THE ICE NUCLEATION GENE FROM PSEUDOMONAS-SYRINGAE AS A SENSITIVE GENEREPORTER FOR PROMOTER ANALYSIS IN ZYMOMONAS-MOBILIS

The expression of the ice nucleation gene inaZ from Pseudomonas syring ae in Zymomonas mobilis strains under the control of three different p romoters was investigated to establish the utility of the gene as a re porter and examine the possible use of the organism as a source of ice nuclei for biotechnological applications. A promoterless version of t he inaZ gene was placed under the control of three different prompters : P-pdc (pyruvate decarboxylase), a homologous strong promoter from Z. mobilis; P-bla (beta-lactamase) of plasmid pBR325; and P-hrpR, the pr omoter of hrpR, a regulatory gene from P. syringae pv. phaseolicola. T he apparent strengths of all three promoters, measured by quantifying the ice nucleation activity at -9 degrees C, were lower in Z. mobilis than in Escherichia coti. The levels of ice nucleation activity expres sed under the P-pdc promoter were significantly higher than those obta ined with the two heterologous promoters in Z. mobilis. Plasmid pCG452 1 (RK2 replicon) gave much lower levels of ice nucleation activity whe n propagated in strain uvs-51, a plasmid instability mutant of Z. mobi lis, compared with the wild-type strain. The ice nucleation activity i n Z. mobilis cultures showed unusual partitioning in that the culture supernatants obtained after low-speed centrifugation contained the maj ority of ice nuclei, Analysis of the ice nucleation spectra revealed t hat the cell pellets contained both ''warm'' and ''cold'' nuclei, whil e the culture supernatant contained primarily cold nuclei, suggesting that the cold nucleus activity may be extracellular. However, all nucl eation activity was retained by 0.22-mu m-pore-size filters.