Phylogenetic Studies of Pasteuria penetrans Looking at the Evolutionary History of Housekeeping Genes and Collagen-like Motif Sequences.

Abstract

Pasteuria penetrans, a gram-positive, endospore-forming eubacterium, is an obligate parasite of the root-knot nematodes, Meloidogyne spp. These nematodes are common root pests of economically important crop plants, making P. penetrans a potential biocontrol agent. Currently, the genome of the bacterium is being sequenced from spores and the data recovered utilized to better understand its mechanisms of parasitism.

Phylogenic analysis, at the protein level, has been done on a total of thirty-three bacterial species using the concatenation of forty housekeeping genes, two subsets of these involving twenty-eight taxa and twenty-seven genes, with and without indels removed, and each gene individually. Through the application of maximum likelihood, maximum parsimony, and Bayesian methods on these datasets, P. penetrans is found to cluster tightly within the class Bacilli of the gram-positive, low G+C content eubacteria with a high level of confidence, excluding seven gene trees. The exact placement of the bacterium as ancestral to Bacillus spp. has been resolved. Surprisingly, it is more closely related to the saprophytic extremophile Bacillus haladurans and B. subtilis than to the parasitic B. anthracis and B. cerus. These findings facilitate the phylogenetic context of Pasteuria to be exploited through further research in comparative genomics towards the development of biocontrol strategies.

Collagen-like genes, recently discovered in bacteria, are predicted to be virulence factors in Bacillus anthracis and B. cereus, close relatives to Pasteuria penetrans. These genes are identified by a GXY triple helix repeat motif that has only been found in the parasitic Bacillus spp., namely the above two species and B. thuringenisis. They have, however, been studied in depth in higher eukaryotes, such as humans, leading to assumptions about the content and structural stability of the triple helix GXY-repeats. As genomes are sequenced and in silico analyses deem possible, the true nature of collagen-like repeats have come to light along with their presence in other types of organisms, ranging from fungi to bacteria. Taking this approach to study Pasteuria penetrans for the possible role in virulence of collagen-like genes, more information has been revealed about the true characteristics and evolution of these motifs in bacteria, vertebrates, invertebrates, and fungi.

The percentage of each amino acid in the X and Y position of the GXY-repeat motif of 85 vertebrate, 228 invertebrate, 17 fungus, and 76 bacterial sequences, including 21 Pasteuria penetrans and 45 Bacilli, were examined. In the X-position, the following amino acids were preferred by each group: proline and then alanine for Bacteria; proline and then threonine, alanine, and cysteine for P. penetrans; proline and then alanine and glutamine for invertebrates and vertebrates; and surprisingly, glycine and alanine with proline as a second choice for fungi. In the Y-position were the following results: Bacteria utilize threonine then glycine and alanine; P. penetrans uses alanine, glycine, proline, and then threonine; Invertebrates choose proline followed by alanine; Vertebrates prefer proline then alanine and arginine; and Fungi use glycine, proline then serine and alanine.

Maximum Parsimony Analyses were performed on the collagen genes due to the heterogeneity in sites seen in the evolution across kingdoms. P. penetrans sequences were first paired with each of the other groups, including the other bacteria, invertebrates, vertebrates, and fungi. Then, a full phylogenetic tree was done on 15 P. penetrans, 40 other bacteria, 61 invertebrates, 16 vertebrates, and 17 fungi GXY-repeat motifs. These results strengthened the similarity of Xaa and Yaa amino acid usage within groups, except for the fungi which paired closest to invertebrates. The tree also shows a pattern of evolution from fungi, invertebrates, vertebrates, to P. penetrans and other bacteria, mainly Bacilli, which cluster adjacent to one another.