1012 low amounts of positive hits yet give the guarantee that the gene is functional in the metagenome host. However, a prob- lem is that the target enzymes are only expressed intracellu- larly in the metagenome host and the cell membrane might not necessarily be permeable to the indicator substances present in the medium. Hence, secretion of the gene product is necessary for detection. Moreover, other problems can arise, such as enzymatic breakdown or intracellular product accumulation (Sørensen and Mortensen 2005), which can result in toxicity. Forced cell lysis may hold the solution, as it may bring the indicator substance into contact with the intracellular target proteins (Bao et al. 2011). However, care should be taken to not denature the expressed proteins by the lysing agents since otherwise protein activity as well as its interaction with the indicator substrate might be lost. Furthermore, mechani- cal cell disruption can be time-consuming and labor- intensive. A possible way of avoiding this is to use an autolytic vector. Li et al. (2007) developed a UV- inducible autolytic vector for use in high-throughput screenings. An SRRz lysis gene cassete was inserted downstream of a UV-inducible promotor in E. coli. Cell lysis efficiency was tested by expressing β-galactosidase in E. coli prior to UV induction. After UV-induced lysis, extracellular (supernatant) β-galactosidase activity was compared to the total of intracellular (pellet) and extracellular β-galactosidase activity to quantify lysis efficiency. A lysis effiencency of 60% or more was observed at a temperature of 30 °C. This is comparable to conventional lysozyme treat- ment. However, at 37 °C, the lysis rate was less consistent. Thus, use of such an autolytic vector might provide a simple alternative to existing lysis techniques (Li et al. 2007). In addition to the beta-galactosidase-based screen, several other chromogenic and fluorogenic reporter techniques were proven to be efficient in the identification of the activities of enzymes encoded by the inserted gene(s). LeCleir et al. (2007) thus showed the presence of chitinolytic enzymes in an estuarine metagenomic library by cleavage of fluorogenic analogs of chitin. MD MD does not rely on the direct detection of an expressed gene, but it uses a predesigned expression route. By modulating the expression host and/or vector systems, selection and detection of inserted genes can be manipulated, for instance by the coexpression of reporter genes or heterologous comple- mentation. This results in more specific screenings and standardized detectable signals. Reporter genes The use of reporter genes is a suitable method for high-throughput screenings. The lacZ gene encoding beta-galactosidase (resulting in colony coloring upon growth on X-Gal-containing medium) is frequently Appl Microbiol Biotechnol (2012) 93:1005–1020 used as a reporter gene. In an experiment to screen for metagenomic clones containing genes that interfere with quorum sensing (QS), this reporter gene was used. Screening was achieved by measuring the potential degradation of the QS signaling molecules. An A. tumefaciens strain containing a traI-lacZ gene fusion was used in the screening. By inducing the traI gene with the QS signal molecule homoserine lactone 3-oxo-C8-HSL, lacZ is activated. This results in beta-galactosidase production, yielding blue colonies. If, however, 3-oxo-C8-HSL is broken down by the host, lacZ induction is inhibited and no blue color appears. This would be an indication of a quorum sensing-inhibitory or degradation activity of the clone. The experiment yielded 438 positive clones showing QS inhibition (Schipper et al. 2009). A great advantage of this approach is that detection of positive clones does not rely on successful expression of a gene product downstream of transcription. Nor does a pos- sibly faint expression of the inserted gene hamper detection, as it might in other detection methods. This can be of great benefit when searching for genes that might be hard to express in the host. Heterologous complementation (HC) Heterologous com- plementation (HC) relies on exploring foreign genes to achieve genetic complementation in the host, resulting in the expression of a gene product that is vital for growth under selective conditions. The technique allows for great selectivity and, thereby, a screen can be precisely directed to search for specific genes (Kellner et al. 2011; Simon et al. 2009). An example is presented by Simon et al. (2009) who screened metagenomic plasmid and fosmid libraries derived from glacial ice for DNA polymerase encoding genes. This was achieved by using an E. coli strain that carries a cold-sensitive mutation in the 5′–3′ exonuclease domain of DNA polymerase I, which is lethal at temperatures below 20 °C. By growing the clones on antibiotic-containing plates compatible with vector resistance, at a temperature of 18 °C, positive clones can be identified that complemented the lethal mutation. Using this approach, 17 plasmids and 1 fosmid with the desired phenotype were retrieved from the clone library. Sequence analysis of nine positive clones indi- cated that indeed DNA polymerase genes had been isolated from the library. Taking into account the conserved nature of DNA polymerase genes and the degree of homology to known DNA polymerase genes, this result led to the conclusion that the genes had been recovered from as-yet-unexplored microorganisms (Simon et al. 2009). Another example of HC is provided by a study that attempted to isolate novel lysine racemase genes from a metagenome (Chen et al. 2009). An E. coli strain carrying a lysine auxotrophy mutation was used to screen for the aforementioned genes in a metagenomic library derived from garden soil. Clones were grown on D-lysine-supplemented