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Copyright © 2000, American Society for Microbiology Design and Evaluation of a Lactobacillus manihotivorans Species-Specific rRNA-Targeted Hybridization Probe and Its Application to the Study of Sour Cassava Fermentation *Mailing address: LBMRPM, CNRS-INRA BP 27, 31326 Castanet-Tolosan Cedex, France. Phone: 33 5 61 28 50 54. Fax: 33 5 61 28 50 61. E-mail: Frederic.Ampe/at/mpl.ird.fr. Received November 15, 1999; Accepted February 23, 2000. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Abstract Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cassava plays an important role in tropical agriculture; it is a staple food for many countries and is a source of raw material for agroindustrial development (9, 10). However, once the roots have been harvested, they are quickly oxidized and their storage is difficult. To solve this problem, a large part of the cassava roots destinated for human consumption is transformed by spontaneous lactic acid fermentations (6, 15). In Colombia and Brazil, the roots are washed, peeled, and grated, and the starch suspended in water is separated, by sieving, from the pulp or bran. The wet starch is passed through a series of tanks, where a natural lactic microflora develops, and, finally, the fermented starch is sun dried (11, 19). Interestingly, this process yields a cassava sour starch capable of going through a bread making process, whereas cassava starch that was not fermented and sun dried is not suitable for making bread. Recently, two strains of a new amylolytic species, Lactobacillus manihotivorans, have been isolated from this fermented food (14). Random amplified polymorphic DNA analysis was capable of discriminating strains isolated from sour cassava, including L. manihotivorans (N. ben Omar, F. Ampe, M. Raimbault, J.-P. Guyot, and P. Taillez, submitted for publication). However, such fingerprints cannot yield quantitative information on the ecological importance of this species, especially because (i) the isolation procedure is selective and only a fraction of the strains are isolated, and (ii) the study of a great number of samples would require the isolation and characterization of thousands of strains (4). So far, it is not known whether L. manihotivorans plays a significant role in this fermentation. Molecular tools such as hybridization probes based on rRNA sequences are well suited to overcome these limitations (8, 18). Therefore, we designed and validated a hybridization probe specific for L. manihotivorans to monitor this species in cassava fermentations. Probe design. The 16S rDNA sequences of L. manihotivorans and closely related lactic acid bacteria were aligned as described by Morlon-Guyot et al. (14), and the target region used for probe design is presented in Fig. 1. The four sequences available for L. manihotivorans strains had identical target sequences (region bp 207 to 227 in Escherichia coli consensus numbering). In that region, at least three mismatches and gaps were found for all the sequences of the closely related lactobacilli. On the basis of these observations, we developed an L. manihotivorans species-specific probe (5′-CAAAAGCGACAGCTCGAAAG-3′), named S-S-Lbma-0207-a-A-20 according to the Oligonucleotide Probe Database nomenclature (1). A computer-assisted specificity control of the S-S-Lbma-0207-a-A-20 probe was then performed (the last control was in September 1999) by using the probe match facility function available with the Ribosomal Database Project facilities (13). The probe sequence excludes by at least three mismatches all nontarget rRNA sequences available in the database.
Probe validation with rRNA from pure cultures. Probe specificity was assessed by using total RNA extracted from pure cultures of 64 microbial strains, including reference strains and strains isolated during sour cassava fermentation. All strains were grown in appropriate rich media, and total RNA was extracted as previously described (2). RNA (100 ng) was blotted on two identical nylon membranes and was hybridized with two oligonucleotide probes: probe S-S-Lbma-0207-a-A-20 and probe S-*-Univ-1390-a-A-18 (5′-GACGGGCGGTGTGTACAA-3′), targeting all organisms (20). Synthetic high-pressure liquid chromatography-purified oligonucleotides (Eurogentec, Seraing, Belgium) were 3′ end labeled with digoxigenin by following the instructions of the manufacturer (Boehringer Mannheim). After hybridization, two stringent washes with 1× SSC (0.15 M NaCl plus 0.015 M sodium citrate) and 1% sodium dodecyl sulfate (washing temperature [Tw] = 44°C for both probes) were performed as described elsewhere (18), and chemiluminescence was detected according to the manufacturer's instructions (Boehringer Mannheim). Without exception, the probe S-S-Lbma-0207-a-A-20 was specific for L. manihotivorans in the experimentally determined stringent washing conditions (Tw = 44°C) in comparison with similar hybridizations with the universal probe (Table 1). This was true for strains from culture collections, but also for strains isolated from sour cassava fermentation. Probe S-S-Lbma-0207-a-A-20 hybridized with the eight strains identified as L. manihotivorans and with none of the other 56 strains that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Oenococcus, Streptococcus, and Weissella, as well as some non-lactic-acid bacterium microorganisms. Nontarget Lactobacillus species were L. amylophilus, L. amylovorus, L. brevis, L. buchneri, L. casei, L. cellobiosus, L. curvatus, L. delbrueckii, L. fermentum, L. hilgardii, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, L. sakei, L. salivarius, and L. sharpeae. TABLE 1 Membrane-based analysis of S-S-Lbma-0207-a-A-20 probe specificity with RNA from reference strains and strains isolated from sour cassava starch
aUniv, probe S-*-Univ-1390-a-A-18; Lbma, probe S-S-Lbma-0207-a-A-20; +, hybridization signal; −, no signal; *, strains isolated from sour cassava fermentation. Application of the probe to quantify L. manihotivorans in sour cassava fermentation. The designed probe was then used to quantify L. manihotivorans in samples taken at different times in four independent fermentations of cassava. Samples were taken from traditional sour starch fermentation tanks in Marilopez, Colombia, with four different varieties of cassava (Mper 183, CM 6740-7, Mbra 383, and Venenosa). The strategy first developed by Stahl et al. (18) and successfully applied to the fermentation of maize (3) was used to quantify L. manihotivorans rRNA. Total RNA was extracted from sour cassava starch by using a previously optimized method (2). As cassava RNA may be present in the sample, we could not use a universal probe to quantify total microbial RNA. Also, the very high majority of microorganisms responsible for the fermentation of cassava sour starch are lactic acid bacteria (5). Therefore, we reported the hybridization signals obtained with probe S-S-Lbma-0207-a-A-20 to those given by a probe targeting all lactic acid bacteria (probe S-*-Lab-0722-a-A-25 [5′-YCACCGCTACACATGRAGTTCCACT-3′], Tw = 54°C) (17). Pure RNA samples from L. manihotivorans and L. plantarum were used as standards. The RNA content of these standards was estimated by hybridization with the universal probe S-*-Univ-1390-a-A-18 (20) with E. coli RNA (Boehringer Mannheim) as absolute standard. Serial twofold dilutions of the standards and the sample RNAs were blotted on two identical membranes and were hybridized with probes S-S-Lbma-0207-a-A-20 and S-*-Lab-0722-a-A-25. The lower limit for detecting a unique small-subunit (SSU) rRNA in the 2-μg sample of nucleic acid spotted on the membrane was approximately 5 ng of SSU-like rRNA (i.e., a threshold of 0.25%), and the quantification of the chemiluminescence signals was linear on a 2-log scale. Results demonstrate that L. manihotivorans was present in the four independent fermentations performed (Table 2). This species could account for up to 20% of the total lactic acid bacterium population (in terms of RNA index). The initially low number of L. manihotivorans apparently increased during the first week to reach maximum values after 6 to 15 days of fermentation. TABLE 2 Importance of L. manihotivorans in cassava sour starch from four independent fermentations using quantitative dot blot measurements of 16S
aThe probes used (S-S-Lbma-0207-a-A-20 and S-*-Lab-0722-a-A-25) were digoxigenin-labeled for hybridization, and the control RNA used for standardization was from strains L. manihotivorans Ond32 and L. plantarum Ond5. Results are given as the relative percentage (± standard deviation) in comparison with total lactic acid bacteria (five repetitions of the quantification procedure were performed on single RNA extractions). The detection limit was 0.25% of total lactic acid bacteria RNA. ND, not determined. The high RNA indexes recovered are the first evidence of the ecological importance of L. manihotivorans in the fermentation of sour cassava starch. This new species, first isolated in the area of Cali, Colombia (14), was found here to represent up to one-fifth of total lactic acid bacteria in four fermentations independently run in another region of Colombia. So far, most of the isolated strains of this species were shown to be amylolytic (Morlon-Guyot, personal communication). This feature may be of primary importance, as the washing of starch prior to fermentation eliminates almost all of the available free sugar, and starch becomes one of the only carbon and energy sources available for lactic acid bacteria (data not shown). This statement is supported by the demonstration of amylolysis in situ (7). In addition, amylolysis by L. manihotivorans contributes to the modification of the structure of starch granules (5), a modification which may be of primary importance for the use of sour cassava starch in bread making. Quantitative rRNA hybridization is a very powerful approach to monitor microbial taxons responsible for traditional fermentations: it does not depend on the biased and time-consuming isolation of microorganisms and may be applied to complex environments such as solid-state cultures. So far, only a limited number of environments have been investigated with this approach, namely gastrointestinal tracts (8, 12, 18) and pozol, another fermented food (3, 4). Nonetheless, the use of rRNA as a target may introduce bias as well. It has been shown that hybridization signals are related to cellular activity (16). During the time course of a cassava lactic acid fermentation where cells are developing, it is likely that a rather comparable physiological status for the various components of the microflora exists. The remaining bias would be due to differential extraction of RNA from various microbial types. A previous work was performed to optimize the extraction procedure of total RNA from lactic-acid-fermented starchy foods (2), and the resulting protocol was found to yield RNA from all the species so far isolated from sour cassava starch, thus minimizing the bias due to the extraction procedure. Therefore, we believe that quantitative rRNA hybridization is a method of primary importance to describe the structure of the microbial communities responsible for the fermentation of food products and that more phylogenetic probes should be developed and extensively tested in this way. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Acknowledgments I thank Nabil ben Omar and Audrey Sirvent for technical assistance, Nadine Zakhia for providing the sour cassava starch samples, Edouard Miambi for critical reading of the manuscript, and Juliette Morlon-Guyot for help with sequence alignments. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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