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EPICENTRE® Biotechnologies Forum
Molecular Analysis of Lactic Acid Bacteria
in an Inhospitable Environment: Beer
Les M. Hoffman and Bruce W. Jarvis, EPICENTRE Biotechnologies
Beer, that fermented drink many of us
enjoy after hours, is inhospitable to many
microorganisms. Unwanted microbial
growth is discouraged not only by the
absence of oxygen, high levels of ethanol
and carbon dioxide but also by the
bittering compounds in hops, which
possess potent antibacterial activity.
Unfortunately for the brewing industry, a
variety of Lactobacillus spp. strains have
evolved a plasmid-encoded defense
mechanism that enables them to transport
these antimicrobial compounds out of the
cell. Since the ability to accurately differentiate these beer-spoilage strains is
critical to quality control, we describe
here how EPICENTRE Biotechnologies’
products were used to identify a potentially novel Lactobacillus species in a
home-brewed pale ale gone bad.
Our study began with genomic DNA
isolated from the sediment in our spoiled
ale. To generate a phylogenetic tree,
the 16S rRNA gene sequences were first
amplified by PCR using EPICENTRE’s
FailSafe™ PCR System, cloned, and then
sequenced. As shown in FIG 1, two
clades (a clade is a monophyletic group
of species with a common evolutionary
ancestor) were present.
The same spoiled beer was also cultured
on a nutrient-rich medium commonly
used for lactic acid bacteria and incubated
anaerobically at 30°C. Two distinct colony
spobac19fp
spobac15fp
spobac24fp
tightcol16S
spobac20fp
spobac2fp
}
}
FIG 1. A phylogenetic tree
based on 16S rRNA gene
spobac18fp
sequences present in
spobac16fp
spoiled beer. The two colony
spobac17fp
types are denoted as “flat”
(purple) or “tight” (green),
L. brevis
spobac1fp
and grouped phylogenetiflatcol16S
cally with the clades from
spobac23fp
total sediment DNA. The
spobac3fp
ClustalX algorithm and
TreeView were used to align
spobac4fp
sequences and to construct
spobac25fp
0.1
a neighbor-joining tree with
1000 bootstrap iterations.
Blue bar at bottom of tree, number of nucleotide
substitutions per base pair, indicating the degree
of phylogenetic relatedness.
spobac22fp
20
www.EpiBio.com
FIG 2. Both Lactobacillus
species contain plasmids that
range in size from 2 to 12 kb.
Cells were pretreated with
EPICENTRE’s Ready-Lyse™
Lysozyme Solution and plasmids were isolated using the
PlasmidMAX™ DNA Isolation
Kit. Lane 1, BAC-Tracker™
Supercoiled DNA Ladder;
Lane 2, L. brevis plasmids;
Lane 3, L. gambrinii plasmid;
Lane 4, Supercoiled kb ladder.
morphologies, denoted as “flat” or “tight”,
were isolated. Comparative analysis of
16S rRNA gene sequences from these
isolates grouped them phylogenetically
with the clades from the sediment DNA
(FIG 1). The “flat” colony type was identified by BLAST analysis as Lactobacillus
brevis, the most common beer spoilage
isolate. The 16S rRNA gene sequence of
the “tight” isolate, however, did not match
any GenBank sequences by more than
97%. Such high levels of 16S sequence
heterogeneity suggest this bacterium be
classified in the genus Lactobacillus as a
new species.1 To explore this further, the
recA gene, which is highly conserved and
has a phylogeny congruent with that of
the ribosomal RNA sequences, was also
analyzed. The highest homology in
GenBank to this isolate’s chromosomal
recA gene was only 82%.
Finally, plasmids were extracted from
both Lactobacillus isolates. There are
at least four plasmids found in each
isolate, varying in size from 2 to 12 kb
(FIG 2). Using EPICENTRE’s EZ-Tn5™
<R6Kγori/KAN-2> Insertion Kit, we were
able to propagate the 3.3 kb plasmid from
the “tight” isolate into EPICENTRE’s
TransforMax™ EC100D™ pir+ E. coli cells
following in vitro insertion of an EZ-Tn5™
Transposon containing an R6Kγ origin of
L. gambrinii
Gene
Chromosomal/
Episomal
16S rDNA
recA
replication and a kanamycin selectable
marker. A Mu-based transposon from
EPICENTRE’s HyperMu™ <CHL-1> Kit
was then randomly inserted into the
“rescued” plasmid, and the template from
the resulting insertion clones was
sequenced bidirectionally from the ends
of the HyperMu transposon.
Homology between replication genes
identified on the 3.3 kb plasmid and
similar Lactobacillus genes averaged only
about 80%. In contrast, the ABC transporter gene, horA, which is required for
growth of Lactobacillus species in hopped
beer, was highly conserved (Table 1). The
strong conservation of horA is thought to
be due to lateral gene transfer between
spoilage bacteria, allowing “recruitment”
of new strains for survival in beer.
Taken collectively, these results suggest
that the “tight” isolate is a novel species,
for which we have proposed the name
Lactobacillus gambrinii. Gambrinus is the
legendary “king of beer”, with an enormous appetite for fermented beverages.
A more complete description of this work
was presented as a poster at the 13th Annual
International Conference on Microbial
Genomes in September, 2005. The poster is
available online at: www.EpiBio.com/posters/
microbialgeneticsposter.pdf.
Reference
1. Lan, R. and Reeves, P.R. (2001) Trends.
Microbiol. 9(9), 419.
Genomic DNA of L. gambrinii or a Fosmid library
are available for parties interested in its further
characterization or sequencing.
Go to www.EpiBio.com and enter this
code: LANX1 for more product information.
Nucleotide
or Protein
Closest BLAST match
Percent
chromosomal
nt
L. bifermentans, L. coryniformis
97
"
protein
Pediococcus pentosaceus
84
recA
"
nt
Pediococcus dextrinicus
82
horA
plasmid
nt
L. lindneri, l. paracollinoides, L. brevis
99-100
horA
"
protein
L. lindneri
100
repA
"
protein
L. plantarum
91
repB
"
protein
L. plantarum
86
mob
"
protein
L. plantarum
~60
Table 1. Comparison of various L. gambrinii genes and the deduced proteins.
Volume 13 • Number 1