Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000

Abstract

The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the Hindlll site of plasmid pBR325, and the two resulting plasmids, pCB120 and pCB121, were subjected to mutagenesis with Tn1000. The insertion sites of TnlOOO into pCB121 were evenly distributed throughout the plasmid, but the insertion sites into pCB120 were not. There was a large excess of TnlOOO insertions in the "plus" or y8 orientation in a small, ca. 3.5-kilobase region of the plasmid. Genetic analysis of the TnlOOO insertions in pCB120 and pCB121 showed that the hutUH genes form an operon transcribed from hutU and that the hutC gene (encoding the hut-specific repressor) is independently transcribed from its own promoter.The hutlG cluster appears not to form an operon. Curiously, insertions in hutI gave two different phenotypes in complementation tests against hutG504, suggesting either that hutI contains two functionally distinct domains or that there may be another undefined locus within the hut cluster. The set of TnlOOO insertions allowed an assignment of the gene boundaries within the hut cluster, and minicell analysis of the polypeptides expressed from plasmids carrying insertions in the hut genes showed that the hutI, hutG, hutU, and hutH genes encode polypeptides of 43, 33, 57, and 54 kilodaltons, respectively.The histidine utilization (hut) gene cluster of Klebsiella aerogenes encodes the structural and regulatory proteins responsible for the inducible degradation of the amino acid L-histidine to glutamate, ammonia, and formamide (13). The hut genes are located between gal and bio on the K. aerogenes genetic map and are arranged in the order hut(M) IGC(P)UH (4, 7), where hut(M) and hut(P) are defined as the regions required for regulated transcription of hutI and hutU, respectively; hutl, hutG, hutU, and hutH encode the four enzymes of the pathway; and hutC encodes the hutspecific repressor which negatively regulates the entire pathway. In the closely related enteric bacterium Salmonella typhimurium, the hut cluster is arranged as two independent transcription units, one originating at hutIp [within the hut (M) region] and extending through hutIGC, and the other originating at hutUp [within the hut(P) region] and extending through hutUH (26,27). Several lines of evidence led us to expect that the arrangement of transcription units in K. aerogenes would be the same as that in S. typhimurium: (i) the gene order is identical; (ii) hutU (but not hutI) expression is coordinate with hutH (7, 14); (iii) hutU and hutH do not require the hutIGC region for expression (4); and (iv) the hut DNA from K. aerogenes and S. typhimurium are so similar that under appropriate conditions, a continuous heteroduplex molecule can be formed across the entire length of the cluster (3).To identify the transcription units within the hut cluster and to provide a mobile priming site for later DNA sequence analysis of the hut genes, we isolated and characterized a set of transposon Tn1OOO (-y)

Regulation of the Histidine Utilization (Hut) System in Bacteria

“…The sequence corresponding to the Ϫ10 region is missing from the shorter intergenic region in S. enterica, which is otherwise similar to the corresponding region from K. pneumoniae. A more troublesome anomaly is the fact that strains with Tn1000 insertions in the hutI gene from K. pneumoniae still produce both HutG activity and HutG polypeptide at nearly normal levels (142). Nevertheless, the same 3-bp overlap between hutI and hutG is seen in both organisms, suggesting that hutI and hutG are probably cotranscribed.…”

“…The hutUH genes of K. pneumoniae form an operon (142). Strains with insertions of Tn1000 in hutU fail to make either active histidase or a HutH polypeptide.…”

“…In K. pneumoniae, the hutIGC operon is strongly repressed by HutC (45), so a different strategy is needed to prevent repressor levels from falling too low to keep hutUHT expression below the level of hutIGC expression. Thus, in K. pneumoniae, hutC can be expressed from a separate promoter (142). Binding studies with the K. pneumoniae operator regions are lacking.…”

The nac (nitrogen assimilation control) gene from Klebsiella aerogenes

“…Transconjugants were selected on glucose minimal medium (to counterselect the donor) containing kanamycin and tetracycline (to counterselect the recipient). The result-ing transconjugants invariably contained a TnOOO insertion randomly positioned in pEC217, whose location and orientation were determined by restriction endonuclease mapping (33). Strains were made competent for transformation by treatment with calcium chloride (26).…”