Transformation in E. coli K12: Relation of linkage to distance between markers

Hoekstra, W. P. M.; Haan, P. G. de; Bergmans, J.; Zuidweg, E. M.

doi:10.1007/bf00331565

Cited by 13 publications

(7 citation statements)

References 7 publications

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“…For this purpose, plasmid pSN507 (2) was digested with MunI and SnaBI, thereby removing a DNA segment encompassing approximately the 3Ј half of pstS, the entire pstC, pstA, and pstB genes, and approximately two-thirds from the 5Ј end of phoU, and ligated with a Cam r cassette (1). The resulting plasmid was digested with EcoRI, and the 6.4-kb DNA fragment with the ⌬pstSCAB-phoU mutation was used to transform recBC sbc strain AM1095 (8). One Cam r transformant, which expressed alkaline phosphatase constitutively and did not produce PstS and PhoU as verified by Western blotting, was designated CE1489.…”

Section: Fig 2 Alkaline Phosphatase Activities In K10mentioning

confidence: 99%

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P _i -Regulated Expression of the pho Regulon

Hoffer

Tommassen

2001

J Bacteriol

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Disruption of pstS encoding the P i -binding protein in Escherichia coli generally leads to the constitutive expression of the pho regulon. We demonstrate that P i -controlled expression is restored when the activity of the P i transporter PitA or PitB is increased. Apparently, PstS is not an essential component of the signal transduction pathway.Growth of Escherichia coli under P i limitation results in the induction of the pho regulon, which includes phoA encoding alkaline phosphatase (18) and the pst operon encoding a P i transporter. This P i transporter consists of a periplasmic P ibinding protein (PstS), two integral membrane proteins (PstA and PstC), and an ATP-binding protein (PstB) (4, 16). Central to the regulation of the pho regulon is a two-component regulatory system encoded by the phoBR operon (21). In addition, the Pst system plays a role in P i regulation, since mutations in any of the genes of the pst operon generally lead to constitutive expression of the pho regulon (21-23). This constitutive expression is not due to decreased intracellular phosphate levels, which were reported to be maintained at a high level under high-P i conditions by a secondary P i transporter, PitA (11,24). Furthermore, the regulatory and transport roles of the Pst system could be uncoupled by specific amino acid substitutions in PstC or PstA (5, 6). Since periplasmic PstS binds P i with high affinity, it could potentially act as the primary sensor of external P i (20). The interaction of P i -loaded PstS with the membrane components of the Pst system might lead to a conformational change, which is sensed by the product of the fifth gene of the pst operon, PhoU. PhoU is not involved in P i transport (15), but probably forms the regulatory link between the P i transporter and the PhoBR system (20).To study the role of PstS in signal perception, we wished to isolate missense mutations in pstA or pstC that allow the Pst system to transport P i in the absence of PstS. In a previous study, such mutants were not obtained, but a third P i transporter, PitB, was discovered (9). In this study, we demonstrate that PitA or PitB activity can restore P i regulation of the pho regulon in the absence of PstS.Pseudorevertants in pitA restore P i regulation in a pstS mutant. To study the role of PstS in P i regulation of the pho regulon, we attempted to isolate mutants in pstA or pstC, allowing the Pst system to transport P i in the absence of PstS. Therefore, strain CE1491, which lacks all three known P i transporters (Table 1), was mutagenized with ethylmethane sulfonic acid, and mutants that could grow on minimal medium plates (9) with 660 M P i as the sole source of phosphate were selected. Emanating from the idea that restored P i transport via the Pst system might also restore P i control of the pho regulon, the mutants obtained were tested for expression of alkaline phosphatase on L broth plates containing the chromogenic substrate 5-bromo-

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Section: Fig 2 Alkaline Phosphatase Activities In K10mentioning

confidence: 99%

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P _i -Regulated Expression of the pho Regulon

Hoffer

Tommassen

2001

J Bacteriol

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“…A pstS::kan mutation was constructed by ligating a kanamycin resistance cassette from pUC18K (13) into the PvuI site of pSN5182 (15). The resulting plasmid, pSL15, was digested with NruI and BamHI, and the 5.8-kb DNA fragment carrying the pstS::kan allele was used to transform recBC sbcB strain AM1095 (8), to disrupt the chromosomal pstS gene. A pstS::kan derivative of strain K10, designated CE1485, was subsequently constructed by P1 transduction (14).…”

mentioning

confidence: 99%

Activation by Gene Amplification of pitB , Encoding a Third Phosphate Transporter of Escherichia coli K-12

et al. 2001

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Two systems for the uptake of inorganic phosphate (P i ) in Escherichia coli, PitA and Pst, have been described. A revertant of a pitA pstS double mutant that could grow on P i was isolated. We demonstrate that the expression of a new P i transporter, PitB, is activated in this strain by a gene amplification event.Transport of inorganic phosphate (P i ) across the cytoplasmic membrane of Escherichia coli is mediated by the PitA protein and Pst system. PitA, which transports metal phosphates (28) and is constitutively expressed (17), is driven by the proton motive force (PMF) (18, 28). The Pst system, which transports P i at the expense of ATP (6, 9), is composed of a periplasmic P i -binding protein (PstS), two integral membrane proteins (PstC and PstA), and an ATP-binding protein (PstB) (24). The genes encoding these four proteins constitute an operon (24), together with phoU, which encodes a protein not required for P i transport (23). Under P i limitation, the expression of the Pst system, which is produced at a basal level under P i -replete conditions, is further induced. Like, for example, phoA, which encodes the periplasmic enzyme alkaline phosphatase (26), the pst-phoU operon is part of the pho regulon, which is under the control of a two-component regulatory system consisting of the proteins PhoB and PhoR (29). Furthermore, the Pst system appears to be involved in regulation, since mutations in the genes of the pst-phoU operon generally result in constitutive expression of the pho regulon (29-31). The exact mechanism by which the Pst system controls the expression of the pho regulon is not known. To study the role of PstS in regulation, we attempted to isolate mutants with mutations in the membrane components of the Pst system that can transport P i in the absence of the PstS protein. In one of the mutants obtained, a new P i transporter, PitB, appeared to be expressed.Construction of a pstS pitA double mutant. A pstS pitA double-mutant strain is expected to behave as an organic phosphate auxotroph (22), but previously described pstS pitA double mutants, such as strain C86, appear to take up P i and to grow on P i as the sole source of phosphate (reference 30 and data not shown). Western blotting revealed that the PstS protein was produced in this strain, although at much lower levels than in its parental strain, K10, grown under P i limitation (data not shown). To characterize the pstS mutation in strain C86, a DNA fragment was amplified by PCR. The amplified fragment was considerably larger than the expected 1.4 kb, which was found when strains MC4100 and K10 were analyzed (Fig. 1A). An enlarged PCR fragment was also found in another pitA pstS strain, C78 (Fig. 1A). Sequencing of the PCR fragment from strain C86 revealed the presence of an IS2 element in the promoter region of pstS (Fig. 1B), whereas no other mutation was found in the pstS gene or the other genes of the pst-phoU operon. Hence, strain C86 and probably also strain C78 contain a Pst system, which is expressed at a lower level due to the ...

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“…PC0031 is a prototrophic strain. AM1095 (leu pdxA ara car thi his trp recB21 recC22 sbcB15) is a transformable strain, described previously (10). AM1283 is a derivative of AM1170 (AM1095 rpoB) carrying plasmid Rldrd-19 (Apr) (1).…”

mentioning

confidence: 99%

“…A transformation mixture, composed as described in the text, was heat shocked, incubated at 0°C for 60 min, and then treated with DNase (30 min, 0°C). Portions of this mixture were diluted into MOPS buffered minimal medium that was not supplemented or was supplemented with 10 mM CaCl2 and10 mM MgCl2. Time zero is the time of dilution into MOPS-buffered medium.…”

mentioning

confidence: 99%

Transformation in Escherichia coli: stages in the process

Bergmans¹,

Die²,

Hoekstra³

1981

J Bacteriol

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Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0°C -* 42'C 0°C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2" is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0°C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2' and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.buffer was replaced by MOPS buffer (1.2 x 10-' M, pH 7.0). Media were solidified with 1.6% agar.Growth requirements were added in the following concentrations (milligrams per liter): DL-leucine, 40; DL-arginine, 40; uracil, 20; pyridoxine, 10; DL-trypto-564 on August 5, 2020 by guest

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Transformation in E. coli K12: Relation of linkage to distance between markers

Cited by 13 publications

References 7 publications

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P _i -Regulated Expression of the pho Regulon

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P _i -Regulated Expression of the pho Regulon

Activation by Gene Amplification of pitB , Encoding a Third Phosphate Transporter of Escherichia coli K-12

Transformation in Escherichia coli: stages in the process

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Transformation in E. coli K12: Relation of linkage to distance between markers

Cited by 13 publications

References 7 publications

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P i -Regulated Expression of the pho Regulon

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P i -Regulated Expression of the pho Regulon

Activation by Gene Amplification of pitB , Encoding a Third Phosphate Transporter of Escherichia coli K-12

Transformation in Escherichia coli: stages in the process

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The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P _i -Regulated Expression of the pho Regulon

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for P _i -Regulated Expression of the pho Regulon