Bidirectional promoter in the hut(P) region of the histidine utilization (hut) operons from Klebsiella aerogenes

Abstract

The hut(P) region (i.e., the region responsible for regulation of hutUH expression) of the Klebsiella aerogenes histidine utilization (hut) operons contains a bidirectional promoter. One transcript from this promoter encodes the hutUH operon; the role of the oppositely directed transcript is unknown, although it appears to be involved in regulating hutUH expression (A. J. Nieuwkoop, S. A. Boylan, and R. A. Bender, J. Bacteriol. 159:934-939, 1984 Klebsiella aerogenes can degrade the amino acid histidine enzymatically to ammonia, glutamate, and formamide (17). The histidine utilization (hut) genes are arranged in two tandem operons in the order hut(M)IGC(P)UH, where I, G, U, and H encode the four enzymes of the pathway and hutC encodes a repressor which regulates both operons (5, 11). The hut(M) and hut(P) regions represent the sequences controlling expression of the left and right operons, respectively. Therefore, the hut(P) region would be expected to include the binding site for RNA polymerase (hutUp), the binding site for repressor (hutUO), and the binding site(s) for positive effectors.Histidine can serve as the sole carbon or sole nitrogen source for K. aerogenes, and high-level expression of the hut operons requires not only the inactivation of the repressor but also the presence of a positive activator. This activator can be either the catabolite gene activator protein (CAP) in the presence of cyclic AMP (cAMP), signaling carbon or energy limitation, or an unknown factor(s) signaling nitrogen limitation (4, 23).We have previously suggested that the positive regulation of hutUH expression by CAP-cAMP might actually be a double-negative control (22) In this report we present the complete nucleotide sequence of the wild-type hut(P) region and identify the two RNA-polymerase-binding sites, Pc and PUH, within this sequence. In addition we identify consensus sequences suggesting binding sites for carbon and nitrogen effectors of hutUH expression (2,8). Thus, the data presented here support our previous double-negative control model of a bidirectional, mutually antagonistic promoter within hut(P). MATERIALS AND METHODSGrowth conditions. Plasmid-containing strains were grown in L broth or on L agar (3) with ampicillin at 100 jig/ml (LBamp). Minimal medium consisted of W salts (3) supplemented with 0.4% histidine as the sole carbon and nitrogen source. For S1 nuclease mapping, minimal medium was 2W5 salts (double-strength W salts adjusted to pH 7.5) supplemented with 0.4% glucose and 0.2% histidine for nitrogenlimited growth and 0.4% histidine alone for carbon-limited growth.Chemicals and enzymes. Restriction enzymes were purchased from

Roles of catabolite activator protein sites centered at -81.5 and -41.5 in the activation of the Klebsiella aerogenes histidine utilization operon hutUH

“…Thus, the region between positions 184 and 173 contained the rightmost boundary of the sequence necessary for hutUp activity. This is in agreement with our identification of the sequence TATATG at positions 179 to 184 as the hutUp -10 promoter sequence (33).…”

“…2B and C), demonstrating that the hutUp promoter sequences are located to the right of nucleotide 145. This is consistent with our identification of the sequence TIGCGC from nucleotides 156 to 161 as the hutUp -35 region (33).…”

“…As noted previously (39), the transcriptional activity of Pc was a mirror image of that of hutUp, with a similar amount of CAP required to repress Pc as to activate hutUp. The fact that the CAP concentration required for half-maximal hutUp transcription was about 3.7 times higher than that required for lac P, even when their CAP binding affinities were comparable (38) (33,39). Thus, CAP-cAMP activates the same promoter in vivo and in vitro.…”

“…Both pCB611 and LA150 lack the wild-type sequence from nucleotides 142 to 149, which suggests that this sequence may play an important role in achieving maximum transcription of hutUp in a manner that is independent of CAP-cAMP. In K aerogenes, activation of hutUp can also be achieved by nitrogen starvation (33). When cells are grown with limited nitrogen source, a nitrogen-controlled transcription activator, NAC, binds to a single site in hut(P) centered at -64 to activate hutUp (5,14,49).…”

“…Our identification of the promoter sequences hutUp and Pc, the two promoters residing within the hut(P) region ( Fig. 1), was based on (i) the sizes of the run-off transcripts obtained from each promoter (35), (ii) exonuclease III protection studies of RNA polymerase bound to hutUp and Pc (33,34), and (iii) resemblance of DNA sequences to the canonical promoter sequence TTGACAN17 TATAAT (33). However, no mutations that abolish the activity of either hutUp or Pc promoters directly have been obtained.…”