Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli

Wanner, Barry L.; Boline, J A

doi:10.1128/jb.172.3.1186-1196.1990

Cited by 72 publications

(94 citation statements)

References 44 publications

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“…Thirteen psi::lacZ(Mu dl-1734)s were molecularly cloned by using the mini-Mu cloning procedure (23 (46). Therefore these data show a requirement for the phnD ORF in phosphonate assimilation or a polarity effect of the phn (psiD)::lacZ(Mu dl)s on the expression of downstream phn genes in an operon structure.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore these data show a requirement for the phnD ORF in phosphonate assimilation or a polarity effect of the phn (psiD)::lacZ(Mu dl)s on the expression of downstream phn genes in an operon structure. The phn (psiD) operon probably encodes systems for both the transport and degradation of phosphonates for use as a sole P source (13,46). Since (47).…”

Section: Resultsmentioning

confidence: 99%

“…The plasmid pEG5294 was from E. Groisman (E. Groisman and M. Casadaban, personal communication), pHP45Q1 (38) was from H. E. Umbarger, and pKUN19 (28) was from R. N. H. Konings. Plkc, the replication-defective A: :TnS-132 (cI857 b221 Oam29 Pam8O rex::TnS-132), and M13mpl8 phages were from laboratory stocks whose sources were previously reported (43,44,46). Strain 71-18 was transformed when testing for lacZa complementation, and BW5104 was transformed when selecting for mini-Mu recombinant plasmids ( Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…The protocols for Plkc and X transductions were described previously (43). Mini-Mu plasmid libraries were made as described previously (46), except that the mini-Mu cloning vector pMW11 was used and transductants of the Mu-1 lysogen BW5104 were selected at 30°C. transformations were done as previously described (44).…”

Section: Methodsmentioning

confidence: 99%

“…Molecular cloning (46) and DNA sequencing (13) of the phn DNA showed that the phn region is at least 10 kilobases long and may encode as many as 17 open reading frames (ORFs) in an operon structure. A precise definition of the sites of the phn (psiD): :lacZ(Mu dl)s 3192 METCALF ET AL. could aid in the assignment of ORFs that are essential for various functions and that are associated with mutations in the phn (psiD) operon (W. W. Metcalf and B. L. Wanner, unpublished data).…”

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confidence: 99%

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

1990

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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 ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

1990

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Answers to the Carbon–Phosphorus Lyase Conundrum

Zhang

Donk

2012

ChemBioChem

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Gene regulation by phosphate in enteric bacteria

Wanner

1993

J of Cellular Biochemistry

405

346

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The Escherichia coli phosphate (PHO) regulon includes 31 (or more) genes arranged in eight separate operons. All are coregulated by environmental (extra-cellular) phosphate and are probably involved in phosphorus assimilation. Pi control of these genes requires the sensor PhoR, the response regulator PhoB, the binding protein-dependent Pi-specific transporter Pst, and the accessory protein PhoU. During Pi limitation, PhoR turns on genes of the PHO regulon by phosphorylating PhoB that in turn activates transcription by binding to promoters that share an 18-base consensus PHO Box. When Pi is in excess, PhoR, Pst, and PhoU together turn off the PHO regulon, presumably by dephosphorylating PhoB. In addition, two Pi-independent controls that may be forms of cross regulation turn on the PHO regulon in the absence of PhoR. The sensor CreC, formerly called PhoM, phosphorylates PhoB in response to some (unknown) catabolite, while acetyl phosphate may directly phosphorylate PhoB. Cross regulation of the PHO regulon by CreC and acetyl phosphate may be examples of underlying control mechanisms important for the general (global) control of cell growth and metabolism.

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Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli

Abstract: The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn)

Cited by 72 publications

References 44 publications

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

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

Answers to the Carbon–Phosphorus Lyase Conundrum

Gene regulation by phosphate in enteric bacteria

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