Summary: Engineered variants of the transposon Tn917 have been widely used to obtain insertion mutations and transcriptional fusions in Bacillus subtilis and other Gram-positive bacteria. We have developed a novel Tn917-based methodology useful for isolation and characterization of mutants resulting from gene over-expression. A Tn917 variant was constructed which contains a strong out-facing promoter near one end, able to promote transcription of genes in the vicinity of its insertion target. This transposon, designated Tn917PF1, was tested in model conditions. Three Tn917PF1 mutants of B. subtilis, with phenotypes presumed to result from gene over-expression, were analysed. Their phenotypes were shown to be due to transcription from the transposon promoter. In one mutant the promoter activated a deg gene, probably degQ. The other two contained different insertions decryptifying a B. subtilis gene encoding β-galactosidase.
AmoryA.,
KunstF.,
AubertE.,
Klier,
RapoportG.1987; Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis
. Journal of Bacteriology 169:324–333
AymerichS.,
Gonzy-TreboulG.,
SteinmetzM.1986; 5′-Noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis
. Journal of Bacteriology 166:993–998
BergC.,
BergD.,
GroismanE.1989; Transposable elements and the genetic engineering of bacteria. In Mobile DNA pp. 879–925 Edited by
BergD.,
HoweM.
Washington, DC: American Society for Microbiology;
BolivarF.,
RodriguezR. L.,
GreeneP. J.,
BetlachM. C.,
HeyneckerH. L.,
BoyerH. W.,
CrosaJ. H.,
FalkowS.1977; Construction and characterization of new cloning vehicles. II. A multipurpose system. Gene 2:95–113
CrutzA. M.,
SteinmetzM.,
AymerichS.,
RichterR.,
Le CoqD.1990; Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phospho-transferase system. Journal of Bacteriology 172:1043–1050
ErringtonJ.,
VogtC.1990; Isolation and characterization of mutations in the gene encoding an endogenous Bacillus subtilis β-galactosidase and its regulator. Journal of Bacteriology 172:488–490
HorinouchiS.,
WeisblumB.1982; Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible resistance to chloramphenicol. Journal of Bacteriology 150:815–825
Le CoqD.,
AymerichS.,
SteinmetzM.1991; Dual effect of a Tn917 insertion into the Bacillus subtilis sacX gent. Journal of General Microbiology 137:101–106
NagamiY.,
TanakaT.1986; Molecular cloning and nucleotide sequence of a DNA fragment from Bacillus natto that enhances production of extracellular proteases and levansucrase in Bacillus subtilis
. Journal of Bacteriology 166:20–28
PeregoM.,
SpiegelmanG. B.,
HochJ. A.1988; Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spoOA sporulation gene in Bacillus subtilis
. Molecular Microbiology 2:689–699
PerkinsJ. B.,
YoungmanP. J.1986; Construction and properties of Tn917-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis
. Proceedings of the National Academy of Sciences of the United States of America83140–144
SchaefferP.,
MilletJ.,
AubertJ. P.1965; Catabolite repression of bacterial sporulation. Proceedings of the National Academy of Sciences of the United States of America54704–711
SteinmetzM.,
AymerichS.1990; The Bacillus subtilis sac-deg system: how and why?. Genetics and Biochnology of Bacilli3ZukowskiM.,
GanesanA. T.,
HochJ. A.
New York: Academic Press (in the press);
SteinmetzM.,
Le CoqD.,
AymerichS.1989; Induction by sucrose of saccharolytic enzymes in Bacillus subtilis: evidence for two partially interchangeable regulatory pathways. Journal of Bacteriology 171:1519–1523
Trieu-CuotP.,
KlierA.,
CourvalinP.1985; DNA sequences specifying the transcription of the streptococcal kanamycin resistance gene in E coli B. subtilis
. Molecular and General Genetics 198:348–352
VandeyarM. A.,
MackeyC. J.,
LipskyR. H.,
ZahlerS. A.1986; The HvBC-leu operon of Bacillus subtilis
. Bacillus Molecular Genetics and Biotechnology Applications295–306GanesanA. T.,
HochJ. A.
New York: Academic Press;
YoungmanP.1987; Plasmid vectors for recovering and exploiting Tn917 transpositions in Bacillus and other Gram-positive bacteria. Plasmids: a Practical Approach79–103HardyK.
Oxford: IRL Press;
ZukowskiM. M.,
MillerL.,
CogswellP.,
ChenK.,
AymerichS.,
SteinmetzM.1990; Nucleotide sequence of the sacS locus of Bacillus subtilis reveals the presence of two regulatory genes. Gene 90:153–155