Genomic insights into the thiamin metabolism of Paenibacillus thiaminolyticus NRRL B-4156 and P. apiarius NRRL B-23460

Sannino and Angert Standards in Genomic Sciences Genomic insights into the thiamin metabolism of Paenibacillus thiaminolyticus NRRL B-4156 and P. apiarius NRRL B-23460 David Sannino 0 Esther R. Angert 0 0 Cornell University , Ithaca, NY , USA Paenibacillus thiaminolyticus is the model organism for studying thiaminase I, an enigmatic extracellular enzyme. Originally isolated from the feces of clinical patients suffering from thiamin deficiency, P. thiaminolyticus has been implicated in thiamin deficiencies in humans and other animals due to its ability to produce this thiamindegrading enzyme. Its close relative, P. apiarius, also produces thiaminase I and was originally isolated from dead honeybee larvae, though it has not been reported to be a honeybee pathogen. We generated draft genomes of the type strains of both species, P. thiaminolyticus NRRL B-4156 and P. apiarius NRRL B-23460, to deeply explore potential routes of thiamin metabolism. We discovered that the thiaminase I gene is located in a highly conserved operon with thiamin biosynthesis and salvage genes, as well as genes involved in the biosynthesis of the antibiotic bacimethrin. Based on metabolic pathway predictions, P. apiarius NRRL B-23460 has the genomic capacity to synthesize thiamin de novo using a pathway that is rarely seen in bacteria, but P. thiaminolyticus NRRL B-4156 is a thiamin auxotroph. Both genomes encode importers for thiamin and the pyrimidine moiety of thiamin, as well as enzymes to synthesize thiamin from pyrimidine and thiazole. Thiaminase I; Paenibacillus thiaminolyticus; Paenibacillus apiarius; Paenibacillus dendritiformis; Thiamin; Hydroxymethyl pyrimidine Introduction Prior to World War II, beriberi and other vitamin deficiencies were prevalent in Japan and linked to a diet composed almost entirely of polished rice [ 1 ]. Additionally, it was discovered that certain fish and shellfish contained no thiamin and moreover any thiamin added to these raw foodstuffs was quickly destroyed [ 2 ]. While investigating potential links between the intestinal microbiota and beriberi, Shibata and colleagues found that when thiamin was added to feces or infused in the colon of patients suffering thiamin deficiency, the added thiamin disappeared [ 2, 3 ]. The thiaminase enzyme responsible for the destruction of thiamin in feces and in animal tissues was discovered shortly thereafter. Several bacteria, including Paenibacillus thiaminolyticus, were isolated by Matsukawa and Misawa from patient fecal samples with thiaminase activity [2]. The discovery of thiaminase producing bacteria facilitated extensive research efforts to understand the biochemistry of thiaminase and the biology of P. thiaminolyticus [ 4 ]. Paenibacillus thiaminolyticus became a model system for studying the secreted bacterial thiaminase now known as thiaminase I [ 5–10 ]. Thiaminase I catalyzes the base substitution of the thiazole moiety of thiamin with numerous organic nucleophiles such as pyridine, quinolone, or compounds containing a sulfhydryl group, like cysteine [ 2, 10, 11 ]. Early studies of this extracellular enzyme found that thiaminase I activity is repressed when high concentrations of thiamin are added to cultures and culture supernatant [ 8, 9 ]. The crystal structure of P. thiaminolyticus thiaminase I revealed that the 42 kDa protein has a catalytic cysteine residue and the protein is structurally similar to the group II periplasmic binding proteins, particularly the thiaminbinding protein TbpA in E. coli [ 12 ]. We recently found that Paenibacillus apiarius also has thiaminase I activity (unpublished). This close relative of P. thiaminolyticus was originally isolated from the larvae of dead honeybees, although it was not the causative agent of their death [ 13 ]. Despite the extensive biochemical and mechanistic understanding of the enzyme, the biological function and context in which P. apiarius, P. thiaminolyticus and other thiaminase I producers use thiaminase I remains a mystery [ 14 ]. Although thiaminase I activity is found in plants such as bracken fern [ 15 ] and nardoo [ 16 ], as well as in animals such as crustaceans, ruminants, and fish, the only confirmed producers of thiaminase I are microbial, including one eukaryote, the amoeba Naegleria gruberi [ 15, 17, 18 ]. Thiaminase I activity in food contributes to thiamin deficiency in animals and is implicated in Early Mortality Syndrome in salmonids in the Great Lakes and Baltic Sea [ 18 ]. A link between P. thiaminolyticus and this thiamin deficiency syndrome has been suggested, as P. thiaminolyticus has been isolated from the viscera of alewife, a fish with high thiaminase activity that is a food source for Great Lakes salmonids. Additionally, it was demonstrated that Early Mortality Syndrome could be induced in lake trout fed an experimental diet supplemented with P. thiaminolyticus [ 18, 19 ]. As with humans, P. thiaminolyticus is not always isolated fro (...truncated)