Nisin Tolerance of DnaK-overexpressing Lactococcus lactis Strains at 40°C
The effect of different concentrations of nisin on DnaK-overproducing Lactococcus lactis strains while growing at 40°C was examined. The plasmid pNZ-8048 which has a nisin promoter was used as a vector for transformation and expression of heterologous or homologous DnaK into L. lactis NZ9000. The transformants were then induced with different concentration of nisin and allowed to grow in presence of nisin. It was found that nisin, the antimicrobial peptide used for induction of DnaK-overexpression, itself conferred a lower but significant stress to the strains. Escherichia coli DnaK (DnaKEco)-overexpressing cells showed better tolerance to nisin than L. lactis DnaK (DnaKLla) and T. halophilus DnaK (DnaKTha)-overexpressing cells. However, all DnaK-overexpressing strains showed better tolerance than the cells without DnaK overexpression. These findings suggest the possibility for a relationship of DnaK protein and tolerance to different antimicrobial peptides.
Received: September 06, 2011;
Accepted: December 23, 2011;
Published: February 21, 2012
L. lactis, a spherical-shaped gram-positive lactic acid bacterium, has
been used for long time for industrial production of fermented dairy products
such as milk, cheese and yogurt. Microbial biomass of Lactic Acid Bacteria (LAB)
are also widely used in the food and pharmaceutical industries (Lee
et al., 2007). Because of the importance of lactic acid bacteria
in food industry extensive research has been done on their metabolic pathway
to increase its efficiency for fermentation. Recently, the bacterial group is
getting much attention even in medical science (Anukam,
2007). Food supplemented with LAB have been shown to work against cancer
development (Gursoy and Kinik, 2006). Some of their
special characteristics such as faster growth, acidification and resistance
to bacteriocin made them suitable for selection in the dairy industries (Van
de Guchte et al., 2002; Yateem et al.,
2008; Ali, 2011). On the other hands, these strains
need to overcome the stress conditions encountered in the fermentation processes
for better productivity. Many efforts have been made to overcome these problems.
With the recent developments of molecular genetics and proteomics, some genes
are figured out to be involved in stress responses of LAB, other bacteria and
mammalian cells (Abdullah-Al-Mahin et al., 2010;
Fiocco et al., 2007; Prasad
et al., 2003; Walker et al., 1999;
Walter et al., 2003; Kullen
and Klaenhammer, 1999; Lentze and Narberhaus, 2005;
Hsp70s play a very important role for folding of newly synthesized proteins,
refolding of misfolded proteins and transportation of proteins through biological
membranes both under normal and stressed conditions (Mayer
et al., 2001). DnaK, a 70-kDa heat shock protein homolog in bacteria,
transduces signals to other cellular factors when a shift of temperature increase
occurs (Craig and Gross, 1991). The hsp70 genes are
widely conserved in all organisms except some archaeal strains. However, the
functional studies of this heat responsive gene were widely carried out with
Saccharomyces cerevisiae, E. coli and Bacillus subtilis
(Homuth et al., 1997; Schulz
et al., 1995). DnaK functions in cooperation with DnaJ and GrpE and
the complex of DnaK-DnaJ-GrpE plays a significant role in the refolding of thermally
damaged proteins. However, the complex also assists in the folding of nascent
protein chains under normal growth conditions (Bukau, 1993;
Bukau and Walker, 1989, 1990;
Cegielska and Georgopoulos, 1989). Liberek
et al. (1991) reported that DnaK requires ATP for its activity in
vitro and this ATPase activity is stimulated by its co-chaperone DnaJ and
GrpE. Like other bacteria, L. lactis also exhibits a heat shock response
in which molecular chaperones play key roles (Arnau et
al., 1996; Kilstrup et al., 1997; Whitaker
and Batt, 1991). Previously, overproductions of GroEL/ES and small Hsps
have been reported to improve the stress tolerance of lactic acid bacteria (Desmond
et al., 2002, 2004). We reported earlier
that both heterologous and homologous expression of DnaK in L. lactis
improves heat, salt, ethanol and acid (low pH)-stress. We also found that nisin,
the antimicrobial peptide used for induction of DnaK-overexpression also conferred
stress both at normal physiological conditions and high temperature (Abdullah-Al-Mahin
et al., 2010). Therefore in this report, we aimed to investigate
more in detail the effect of DnaK-overexpression in L. lactis strains
to nisin when growing at 40°C.
MATERIALS AND METHODS
Bacterial strains and media: L. lactis subsp. cremoris
NZ9000 and E. coli JM109 were used throughout the study. L. lactis
NZ9000 was grown in GM17 medium (M17 broth supplemented with 0.5% glucose) at
30°C, unless stated otherwise. E. coli JM109 was grown aerobically
in Luria-Bertani (LB) broth at 37°C, unless stated otherwise. T. halophilus
JCM5888 was grown in MRS medium (Oxoid, Hampshire, UK) containing 1 M NaCl
at 30°C. The medium was adjusted to pH 7.5 before sterilization. In order
to facilitate clonal selection, 5 μg mL-1 chloramphenicol was
added to the media.
Construction of DnaK-expressing L. lactis: Chromosomal DNA was
isolated from E. coli JM109, L. lactis NZ9000 and T. halophilus
JCM5888 using combination of two methods as described earlier (Berns
and Thomas, 1965; Marmur, 1961). The DnaKEco
gene was amplified from E. coli chromosome using primers 5-CCCCTATTAGGATCCCACAACCACATGATGACCGAATATAT-3
and 5-GTCAGTATAATTACCCGTTTATAGAGCTCTTATTT-3. The BamHI and
SacI sites were simultaneously inserted into the amplified DnaKEco
gene. A BamHI restriction endonuclease site was inserted into the plasmid
pNZ8048 (De Ruyter et al., 1996) by inverse PCR
using primers 5-CTAGAGAGCTCAAGCTTTCTTTGAACCAAA-3 and 5-TTTTGTGGATCCTTTCGAACGAAATC-3.
The DnaKLla was amplified from L. lactis NZ9000 using primers
5-ATATTGACCGCCATGGCTTTAAACTATTC-3 and 5- ACTGACGAAACGATGAGCTCTTTTTTAAA-3,
The NcoI and SacI sites were simultaneously inserted into the
amplified DnaKLla gene. The DnaKTha gene was amplified
from T. halophilus JCM5888 chromosome using primers 5-AGATCAATATCATGAGTAAGATAATTGGTATTGACT-3
and 5-ATTTCCCAAATAGAGCTCTTATTGATTATCGTT-3. The PagI and SacI
sites were simultaneously inserted into the amplified DnaKTha gene.
PCR was performed with KOD plus Dna polymerase (Toyobo, Osaka, Japan). The amplified
PCR products were purified with the QIAquick PCR Purification Kit (Qiagenne
West Sussex, United Kingdom). All the amplified DnaK genes and the plasmid were
digested with their respective restriction enzymes. For insertion of DnaKLla
and DnaKTha the plasmid pNZ8048 was enzymatically digested with NcoI
and SacI restriction enzymes. Enzymatically digested products were then
ligated using Ligation High ver. 2 (Toyobo), according to the manufacturers
instructions. The resulting plasmids which contained DnaKEco DnaKLla
and DnaKTha were named as pNZ-EDnaK, pNZ-LDnaK and pNZ-TDnaK. All
these three plasmids were then transformed into L. lactis NZ9000, according
to the method developed by Holo and Nes (Holo and Nes, 1989)
and the transformants were designated as NZ-EDnaK, NZ-LDnaK and NZ-TDnaK, respectively.
An empty plasmid pNZ8048 were also transformed into L. lactis NZ9000
and the transformant was named as NZ-Vector which is used as the control strain
throughout the study.
Overexpression of DnaK and investigation of stress tolerance: All the transformants were grown in GM17 medium (10 mL) containing 5 μg mL-1 chloramphenicol at 30°C. When optical density at 600 nm (OD600) reached at 0.5-0.6, the expression of DnaK was induced with different concentrations of nisin (0, 0.05, 1.0, 10.0, 25.0 and 50 ng mL-1) for overnight. Nisin solution was prepared with commercial nisin of Streptococcus lactis (Sigma). Aliquots of the nisin-induced cultures were then transferred to fresh GM17 medium (30 mL) containing 5 μg mL-1 chloramphenicol and the same concentrations of nisin that was used for induction to obtain an OD600 of 0.04-0.05. Bacterial strains were then allowed to grow at 40°C and growths were measured at OD600. Bacterial strains that survived combined stresses of nisin and heat were detected using the same method. All the experiments were repeated at least three times to check the reproducibility of the results.
Confirmation of DnaK production by Western blotting: To check the production
level of DnaK proteins after induction with different nisin concentration, overnight
bacterial cultures (10 mL) were harvested by centrifuging at 6,000x g for 5
min at 4°C. Cell-free extracts were prepared from the pellets suspended
in chilled 50 mM potassium phosphate buffer (pH 7.4) by using a Multi-Beads
Shocker (Yasui Kikai, Osaka, Japan) at 2,500 rpm for 1 min at 4°C; this
process was repeated 5 times with 1 min intervals. The cell-free extracts were
obtained by centrifuging at 2,000x g for 15 min to remove the cell debris and
the supernatant that contained the soluble proteins was collected. The protein
concentration in the supernatant was determined by using the Bradford assay
kit (Nacalai Tesque, Kyoto, Japan). The soluble proteins (10 μg) were then
subjected to separation on a 12% (w/v) SDS-PAGE gel. The proteins were transferred
onto a polyvinylidene difluoride membrane. Immunoblotting and detection of DnaK
proteins were performed as previously described using anti-T. halophilus
DnaK antibody (Sugimoto et al., 2008).
Overexpression of DnaK in L. lactis: To check the production
of DnaK upon nisin induction, cell lysates of all four transformants induced
with different concentrate of nisin (0-50 ng mL-1 nisin) was used
for Western blot analysis (Fig. 1a-c). We
could detect L. lactis DnaK (DnaKLla) in all the transformants
including control strain (NZ-Vector). An additional band was detected in NZ-EDnaK
only in presence of 10 ng mL-1 nisin which had an equal mobility
of that of DnaK from E. coli cell extract used as a control (Fig.
1b). DnaKEco could be clearly distinguished from DnaKLla
in NZ-EDnaK based on the difference in their mobilities. Between the two protein
bands obtained from the cell lysate of NZ-EDnaK, the upper band was identified
as DnaKEco. It was also observed that the amount of heterologously
produced DnaKEco is very low compared to the NZ-LDnaK`s own DnaKLla.
When NZ-LDnaK and NZ-TDnaK were induced with different nisin concentration overproduction
of L. lactis DnaK (DnaKLla) and T. halophilus DnaK
(DnaKTha) were detected in cell lysates of NZ-LDnaK and NZ-TDnaK,
respectively (Fig. 1b).
||DnaK production after nisn induction. Cell-free extracts of
NZ-Vector (NZ-V), NZ-EDnaK (NZ-E), NZ-LDnaK (NZ-L) and NZ-TDnaK (NZ-T) prepared
from cultures that were induced overnight with indicated concentration of
nisin in GM17 liquid medium supplemented with 0.5% glucose at 30°C were
separated using a 12% SDS gel for Western blotting. In cell free extracts
production of DnaK was confirmed using DnaK polyclonal antibody raised against
T. halophilus DnaK. (a) DnaK productions after induction with 0-10
ng mL-1 nisin were checked by western blot where a single blot
was use for NZ-Vector and NZ-EDnaK. Here cell free extract of E. coli
(Ecoext) was used as control for DnaKEco. (b) DnaKLla
and DnaKTha production were checked in another blot in presence
of 0-10 ng mL-1 nisin where same concentration of NZ-Vector cell
free extract was used to compare the amount of these DnaKs. (c) DnaK productions
in cell free extracts of NZ-Vector, NZ-EDnaK and NZ-LDnaK were compared
in another blot after induction with 10-50 ng mL-1 nisin. Ecoext
was used as control. Equal amounts of proteins were used in all lanes
||Generation time and maximum growth yields of L. lactis
strains in presence of different concentration of nisin GM17 media at 40°C
|aGeneration time was determined in the exponential
growth phase. bND: Not detected (since the cells were not in
exponential phase). c: Uncalculated
Although chromosomal- and plasmid-borne DnaK could not be separated in NZ-LDnaK
and NZ-TDnaK the increased band intensity confirmed the overproduction of DnaKLla
and DnaKTha in cell lysates of NZ-LDnaK and NZ-TDnaK, respectively.
Figure 1 clearly shows that although 0.05 ng mL-1
nisin was quite enough to induce DnaK production in NZ-LDnaK and NZ-TDnaK (Fig.
1b) production of E. coli DnaK was induced only after induction with
10 ng mL-1 nisin (Fig. 1a). To check whether increased
nisin induce further production of DnaK in the studied strains cell lysates
of the strains after induction with 25 and 50 ng mL-1 nisin were
compared with the cell lysates induced with 10 ng mL-1 nisin. It
was then detected that nisin concentration more than 10 ng mL-1 did
not induce more DnaK production in any of the strains of NZ-Vector, NZ-LDnaK,
NZ-EDnaK (Fig. 1c) and NZ-TDnaK (Data not shown).
Tolerance to nisin stress by DnaK-overexpression at 40°C: While
comparing the nisin stress tolerance of all heterologous or homologous DnaK-producing
cells at 40°C, no growth difference of the strains in absence of nisin (Fig.
2a) and no visible growth of the strains in presence of 25 and 50 ng mL-1
nisin (Fig. 2e, f) clearly indicates a stress
effect of this antimicrobial agent in presence of 40°C temperature. In presence
of 0.05 ng mL-1 nisin, there were no significant difference in growth
pattern (Fig. 2b), generation time and maximum OD600
(Table 1) although in this concentration of nisin NZ-LDnaK
and NZ-TDnaK over-produced DnaKLla and DnaKTha (Fig.
1b). Again, in presence of 1 ng mL-1 nisin, the generation time
and maximum OD600 of NZ-Vector, NZ-EDnaK and NZ-LDnaK were also not
significantly different. However, DnaKTha producing cells showed
a little higher generation time (1.11-fold) and lower maximum OD (0.86-fold)
than the corresponding values of NZ-Vector (Table 1). A clear
difference in stress tolerance by the studied strains was visible in presence
of nisin concentration of 10 ng mL-1 only (Fig. 2c).
||Effect of nisin on growth at 40°C. NZ-Vector, NZ-EDnaK,
NZ-LDnaK and NZ-TDnaK were grown in GM17 medium containing 0.5% glucose
as the sole carbon source containing without pH control. Nisin concentrations
of (a) 0 ng mL-1, (b) 0.05 ng mL-1, (c) 1 ng mL-1,
(d) 10 ng mL-1, (e) 25 ng mL-1 and (f) 50 ng mL-1
were used in the media and the periodic growth was measured at 600 nm. The
values shown are the Means± standard errors (error bars) for three
At 10 ng mL-1 nisin concentration, the concentration which is sufficient
for the DnaK-overproduction in all the strains, NZ-EDnaK showed maximum growth
yields among the strains indicating highest tolerance to the combined effect
of nisin and heat. Since NZ-Vector was not in log phase generation time could
not be compared. However, the maximum OD of NZ-EDnaK, NZ-LDnaK and NZ-TDnaK
were 7.81, 4.75 and 4.44 fold higher than that of NZ-Vector (Table
1) suggesting the potency of overproduced DnaK to make the strains capable
to grow even the conditions when NZ-Vector showed a very negligible growth.
Although recent developments in fermentation technology are capable of minimizing
the stress conditions during fermentation, scientists are conducting research
on stress-stable fermentation microorganisms and overexpression of molecular
chaperone genes is now considered to be one of the most promising approaches
to achieve that goal. We successfully overexpressed DnaKEco in L.
lactis which showed multiple stress tolerance and higher lactic acid production
at high temperature (Abdullah-Al-Mahin et al., 2010).
However, the problem that was found is that nisin, the antimicrobial peptide
used for DnaK overproduction, itself conferred stress to L. lactis strains
(Abdullah-Al-Mahin et al., 2010). Antimicrobial
agents were also reported to effect on the production of lactic acid and other
fermentation products by LAB (Abou Ayana et al.,
2011). Therefore, this study was aimed at evaluating the stress tolerance
to nisin after expression of DnaK in lactic acid bacterium L. lactis
NZ9000. Our study concluded with the finding that the expression of DnaK conferred
increased tolerance to combined stress effect of heat and nisin to L. lactis
NZ9000. The role of molecular chaperone in stress tolerance has already been
reported by many researchers. Susin et al. (2006)
reported the importance of DnaK/DnaJ for the survival of Caulobacter crescentus
when exposed to heat stress. Previously, GroESL-overproducing L. lactis
was reported to be more tolerant to heat (54°C for 30 min), salt (5 M NaCl
for 1 h), or solvent (0.5% butanol) stress (Desmond et
al., 2004). Sugimoto et al. (2003) earlier
reported the remarkable suppression of the 5% (0.86 M) NaCl-induced protein
aggregates by the overproduction of T. halophilus DnaK in E. coli.
Tomas et al. (2003) reported that the overexpression
of groESL in Clostridium acetobutylicum resulted in a 38 and 30%
increase in acetone and butanol production, respectively, relative to the plasmid
control strain during pH-controlled glucose-fed batch acetone-butanol fermentation.
The groESL-overexpressing strain also showed increased tolerance against
butanol than plasmid-controlled strain. Involvement of heat shock proteins to
protect plants against heat and salt stresses has also been reported (Essemine
et al., 2010; Mudgal et al., 2010;
Joseph and Jini, 2010). These findings with regard to
improved stress tolerance efficiency due to the expression of chaperone genes
were consistent with the findings of present study. Further, the most important
finding of this study was the tolerance of all the DnaK-overproducing strains
to combined effects of heat and nisin.
Nisin is known to effect on cell membrane of gram-positive bacteria having
the target site of lipid II (Guder et al., 2000;
Sahl and Bierbaum, 1998). Increased sensitivity of heat
stressed B. cereus and L. lactis to nisin was reported earlier
(Beuchat et al., 1997; Kalchayanand
et al., 1992, 1994). It was suggested that
nisin prevented the repair of heat damaged membrane. In this study, combined
action of heat and nisin (10 ng mL-1) effected on the cell growth
of NZ-Vector. Homologously or heterologously overproduced DnaK, especially DnaKEco
helped the strains to overcome the combined stresses.
Although DnaKLla and DnaKTha were produced higher than
DnaKEco, more potentiality for combined stress tolerance was found
by DnaKEco. These differences in DnaK overproduction ability can
be explained by differences in codon usase (Abdullah-Al-Mahin
et al., 2010). Our findings also suggested that DnaKEco
was more efficient than DnaKLla and DnaKTha to overcome
the stress at early growth period (Fig. 1d). Difference in
stress tolerance ability by different DnaK has already been reported earlier
(Abdullah-Al-Mahin et al., 2010; Sugimoto
et al., 2003). However, despite higher DnaK production the failure
to confer better stress tolerance by NZ-LDnaK and NZ-TDnaK suggested the importance
of functional potency rather than the level of production.
Finally, it can be conclude that both homologously or heterologously overproduced DnaK had the effect to rescue the growth inhibition due to nisin at high temperature. This finding could have important implication for exploring the scope for further study to find the relationship of DnaK/chaperones and tolerance to different antimicrobial peptides. Since, the target site of nisin is cell membrane, the tolerance to nisin thereby opens a scope to research about the role of DnaK in membrane stability against bacteriocins.
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