Cloning of phoM, a gene involved in regulation of the synthesis of phosphate limitation inducible proteins in Escherichia coli K12

The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated. Mutations that inactivate sbcC improve the ability to propagate Ared gam phage that carry a palindromic sequence in their DNA. They also act with sbcB mutations as cosuppressors of the defects in recombination, DNA repair, and cell viability associated with recBC mutations. A 1,282-bp cassette encoding resistance to kanamycin was used to disrupt orf-45. The mutation, which has a polar effect on the expression of sbcC, allowed stable propagation of palindromic k phage even when the sbcC gene product was provided in trans. Additional nonpolar mutations in orf-45 were isolated on the basis of their ability to improve the growth of recBC sbcB strains. These mutations also confer resistance to mitomycin C, allow efficient recombination in Hfr crosses, and facilitate stable propagation of palindromic phage. It is concluded that the products of orf-45 and sbcC are functionally related. The orf-45 gene is therefore renamed sbcD.The sbcC gene of Escherichia coli was defined originally by mutations that arise spontaneously in recBC sbcB strains (16). These mutations act with mutations in sbcB to suppress the defects in recombination and DNA repair associated with the recBC genotype and are selected, therefore, during normal growth because of the improved viability. Subsequently, sbcC single mutants were shown to be good hosts for a Xred gam phage carrying a DNA sequence with palindromic symmetry (Xpal) (5). Phage of this type grow poorly in sbcC+ cells. Leach and Stahl (13) had observed this feature of the sbcC phenotype in their earlier work with recBC sbcB sbcC strains but, because isogenic strains were not constructed and used for comparisons, had attributed it to the absence of exonuclease V (RecBCD enzyme) and exonuclease I (the product of sbcB). This seemed reasonable since palindromes might form hairpins or cruciforms which could be recognized and cleaved by nucleases that normally resolve Holliday intermediates in genetic recombination. Kulkarni and Stahl (11) MATERIALS AND METHODSBacterial strains and X phages. The E. coli K-12 strains and A phages used are described in Table 1. Phage stocks were prepared on strain JC7623, which allows stable propagation of the 571-bp palindrome carried by XMMS1632.Plasmids. Plasmid constructs carrying DNA inserts from the sbcC-orf-45 region of the chromosome are shown in Fig. 1. pSB43, pJP77, and pIN510 are described in detail elsewhere (3,23,28). pIN507 corresponds to pJP77 sbcC:: TnlOOO no. 5 described by Naom et al. (23). pFG103 was made by cloning a BamHI-HindIII fragment of approximately 14.5 kb from pJP77 into pACYC184 (6). A 1,282-bp cassette (kan) encoding resistance to kanamycin was cloned from pUC4K (29) into the unique PstI site of pFG103 to give pFG103K. pFG106 was constructed by removing the Kmr insertion from pFG103K by digestion with EcoRI and inserting it into the EcoRI site of the temperature-sensitive replicon pHSG415 (10). The sbcC+ construc...

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“…1. pSB43, pJP77, and pIN510 are described in detail elsewhere (3,23,28). pIN507 corresponds to pJP77 sbcC:: TnlOOO no.…”

Section: Methodsmentioning

confidence: 99%

Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12

1992

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“…The plasmid constructs used are shown in Figure 1. pJP71 and pJP77 are derivatives of pACYC184 and have been described elsewhere (9), as has pSB43 (10). pBL1 18 and pBL121 are derivatives of pBR322 (1).…”

Section: Nucleic Acids Researchmentioning

confidence: 99%

Molecular organization ofsbcC, a gene that affects genetic recombination and the viability of DNA palindromes inEscherichia coliK-12

Naom

Morton²,

Leach

et al. 1989

Nucl Acids Res

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“…The protein is also produced constitutively inphoR strain CE1238 (lane e), and the constitutive synthesis in aphoR strain is dependent on the phoM product (lane f). In all these respects, the expression of the E. cloacae PhoE protein is exactly the same as that of the E. coli K-12 PhoE protein (Tommassen and Lugtenberg, 1980;Tommassen et al, 1984).…”

Section: (E) R~uiation Of the Expression Of The E Czech Phue Gene Inmentioning

confidence: 96%

“…F -, thi pyrF thy ilvA his argG tonh rpsL deoC vtr g&R ompR47 1 phoR69 phoh8 &proh,B-phoE) Tommassen et al, 1984Korteland et al, 1982 CEi304 F' kc', thr thi recA 17 1 IacZ22 farI rpsL rpo3 supE…”

Section: Ce1238mentioning

confidence: 99%

“…The synthesis of PhoE protein is subject to a complex regulatory system in which three regulatory genes are involved, namely phoB, phoR and phoM (Tommassen and Lugtenberg, 1980;Tommassen et al, 1982aTommassen et al, , 1984W~ner and Latterell, 1980). Also, mutations in the genes phoS, phoT, phoU and pst, which are primarily involved in phosphate transport, affect the expression of PhoE protein, in that the protein is produced constitutively in strains carrying any of these mutations (Tommassen and Lugtenberg, 1980).…”

mentioning

confidence: 99%

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Cloning and expression in Escherichia coli K-12 of the structural gene for outer membrane PhoE protein from Enterobacter cloacae

Verhoef

Koppen

Overduin

et al. 1984

Gene

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SUMMARYIn Escherichia coli K-12, the phoE gene encodes an outer membrane pore protein, which is induced by phosphate starvation. The co~esponding gene of ~~tero~acter cfoucae was transferred to E. coli K-12 by using RP4 : : mini Mu plasmid pULB 113 and selecting for R-prime plasmids that carry the genesproA andpro& which are closely linked to phoE in E. coli K-12. The phoE gene was subcloned into the multicopy vector pACYC 184, and the location of the gene was determined by analysis of in vitro constructed deletion plasmids and mutant plasmids generated by $5 insertions. The E. CioacaephoE gene is normally expressed in E. coli K-12, and the regulation of the expression is similar to that of the E. coiipho~ gene. Function~ly, the products of the phoE genes of E. coli K-12 and E. cloacae behave very similarly since they form pores in the outer membrane with a recognition site for negatively charged compounds and they serve as (part of) the receptor for phage TC45.The outer membrane of Enterobacteriaceae functions as a barrier for harmful compounds. The

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Cloning of phoM, a gene involved in regulation of the synthesis of phosphate limitation inducible proteins in Escherichia coli K12

Cited by 14 publications

References 31 publications

Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12

Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12

Molecular organization ofsbcC, a gene that affects genetic recombination and the viability of DNA palindromes inEscherichia coliK-12

Cloning and expression in Escherichia coli K-12 of the structural gene for outer membrane PhoE protein from Enterobacter cloacae

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