High-Throughput Identification and Validation of In Situ-Expressed Genes of Lactococcus lactis

Abstract

Understanding the functional response of bacteria to their natural environment is one of the current challenges in microbiology. Over the past decades several techniques have been developed to study gene expression in complex natural habitats. Most of these methods, however, are laborious, and validation of results under in situ conditions is cumbersome. Here we report the improvement of the recombinase-based in vivo expression technology (R-IVET) by the implementation of two additional reporter genes. The first one is an ␣-galactosidase gene (melA), which facilitates the rapid identification of in vivo-induced genes. Second, the bacterial luciferase genes (luxAB) are transcriptionally coupled to the resolvase gene, which allows rapid validation and characterization of in vivo-induced genes. The system is implemented and validated in the industrially important lactic acid bacterium Lactococcus lactis. We demonstrate the applicability of the advanced R-IVET system by the identification and validation of lactococcal promoter elements that are induced in minimal medium compared to the commonly used rich laboratory medium M17. R-IVET screening led to the identification of 19 promoters that predominantly control expression of genes involved in amino acid and nucleotide metabolism and in transport functions. Furthermore, the luciferase allows high-resolution transcription analysis and enabled the identification of complex medium constituents and specific molecules involved in promoter control. Rapid target validation exemplifies the high-throughput potential of the extended R-IVET system. The system can be applied to other bacterial species, provided that the reporter genes used are functional in the organism of interest.Understanding the responses of microorganisms in their natural environments is one of the big challenges of the postgenomic era. Complex niches such as soil, decaying plant material, and the (mammalian) gastrointestinal tract show a rich bacterial diversity and complex microbial activity patterns. Sampling complexity and environmental dynamics hamper comprehensive transcriptional analysis of a specific microorganism residing in such a complex environment. To overcome these challenges, a number of tools to study in vivo gene expression were developed during the past decades (25). One of the most powerful technologies is termed in vivo expression technology (IVET) and is based on transcriptional fusions of genomic DNA fragments to a selectable reporter gene (32). Recombinase-based IVET (R-IVET) is an IVET variation that employs a resolvase as the primary reporter gene. Upon expression of the resolvase, a chromosomally located marker, situated between two recombination sites, is excised from the genome (1). This irreversible step "records" in situ promoter activity and acts as a promoter-trap system, allowing the accumulation of promoter activity detection over extended in situ incubation periods. Moreover, the system is able to detect very low and transient gene expression in complex environmental condi...

Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation

“…100 l of this culture was used to suspend the dried fraction and then dispensed to a 96-well clear bottom plate. Decanal, the aldehyde substrate required for luciferase, was provided as a 1% solution in mineral oil and was included in the plate in spaces outside of wells, as has been described previously (19,20). The plate was lidded, sealed, and read in a Synergy 2 plate reader (BioTek) set to 37°C with continuous shaking to prevent cells from settling at the bottom of the plate.…”

“…Filtered spent-culture supernatants were loaded onto 2000-mg HyperSep C18 cartridges (Thermo Scientific) and washed with 5% acetonitrile and 0.1% trifluoroacetic acid (TFA). Fractions were eluted with 10 ml of each acetonitrile concentration (20,40, and 70%, each containing 0.1% TFA). 100 l (1% of total volume) of each fraction was tested for activity in the luciferase reporter assay.…”

Indigenous and Environmental Modulation of Frequencies of Mutation in Lactobacillus plantarum

“…Isolation of L. lactis plasmid DNA was performed using Jetstar columns according to the manufacturer's recommendations, with modifications described previously (5). Transformation of L. lactis was performed as described by Holo and Nes (23).…”

Redundant Group A Streptococcus Signaling Peptides Exhibit Unique Activation Potentials

“…Aliquots of the dilute culture were then distributed into the wells of a clearbottom 96-well plate containing peptides of interest. Volatile decyl aldehyde was provided as a 1% solution in mineral oil between the wells of the microplate as previously described (24), and the plate was lidded and parafilmed to ensure entrapment of the volatile content. Cells were grown at 37°C in a Synergy 2 plate reader (BioTek) with continuous fast shaking to deter cell sedimentation and facilitate accurate evaluation of the culture density, and OD 600 and cps were measured every 10 min throughout growth.…”