Research in Microbiology 153 (2002) 115123

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Erratum

Erratum to
Lactobacillus sakei: recent developments and future prospects
[Research in Microbiology 152 (2001) 839] ,
Marie-Christine Champomier-Vergs , Stphane Chaillou, Monique Cornet, Monique Zagorec
Unit Flore Lactique et Environnement Carn (FLEC), INRA, Domaine de Vilvert, 78 350 Jouy en Josas, France
Received 2 July 2001; accepted 20 August 2001
First published online 17 January 2002

Abstract
Lactobacillus sakei is one of the most important bacterial species involved in meat preservation and meat fermentation. In the last fifteen
years, numerous studies have focused on this species due to its important role in food microbiology. The present paper reviews current
knowledge of this emerging species in the fields of taxonomy, phylogeny and physiology, and metabolism. Recent developments in genetic
tools and molecular genetics will also be emphasized to evaluate future prospects. 2002 ditions scientifiques et mdicales Elsevier SAS.
All rights reserved.

1. Introduction
Lactobacillus sakei has been isolated from several raw
fermented food products of plant and animal origin. It is
found in silage, sauerkraut, sourdough, and smoked fish, but
is mainly found in meat products ([17,26] and references
herein). It belongs to the main flora of fresh meat and be-
comes dominant flora when meat is stored under a vacuum.
It is widely used in France and Western Europe in associ-
ation with micrococci and yeasts as a starter for the man-
ufacture of fermented sausages. Added at the beginning of
the fermentation process at about 106 bacteria per gram and
reaching 108 bacteria per gram, L. sakei is present through-
out the ripening process. Its crucial role in meat preserva-
tion and fermentation is mainly due to lactic acid produc-
tion or to the synthesis of compounds inhibitory towards
pathogenic or spoilage bacteria. In Western Europe, L. sakei
is generally considered as beneficial. Nevertheless, some
exopolysacharide-producing strains have been reported in
meat products [33] and fish products [32]. They are consid-
ered to be deleterious for these products, especially in the
Finnish meat and fish industries ([5] and references herein).
PII of original article: S0923-2508(01)01267-0.
The article is re-published. Due to a technical incident, references in
the original article were incorrect.
* Correspondence and reprints.
E-mail address: verges@jouy.inra.fr (M.-C. Champomier-Vergs).
0923-2508/02/$ see front matter 2002 ditions scientifiques et mdicales Elsevier SAS. All rights reserved.
PII: S 0 9 2 3 - 2 5 0 8 ( 0 1 ) 0 1 2 9 6 - 7
Strains harboring plasmids associated with hydrogen perox-
ide production have also been reported [47].
Due to this important role in meat technology, its taxo-
nomic status, physiology and metabolism have been studied
in relation to its particular habitat.
2. Taxonomy and phylogeny of L. sakei group
L. sakei, first described as Lactobacillus sake, a spoilage
microorganism in the fermented beverage, sake, in 1934,
was renamed following recommendations of Trpper and
De Clari [55]. It is a lactic acid bacterium (LAB). This
group of bacteria contains many other species involved in
food fermentation and preservation. It is a Gram-positive
bacillus which groups by pair or short chain (Fig. 1). The
GC content is 4244%. Phenotypic differentiation between
strains and other species is usually based on the type of
lactic acid isomer produced, sugar fermentation pattern
and ammonia production from arginine. Wide phenotypic
heterogeneity within strains of L. sakei isolated from meat
has been reported in many studies based on biochemical
and physiological features, especially in sugar fermentation.
Difficulties in correctly identifying these strains are also due
to the close relatedness between L. sakei and Lactobacillus
curvatus, also found in meat products. The need for correct
identification, especially of starter strains, has led over the
116 M.-C. Champomier-Vergs et al. / Research in Microbiology 153 (2002) 115123
Fig. 1. Scanning electron microscopy of L. sakei strain 23K grown at 30 C.
last ten years to several taxonomic studies dealing with
the taxonomic status of these species using molecular and
genomic methods, thus enabling a more clear distinction
between L. sakei and L. curvatus.
DNADNA hybridization revealed that despite pheno-
typic heterogeneity, L. sakei strains were closely related at
the genomic level (
75% identity) [8,23]. L. sakei and
L. curvatus are clearly separated at the genomic level (40 to
50% identity) but are phenotypically highly related and dif-
ficult to separate. Nevertheless, these two species have been
split into two subgroups by different studies using different
methods. On the basis of whole soluble protein analysis, two
groups were found within L. sakei strains and L. curvatus
strains. The two L. sakei groups could not be differentiated
by phenotypic features, whereas the two L. curvatus groups
were differentiated by their ability to ferment melibiose [54].
Analysis of randomly amplified polymorphic DNA(RAPD)-
PCR profiles confirmed this subgrouping and the creation of
two subspecies within each species. L sakei was split into
L. sakei subsp sakei and L. sakei subsp carnosus. L. curva-
tus was split into L. curvatus subsp curvatus and L. curvatus
subsp melibiosus [23,54]. Another study also using RAPD
enabled separation of the two species into two subgroups [4].
The two L. curvatus RAPD subgroups were differentiated on
the basis of catalase activity but could not be differentiated at
the genomic level by specific primers. The two L. sakei sub-
groups could not be phenotypically differentiated, although
PCR primer pairs specific for each group were developed.
The separation into two subgroups within each species has
been validated by the subcommittee on taxonomy of Bifi-
dobacterium, Lactobacillus and related organisms. Never-
theless, for some strains, this separation was not validated
by further studies [4,32], and no study correlates these sub-
groups with interesting properties for industrial purposes.
Specific oligonucleotidic probes based on 5 termini of
23S RNAs have been designed [19]. Primers for PCR
amplification of the large intergenic region of rDNA 16/23S
were designed which allowed specific species identification
of L. sakei and L. curvatus [3]. No strain-specific primers
are yet available. Strains are now generally identified by
combined methods using RAPD-PCR profiles, RFLP, whole
protein analysis and some phenotypic features. It seems that
the main phenotypic differentiating trait between L. sakei
and L. curvatus remains the ability to hydrolyze arginine by
the arginine deiminase pathway.
Phylogeny based on 16S rRNA sequencing showed that
L. sakei belongs to cluster 2 of LAB constituted by the
Lactobacillus casei-Pediococcus group which is the most
phylogenetically heterogenous group of the LAB. This study
also confirmed the close relationship between L. sakei and
L. curvatus [10].
3. Physiology and metabolism
Physiology of dairy LAB is now well documented.
However, some of this knowledge cannot be extrapolated
to LAB multiplying on meat. Unlike milk, meat products
have a poor carbohydrate content and are rich in myofibrillar
proteins. They consist of a solid matrix which influences
the availability of nutrients. Treatments related to meat
technology such as drying, salting or modified atmosphere
packaging are additional environmental factors influencing
the physiology of L. sakei.
3.1. Nutritional requirements
Like other LAB, L. sakei has multiple nutritional require-
ments. Strains are generally prototrophic for most amino
acids. Chemically defined media suitable for L. sakei strains
contain 18 amino acids, except for glutamic acid and as-
partic acid [29]. Biosynthetic pathways have not been sys-
tematically explored but an incomplete or mutated biosyn-
thetic operon has been found in L. sakei and is possibly
involved in arginine biosynthesis (Dudez et al., in prepara-
tion). However, it would appear that, as is known for lacto-
cocci, L. sakei strains have evolved towards auxotrophy due
to their environment, and biosynthetic pathways do not re-
main functional. Little is known about base and vitamin re-
quirements. It is generally admitted that the L. sakei strains
are auxotrophic for most of these compounds. Uracil pro-
totrophy has been studied in more detail for one L. sakei
strain [35]. The classical de novo pyrimidine biosynthetic
pathway would appear to be functional in the absence of
uracil and is governed by a pyrR regulator.
3.2. Carbohydrate metabolism and energy transduction
Glucose and ribose are the main sugars present in meat.
The first originates from glycogen and the second from ATP
hydrolysis. Exogenous glucose and sometimes lactose are
usually added to speed up and improve the ripening process
of fermented meat products. In L. sakei, hexose fermenta-
tion is homolactic and proceeds via anaerobic glycolysis.
 M.-C. Champomier-Vergs et al. / Research in Microbiology 153 (2002) 115123
The resulting formation of lactic acid and subsequent de-
crease in pH is of major importance to the hygienic safety
and quality of the fermented product. Glucose is transported
to the cell via the phosphoenolpyruvate:carbohydrate phos-
photransferase system (PTS) as well as by a secondary
non-PTS route [29]. The mannose-specific PTS permease,
coined EIIman , displays a broad substrate specificity since
glucose, mannose and N-acetylglucosamine are also taken
up by the system. The ptsHI operon (encoding the pro-
tein HPr and enzyme I, common to all PTS complexes) has
been cloned [50]. Mutants with a ptsI mutation no longer
grow on fructose and sucrose, indicating that these two sug-
ars are also transported by the PTS. On the other hand, a
few L. sakei 2 deoxyglucose-resistant mutants defective in
EIIman activity were shown to grow normally on fructose
and sucrose, demonstrating that one or more PTS complexes
distinct from EIIman are present in L. sakei. Indeed, the scrA
gene encoding sucrose-specific enzyme II of the PTS has
been recently characterized (Dudez et al., in preparation).
Lactose and galactose are also fermented by L. sakei but
their transport is not dependent on a PTS complex. The
-galactosidase protein of L. sakei is encoded on the chro-
mosome by two genes, lacL and lacM [41]. The two genes
overlap by 15 nucleotides and are out of frame, suggesting
a coordinate regulation at the level of translation in order to
produce an equal amount of both polypeptides. The lactose
transporter remains unknown. Part of the Leloir pathway
for galactose catabolism has been characterized in L. sakei.
The gene cluster comprises a partial galE gene encod-
ing UDP galactose-4-epimerase, galT encoding galactose-
1-phosphate-uridyltransferase and a partial galM encoding a
mutarotase (C.A. Alpert, personal communication).
Ribose, arabinose and gluconate are fermented via the
heterolactic pathway, also called the phosphoketolase path-
way. Their use for growth requires thiamine in the medium,
a precursor of thiamine pyrophosphate synthesis, which is
a cofactor in phosphoketolase. The genes responsible for ri-
bose catabolism have been cloned and partially character-
ized [51]. The rbsUDKR cluster encodes, respectively, two
putative transport proteins (RbsU and RbsD), ribose kinase
(RbsK) and a repressor (RbsR). The cluster shows striking
differences with that of Escherichia coli or Bacillus sub-
tilis rbs clusters. While the RbsD subunit of the ribose high-
affinity ABC-transporter is present in the L. sakei cluster,
the remaining RbsA, RbsB and RbsC subunits are lacking.
Instead, a protein showing homology with a glucose facili-
tator of Staphylococcus xylosus is present (RbsU). The rea-
son for this chimeric situation, unique so far, is not clear. It
may indicate a strong correlation between glucose and ribose
metabolisms in L. sakei. A knockout rbsR mutation leads to
constitutive expression of the rbsUDK unit, the transcription
of which is normally induced by ribose. Transport and ATP-
dependent phosphorylation activities are two-fold increased
in a ptsI mutant, suggesting allosteric regulation of ribose
utilisation by the PTS.
117
During sugar fermentation, D- and L-lactate are produced,
though only L-lactate dehydrogenase is present in L. sakei.
L -LDH is encoded by the monocistronic ldhL gene the
disruption of which suffices to prevent production of both
D - and L -lactate [34]. The conversion of L - to D -lactate is
catalyzed by a lactate racemase, still uncharacterized at the
genetic level [21].
3.3. Secondary ATP-generating metabolism
Malolactic fermentation has been identified in L. sakei
[24]. At first sight, the fermentation process does not provide
an obvious advantage to the bacteria since the conversion of
malate to lactate does not yield energy for growth. At pH
4.5 however, the malate/lactate exchange transport reaction
is coupled with the creation of a proton motive force strong
enough to generate 1 mol of ATP per mol of malate. In gen-
eral, a pH of 4.5 is reached when most of the sugar present
in the growth medium has been catabolized consequently to
cells entering stationary phase. The genes encoding the mal-
olactic pathway in L. sakei are not yet characterized.
Arginine is also catabolized via a pathway generating
energy. Nevertheless, as for malate, L. sakei is not able to use
arginine as the sole carbon source for growth (see below).
3.4. Stress response
Various factors associated with the technological process
of fermented meat products represent stress conditions for
starter strains. These include curing with salts, nitrite/nitrate
or the creation of an artificially modified atmosphere af-
fecting both redox potential and toxicity of oxygen. Fur-
thermore, starter cultures usually undergo steps of chilling
or freeze-drying representing additional stress. L. sakei is
known to be one of the most psychrophilic species of lac-
tobacilli since some strains grow at 24 C. Unfortunately,
no study of cold stress response in L. sakei is currently avail-
able. On the other hand, the heat shock genes hrcA, groE,
dnaK and dnaJ, encoding proteins highly conserved in all
microorganisms, have been identified [46]. The transcription
of these genes is induced by heat shock, salt and ethanol. An-
other heat shock protein, ClpE, encoding a new type of Clp
ATP protease, was identified in L. sakei [52].
Most lactobacilli are aerotolerant and have inducible ox-
idative stress responses to deal with the resulting oxygen rad-
icals and H2 O2 . L. sakei possesses a heme-dependent cata-
lase (KatA) responsible for the efficient decomposition of
H2 O2 . Expression of the katA gene is induced by H2 O2 or
by an anaerobiosis/aerobiosis shift [20]. Regulation takes
place at the transcriptional level and may involve a stem-loop
structure located 32-bp downstream of the transcription start
point. This sequence showed some similarity to the consen-
sus sequence of potential binding sites for FNR homologues.
Five L. sakei two-component regulatory systems (rrp/
hpk1, rrp/hpk2, rrp/hpk3, rrp/hpk31 and rrp/hpk48) were
also isolated by degenerated PCR amplification. The genes
118 M.-C. Champomier-Vergs et al. / Research in Microbiology 153 (2002) 115123

encoding the respective response regulators (rrp) of the five 3.6. Secondary metabolism
systems were disrupted and the mutants were studied for
their behavior towards external stress stimuli [38]. Mutants 3.6.1. Bacteriocins
from the rrp3 and rrp48 genes showed wild type behav- Three bacteriocins, sakacin A, sakacin P and lactocin S,
ior whereas the rrp2 mutant was temperature-sensitive and have been isolated and well characterized in L. sakei [40].
the rrp1 mutant had a weaker acid tolerance. The mutant Their biochemical characteristics and inhibitory spectrum
in the rrp31 gene showed a pleiotropic phenotype. This are summarized in Table 1. Sakacin A and lactocin S
mutant showed susceptibility to heat, acid, pH, aerobiosis, production and immunity are associated with a 28-kb and
H2 O2 and grew poorly even under normal growth condi- a 50-kb (pCIM1) plasmid, respectively. The 50-kb pCIM1
tions. Unexpectedly, the rrp31 mutant was also character- plasmid in L. sakei 45 is prone to instability or changes
ized by higher tolerance to the antibacterial glycopeptide affecting bacteriocin production, in contrast to the 28-kb
vancomycin. The target genes of these five regulatory sys- plasmid of L. sakei Lb 706 which is rather stable. On the
tems are not yet characterized and the cellular functions in- other hand, no plasmids were detected in L. sakei LTH 673,
volved in the stress response await identification. suggesting the chromosomal location of the genes involved
in production of sakacin P and immunity to this bacteriocin.
3.5. Exopolysaccharide (EPS) production The genes necessary for sakacin A production and im-
munity [1] are organized into two divergently transcribed
Some L. sakei strains are associated with the production operons, one large operon (sapKRTE) including genes for
of ropy slime. Despite the relative importance of EPS pro-
duction in the food industry, as food-grade additive for in-
stance, only a few studies have been published on EPS syn-
thesis in this species. As mentioned above slime production
in L. sakei is not always a desirable property since it may
cause important spoilage in vacuum-packed cooked meat. L.
sakei EPS consists of glucose and rhamnose at a ratio of 3:2
[56]. EPS containing glucose and galactose has also been re-
ported [25]. One study showed that the composition of the
EPS produced is not altered when cells are grown in differ-
ent energy sources, but the yield of production was highest
with glucose, reaching a maximum of 1.4 g/L [56]. Mutants
in the rhamnose biosynthesis pathway are defective in EPS
production, which confirms the key role of this pathway in Fig. 2. Organization of the gene clusters involved in the bacteriocin
EPS synthesis [6]. Thus far, however, no eps gene cluster has production and immunity of L. sakei (according to [13,48]). Promoters and
been characterized in L. sakei. terminators of transcription are shown (see text for gene nomenclature).

Table 1
Biochemical characteristics and inhibitory spectrum of bacteriocins produced by L. sakei
Bacteriocin Producers Biochemical characteristics Inhibitory spectrum References
sakacin A L. sakei L706 class IIa bacteriocin Carnobacterium piscicola [1,11,18,39]
4309 Da Enterococcus spp.
41 amino acids L. sakei, L. curvatus,
hydrophobic Lactobacillus brevis
identical to curvacin A produced Leuconostoc paramesenteroides
by L. curvatus LTH1174 Listeria monocytogenes
Staphylococcus aureus
Lactococcus cremoris
sakacin P L. sakei LTH673 class IIa bacteriocin L. curvatus, Lactobacillus delbrueckii, L. sakei [18,22,40,53]
(also called L. sakei L674 4436 Da L. pentosus, L. plantarum, Lactobacillus reuteri
sakacin 674) 43 amino acids L. fructivorans, Listeria ivanovii,
hydrophobic E. faecalis,
Carnobacterium spp.
Listeria monocytogenes
lactocin S L. sakei L45 antibiotic Lactobacillus spp. [18,43,48,49]
(also called L. sakei 148 3769 Da Leuconostoc spp.
sakacin M) 33 amino acids Pediococcus spp.
50% nonpolar amino acids
 M.-C. Champomier-Vergs et al. / Research in Microbiology 153 (2002) 115123
the signal-transducing system, and one divergently tran-
scribed operon sapA-saiA (Fig. 2). Between these two oper-
ons there are several orfs encoding putative peptides, espe-
cially orf4 which encodes the putative precursor of a 23-
amino-acid cationic peptide termed Sap-Ph. The sakacin
P gene cluster [39] comprises seven consecutive genes,
sppIp, sppK, sppR, sppA, spiA, sppT and sppE, all tran-
scribed in the same direction.
Sakacin A and sakacin P are ribosomally synthesized as
precursors or prepeptides which appear to be biologically in-
active, and share with the bacteriocins of the same group a
consensus sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys in their
N-terminal region, the so-called double-glycine leader se-
quence [13]. The genes encoding sakacin A and P are sapA
and sppA respectively. The double-glycine leader-containing
bacteriocins are processed concomitantly with their exter-
nalization. The leader peptide serves as a recognition signal
for both cleavage of the bacteriocin prepeptide and trans-
membrane translocation of the mature protein. This trans-
membrane translocation is mediated by an ABC transporter
which possesses an N-terminal proteolytic domain and an
accessory protein. The sakacin A ABC transporter and ac-
cessory protein are encoded by sapE and sapT genes, re-
spectively. The genes ecoding for sakacin P ABC transporter
and accessory protein are termed sppE and sppT. Sakacin
P and sakacin A production is transcriptionally regulated
through a signal transducing system which consists of three
components: a histidine protein kinase, a response regula-
tor, and an induction factor also called pheromone [39]. The
secretion of the peptide pheromone is directed by a typical
double-glycine leader peptide. Its gene is cotranscribed with
the two other components of the signal transducing system.
In sakacin A production, such a three-component regula-
tory unit is encoded by the gene cluster orf4-sapKR. The
products of sppIP, sppK and sppR genes form the three-
component signal transducing system in sakacin P produc-
tion and sppK encodes the pheromone receptor [7]. Growth
conditions were shown to have considerable effects on the
functionality of this regulatory system [11]. The putative im-
munity proteins to sakacin A and sakacin P are encoded by
saiA and spiA genes, respectively.
The plasmid region of lactocin S production [48,49] con-
tains nine colinearly arranged open reading frames (ORFs)
that are transcribed as a unit which initiates upstream of
lasA and terminates downstream of lasW. The prelactocin
S is encoded by the lasA gene. Based on sequence similar-
ity to other lantibiotic systems, three genes in the las operon
are identified. The lasM gene may be involved in prepeptide
modification. The lasT gene encodes a typical ABC trans-
porter and is probably involved in secretion of lactocin S.
The lasP product which contains the three active site amino
acid motifs conserved in the subtilisin-like serine proteases
may be the lactocin S leader peptidase. The five remain-
ing orfs encode proteins which show apparently unique se-
quences and their significance in lactocin S production re-
mains to be established.
119
3.6.2. Amino acid catabolism
3.6.2.1. Arginine catabolism. Arginine catabolism is doc-
umented in L. sakei. Arginine degradation by L. sakei is es-
sential since this feature enables differentiation from L. cur-
vatus, and arginine is the only amino acid in LAB which is
phosphorylated at the substrate level with energy generation.
Arginine degradation in L. sakei is governed by the argi-
nine deiminase pathway which leads to NH3 and ATP pro-
duction. This pathway is composed of three enzymes: argi-
nine deiminase, catabolic ornithine transferase, carbamate
kinase (CK) and an arginine/ornithine antiporter. The struc-
ture of the arcABCTD operon encoding these four proteins
in L. sakei revealed the existence of an additional gene, arcT,
possibly encoding a transaminase [58]. Such an organization
has recently been found in the L. lactis chromosome.
Transcriptional regulation by arginine under anaerobiosis
has been shown. Furthermore, a cre element upstream from
the arcA gene is present. This regulator signal indicates that
arginine degradation is submited to Ccpa-mediated catabolic
repression. This is in accordance with the observed arginine
catabolism only at low glucose concentration. The analysis
of arc mutants revealed that no other route exists in L. sakei
for arginine degradation. This degradation is associated with
enhanced survival during the stationary phase. A mutant
unable to lower the pH also exibits the same behavior,
showing that cell death is not due to low pH and that
survival due to arginine catabolism is mainly due to energy
generation by ATP generation rather than a rise in pH [9].
3.6.2.2. Other amino acids. Due to a putative role of
amino acid catabolism products in flavor development, these
activies were screened in starter strains. Thus, leucine seems
to be weakly catabolized via an amino transferase, but genes
for such activity have not yet been identified [28]. Histidine
and tyrosine decarboxylation activities have also been in-
vestigated as they can lead to histamine production, a major
risk in food products. No strain showed significant capacity
for amino acid decarboxylation [42] or tyramine production
[36]. Nevertheless, information is lacking concerning the ge-
netic determinism of enzymatic pathways involved in these
catabolisms.
3.6.3. Proteolytic activities and peptidasic activities
Proteolytic activities of LAB are generally well docu-
mented for dairy LAB since these activities play a major role
in growth and also in the development of flavor and texture
of fermented products. Such an involvement has also been
suspected for L. sakei since production of peptides and free
amino acids has been reported during the ripening process
of fermented sausages. Nevertheless, it seems difficult to es-
tablish the microbial role and the enzymatic role of this ac-
cumulation of protein catabolism products.
Global proteinase and aminopeptidase activities have
been demonstrated for some L. sakei strains in pork sar-
coplasmic and myofibrillar proteins [14]. Inoculation of cell
extract in meat extract also resulted in free amino acid accu-
120 M.-C. Champomier-Vergs et al. / Research in Microbiology 153 (2002) 115123
Table 2
Characteristics of purified peptidases of L. sakei
Main substrate specificity
dipeptidase broad
Tyr-, Val-, Leu-X
dipeptidase X-Pro
X-prolyl dipeptidyl peptidase
tripeptidase broad
100% against Ala-Ala-Ala
high against basic or aromatic
amino acids at N-terminal
position
aminopeptidase broad
Leu-, Ala-AMC(a)
(a) Aminocoumarine derivative.
mulation, mainly glutamic acid and alanine, which are both
known to participate in flavor development. Besides this gen-
eral approach, only three peptidases have been purified and
studied in L. sakei (Table 2): a general aminopeptidase with
broad specificity, a dipeptidase with primary specificity to-
wards Ala-X peptides and neutral amino acids, an X-prolyl
dipeptidyl aminotransferase with X-Pro specificity and a
tripeptidase with broad specificity against di-and tripeptides.
Nevertheless, their involvement in growth and physiology
and their possible impact on meat technology have not been
established.
Proteolysis of muscle myofibrillar proteins and also lipol-
ysis and lipid oxidation are known to be key activities in fla-
vor formation in fermented meat products. Although such
activities were partially identified in L. sakei (see above),
data are rather scattered and no clear conclusion can be
drawn as to the relationship between L. sakei metabolism
and aroma formation. It seems clear that the metabolism
of other microbial species (mainly Staphylococci and yeast)
together with endogenous meat enzymes are also impor-
tant components of flavor development (for a review see
ref. [17]). However, this aspect of L. sakei metabolism is still
poorly understood and widely underestimated; it could thus
constitute one of the interesting challenges for starter strain
improvement.
4. Molecular genetics of L. sakei: genetic tools and
recent developments
A prerequisite to molecular genetic investigations in
microorganisms is a set of basic tools allowing DNA
transfer and subsequent genetic constructs. Several attempts
to transform L. sakei strains by electroporation yielded
various efficiencies ranging from 102 to more than 107
transformants per g of DNA, depending on strains and
plasmids used ([2] and references therein). Conjugal DNA
transfer could also be observed [27].
MW References
50 kDa [37]
88 kDa [45]
55 kDa [44]
35 kDa [43]
Highly efficient DNA transfer by electroporation allowed
the construction of mutants by chromosomal integration of
a nonreplicating plasmid carrying the erythromycin resis-
tance marker of pAM1, through Campbell-like recombi-
nation [30]. It was shown that the minimal size of homology
between the suicide vector and the chromosome, necessary
for chromosomal integration, was 300 bp. In the mutants ob-
tained by single-crossover, the integrated plasmid is flanked
by two copies of the homologous DNA used for recombina-
tion, leading to an unstable structure unless selective pres-
sure is maintained by erythromycin. It then became possible
to construct a second generation of mutants obtained by two
successive crossovers, thus allowing stable gene replace-
ment. Such a point mutation was constructed in ptsI [51], as
well as a deletion of the lacLM operon [52]. The construc-
tion of a mutant selected directly for a double crossover by
the use of the chloramphenicol resistance marker of pC194
was also reported [34].
Reporter genes are frequently used in bacteria in order to
facilitate gene expression studies. The lacZ gene, encoding
-galactosidase, is largely used in many organisms but its
use in L. sakei was made possible only after the deletion
of the endogenous lacLM operon [52]. The gusA gene
encoding -glucuronidase and the green fluorescent protein
gene (gfp) were also successfully used in L. sakei [16,20]. In
these studies, the reporter genes were either integrated into
the chromosome or expressed from a multicopy replicating
plasmid: gusA was fused to the katA promoter and carried
by the replicating plasmid pNZ272, thereby revealing the
oxygen-dependent regulation of katA [20]; lacZ was fused
to a copper-inducible promoter and its expression could be
measured after chromosomal integration [52]; gfpuv , fused to
the constitutive ldhL promoter, could be detected both as a
single chromosomal copy and when carried by a replicating
plasmid [16].
The plasmid content of several L. sakei strains has
been investigated but little information on their function is
available. Most L. sakei strains contain at least one plas-
 M.-C. Champomier-Vergs et al. / Research in Microbiology 153 (2002) 115123
mid, the size of which ranges from 2 to more than 50
kb, and the restriction map of three small L. sakei cryp-
tic plasmids was published but no information on their
function was reported [57]. Some of the L. sakei plas-
mids could be cured by plasmid incompatibility or by
antibiotic treatments combined to high temperature, such
as the 42-kb cryptic plasmid of L. sakei 64F which was
lost after establishment of pIL205 by conjugal transfer [2,
27]. The resulting strain had lost its kanamycin resistance
and its ability to grow on maltose (R. Lauret, F. Morel-
Deville, unpublished results). Recently, L. sakei strains re-
sistant to tetracycline were isolated from meat products al-
though this species is naturally tetracycline-sensitive. How-
ever, plasmid-associated resistance as well as the mecha-
nism by which it was acquired have not yet been investi-
gated [15].
Finally, the most valid information on the function of
L. sakei plasmids comes from bacteriocin production stud-
ies (see above). A system designed for plasmidic expression
of bacteriocin genes in L. sakei was developed using L. sakei
genetic elements carried by a replicon isolated from Lacto-
bacillus plantarum [1]. Information on phages of L. sakei
is even more scarce, undoubtedly because investigations in
this field have not been encouraged since phage attacks have
never been described as posing a technological problem dur-
ing the meat fermentation process. One phage, PWH2, was
described, but as far as we know, its 35-kb linear genome has
never been sequenced [31]. Because of the lack of informa-
tion on extrachromosomal DNA in this species, no plasmid
vector based on L. sakei elements has yet been reported.
The use of transposons for mutagenesis provides an
excellent tool for targeting, localizing and cloning of specific
genes. Again, most of the information on transposition
and on IS elements in L. sakei comes from bacteriocin
production studies. Two similar IS elements belonging to the
IS3 family, IS1520 and IS1163, were characterized through
the isolation of spontaneous mutants which had lost the
capacity to produce lactocin S [48,49].
5. Concluding remarks
During the last decade, many genetic tools have been de-
veloped and used in L. sakei and an increasing knowledge of
this LAB has become available. In 1998 less than 15 genetic
loci from L. sakei had been sequenced [18]. We recently de-
termined the size of the chromosome and approximately 40
loci have been mapped [12]. The chromosome of our model
strain is currently being sequenced and the use of bidimen-
sional electrophoresis is helping to detect approximately 400
protein spots [35]. After completion of the sequence, these
spots will be assigned to known DNA sequences and a fur-
ther step in the development of L. sakei investigations can be
expected.
121
Acknowledgements
We thank Anika Marceau and Carl Alfred Alpert for
scanning electron microscopy of L. sakei.
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