Unusual pattern of bacterial ice nucleation gene evolution.

Abstract

Bacterial ice nucleation activity (INA+ phenotype) can be traced to the product of a single gene, ina. A remarkably sparse distribution of this phenotype within three bacterial genera indicates that the ina gene may have followed an unusual evolutionary path. Southern blot analyses, coupled with assays for ice-nucleating ability, revealed that within four bacterial species an ina gene is present in some strains but absent from others. Results of hybridization experiments using DNA fragments that flank the ina gene suggested that the genotypic dimorphism of ina may be anomalous. A phylogenetic analysis of 16s ribosomal RNA gene sequences from a total of 14 ina+ and inabacterial strains indicated that the ina+ bacteria are not monophyletic but instead phylogenetically interspersed among ina-bacteria. The relationships of ina+ bacteria inferred from ina sequence did not coincide with those inferred from the 16s data. These results suggest the possibility of horizontal transfer in the evolution of bacterial ina genes.

Comprehensive characterization of an aspen (<i>Populus tremuloides</i>) leaf litter sample that maintained ice nucleation activity for 48 years

“…The suspensions and 10 −1 and 10 −2 dilutions described above were plated on R2A and LEM (Lysinibacillus enrichment medium). LEM is based on a medium originally developed for Lysinibacillus sphaericus (Russell et al, 1989) to enrich for Lysinibacillus strains similar to the Lysinibacillus strain with INA that we recently isolated (Failor et al, 2017). LEM contains, per liter, Na 2 HPO 4 , 5.57 g; KH 2 PO 4 , 2.4 g; (NH 4 ) 2 SO 4 , 2.0 g; MgSO 4 • 7H 2 O, 50 mg; MnCl 2 • 4H 2 O, 4.0 mg; FeSO 4 •7H 2 O, 810 µg; CaCl 2 •2H 2 O, 1.5 mg; H 2 SO 4 , 0.3 µL; sodium acetate, 321.8 mg; thiamine 200 mg; and biotin, 20 µg.…”

“…Unfortunately, the P. syringae and the two Pa. ananatis strains isolated from the leaf litter were lost and could not be further characterized. However, we characterized the cumulative ice nucleation spectrum of M. alpina LL118 and of Pa. ananatis strain BAV 3057, which was previously isolated from rain (Failor et al, 2017) and that had over 99 % identity in its 16S rRNA sequence to one of the two Pa. ananatis strains from leaf litter. Because of the genetic similarity of Pa. ananatis BAV 3057 to the two Pa. ananatis strains isolated from the leaf litter, these strains can be expected to have very similar cumulative ice nucleation spectra.…”

“…Because of its ubiquitous presence in compartments of the water cycle and other circumstantial evidence, P. syringae might contribute to the formation of precipitation in clouds (Morris et al, 2013). Other bacteria with INA have been identified in additional Pseudomonas species (Failor et al, 2017) and in additional genera within the Gammaproteobacteria: Erwinia (Phelps et al, 1986), Pantoea (Failor et al, 2017), a genus that includes several species classified in the past as members of the genus Erwinia, and Xanthomonas (Kim et al, 1987). A strain belonging to the genus Lysinibacillus was recently identified and characterized as the first ice-nucleating Gram-positive bacterium (Failor et al, 2017).…”

“…Other bacteria with INA have been identified in additional Pseudomonas species (Failor et al, 2017) and in additional genera within the Gammaproteobacteria: Erwinia (Phelps et al, 1986), Pantoea (Failor et al, 2017), a genus that includes several species classified in the past as members of the genus Erwinia, and Xanthomonas (Kim et al, 1987). A strain belonging to the genus Lysinibacillus was recently identified and characterized as the first ice-nucleating Gram-positive bacterium (Failor et al, 2017). Some fungal species have been found to have INA as well: Fusarium acuminatum and F. avenaceum (Pouleur et al, 1992), F. tricinctum and F. oxysporum (Richard et al, 1996), F. sporotrichioides (Huffman et al, 2013), Mortierella alpina (Fröhlich-Nowoisky et al, 2015), Isaria farinosa, and Acremonium implicatum (Huffman et al, 2013).…”

“…The INPs produced by the INA isolate of the genus Lysinibacillus are moreover very heat stable (remaining partially active even after an hour of boiling), are proteinase resistant, and do not pass through a 100 kDa protein filter with a pore size of approximately 50 nm. This suggests that they consist of relatively large nonproteinaceous secreted single molecules or stable aggregates of multiple smaller molecules (Failor et al, 2017). Fungal INPs are also mostly retained by 100 kDa filters, suggesting that they also consist of large secreted single molecules or of large aggregates of smaller molecules O'Sullivan et al, 2015).…”