Mutational analysis of the Escherichia coli glpFK region with Tn5 mutagenesis and the polymerase chain reaction

Lupski, James R.; Zhang, Y H; Rieger, Michael A.; Minter, Mikeanne; Hsu, B; Ooi, Beng Guat; Koeuth, Thearith; McCabe, Edward R.B.

doi:10.1128/jb.172.10.6129-6134.1990

Cited by 11 publications

(2 citation statements)

References 32 publications

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“…The function of GlpX is not yet known. It is possible that some of the chromosomal TnS insertions isolated by Lupski et al (25) were located in glpX. They described mutants with reduced growth on glycerol despite wild-type glycerol kinase activity and no glycerol transport defect.…”

Section: Discussionmentioning

confidence: 99%

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri

1992

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We have identified a new gene, glpX, belonging to the glp regulon of Escherichia coli, located directly downstream of the glpK gene. The transcription of glpX is inducible with glycerol and sn-glycerol-3-phosphate and is constitutive in a glpR mutant. glpX is the third gene in the glpFKX operon. The function of GlpX remains unknown. GlpX has an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gels. In addition to determining the E. coli glpX sequence, we also sequenced the corresponding glpFKX region originating from Shigella flexneri, which after transfer into E. coli was instrumental in elucidating the function of glpF in glycerol transport (D. P. Richey and E. C. C. Lin, J. Bacteriol. 112:784-790, 1972). Sequencing of the glpFKX region of this hybrid strain revealed an amber mutation instead of the tryptophan 215 codon in glpF. The most striking difference between the E. coli and S. flexneri DNA was found directly behind glpK, where two repetitive (REP) sequences were present in S. flexneri, but not in the E. coli sequence. The presence or absence of these REP sequences had no effect on transport or on growth on glycerol. Not including the REP sequence-containing region, only 1.1% of a total of 2,167 bp sequenced was different in the two sequences. Comparison of the sequence with those in the EMBL data library revealed a 99% identity between the last third of glpX and the first part of a gene called mvrA. We show that the cloned mvrA gene (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988) originated from the 88-min region of the Escherichia coli chromosome and not, as reported, from the 7-min region and that the gene product identified as MvrA is in fact encoded by a gene distal to glpX.

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Section: Discussionmentioning

confidence: 99%

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri

1992

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“…Glycerol-3-phosphate is also a primary precursor of phospholipid biosynthesis. The glycerol repressor, GlpR, controls expression of both glpFK and glpD, but the cyclic AMP receptor protein, Crp, controls transcription only of the glpFK operon, not glpD (28, 29). Thus, the glpFK operon, but not glpD, is subject to cyclic AMP control.…”

Section: Introductionmentioning

confidence: 99%

Transposon-mediated directed mutation in bacteria and eukaryotes

2017

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Transposon-mediated “directed” mutations occur at higher frequencies when beneficial than when detrimental and relieve the stress that causes them. The first and best-studied example involves regulation of Insertion Sequence-5 (IS5) insertion into a specific activating site upstream of the glycerol utilization operon in Escherichia coli, glpFK. This event promotes high level expression of the glpFK operon, allowing glycerol utilization in wild type cells under inhibitory conditions. The phosphoenolpyruvate-dependent, sugar transporting, phosphotransferase system (PTS) influences this process by regulating cytoplasmic glycerol-3-phosphate and cyclic AMP concentrations. Insertion frequencies are determined by IS5-specific tetranucleotide target sequences in stress-induced (DNA) duplex destabilization (SIDD) structures counteracted by two DNA binding proteins, GlpR and Crp which directly inhibit insertion, responding to cytoplasmic glycerol-3-phosphate and cyclic AMP, respectively. Expression of the E. coli master regulator of flagellar gene control, flhDC, is subject to activation by IS elements by a directed mechanism, and zinc-induced transposon-mediated zinc resistance has been demonstrated in Cupriavidus metallidurans. The use of DNA conformation and DNA binding proteins to control transposon hopping also occurs in eukaryotes.

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Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA

1991

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The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 bp away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam- mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.

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Mutational analysis of the Escherichia coli glpFK region with Tn5 mutagenesis and the polymerase chain reaction

Cited by 11 publications

References 32 publications

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri

Transposon-mediated directed mutation in bacteria and eukaryotes

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA

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