Introduction

A vast quantity of microorganisms is present on our planet. Researchers have estimated that the total number of microorganisms stands at 4 to 6 × 10³⁰ cells (1) and that there are approximately 10⁸ different species (2). This diversity represents a huge genetic and biological resource that has been exploited for the recovery of useful genes, metabolic pathways, and their products (3). Traditionally, screening of natural microorganisms has been carried out based on cultivation and isolation techniques; however, only a tiny fraction of natural microorganisms can be cultured by conventional microbiological techniques (2). Screening of environmental metagenomes without isolating individual microorganisms has recently been recognized as an attractive approach to mining novel genetic resources in the natural environment (3,4,5,6,7).

Currently, three approaches are available for screening of environmental metagenomes for isolating novel genes: (i) function-based screening; (ii) nucleotide sequence-based screening; and (iii) gene expression-based screening. In the first approach, a shotgun metagenomic library is constructed and used for screening for acquired phenotypes expressed by a cloning host (8,9,10). The second strategy is based on conserved nucleotide sequences from which hybridization probes and/or PCR primers are designed to detect and amplify target genes (11,12,13,14). In addition to these approaches, we have recently proposed a third option, called substrate-induced gene expression screening (SIGEX) (7). This method is based on the knowledge that catabolicgene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes.

It is important to realize that each approach has its intrinsic advantages and disadvantages. For example, the second and third approaches are considered to be less laborious than the first approach, although they may fail to clone complete genes necessary for expressing their functions. This is particularly true for PCR-mediated methods that utilize primers designed from inner conserved sequences. It is therefore necessary to combine these approaches with methods for walking across flanking genomic regions.

Several PCR-based methods are available for genome walking, including adaptor-ligated PCR (15), randomly primed PCR (16), suppression PCR (17), and inverse PCR (18). These methods are potentially applicable to walking across genome fragments in an environmental metagenome (12), but so far, success has been limited due to inefficient amplification from genome fragments at low copy numbers. For instance, in previous work, we have tried to apply inverse PCR for recovering flanking regions of SIGEX-derived fragments (obtained in Reference 7) from an original ground-water metagenome. These trials were, however, unsuccessful, probably because the copy number of the target fragment was too low to apply inverse PCR (our unpublished results). Here we describe an improved inverse PCR scheme (inverse affinity nested PCR or IAN-PCR) that enables walking across a rare genomic fragment in an environmental metagenome. Its utility was demonstrated by fishing chitinase genes from a groundwater metagenome.

Materials and Methods

Bacterial Strains and Growth Conditions

Escherichia coli JM109 was grown in LB medium (19) at 37°C, while Ralstonia eutropha E2 was grown in dLB medium (1/10 diluted LB medium) (20) at 30°C. When necessary, ampicillin was added at 100 µg/mL, while kanamycin was added at 50 µg/mL.

Groundwater Sample and Collection of Microorganisms

Oil-contaminated groundwater was obtained from a sampling facility of the TK101 underground crude oil storage cavity at Kuji in Iwate, Japan. Characteristics of this groundwater have been reported previously (21). Microorganisms in the groundwater (460 L) were collected on a 0.22-m pore size membrane filter (Millipore, Billerica, MA, USA) by filtration within 5 h after sampling. Microorganisms were removed from the filter by washing it twice with 100 mL TE buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA) and collected by centrifugation at approximately 9000× g for 30 min at 4°C.

Purification, Digestion, and Self-Ligation of Metagenome Fragments

Genomic DNA was extracted from the collected microorganisms as described by Silhavy et al. (22). After 3 µg chromosomal DNA were digested with a restriction enzyme, 0.75 µg digested DNA was circularized by self-ligation in 50 µL reaction mixture at 16°C for 16 h. The reaction mixture was composed of 66 mM Tris-HCl, pH 7.5, 6.6 mM MgCl₂, 10 mM dithiothreitol (DTT), 0.1 mM ATP, and 1 U/mL T4 DNA ligase (Takara, Ohtsu, Japan). The self-ligated DNAs were purified using the QIAquick® PCR Purification kit (Qiagen, Valencia, CA, USA).