Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions

Abstract

Twenty-four independent phosphate starvation-inducible (psi) transcriptional fusions made with Mu dl(lacZ bla) were analyzed by sequencing the psi::lacZ(Mu dl) chromosomal junctions by using DNAs amplified with the polymerase chain reaction or mini-Mu cloning. Our DNA sequence analysis showed that the MuR DNA in Mu dl has an unexpected structure that is comprised of 104 bases of MuR DNA in the form of a large inverted repeat, which we denoted Mu dl-R. Also, Mu dls in the phoA and phn (psiD) loci of the phosphate regulon showed regional specificities for the insertion sites despite the randomness of Mu dl insertions into the genome as a whole. Gene products or open reading frames were identified for seven unknown psi::lacZ(Mu dl) transcriptional fusions by searching DNA data bases with the sequences adjacent and upstream of the Mu dls. One psiC::lacZ(Mu dl) lies in the ugpB gene of the ugpBAEC operon, which encodes a periplasmic sn-glycerol-3-phosphate-binding protein; two psiQ::lacZ(Mu dl)s lie in the gltB gene, and one psiQ::lacZ(Mu dl) lies in the gltD gene of the gltBDF operon, encoding the large and small subunits of glutamate synthase, respectively; and the psi-51::lacZ(Mu dl) lies in the glpB gene of the glpABC operon, which codes for the anaerobically regulated glycerol-3-phosphate dehydrogenase. psiE and psiF::lacZ(Mu dl)s lie in uncharacterized open reading frames near the xyLE and phoA genes, respectively. Six other psi::lacZ(Mu dl)s lie in yet unreported Escherichia coli sequences.Transcriptional regulation is one of the most intensively studied topics in modern biology. These studies rely upon a number of methods for detection and quantitation of the protein products of a gene or operon. The development of transcriptional and gene (protein) fusion technology has proved valuable in the study of genetic regulatory systems in novel ways, especially in cases where the gene products are difficult to assay directly or are themselves unknown. In such cases, gene regulation can be studied by fusing a reporter gene, minus its own regulatory sequences, to the gene or promoter of interest. The most commonly used reporter gene is lacZ, which encodes 3-galactosidase, for it can be easily assayed. Although numerous techniques for making lacZ transcriptional and translational fusions now exist, one of the more common methods employed in gramnegative bacteria involves in vivo construction with transposons like Mu dl (8).Mu dl is a defective Mu-1 bacteriophage that, like Mu-i, transposes somewhat randomly into Escherichia coli chromosomal or plasmid DNAs (8). The MuR and MuL ends of Mu DNA and the Mu A and B genes are required for transposition, although the Mu A and B products can be supplied in trans (10). In Mu dl the lacZ reporter gene is near MuR so that insertion of Mu dl into a gene in the correct orientation places the lacZ gene under the control of the regulatory sequences of the interrupted gene. The precise physical structure of the MuR end in Mu dl was not determined.Mu dl was used to study Pi control of ...

Effect of cell growth rate on expression of the anaerobic respiratory pathway operons frdABCD, dmsABC, and narGHJI of Escherichia coli

“…coli MC4100 strain used for the above studies was representative of other E. coli strains, the frdA-lacZ fusion contained on XJ100 was introduced into BW13711 which carries AlacZ but otherwise has the wild type genes (19). The pattern for frdA-lacZ expression was essentially identical to that seen in MC4100 at all growth rates examined ( Fig.…”

“…All experiments were performed with the E. coli strain, MC4100 (25), an isogenic Afhr strain, PC2 (7), or BW13711 (19). The phages used were XJ100 which contained afrd4-lacZ fusion (16), XPC25 dmsA-lacZ (9), or XPC50 narG-lacZ (8).…”

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

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Why does Escherichia coli have two primary pathways for synthesis of glutamate?

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