161 , No. 2 onella typhimurium Contains an Anion-Selective Ou Membrane Porin Induced by Phosphate Starvation KATHARINA BAUER , ROLAND BENZ , JOHANN BRASS , AND WINFRIED BOOS * Department of Biology , University of Konstanz , D-7750 Konstanz , West Germany Received 21 September 1984/Accepted 14 November 1984 ter Sa lm A mutant of Salmonella typhimurium was selected that is constitutive for the pho regulon .
It exhibited constitutive glycerol-3-phosphate transport activity and synthesized a new outer membrane porin .
Upon measurement of porin activity in black lipid films , it exhibited anion selectivity .
It therefore appears analogous to the Escherichia coli PhoE porin .
The genetic organizations of Escherichia coli and Salmo - nella typhimurium are rather similar ( 18 , 21 ) .
Known differences include the absence in S. typhimurium of lac operon genes as well as the phoA gene coding for the periplasmic alkaline phosphatase .
Even though phoA is missing , the regulatory genes phoR and phoB are present ( 25 ) .
Recently , we described the presence of a pho-regulated system in S. typhimurium , the ugp-dependent transport system for sn-glycerol-3-phosphate ( G3P ) ( 10 ) .
Transport through this system is mediated by a periplasmic binding protein and is distinct from a glpT-dependent transport system for G3P that is active in membrane vesicles ( 10 ) .
In the present paper , we describe the isolation of a mutant that is constitutive for the ugp-dependent transport system .
This mutant has the phenotype of a pho constitutive strain .
It synthesizes a new outer membrane protein that exhibits properties similar to those of the PhoE porin of E. coli .
This new protein is a murein-associated porin and forms anion-selective pores in black lipid films .
Isolation of a pho constitutive mutant of S. typhimurium .
The starting strain was SH6264 ( 14 , 15 ) .
This strain had been isolated as a mutant resistant to phages PA105 and PH51 and is therefore deficient in two outer membrane porins ( the OmpD and OmpC proteins ) , the phage receptors .
It contains as its only porin the 35-kilodalton OmpF outer membrane protein ( 2 , 15 , 22 ) .
With the help of a nearby TnJO insertion , we transduced glpT , coding for a defective glpT-dependent transport system for G3P , from strain RH8 ( 10 ) into strain SH6264 via P22-mediated transduction .
The only remaining transport system for G3P in the resulting strain ( KB66 ) should then be the pho regulon-dependent ugp system .
In E. coli , G3P transported by Ugp can serve as the sole source of phosphate but not of carbon .
Like all other pho-dependent genes , ugp in the wild type is expressed only under growth of phosphate-starvation .
To select a mutant conditions in G plus L constitutive for ugp , we grew KB66 alternately medium ( 9 ) with 1 mM Pi or 2 mM G3P as the only source of phosphate and with glucose as the carbon source , diluting the culture each time 100-fold .
The rationale for this selection was that ugp constitutive mutants would not experience a lag in growth after being shifted from 1 mM Pi to 2 mM G3P .
We monitored the success of the selection procedure measuring ugp-dependent G3P transport after growth at 1 by mM Pi .
We observed the first increase of G3P transport 20-fold increase after 3 activity after 12 growth cycles , and a additional cycles .
From this last culture , single cell colonies .
Polyacrylamide gel electrophoresis of outer membrane proteins .
Gels ( 9 % ) containing sodium dodecyl sulfate and 8 M urea were run according to Pugsley and Schnaitman ( 17 ) .
Outer membranes were isolated and separated from inner membranes as described by Hengge et al. ( 10 ) .
Murein-associated proteins were prepared as described by Nikaido ( 13 ) .
Periplasmic proteins were isolated by the cold osmotic shock procedure of Neu and Heppel ( 12 ) ; for S. typhimurium , the procedure was modified as described by Aksamit and Koshland ( 1 ) .
Purified PhoE porin from E. coli was obtained from R. Hancock , and purified OmpD , OmpF , and OmpC outer membrane proteins were obtained from T. Nakae .
Lanes : a , OmpD , OmpF , and OmpC outer membrane proteins from S. typh-imurium ; b , purified PhoE porin of E. coli ; c and d , periplasmic proteins ; e and f , murein-associated proteins ; g to i , outer membrane proteins ; c , e , and g , pho constitutive strain KB17 grown in Luria KB66 in broth ( LB ) ( 11 ) ; d , f , and i , pho wild-type strain grown LB ; h , strain grown in G plus L medium and 60 , uM Pi .
Each slot contained 20 p , g of protein .
The arrowhead indicates the position of the pho-dependent porin of S. typhimurium ; the arrow indicates the position of the E. coli PhoE porin .
Strain KB17 was chosen for further studies .
At 0.25 jxM external substrate concentration , it transported G3P with an initial rate of 48 pmol/min and 108 cells .
Under the same conditions , a pho constitutive E. coli strain transported 30 pmol/min and 108 cells .
The parent strain KB66 transported less than 0.1 and 6 pmol/min and 108 cells after growth at high ( 1 mM ) and limiting Pi concentration , respectively .
The ugp constitutive strain contains a new outer membrane protein that is murein associated .
Since the ugp transport system is under pho control ( 10 ) , it was likely that the ugp constitutive strain KB17 was constitutive also for other pho-controlled genes .
E. coli contains a pho-regulated outer membrane protein , the PhoE porin ( 16 ) .
We therefore analyzed membranes of the ugp constitutive strain KB17 by protein in this strain , and it is not identical to the three known murein-associated porins of S. typhimurium ( Fig. 1 ) .
In the gel system used , containing sodium dodecyl sulfate as well as urea ( 17 ) , this protein exhibits an electrophoretic mobility similar , but not identical , to the mobility of the E. coli PhoE porin .
The gel in Fig. 1 also shows the periplasmic proteins of the constitutive strain KB17 and its parent KB66 .
At least two proteins absent in KB66 appear in KB17 .
In analogy to periplasmic proteins isolated from pho constitutive strains in E. coli , these are likely to correspond to the phosphate-bind-ing ( 4 ) and G3P-binding proteins ( 3 ) of E. coli .
Therefore , it is clear that KB17 carries a mutation leading to a constitutive pho regulon .
We have not characterized the nature of this mutation .
In particular , it is not clear whether it is of the phoR or phoSIT type ( 23 ) .
Also , it is not clear that codes for the pho-controlled where the gene is located outer membrane protein in S. typhimurium .
The phoE gene coding for the corresponding E. coli porin maps between the markers gpt and proA , at 6 min on the linkage map ( 5 , 24 ) .
This region from S. typhimurium has been cloned ( 19 ) .
We have introduced the relevant plasmids pMR102 , pSG7 , and pSP1 ( 19 ) into E. coli CE1194 , which lacks the PhoE porin but is otherwise constitutive for the pho regulon ( 24 ) .
Membranes of these plasmid-carrying strains did not synthe-strain size the pho-controlled outer membrane protein of KB17 ( data not shown ) .
Either the structural gene for this porin does not map between gpt and proA in S. typhimurium or the plasmids acquired secondary mutations preventing the probably stressful overproduction of this protein in the pho constitutive strain CE1194 ( 24 ) .
Alternatively , the phoE gene of S. typhimurium may not be correctly regulated in E. coli ( Tommassen , personal communication ) .
The pho-controlled outer membrane protein forms anionselective pores in black lipid films .
When incorporated into black lipid membranes , porins form water-filled channels that increase the ion conductivity across the membrane ( 7 , 8 ) .
By applying different concentrations of the same salt on opposite sides of the membrane , one obtains a zero current potential across the membrane if the pores exhibit a selectivity for cations or anions .
The major porins of E. coli , OmpF and OmpC , as well as the major porins of S. typh-imurium , the OmpD , OmpF , and OmpC proteins , exhibit R. E. W. cation selectivity ( 7 ; R. Benz , A. Schmid , and Hancock , submitted for publication ) , whereas the E. coli PhoE pore exhibits anion selectivity ( 6 ) , consistent with the view that this protein participates in the permeation of phosphates through the outer membrane .
Consequently , we used the murein-associated porins of the pho constitutive strain KB17 as well as its parent KB66 and tested their ion selectivities in black lipid films .
Both preparations still contained the cation-selective OmpF porin , whereas the preparation of strain KB17 contained , in addition and as the major component , the pho-controlled porin ( Fig. 1 ) .
The amount of protein used in the assay was adjusted so that the black lipid membrane incorporated ca. 100 channels in each experiment .
The preparation of the parent strain KB66 exhibited cation selectivity ; the preparation of the pho constitutive strain exhibited anion selectivity ( Fig. 2 ) .
In conclusion , we isolated an S. typhimurium strain , KB17 , that carries a mutation leading to a constitutive pho regulon .
The consequences of this mutation include the appearance of a new anion-selective outer membrane protein , a transport system for G3P , and two new periplasmic proteins analogous to pho regulon products in E. coli .
Tokunaga , H. , M. Tokunaga , and T. Nakae .
Characterization of porins from the outer membrane of S. typhimurium .
Tommassen , J. , and B. Lugtenberg .
Pho-regulon of E. coli K12 : a minireview .