SUPPORTING INFORMATION
APPENDIX S1
Experimental procedures
Bacterial strains, plasmids, primers and growth conditions
The bacterial strains and plasmids used in this study are listed in Table S1. All primers are listed in Table S4.
L. plantarum was routinely grown at 30°C in MRS medium (Oxoid Ltd., Hampshire, UK) without shaking. Escherichia coli strains were grown at 37°C in BHI medium (Oxoid) with vigorous shaking. BHI agar and MRS agar (both Oxoid) were used as solid media for E. coli and L. plantarum, respectively. When appropriate, 10 mg ml-1 chloramphenicol and 10 mg ml-1 or 30 mg ml-1 erythromycin was used for L. plantarum, while for E. coli, 200 mg ml-1 erythromycin was used.
In the growth experiments, a defined medium for lactobacilli (DML) (Møretrø et al., 1998) was used, with the following modification: The succinate buffer was replaced with 0.1 M MES buffer, and 12 mM glucose or 12 mM ribose was used as carbon source. Growth on ribose was performed with 2x DML, except for 1x MES, 1x L-cysteine and 4,5x Tween-80. Adaptation to ribose fermentation was done by adding 0.02 % glucose to the 2xDML with 51 mM ribose, and when the cells reached the exponential phase (after approximately 48 hours) they were re-incubated once in 2x DML with 12 mM ribose before inoculation in the growth experiment. The growth experiments were performed with batch cultures (flasks) in 100 ml of DML with biological duplicates of each strain, and followed in the exponential phase. Slow stirring with magnets was used to keep the cultures homogenous.
DNA isolation and transformation
Total DNA from L. plantarum was isolated using DNeasy Tissue Kit (Qiagen N.V., Venlo, The Netherlands) according to manufacturer’s procedure. Lysozyme (20 mg/ml) (Sigma Aldrich) and mutanolysin (40 U/ml) (Sigma Aldrich) were used in the lysis step. Plasmid DNA from E. coli and L. plantarum was isolated using the QIAprep Miniprep Kit (Qiagen) as previously described (Rud et al., 2006). E. coli XL Gold cells were transformed according to manufacturer’s procedure and Lactobacillus strains were transformed by electroporation using previously described procedures (Aukrust and Blom, 1992).
Construction of strains over-expressing cggR
The cggR gene was cloned into pSIP409, harbouring the gusA reporter gene, using standard molecular cloning techniques (Sambrook et al., 1989). PCR was performed with the PCR phusion (Phusion DNA polymerase, Finnzymes) using standard procedure. Due to NcoI sites in the cggR gene, which is the typical 5’-end cloning site of the pSIP409, the PorfX promoter of pSIP409 and the cggR’ were amplified separately (PCR 1: orf-bglII and pcggr-orf and PCR 2: pygap-start and pcggr-nco2, respectively) before they were fused by a fusion PCR (PCR 3: orf-bglII and pcggr-nco2). PorfX was amplified with pSIP409 as template, whereas genomic DNA isolated from
L. plantarum NC8 was used for the cggR’ amplification. A PCR of the gusA’ in pSIP409 was performed (PCR 4: gus-start and gusRT3) in order to include a Shine-Dalgarno sequence upstream of the gusA gene start, which further was fused to the PCR 3 product by fusion PCR (PCR 5: orf-bglII and gusRT3). The final amplicon, PCR 5, was cloned into the BglII and the SnaBI restriction sites of pSIP409 and the resulting plasmid (pSIP409/cggR) was transformed into E. coli and L. plantarum NC8, and subsequently sequenced.
A preliminary study of over-expressed cggR was performed by dose-dependent induction with SppIP (Molecular Biology Unit, University of Newcastle, UK), in order to evaluate the effect of CggR before selecting the level of constitutive cggR over-expression: The inducible PorX promoter of pSIP409/cggR was replaced by the synthetic promoter, P25 (Rud et al., 2006). P25 was obtained by PCR with the pSIP409-P25 plasmid as template using primers; sip3 and pcggr-orf, and was further fused to a PCR product (pygap-start and pygap3) containing a region of the cggR gene with the following primers, sip3 and pygap3. The fused product was cloned into the BglII and the PvuI restriction sites of pSIP409/cggR and the resulting plasmid (pSIP409/cggR-P25) was transformed into L. plantarum (NC8 and WCFS1).
Construction of cggR mutants by double-crossover
L. plantarum cggR mutant strains were constructed in both NC8 and WCFS1 by in-frame double-crossover gene replacement and removal of selection marker using a Cre-lox-based system developed for L. plantarum (Lambert et al., 2007). The gene-specific mutagenesis vector pNZ5319 was used to construct the cggR mutagenesis vector, pNZ5319-cggR. 650-bp fragments were amplified by PCR, overlapping the 5’-region and the 3’-region of cggR of NC8 using the rpoN-FP2 and pcggr-RPcre primers, and the pcggr_FPcre2 and pgapRT1 primers, respectively. The primers were designed to obtain in-frame deletion of cggR. Blunt-end cloning of the fragments into pNZ5319 was achieved by digesting the mixture with PmeI and EcoICRI (Ecl136II) restriction enzymes before ligation. Self-ligated vector in the ligation mixture was removed by digestion with PmeI/EcoICRI before transformation. Positive transformants were identified by colony PCR using vector primers (upsp35-RP/cat-FP) and insert primers (pygap-start/pgapRT2).
Double-crossover mutants (cggR-mutant-cat) in NC8 and WCFS1 were constructed by transformation with the cggR mutagenesis vector (pNZ5319-cggR), and chloramphenicol-resistant (Cmr) integrants were selected and checked for an erythromycin-sensitive (Ems) phenotype. The correct integration of the lox66-P32-cat-lox71 cassette into the cggR locus was verified in candidate positive double-crossover clones (Cmr, Ems) by PCR using primers flanking the crossover region (rpoN-FP1/pgapRT3) and primers specific for the integration cassette (upsp35-RP/cat-FP).
The cre expression vector pNZ5348 was transformed to cggR derivatives of L. plantarum NC8 and WCFS1, in order to prevent eventual polar effects of the mutation introduced by the P32-cat selection marker in the Cre-lox recombination sites. After 72 h of growth, pNZ5348 transformants (Emr) were checked for chloramphenicol sensitivity (Cms) and subsequently pNZ5348 was cured from these colonies by growth without erythromycin for 10 generations. The resulting, resolved in-frame cggR mutant derivatives of NC8 and WCFS1 were re-checked for the absence of pNZ5348 (Ems, and by PCR), and by verification of the appropriate Cre-mediated resolution of the lox66-P32-cat-lox71 cassette into a single lox72 site by amplification of the resolution region using primers pygapRT2, pygap-start and pgapFP2 as well as amplicon sequencing (data not shown)
Sequence analysis
All plasmids and genome sequences were verified by sequence analysis in the altered regions using an ABI Prismâ 3100 Genetic Analyzer with the ABI Prismâ BigDyeTM Terminator Cycle Sequencing Ready Reaction kit, following the manufacturer’s recommendations (PE, Biosystems). Sequencing primers are listed in Table S4.
Promoter prediction analysis was done by the Neural Network Promoter Prediction program (Reese MG, 2001) at the Berkeley Drosophila Genome Project, with the analysis restricted to prokaryotes.
Manual search for putative CRE sites was done in the vicinity of atg start sites (200 basepairs upstream and downstream) of specifically selected genes. Some genes were organized in an operon, and thus the first gene in the operon was evaluated for CRE sites. The consensus sequence TGWNANCGNTNWCA (Weickert and Chambliss, 1990) was used in the search, and only sequences with two or less mismatches compared to the consensus sequence were accepted as putative CRE sites. Mismatch in the middle bases “CG” was not accepted.
b-glucuronidase assay
The promoter activities of L. plantarum strains harboring pSIP409/cggR and pSIP409/cggR-P25 were investigated with regard to b-glucuronidase (GusA) activity, which was expressed from the gusA reporter gene, fused to the cggR gene. The samples were collected in the growth experiments, and a modified GusA assay (Miller, 1972) was used for this purpose as previously described (Axelsson et al., 2003). Activity was calculated and expressed as Miller Unit equivalents (MU).
HPLC measurements, calculation of metabolic flux and API test
HPLC samples were collected in the exponential growth phase and filtered through a 0.22 mm filter (Millipore) and stored at –20°C. The concentration of glucose and lactate were determined by a Shimadzu HPLC on an Aminex HPX-87-H column (BioRad Laboratories) at 45°C, with refractive index detection (RID-6A, Shimadzu). H2SO4 (5 mM) was used as mobile phase at 0.6 ml/min. The cell density was correlated to the cell mass of L. plantarum NC8 to be 0.38 g (dry weight)*L-1 DML medium for an OD600 of 1. Glycolytic flux and lactate flux (mmol of metabolite*h-1*g-1 (dry weight)) of L. plantarum were calculated from changes in the concentration of metabolites measured by HPLC relative to the specific growth rate (h-1) and the cell density. Fermentation of carbohydrates (e.g. glycerol) in the
L. plantarum strains was determined using API 50 CH assay (BioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Changes in color from purple were monitored after 24 h and verified after 48 h.
FBP
Extracts were prepared from batch cultures at OD600 of 0.65 by quenching in 80oC buffer-saturated phenol supplemented with 0.6 g of glass beads as previously described (Koebmann et al., 2002). Analysis of fructose-1,6-bisphosphate (FBP) was carried out by an enzymatic assay according to Bergmeyer and Grassl (1984), where FBP was determined from the decrease in A340 after addition of NADH and subsequent conversion with glycerol-3-phosphate dehydrogenase, triosephosphate isomerise and aldolase. Intracellular concentration of FBP was calculated by assuming that 1 g (dry weight) corresponded to 1.67 ml of intracellular volume.
RNA extraction
Total RNA was extracted from exponentially growing L. plantarum strains (OD600 of 0.65) using the RNeasy Protect Bacteria Mini Prep Kit (Qiagen). Samples (3 ml) were collected and added to RNA Protect Bacteria Reagent (Qiagen) (6 ml), vortexed for 5 s, incubated at room temperature for 5 min and then centrifugated for 10 min at 6,000 x g. The supernatant was discarded, and the pellet was frozen at -20oC for later RNA purification. The pellet was thawed at room temperature and resuspended in 200 ml of TE buffer containing lysozyme (40 mg/ml) and 50 ml of mutanolysin (5,000 U) by vortexing for 10 s. The suspension was divided into two tubes and the lysis step lasted for 30 min at 37 oC. The remaining purification was continued according to manufacturer’s protocol, including the DNease treatment on the column. RNase-free water (35 ml) was used in the elution step and the eluate was used in a second elution step to obtain a higher total RNA concentration. The concentration and the purity of the total RNA was analyzed using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.) and the quality using the Agilent 2100 Bioanalyser (Agilent Technologies). Samples with the A260/A280 ratio superior to 1.9 and the 23S/16S RNA ratio superior to 1.6 were further used in the transcriptome analysis.
Global transcriptome analysis
The first strand cDNA synthesis was performed with the CyScribe Post-Labelling Kit (Amersham, United Kingdom) according to the manufacturer’s procedure with the following modifications: Five micrograms of total RNA were used per sample and the cDNA synthesis was incubated for 3 h at 42oC. The CyScribe Purification kit (Amersham) was used prior and subsequent to the labeling of the amino allyl-modified cDNA, performed by the CyScribe first-strand cDNA labeling kit (Amersham). Two labeled cDNA (cyanine 3/cyanine 5) of
0.5 mg were hybridized on the custom designed L. plantarum WCFS1 11K Agilent oligo microarray using the Agilent in situ Hybridization Kit Plus. The arrays were hybridized, washed, dried and scanned as described by Saulnier et al. (2007).
Several relevant genes of the sequenced L. plantarum WCFS1 were missing on the array (LPAG1) (e.g. ack1-3, xpk1-2, rbsk2-3, adh1-2, glpK1-2, glpF1 and glpF4), which was because the probe design program was unable to select probes that uniquely targeted each of these genes, due to homologues in the genome.
Data analysis
The hybridization schemes were loop designed (Supporting information, Figure S1) that allowed for evaluation of putative dye effects. Significant dye effects were not observed (False Discovery Rate (FDR) corrected p-value <0.05). Transcriptome data analysis and evaluation of the size and significance of gene regulation was performed as previously described (Saulnier et al., 2007) using the Limma package (Smyth et al., 2003) in R (http://www.r-project.org). Contrasts were calculated as in a standard two-way ANOVA, namely as two main effects originating from the carbon source and mutation factors (carbon source effect and mutation effect; CE and ME, respectively) and an interaction effect (IE), all including degree of significance presented as FDR corrected p-value. The CE is the effect of growth on ribose relative to glucose regardless of the cggR mutation. The ME is the effect of cggR mutation regardless of which carbon source is used. The IE shows when there are significant differences between the way in which the wild-type and the cggR mutant respond to the carbon source. The three effects (CE, ME and IE) can theoretically be divided into separate effects, albeit with significance calculations that are based on the overall analysis (including all effects) rather than the individual effects. Nevertheless, the individual effects are mentioned here to facilitate the interpretations of the microarray data, and a summary of all data can be found in Supporting information (Table S2): {CE: CE (mutant) + CE (wild-type); ME: ME (ribose) + ME (glucose); IE: ME (ribose) – ME (glucose) = CE (mutant) – CE (wild-type)}. Over-expression effect (OE) and over-expression interaction effect (OIE) was calculated for WCFS1 in a similar manner as the ME and the IE. No significance of the cggR over-expression could be assessed for NC8 due to limited hybridization data (only a single hybridization represents the cggR-P25 versus wild-type strain of NC8 grown on glucose).
The genes were represented on the array with varying numbers of probes. Probe outliers in term of log2-value, were deleted and the average log2-value of the other probes were displayed in the tables. When 3 or more probes were represented, at least 2 of the probes need to have a FDR corrected p-value <0.05 in order to be displayed in the tables. Both of the probes needed to have a p-value <0.05 when less than 3 probes were represented.
Microarray data accession numbers
The L. plantarum WCFS1 11 K Agilent oligo microarrays used has the GEO accession no. GPL4318 in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo). The microarray data have been deposited and received the GEO accession no. GSE18435.
Table S1. Bacterial strains and plasmids
Strains
Escherichia coli XL10 Gold / Cloning host strain / Stratagene, USA
Lactobacillus plantarum
NC8 / Host strain, silage isolate / (Aukrust and Blom, 1992)
NC8/pSIP409/cggR / Derivative of NC8 containing pSIP409/cggR / This work
NC8/pSIP409/cggR-P25 / Derivative of NC8 containing pSIP409/cggR-P25 / This work
NC8/cggR-mutant / Derivative of NC8/cggR-mutant-cat containing a lox72 replacement of cggR / This work
WCFS1 / Single-colony isolate of L. plantarum NCIMB8826 / (Kleerebezem et al., 2003)
WCFS1/pSIP409/cggR-P25 / Derivative of WCFS1 containing pSIP409/cggR-P25 / This work
WCFS1/cggR-mutant / Derivative of WCFS1/cggR-mutant-cat containing a lox72 replacement of cggR / This work
Plasmids
pSIP409 / Emr; spp-based expression vector with PorfX::gusA / (Sørvig et al., 2005)
pSIP409/cggR / Emr; pSIP409 derivative containing cggR (NC8) / This work
pSIP409/cggR-P25 / Emr; pSIP409 derivative containing cggR (NC8) under control of P25 (Rud et al., 2006) / This work
pNZ5319 / Cmr Emr; multiple gene replacement vector for gram-positive bacteria / (Lambert et al., 2007)
pNZ5348 / Emr; cre under the control of the lp_1144 promoter / (Lambert et al., 2007)
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