Functional analysis of 1440 Escherichia coli genes using the combination of knock-out library and phenotype microarrays
Introduction
Recent advance in the field of microbial genomics seems to open the way to comprehensive elucidation of the life of microbes. However, we have been still confronting functionally unknown genes. Even in smaller genomes, genes were reported as unknown to the extents of 16%, 26%, 12%, and 14% for Wigglesworthia glossinidia (698 kbp), Tropheryma whipplei TW08/27 (926 kbp), Blochmannia floridanus (706 kbp), and Buchnera sp. APS (641 kbp), respectively (Akman et al., 2002; Bentley et al., 2003; Gil et al., 2003; Shigenobu et al., 2000). Though Mycoplasma genitalium G-37 is thought to be the self-replicating organism with the smallest genome of 580 kbp, 20% of CDSs were reported to have no matches to protein sequences from the database (Fraser et al., 1995). We can still recognize unknown genes in the genome of M. genitalium G-37 almost to the same extent even in the case that we use the ERGO database constructed with the recent knowledge (Overbeek et al., 2003). Last year genome sequence of Mycoplasma hyopneumoniae strain 232 was reported (Minion et al., 2004). The 893-kbp genome of the bacterium was found to include functionally unknown genes up to 56% of its CDSs. It is still difficult to comprehensively understand whole content of genes in a bacterium even in the case of one with the genome smaller than 1 Mbp. Furthermore about 40% of Escherichia coli genes have not been assigned to their function yet, though E. coli is one of the best elucidated organisms. It is still difficult to elucidate function of so-called y-genes having no assigned function.
A new technology for phenotypic analysis was developed by Bochner and colleagues (Bochner et al., 2001) and called as Phenotype MicroArrays (PM). PM technology was designed to test a large number of cellular phenotypes simultaneously. Nearly 2000 phenotypic tests can be performed in wells of 20 96-well microplates. Each well contained a particular chemical and tetrazolium violet and develops a purple color quantitatively according to the respiration of inoculated cells. Panoramic view of the color change was reported to be quantitative and highly reproductive. PM technology was already applied to analyze E. coli mutants with deletions of all two-component systems (Zhou et al., 2003). In the analysis, many phenotypes were shown to be as expected, and several new phenotypes were also revealed. PM technology would be useful for phenotypic analysis of knock-out mutants. On the other hand, Blattner and his coworkers have been constructing a set of mutant strains of MG1655, designated as Sce-poson mutants, and analyzing them with PM plates. Current number of mutants for individual CDSs is about 2000 (Kang et al., 2004; http://www.genome.wisc.edu/functional/tnmutagenesis.htm). Part of the dataset of PM analysis was used to assist the construction of in silico E. coli strain (Covert et al., 2004). However, clustering of mutant phenotypes has not been studied.
Independent from the Sce-poson mutants, another library of gene knock-out mutants has been established in Japan and designated as the KO library (Mori, 2004). It consists of more than 4000 gene knock-outs. We have been analyzing phenotypic changes of the KO library using the GN2-MicroPlate™ (GN2) that is a part of PM plates (Biolog Inc., CA). The GN2 plate can qualify 95 carbon-source utilizations of a particular mutant simultaneously. First we selected 1440 strains, 48% of which are knock-outs of poorly characterized genes in the COGs database. The raw dataset of GN2 analysis was processed by the GeneSpring™ software (Silicon Genetics, CA) in order to find clusters of interrelated genes that showed similar phenotypic changes when they were deleted. In the resulting dendrogram of genes, genes of known and related function tended to be assembled into a cluster. This result indicated that our clustering method would be useful for functional assignment of y-genes, since y-genes could be connected to phenotype and function of well-known genes in the same cluster.
Section snippets
Strain
E. coli BW25113 [lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78] was used as the parent for constructing in-frame null mutants for 1440 open reading frames (Datsenko and Wanner, 2000). Gene disruptants were constructed systematically by replacing a target gene by PCR-amplified kanamycin resistance gene with short homologous regions to the target at both ends (Datsenko and Wanner, 2000; Mori, 2004).
Phenotype analysis using the GN2-MicroPlate
The GN2-MicroPlate (Biolog Inc., CA) was used for the phenotypic analysis of 1440 mutants
Used gene knock-out mutants
We used 1440 knock-out mutants that are the part of the KO library. In the library, the target gene was replaced by kanamycin resistance gene (kan) to make null mutants (Mori, 2004). Content of the deleted genes are summarized in Table 1 according to their function described in the COGs database (http://www.ncbi.nlm.nih.gov/COG/new/) and the GenoBase (http://ecoli.aist-nara.ac.jp/GB5/search.jsp/). These mutants included genes of energy production process, regulation, and especially poorly
Acknowledgments
We thank Natsuka Shimodate for her excellent technical assistance. This study was carried out as a part of The Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers by Ministry of Economy, Trade & Industry (METI), and supported by New Energy and Industrial Technology Development Organization (NEDO).
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