National Institute of Sericultural and Entomological Science, 1-2 Ohwashi, Tsukuba, lbaraki 305-8634, Japan

Background and objectives
Comparison of the sequence data of the ice nucleation (IN) genes revealed a moderate similarity in amino acid identity (60.6-65.0%) between the IN genes from different bacterial species [1]. By contrast, surprisingly, a very high similarity in amino acid identity (98.3%) was detected between InaA (Erwinia ananas, 3966 bp) and lceE (Erwinia herbicola, 3774 bp) [1]. In our preliminary study, we identified all INA 'herbicola-group bacteria' isolated from plants and insects as E. ananas but not E. herbicola. These findings suggest that all the INA strains of 'herbicola group bacteria', including those previously reported as E. herbicoia [2], may be E. ananas [3]. In addition, it was suggested that the size or structure of the IN genes distributed in the same bacterial species may be variable. To analyse the evolution or functions of IN genes, we carried out comparative studies of IN genes using a number of strains of E. herbicola-group bacteria.

Materials and methods
The structure of IN genes was compared among 20 strains of E. herbicola-group bacteria of plant and insect origin. The IN genes were amplified by PCR using several primer sets designed from sequence data of iceE and inaA. PCR was carried out under the following conditions: 1 min at 94C, 1 min at 55C, 1 min at 72C, 20 cycles to amplify the N-domain or C-domain; 1 min at 94C, 1 min at 47C, 3 min at 72C, 40 cycles to amplify the whole INA gene. For RFLP analysis of IN genes, PCR products were digested with several restriction enzymes. Southern blot hybridization was performed according to a standard method using PCR products amplified from the N-domain or C-domain of E. ananas IN10 or oligonucleotide probe [4]. IN activity was measured by the test tube method [3]. To identify E. ananas and E. herbicola among strains of 'herbicola-group bacteria', six bacterial properties by which E. ananas can be distinguished from E. herbicola [5] were investigated.

Results and conclusions
When the DNAs of N- or C-domains were amplified, PCR products of similar size were obtained in all the strains, respectively, while the size of the PCR products from the whole genes containing the R domain varied remarkably within a range of 3.8-4.4 kb. IN activities of 20 strains, however, were almost the same level. RFLP analysis of the IN genes revealed that the sizes of the R domains varied within the region from the PvuII site to the DraI site. Based on the restriction maps of the R-domain, 20 IN genes were classified into 12 groups designated as group I to group XII. Furthermore, all the strains were identified as E. ananas based on six bacteriological tests differentiating E. herbicola. These results suggest that the IN genes may be distributed in only E. ananas strains among 'herbicola group bacteria'.

1. Lee RE Jr, Warren GJ, Gusta LV, eds, 1995. Biological Ice Nucleation and its Applications. American Phytopathological Society, St Paul, Minnesota, pp. 85-99.
2. Warren GJ, Corotto L, 1989. Gene 85, 239-242.
3. Abe K, Watabe S, Emori Y et al., 1989. FEBS Letters 258, 297-300.
4. Arai S, Abe K, Watabe S et al., 1989. FEMS Microbiology Letters 61, 53-56.
5. Dye DW, 1983. in Fahy PC, Persley GJ, eds, Plant Bacterial Diseases, pp. 67-86.