Induction of the Escherichia coli aidB Gene under Oxygen-Limiting ...

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FERRIC C. FANG,2 AND ROBERT PRINCE2 ...... Gross, Regine Hengge-Aronis, Peter Loewen, and Robert Tuveson for .... Vogel, H. J., and D. M. Bonner. 1956.
JOURNAL OF BACrERIOLOGY, Dec. 1994, p. 7638-7645

Vol. 176, No. 24

0021-9193/94/$04.00+0

Copyright © 1994, American Society for Microbiology

Induction of the Escherichia coli aidB Gene under Oxygen-Limiting Conditions Requires a Functional rpoS (katF) Gene MICHAEL R. VOLKERT,l* LAUREL I. HAJEC,' ZDENKA MATIJASEVIC,'t FERRIC C. FANG,2 AND ROBERT PRINCE2

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655,1 and Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 802622 Received 25 July 1994/Accepted 14 October 1994

The Escherichia coli aidB gene is regulated by two different mechanisms, an ada-dependent pathway triggered by methyl damage to DNA and an ada-independent pathway triggered when cells are grown without aeration. In this report we describe our search for mutations afecting the ada-independent aidB induction pathway. The mutant strain identified carries two mutations affecting aidB expression. These mutations are named abrB (aidB regulator) and abrD. The abrB mutation is presently poorly characterized because of instability of the phenotype it imparts. The second mutation, abrDl, reduces the expression of aidB observed when aeration is ceased and oxygen becomes limiting. Genetic and phenotypic analysis of the abrDI mutation demonstrates that it is an allele of rpoS. Thus, aidB is a member of the family of genes that are transcribed by a crs-directed RNA polymerase holoenzyme. Examination ofaidB expression in an rpoS insertion mutant strain indicates that both rpoSl3::TnlO and abrDl mutations reduce aidB expression under oxygen-limiting conditions that prevail in unaerated cultures, reduce aidB induction by acetate at a low pH, but have little or no elect on the adadependent alkylation induction of aidB.

The Escherichia coli aidB gene is one of several genes induced in response to alkylation damage caused by treatments with agents that methylate DNA. The aidB gene encodes a protein that has a high degree of homology to several mammalian coenzyme A dehydrogenases (12). In agreement with this homology, it has been shown that the aidB gene product has isovaleryl coenzyme A dehydrogenase activity (12), an activity known to be required for leucine metabolism in mammalian cells (8, 9). Overexpression of aidB protein has also been shown to reduce the mutagenic effects of the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, although the mechanism responsible for this resistance is unclear (12). Thus, aidB has roles in basic metabolism as well as defenses against methylation damage. In E. coli, the Ada protein mediates the response to alkylation damage, which has been called the adaptive response. Adaptive-response induction occurs when the Ada protein transfers a methyl group from methylphosphotriesters formed in DNA by methylating agents to its own Cys-69 residue (for reviews, see references 12, 15, and 36). Methylation of Ada converts this protein to a positive regulatory factor that binds sequences upstream of the ada, alkA, and aidB promoters, stimulating their transcription (12, 15, 36). Unlike the ada-alkB operon and the alkA gene, aidB is also subject to a second form of regulation (18, 37, 39). This second aidB induction pathway is activated when cells are grown in the absence of aeration and does not require a functional ada gene (37). Thus, there are two independent aidB regulatory pathways: an ada-dependent alkylation induction pathway and an ada-independent pathway induced when aeration is ceased and oxygen becomes limiting. To examine further the regulatory pathway triggered in

unaerated cultures, random mini-TnlO-Kan insertions were screened for mutations that specifically block this aidB induction pathway. This screening resulted in the isolation of a strain carrying two mutations which we call abrB and abrD (aidB regulators B and D). The abrBl::TnlO-Kan mutation is presently poorly characterized because of strong selective pressure for second-site suppressor mutations which we call abrD. In this report we characterize the abrDl suppressor mutation and demonstrate that the abrDl mutation is responsible for the reduction in aidB expression normally seen in unaerated cultures, that abrD1 is also required for the low-pH-acetate induction of aidB recently demonstrated by Smirnova et al. (30), and that abrDl is an allele of the rpoS gene. The rpoS gene encodes an alternative or factor called as (21, 32) which is required for the expression of a wide variety of genes expressed in stationary-phase cultures or upon carbon starvation (1, 2, 11, 13, 24, 25, 28, 33, 34). The rpoS gene is present at low levels in exponentially growing cells (32). As cells enter stationary phase, rpoS levels increase and expression of rpoS-dependent genes is stimulated. Expression of rpoS itself appears to be regulated at both the level of transcription and the level of translation (17, 19).

MATERUILS AND METHODS Bacterial strains and media. All bacterial strains designated CAG have been described by Singer et al. (29), and strain A594 has been described by Matijasevic et al. (18). All other strains derivatives of the E. coli K-12 strain AB1157 and are described in Table 1. All media have been described elsewhere (39).

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assays were per-

formed with cells grown in minimal medium containing E salts (35), glucose (0.4%), Casamino Acids hydrolysate (0.2%; Difco), and thiamine (0.2 pug/ml). Cells were grown with aeration and maintained in log phase (