Vol. 174, No. 2
JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 486-491
0021-9193/92/020486-06$02.00/0 Copyright © 1992, American Society for Microbiology
Molecular Genetic Analysis of the Escherichia coli phoP Locus EDUARDO A. GROISMAN,l 2* FRED HEFFRON,2t AND FELIX SOLOMON' Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8230, St. Louis, Missouri 63110-1093,1* and Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 920372 Received 6 August 1991/Accepted 31 October 1991
We have cloned the Escherichia coli phoP
gene, a
member of the family of environmentally responsive
two-component systems, and found its deduced amino acid sequence to be 93% identical to that of the Salmonella typhimurium homolog, which encodes a major virulence regulator necessary for intramacrophage survival and resistance to cationic peptides of phagocytic cells. The phoP gene was mapped to kilobase 1202 on the Kohara map (25-min region) of the E. coli genome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) and found to be transcribed in a counterclockwise direction. Both E. coli and S. typhimurium phoP mutants were more sensitive than their isogenic wild-type strains to the frog-derived antibacterial peptide magainin 2, suggesting a role for PhoP in the response to various stresses in both enteric species.
Bacteria modulate expression of their genes in response to environmental changes. This modulation is frequently controlled at the transcriptional level by members of the family of two-component systems, in which a membrane-bound sensor-transmitter can detect changes in the environment, such as changes in osmolarity or concentrations of essential nutrients, and mediate the phosphorylation-dephosphorylation of the regulatory-receiver component (30, 31). We and others have recently identified the PhoP protein of Salmonella typhimurium as a transcriptional regulator which controls the expression of genes essential for virulence, survival within macrophages (5, 11, 26), the ability to withstand an acid pH (7), and resistance to antimicrobial peptides of mammals (5), frogs, and insects (14; see reference 12 for a review). Genetic and DNA sequence analyses revealed that phoP is part of an operon with phoZ (also called phoQ), which encodes a protein which exhibits homology to the sensors-transmitters (11, 26). To date, five PhoP-regulated loci in S. typhimurium have been identified: phoN, encoding a nonspecific acid phosphatase (NSAP) (13, 17), and psiD (11), pagA, pagB, and pagC, all of unknown function (26). Because most PhoP-regulated loci identified so far are not essential for virulence of S. typhimurium, and because phoP homologs have been detected in several bacterial species (11), PhoP may play a central role in the normal physiology of gram-negative bacteria. To gain more insights into the role of PhoP in regulation, we began a molecular genetic analysis of the Escherichia coli phoP locus. Here, we present a phenotypic characterization of a phoP mutant of E. coli, and we also report the cloning, nucleotide sequence, and map position of phoP.
pLg/ml, chloramphenicol was used at 25 ,ug/ml, kanamycin used at 40 p.g/ml, and tetracycline was used at 10 p.g/ml. Peptides. Magainin 2, mastoparan, and melittin were purchased from Bachem Inc. (Torrance, Calif.), and cecropin P1 was purchased from Peninsula Laboratories, Inc. (Belmont, Calif.). Peptides were dissolved in distilled doubly deionized sterile water to a final concentration of 1 mg/ml and kept at -20°C. Bacterial genetic techniques. Transformation of S. typhimurium with plasmid DNA was carried out as described by MacLachlan and Sanderson (22). Transformation of E. coli JC7623 was performed by electroporation with a Bio-Rad apparatus according to the manufacturer's recommendations. Lysates of mini-Mu replicon phage were prepared and used as described previously (8). Phage P1 transductions were carried out with Plcml c1rlOC as described previously (25). Bactericidal assays. Log-phase cells grown in LB broth were diluted to 5 x 104 to 1 x 105 CFU/ml in LB broth. Diluted material (50 pl) was placed in 96-well microtiter dishes and added to different amounts of peptide (50 ,ul) diluted in phosphate-buffered saline (PBS) to the final concentration indicated in Fig. 3. Cells and peptides were incubated fdr 1 h at 37°C with shaking, and then a portion of each sample was diluted in PBS and plated on LB agar plates to assess bacterial viability. Data are presented as percent survival relative to the original inoculum. Controls contained PB3S in place of peptide. DNA biochemistry. Restriction endonucleases and phage T4 DNA ligase were purchased from Bethesda Research Laboratories, Inc.; Boehringer Mannheim Biochemicals; and New England BioLabs, Inc., and used according to the suppliers' specifications. Large-scale isolation of plasmid DNA was carried out by the procedure of Kupersztoch and Helinski (19). Small-scale preparation of plasmid DNA was done as described by Holmes and Quigley (15). Dot blot hybridization was performed as follows: approximately 250 ng of purified plasmid DNA was denatured, transferred to a nylon membrane (Nytran; Schleicher & Schuell), and hybridized under stringent conditions to a 32P-labeled probe corresponding to the 514-bp EcoRV fragment internal to the S. typhimurium phoP gene (11). Plaque hybridization of recombinant phages harboring DNA segments of the 25-min region of the E. coli genome to a 32P-labeled pEG5424 probe was
MATERIALS AND METHODS Bacterial strains and plasmids. Bacterial strains and plasmids are described in Table 1. Media. Luria-Bertani (LB) and M63 minimal media have been described previously (25). Ampicillin was used at 50 * Corresponding author. t Present address: Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201.
486
ESCHERICHIA COLI phoP LOCUS
VOL. 174, 1992
TABLE 1. Strains and plasmids used in the study Strain or
Description or
plasmid
genotypea
E. coli FS1000 FS1002
JC7623
MC1061
MC4100 MH2923
MC1061 with phoP::kan MC4100 with phoP::kan thr-l ara-14 leuB6 A(gptproA)62 lacYl sbcC201 tsx33 supE44 galK2 lambdarac sbcBIS hisG4 rfbDl recB21 recC22 rpsL31 kdgK51 xyl-S mtl-l argE3 thi-i F- araDi39 A(ara-leu)7697 A(lac)X74 galU galK hsdR2 (rK mK ) supE44 thi-i relAl? F- araDJ39 A(lac)U169 rpsL150 relAl thiflbBS301 deoCi ptsF25 rbsR F+ Mu cts62 hPl-1 araD
TA2328
Source or reference
pEG5424 pEG5425
pEG5426 pEG5484 pEG5513 pUC4-K
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v
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This work This work 21 pEGS420 pEGS424 pEG5426
pEG5423 pEG5425
1200
400
proc
phoP
3
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(8.9)
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.
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.
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7F9 (239) SF5
Laboratory collection
20E6 240) ISAS (237) E
JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 486-491
0021-9193/92/020486-06$02.00/0 Copyright © 1992, American Society for Microbiology
Molecular Genetic Analysis of the Escherichia coli phoP Locus EDUARDO A. GROISMAN,l 2* FRED HEFFRON,2t AND FELIX SOLOMON' Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8230, St. Louis, Missouri 63110-1093,1* and Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 920372 Received 6 August 1991/Accepted 31 October 1991
We have cloned the Escherichia coli phoP
gene, a
member of the family of environmentally responsive
two-component systems, and found its deduced amino acid sequence to be 93% identical to that of the Salmonella typhimurium homolog, which encodes a major virulence regulator necessary for intramacrophage survival and resistance to cationic peptides of phagocytic cells. The phoP gene was mapped to kilobase 1202 on the Kohara map (25-min region) of the E. coli genome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) and found to be transcribed in a counterclockwise direction. Both E. coli and S. typhimurium phoP mutants were more sensitive than their isogenic wild-type strains to the frog-derived antibacterial peptide magainin 2, suggesting a role for PhoP in the response to various stresses in both enteric species.
Bacteria modulate expression of their genes in response to environmental changes. This modulation is frequently controlled at the transcriptional level by members of the family of two-component systems, in which a membrane-bound sensor-transmitter can detect changes in the environment, such as changes in osmolarity or concentrations of essential nutrients, and mediate the phosphorylation-dephosphorylation of the regulatory-receiver component (30, 31). We and others have recently identified the PhoP protein of Salmonella typhimurium as a transcriptional regulator which controls the expression of genes essential for virulence, survival within macrophages (5, 11, 26), the ability to withstand an acid pH (7), and resistance to antimicrobial peptides of mammals (5), frogs, and insects (14; see reference 12 for a review). Genetic and DNA sequence analyses revealed that phoP is part of an operon with phoZ (also called phoQ), which encodes a protein which exhibits homology to the sensors-transmitters (11, 26). To date, five PhoP-regulated loci in S. typhimurium have been identified: phoN, encoding a nonspecific acid phosphatase (NSAP) (13, 17), and psiD (11), pagA, pagB, and pagC, all of unknown function (26). Because most PhoP-regulated loci identified so far are not essential for virulence of S. typhimurium, and because phoP homologs have been detected in several bacterial species (11), PhoP may play a central role in the normal physiology of gram-negative bacteria. To gain more insights into the role of PhoP in regulation, we began a molecular genetic analysis of the Escherichia coli phoP locus. Here, we present a phenotypic characterization of a phoP mutant of E. coli, and we also report the cloning, nucleotide sequence, and map position of phoP.
pLg/ml, chloramphenicol was used at 25 ,ug/ml, kanamycin used at 40 p.g/ml, and tetracycline was used at 10 p.g/ml. Peptides. Magainin 2, mastoparan, and melittin were purchased from Bachem Inc. (Torrance, Calif.), and cecropin P1 was purchased from Peninsula Laboratories, Inc. (Belmont, Calif.). Peptides were dissolved in distilled doubly deionized sterile water to a final concentration of 1 mg/ml and kept at -20°C. Bacterial genetic techniques. Transformation of S. typhimurium with plasmid DNA was carried out as described by MacLachlan and Sanderson (22). Transformation of E. coli JC7623 was performed by electroporation with a Bio-Rad apparatus according to the manufacturer's recommendations. Lysates of mini-Mu replicon phage were prepared and used as described previously (8). Phage P1 transductions were carried out with Plcml c1rlOC as described previously (25). Bactericidal assays. Log-phase cells grown in LB broth were diluted to 5 x 104 to 1 x 105 CFU/ml in LB broth. Diluted material (50 pl) was placed in 96-well microtiter dishes and added to different amounts of peptide (50 ,ul) diluted in phosphate-buffered saline (PBS) to the final concentration indicated in Fig. 3. Cells and peptides were incubated fdr 1 h at 37°C with shaking, and then a portion of each sample was diluted in PBS and plated on LB agar plates to assess bacterial viability. Data are presented as percent survival relative to the original inoculum. Controls contained PB3S in place of peptide. DNA biochemistry. Restriction endonucleases and phage T4 DNA ligase were purchased from Bethesda Research Laboratories, Inc.; Boehringer Mannheim Biochemicals; and New England BioLabs, Inc., and used according to the suppliers' specifications. Large-scale isolation of plasmid DNA was carried out by the procedure of Kupersztoch and Helinski (19). Small-scale preparation of plasmid DNA was done as described by Holmes and Quigley (15). Dot blot hybridization was performed as follows: approximately 250 ng of purified plasmid DNA was denatured, transferred to a nylon membrane (Nytran; Schleicher & Schuell), and hybridized under stringent conditions to a 32P-labeled probe corresponding to the 514-bp EcoRV fragment internal to the S. typhimurium phoP gene (11). Plaque hybridization of recombinant phages harboring DNA segments of the 25-min region of the E. coli genome to a 32P-labeled pEG5424 probe was
MATERIALS AND METHODS Bacterial strains and plasmids. Bacterial strains and plasmids are described in Table 1. Media. Luria-Bertani (LB) and M63 minimal media have been described previously (25). Ampicillin was used at 50 * Corresponding author. t Present address: Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201.
486
ESCHERICHIA COLI phoP LOCUS
VOL. 174, 1992
TABLE 1. Strains and plasmids used in the study Strain or
Description or
plasmid
genotypea
E. coli FS1000 FS1002
JC7623
MC1061
MC4100 MH2923
MC1061 with phoP::kan MC4100 with phoP::kan thr-l ara-14 leuB6 A(gptproA)62 lacYl sbcC201 tsx33 supE44 galK2 lambdarac sbcBIS hisG4 rfbDl recB21 recC22 rpsL31 kdgK51 xyl-S mtl-l argE3 thi-i F- araDi39 A(ara-leu)7697 A(lac)X74 galU galK hsdR2 (rK mK ) supE44 thi-i relAl? F- araDJ39 A(lac)U169 rpsL150 relAl thiflbBS301 deoCi ptsF25 rbsR F+ Mu cts62 hPl-1 araD
TA2328
Source or reference
pEG5424 pEG5425
pEG5426 pEG5484 pEG5513 pUC4-K
en
en
a
vL
v e1
n
v w
O
C.
C. C.>
"
v In
CI
v
v
v e n
v
Cla
Q
This work This work 21 pEGS420 pEGS424 pEG5426
pEG5423 pEG5425
1200
400
proc
phoP
3
;
,-
(8.9)
-.. I*
'- '
' . .'.
w
.
A
..
a
in .
.
i_. .
.
_
7F9 (239) SF5
Laboratory collection
20E6 240) ISAS (237) E
