池上 昭彦

Potential regulatory elements were identified upstream of the pressure-regulated operon in the deep-sea barophilic bacterium Shewanella violacea strain DSS12 and binding of cellular proteins to this region was studied under different pressure conditions. Sequence analysis revealed the existence of a region, designated region A, showing similarity to the consensus sequence for sigma(54) binding, when the region upstream of the operon was compared with several consensus sequences for sigma factors. A palindromic sequence AGTTAAAGATTAAACT, designated region B, was found further upstream beyond region A. In a region designated region C, just upstream of region B; a unique octamer motif AAGGTAAG, tandemly repeated 13 times, was found. By means of an electrophoretic mobility shift assay it was demonstrated that a sigma(54)-like factor recognized region A while other unknown factors recognized the sequences in regions B and C. Different shift patterns of protein-DNA complexes were observed when extracts of cells cultured at 0.1 MPa and 50 MPa were incubated with P-32-labeled DNA probes corresponding to region B or C. These results indicate that barophilic strain DSS12 expresses different DNA-binding factors capable of recognizing these elements upstream of the pressure-regulated operon under different pressure conditions. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

A pressure-regulated operon has been cloned and sequenced from deep-sea barophilic Shewanella strains. To understand pressure-regulated mechanisms of gene expression, a regulatory element upstream of the pressure-regulated operon from Shewanella sp. strain DSS12 was studied. Regions A and B were classified by sequence analysis. A unique octamer motif, AAGGTAAG, was found to be repeated in tandem 13 times in region B. An electrophoretic mobility shift assay demonstrated that a sigma(54)-like factor recognizes region A and other unknown factors recognize region B. Different shift patterns of the protein-DNA complexes were observed when extracts of cells cultured at 0.1 MPa or 50 MPa were incubated with a DNA probe specific for region B. These results indicate that the deep-sea strain DSS12 expresses different DNA-binding factors under different pressure conditions.

A pressure-regulated operon that displayed high homology with one present in the barophilic bacterium strain DB6705 was cloned and sequenced from a barotolerant bacterium strain DSS12. Two open reading frames, ORF1 and ORF2, which constituted the pressure-regulated operon, were identified within the sequence, DNA fragments from regions upstream and downstream of the pressure-regulated operon in strain DSS12 were also cloned and sequenced. Downstream of the pressure-regulated operon, three open reading frames, designated ORF3, ORF4, and ORF5 were found, and part of another ORF, designated UORF6, was located upstream of the operon. From the results of primer extension analysis, the gene expressions of the pressure-regulated operon (ORF1, 2) and downstream operon (ORF3, 4) were clearly regulated by elevated pressure at the transcription level, The deduced amino acid sequences of the gene products of ORF3, 4, 5, and UORF6 showed high homology with CydD, CydC, proline dehydrog enase, and ATP-dependent RNA helicase from Escherichia coli, respectively. These results indicate that the genes identified in the regions flanking the pressure-regulated operon are homologous to E. coli genes, although the genes within this operon are not.

Escherichia coli strain JD518, a cydD-deficient mutant, displayed temperature-sensitive and pressure-sensitive growth, The defective cydD gene in this strain was complemented by open reading frame 3 (ORF3), previously identified in DNA from a barotolerant bacterium, strain DSS12, allowing growth of the cydD mutant under high temperature and high pressure conditions, Spectrophotometrical analysis indicated that the cytochrome bd complex which is assembled by the CydD protein was expressed in E. coli strain JD518 carrying the ORF3 gene at the same level as occurred in the wild-type strain, Our results indicate that the cydD gene functions are required for cell stability under the condition of high pressure stress in bacteria.