Synergistic use of plant-prokaryote comparative genomics for functional annotations

Abstract

Background: Identifying functions for all gene products in all sequenced organisms is a central challenge of the post-genomic era. However, at least 30-50% of the proteins encoded by any given genome are of unknown or vaguely known function, and a large number are wrongly annotated. Many of these 'unknown' proteins are common to prokaryotes and plants. We set out to predict and experimentally test the functions of such proteins. Our approach to functional prediction integrates comparative genomics based mainly on microbial genomes with functional genomic data from model microorganisms and post-genomic data from plants. This approach bridges the gap between automated homology-based annotations and the classical gene discovery efforts of experimentalists, and is more powerful than purely computational approaches to identifying gene-function associations.

Results: Among Arabidopsis genes, we focused on those (2,325 in total) that (i) are unique or belong to families with no more than three members, (ii) occur in prokaryotes, and (iii) have unknown or poorly known functions. Computer-assisted selection of promising targets for deeper analysis was based on homology-independent characteristics associated in the SEED database with the prokaryotic members of each family. In-depth comparative genomic analysis was performed for 360 top candidate families. From this pool, 78 families were connected to general areas of metabolism and, of these families, specific functional predictions were made for 41. Twenty-one predicted functions have been experimentally tested or are currently under investigation by our group in at least one prokaryotic organism (nine of them have been validated, four invalidated, and eight are in progress). Ten additional predictions have been independently validated by other groups. Discovering the function of very widespread but hitherto enigmatic proteins such as the YrdC or YgfZ families illustrates the power of our approach.

Conclusions: Our approach correctly predicted functions for 19 uncharacterized protein families from plants and prokaryotes; none of these functions had previously been correctly predicted by computational methods. The resulting annotations could be propagated with confidence to over six thousand homologous proteins encoded in over 900 bacterial, archaeal, and eukaryotic genomes currently available in public databases.

Figure 1

Project workflow. The overall strategy that combined in silico and experimental validation is presented showing the number of genes that were analyzed at each stage.

Figure 2

Clustering arrangements of genes encoding COG0354 and functional complementation of an E. coli COG0354 deletant by an Arabidopsis COG0354. (A) Clustering of COG0354 genes with Fe/S-related genes. Blue, COG0354; red, Fe/S proteins; rose, proteins in same complex or pathway as Fe/S proteins; turquoise, Fe/S cluster assembly proteins. Rx, Rubrobacter xylanophilus; Sm, Stenotrophomonas maltophilia; Pu, Pelagibacter ubique. (B) Growth of an E. coli COG0354 (ygfZ) deletant harboring plasmid-borne E. coli ygfZ, Arabidopsis mitochondrial COG0354, or vector alone on LB medium or LB plus the oxidative stress agent plumbagin (OX) (30 μM), arabinose (0.02% w/v), and appropriate antibiotics.

Figure 3

COG3643 in relation to the Hut pathway. (A) Hut pathway; note the three different routes. (B) The distribution of histidine utilization genes among bacterial and eukaryal genomes in relation to that of the ygfA gene for 5-formyltetrahydrofolate disposal. Gene colors correspond to different parts of the pathway as in part A. Lines between boxes denote gene fusions. (C) Growth of an E. coli ygfA deletant harboring plasmid-borne E. coli ygfA, Acidobacterium COG3643, or vector alone on minimal medium with NH₄Cl or glycine as sole nitrogen source. The medium contained 1 mM IPTG and appropriate antibiotics.

Figure 4

Separation of the COG009 family into two subgroups YrdC and YciO based on motifs and functional assays. (A) Complementation of the t⁶A^- phenotype of the yeast Δsua5 (YGN63) strain by the E. coli yrdC gene but not the E. coli yciO gene. (B) Complementation of the yrdC essentiality phenotype in E. coli by yrdC subfamily members from E .coli (EcyrdC), Bacillus subtilis (BsywlC), Methanococcus maripaludis (MmyrdC) and yeast (Scsua5) but not by yciO from E. coli (EcyciO). All genes were cloned in pBAD24 [95] and were therefore expressed in the presence of arabinose (Ara, 0.2%) and transformed in an E.coli strain carrying the chromosomal copy of yrdC under P_Tet control [96] that does not grow in the absence of anhydrotetracycline (ATc, 50 ng/ml). (C) Signature motif of the functional homologs of YrdC (KxR/SxN) that are not found in the YciO subfamily. In green are the two homologs from Arabidopsis and their distribution.