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.

FIG. 1.

Schematic representation of the basic IVET strategy. This strategy involves the construction of a conditionally compromised strain that is mutated in a gene encoding an essential growth factor (egf). This mutant strain is not able to grow in the environment under study. The second component of IVET is the promoter trap, consisting of a promoterless egf gene and a transcriptionally linked reporter gene (rep). Bacterial DNA is cloned randomly into the promoter trap (step 1) and integrated in the chromosome of the egf mutant strain (step 2). Only in strains that carry a promoter active in the specified niche can the egf mutation be complemented (step 3). After selection in this environment, bacteria are reisolated and spread on a general growth medium that is suitable for monitoring reporter gene activity in vitro (step 4). Accordingly, constitutive promoters are distinguished from promoters that are specifically induced in the wild. Colonies bearing the latter type of transcriptional fusion are subjected to a second IVET screening to eliminate false positives (step 5).

FIG.2.

Schematic overview of the three main IVET selection strategies. Depending on the chosen IVET selection, the promoter trap contains a promoterless reporter gene (rep) transcriptionally linked to (1) a promoterless egf gene, encoding an essential growth factor (auxotrophy-based selection); (2) a promoterless Ab^R gene, conferring antibiotic resistance (antibiotic resistance-based selection); or (3) a promoterless site-specific recombinase gene (rec), which, when expressed, will splice out the antibiotic resistance (Ab^r) gene that is integrated elsewhere in the bacterial genome. Fusion libraries are constructed by ligating random genomic fragments (designated gene X) into the IVET vector of choice. Subsequently, the fusion library is transferred to an auxotrophic (egf) mutant strain (1) or a strain harboring the Ab^r gene flanked by recognition sites for the recombinase (indicated by flags) (3) in the case of auxotrophy-based and RIVET selection, respectively. After transfer of the transcriptional fusions into the microorganism of interest, the suicide plasmid is, in most cases, integrated into the chromosome at the sites of homology to gene X, thereby creating a merodiploid and retaining a functional copy of gene X (indicated with X⁺). In the case of RIVET, prescreening is required to remove strains harboring in vitro active gene fusions by selecting for Ab^R during construction of the fusion library. Subsequently, strains carrying the fusions are passed through the specific environment of interest and collected after a period of time. For antibiotic resistance-based selection, the antibiotic must be administered to the environment at a sufficient dose. Strains containing genes induced in the wild are selected by the ability to sustain growth in the environment (auxotrophy- or antibiotic resistance-based selection) or by screening for the loss of antibiotic resistance after recovery (RIVET selection). In the case of auxotrophy-based and antibiotic resistance-based selection, constitutive promoters can be discarded by monitoring the activity of the reporter gene in vitro and for antibiotic sensitivity, respectively, on a general growth medium.

FIG. 3.

Distribution of promoter trap-isolated host-induced genes among different functional classes. The percentages of genes involved in chemotaxis and motility (class I), nutrient scavenging (class II), central metabolism (class III), adaptation to environmental stresses (class IV), regulation (class V), cell envelope structure and modification (class VI), virulence and secretion (class VII), nucleic acid metabolism (class VIII), and transposition and site-specific recombination (class IX) and FUN genes (genes with unknown function or without significant similarity with known genes) are presented in the diagram.

FIG. 4.

Schematic overview of bacterial genes that are induced during life in the host environment. The host environment (black box) is a complex system of environmental parameters that activate or repress expression of several microbial genes. Activated genes can be isolated with promoter-trapping techniques such as IVET and DFI. Genes involved in chemotaxis and motility (1) are specifically expressed during (early stages of) interaction with the host. IVET and DFI studies of plant- and animal-associated microorganisms reveal several parallels and dissimilarities. Genes involved in amino acid uptake (2) and in direct inactivation of reactive oxygen species (3) were predominantly isolated from plant-associated microorganisms, while genes involved in suppression of chemotaxis and motility (4) and the acid stress response (5) were predominantly found in animal-associated microorganisms. In both ecological niches, genes involved in TTSS (6), DNA modification (7), cell surface modification (8), nutrient scavenging (9), and more specifically, iron acquisition (10) are upregulated. A significant number of the genes that are specifically induced in the wild have so far unknown functions (indicated with a question mark).