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Applied and Environmental Microbiology, May 2000, p. 2224-2226, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Design and Evaluation of a Lactobacillus
manihotivorans Species-Specific rRNA-Targeted Hybridization Probe
and Its Application to the Study of Sour Cassava Fermentation
Frédéric
Ampe*
Institut de Recherche pour le
Développement (IRD), Laboratoire de Biotechnologie
Microbienne Tropicale, 34032 Montpellier, France
Received 15 November 1999/Accepted 23 February 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.
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TEXT |
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.
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FIG. 1.
Definition of the L. manihotivorans
species-specific probe S-S-Lbma-0207-a-A-20 based on the 5'-to-3' 16S
rRNA sequence alignment (region bp 198 to 234 in E. coli
consensus numbering). Nucleotides identical to those of L. manihotivorans are represented by dots, mismatches are indicated
by the nucleotide letter, and gaps are represented by dashes. At least
three mismatches or gaps can be seen for all the nontarget
microorganisms.
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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.
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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 fermentationa
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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.
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TABLE 2.
Importance of L. manihotivorans in cassava
sour starch from four independent fermentations using quantitative dot
blot measurements of 16S rRNAa
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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.
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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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FOOTNOTES |
*
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.
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Applied and Environmental Microbiology, May 2000, p. 2224-2226, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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