in BCG Pasteur, Copenhagen and Tice (low-producer substrains) it is detected at 1 % (w/w) or less of the concentration of BCG Tokyo. To investigate why this protein is secreted differently, the MPB70 genes of BCG Tokyo and Pasteur were cloned, sequenced and compared. The MPB70 genes in two substrains showed exactly the same sequence. Even the upstream and downstream regions of the MPB70 gene were identical. MPB70 gene expression was assessed by means of Northern hybridization analysis and reverse transcriptase polymerase chain reaction. The mRNA was clearly detected in BCG Tokyo, but at a very low level in BCG Pasteur. On the basis of these results, the difference in the secretion of the MPB70 protein between BCG Tokyo and Pasteur was attributed to differential transcription efficiencies.
BoddinghausB.,
RogallT.,
FlohrT.,
BlockerH.,
BottgerE.C.1990; Detection and identification of mycobacteria by ampli-fication of rRNA.. J Clin Microbiol 28:1751–1759
BorremansM.,
De WitL.,
VolckaertG.,
OomsJ.,
de BruynJ.,
HuygenK.,
van VoorenJ.,
StelandreM.,
VerhofstadtR.,
ContentJ.1989; Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis.
. Infect Immun 57:3123–3130
De WitL.,
de la CuvellerieA.,
OomsJ.,
ContentJ.1990; Nucleotide sequence of the 32 kDa-protein gene (antigen 85 A) of Mycobacterium bovis BCG.. Nucleic Acids Res 18:3995
FomukongN.G.,
DaleJ.W.,
OsbornT.W.,
GrangeJ.M.1992; Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains.. J Appl Bacteriol 72:126–133
LehrachH.,
DiamondD.,
WozneyJ.M.,
BoedtkerH.1977; RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination.. Biochemistry 16:4743–4751
LiH.,
UlstrupJ.C.,
JonassenT.O.,
MelbyK.,
NagaiS.,
HarboeM.1993; Evidence for absence of the MPB64 gene in some substrains of Mycobacterium bovis BCG.. Infect Immun 61:1730–1734
MatsumotoS.,
MatsuoT.,
OharaN.,
NaitoM.,
MinamiJ.,
YamadaT.1995; Cloning and sequencing of the MPT70 from Mycobacterium tuberculosis H37Rv and expression in mycobacteria using E. coli-Mycobacteria shuttle vector.. Scand J Immunol 41:281–287
MiuraK.,
NagaiS.,
KinomotoM.,
HagaS.,
TokunagaT.1983; Comparative studies with various substrains of Myco-bacterium bovis BCG on the production of an antigenic protein, MPB70.. Infect Immun 39:540–545
RadfordA.J.,
WoodP.R.,
BillmanJ.H.,
GeysenH.M.,
MasonT.J.,
TribbickG.1990; Epitope mapping of the Mycobacterium bovis secretory protein MPB70 using overlapping peptide analysis.. J Gen Microbiol 136:265–272
TholeJ.E.,
KeulenW.J.,
DeB.J.,
KolkA.H.,
GroothuisD.G.,
BerwaldL.G.,
TiesjemaR.H.,
van EmbdenJ.D.1987; Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG expressed in Escherichia coli K-12.. Infect Immun 55:1466–1475 [erratum inInfect Immun55, 1949]
WikerH.G.,
HarboeM.,
NagaiS.1991; A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis.
. J Gen Microbiol 137:875–884
YamaguchiR.,
MatsuoK.,
YamazakiA.,
AbeG,
TerasakaK.,
NagaiS.,
YamadaT.1989; Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis BCG.. Infect Immun 57:283–288
YamaguchiM.,
DieffenbachC.W.,
ConnollyR.,
CruessD.F.,
BaurW.,
SharefkinJ.B.1992; Effect of different laboratory techniques for guanidinium-phenol-chloroform RNA extraction on A260/A280 and on accuracy of mRNA quantitation by reverse transcriptase-PCR.. PCR Methods Appl 1:286–290