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

Osuna, Robert; Janes, Brian K.; Bender, R.

doi:10.1128/jb.176.17.5513-5524.1994

Cited by 12 publications

(16 citation statements)

References 52 publications

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“…The hutUp region contains two CRP-cAMP-binding sites: a stronger site centered at position Ϫ82.5 (relative to the start of transcription) and a weaker site centered at position Ϫ42.5 (116,117). The stronger site is essential for activation of hutUp by CRP-cAMP (117). The weaker site also appears to play a role in activation, but this is not well characterized (117).…”

Section: Enteric Bacteriamentioning

confidence: 99%

“…In vitro transcription with purified components showed that both CRP and cAMP were necessary (and sufficient) to activate transcription from hutUp, the hutU promoter (115,116). The hutUp region contains two CRP-cAMP-binding sites: a stronger site centered at position Ϫ82.5 (relative to the start of transcription) and a weaker site centered at position Ϫ42.5 (116,117). The stronger site is essential for activation of hutUp by CRP-cAMP (117).…”

Section: Enteric Bacteriamentioning

confidence: 99%

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Regulation of the Histidine Utilization (Hut) System in Bacteria

Bender

2012

Microbiol Mol Biol Rev

125

128

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SUMMARY The ability to degrade the amino acid histidine to ammonia, glutamate, and a one-carbon compound (formate or formamide) is a property that is widely distributed among bacteria. The four or five enzymatic steps of the pathway are highly conserved, and the chemistry of the reactions displays several unusual features, including the rearrangement of a portion of the histidase polypeptide chain to yield an unusual imidazole structure at the active site and the use of a tightly bound NAD molecule as an electrophile rather than a redox-active element in urocanase. Given the importance of this amino acid, it is not surprising that the degradation of histidine is tightly regulated. The study of that regulation led to three central paradigms in bacterial regulation: catabolite repression by glucose and other carbon sources, nitrogen regulation and two-component regulators in general, and autoregulation of bacterial regulators. This review focuses on three groups of organisms for which studies are most complete: the enteric bacteria, for which the regulation is best understood; the pseudomonads, for which the chemistry is best characterized; and Bacillus subtilis , for which the regulatory mechanisms are very different from those of the Gram-negative bacteria. The Hut pathway is fundamentally a catabolic pathway that allows cells to use histidine as a source of carbon, energy, and nitrogen, but other roles for the pathway are also considered briefly here.

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Section: Enteric Bacteriamentioning

confidence: 99%

Section: Enteric Bacteriamentioning

confidence: 99%

Regulation of the Histidine Utilization (Hut) System in Bacteria

Bender

2012

Microbiol Mol Biol Rev

125

128

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“…For example, the hutUH operon, whose products provide glutamate and energy, is activated by catabolite gene activator protein (CAP)-cyclic AMP (cAMP) (18) and is repressed during anaerobic growth (10). Thus, it is attractive to speculate about NAC's role in the evolution of the global response to nitrogen deprivation.…”

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confidence: 99%

“…Ampicillin and kanamycin (Sigma) were added at concentrations of 100 and 40 g/ml, respectively. ␤-Galactosidase assays were performed as described previously (18).…”

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confidence: 99%

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

Pomposiello

Bender

1995

J Bacteriol

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A chimeric promoter with the nitrogen assimilation control protein binding site from hutUp of Klebsiella aerogenes fused to the lacZ core promoter from Escherichia coli was built and cloned in a lacZ reporter plasmid. This construct showed a 14-fold increase of ␤-galactosidase activity upon nitrogen limitation. Primer extension experiments showed that the nitrogen assimilation control protein activates lacZp 1 in a position-dependent manner.The enteric bacterium Klebsiella aerogenes responds to a decrease in the concentration of ammonia, the preferred nitrogen source, by modifying transcription rates at many operons. Some of these nitrogen-regulated operons allow the cell to assimilate ammonia in low concentration or to use alternative nitrogen sources. This global response to nitrogen starvation is regulated by the Ntr system, a regulatory network built around a two-component system (see references 16 and 27 for reviews). Essentially, nitrogen starvation results in the activation of NtrC, a DNA-binding protein that enhances transcription from 54 -dependent promoters. Although the Ntr system is necessary for the modulation of all nitrogen-regulated operons, transcriptional activity of a subset of these operons is also dependent on the nitrogen assimilation control protein (NAC) (4,5,15). NAC is a member of the LysR family of DNA binding transcriptional factors (23,24). The transcription of the nac gene is 54 dependent and under positive control by the Ntr system (8, 15). The apparent paradox posed by a second, positively regulated transcription factor in the Ntr cascade is explained by the functions NAC adds to the Ntr repertoire: NAC regulates 70 -dependent promoters and can either activate or repress transcription (see reference 2 for a review). Therefore, we see NAC as the coupling agent between 54 -dependent Ntr regulation and the 70 -dependent operons. NAC activates the expression of operons involved in the catabolism of alternative nitrogen sources: histidine utilization (hut), proline utilization (put), and urease (ure) (15). The bestcharacterized NAC-activated system is that of the hutUH operon (25). The transcriptional activation of the hut operon by NAC has been reproduced in vitro with purified components (11). These in vitro transcription experiments have shown that NAC is sufficient for the activation of 70 -directed transcription. In addition, the binding site for NAC has been determined by gel mobility retardation and footprinting assays (11). NAC protects a 26-bp fragment of the hutU promoter centered 64 bp upstream of the hutU transcriptional initiation site. A sequence comparison of the NAC-protected regions from the hutU, putP, and ureD promoters revealed a minimal NAC binding consensus: 5Ј-ATA-N 9 -TAT-3Ј.Interestingly, some NAC-regulated operons have functions other than the generation of reduced nitrogen, such as carbon and energy provision, and are regulated independently by other control signals. For example, the hutUH operon, whose products provide glutamate and energy, is activated by c...

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“…3 are also indicated as follows: +, functional hutUo; +/-, partial hutUo function; -, nonfunctional hutUo. protein (25), the two mutually competitive RNA polymerase binding sites (25), and the binding site for NAC, the protein responsible for mediating activation of hut transcription by nitrogen starvation (11 …”

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Identification of the hutUH operator (hutUo) from Klebsiella aerogenes by DNA deletion analysis

1994

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Expression of Klebsiella aerogenes histidine utilization operons hutUH and hutIG is negatively regulated by the product of hutC. Multiple copies of the hutUH promoter region [hut(P)] present in trans were able to titrate the limited amount of host-encoded hut repressor (HutC). Thus, the hut(P) region contains a specific binding site for HutC. To The histidine utilization (hut) operons of Klebsiella aerogenes, Salmonella typhimurium, and Pseudomonas putida are negatively controlled by the product of the hutC gene (4,18,26), and this negative control resembles other well-characterized repressor-operator interactions. Mutations within hutC result in either constitutive expression or superrepression (noninducibility) of the hut operons (4, 12). Urocanate (the first intermediate in the hitt pathway) eliminates the binding of the hut repressor protein (HutC) to its operator site, and addition of urocanate to growing cells results in a 50-to 100-fold induction of hut operon expression (19). Purified HutC from S. typhimurium and P. putida binds to DNA fragments containing the hut regulatory regions in the absence of urocanate but not in its presence (13,15). The K aerogenes hut repressor-operator system has significant similarity with that from S. typhimurium, since hutC can be interchanged between the two species without loss of repressor activity (8).Operator of their failure to titrate the repressor in vivo (7,16). We have made use of this repressor titration approach to screen a set of plasmids lacking portions of the hut(P) region for their ability to titrate hut repressor and thereby identify the hutUH operator (hutUo) from K aerogenes.Deletions with endpoints throughout the hut(P) region. A SalI-PvuII restriction fragment from kb 3.35 to 3.59 on the standard restriction map of pCB101 (3) contains the entire hutUH promoter region, hut(P) (21, 23). The ends of this fragment were blunted by using Klenow enzyme, Sall linkers were added to each end as described before (20), and the resulting fragment was cloned into the SalI site of pUC18 as illustrated in Fig. 1. Plasmid pOS1 contains the fragment with the hutUH promoter (hutUp) oriented toward the EcoRI site of the pUC18 polylinker region; pOS2 contains the same fragment in the opposite orientation. Unidirectional exonuclease III deletions were generated essentially as described by Hennikoff (14), taking advantage of the flanking restriction sites for XbaI (which creates a 5' extension) and SstI (which creates a 3' extension) to provide asymmetry of flanking ends (Fig. 1). The extent of each deletion and the integrity of the remaining nucleotides in the region were confirmed by DNA sequence analysis (2, 29).In each of the deletions, a block of nucleotides at either the right or left end of the hut(P) region was removed by exonuclease III digestion. The DNA ends were then made blunt (by S1 nuclease cleavage followed by filling in any remaining ends with Klenow enzyme in the presence of all four deoxyribonucleotides) and ligated to recircularize the plasmid. In this...

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

Cited by 12 publications

References 52 publications

Regulation of the Histidine Utilization (Hut) System in Bacteria

Regulation of the Histidine Utilization (Hut) System in Bacteria

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

Identification of the hutUH operator (hutUo) from Klebsiella aerogenes by DNA deletion analysis

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