Genetic analysis, using P22 challenge phage, of the nitrogen activator protein DNA-binding site in the Klebsiella aerogenes put operon

Abstract

The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons from Klebsiella aerogenes and Escherichia coli. We used P22 challenge phage carrying the put control region from K. aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium, which is not regulated by NAC.

FIG. 1

Physical map of the K. aerogenes put regulatory region and subcloned DNA fragments. The positions of relevant restriction sites are indicated with the distances in base pairs from the putP transcriptional start site shown in parentheses. Subcloned regions used for construction of challenge phage are shown as shaded bars directly below the corresponding region of the restriction map.

FIG. 2

DNA sequences of the K. aerogenes put regulatory region and challenge phages. The wild-type K. aerogenes put regulatory region and DNA sequences from the wild-type K. aerogenes put regulatory region present in challenge phages SD1-1, SD2-1, SH4-1, and SH26-10 are shown. The sequence of the put DNA is shown in capital letters. The adjacent sequences in the challenge phage are shown in lowercase letters. Note that the substitution is inverted in challenge phage SD2-1 versus SD1-1 and SH4-1 versus SH26-10. Underlined nucleotides represent the −10 and −35 region of P_ant.

FIG. 3

Results of challenge phage assays with wild-type SH2-1 and SH4-1 and mutant derivatives of SH4-1 shown as follows: wild-type SH2-1, ⧫; wild-type SH4-1, □; 6G mutant, •; 8C mutant, ○. The percent lysogeny of the negative control which lacks the nac gene (MST1762) was less than 10⁻⁷ at all IPTG concentrations tested. The percent lysogeny indicates the number of Kan^r lysogens formed per viable cell infected. NAC is expressed from the P_tac promoter under the control of LacI^q, and thus the intracellular concentration of NAC increases as the concentration of IPTG is increased.

FIG. 4

Gel retardation assay of the interactions between NAC and DNA fragments derived from the putAP control region. The indicated amounts of NAC were incubated with a mixture of digested pPC47 and DNA amplified from challenge phages SH4-1 (wild-type), 90-32 (P_ant promoter mutation), 90-40 (6G mutation), and 90-2 (8C mutation). Digestion of pPC47 with HincII and BamHI yielded a 215-bp control fragment containing nucleotides 406 to 621 of the putAP control region and a larger fragment containing the vector pTZ18U plus nucleotides 381 to 405 of the putAP control region. The migration position of the unretarded 215-bp control and 150-bp test fragments are indicated at the right.