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

Molecular toolDescriptionPotential usePitfallsKey reference(s)
Dedicated mutant analysisTargeted gene mutation by gene deletion or insertional inactivationIdentification 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 conformationRequires 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 relationships53, 97a
In silico analysisAlgorithm-based analysis of sequencesMining for yet-undocumented genetic elements and interactions; predictions on evolutionRequires the availability of the genome sequence; assigned functions are putative and need to be confirmed experimentally; erroneously annotated sequences can easily be spread4, 39, 50, 128, 196
CGHDNA-DNA hybridization-based comparison of sequencesEnables identification of unique sequences in the reference strainGives information only about genes present in the reference strain; cross-hybridization of similar sequences is possible, complicating interpretation67, 172
DNA microarray for transcription profilingDNA-cDNA hybridization-based method for analyzing transcriptionProvides a global view of transcription under specific conditionsRequires 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 analyses34, 66, 67, 266, 277
ProteomicsLarge-scale analysis of protein profiles (gel or non-gel based)Proteins are most directly related to functions of the cell; can reveal posttranslational modificationsIdentification of proteins requires mass spectrometry facility; coverage is mostly only partial; difficult to obtain sufficient protein from in vivo samples; downstream analyses are needed141, 149
IVET and R-IVETPromoter-trap technique that allows selection of active promoters in vivoAllows in vivo identification of putative adaptation factors; with R-IVET, weak and transiently expressed genes can be identifiedRequires a genetically accessible strain; only promoter activities are detected; downstream analyses are needed by, e.g., construction of knockout mutants31, 269
DD-PCRBased on PCRs of reverse transcribed RNA (cDNA) with random primersAvailability of genome sequence or special genetic tools is not requiredOverrepresentation of structural RNA in total RNA can result in false positives; isolation of bacterial RNA from in vivo samples is difficult130
  • a See Tables 2 to 5.