Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

Abstract

A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms

An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers

“…We identified 217 V. cholerae genes that are putatively induced during human infection (SI Tables 3 and 4). When categorized by functional roles (data not shown) the largest class of genes in the set (26%) encodes hypothetical and conserved hypothetical proteins, which is typical of most genomewide analyses, including IVET studies (1). Fifteen of these hypotheticals were isolated from at least three volunteers and therefore seem more likely to be induced in vivo than genes isolated from only one infection.…”

“…The next largest classes are of genes encoding transport and binding proteins and proteins involved in energy metabolism and amino acid biosynthesis. These classes also tend to be heavily represented in IVET screens in animal models, underscoring the key roles of metabolic processes in colonization and infection (1).…”

Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions

“…In vivo expression technology is now established as a method of analyzing bacterial gene expression in the rhizosphere as an approach for the identification of genes involved in microbe-plant interactions (22,23,29,35). One clear advantage of in vivo expression technology is that investigations are carried out in the appropriate environment in the organism of interest; there is no extrapolation from in vitro model systems.…”

Identification of Burkholderia multivorans ATCC 17616 genetic determinants for fitness in soil by using signature-tagged mutagenesis

“…However, it is now strongly suggested that the bacterial activities in natural environments are quite different from those under laboratory conditions (van Veen et al, 1997;Rediers et al, 2005). In fact, we are now encountering problems with the practical use of bacteria in complex natural environments (e.g.…”

“…surface and internal parts of plants and animals, and the plant rhizosphere) (Handfield & Levesque, 1999). IVET can identify genes specifically expressed under a particular condition (Rediers et al, 2005), while STM can identify the genes or loci important for growth and/or survival under a specific condition (Mazurkiewicz et al, 2006). Many studies using STM employ a set of transposons, each containing a tag signature with a unique oligonucleotide sequence.…”