This study focused on defining the differences in L. plantarum gene expression levels in different media and in different growth phases using an easy and cost-efficient monitoring of gene expression. A macroarray based on a group of selected L. plantarum genes, 178 genes belonging to 18 main groups, printed onto a nitrocellulose filter was designed in this work. Using the macrofilters designed, the expression of a selected set of L. plantarum genes was assayed in synthetic MRS medium and in extracted carrot juice. To compare the potential differences of starter gene expression in hygienic and contaminated cultivation media, the L. plantarum strain was cultivated in both sterile and contaminated (yeast and Escherichia coli) MRS and carrot juice. The number of genes found to be regulated as a function of growth was clearly higher in MRS-based growth medium than in carrot juice, In carrot juice, expression of the gene encoding malolactic enzyme (MLE), which makes L. plantarum an advantageous microbe in e.g. wine making, was found to be upregulated in logarithmic phase of growth. The current study demonstrated that macroarrays printed on nitrocellulose filters with simple robotic systems can be analyzed by standard laboratory equipment and methods usually available in molecular laboratories. Using this technology, rapid and cost-efficient analysis of genome function of L. plantarum can be carried out e.g. in developing regions, where lactic acid fermentation of food and feed matrices is a common practice.
Lactic acid bacteria (LAB) are widely used for the preservation of food and feed raw materials and to intensify the flavour and texture of fermented products. Of the lactobacilli commonly used in food processes, Lactobacil- lus plantarum is important in the production of many fermented foods of both plant (pickled vegetables, silage, sourdough) and animal (dry ferment sausages, fermented fish, cheese) origin [
Due to the ability to maintain pH homeostasis at low external pH, L. plantarum is tolerant to acidic environ- ment and often becomes the dominant LAB at the end of spontaneous vegetable fermentation [
Previously, the technological properties of potential starter LAB could be determined almost exclusively in pilot- and full-scale food and feed production experiments. Today, the development of molecular techniques has made possible the exploitation of genomic and proteomic data for the observation of potential genotypic and phenotypic differences between individual strains in specific growth conditions. Studies based on L. plantarum DNA-microarrays [
Both microarray and proteomic studies require specific laboratory facilities and expertise, which may not be available in all research laboratories. Yet, L. plantarum is used worldwide for food and feed fermentations and there is a growing demand for the design of starter cultures with well-characterized technological properties. For easy and cost-efficient monitoring of gene expression in L. plantarum, a macroarray based on a group of se- lected L. plantarum genes printed onto a nitrocellulose filter was designed in this work. Using the macrofilters designed, the expression of a selected set of L. plantarum genes was assayed in synthetic MRS medium and in extracted carrot juice. To compare the potential differences of starter gene expression in hygienic and contami- nated cultivation media, the studied L. plantarum strain was cultivated in both sterile and contaminated (yeast and Escherichia coli) MRS and carrot juice.
L. plantarum strain MLBPL1 has been isolated from sauerkraut [
For macroarray analyses, L. plantarum strain MLBPL1 was grown in synthetic medium MRS and carrot juice. To simulate contaminated growth conditions, the spoiling E. coli and yeast strains were inoculated into MRS and carrot juice. Cultivations were performed using Spectra/Por Float-A-Lyzer dialysis tube (MWCO 100 kDa, Spectrum Laboratories, Rancho Dominguez, CA, USA) in order to make it easier to separate the L. plantarum cells, the vegetable matrix and the spoiling strains of yeast and E. coli. By using the dialysis tube, no filtering of plant material was needed and in addition, the cells of contaminating strains didn’t interfere with the extraction of RNA. Carrot juice was prepared from fresh vegetables with a juice extractor. The extracted juice was centri- fuged at 18,500 g for 40 min and pasteurized in a water bath at 95˚C for 30 min.
An overnight culture of MLBPL1, grown in MRS-medium at 32˚C, was used to inoculate MRS broth and carrot juice. For MRS cultivation, a 1% inoculumn was used. For carrot juice cultivation and contamination cul- tivations, the inoculumn was centrifuged at 13,000 g for 3 min and the pellet was suspended into centrifuged (18,500 g for 40 min) and filter-sterilized (0.8/0.2 µm pore sizes) carrot juice (carrot cultivations) or MRS broth (MRS contamination cultivation), after which the suspension was transferred to a Spectra/Por Float-A-Lyzer dia- lysis tube. The tube was then transferred to a bottle containing carrot juice, contaminated MRS broth or con- taminated carrot juice. In contamination cultivations, MRS broth and carrot juice were contaminated by inocu- lating them with 1% E. coli and yeast. The cultivations were performed at 32˚C. The growth was determined by plating onto MRS agar plates appropriate dilutions from the samples taken during the growth. The plates were incubated at 32˚C for 48 h, until single bacterial colonies appeared.
Bacterial cells of the L. plantarum strain MLBPL1 grown in MRS broth and carrot juice were harvested at ex- ponential (6 h) and stationary phase (14 h) of growth by centrifugation for 3 min at 4˚C at 11,000 g. The col- lected cells were frozen immediately in liquid nitrogen and stored at −70˚C. Extraction of total RNA was carried out with SV total RNA isolation system (Promega, Madison, WI, USA) with some modifications to the protocol on the disruption of the cells. Briefly, bacterial cells thawed slowly on ice were first washed with sterile water treated with diethyl pyrocarbonate (DEPC) and collected by centrifugation. Next, the pellet was resuspended into 225 µl of SV RNA lysis buffer of the Promega kit and transferred to an eppendorf tube containing 100 µl of nitric acid-washed glass beads. The cells were disrupted with glass beads in a cell homogenizer as described be- fore (Kahala, et al., 2008). After that, the lysate was transferred to a new tube and 350 µl of SV RNA dilution buffer of the Promega kit was added per 175 µl of lysate. From this step on, the extraction was carried on as recommended by the manufacturer. Two technical duplicates from the RNA extraction on each culture medium and harvesting point were made. mRNA was enriched from total RNA samples by removing the 16S and 23S rRNAs with MICROB Express Bacterial mRNA Purification kit (Ambion, Austin, TX, USA) according to the instructions of the manufacturer. The RNA concentration was determined spectrophotometrically at 260 nm.
The integrity of the isolated prokaryotic RNA was determined by total RNA gel electrophoresis and Northern blot carried out as described by [
cDNA was synthesized by RT from DNA-free mRNA and cDNA labelling was performed with an alkalilabile digoxigenin-11-dUTP (DIG) (Roche, Basel, Switzerland) in a reverse transcription reaction with Im-Prom-II Reverse Transcription System kit (Promega) as follows: 1 µg of mRNA was mixed with 0.5 µg of random hexamer primers provided by the kit manufacturer. The mixture was heated at 70˚C for 5 min and chilled on ice for 5 min. cDNA synthesis was carried out by combining RNA-primer mixture with 1 × ImProm-II reaction buffer, 5 mM MgCl2, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.325 mM dTTP, 0.175 mM DIG-11- dUTP, 1 U/µl RNasin Ribonuclease Inhibitor, and 1 µl ImProm-II Reverse Transcriptase. Annealing was per- formed at 25˚C for 5 min, followed by extension at 43˚C for 1h and enzyme inactivation at 70˚C for 15 min. The labeled cDNA was purified with Microarray Target Purification kit (Roche) according to the instructions of the manufacturer.
Human-based HbGAM (heparin-binding growth-associated molecule) gene [
Primers were designed for the amplification of selected genes from the fully sequenced genome of L. plantarum WCFS1 [
PCR fragments in the 96 well plates were transferred to 384 plates for printing on the nitrocellulose macroar- ray (Supplement 2). Purified PCR fragments were gridded in duplicate on nitrocellulose membranes with a QPix automated colony picker (Genetix Ltd., UK) using a 384-pin gridding head as described in [
Macroarrays were prehybridized for 2 h at 60˚C with 20 ml of DIG Easy Hyb buffer (Roche). Hybridizations were performed overnight at 60˚C with 6 ml DIG Easy Hyb buffer (Roche) containing 5 µl of labeled cDNA probe and HbGAM which was used as a positive control in hybridization reactions. After hybridization, macroarrays were washed twice at room temperature for 5 min with washing solution containing 2 × SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) and 0.1% sodium dodecyl sulphate (SDS) and twice at 68˚C for 15 min with washing solution (0.1 × SSC, 0.1% SDS). Hybridized spots were detected with chemilumi- nesence-based DIG detection kit (Roche) using CDP-Star (Roche) as a substrate and chemiluminescence produced was detected by FluorChem (Alpha Innotech Corp., San Leandro, CA) gel image system. For re- probing, the DIG-labelled probe was removed with a following procedure. The membrane was rinsed thor- oughly in sterile water, washed twice with 0.2 M NaOH containing 0.1% SDS at 37˚C for 20 min and rinsed with 2 × SSC for 5 min.
The DNA probes spotted on the macroarray were selected using results from the previous proteomics results us- ing 2-DE and HPLC-ESI-MS/MS [
where f(x,y,z) denotes a normalized frequency of the codon triplet (x,y,z) coding for an amino acid a in a gene g, g(x,y,z) denotes the frequency of the codon triplet (x,y,z) in the gene set G, and pa(g) is the fraction of the amino acid a in the gene g [
The gene g was predicted as highly expressed if the relative codon bias
exceeded 1.05. The genes obtaining the greatest RCB values were chosen for the DNA macroarray filter in addi- tion to those identified by the HPLC-ESI-MS/MS.
After scanning of the macroarray images, the quantification of the hybridized signals and background sub- traction were done by the TIGR Spotfinder image processing software [
Growth rate of L. plantarum MLBPL1 cells was similar in MRS and carrot juice (
The macroarray included 178 genes belonging to 18 main groups. The largest groups were energy metabolism (59 genes), protein synthesis (30 genes), protein fate (10 genes), regulatory functions (10 genes), cell envelope (9 genes) and DNA metabolism (9 genes). Of the 178 genes tested, 18 (10%) showed a mean fold change greater than 2.0 in at least one of the ten comparisons between growth media or growth phases (
The majority of the genes studied on the membranes showed no significant change in levels of expression during the growth or between the growth media. The number of the genes found to be regulated as a function of growth was clearly higher in MRS-based growth medium than in carrot juice, in which only genes involved in fatty acid and phospholipid metabolism showed differential expression in different growth phases. The function of the genes showing upregulation in logarithmic phase in MRS medium was mostly related to energy metabolism, but also to cell division, cell envelope biosynthesis and pyrimidine ribonucleotide biosynthesis. Generation of suffi- cient energy for growth in logarithmic phase is important and was evidenced in the MRS based medium.
In MRS cultivation, when entering in the stationary phase of growth, transcription of genes involved in energy metabolic pathways decreased and higher expression levels were found for genes involved in protein fate, pro- tein folding and stabilization, like “folding” chaperones DnaK and GroEL. In contaminated MRS medium, es- pecially the expression levels of genes involved in sugar metabolism pathways (galK, lacM) were found to be higher in logarithmic phase. This reflects higher demand for energy in the logarithmic phase and probably competition between the Lactobacillus and contaminating strains in the utilization of sugars that are needed for growth.
Main role | gene ORF | gene product | MRS vs MRS cont. 6 h | MRS vs MRS cont. 14 h | Carrot juice vs Carrot juice cont. 6 h | Carrot juice vs Carrot juice cont. 14 h | MRS vs Carrot juice 6 h | MRS vs Carrot juice 14 h | MRS 6h vs 14 h | MRS cont. 6 h vs 14 h | Carrot juice 6 h vs 14 h | Carrot juice cont 6 h vs 14 h |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Energy metabolism— Pyruvate dehydrogenase | pdhB lp_2153 | pyruvate dehydrogenase complex, E1 component, beta subunit | −2.20 | - | - | - | - | - | - | - | - | - |
Energy metabolism— Sugars | galK lp_3482 | galactokinase | −3.01 | - | - | - | - | - | - | +2.13 | - | - |
lacM lp_3484 | beta-galactosidase, small subunit | −2.30 | - | - | - | - | - | - | +2.43 | - | - | |
Energy metabolism— Glycolysis/ gluconeogenesis | pyk lp_1897 | pyruvate kinase | - | - | - | - | - | −2.32 | - | - | - | - |
Energy metabolism— Pentose phosphate pathway | rpiA1 lp_0602 | ribose 5-phosphate epimerase | - | - | - | - | - | - | +2.02 | - | - | - |
DNA metabolism— DNA replication, recombination | dnaN lp_0002 | DNA-directed DNA polymerase III, beta chain | - | - | - | - | - | - | - | - | - | +2.15 |
Cellular processes— Cell division | ftsH lp_0547 | cell division protein FtsH, ATP-dependent zinc metallopeptidase | - | −2.06 | - | - | - | - | +2.28 | - | - | - |
Cell envelope— Biosynthesis and degradation | lp_0304 | extracellular protein | - | - | - | - | - | - | +2.16 | - | - | - |
Cell envelope—Other | lp_2290 | integral membrane protein | - | - | - | - | - | - | +2.41 | - | - | - |
Signal transduction— PTS | pts16ABC lp_2097 | fructose PTS, EIIABC | - | −2.50 | - | - | - | −2.86 | - | - | - | - |
Enzymes of unknown specificity | mleS lp_1118 | malolactic enzyme | - | - | +2.32 | +2.17 | −2.56 | −2.28 | - | - | - | - |
Fatty acid and phospholipid metabolism— Biosynthesis | fabF lp_1675 | 3-oxoacyl-[acyl-carrier protein] synthase II | - | - | - | - | - | - | - | - | +2.18 | |
Purines, pyrimidines, nucleosides, and nucleotides— Pyrimidine ribonucleotide biosynthesis | pyrD, lp_2697 pyrC, lp_2699 | dihydroorotate oxidase, dihydroorotase | - - | - - | - - | - - | +3.74 +3.36 | - - | - +2.54 | +2.02 - | - - | - - |
Transport and binding proteins—Amino acids, peptides | oppA lp_1261 | oligopeptide ABC transporter, substrate binding protein | - | - | - | - | −2.41 | - | - | - | - | - |
Protein fate— Protein folding and stabilization | groEL lp_0728 dnaK lp_2027 | GroEL chaperonin heat shock protein DnaK | - - | - - | - - | - - | - - | - - | −2.25 −2.17 | - - | - - | - - |
Unknown function | typA lp_2146 lp_3092 | GTP-binding protein TypA fumarate reductase, flavoprotein subunit precursor, N-terminally truncated | - - | - - | - - | - - | - - | - - | +2.45 −2.13 | - - | - - | - - |
The mRNA level of several genes was shown to be regulated in response to different growth media. At the ex- ponential (6 h) phase of growth, the genes encoding dihydroorotate oxidase and dihydroorotase enzymes were differentially expressed in MRS and carrot juice. They showed 3.7- and 3.4-fold higher expression in the MRS compared to carrot juice growth medium, respectively (
Expression of malolactic enzyme (mle) gene was clearly higher in logarithmic phase when grown in plant based medium. Upregulation of cell division protein FtsH was observed in contaminated MRS 14 h compared to MRS 14 h, probably indicating higher stress response in contaminated MRS.
This study focused on defining the differences in L. plantarum gene expression levels in different media and in different growth phases by the use of a simple and low-cost macroarray technique. Previously described DNA macroarray technique [
Fermentation conditions may dramatically affect functional characteristics of LAB [
Proteomic studies by [
Higher expression of cell division protein FtsH in contaminated MRS compared to MRS probably indicated higher stress response in contaminated MRS. Functional studies have revealed an important role for FtsH in the bacterial stress response. In several bacteria, including E. coli, B. subtilis, Lactococcus lactis, O. oeni, Helico- bacter pylori, and L. plantarum, ftsH expression is induced in response to heat and other stress factors controlled by additional regulators [
Macroarray was found to be an applicable method for studying expression of defined genes of L. plantarum during fermentation. Macroarray technology has been successfully applied also e.g. for the detection of patho- gens in chicken samples [
L. plantarum is encountered in a variety of environmental niches, which include dairy, meat and many vege- table or plant fermentations as well as the human gastrointestinal tract. Because of this flexibility and versatility, strains of this species have been traditionally used for food and feed preservation and as starters in the manufac- ture of fermented products. Formerly, the technological properties and suitability of certain strains to selected applications could be ensured almost exclusively by laborious and time-consuming food processing and preser- vation experiments. Today, the long history of use and on the other hand the development of molecular and ge- nomic techniques have made L. plantarum one of the most studies food microbes. Modern DNA microarray [
Tekes, the Finnish Funding Agency for Technology and Innovation, is gratefully acknowledged for the financial support of this work. The authors wish to thank Anneli Paloposki for the skilful technical assistance, Ari-Matti Sarén for designing the primers, Markku Ala-Pantti and Hannu Väänänen for printing the membranes.