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Applied and Environmental Microbiology, June 2007, p. 3850-3858, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00243-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of a Gene Cluster Enabling Lactobacillus casei BL23 To Utilize myo-Inositol ^,

María Jesús Yebra,¹ Manuel Zúñiga,¹ Sophie Beaufils,² Gaspar Pérez-Martínez,¹ Josef Deutscher,² and Vicente Monedero^1*

Laboratorio de Bacterias Lácticas y Probióticos, IATA-CSIC, P.O. Box 73, 46100 Burjassot, Valencia, Spain,¹ Laboratoire de Microbiologie et Génétique Moleculaire, INRA-AgroParisTech-CNRS, F-78850 Thiverval-Grignon, France²

Received 31 January 2007/ Accepted 11 April 2007

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Genome analysis of Lactobacillus casei BL23 revealed that, compared to L. casei ATCC 334, it carries a 12.8-kb DNA insertion containing genes involved in the catabolism of the cyclic polyol myo-inositol (MI). Indeed, L. casei ATCC 334 does not ferment MI, whereas strain BL23 is able to utilize this carbon source. The inserted DNA consists of an iolR gene encoding a DeoR family transcriptional repressor and a divergently transcribed iolTABCDG1G2EJK operon, encoding a complete MI catabolic pathway, in which the iolK gene probably codes for a malonate semialdehyde decarboxylase. The presence of iolK suggests that L. casei has two alternative pathways for the metabolism of malonic semialdehyde: (i) the classical MI catabolic pathway in which IolA (malonate semialdehyde dehydrogenase) catalyzes the formation of acetyl-coenzyme A from malonic semialdehyde and (ii) the conversion of malonic semialdehyde to acetaldehyde catalyzed by the product of iolK. The function of the iol genes was verified by the disruption of iolA, iolT, and iolD, which provided MI-negative strains. By contrast, the disruption of iolK resulted in a strain with no obvious defect in MI utilization. Transcriptional analyses conducted with different mutant strains showed that the iolTABCDG1G2EJK cluster is regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor IolR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This is the first example of an operon for MI utilization in lactic acid bacteria and illustrates the versatility of carbohydrate utilization in L. casei BL23.

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myo-Inositol (MI) is the most abundant stereoisomer of inositol (1,2,3,4,5,6-cyclohexanehexol). It is common in soils, and it is a constituent of phytic acid (inositol hexaphosphate), which in the form of various salts (phytates) provides a major phosphate storage molecule in plant seeds. Several microorganisms, mostly inhabitants of soil, can utilize MI as a carbon source. For bacteria, the MI catabolic pathway has been elucidated for Enterobacter aerogenes (formerly Aerobacter aerogenes) (3). MI is important for the establishment of symbiosis in legume nodules. The metabolism of MI in Sinorhizobium fredii and Rhizobium leguminosarum is required for efficient nitrogen fixation and nodulation of soybeans (9, 15). MI dehydrogenase genes (e.g., iolG) and inosose dehydratase genes (iolE) have been characterized in S. fredii and Sinorhizobium meliloti (10, 15, 36), and iolA and iolDEB genes have been studied in R. leguminosarum (9). An MI utilization gene cluster has also been described for Clostridium perfringens, which was induced by MI via the IolR regulator (16), and recently, transcriptome analysis permitted the identification of an iol cluster which allowed the rapid growth of Corynebacterium glutamicum on MI (19). However, the first MI catabolic gene cluster was characterized in Bacillus subtilis, and detailed molecular data are available only for this bacterium. The B. subtilis iolRS and iolABCDEFGHIJ divergon and the iolT gene are involved in MI catabolism (39, 40). These genes are regulated by the product of iolR, a repressor which binds to the promoter regions and blocks transcription of the three transcriptional units in the absence of MI (41). In the presence of MI, a molecule derived from its metabolism binds IolR, thereby preventing its interaction with the DNA. The B. subtilis iol regulon is also subject to carbon catabolite repression (CCR) mediated by the catabolite control protein A (CcpA) and its corepressor, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) phosphorylated at serine residue 46 (11, 24).

MI utilization is not a common feature among lactic acid bacteria (LAB), which are defined as a group of microaerophilic, gram-positive organisms that ferment carbohydrates to produce primarily lactic acid. Taxonomically, LAB are classified within the order Lactobacillales, which includes, among others, the genus Lactobacillus. Although many lactobacilli can grow on plant-derived material, ferment hexitols, or possess phytase activity able to release phosphate from phytates (21, 28), there is no report on MI catabolism for this bacterial group. Furthermore, no genetic locus involved in MI catabolism has been characterized for members of the Lactobacillales. For example, the genome sequence of Lactobacillus plantarum reveals only the presence of an incomplete MI catabolic pathway (18), whereas genes related to MI catabolism are absent from the other 45 lactobacillales whose genomes have been partially or completely sequenced (22), including Lactobacillus, Lactococcus, Oenococcus, Pediococcus, Streptococcus, and Enterococcus strains. Nevertheless, we report here the characterization of an operon of Lactobacillus casei strain BL23 involved in MI catabolism.

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Strains and growth conditions.
Lactobacillus strains used in this study are listed in Table 1. They were grown in MRS broth (Oxoid) or MRS fermentation medium (Scharlau Chemie S.A., Barcelona, Spain) plus 0.5% (wt/vol) of different sugars at 37°C under static conditions. When required, growth was performed under agitation at 200 rpm in 50-ml Erlenmeyer flasks containing 10 ml of medium. Escherichia coli DH5 was used as a cloning host and was grown in LB medium at 37°C under agitation. The antibiotics used were 100 µg/ml ampicillin for E. coli and 5 µg/ml erythromycin for L. casei.

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TABLE 1. Strains and plasmids used in this study

Construction of strains.
To construct different L. casei mutants, internal fragments of iolR, iolT, iolD, and iolK were amplified by PCR and cloned in the integrative vector pRV300 (20). Oligonucleotide pairs were as follows (with restriction sites in italics): 5'-TTCGGTACCTTCAGAGTTTGA/5'-AACGAATTCGATGCTGGTACTG for iolR, 5'-GGCGAATTCTCGGTGATCGTG/5'-AATGGTACCAACGACAGACGT for iolT, 5'-GAAGCGTGTTCATGTTGTTA/5'-CAATAACTGCCTCAGTTTGT for iolD, and 5'-CGATCACCTGAAGAGATTAA/5'-GATCATCATTGAATGAAACG for iolK. The PCR products corresponding to iolR and iolT fragments were digested with EcoRI/KpnI and cloned into pRV300 digested with the same enzymes. iolD and iolK fragments were directly cloned into SmaI-digested pRV300. PCR amplifications were carried out with Platinum Pfx polymerase (Invitrogen). The resulting plasmids (pRViolR, pRViolT, pRViolD, and pRViolK) were used to transform L. casei BL23, and single-crossover integrants were selected by resistance to erythromycin and confirmed by PCR. To construct an iolA mutant, a 5.8-kb DNA fragment expanding from the 5' half of iolT to the 3' half of iolD was amplified with oligonucleotides 5'-GGCGAATTCTCGGTGATCGTG and 5'-CAATAACTGCCTCAGTTTGT by using the Expand-High Fidelity kit (Roche) and cloned into EcoRV-digested pMOSBlue (Amersham). The obtained plasmid (pMiolTD) was partially digested with SpeI, treated with the Klenow fragment of DNA polymerase I, ligated, and transformed. One plasmid-containing clone in which a frameshift was introduced at the SpeI site in iolA (pMiolTDSpeI) was selected. This plasmid was digested with EcoRI-HindIII, and a 2.2-kb fragment carrying the mutated iolA gene was cloned into pRV300 digested with the same enzymes, giving pRViolASpeI. L. casei was transformed with this plasmid, and one erythromycin-resistant clone carrying the plasmid integrated by a single-crossover event was grown in MRS without erythromycin for 200 generations. Cells were plated on MRS and replica plated on MRS plus erythromycin. Antibiotic-sensitive clones were isolated, and among them, one was selected (the BL276 strain) in which a second recombination event led to the excision of the plasmid leaving a mutated iolA copy, as was confirmed by sequence analysis of appropriate PCR products. Standard methods were used for cloning in E. coli. Restriction enzymes, Klenow enzyme, and T4 DNA ligase were purchased from New England Biolabs. Taq DNA polymerase for PCR screening was from Biotools (B & M Labs, Madrid, Spain). Plasmids were isolated with a GFX micro plasmid prep kit (Amersham). DNA from L. casei was isolated with an UltraClean microbial DNA isolation kit (MoBio Laboratories, Solana Beach, CA). L. casei BL23 was transformed by electroporation with a Gene Pulser apparatus (Bio-Rad) as previously described (27).

Northern blots.
For RNA isolation, strains were grown in 10 ml of MRS fermentation medium with different sugars under agitation until an optical density at 550 nm of 0.8 to 0.9 was reached. Cells pregrown in MRS fermentation medium plus either ribose or MI were used as inocula (starting optical density at 550 nm was 0.05). Cells were collected by centrifugation, washed with 10 ml of 50 mM EDTA, pH 8, and resuspended in 1 ml of TRIzol (Gibco). One gram of 0.1-mm glass beads was added, and cells were broken with a MiniBeadbeater apparatus (Biospec Products, Bartlesville, OK). RNA was isolated as described by the manufacturers of TRIzol, and 5-µg aliquots were separated on formaldehyde-agarose (1%) gels in a buffer with a pH of 7 and containing 40 mM MOPS (morpholinepropanesulfonic acid), 10 mM acetate, and 1 mM EDTA. RNA was transferred to Hybond N+ membranes (Amersham) and hybridized with digoxigenin-labeled probes. Probes were synthesized by PCR using the same oligonucleotides designed for gene disruption and the PCR-digoxigenin labeling mix from Roche. Hybridization, washing, and detection were performed as recommended by the supplier with the CDP-Star (Roche) chemiluminescent reagent.

MI dehydrogenase assays.
Cells of strain BL255 were grown in the presence of either ribose, glucose, ribose plus MI, or ribose plus MI plus glucose. The preparation of crude extracts and enzyme assays were carried out as previously described (6), except that 500-µl instead of 100-µl volumes of crude extracts were used for each assay.

Nucleotide composition similarity and phylogenetic analysis.
In order to determine the nucleotide composition similarity between the iol gene cluster and the complete L. casei ATCC 334 genome sequence, dinucleotide composition differences were estimated by comparing their * values calculated using the -web server (31). Briefly, the * value of the iol gene cluster was compared to the * values of fragments of the host genomes that were equal in size. A high compositional dissimilarity between the gene cluster and the host genome is reflected by a large percentage of genomic fragments with lower * values than the * value for the cluster.

Bacterial genes encoding homologues of IolD, IolE, IolG, and IolK were retrieved from whole genomes (NCBI repository, June 2006) by using BLASTP and TBLASTN (1, 2), and the genes carried by L. casei were used as query sequences. The data sets were refined by excluding redundant sequences, sequences that shared significant similarities in fewer than 80% of their coding sequences, and sequences with expected values (E) of more than 10^–102 (iolD), 10^–9 (iolE, iolG1, and iolG2), and 10^–1 (iolK). Some sequences were modified as follows. A possible frameshift in the putative iolE sequence of Bacillus anthracis strain Sterne (GenBank accession number AAT54648) was corrected by inserting one A between positions 2339181 and 2339186 and moving the start site to position 2339008 in the genome nucleotide sequence (accession number NC_005945). The start site of B. subtilis iolD was moved to position 4080027 in the sequence corresponding to accession number NC_000964. The start site of the iolE homologue RHE_PF00446 of Rhizobium etli (labeled as retli1 [see Fig. 3]; YP_473063) was moved to position 499306 in the sequence corresponding to accession number NC_007766. The start site of the iolG homologue RHE_CH01209 of Rhizobium etli (labeled as retli1; YP_468741) was moved to position 1261472 in the sequence corresponding to accession number NC_007761. A possible frameshift in the putative coding sequence of Salmonella enterica serovar Typhimurium LT2 iolD (accession numbers AAL23251 and AAL23252) was corrected by inserting one N after position 1342 in the sequence corresponding to accession number AE008908. The resulting translated products were used in this study. Multiple alignments were obtained using ClustalW (29) and manually corrected where necessary. Positions of doubtful homology or that introduced phylogenetic noise were removed by using Gblocks software (4). Phylogenetic reconstructions were performed by using maximum-likelihood inference as implemented in PHYML version 2.1b (14). We used the WAG substitution matrix (34), with a discrete gamma function with four categories plus invariant sites to account for substitution rate heterogeneity among sites and empirically estimated amino acid frequencies.

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FIG. 3. Phylogenetic reconstruction of iolE genes. The tree is arbitrarily rooted as indicated by the black triangle. Asterisks indicate sequences that have been modified as indicated in Materials and Methods. Paralogous genes harbored by the same organism are indicated by labels following the accession numbers. The arrows indicate the positions of genes harbored by lactobacilli.

Nucleotide sequence accession number.
The nucleotide sequence reported in this work has been deposited in the EMBL-GenBank under accession number EF382358.

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Identification and analysis of the L. casei BL23 iol operon.
Comparison of the incomplete genome sequence of L. casei strain BL23 (17) with that of the newly proposed type strain L. casei ATCC 334 (GenBank accession number CP000423.1) (22) revealed that BL23 carries numerous DNA insertions, which accounts for the difference in genome size between the two strains. One of these insertions consisted of a 12.8-kb-long DNA stretch located between a B. subtilis yvgN homologue and a putative gluconate utilization operon (Fig. 1). At both ends, the insert was flanked by a 19-bp sequence (CAACGGATTTCTAAGGCAA), whereas only a single copy of the 19-bp sequence was found in the ATCC 334 strain between yvgN and the gluconate operon (Fig. 1). This suggests that insertion in BL23 probably took place by recombination or that excision in ATCC 334 was caused by homologous recombination between the two direct repeats. Sequence analysis revealed that the inserted DNA probably contains genes involved in MI metabolism. The insertion starts with a gene encoding a DeoR-type transcriptional repressor (iolR). Ten genes were found to be transcribed in the opposite direction: iolT (MI transporter), iolA (malonate semialdehyde dehydrogenase), iolB (unknown function), iolC (2-deoxy-5-keto-D-gluconate kinase), iolD (encoding an enzyme resembling acetolactate synthase), iolG1 and iolG2 (encoding two MI dehydrogenases exhibiting similarity to each other), iolE (inosose dehydratase), iolJ (2-deoxy-5-keto-D-gluconic acid-6-phosphate dihydroxyacetone-phosphate lyase), and iolK (encodes a protein belonging to the tautomerase family). The phylogenetic analysis of iolK and homologous sequences (see Fig. S1 in the supplemental material) revealed a close relationship between the product of iolK and a malonate semialdehyde decarboxylase encoded by Pseudomonas pavonaceae (26). This result suggests that IolK also has malonate semialdehyde decarboxylase activity.

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FIG. 1. Structural organization of the L. casei BL23 iol operon. The structure of the genes in the inserted DNA region of L. casei BL23 (12.8 kb) is presented in comparison to that of L. casei strain ATCC 334. The structures of the L. plantarum, B. subtilis, and C. glutamicum iol clusters are also shown. The positions of the 19-bp direct-repeat sequences flanking the inserted DNA are marked by boxes with diagonal stripes. Stem-loop structures in L. casei DNA represent putative transcriptional terminators. Horizontal solid bars represent DNA fragments used as probes and for gene disruption.

Hence, the L. casei iol gene cluster codes for all the enzymatic activities comprising the classical MI bacterial catabolic pathway, leading to the production of acetyl-coenzyme A (CoA) and dihydroxyacetone-phosphate (Fig. 2). However, the presence of a putative malonate semialdehyde decarboxylase (encoded by iolK) suggests that malonic semialdehyde, one of the products of the activity of the IolJ enzyme, can be metabolized through two different reactions. The first one would be catalyzed by the product of iolA with the formation of acetyl-CoA, whereas the second reaction would involve the product of iolK and lead to the formation of acetaldehyde (Fig. 2).

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FIG. 2. Proposed MI catabolic pathway in L. casei BL23. For the B. subtilis iol genes, the functions of only iolT, iolG, and iolE have been experimentally elucidated. The functions of the other genes are assigned based on sequence homology, and their gene products are underlined. The function of iolB is unknown, but in Legionella pneumophila, iolB is fused to iolC (accession number YP_123940), which is probably coding for a 2-deoxy-5-keto-D-gluconate kinase. In L. casei, iolK (encoding a probable malonic semialdehyde decarboxylase) might define an alternative pathway for the conversion of malonic semialdehyde to acetaldehyde. The metabolites are numbered as follows: (1), MI; (2), inosose; (3), D-2,3-diketo-4-deoxy-epi-inositol; (4), 2-deoxy-5-keto-D-gluconate; (5), 2-deoxy-5-keto-D-gluconate-6-phosphate; (6), dihydroxyacetone-phosphate; (7), malonic semialdehyde; (8) acetyl-CoA; (9), acetaldehyde.

L. casei BL23 was able to ferment MI on MRS indicator agar plates containing 0.5% (wt/vol) of the polyol, whereas ATCC 334 was not. However, fermentation was observed only under aerobic conditions. Growing the cells in MRS liquid medium plus MI under static conditions did not result in acidification and gave only poor growth, whereas strong agitation resulted in acid production. MI fermentation was also tested on MRS indicator plates with seven other L. casei strains. Aside from BL23, three other strains (ATCC 4646, ATCC 11578, and 61BG) were able to produce acid from MI (Table 1). Amplification by PCR of iolT and iolD by using BL23-derived oligonucleotides was also possible in these MI-proficient L. casei strains (data not shown), suggesting that homologous iol genes are involved in MI catabolism in these strains.

To ascertain that the iol genes were responsible for the observed phenotype in BL23, disruptions of iolT (BL254), iolD (BL264), and iolK (BL265) were introduced in BL23 by integration of the pRV300 plasmid (20) carrying internal fragments of the respective genes. The iolT and iolD mutants failed to ferment MI, demonstrating that these genes are essential for MI catabolism. However, disruption of iolK had no significant effect on MI fermentation. A third mutant was constructed by a double-crossover event, which introduced a frameshift at the level of iolA (BL276). In this strain, no plasmid vector remains integrated in the operon, and therefore minor effects on the expression of downstream iol genes are expected. This mutant was unable to produce acid from MI on agar plates, suggesting that blocking the conversion of malonic semialdehyde to acetyl-CoA prevents MI utilization.

Phylogenetic analysis of iol genes.
Among all LAB genomes present in databases, including the recently published genomes of nine members of the Lactobacillales (22), homologues of iol genes were detected only in L. plantarum. The iol gene cluster harbored by L. plantarum shows significant differences from the iol cluster of L. casei BL23 (Fig. 1): L. plantarum has four iolG paralogues, iolE, and two genes encoding putative sugar permeases, while it lacks iolA, iolB, iolC, iolD, iolJ, and iolK. In agreement with this fact, L. plantarum NCIMB8826 and L. plantarum ATCC 14917 (type strain) were not able to ferment MI (data not shown).

In order to estimate whether L. casei recently acquired the iol gene cluster by horizontal gene transfer (HGT), the genome signatures were compared by calculating the genome signature dissimilarity scores (* values) between the iol gene cluster and the genome sequence of L. casei ATCC 334 (31). The * value of the iol cluster was 0.029. The percentage of genomic fragments of equal lengths exhibiting less genomic dissimilarity to the genome as a whole was 62.95%. This value indicates that the iol cluster does not differ significantly from the whole-genome composition (31). By contrast, similar analyses using the genome sequences of Bacillus subtilis 168, Bacillus halodurans, or Clostridium perfringens 13 instead of the L. casei BL23 genome sequence gave significant differences (percentages of less-dissimilar fragments were 88.99%, 84.7%, and 100%, respectively).

In order to gain insight into the origin and evolution of the L. casei iol cluster, a phylogenetic analysis of iolD, iolE, and iolG homologues was carried out. Both iolE and iolD genes rendered phylogenetic reconstructions with high support values even in deep nodes (Fig. 3 and see Fig. S2 in the supplemental material). In contrast, the iolG phylogenetic tree was poorly resolved (see Fig S3 in the supplemental material). The fact that many strains contain several iolG paralogues may partly account for this result. The phylogenetic trees show that none of the three genes studied grouped according to the order of descent based on phylogenetic markers such as 16S rRNA. Furthermore, the congruence among the three trees is also low. HGT possibly accounts for many of the observed discrepancies. For example, the location of the iolE and iolG genes of Salmonella enterica serovar Typhimurium LT2 close to their homologues from L. plantarum in clusters of genes of the Firmicutes indicates that iolE and both iolG paralogues were probably acquired from L. plantarum (Fig. 3 and see Fig. S2 in the supplemental material). In contrast, Salmonella serovar Typhimurium iolD is closely related to genes from other members of the Gammaproteobacteria (see Fig. S3 in the supplemental material). This result, together with the great variation observed in both gene content and order in iol clusters (see Fig. S4 in supplemental material), makes it very difficult to track down the origin of these clusters.

The positions of the L. casei genes analyzed here show a close relationship with those of their counterparts from the Firmicutes. However, significant differences can be observed from one gene to another: iolD appears as a basal branch of a cluster with strong support which contains genes from Bacillus and Listeria (see Fig. S3 in the supplemental material). In contrast, L. casei iolE occupies an uncertain position in a cluster which includes all genes from the Firmicutes as well as genes from the Gammaproteobacteria, one of the iolE genes present in Rhizobium etli, and two iolE homologues from the Cyanobacteria (Fig. 3), whereas L. plantarum iolE is closely related to its counterparts harbored by Bacillus clausii, B. halodurans, Bacillus licheniformis, and B. subtilis. Finally, the two L. casei iolG genes are only distantly related to each other (see Fig. S2 in the supplemental material). Therefore, these genes did not originate from a recent duplication event. Gene iolG1 is closely related to idh genes from B. subtilis and other bacilli as well as to iolG2 from L. plantarum. On the other hand, L. casei iolG2 is related to genes harbored by clostridia, B. cereus, and B. halodurans and more distantly related to L. plantarum iolG1 and one of the iolG homologues harbored by Mycoplasma hyopneumoniae (mioJ1).

Transcription of iol genes is induced by MI and repressed by IolR.
Northern blot analysis showed that iol genes were transcribed only in the presence of MI and that their transcription was repressed by the presence of glucose (Fig. 4A). Similar results were obtained when either iolT, iolD, or iolK probes were used (not shown). On the contrary, Northern blot analysis with an iolR probe showed that the iolR messenger was present at similar levels irrespective of the carbon source used for growth (Fig. 4B). The size of its transcript (1 kb) was in agreement with an mRNA covering the whole iolR gene and expanding up to the position of a putative rho-independent terminator (–23.2 kcal/mol) placed at the end of the insertion point (Fig. 1). Except for iolR, no putative transcriptional terminator was found in the inserted region, and some of the stop codons of the iol genes overlapped start codons or Shine-Dalgarno sequences of the following genes, suggesting translational coupling. This indicates that the iolTABCDG1G2EJK cluster is transcribed as a long mRNA (11 kb) that terminates at a stem-loop structure (–19.5 kcal/mol) located 36 bp downstream from the insertion point (Fig. 1). This stem-loop could also serve as a rho-independent terminator for the gntR gene, which is transcribed in the opposite direction in both the BL23 and ATCC 334 strains. Northern blot analysis did not allow detection of the putative long iol mRNA, and most of the signal was at the level of ribosomal RNAs (Fig. 4A and 4C), indicating comigration or degradation of the RNA, a problem usually found when analyzing long Lactobacillus transcripts (35).

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FIG. 4. Northern blot analysis of the transcription of iol genes in L. casei BL23. RNA was isolated from strains grown on glucose (g), MI (i), glucose plus MI (g+i), MI plus ribose (i+r), and ribose (r) and hybridized with probes covering iolR and iolT. The culture medium for RNA isolation was inoculated with cells pregrown on MI. A stain of the rRNA in each membrane is shown below the blots. Strains used were BL23 (wild type), BL121 (ptsH1), BL190 (ccpA), BL255 (iolR), and BL256 (ptsH1 iolR).

The iolR gene was inactivated by the integration of a suicide plasmid, giving strain BL255. Transcription of the other iol genes in this mutant occurred independently of the presence of MI, thus supporting the concept that IolR is a transcriptional repressor (Fig. 4A). In this mutant, the presence of glucose in the culture medium caused a decrease in messenger levels, indicating that transcription was also regulated by CCR (Fig. 4A).

Unfortunately, no quantitative measurements of MI dehydrogenase activity were possible, owing to the high NADH oxidase activity present in L. casei crude extracts. In contrast to that in B. subtilis strains (6), MI oxidation was not measurable in crude extracts prepared from BL23 cells grown in the presence of MI. Nevertheless, MI dehydrogenase activity was clearly measurable in the iolR mutant grown either on ribose or ribose plus inositol (enzyme activities within the first 30 seconds were in the order of 20 nmol of product formed per min and mg of protein but rapidly leveled off). Under repressing conditions, when the growth medium was supplemented with MI and glucose, MI dehydrogenase activity was no longer measurable, not even during the first 30 seconds. The enzymatic assays at least qualitatively confirmed the results obtained by Northern blot analyses.

Regulation of iol genes by the PTS/CcpA pathway.
A sequence resembling a cre site (AGTAAGCGCTTAAT), the binding site of the CcpA/P-Ser-HPr complex (30), was found in the iolT promoter region, suggesting that CCR of the iol operon was mediated via the PTS/CcpA pathway (5). To prove this hypothesis, RNA was isolated from strains BL190 (ccpA deletion mutant) and BL121 (a strain carrying the ptsH1 mutation and therefore producing HPr, which cannot be converted to P-Ser-HPr, the CcpA corepressor, as the Ser-46 residue is replaced with Ala) (32). None of these mutations provided relief from the glucose repression of iol genes, as no expression was detected in a medium containing glucose plus MI (data not shown). To avoid the possibility that the iol genes were not yet induced at the moment of RNA isolation due to the rapid growth provided by glucose, similar experiments were repeated by inoculating liquid medium with cells which had previously been grown on MI, a condition where the iol genes should be fully induced. Again, no transcription was detected in the presence of glucose in any of the CCR mutants, whereas the addition of ribose (nonrepressing sugar) allowed the normal induction of the iol genes (Fig. 4C). Glucose repression in a ccpA or ptsH1 background might be mediated by an inducer exclusion effect on MI that would act at the level of IolR (the presence of glucose blocks MI transport and hence prevents the induction of the iol genes). In order to clearly demonstrate the involvement of the PTS/CcpA pathway in the CCR of iol genes, the iolR gene was inactivated in a ptsH1 background, providing the BL256 (ptsH1 iolR) double mutant. A complete relief of glucose repression was found in this strain (Fig. 4C). Unfortunately, we could not study the CCR effect in a ccpA iolR double mutant because, for unknown reasons, this strain could not be constructed.

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We showed that, in contrast to ATCC 334, L. casei BL23 carries a complete set of genes for the metabolism of MI. However, L. casei BL23 was able to metabolize MI only under aerobic conditions. This is in agreement with the finding that the fermentation of other hexitols (sorbitol, mannitol) was improved when the L. casei cells were grown under aeration (M. J. Yebra, unpublished observations). In addition, an L. casei mutant with impaired NADH oxidation (lactate dehydrogenase-deficient mutant) grew significantly faster when the culture was aerated (33). The degradation of one MI molecule produces two NADH equivalents in the course of the first enzymatic reactions, leading to the formation of acetyl-CoA and dihydroxyacetone phosphate (the dehydrogenase step catalyzed by IolG and the conversion of malonic semialdehyde to acetyl-CoA catalyzed by IolA). Therefore, an elevated NADH/NAD⁺ ratio was possibly responsible for the poor growth on MI under static (anaerobic) conditions. By contrast, growth under aeration can be enhanced by the activity of O₂-dependent NADH oxidases that could reoxidize excess NADH. Although there is no experimental evidence of O₂-dependent NADH oxidase activity in L. casei, the inspection of the available genome sequence of L. casei BL23 shows that this organism encodes at least three putative NADH oxidases (data not shown). Furthermore, there is evidence of the involvement of NADH oxidases in NAD⁺ regeneration in other lactobacilli (13, 23).

The scarceness of iol clusters in LAB, together with the relative similarity of the L. casei iol cluster to iol clusters harbored by other Bacillus species (Fig. 1), suggested that L. casei might have acquired iol genes from Bacillus by HGT. Nevertheless, although phylogenetic analyses place the origin of the L. casei iol cluster in the Firmicutes, our results do not support the idea that L. casei acquired the iol cluster by a recent HGT from one of the organisms discussed here. First, the calculation of the * values showed that the L. casei iol cluster does not significantly differ from the whole-genome composition, thus arguing against a recent acquisition of the iol cluster by HGT. This result also suggests that the absence of the iol cluster in L. casei ATCC 334 is due to a deletion in this strain. Furthermore, the phylogenetic analyses suggest that the iol clusters harbored by L. casei and L. plantarum either do not come from a common ancestor or have sustained major modifications during the evolutionary history of these species. These changes would at least include the loss and/or gain of genes and nonorthologous displacements (iolE, for example). The data presently available do not allow the determination of which possibility is more probable. In summary, our results indicate that the iol cluster was acquired by L. casei from a Firmicutes donor in a relatively early stage of the evolution of this species, and it was subsequently lost in some strains. This is in agreement with the current view of the evolution of the Lactobacillales, in which the adaptation of LAB to nutrient-rich habitats was accompanied by significant gene losses and the emergence of new genes via duplication and HGT (22).

Despite its similarity to that of B. subtilis, the L. casei iol operon contains distinct features related to gene content and regulation. (i) In L. casei, a putative malonic semialdehyde decarboxylase gene is transcribed with the other iol genes. The gene encoding this enzyme was first described in Pseudomonas pavonaceae and was clustered in an operon involved in the degradation of the xenobiotic trans-1,3-dichloropropene (26). The presence of this gene in L. casei suggests an alternative pathway for the metabolism of malonic semialdehyde different from the classical oxidative decarboxylation catalyzed by the product of iolA (Fig. 2). The B. subtilis genome contains two iolK homologues (yrdN and yodA) which are not clustered with iol genes. The phylogenetic analysis suggests that the products of these genes also have malonate semialdehyde decarboxylase activity. However, it remains unknown whether these genes are involved in MI catabolism in B. subtilis. Inactivation of iolK and iolA showed that, while iolA was indispensable for MI fermentation, a mutation in iolK caused no apparent phenotype under our laboratory conditions. The physiological relevance of each gene is not known, but it can be postulated that each pathway could make a different contribution to the catabolism of malonic semialdehyde depending on the growth conditions (anaerobic conditions might favor the IolK pathway). (ii) Three genes belonging to the B. subtilis MI regulon are absent from L. casei BL23. L. casei does not possess iolH, iolI, or iolS homologues. The functions of iolH and iolS are not known, but iolI encodes an inosose isomerase, which converts inosose to 1-keto-D-chiro-inositol (38). In any case, none of these genes is essential for MI utilization. Furthermore, B. subtilis has two MI transporters encoded by iolF and iolT (39). The transporter encoded by iolT has a higher affinity, but a complete loss of MI utilization is obtained only upon the mutation of both genes. Similarly, two MI transporters which exhibited comparable kinetic parameters (IolT1 and IolT2) have been identified in C. glutamicum (19). It is not known whether iolT is the only gene involved in MI uptake in L. casei BL23. The disruption of iolT renders an MI-negative phenotype, but the integration of a nonreplicative plasmid at this locus (strain BL254) probably leads to polar effects on the other iol genes (except iolR). In addition, L. casei carries two different inositol dehydrogenase-encoding genes (iolG1 and iolG2). Phylogenetic analysis does not support the idea that duplication would be the cause for the two iolG genes. It is not known at this stage whether they have redundant functions or possess different substrate affinities toward inositol isomers or related molecules. (iii) The B. subtilis iol regulon is repressed by IolR, and it is subject to CCR regulation mediated by CcpA. Both mechanisms result in a repression ratio (i.e., of the expression on MI to the expression on MI plus glucose) of greater than 100 (6, 11, 24). iolR transcription in B. subtilis and C. perfringens is induced by MI (16, 40), whereas its transcription in L. casei was constitutive. Our results suggest that IolR and CcpA act synergistically to regulate iol genes in L. casei. However, regulation in L. casei occurs mainly through IolR, whereas CcpA-dependent repression plays a minor role. A cre sequence is present in the promoter region, but the CCR effect triggered by CcpA was weak and became visible only after the comparison of the iolR and iolR ptsH1 mutants. By contrast, Deutscher et al. (6) reported that a ccpA mutation completely abolished the glucose repression of iol genes in B. subtilis. However, residual repression (about 10% to that of the wild type) was detected in a ccpA background by Yoshida et al. (37). The authors claimed that this discrepancy might be due to the different compositions of the growth media and pointed to inducer exclusion as the phenomenon responsible for the residual CcpA-independent glucose repression. Inducer exclusion might also be responsible for glucose repression in the L. casei ccpA or ptsH1 mutants. This regulatory mechanism has been studied in LAB for the transport of maltose, ribose, and galactose (7, 32) and shown to be dependent on P-Ser-HPr, which interacts with and blocks non-PTS transporters. Because the repression of iol genes is still operative in an L. casei ptsH1 mutant unable to form P-Ser-HPr, a P-Ser-HPr-independent mechanism needs to be operative to explain the regulation of the iol operon in L. casei.

Despite the fact that a large number of putative iol genes are found in genome sequences, there is only limited information available about their functionality, and only a few of them have been characterized. In this work, we identified and analyzed an iol gene cluster of L. casei BL23 and demonstrated its implication in MI utilization in this bacterium. The physiological role of the L. casei BL23 MI pathway is probably not related to phytate utilization, as no phytase-encoding gene has been found in ATCC 334 or BL23 genomes. Phytates have been reported to enhance the growth of certain lactobacilli, but this result might be a consequence of the increased availability of Mg²⁺ or Fe³⁺ generated by phosphate removal from phytates (21, 25). L. casei and lactobacilli in general thrive in complex niches characterized by the availability of numerous fermentable sugars, such as the gastrointestinal tract and decaying plant material. The iol genes may offer some advantage to L. casei BL23 in the latter niche, where MI is abundant.

 
This work was partially supported by CSIC/CNRS cooperation project grant 2004FR0018. Genome sequencing of L. casei BL23 was carried out at the University of Caen, Laboratoire de Microbiologie de l'Environnement; at the INRA Thiverval-Grignon, Microbiologie et Génétique Moléculaire; and at the Instituto de Agroquímica y Tecnología de Alimentos, CSIC, with financial aid from the Region Basse Normandie and the INRA. During the preparation of this paper, Josef Deutscher spent a sabbatical at the University of Göttingen, Abteilung für Allgemeine Mikrobiologie, and was supported by the Alexander von Humboldt Foundation.

 
* Corresponding author. Mailing address: Laboratorio de Bacterias Lácticas y Probióticos, IATA-CSIC, P.O. Box 73, 46100 Burjassot, Valencia, Spain. Phone: 34 963900022. Fax: 34 963636301. E-mail: btcmon{at}iata.csic.es

Published ahead of print on 20 April 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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