Biomedical Engineering Reference
In-Depth Information
sourdough LAB strains by DNA fingerprinting, however, AFLP fingerprinting and
pulsed-field gel electrophoresis (PFGE) are the most powerful. Essentially, PFGE
involves the electrophoretic separation of genomic macrorestriction fragments
obtained by digestion with rare-cutting enzymes in an alternating electric field. In
sourdough studies, PFGE has been used as a typing method to differentiate among
strains within
Lb. plantarum
and
Lb. sanfranciscensis
[
126,
135,
174
] .
Sequence-based analysis approaches for identification of sourdough LAB have
long relied on the use of 16S rRNA genes, and this has become a standard approach
to obtain a first preliminary view of the taxonomic diversity among a set of unknown
isolates recovered from a sourdough ecosystem [
72,
80,
148
]. In many of these
studies, only partial 16S rRNA gene sequences are determined and used in compari-
sons with public sequence databases. In many cases, the use of partial sequences
will only allow a tentative identification, of which the reliability is likely to improve
when the entire 16S rRNA gene is sequenced [
228
]. Despite its established use as a
standard method for identification of LAB species, 16S rRNA gene sequencing
does not allow differentiation of phylogenetically closely related species [
63-
66,
71
]. The growing availability of whole-genome sequences has triggered the search
for alternative genes that offer a higher taxonomic resolution than the 16S rRNA
gene. The use of protein-encoding genes or so-called housekeeping genes essen-
tially combines the technological advantages of 16S rRNA gene sequencing and the
taxonomic resolution offered by a number of fingerprinting methods. Sequencing of
one or preferably multiple of these genes as taxonomic markers is a crucial step
forward in the development of standardized and globally accessible methods for the
identification of LAB. Housekeeping genes such as
pheS
(encoding the phenylala-
nyl-tRNA synthase) and
rpoA
(encoding the DNA-dependent RNA polymerase
alpha-subunit) display higher divergence rates than the 16S rRNA gene, and allow
discrimination between closely related LAB species with almost identical 16S
rRNA gene sequences [
67,
229
]. Several studies on sourdough LAB species diver-
sity have used such protein-encoding genes as phylogenetic markers in a single-
locus sequence approach. In conjunction with (GTG)
5
-PCR fi ngerprinting, the
pheS
gene has been successfully used for the identification of LAB species from sour-
dough fermentations at laboratory scale [
107
] and from Belgian artisan bakery sour-
doughs and their environment [
105,
106,
155
], as well as for unraveling the
intraspecific diversity in the sourdough species
Lb. rossiae
[
74
] . Settanni and co-
workers [
156
] used the
recA
gene, encoding a protein essential for repair and main-
tenance of DNA, in a multiplex PCR assay to discriminate between the
phylogenetically highly related
Lb. plantarum
,
Lb. pentosus
and
Lb. paraplantarum
in sourdough ecosystems. The
recA
gene has also been used in combination with the
16S rRNA gene to unravel the identity of LAB isolates recovered during wheat flour
sourdough type I propagation [
135
]. Sequences derived from the
tuf
gene, which
encodes the elongation factor Tu, have revealed a higher discriminatory power com-
pared to 16S rRNA gene sequences and have been used to support the delineation
of the new sourdough species
Lb. secaliphilus
[
85
] . Although single-locus sequence
analysis approaches are now commonly used within specific LAB groups, it has
been argued that the phylogenetic information obtained from only a single gene
