Sam McCarthy, Caitlin Olmstead & Ryan Williams

1. A project of the and the Center for Deciphering Life’s Languages at Hiram College, in collaboration with undergraduates at 10 other colleges and universities as part of the Undergraduate Research in Microbial Genome Annotation project of the U.S. Dept. of Energy Joint Genome Institute. Detailed Bioinformatics Analysis of the Branched Chain Amino Acid Biosynthesis Pathway in Planctomyces limnophilus Sam McCarthy, Caitlin Olmstead & Ryan Williams • Within our entire pathway, there were three separate pathways that formed the entire Branched Chain Amino Acid Biosynthesis Pathway in the organism P. limnophilus. The first pathway was Valine synthesis and had 6 steps, with step one having two alternate genes and step 6 having three alternate genes. The second pathway was for Leucine synthesis and had within it’s pathway a total of five steps. The third pathway, Isoleucine synthesis, had 5 complete steps, with the final step having two alternates. There was a total of ten proteins involved in the entire branched chain amino acid biosynthesis pathway. • We did find all of the genes within out pathway, with the exception of three. One gene, with the EC number 2.6.1.66, was not confidentaly identified because this gene, which encodes the enzyme Alanine-alpha-ketoisovalerate aminotransferase, was found only in an organsim unrelated to P. limnophilus and had a very low percent identity. The other two genes, one with an EC number of 1.4.1.9 and the other with an unknown identity, were also not found. However, all three of these genes offer an alternate enzyme to the pathway, so they are not needed to undergo branched chain amino acid biosynthesis and may not even be present in P. limnophilus. • Gene 2500605348’s start codon had to be changed dramatically. It was moved about 200 base pairs downstream. This put it in perfect or near perfect alignment with its best BLAST hits. • Genes 2500605348 and 2500605350 appear to be in an operon. This makes biological sense because they encode the large and small subunits of the same enzyme, 3-isopropylmalate dehydratase, respectively. Really interesting diagram or figure of your pathway or process (make sure you add a figure legend) The position of gene 2500605348 is shown in red. Directly to the right of it is the gene hypothetical protein conserved in cyanobacteria, followed by gene 2500605350. The close proximity of these genes going in the same direction indicates that they are part of an operon. • Genes 2500605348 and 2500605350 both have many of their best BLAST hits with bacteria in the Cyanobacteria family (order?). Further evidence for this can be seen in the proposed operon mentioned previously. A gene for an uncharacterized protein conserved in Cyanobacteria is located in between the other two genes (see figure 2). • It was evident when first looking at our genes that some enzymes were used in more than one pathway. It was fascinating to find that an enzyme coded from one gene could have two completely different substrates and from that could yield two completely different products. The five best BLAST hits to gene 2500605350 are all to organisms unrelated to P. limnophilus, indicating a lateral gene transfer >637876418 YP_478232 CYB_2021 3-isopropylmalate dehydratase, small subunit [Synechococcus sp. JA-2-3Ba(2-13)] >637313855 NP_682024 tlr1234 isopropylmalate isomerase small subunit [Thermosynechoccus elongatus BP-1] >637011173 NP_441618 sll1444 isopropylmalate isomerase small subunit [Synechocystis sp. PCC 6803] >637873736 YP_475604 CYA_2201 3-isopropylmalate dehydratase, small subunit [Synechococcus sp. JA-3-3Ab] >637460828 NP_926364 glr3418 isopropylmalate isomerase small subunit [Gloeobacter violaceus PCC 7421]