Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Apdo. Correos 73, 46100 Burjassot, Spain1
Author for correspondence: Gaspar Pérez-Martínez. Tel: +34 96 390 0022. Fax: +34 96 363 6301. e-mail: gaspar.perez{at}iata.csic.es
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ABSTRACT |
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Keywords: sorbitol-6-phosphate dehydrogenase, sugar catabolism, gene regulation
Abbreviations: PTS, phosphotransferase system; Sor-PR, L-sorbose-1-phosphate reductase; Stol-PDh, D-sorbitol-6-phosphate dehydrogenase
a The GenBank accession number for the sequence reported in this paper is AF396831.
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INTRODUCTION |
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METHODS |
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DNA manipulation and sequencing.
To amplify genes encoding Stol-PDh enzymes from L. casei by PCR, total DNA was used as the template with two synthetic primers, gut1 (5'-GGSGGHKCMTCDGGWATTGG-3') and gut2 (5'-ACKCCVKKRATRTARCTDGC-3'), designed from the conserved sequence regions of Stol-PDhs from E. coli (Yamada & Saier, 1987 ), Klebsiella pneumoniae (Wehmeier & Lengeler, 1994
), C. beijerinckii (Tangney et al., 1998
) and L. casei (Yebra et al., 2000
). Amplified DNA fragments of the expected size were cloned into pRV300 and transformed into E. coli. Recombinant DNA techniques, plasmid DNA isolation and reverse PCR experiments were performed by standard procedures (Sambrook et al., 1989
). DNA sequencing was carried out by using the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase and an automatic ABI 310 DNA sequencer (Applied Biosystems). M13 universal and reverse primers or primers annealing within the cloned DNA were used to sequence both strands of the DNA. Alignment of the sequences and analysis of the sequence for ORFs were carried out with version 4.0 of the DNAMAN for windows (Lynnon BioSoft). Sequence similarities were analysed with the BLAST program.
Chromosomal inactivation of the Stol-PDh genes.
The 490 bp PstIHindIII sorF fragment was isolated from the original clone in pJDC9 (Chen & Morrison, 1988 ) and subcloned into the integrative vector pRV300. The resulting plasmid was digested with NheI, the generated 3' recessed ends were filled-in with the DNA Polymerase I Large (Klenow) Fragment and then it was self-ligated to render a frameshift in the sorF gene. The plasmid was then used to transform L. casei and the transformants were selected on agar plates with erythromycin. Chromosomal integration of this plasmid was confirmed by PCR analysis. Subsequently, one of the integrants was selected and used to inoculate fresh MRS medium without antibiotic. After growth for approximately 200 generations, appropriate dilutions were replicated onto agar plates with and without erythromycin. Strains that had undergone the second recombination and had therefore lost the integrated plasmid were selected as erythromycin sensitive. The frameshift mutation introduced in sorF was confirmed by PCR analysis and DNA sequencing, and one mutant strain named BL23F1 was selected.
The plasmid pRVgut3 (Table 1) containing a 720 bp PCR fragment corresponding to gutF was electroporated into L. casei and the transformants were selected on agar plates with erythromycin. Chromosomal integration of this plasmid was checked by PCR analysis and DNA sequencing. The sugar fermentation pattern of the integrants was analysed and one of them, designated BL23F2, was selected. The same procedure was used to inactivate the gutF gene in the sorF mutant (BL23F1) obtaining the double mutant gutF sorF (BL23F1-2).
Enzyme activity tests.
L. casei strains were grown in MRS fermentation medium supplemented with 0·5% of the appropriate sugar to OD550 0·8, then cells were harvested, washed and resuspended in 1 ml buffer (10 mM Tris/HCl pH 7·5, 1 mM DTT, 0·1 mM PMSF). Crude extracts were prepared by disruption of the cells with glass beads in a Fastprep apparatus (Savant Instruments) for four periods of 1 min with intervals of 1 min on ice. Sor-PR activity was determined by the method of Wöhrl & Lengeler (1990) , using 2·5 mM D-fructose 1-phosphate as substrate. Stol-PDh activity was determined as described by Yebra et al. (2000)
using 2 mM D-sorbitol 6-phosphate (D-glucitol 6-phosphate) as substrate. The rates of NADH oxidation (for Sor-PR) and NAD+ reduction (for Stol-PDh) were determined by measuring the change of absorbance at 340 nm. NADH oxidase activity was determined in Tris/HCl buffer (pH 7·5) and 0·1 mM NADH. NADH oxidation was monitored in the reaction mixture (0·5 ml) by following the absorbance at 340 nm. Specific enzyme activities are given in nmol min-1 (mg protein)-1. Protein concentrations were measured by the method of Bradford (1976)
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Southern and Northern blot analysis.
DNA probes were synthesized by PCR using L. casei chromosomal DNA as template, deoxynucleoside triphosphate mix with digoxigenin-dUTP from the Roche digoxigenin-labelling kit and Taq (Expand High Fidelity PCR System) from Roche. Each probe was amplified with two internal primers: sorR probe with sorR1 (5'-AAGGCGTTGTTTCAATTGCC-3') and sorR3 (5'-ACACGTGTCCACAGGCTAAG-3'), sorF probe with sorF1 (5'-AGCAGAGAAGATTAATGGC-3') and manR21 (5'-TAGGATAATTGGTGCGCTAAAGG-3'), gutF with gut3 (5'-CGACTTTAAGGACCCTAA-3') and gut4 (5'-GACGACAACCCCATGTCC-3'). Chromosomal DNA from L. casei (6 µg) was digested with the restriction enzymes HindIII or SphI and the same amount of chromosomal DNA from E. coli was digested with SphI. The cleaved DNAs were separated on a 0·7% agarose gel, transferred to a nylon membrane and hybridized with the probes as described in Sambrook et al. (1989) . Total RNA was isolated from L. casei cells grown in MRS fermentation medium with 0·5 % appropriate sugars to OD550 0·8. Cells were collected by centrifugation, washed with 50 mM EDTA, pH 8·0, resuspended in 1 ml TRIZOL Reagent (Gibco-BRL) and then mechanically disrupted with glass beads in a Cell Disruptor (Savant Instruments). Total RNA was isolated according to the protocol of the TRIZOL manufacturer. Sample preparation, denaturing agarose gel electrophoresis and RNA transfer were performed by standard methods (Sambrook et al., 1989
).
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RESULTS |
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Southern blot analysis
To corroborate the presence of sorF and gutF in the L. casei genome, we used internal DNA fragments of each gene as probes in Southern hybridization experiments (Fig. 2). Chromosomal DNA of L. casei cleaved with HindIII showed bands of approximately 4 kb and 1·9 kb in size when sorF was used as a probe, and a single band of about 5 kb with gutF as a probe (Fig. 2
, lanes 1a and 1b). Lanes containing SphI digests of L. casei DNA showed bands of 6 kb and 2·3 kb when sorF and gutF fragments were used as probes, respectively (Fig. 2
, lanes 2a and 2b). The bands obtained with HindIII and SphI digests had fragments with expected sizes in the regions of the L. casei genome that contain the sorF gene (Yebra et al., 2000
) or the gutF gene (genomic region known by reverse PCR analysis). The probes did not hybridize with E. coli chromosomal DNA (Fig. 2
, lanes 3a and 3b). Although the alignment of the DNA sequences of sorF and gutF genes showed a high homology (74% identity), there was no cross-hybridization after overexposing the membranes to X-ray films (data not shown).
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Growth pattern of L. casei strains
The growth pattern of L. casei wild-type (BL23) and mutant strains on MRS basal medium supplemented with L-sorbose, D-sorbitol, D-ribose or a mixture of these sugars is shown in Fig. 3. BL23F1 (sorF), BL23F2 (gutF::pRV300) and BL23F1-2 (sorF gutF::pRV300) showed the same pattern of growth on D-ribose as the wild-type (data not shown). Both BL23 and BL23F2 could grow efficiently on L-sorbose, but BL23F1 and BL23F1-2 hardly proliferated (Fig. 3a
). On MRS basal medium with D-sorbitol, BL23F1 displayed a slower growth rate than the wild-type, and BL23F2 and BL23F1-2 could barely grow (Fig. 3b
). This indicated that sorF may also participate in D-sorbitol catabolism. When L-sorbose was supplemented with D-ribose, again BL23 and BL23F2 had a similar growth pattern; however, BL23F1 could grow to a high density, although with a lower growth rate than the wild-type on D-ribose (Fig. 3c
). This could be due to a transient accumulation of sorbose 1-phosphate and sorbitol 6-phosphate in the cells, which could have a toxic effect. Growth of BL23F1-2 on L-sorbose plus D-ribose was very limited. In this strain, sorbose 1-phosphate may be accumulated with consequent toxicity for the cells. However, in the presence of D-sorbitol plus D-ribose, the double mutant showed a growth pattern similar to the wild-type with only D-ribose (Fig. 3d
).
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DISCUSSION |
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Certain Lactobacillus species can utilize L-sorbose and D-sorbitol, among them some strains of the L. caseiparacasei group (Acedo & Perez-Martínez, unpublished). During the study of the sorbose operon of L. casei CECT 5275 (formerly ATCC 393 [pLZ15-]), different mutants were obtained lacking the PTS transport elements (sorBC), the activator of the operon (sorR) and Stol-PDh (sorF) (Yebra et al., 2000 ). They constituted an excellent ground to start the study of the biochemical and possible regulatory links with the D-sorbitol metabolic pathway. In this work, it soon became evident that in L. casei BL23F1 (sorF) there was at least another Stol-PDh activity directly related to the consumption of D-sorbitol and induced by this sugar-alcohol. Then, a gene encoding a new Stol-PDh in L. casei was identified and sequenced. This gene, gutF, showed sequence differences with the previously characterized sorF, encoding the Stol-PDh of the sorbose operon (Yebra et al., 2000
). Chromosomal inactivation of these two genes and analysis of the growth patterns on L-sorbose or D-sorbitol suggested that sorF could also be involved in the metabolism of D-sorbitol and gutF would only be involved in the catabolism of D-sorbitol. These data were confirmed by the transcription analysis in the wild-type, where sorF was transcribed at a very high rate on L-sorbose and on D-sorbitol (with and without D-ribose), while gutF was only transcribed on D-sorbitol. This may indicate that sorF provides the preferred Stol-PDh in L. casei. Also a double sorF gutF mutant was generated that showed a Sor-Gut- phenotype, but interestingly, this strain still had a significant Stol-PDh activity, suggesting that yet another enzyme could be present in this micro-organism with this activity profile. Then, the negative phenotype of gutF mutants on D-sorbitol might be explained by a likely polar effect of the plasmid insertion in gutF on the transcription of other D-sorbitol metabolic genes, such as PTS transporters (Yamada & Saier, 1987
; Aldridge et al., 1997
; Boyd et al., 2000
).
The growth pattern of the different strains on liquid broth further illustrated the data obtained by growth on agar plates. BL23F1 (sorF) was unable to grow on L-sorbose; however, addition of D-ribose favoured a slow but steady growth, suggesting that L-sorbose may be transported by its own PTSSor elements or even through PTSMan (Yebra et al., 2000 ) and metabolized through the weak expression of sorE (M. J. Yebra, unpublished) and gutF (Fig. 4c
). A poisoning effect has been attributed to sorbose 1-phosphate in E. coli (Wehmeier et al., 1992
). Hence, transient accumulation of this sugar phosphate could prevent growth of this mutant on L-sorbose and may explain the growth delay when D-ribose was added. The difference in growth pattern between D-ribose plus L-sorbose and D-ribose plus D-sorbitol observed in the sorF gutF double mutant could also be explained by the toxic accumulation of sorbose 1-phosphate.
D-Ribose was chosen in this work as neutral carbon source (not inducing nor repressing sugar) to facilitate certain strains to grow on L-sorbose or D-sorbitol when they carried mutations in their respective pathways. It was also required to obtain cell batches for Stol-PDh quantification, where NADH oxidase activity was not induced. However, these experiments had an unexpected interference due to the inhibitory effect of D-ribose on the metabolism of D-sorbitol detected in the growth curves. This effect was taking place at the transcriptional level, since gutF mRNA was hardly expressed in the wild-type and sorR strains when D-ribose was present.
Additional difficulties were encountered in this work to determine Stol-PDh activity in D-sorbitol-grown cells by a standard NADH-consumption reaction, because the indigenous NADH-oxidase levels were 103-fold higher than in the control. The remarkable differences between the Stol-PDh activities of different strains grown on L-sorbose in Table 3 were possibly due to L-sorbose induction of sorbose-1-phosphate reductase in the sorbose operon. This enzyme would regenerate NAD+ and partially restore the redox balance in the cell. Full expression of sorF under these conditions, and especially of that of gutF, was also confirmed by the Northern blot assay, indicating an interesting relationship between the expression of Stol-PDh enzymes and the intracellular redox potential. This will be expanded in future studies.
The transcriptional activator of the sorbose operon in L. casei, SorR, showed significant amino acid sequence similarity to regulatory proteins such as SorC from K. pneumoniae, DeoR from Bacillus subtilis and YgaP from Bacillus megaterium (Yebra et al., 2000 ). These transcriptional regulators control the transcription of genes involved in phosphorylated sugar catabolism and, in the case of DeoR from B. subtilis, its binding to DNA is modulated by D-ribose 5-phosphate (Zeng et al., 2000
). This work confirmed previous results (Yebra et al., 2000
) indicating that SorR positively regulated expression of sorF at the transcriptional level during growth on L-sorbose. D-Sorbitol uptake in L casei occurs through PTS elements (Viana et al., 2000
), possibly yielding sorbitol 6-phosphate, as in other bacteria (Yamada & Saier, 1987
; Aldridge et al., 1997
; Tangney et al., 1998
; Boyd et al., 2000
). Findings of this work are not just related to D-sorbitol metabolism, but have a significant impact on the mechanism of regulation of the sorbose operon. Three facts indicated that D-sorbitol 6-phosphate could be the actual effector of SorR: (i) in the wild-type, D-sorbitol induced expression of sorF as strongly as L-sorbose; (ii) inactivation of sorR inhibited expression of sorF on D-sorbitol and L-sorbose, indicating that both sugars induce SorR activation through the same effector; and (iii) in strain BL23F1 (sorF), D-sorbitol led to a higher expression of sorR (SorR is known to activate also its own transcription (Yebra et al., 2000
)) possibly due to accumulation of D-sorbitol 6-phosphate.
In relation to the likely regulatory mechanism of gutF, it has been stated that D-sorbitol induced expression of gutF. In addition, when grown on D-ribose plus L-sorbose, or D-ribose plus D-sorbitol, gutF is only induced in the sorF mutant, suggesting that sorbitol 6-phosphate could also be the actual inducer of this gene. However, inactivation of sorR had no effect on the expression of gutF during growth on D-sorbitol, which indicated that this gene may rely on a regulator different from SorR.
In this paper, the genetic and functional characterization of the genes encoding two Stol-PDh, sorF and gutF, from L. casei has been achieved. Results of these studies indicated that cross-talk occurs between these metabolic pathways and the redox state of the cell and also the genes involved in the L-sorbose and D-sorbitol catabolism interact through a common effector in their regulatory mechanisms.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 January 2002;
revised 10 April 2002;
accepted 8 May 2002.
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