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Regulation of Components of the Pseudomonas Aeruginosa Phosphate-Starvation-Inducible Regulon in Escherichia Coii

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Regulation of Components of the Pseudomonas Aeruginosa Phosphate-Starvation-Inducible Regulon in Escherichia Coii Molecular Microbiology (1988) 2(3), 347-352 Regulation of components of the Pseudomonas aeruginosa phosphate-starvation-inducible regulon in Escherichia coii R. J. Siehnel, E. A. Worobec and R. E. W. Hancock* of the structural elements of the Pho regulon since, in such Department ot Microbioiogy, University ot British mutants, another regulatory gene product, PhoM, will tran- Columbia, Vancouver, British Columbia, Canada scriptionally activate PhoB. The PhoM activator is not con- V6T1W5. trolled by changes in phosphate levels and is repressed when PhoR is functional. Summary Conditions of low environmental phosphate stimulate the co-induction, in E. coli, of an outer membrane porin Plasmids pPBP and pRS-XP containing the cloned (PhoE), a periplasmic phosphate-binding protein (PhoS), genes for the Pseudomonas aeruginosa phosphate- and a periplasmic alkaline phosphatase (PhoA). JUephoB starvation-inducible periplasmic phosphate-binding gene product appears to activate the transcription of each protein and outer membrane porin P {oprP), respec- of these genes by interacting with specific regulation tively, were introduced into various Escherichia coti sequences located in analogous regions upstream from Pho-regulon regulatory mutants. Using Western the structural genes. These sequences are highly hom- immunoblots and specific antisera, the production of ologous and have been termed the 'Pho box' (Shinagawa both gene products was observed to be under the etai., 1987). control of regulatory elements of the E. coti Pho regu- Phosphate transport has been examined in several other lon. Sequencing of the region upstream of the trans- bacterial species and recent evidence suggests that a lationai start site of the oprP gene revealed a 'Pho phosphate-starvation-inducible regulon, similar to that box' with strong homology to the E. coti consensus found in £. coli, exists in these other species. For example, 'Pho box', the putative binding site of the PhoB a PhoB analogue has been identified in Baciitus subtilis. activator. Since P. aeruginosa and E. coli belong to This gene product regulates components of this different families and have quite different GC contents, organism's Pho regulon in a similar fashion to that of these data suggest strong evolutionary conservation £ coii (Seki et al., 1987). The genes for the phosphate- of regulatory elements of the Pho reguion. regulated porins of Vibrio parahaemoiyticus (McCarter and Silverman, 1987), Ktebsietia pneumoniae, and Entero- bacter cloaoae (van der Ley ef al., 1987) are all regulated Introduction in E. coii in the same manner as the E. coii phosphate- The control of gene expression of the components of the regulated porin PhoE, with the latter two genes having E. coli high-affinity phosphate transport system (Pst) is distinct Pho boxes. P. aeruginosa also has a high-affinity very complex, involving at least four regulatory genes, phosphate-starvation-inducible regulon (Gray ef a/., 1982; phoB, phoR, phoM and phoU, and their gene products Poole and Hancock, 1983; 1984), which appears to have PhoB, PhoR, PhoM and PhoU, respectively (see Wanner, several components analogous to those found in E. coti. 1987 for a review). The ultimate activator is PhoB since Under conditions of phosphate limitation, P. aeruginosa mutations in phoB result in a pleiotropically negative demonstrates induction of an outer membrane phosphate- phenotype with respect to the components of the phos- specific porin protein P (Hancock ef al., 1982), which phate-starvation-inducible (Pho) regulon. The PhoB acti- immunologically cross-reacts with PhoE trimers (Poole vator is either transcriptionally activated by PhoR when and Hancock, 1986a) but which has substantially different phosphate is limiting, or transcriptionally repressed by channel properties (Hancock and Benz, 1986), a periplas- PhoR when phosphate is non-limiting. Because phosphate mic phosphate-binding protein (Poole and Hancock, limitation favours production of the PhoB activator, mutants 1984), an alkaline phosphatase, and several other in Pst have constitutive phenotypes for Pho-regulon pro- polypeptides (Gray etal., 1982). The P. aeruginosa phos- ducts. PhoU is thought to function in the conversion of phate-starvation-inducible products also include two PhoR from the activator form to the repressor form. Muta- haemoiysins, a rhamnolipid, and a phospholipase C (Liu, tions in phoU or phoR result in the constitutive production 1979), which are not produced in E. coli. The regulation of this system has not been examined in detail but mutant strains that are either constitutive (Gray etal., 1982; Poole Received 21 December, 1987. 'For correspondence. 348 R. J. Siehnel, E. A. Worobec and R. E. W. t-lancoct< and Hancock, 1984) for all the phosphate-starvation- Table 1. Esctierichia coli strains and plasmids. inducible gene products (i.e. PhoU-, PhoS- or PhoR-like Strain/ Source/ mutations) or pleiotropically negative (Poole and Hancock, Plasmid Description" Reference 1986b) for all these components (i.e. PhoB-like mutation) have been identified. Bacterial strains In this communication we have addressed the analogy LE392 F",/7sdR-514, (rk-, m^-)supE-44 (Murray ef a/., 1977) between the P. aeruginosa and the E. coli Pho regulons. supF•58.lacY^ or A(/ac/Zy)6. galK-2, ga/r-22, metB-1. trpR-55. X" We introduced plasmids containing the cloned structural K10 HfrC, relA^. tonA22, p/f-10, spofi, B. Bachmann genes for the P. aeruginosa phosphate-specific porin P T^w' and the periplasmic phosphate-binding protein into wild- C86 p/io-21 derivative of K10 J. Tommassen AB1157 F", thr. leu. proA2. A{proA-phoE-gpt) J. Tommassen type E. coli and several £. coii strains bearing mutations his. thi. argE. IacY, galK, xyl. rpsL in the regulatory components of the Pho regulon and found CE1194 pho-2^ derivatlveof AB1157 J. Tommassen that the expression of these genes was regulated by the S3 phoS63 derivative of Kl 0 B. Bachmann BW256 thi, crp-72,rpsLpho-510 B. Wanner £. coli Pho-regulon control elements. The identification BW255 phoR68 derivative of BW 256 B. Wanner of a Pho box upstream of the porin P structural gene BW705 lac proL::Jn5 phoR6B phoM 453 B. Wanner suggested strong evolutionary conservation of Pho regulon derivative of BW256 BW3908 thi. /ac-169.p/7o-510, rpst.267 B. Wanner regulation elements. BW3212 ^(psIFproC avoLM phoBR) B. Wanner derivative of BW3908 BW6504 phoU35 derivative of BW3908 B. Wanner Results Plasmids Regulation of P. aeruginosa porin P and pPBP IncPI :Tc'rixXcos. pLAFri This iaboratory phosphate-binding protein gene products in E. coli derivative containing the cloned P. aeruginosa phosphate-binding The plasmids containing the cloned P. aeruginosa genes protein gene pRS-XP Amp', pUC18 derivative containing This iaboratory for the phosphate-porin protein P (pRS-XP) and the phos- the cioned P. aeruginosa phate-binding protein (pPBP) were transformed into £. ooli oprPgene wild-type strains and several strains with regulatory muta- a. Abbreviations: Tc', tetracycline resistance; Amp', ampicillin resistance; tions (Table 1). These £. ooli strains produce the compo- IncPI, incompatibility group PI. nents of the £. coli Pho regulon constitutiveiy in the case of phoU, phoS and phoR mutants, whereas strains with mutations in phoB or phoR plus phoM are pleiotropically negative for the same components. The strains trans- pPBP were examined for alkaline phosphatase production formed with plasmids pRS-XP and pPBP were grown in to confirm the phenotypes of the strains used and the phosphate-sufficient and phosphate-deficient media; inability of the plasmids' insert DNA to complement the £. cell pellets were collected and cell lysates examined by coii mutations (Table 2). SDS-polyacrylamide gel electrophoresis and Western immunoblots using antisera specific for protein P or the phosphate-binding protein. Phosphate-binding protein, Regulatory region of porin protein P with the same molecular weight and antigenic properties as In the £. coli Pho regulon, gene expression is dependent authentic P. aeruginosa phosphate-binding protein, was on the PhoB activator (Makino ef al., 1986). It has been found to be produced and regulated by the control postulated that the PhoB protein interacts with a family of elements of £. coli phosphate regulon (Table 2). Examples 18 nucleotide sequences termed the phosphate box or of this regulation are demonstrated in Fig. 1 for a wild-type Pho box, which are located exactly 10 nucleotides up- E. coli, a phoU constitutive mutant, and a pleiotropically stream from the deduced Pribnow (-10) boxes (Makino negative phoB, phoR double mutant. ef al., 1986; Shinagawa etal., 1987). The amino-terminus Protein P monomer was only detected in whole cell of the protein P (oprP) gene had been identified by lysates of strains with mutations preventing production of hybridization of subclones of plasmid pRS-XP with an the equivalent £. ooli outer membrane protein PhoE. In oligonucleotide probe synthesized on the basis of the PhoE* strains, protein P was more readily identified in known amino-terminal amino acid sequence of the mature outer membrane fractions. As with the P. aeruginosa P (R.J. Siehnel etal., submitted manuscript). The direction phosphate-binding protein, production of porin protein P of transcription of the oprP gene was deduced from the site was also found to be controlled by the £. coii regulatory of insertion of a transposon (Tn507) in a protein-P-deficient components (Table 2). mutant and from fusion of downstream sequences to
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