TABLE 1.

Genetic tools used so far in the identification of adaptation and probiotic factors of lactobacilli

Molecular tool	Description	Potential use	Pitfalls	Key reference(s)
Dedicated mutant analysis	Targeted gene mutation by gene deletion or insertional inactivation	Identification of cause-consequence relationships linking genes and their functions; allows in vivo confirmation of proposed functions; allows studies on the role of surface components in their native conformation	Requires a hypothesis from which to start (“bottom-up” approach); requires that a strain is genetically accessible; pleiotropic effects of a certain mutation can occur, complicating cause-consequence relationships	53, 97a
In silico analysis	Algorithm-based analysis of sequences	Mining for yet-undocumented genetic elements and interactions; predictions on evolution	Requires the availability of the genome sequence; assigned functions are putative and need to be confirmed experimentally; erroneously annotated sequences can easily be spread	4, 39, 50, 128, 196
CGH	DNA-DNA hybridization-based comparison of sequences	Enables identification of unique sequences in the reference strain	Gives information only about genes present in the reference strain; cross-hybridization of similar sequences is possible, complicating interpretation	67, 172
DNA microarray for transcription profiling	DNA-cDNA hybridization-based method for analyzing transcription	Provides a global view of transcription under specific conditions	Requires genome sequence; depends on the annotation of open reading frames for a classical microarray (not for a tiling array); only a “snapshot” view of transcription; difficult to obtain sufficient RNA from in vivo samples; good analytical tools are needed; role of identified genes needs to be confirmed by downstream analyses	34, 66, 67, 266, 277
Proteomics	Large-scale analysis of protein profiles (gel or non-gel based)	Proteins are most directly related to functions of the cell; can reveal posttranslational modifications	Identification of proteins requires mass spectrometry facility; coverage is mostly only partial; difficult to obtain sufficient protein from in vivo samples; downstream analyses are needed	141, 149
IVET and R-IVET	Promoter-trap technique that allows selection of active promoters in vivo	Allows in vivo identification of putative adaptation factors; with R-IVET, weak and transiently expressed genes can be identified	Requires a genetically accessible strain; only promoter activities are detected; downstream analyses are needed by, e.g., construction of knockout mutants	31, 269
DD-PCR	Based on PCRs of reverse transcribed RNA (cDNA) with random primers	Availability of genome sequence or special genetic tools is not required	Overrepresentation of structural RNA in total RNA can result in false positives; isolation of bacterial RNA from in vivo samples is difficult	130

^aSee Tables 2 to 5.