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DOI 10.1099/mic.0.2007/006452-0
Department of Food Science, 1605 Linden Dr., University of Wisconsin, Madison, WI 53706, USA
Utah Veterinary Diagnostic Laboratory, 950 East 1400 North, Logan, UT 84322, USA
National Center for Food Safety and Technology, Illinois Institute of Technology, Summit,
IL 60501, USA
5 April 2007
Lactobacillus casei strains are lactic acid bacteria (LAB) that colonize diverse ecological niches,
and have broad commercial applications. To probe their evolution and phylogeny, 40 L. casei
strains were characterized; the strains included isolates from plant materials (n59), human
gastrointestinal tracts (n57), human blood (n51), cheeses from different geographical locations
(n522), and one strain of unknown origin. API biochemical testing identified niche-specific
carbohydrate fermentation profiles. A multilocus sequence typing (MLST) scheme was developed
for L. casei. Partial sequencing of six housekeeping genes (ftsZ, metRS, mutL, nrdD, pgm and
polA) revealed between 11 (nrdD) and 20 (mutL) allelic types, as well as 36 sequence types.
Phylogenetic analysis of MLST data by Reticulate and split decomposition analysis indicated
frequent intra-species recombination. Purifying selection was detected, and is likely to have
contributed to the evolution of certain L. casei genes. Pulsed-field gel electrophoresis (PFGE)
using SfiI was able to discriminate all the isolates, even those not differentiated by MLST.
Phylogenetic trees reconstructed based on the MLST data using minimum evolution algorithm,
and the SfiI-PFGE restriction patterns using the unweighted-pair group method with arithmetic
mean (UPGMA), revealed consensus clusters of strains specific to cheese and silage. Topological
discrepancies between the MLST and PFGE trees were also observed, suggesting that intragenic
point mutations have accumulated at a slower rate than indels and genome rearrangements in L.
casei. The L. casei population analysed in this study demonstrated both a high level of phenotypic
and genotypic diversity, as well as specificity to different ecological niches.
INTRODUCTION
Lactobacillus casei strains are Gram-positive, facultatively
anaerobic, industrially important lactic acid bacteria (LAB)
that have been primarily used as probiotics and speciality
Abbreviations: DI, discrimination index; dN, number of non-synonymous
substitutions per non-synonymous site; dS, number of synonymous
substitutions per synonymous site; GI, gastrointestinal; HGT, horizontal
gene transfer; LAB, lactic acid bacteria; ME, minimum evolution; MLST,
multilocus sequence typing; PFGE, pulsed-field gel electrophoresis; ST,
sequence type; SNP, single nucleotide polymorphism; UPGMA,
unweighted-pair group method with arithmetic mean.
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are EF538428EF538467 (ftsZ), EF538468
EF538507 (metRS), EF538508EF538547 (mutL), EF538548
EF538587 (nrdD), EF538588ER538627 (pgm) and EF538628
EF538667 (polA).
METHODS
Bacterial strains. A total of 40 L. casei strains were selected and
were selected for the MLST analysis (Table 2). General criteria for
gene selection included the chromosome locations (preferably evenly
separated across the entire genome), functions of the encoded
proteins (preferably conserved and well characterized), presence in all
the strains as a single copy, and size of at least 1 kb (convenience of
PCR primer design). In addition, pgm was selected based on the
results of a previous study on L. plantarum (de las Rivas et al., 2006),
while ftsZ has been shown to be polymorphic in several LAB strains
(Zhang & Dong, 2005). Selection of the remaining loci (polA, mutL,
metRS and nrdD) was based on the presence of single nucleotide
polymorphisms (SNPs) between L. casei ATCC 334 and L. casei 12A.
These SNPs were identified in a previous study using comparative
genome microarrays (H. Cai, J. R. Broadbent & J. L. Steele,
unpublished data).
PCR amplification and DNA sequencing. Genomic DNA was
ST
Allele
pgm
Origin
polA
nrdD
metRS
mutL
Reference or source*
ftsZ
L3
L6
L9
L14
L19
L25
L30
CRF28
12A
32G
13/1
21/1
33/1
A2-309
A2-362
BI0231
USDA-P
UW-1
UW-4
120501-1/6M
120501-3/6M
M36
1
2
3
4
5
6
7
8
1
9
10
11
12
13
14
15
16
17
18
19
20
21
7
1
1
8
1
1
1
2
7
11
7
1
1
3
9
1
8
4
5
1
5
1
14
11
3
1
3
3
11
3
14
7
14
14
14
6
8
3
5
1
12
2
12
3
11
5
3
9
3
3
9
6
11
8
11
11
11
10
4
3
7
1
2
3
2
3
2
5
7
3
4
10
5
5
2
6
2
2
2
5
5
5
5
1
1
10
1
10
12
7
16
18
11
8
16
9
12
12
3
9
12
6
5
2
3
1
13
19
13
19
13
6
1
12
1
1
6
1
13
2
13
1
1
4
16
1
5
15
9
1
3
1
ATCC 334
ASCC 428
ASCC 477
ASCC 1087
ASCC 1088
ASCC 1123
DPC 3971
DPC 3968
DPC 4108
DPC 4249
DPC 4748
4R4
43M3
7A1
7R1
83M4
8I2
MI280
22
23
24
25
25
26
27
26
28
26
29
30
31
32
33
34
35
36
12
5
5
5
5
5
10
5
1
5
6
8
1
5
5
5
4
10
6
5
5
10
10
10
1
10
3
10
10
4
3
5
13
5
9
1
10
1
1
2
2
2
8
2
3
2
2
8
3
1
1
1
1
8
5
1
1
1
1
1
3
1
5
1
1
3
9
1
8
1
1
3
4
14
13
13
13
13
10
13
15
13
13
2
19
20
9
17
1
3
7
3
3
11
11
3
12
3
1
3
3
8
1
14
9
10
15
12
*USDA-ARS, US Department of Agriculture, Agricultural Research Service. M. Johnson, Wisconsin Center for Dairy Research, Madison, WI, USA;
I. Powell, Australian Starter Culture Research Center Limited, Werribee, Victoria 3030, Australia; F. Vogensen, Dept of Food Science, Royal
Veterinary and Agricultural University, Frederiksberg C, Denmark.
(Beckman Coulter), and then sent to the Biotech Center for sequence
determination.
MLST data analysis. Multiple sequence alignments were performed
using molecular evolutionary genetic analysis (MEGA) software version
3.1 (http://www.megasoftware.net). Descriptive evolutionary analyses
such as mol% G+C content, dS/dN ratios (where dS is the number of
synonymous substitutions per synonymous site, and dN is the number
of non-synonymous substitutions per non-synonymous site), and
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Gene function
ftsZ
polA
DNA polymerase I
mutL
metRS
Methionyl-tRNA synthetase
nrdD
pgm
Phosphoglucomutase
GGCATTGCACAACTGAAAGA;
GCATCGTCTGCGTTAGTTTG
TTATCATGTGGCCGAACAAA;
GTTTGCGTCAAAGTCTGCAA
ATCGGCAACATTAAGCAACC;
GATGACGCCCATTGGATAAC
CGGTATTTTGCCAGCCTTTA;
CATTTCGCCTTTTAGCTTGC
GCTTGAAGCGTGATTTAGCC;
ACATTCGATCGCCAATTGTT
AGGCATTTGCTGCTCCTATG;
GGGATCAGTCGCGATTAAGA
s
X
1
nj nj{1
N N {1 j~1
1
RESULTS
API biochemical testing
Analysis of carbohydrate fermentation patterns by API
biochemical testing demonstrated that all 40 L. casei strains
could ferment galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine and tagatose, but they could not
ferment glycerol, erythritol, arabinose, L-xylose, melibiose,
raffinose, glycogen, xylitol, fucose, D-arabitol, potassium 2ketogluconate and potassium 5-ketogluconate. Differences in
carbohydrate utilization by L. casei strains are summarized in
Table 3, and some niche-specific phenotypic traits were
identified. For example, the ability to utilize some C5 sugar
alcohols (e.g. adonitol), C5 sugars (e.g. ribose) and C6 sugar
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Plant
(n59)
D-Ribose
77
100
100
D-Xylose
11
D-Adonitol
22
43
100
32
33
57
11
9
0
46
56
0
89
57
14
86
0
0
100
100
22
29
59
86
96
91
22
67
100
78
57
86
100
86
100
100
100
100
D-Maltose
100
78
100
100
D-Lactose
83
22
71
100
Sucrose
73
96
67
100
86
100
100
100
D-Melezitose
27
96
67
78
86
100
100
100
Starch
Gentiobiose
D-Turanose
0
64
83
11
78
89
0
57
100
0
100
100
D-Lyxose
L-Arabitol
29
14
11
D-Sorbose
L-Rhamnose
Dulcitol
Inositol
D-Sorbitol
Methyl-a-D-mannopyranoside
Methyl-a-D-glucopyranoside
Amygdalin
Arbutin
Salicin
D-Cellobiose
D-Trehalose
Inulin
Potassium gluconate
GI
Blood
(n57) (n51)
100
polA
mutL
metRS
nrdD
pgm
ftsZ
Fragment
analysed (bp)*
731
747
636
735
734
676
(27.0)
(38.2)
(32.1)
(33.7)
(40.2)
(53.8)
G+C
content
(mol%)
44.8
49.8
47.5
48.7
46.9
49.3
No. of
Polymorphic
sites
36
29
50
44
14
26
p/siteD
SNPs
Alleles
39
29
50
45
14
26
14
20
10
11
12
16
0.0147
0.00811
0.0276
0.0146
0.00418
0.00829
kd
10.7
6.06
17.6
10.7
3.07
5.61
Tajimas D
value
0.590
20.381
1.76
0.0545
20.213
20.281
No. of
Syn.
31
21
42
41
11
22
dS/dN
Nonsyn.
8
8
8
4
3
4
13.1
7.9
18.3
33.6
11.8
18.8
strain, and the estimated rate of single nucleotide substitutions between Escherichia coli and Salmonella enterica
of 4.761029 per site per year (Doolittle et al., 1996; Lawrence & Ochman, 1998). Results indicated that the
divergence of the three clusters of L. casei occurred
approximately 1.5 million years ago, whereas most cheese
and silage strains in clusters III and II seemed to have
diversified more recently (Fig. 4a).
Comparison to PFGE
The 40 L. casei strains were analysed by PFGE, and a
UPGMA tree was constructed based on SfiI restriction
patterns (Fig. 4b). PFGE discriminated all the strains,
including those not differentiated by MLST. When
compared with the ME tree, the PFGE tree showed a
similar topology for the L. casei strains, including a
relatively large cluster of cheese-derived strains. However,
some human GI tract strains (L9 and L6) and wine strains
(A2-309 and A2-362) seemed to be closely related to the
main clusters of cheese strains on bifurcating branches in
the PFGE tree, conflicting with relationships shown in the
ME tree. Also similar to the ME tree, strains from blood,
pickle, human GI tract and corn silage appeared to be
genetically diverse, and grouped in different clusters. In
both the ME tree and the PFGE tree, cheese strains did not
cluster based on their geographical origin.
DISCUSSION
Fig. 4. (a) Linearized ME tree based on 1419 allelic codons of the 40 L. casei strains. The bottom scale shows the divergence
time frame and the number of synonymous substitutions per nucleotide site. Bootstrap values on bifurcating branches are based
on 1000 random bootstrap replicates for the consensus tree. (b) UPGMA tree based on SfiI-PFGE macrorestriction patterns.
Geographical locations of cheese strains are labelled.
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Microbiology 153
http://mic.sgmjournals.org
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ACKNOWLEDGEMENTS
We thank Ron Agee for technical assistance with PFGE, and Lenese
Grant for help with DNA sequencing. We thank Finn Vogensen (Dept
of Food Science, Royal Veterinary and Agricultural University,
Frederiksberg C, Denmark), Mark Johnson (Wisconsin Center for
Dairy Research, Madison, WI, USA), Tom Beresford (Teagasc, Oak
Park Research Centre, Carlow, Ireland), Ian Powell (Australian Starter
Culture Research Center Limited, Werribee, Victoria 3030, Australia),
Kurt Reed (Marshfield Clinic Research Foundation, Marshfield, WI,
USA), Gerald Tannock (Dept of Microbiology and Immunology,
University of Otago, Dunedin, New Zealand) and Fred Breidt (Dept
of Food Science, North Carolina State University, Raleigh, NC, USA)
for providing the L. casei strains. Funding has been provided for this
research and publication from Dairy Management, Inc. through the
Center for Dairy Research, the College of Agricultural and Life
Sciences at the University of Wisconsin, and the USDA Cooperative
State Research, Education and Extension Service (CSREES) project
WIS04908.
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