A large scale analysis of protein-protein interactions in the nitrogen-fixing bacterium Mesorhizobium loti

Abstract

Global viewing of protein-protein interactions (PPIs) is a useful way to assign biological roles to large numbers of proteins predicted by complete genome sequence. Here, we systematically analyzed PPIs in the nitrogen-fixing soil bacterium Mesorhizobium loti using a modified high-throughput yeast two-hybrid system. The aims of this study are primarily on the providing functional clues to M. loti proteins that are relevant to symbiotic nitrogen fixation and conserved in other rhizobium species, especially proteins with regulatory functions and unannotated proteins. By the screening of 1542 genes as bait, 3121 independent interactions involving 1804 proteins (24% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure and the general features of the interacting partners. Most PPIs detected in this study are novel interactions revealing potential functional relationships between genes for symbiotic nitrogen fixation and signal transduction. Furthermore, we have predicted the putative functions of unannotated proteins through their interactions with known proteins. The results described here represent new insight into protein network of M. loti and provide useful experimental clues to elucidate the biological function of rhizobial genes that can not be assigned directly from their genomic sequence.

Figure 1

Flow chart of the sequential steps in the YTH analysis of M. loti (see Materials and Methods for details). BD and AD indicate the GAL4 DNA-binding domain and GAL4 activation domain, respectively.

Figure 2

Global view of the PPIs of M. loti. (A) All detected PPIs. Proteins (circles) are color-coded according to their functional category assigned by Kaneko et al.¹ Interactions (lines) are colored according to the interaction category (A–D), which is classified based on their frequency of detection of identical pairs. (B) Number of identified interactions in each function category. The white bar indicates the total number of genes in each functional category assigned in the M. loti genome and the black bar indicates the number of genes shown to have interactions. The red bar indicates the number of genes used as bait in the screening. Percentages represent the coverage of interacting proteins in each function category.

Figure 3

Interaction pairs of two-component signal transducers. Sensor HK and RR are shown by the blue and the orange boxes, respectively. Boxes marked with a red line indicate the interactions between HK and RR that are encoded by the same operon. The arrow in each interaction indicates the direction of bait protein to prey protein and the reliability of each interaction. HK and RR are designated according to the classification described in Hagiwara et al.³⁸ hHK, hybrid sensor HK; CheA, CheA-type HK; RR(c), NtrC-family RR; RR(l), NarL-family RR; RR(r), OmpR-family RR; RR(y), CheY-family RR; RR(y), unclassified RR.