- Open Access
Functional identification in Lactobacillus reuteri of a PocR-like transcription factor regulating glycerol utilization and vitamin B12 synthesis
- Filipe Santos†1, 2, 6,
- Jennifer K Spinler†3, 4,
- Delphine MA Saulnier†3, 4, 7,
- Douwe Molenaar1, 2,
- Bas Teusink1, 2,
- Willem M de Vos5,
- James Versalovic3, 4 and
- Jeroen Hugenholtz2, 6Email author
© Santos et al; licensee BioMed Central Ltd. 2011
- Received: 3 February 2011
- Accepted: 21 July 2011
- Published: 21 July 2011
Lactobacillus reuteri harbors the genes responsible for glycerol utilization and vitamin B12 synthesis within a genetic island phylogenetically related to gamma-Proteobacteria. Within this island, resides a gene (lreu_1750) that based on its genomic context has been suggested to encode the regulatory protein PocR and presumably control the expression of the neighboring loci. However, this functional assignment is not fully supported by sequence homology, and hitherto, completely lacks experimental confirmation.
In this contribution, we have overexpressed and inactivated the gene encoding the putative PocR in L. reuteri. The comparison of these strains provided metabolic and transcriptional evidence that this regulatory protein controls the expression of the operons encoding glycerol utilization and vitamin B12 synthesis.
We provide clear experimental evidence for assigning Lreu_1750 as PocR in Lactobacillus reuteri. Our genome-wide transcriptional analysis further identifies the loci contained in the PocR regulon. The findings reported here could be used to improve the production-yield of vitamin B12, 1,3-propanediol and reuterin, all industrially relevant compounds.
- Empty Plasmid
- Functional Assignment
- Chemically Define Medium
- Lactobacillus Reuteri
- Glycerol Utilization
Lactobacillus reuteri is a heterofermentative lactic acid bacterium colonizing the gastrointestinal tract (GI tract) of various mammals, including humans . It is able to convert glycerol to 1,3-propanediol in a two-step enzymatic conversion, yielding NAD+. In the first reaction, glycerol dehydratase (EC 220.127.116.11), converts glycerol to 3-hydroxypropionaldehyde requiring the presence of vitamin B12 as a coenzyme . Reuterin, a mixture of 3-hydroxypropionaldehyde isomers , is a potent antimicrobial, bestowing L. reuteri with an important growth advantage over other residents of the GI tract, such as Gram-negative enteric bacteria [5, 6].
We have shown previously that L. reuteri CRL1098 encodes the complete machinery necessary for de novo synthesis of vitamin B12 in a single chromosomal gene cluster . This cluster was shown to be very similar to that present in various representatives of γ-Proteobacteria, standing out against canonical phylogeny. Complete genome sequence analysis of the type strain of L. reuteri revealed that the region immediately upstream of the vitamin B12 biosynthesis cluster maintains a gene order similar to that of Salmonella. The functionality of this upstream region was demonstrated to also match Salmonella where the pdu gene cluster is located. The latter encodes the assembly machinery of metabolosomes and the several subunits of a large diol dehydratase that can metabolize both glycerol and 1,2-propanediol .
Also within this cluster resides a gene (lreu_1750) predicted to encode a 359 amino acid long putative transcription factor of the AraC type family, containing a typical helix-turn-helix domain. Based strictly on its conserved genomic context, this gene has been suggested to encode PocR, a regulatory protein that modulates propanediol utilization (pdu) and vitamin B12 biosynthesis in enteric bacteria [8–10]. This functional annotation, however, does not seem to be fully supported by sequence homology. And more importantly, to the best of our knowledge, it completely lacks experimental confirmation.
Here we provide the first experimental evidence to support the functional assignment of Lreu_1750. This was achieved by overexpression and inactivation of lreu_1750, assessing its impact on central carbon and energy metabolism, and on reuterin and vitamin B12 synthesis. In addition, we characterized the genome-wide transcriptional response of both constructs in comparison to their parent strains leading to the identification of the genes comprised in the PocR regulon of Lactobacillus reuteri.
Phylogenetic analysis of Lreu_1750
The physiological effects observed for the overexpression and inactivation of Lreu_1750 are all in agreement with its functional assignment as the regulatory protein PocR.
In order to probe the global regulatory role of the putative PocR of L. reuteri, we compared the transcriptomes of the deficient and overexpressing strains relative to their parent strains. Considering that (i) glycerol has been shown to induce the expression of lreu_1750, masking the effect of its overexpression; (ii) consequently the differentiating phenotype of the PocR deficient strain can be best observed under conditions in which its growth kinetics are hampered (such as in the presence of glycerol - Figure 2); and (iii) there is a large redundancy between the different transcriptome analyses carried out; most emphasis in this report has been put on the data related to the lreu_1750 overexpression in the absence of glycerol. The complete list of differentially regulated genes under all conditions assayed is available in Additional file 1: Transcriptome analysis data.
Relative expression levels of loci associated to PocR and not within its flanking region a.
Transcriptional regulator, LacI family
3-hydroxybutyryl-CoA dehydrogenase (EC 18.104.22.168)
Putative cobalt-transporting ATPase d.
Putative cobalt-transporting ATPase d.
Pyruvate dehydrogenase alpha subunit (EC 22.214.171.124)
Pyruvate dehydrogenase beta subunit (EC 126.96.36.199)
Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex (EC 188.8.131.52)
Alpha-galactosidase (EC 184.108.40.206)
Transcription regulator, Crp family
Fumarate hydratase (EC 220.127.116.11)
Histidine decarboxylase (EC 18.104.22.168)
The transcriptome studies carried out for the PocR insertion mutant were consistent with the results obtained through the overexpression of lreu_1750. We mainly observed in the PocR mutant compared to the wild-type strain, a down-regulation of the genes located within the genetic island that comprises the pdu and vitamin B12 operons (Additional file 1, Table S3). Again due to the rarity and fragility of these transcripts [7, 14] only 30 out of 58 loci are differentially expressed significantly (p-value ≤ 0.05) even though the whole region, excluding the transposase, appears co-regulated.
There is strong phylogenetic evidence supporting that the pdu and vitamin B12 synthesis gene clusters have been acquired by L. reuteri through distant horizontal gene transfer [7, 8]. The confinement of the PocR regulon to mostly one continuous stretch of the chromosome (Figure 6), with exception of the putative cobalt transporter, further substantiates this hypothesis.
In this study, we have provided experimental evidence that lreu_1750 encodes a PocR-like regulatory protein, despite its lack of sequence homology to PocR from enteric bacteria. This was achieved by overexpression and inactivation of lreu_1750, and assessment of its impact on central carbon and energy metabolism, and on reuterin, 1,3-propanediol and vitamin B12 biosynthesis. In addition, we characterized the genome-wide transcriptional response of both constructs in comparison to the wild-type leading to the identification of the genes encompassed in the PocR-like regulon of L. reuteri. The latter were found to be similar to the ones present in some representatives of γ-Proteobacteria. Ultimately, the demonstrated stimulatory effects of PocR on vitamin B12, 1,3-propanediol and reuterin synthesis could be applied to improving the production yield of these industrially relevant compounds.
Phylogenetic analysis of Lreu_1750
The sequence of Lreu_1750 (GI:148544956) was entered as a string to search for closely related homologs within available microbial genomes using the protein-protein BLAST algorithm . Relevant sequences were retrieved and aligned using ClustalW with default settings  and visualized in CLC Sequence Viewer 6.5.
Strains, plasmids, primers and cultivation conditions
Strains, plasmids and primers used in this study
Source or reference
Type strain, synonymous to ATCC 23272, DSM 20016 and F275. Human isolate.
Japanese Collection of Microorganisms (Riken, Japan)
ATCC PTA 6475
Synonymous to MM4-1A. Finnish mother's milk isolate.
Biogaia AB (Stockholm, Sweden)
EmR, pocR insertion mutant derivative of L. reuteri ATCC PTA 6475
MG1363 pepN:nisRK, cloning host.
NIZO culture collection (Ede, The Netherlands)
L. delbrueckii subsp. lactis ATCC 7830. Vitamin B12 assay indicator strain.
NIZO culture collection (Ede, The Netherlands)
Used in routine cloning and to construct pJKS100
Invitrogen (Carlsbad, CA)
L. reuteri replication origin used to construct pJKS100
CmR, pNZ8148 derivative with the nisin promoter replaced by the pepN promoter
CmR, pNZ7021 derivative harboring lreu_1750 downstream of the pepN promoter.
CmR, repA-positive temperature-sensitive derivative of pWV01
EmR, repA-negative derivative of pWV01
EmR, pORI28 derivative harboring internal fragment of gene encoding putative PocR
CmR, E. coli-L. reuteri shuttle vector
CmR, pJKS100 derivative expressing 6475 pocR gene under control of its natural promoter
5' - 3'
Amplification of lreu_1750 and addition of Kpn I site
Amplification of lreu_1750
Control of pNZ7748
TGACGGATCC TAA CACAAGCATTACCGGAGCAATTG
Amplification of internal fragment of putative pocR, addition of Bam HI site and translational stop codon
Amplification of internal fragment of putative pocR and addition of Eco RI site
LR0062 FL F
Amplification of wild-type pocR gene and natural promoter
LR0062 FL R
Amplification of wild-type pocR gene and natural promoter
Construction of putative pocR overexpression and deletion mutants
Gene lreu_1750, encoding the putative PocR in JCM1112T, was overexpressed constitutively under control of the pepN promoter in a similar fashion as previously described . A fragment containing lreu_1750 was amplified from chromosomal DNA of L. reuteri using Herculase II DNA polymerase (Stratagene, La Jolla, USA), and primers P180 and P181 (Table 2). After digestion with Kpn I, the modified amplicon was purified and cloned in pNZ7021 making use of the Kpn I and Pml I restriction sites directly downstream of the pepN promoter. The resulting plasmid, termed pNZ7748, was used directly from the ligation reactions to transform Lactococcus lactis NZ9000 by electroporation . Subsequently, pNZ7748 was purified from Lc. lactis as previously described  and, after confirming the sequence of the insert using both P181 and P182, it was used to transform L. reuteri also by electroporation .
The disruption of the putative pocR gene was carried out in L. reuteri ATCC PTA 6475, which shares an identical sequence with the type strain (JCM1112) for this region of the chromosome . This was achieved by site-specific integration of plasmid pORIpocR as described previously  using the temperature-sensitive plasmid pVE6007  as the helper plasmid. The internal fragment of the target gene was amplified by PCR using primers LR0062F-BHI and LR0062R-ERI (Table 2), and inserted into pORI28 by directional cloning using standard techniques . The resulting insertion mutant was designated 6475::pocR.
Complementation of L. reuteri 6475::pocR
An E. coli-L. reuteri shuttle vector (pJKS100) was constructed by combining an L. reuteri replicon from pLEM5 , the chloramphenicol resistance gene (CmR) from pVE6007 , the L. lactis promoter (P23) , and the pUC origin and multiple cloning site (MCS) from pCR®2.1 (Invitrogen, Carlsbad, CA). Each fragment was PCR amplified from their respective template, restriction enzyme digested and subsequently ligated to generate the final shuttle-vector, pJKS100. To create the complementation vector for 6475::pocR, the L. reuteri 6475 pocR gene with its natural promoter was PCR-amplified from genomic DNA using LR0062 FL F and LR0062 FL R primers and cloned into pJKS100 using standard techniques . Both constructs, pJKS100 and pJKS101, were electroporated seperately into L. reuteri 6475::pocR as previously described .
Fermentation conditions and substrate and product analysis
The physiological effects of the overexpression of lreu_1750 were studied in pH-controlled batch cultivations of L. reuteri pNZ7748 (lreu_1750 overexpression) and L. reuteri pNZ7021 (empty plasmid) in CDM in the presence or absence of glycerol carried out as described previously . At different time points, samples were taken for transcriptome, supernatant and vitamin B12 analysis (Figure 2). We determined the extracellular concentration of main fermentation substrates and products by HPLC, as described elsewhere [6, 30].
The comparison between the insertion mutant, 6475::pocR, and its parent strain was established in batch fermentations of LDMIIIG or MRS carried out in an anaerobic chamber (80% N2, 10% H2, and 10% CO2; Microbiology International). Transcriptome comparisons were carried out at the end of the fermentation when biomass concentration became stable.
Vitamin B12 and reuterin determination
Vitamin B12 levels were determined as described in the Official methods of analysis of AOAC International , using a bioassay with L. delbrueckii subsp. lactis ATCC 7830 as the indicator strain. Reuterin production was measured with a bioassay and carried out as previously described .
- Transcriptional analysis of the putative PocR overexpression mutant
The transcriptome of cells transformed with pNZ7748 (lreu_1750 overexpression) and pNZ7021 (empty plasmid) were compared using cDNA microarrays as previously detailed  using a hybridization scheme comprising 17 arrays in a loop-design. The following samples were hybridized per array labeled with cyanine3 and cyanine5, respectively: sta-F6 and sta-F5, sta-F7 and sta-F8, sta-F5 and sta-F7, sta-F8 and sta-F6, sta-F3 and sta-F4, sta-F1 and sta-F3, sta-F2 and sta-F1, sta-F4 and sta-F2, exp-F3 and exp-F4, exp-F1 and exp-F3, exp-F2 and exp-F1, exp-F4 and exp-F2, exp-F4 and sta-F4, sta-F3 and exp-F3, sta-F2 and sta-F8, sta-F4 and sta-F6, exp-F2 and sta-F2. Here, F1 and F5 represent completely independent biological duplicates of L. reuteri pNZ7021 cultured in the absence of glycerol; F2 and F6 represent completely independent biological duplicates of L. reuteri pNZ7748 cultured in the absence of glycerol; F3 and F7 represent completely independent biological duplicates of L. reuteri pNZ7021 cultured in the presence of glycerol; and F4 and F8 represent completely independent biological duplicates of L. reuteri pNZ7748 cultured in the presence of glycerol. The prefix exp- and sta- stand for cells harvested at mid-logarithmic and early-stationary growth phases, respectively. The custom probe design of the Agilent 11 K microarray platform (Agilent Technologies, Santa Clara, CA, USA) used is available at the Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo under accession number GPL6856, and the data obtained were deposited in the same repository under accession number GSE13289.
- Transcriptional analysis of the putative PocR insertion mutant
The transcriptome of the insertion mutant, 6475::pocR, and its parent strain were compared using two-color microarrays as previously detailed . Briefly, oligonucleotides (60-mers) were designed and synthesized for 1,966 open reading frames from a draft genome sequence of L. reuteri ATCC PTA 6475 . For expression analyses, three biological replicates of the insertion mutant and parent strain were compared. Moreover, dye-swap hybridization was performed for each comparison. Following mRNA isolation , cDNA synthesis, labeling, and hybridization were performed as previously described . Information regarding the microarray platform and data obtained is deposited at NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) under GPL7541 and GSE22926, respectively.
GenePix Pro 4.0.12 software was utilized for image analysis of the 6475 microarrays. Normalization within arrays and between arrays was performed by applying the Loess algorithm  using the Limma package  in R http://www.r-project.org. Normalized intensities were used for further analysis. The average signal intensities of three biological replicates were calculated in order to compare the relative gene expression of mutant and wild type strains. The statistical significance of differences was calculated based on variation in biological duplicates, using the eBayes function in Limma (cross-probe variance estimation) and false discovery rate (FDR) adjustment of the p-values. Only genes that were differentially expressed by least 1.5-fold with FDR-adjusted p-values lower than 0.05 were considered significant.
This work was supported by the Department of Defense through the Defense Advanced Research Projects Agency (DARPA), National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Diseases (R01 DK065075), and National Center for Complementary and Alternative Medecine (R01 AT004326). We thank Eammon Connoly for providing the L. reuteri 6475 strain, P. Hemarajata and M. Balderas for their technical efforts, and TA Misttetta for assistance in the statistical analyses of the 6475 microarray data.
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