Development and Validation of a Species-Independent Functional Gene Microarray That Targets Lactic Acid Bacteria

Abstract

During the last few years, genome-related information has become available for many microorganisms, including important food-related bacteria. Lactic acid bacteria (LAB) are important industrially in the production of fermented foods such as dairy products, sausages, sourdoughs, and vegetables. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as flavor and texture. In the present study, a species-independent functional gene microarray was developed that targets 406 genes that play key roles in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in the stress response. Also, genes linked to negative traits, such as antibiotic resistance and virulence, are represented. As LAB ecosystems contain a variety of species, there was a more global focus on these specific functional properties. Thus, an algorithm was used to design gene-specific oligonucleotides that preferably hybridize with multiple LAB species, thereby allowing controlled cross-hybridization. For proof of concept, the microarray composed of 2,269 30-mer oligonucleotides focused on LAB species that are prevalent in sourdough ecosystems. Validation hybridizations using DNA and RNA from 18 LAB strains, covering 86% of all the oligonucleotides, showed that there were wide ranges in intensity and high reproducibility between microarrays.

Adaptation of Lactobacillus plantarum IMDO 130201, a Wheat Sourdough Isolate, to Growth in Wheat Sourdough Simulation Medium at Different pH Values through Differential Gene Expression

“…The aim of the present study was to indicate genes and gene clusters that are involved in cellular responses related to acid adaptation of L. plantarum IMDO 130201, an isolate obtained from a laboratory wheat sourdough fermentation (33), during growth in wheat sourdough simulation medium by using a LAB functional gene microarray (40).…”

“…Given that each oligonucleotide was spotted in triplicate on the microarray slides and that each sample was hybridized twice, once with each fluorescent dye, the intensity of each oligonucleotide was measured six times. The intensity of an oligonucleotide was considered to be above the background level if the intensities for at least three out of six spots were above the background level, as described previously (40). Prior to further data analysis, the local background intensity was subtracted from the foreground intensity and a base 2-log transformation was applied.…”

“…A LAB functional gene microarray version 2 was used, which is an updated version of the microarray described previously (40). This updated version contains in total 6,209 oligonucleotides, of which 795 oligonucleotides are related to L. plantarum.…”

“…To obtain total RNA from L. plantarum IMDO 130201, 5 ml fermentation medium of a mid-exponential-growth-phase culture was collected in 10 ml RNAprotect (Qiagen, Hilden, Germany), mixed, and kept at room temperature for at least 5 min. Subsequently, the sample was centrifuged at 5,000 ϫ g for 15 min and the RNA was isolated from the resulting cell pellet by applying an enzymatic lysis using mutanolysin and lysozyme, after which the RNA was extracted from the resulting mixture by using an RNeasy minikit (Qiagen), following standard instructions and including mechanical disruption of the cells using glass beads, as described previously (40). As the RNA profiles, which were obtained after capillary electrophoresis using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA), showed an unexpected peak at the lower size range, possibly due to the presence of small RNA molecules (e.g., tRNA), an additional RNA cleanup step was performed.…”

“…In addition, 175 oligonucleotides were designed based on gene sequences from other LAB species, but they could hybridize L. plantarum gene sequences based on in silico comparative sequence analysis. The microarray was spotted on CodeLink activated slides as described previously (40).…”