Cloning, Sequencing, and Expression of the Cold-Inducible hutU Gene from the Antarctic Psychrotrophic Bacterium Pseudomonas syringae

Abstract

ABSTRACT A promoter-fusion study with a Tn 5 -based promoter probe vector had earlier found that the hutU gene which encodes the enzyme urocanase for the histidine utilization pathway is upregulated at a lower temperature (4° C) in the Antarctic psychrotrophic bacterium Pseudomonas syringae . To examine the characteristics of the urocanase gene and its promoter elements from the psychrotroph, the complete hutU and its upstream region from P. syringae were cloned, sequenced, and analyzed in the present study. Northern blot and primer extension analyses suggested that the hutU gene is inducible upon a downshift of temperature (22 to 4°C) and that there is more than one transcription initiation site. One of the initiation sites was specific to the cells grown at 4°C, which was different from the common initiation sites observed at both 4 and 22°C. Although no typical promoter consensus sequences were observed in the flanking region of the transcription initiation sites, there was a characteristic CAAAA sequence at the −10 position of the promoters. Additionally, the location of the transcription and translation initiation sites suggested that the hutU mRNA contains a long 5′-untranslated region, a characteristic feature of many cold-inducible genes of mesophilic bacteria. A comparison of deduced amino acid sequences of urocanase from various bacteria, including the mesophilic and psychrotrophic Pseudomonas spp., suggests that there is a high degree of similarity between the enzymes. The enzyme sequence contains a signature motif (GXGX 2 GX 10 G) of the Rossmann fold for dinucleotide (NAD + ) binding and two conserved cysteine residues in and around the active site. The psychrotrophic enzyme, however, has an extended N-terminal end.