Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein

Abstract

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.

FIG. 1

Comparison of the regulatory regions of dadAB from K. aerogenes and dadAX from E. coli. The lower DNA sequence is from dadAX as reported by Lobocka et al. (23). The start of transcription and the proposed −10 and −35 promoter regions are in boldface. Solid underlines indicate putative CAP-cAMP binding sites, ΔΔΔΔΔ indicates the NAC binding site (the asterisk indicates the C-to-T change that most likely is the reason NAC fails to bind to the dad promoter from E. coli), and the broken lines indicate putative Lrp binding sites.

FIG. 2

Mapping the transcriptional start site of dadAB by primer extension. The sequence lanes have been oppositely labeled such that the coding strand sequence is denoted, although the actual reactions were performed with the primer extension primer (RJ800EXT) and is thus the noncoding strand. Below is a control extension reaction of the β-lactamase gene (blaP) also present on the plasmid to ensure equal loading. RJ800EXT hybridizes to plasmid pRJ800 downstream of the multiple cloning site; thus, the extended products shown are from the plasmid-borne promoter (pCB888) only. Total RNA was isolated from the strains grown in glucose-ammonium minimal medium supplemented with ampicillin (100 μg/ml) for plasmid maintenance and with alanine or IPTG as indicated. Lanes: 1, KC3821, no addition; 2, KC3821, supplemented with 0.2% l-alanine; 3, KC3848, no addition; 4, KC3848, supplemented with 1 mM IPTG.

FIG. 3

Interaction of the dad promoters from E. coli and K. aerogenes with NAC. pCB888, which contains the dad promoter from K. aerogenes, and pCB889, which contains the dad promoter from E. coli, were digested and radiolabeled as described in Materials and Methods. Each was then incubated with buffer 6 (19) or increasing amounts of purified NAC (0, 16.5, 22, 33, and 66 nM) for 20 min. The bound and unbound fragments were then separated by electrophoresis on a 4% TE–polyacrylamide gel run for 2 h at 13.3 V/cm at 4°C. The gel was dried, and the DNA was visualized by exposure to X-ray film. Unbound vector and dad promoter bands are indicated by labeled arrows; the unlabeled arrow indicates the NAC-dadAp complex.

FIG. 4

Interaction of Lrp with the promoter region of dadAB from K. aerogenes. pCB888 was digested and radiolabeled as described in Materials and Methods; 0.1 μg of DNA was incubated with increasing amounts of Lrp (lanes 1, 6, and 11, no Lrp; lanes 2, 7, and 12, 8 nM; lanes 3, 8, and 13, 23 nM; lanes 4, 9, and 14, 68 nM; lanes 5, 10, 15, 200 nM) and incubated for 20 min at room temperature; 30 mM l-leucine (lanes 6 to 10) or 30 mM l-alanine (lanes 11 to 15) was added prior to the addition of Lrp. Separation of the bound and unbound fragments by electrophoresis and visualization of the DNA fragments by autoradiography are described in Materials and Methods. The vector and unbound dad promoter bands are indicated by labeled arrows; the two unlabeled arrows indicate the Lrp-dadAp complexes. The unlabeled band that appears in lanes 5, 10, and 15 is most likely the result of an Lrp-vector DNA complex due to the high levels of Lrp present.