The Ice Nucleation Gene from Pseudomonas syringae as a Sensitive Gene Reporter for Promoter Analysis in Zymomonas mobilis.

The expression of the ice nucleation gene inaZ from Pseudomonas syringae in Zymomonas mobilis strains under the control of three different promoters was investigated to establish the utility of the gene as a reporter and examine the possible use of the organism as a source of ice nuclei for biotechnological applications. A promoterless version of the inaZ gene was placed under the control of three different promoters: P(infpdc) (pyruvate decarboxylase), a homologous strong promoter from Z. mobilis; P(infbla) ((beta)-lactamase) of plasmid pBR325; and P(infhrpR), the promoter of hrpR, a regulatory gene from P. syringae pv. phaseolicola. The apparent strengths of all three promoters, measured by quantifying the ice nucleation activity at -9 deg C, were lower in Z. mobilis than in Escherichia coli. The levels of ice nucleation activity expressed under the P(infpdc) promoter were significantly higher than those obtained with the two heterologous promoters in Z. mobilis. Plasmid pCG4521 (RK2 replicon) gave much lower levels of ice nucleation activity when propagated in strain uvs-51, a plasmid instability mutant of Z. mobilis, compared with the wild-type strain. The ice nucleation activity in Z. mobilis cultures showed unusual partitioning in that the culture supernatants obtained after low-speed centrifugation contained the majority of ice nuclei. Analysis of the ice nucleation spectra revealed that the cell pellets contained both "warm" and "cold" nuclei, while the culture supernatant contained primarily cold nuclei, suggesting that the cold nucleus activity may be extracellular. However, all nucleation activity was retained by 0.22-(mu)m-pore-size filters.