Genetic and physical maps of Klebsiella aerogenes genes for histidine utilization (hut)

Abstract

Deletion derivatives of the hut-containing plasmid pCB101 were tested against point mutants defective in individual genes of the histidine utilization (hut) operons using a complementation/recombination assay. Location of the genes of the right operon, hutU and hutH, was confirmed by direct assay of the gene products, urocanase and histidase; location of the repressor gene was identified by measuring the ability of the plasmid-carried genes to repress the formation of histidase from a chromosomal location. The analysis of eight deletion plasmids unambiguously confirms the map order of the hut genes as hutI-G-C-U-H, and demonstrates that, in Klebsiella aerogenes, the hutU and hutH genes are transcribed from their own promoter. In addition, the genetic map of hut can be aligned with the restriction map of the hut DNA in plasmid pCB101. One of the deletion plasmids studied apparently encodes a defective histidase subunit that is trans-dominant to active histidase. Another deletion, which completely removes the left operon, hutIG, allows high level expression of the hutUH operon and thus overproduction of a toxic intermediate.

Regulation of the Histidine Utilization (Hut) System in Bacteria

“…The reason for this toxicity is unknown but might result from accumulation of formylisoglutamine or 4-ketoglutaramic acid derived from the spontaneous decomposition of IP (97,128). That would be consistent with the observation that either a very high concentration of exogenous histidine (48) or a large amount of histidase and urocanase activity (14) is required for histidine to inhibit growth effectively. It has also been suggested that both imidazole propionate and IP or products derived in vivo from them may somehow interfere with the aspartate aminotransferase of Salmonella enterica and that this might explain the toxicity of IP (12).…”

“…4). Genetic experiments (45) and cloning data (14) established the order of the genes to be hutIGCUH, the same as the order established earlier for S. enterica (148). Alignment of sequenced and assembled genomes by use of a simple Web-based tool (http: //www.ecocyc.org/) confirms this order and places the hut operons immediately adjacent to the bio cluster.…”

“…It is assumed that NAC also activates hutIG expression, because at least hutG responds to nitrogen limitation (91). Furthermore, if hutUH expression is activated by inducing nac, no histidine toxicity is observed as would be expected if hutUH expression outstripped hutIG expression (14,48). However, the role of NAC in activation of hutIG expression has not been confirmed.…”

“…Although the details of the reaction mechanism and the nature of the active site metal remain somewhat unclear, two facts are well established: the reaction is essential for utilization of histidine as a carbon source (74,148), and IPase is a hydrolase that cleaves the ring to yield formiminoglutamate, in a different reaction from the nonenzymatic cleavage that yields formylisoglutamine (128). It is interesting that mutants lacking IPase are poisoned by growth in the presence of histidine if histidase and urocanase are both active (14,48). The reason for this toxicity is unknown but might result from accumulation of formylisoglutamine or 4-ketoglutaramic acid derived from the spontaneous decomposition of IP (97,128).…”

“…Mutants lacking urocanase express Hut at constitutively high levels in the absence of any inducer (137). Recall that mutants expressing hutUH but not hutIG are poisoned by histidine (14,48), so it is important that induction of the two operons be coordinated both in time and in degree. In S. enterica, this is achieved by having hutC be part of the hutIGC operon and by the fact that the hutIGC operator region has a lower affinity for HutC than does the hutUH operator (49).…”