Hydrogen peroxide-inducible proteins in Salmonella overlap with-heat-shock and other stress proteins ABSTRACT Hydrogen-peroxide-treatment induces the synthesis of 30 proteins in Salmonella typhimurium .
Five of these proteins are also induced by heat-shock , including the highly conserved DnaK protein .
The induction of one of these five proteins by heat-shock is dependent on oxyR , a positive regulator of hydrogen-peroxide-inducible genes , while the induction of the other four by heat-shock is oxyR independent .
Five of the 30 hydrogen-peroxide-inducible proteins have been identified , and their structural genes have been mapped .
Other stresses such as nalidixic-acid , ethanol , or cumene hydroperoxide treatment also induce subsets of the 30 hydrogen perox-ide-inducible proteins as well as additional proteins .
Hydrogen peroxide-inducible proteins are shown to be largely different from those proteins induced by aerobiosis .
In addition , the expression of the katG ( catalase ) gene is shown to be regulated by oxyR at the level of mRNA .
MATERIALS AND METHODS Bacterial Strains and Labeling of Proteins .
S. typhimurium LT2 and E. coli K12 were the wild-type strains used .
Overnight cultures in VBC salts ( 14 ) supplemented with 0.4 % glucose ( VBC/glucose ) at 280C or 370C were diluted , incubated , and then labeled during exponential-growth ( OD650 of 0.2-0.4 ) .
The labeling reactions were as described ( 12 ) : 0.4-ml aliquots of the cultures were grown in VBC/glucose containing L - [ 35S ] methionine at 200 , ACi/ml ( 1 Ci = 37 GBq ; Dupont-NEN Products ) and unlabeled L-methionine such that the final L-methionine concentration was 10 , AM .
Cultures were labeled for the designated period of time , and the labeling reaction was terminated by the addition of 10 Al of 0.1 M L-methionine .
Cells were harvested by centrifugation for 5 min in a Fisher microfuge and resuspended in 2D gel electrophoresis sample buffer ( 15 ) .
Conditions for Labeling Proteins During Stresses .
Adaptation to hydrogen-peroxide and temperature shift experiments were done as described ( 12 ) .
For ethanol treatment , cells were labeled for 30 min immediately following exposure to 4 % ( vol/vol ) ethanol at 280C .
For nalidixic-acid treatment , cells were labeled for 12 min immediately following exposure to nalidixic-acid ( 60 , ug/ml ) at 370C .
Cells were treated with 125 , aM cumene hydroperoxide at 370C for 60 min .
To compare cellular proteins synthesized during anoxic and aerobic-growth-conditions , cultures were grown at 370C in VBC/glucose medium in tubes that were constantly bubbled with either air ( aerobic ) or 95 % N2/5 % CO2 ( anoxic ) .
2D gel electrophoresis was done as described by O'Farrell ( 15 ) .
Proteins were classified using an alphanumeric system similar to that used by Neidhardt and coworkers ( 16 ) .
The numerical portion of each designation indicates the approximate molecular weight of the protein , and the letter portion indicates the approximate isoelectric point , as follows : C , < 5.10 ; D , 5.10-5.59 ; E , 5.59-5.74 ; F , 5.74-6.18 ; G , > 6.18 ( Figs. 1 and 2 ) .
RNA Isolation and Dot-Blot Hybridization .
Total RNA was isolated from 5-ml aliquots withdrawn from exponentially growing cultures of LT2 and TA4100 ( oxyRI ) in VBC/glucose and Luria broth ( LB ) .
The cells were disrupted by phenol extraction , and the RNA was isolated as described ( 17 ) .
The RNA pellets were resuspended in 10 p1L of distilled RESULTS Protein F52a , which is one component of the alkyl hydroperoxide reductase , is distinctive in that it requires oxyR for both heat-shock induction and for hydrogen-peroxide induction .
In strains containing a deletion of oxyR , the F52a protein can not be induced by heat-shock ( 12 ) .
Deletions of the ahp locus , which eliminate protein F52a from 2D gels , also prevent F52a from being induced by either hydrogen-peroxide treatment or heat-shock ( data not shown ) .
This is strong evidence that the heat-and hydrogen-peroxide-induced spots in the F52a region of the gel are , in fact , the same protein .
F52a is also unusual in that it is not regulated in the same way in E. coli and Salmonella .
In E. coli there is a spot analogous to the Salmonella F52a spot ( unpublished results ) , which is also controlled by oxyR and forms one component of an alkyl hydroperoxide reductase activity .
However , this protein is not heat inducible in E. coli ( data not shown ) .
Analysis of the cloned gene ( s ) ( ahp ) for the F52a protein in Salmonella and E. coli may reveal the reason for this difference .
Other Stresses Induce Some Heat Shock Proteins in Salmo-nella .
In E. coli the DnaK and F84 .1 ( analogous to E79 in Salmonella ) heat-shock proteins can also be induced by ethanol ( 22 ) , by UV-irradiation and nalidixic-acid ( 23 ) , by bacteriophage X infection ( 24 ) , and by amino-acid starvation ( 25 ) .
We have found that DnaK and E79 proteins in Salmo-nella can be induced by ethanol , by nalidixic-acid , and by cumene hydroperoxide ( data not shown ) in addition to hydrogen-peroxide .
Each stress also induces distinct proteins .
These data and data reported for Salmonella are shown schematically in Fig. 3 .
Proteins Synthesized Anoxically Versus Aerobically .
Catalase and Mn-containing superoxide dismutase are regulated by oxyR and have been reported to be induced by anaerobic to aerobic shifts ( 26 , 27 ) .
Therefore , we have investigated whether other proteins that are more abundant during aerobic-growth overlap with hydrogen-peroxide-inducible proteins .
The protein synthesis patterns for wildtype Salmonella cells grown in nonshaking tubes bubbled with either air ( aerobic ) or 95 % N2/5 % CO2 ( anoxic ) were compared .
The steady-state pattern of protein synthesis as determined by 2D gels for nonshaking tubes bubbled with air was essentially identical to cells grown aerobically on a rotary shaker .
Cells bubbled with air differed from cells bubbled with N2/CO2 in the abundance of 26 proteins ( data not shown ) .
There are 12 proteins that are more abundant under air and 14 proteins that are more abundant under N2/CO2 ( Fig. 3 ) .
In E. coli 19 proteins are induced by aerobiosis , and 18 proteins are induced by anaerobiosis ( 28 ) .
Seven of the 26 proteins whose levels are affected by the degree of aeration are hydrogen-peroxide-inducible proteins .
However , of these seven , four are more abundant during aerobic-growth ( G67 , G45 , G55a , and E89 ) , and three are more abundant during anoxic ( N2/CO2 ) growth ( D69 , D71 , and F52a ) .
Therefore , hydrogen-peroxide-inducible proteins are not identical to proteins induced during aerobiosis .