Download PDF

FEMS MicrobiologyLetters 71 (19~0) 105-110
Published by El~'vier
105
FEMSLE 04116
Integratien of an unstable plasmid into the chromosome
of Lactobacillusplantarum
J a n e E. Rixon t, Geoffrey. P. Hazlewood 2 and Ha .try J. Gilbert t
t Departmenlof Agricultural Biochemistry and Nutrition. NewcastleUnit,ersit~'. Newcastleupon Tyne.
and : AFRClnst*tute of Ammat Physiologyand Geneocs Research. Babraham,Cambridge. fLK.
Received 12 March 1990
Revisionreceivedalld accepted I May 1990
Key words: Lactobaeillus plantarum; Plasmid; Integration; Transformation
1, SUMMARY
A 2.3 kb EcoRl restriction fragment of
Lactobacitlus plantarum genomic D N A was cloned
into pSA3 to generate pJRI. Both pgA3 and pJR1
were transformed into L. plantarum. Growth of
the transformants in the absence of the selection
pressure, erythromycin (both plasmids confer
Emr), led to a stable sub-population of Emr
bacteria in which pSA3 and p JR1 had integrated
into the L. plantarum genome. Amplification of
the chromosomally located plasmids was observed
when the organism was grown in the presence of
erytbromycin. The integrated plasmid sequences
were stably maintained in the absence of selection
pressure, and did not affect the growth rate of the
lactic acid bacterium in rich media.
2, I N T R O D U C T I O N
Lactobacillus planmrum is a Gram-positive her• ro-fermentative lactic acid bacterium which plays
Correspondence Io: H.J. Gilbert. Department of Agricultural
Bio~hmalistry and Nutrition, University of Newcastle upon
Tyne, Newcastleupon Tyne, NEt 7RU, U.K.
a major role in forage conservation. Because of its
agricultural significance, several research groups
have been studying the genetics of this organism.
Luchansky et al. [1] described the transformation
of L. plantarum using broad host-range Gram
positive vectors. Recently, Bates et al. [2] and
Shcirlinock et al. [3] have expressed cellulase genes
in L planmrum. The lactic acid bacterium is widely
used in open ecosystems (ensilage, probiotic) and
thus heterologous D N A transformed into L.
planmrum must be stably maintained, in the absence of selection pressure, and not transferred to
other microorganisms in the ecosystems. One approach to achieving this objective is to insert foreign D N A into the Lactobaeillas ger~ome. Shcirlinock et al. 131, using a suicide vector, have integrated heterologous D N A into the chromosome
of at~ L. plantarum strain which can be transformed at a high frequency. However, most strains
of L, plamarum, including those with industrial
potential, take up exogenous D N A at very low
frequencies (1-60 cfu//~g DNA; Bates. Stewart.
Gasson and Warner, personal communications),
precluding the use of suicide vectors to achieve
foreign D N A genomic insertion. This study describes the use of an unstable plasmid to obtain
chromosomal integration of heterologous D N A in
037g-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
106
L plantarurn strains which are transformed at low
frequency. This approach provides a universal
method of achieving foreign DNA genomic integration in L. plantata~m strains. We also report
the genomic integration of the plasmid pSA3 even
though it shows no apparent homology to the L.
plantarum genome.
3. MATERIALS AND METHODS
3. i. Bacterial strains and vectors
E. colt JM83 and L planlarum NCD01193
were grown in MRS [4] and Luria broth respectively. Erythromycin (Era), tetracycline (Te) and
chhiramphenicol (Cm) were added to media at
concentrations of 10 p,g/ml, 12.5 btg/ml and 10
~ g / m ] respectively. The plasmid used in this study
was pSA3 [5], a streptococcal/E, colt shuttle vector which encodes Tc ~, and Cmr genes which function in E. coli, and an Em~ gene which expresses
in .L. plantarum.
3.2. Recombinant DNA techniques
Extraction of nucleic acid .from E. coli and L
plantarum, transformation off the organisms and
growth of the lactic acid bacterium under nonselective conditions were perfcrmed as described
in ref. 2. Restriction digests, restriction fragment
purification and agarose gel electrophoresis were
as described in reL 6. In Southern hybridisations
DNA was transferred from agarose gels to hybond
N filters (Amersham) using a Vacuum blotter
(Hybaid). Probes were labelled by nick-translation
[6] and hybridisations were performed overnight
at 68~C under high stringency conditions [6].
4. RESULTS
The plasmids pSA3 and p JR1 (a recombinant
ol F~A3 containing a 2.3-kb EcoRI restriction
fragment of the L. plamarum chromosome) were
transformed into L. plantarum by dectroporation
at frequencies of 5 and 3 transformants per/~g of
DNA respectively. The stability of each plasmid
in the lactic acid bacterium was assessed by grow-
Table
Percentage of Em~cellsflown in the absence of antibiotic
The bacterial colonit~1-15 were isolatedfrom the sttbpopulation of Erar bacteria after growing L plantarum harbouring
pSA3 and pJR1 non-selectively for 60 generations. ~es¢
organisms were again grown in the absence of antibioticand
the percentage of End"bacteria determined.
Plasmid
Isolateno.
pJRI
6*
9*
1
2
3
4
5•
6
7
8*
9*
10
It
t2
13
14
15
pSA3
No of generations
|0
30
S0
98
98
98
160
100
96
100
160
160
100
100
100
160
96
160
100
160
100
100
100
100
100
]00
100
94
100
100
100
160
1130
70
160
160
96
160
96
94
92
98
100
98
160
9~,
100
10G'
100
160
~6
* Used in S-hybridizationexpls.
lag transformants in the absence of selection pressure. The number of colonies retaining pSA3 and
p./Rl was determined by the percentage of Era'
bacteria (both plasmids contain an Era' gene). The
data revealed a rapid decrease in ',he E~nr of both
p JR1 and pSA3 transformants which stabilized
after 40 generations at 0.5 and 0.01,~ of the
bacterial population, respectively. Ten microorganisms from the Em' sub-population of pJRl
transformants were reinoculaled into non-selective
medium and grown for a further 70 generations.
Progeny of two of these retained the Emr phenotype. Similarly, 15 microorganisms from the Era'
sub-population of pS?,3 transformants c3~',inned
to express Em r when grown for a further 50 generations in the absence of antibiotic (Table 1). These
data show that both pSA3 and derivatives of the
plasuxid can be stably maintained in a small pereentage of L. planmrum cells, probably through
integration into the bacterial chromosome.
107
B
e
o
kb
-9.4
"" ~
;~
-s-s
-4'4
-2"3
-2"0
$
1
2
3
4
5
2
3
4
5
1
2
3
4
5
1 2
3
4
,5
Fig. L Somhem hybridization of L, plantartml DNA. Total DNA was extracted from L platllarum strains, subjected to agarose gel
electrophoresis and probed with pSA3. Filters were exposed to autoradiography for 30 rain. DNAs from four stable L plantarum
isolates harbouring pSA3 (2-5) were run alongside DNA from the non-transformed strain (t), Samples were left uncut (A) or
digested with BamHI (B), EcoRV (C) or SphI (D). Lane 5 contained pSA3 cleaved with BamHI. The position of molecular mass
markecs consisting of ~ DNA digested with Hindl[l are shown.
T o t a l D N A was isolated from the stable E m ¢
bacteria as described in t e l 6. T h e nucleic acid
was subjected t o Southern hybridisation using
pSA3 a n d p JR1 a s probes. Both plasmids hyb d d i s c d to undigested c h r o m o s o m a l D N A (Figs. 1
and 2). T h e r e was n o evidence of homology be-
D
c
B
J
kb
f
-23
-9'4
-6"6
q)'W
-4'4
-2.3
-2'0
I
2
3
1
2
3
1
2
3
1
2
3
Fig. 2. Southern hybridization of L plantarum DNA with pJR1 as the probe (J). Total DNA isolated horn non-transformed £.
plantarum strain NC D01193 H) and two stable transformants carrying pJR] (2 and 3) were cleaved with BamHI (B), EcoRV (C)
and Nrul (D) or left undigested IA~. Filters were exposed to autoradiography for 30 rain. The position of molecular mass markers
consisting of ~ DNA digested with Hmdlll are shown.
108
twecn the probes and extra-ct~romosomal elements. These data indicate that both replicons had
integrated into the L. plantarum genome, although
pSA3 showed no apparent identity with the
bacterial chromosome in the non-transformed
strain. Both pSA3 and pJR1 iiyhridised strongly
to single D N A species and weakly to two other
nucleic acid sequences, when the L. plantarum
D N A had been cleaved with restriction enzymes
which cut each of the two plasmids at a single site.
This is indicative of amplification of the hetcrologous D N A through ap array of tandem repeats.
The number of copies of pSA3 and pJR1 which
had integrated into the L. plantarum genome were
six and three respectively. In the case of pSA3, the
rcpceting unit was larger (12.5 kb) than linear
pSA3 (10.2 kb). This suggests that pSA3 amplification within the L. plantarum chromosome also
incorporates short sequences of the organisms gehome which flanks the plasmid insertion site. This
is not the case for p JR1 where the amplified unit
co-migrates with linear pJR1 which was 12.4 kb in
size.
4. l. Features of integrated pSA3
Total DNA, extracted from L. plantarum containing pSA3 integrated into the bacterium's chromosome, was transformed into competent E. coli
cells. No Tc r or Cm r colonies were observed (pSA3
encodes Gram-negative Tc r and Cm t genes) indicating the absence of extrachromosomal pSA3 in
the Lactobacillus. D N A extracted from L. plantarum in which pSA3 was maintained as a plasmid. discrete from the host's genome, generated
10 3 Tc r bacteria/lag D N A when inserted into E.
coll.
The effect of pSA3 chromosomal insertion on
the growth rate of L plantarum was also investigated. Wild-type and recombinants of the lactic
acid bacterium were co-inoculated into MRS containing no antibiotic. After growth for 20 generations the percentage of Em r microorganisms was
determined. Data showed that the proportion of
Em r bacteria remained constant throughout the
growth phase. This suggests that chromosomal
integration of pSA3, or its derivatives, does not
adversely effect the growth of L. plantarum at
least in rich medium.
5. DISCUSSION
Results presented in this paper show that the
unstable repficon, pSA3, can be used as d vector
to integrate D N A into the L. plantarum genome.
The major advantage of this system, compared to
the use of suicide vectors [3] is that the latter
requires a high transformation frequency. In the
approach described in this report, a tingle transformant, harbouring pSA3, when grown nonselectively will incorporate the plasmid into its
genomc. This is particularly relevant to Lactobacil.
/us spp. which can often only be transformed at
vepj low frequencies, thereby precluding the use of
suicide vectors.
In this study we have shown that pSA3, although not homologous with L plamarum DNA,
will integrate into the bacterium's chromosome.
Possible mechanisms for this insertion are as follows: (i) pSA3 does exhibit homology with the L.
plamarum chromosome, which is not detected by
Southern hybridisation; (ii) pSA3 contains a
transposon or insertion sequcnca which integrates
in a site specific fashion into the L plantarum
genome.
Future work will aim to exploit the chromosomal insertion of pSA3, to study the genetic
organisation of L. plamarum, and develop a reeorr, binant strain of the organism which is suitable
for t~ze in open ecosystems.
ACKNOWLEDGEMENTS
This work was supported by MAFF and ICi
(Open contract CSA 1347).
REFERENCES
Ill Luchansky, J,B., Muriana, P.M. and Klaenhammet, T.R.
(1989) Mol. MierobioL2, 637-646.
[2] Bales. E.E.M,, OilberL H,J., Hazles,0od, G.P.. Huckl¢, J..
Laurie, J,l. and Mann. S.P. (1989) AppL Environ. Microbiol, 55. 2095 2097.
[3l Scheitlink, T., Mahillon, J,, Joos, H., Dhat~m P. and
Michiels, F'. (1989} AppL Environ. Microbiol. 55, 21302137.
109
[4] De Man, J.C., Rogosa, C,M. and Sharp¢, M.E. (1960) J.
Appl. BactefioL 23,130-135.
~5] Dao, M.L and Ferrctti, JJ. (1985) Appl. Environ. Microbiol. 49,115-119.
[6] Manialis. T,. Fritsch, E.F. and Sambrooke, T. (1982)
Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY.