plg-1 gene expressing propionicin by lactic starters ... - ResearchGate

porcupineideaBiotechnologie

16 déc. 2012 (il y a 4 années et 9 mois)

188 vue(s)

Arab Republic of
Egypt

Alexandria
University

Sameh E. Mohamed and Mahmoud K. Tahoun

Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University.

Aflatoon

Street, El
-
Shatby

21545
, Alexandria, Egypt
.

Propionicin

PLG
-
1

is

a

bacteriocin

produced

by

Propionibacterium

thoenii

P
127
.

Such

bacteriocin

inhibits

wide

range

of

food
-
borne

pathogens

such

as

Escherichia

coli,

Pseudomonas

aeruginosa,

Vibrio

parahaemolyticus,

Yersinia

enterocolitica

and

a

strain

of

Corynebacterium

sp
.

In

the

present

study,

plg
-
1

gene

expressing

propionicin

PLG
-
1

was

isolated

for

the

first

time

and

transferred

to

different

lactic

acid

bacterial

[LAB]

strains

using

pLEB
590

to

give

the

modified

vector

pLEBPLG
-
1
.

LAB

transformants

showed

strong

antimicrobial

activity

against

E
.

coli

DH
5
α

(most

affected

strain),

Listeria

monocytogenes

18116
,

and

Salmonella

entirica

25566
.

Such

LAB

transformants

can

be

used

in

dairy

industry

to

control

the

food
-
borne

pathogens

that

are

largely

distributed

in

worm

climates

and

to

feed

school

children

in

the

poor

countries

where

epidemic

diseases

and

diarrhea

prevails
.


DNA manipulations

Isolation

of

plasmids

from

E
.

coli

was

carried

out

using

BioBasic

EZ
-
10

plasmid

spin

column

kit,

whereas

isolation

of

plasmids

from

LAB

was

performed

using

a

mini
-
prep

isolation

method
.

Purification

of

DNA

samples

was

performed

using

Fermentas

DNA

purification

spin

column

kit
.

DNA

digestions,

DNA

ligations,

and

electroporation

of

E
.

coli

strains

were

carried

out

according

to

Sambrook

and

Russell

[
2003
],

while

electroporation

of

lactobacilli

strains

was

carried

out

according

to

Serror

et

al

[
2002
]

and

electroporation

of

lactococci

strains

was

performed

according

to

Holo

and

Nes

[
1989
]
.

Electroporation

and

stability

of

pLEBPLG
-
1

into

LAB

strains

Electroporation

efficiency

of

different

LAB

strains

harboring

pLEBPLG
-
1

was

indicated

as

4
.
2

X

10
9

cfu/µg

DNA

for

L
.

lactis

LL
108
,

while

that

for

S
.

thermophilus

was

found

to

be

4
.
1

x

10
9

cfu/µg

DNA
.

On

the

other

hand,

L
.

bulgaricus

DSM
20080

and

L
.

plantarum

TF
103

recorded

5
.
1

x

10
8

and

5
.
2

x

10
8

cfu
/µg

DNA,

respectively
.


The present work was supported using the fund of the International Center for
Genetic Engineering and Biotechnology [
ICGEB, Italy
].

plg
-
1
gene expressing propionicin by lactic
starters in dairying

Isolation

of

plg
-
1

gene

of

P
.

thoenii

P
127

using

specific

PCR

and

sequencing

of

the

PCR

product

After

PCR,

the

PCR

products

were

exposed

to

electrophoretic

migration

on

agarose

gel

[
1

%
]

in

order

to

visualize

their

bands

compared

to

the

1

Kb

DNA

ladder
.

To

our

knowledge,

this

is

the

first

time

to

isolate

such

gene
.

Furthermore,

a

sample

of

the

purified

PCR

product

is

prepared

to

be

sequenced

using

ABI

Prism

377

DNA

sequencer

[Perkin



Elmer,

Applied

Biosystems
,

USA]
.


Cloning

and

expression

of

plg
-
1

gene

of

P
.

thoenii

P
127

into

LAB

plg
-
1

gene

was

cloned

into

pLEB
590

and

electroporated

into

different

LAB

strains
.

pLEB
590

was

constructed

as

an

expression

vector

for

LAB
.

Such

vector

fulfilled

all

necessary

requirements

to

be

a

food

-

grade

vector
;

hence

it

has

a

small

size

[
3
.
1

Kb],

a

multiple

cloning

site

[MCS],

a

nisin

immunity

gene

[
nisI
]

as

a

dominant

food

-

grade

selection

marker,

a

high

copy

number,

and

a

high

stability

for

several

generations
.

It

has

a

potential

for

use

in

dairy

processes,

in

order

to

construct

improved

LAB

starter

strains


Screening

for

the

antimicrobial

activity

of

LAB

transformants

against

Escherichia

coli

DH
5
α,

Listeria

monocytogenes

18116
,

and

Salmonella

entirica

25566

(Table

1
)

PLG
-
1

has

strong

antimicrobial

activity

against

the

examined

pathogens

with

special

emphasis

on

E
.

coli

DH
5
α

which

was

the

most

sensitive

strain
.

Thus,

propionicin

PLG
-
1

was

expressed

using

pLEB
590

as

a

multi
-
copy

expression

vector
.


Microbial

strains,

plasmids

and

growth

conditions

All

microbial

strains

used

in

this

study

and

their

media

are

listed

in

Table

1
.

E
.

coli

DH
5
α

was

grown

in

LB

medium

at

37
°
C
.

L
.

monocytogenes

18116

and

S
.

entirica

25566

was

grown

in

BHI

at

37
°
C
.

Lactobacilli

strains

were

grown

in

MRS

medium,

whereas

lactococci

strains

were

grown

in

Elliker

medium

containing

0
.
5
%

glucose

[G

Elliker]
.

L
.

lactis

MG
1614

harboring

the

vector

pLEB
590

was

grown

in

G

Elliker

containing

nisin

(
60

IU/ml)
.

All

lactococci

and

lactobacilli

were

grown

at

30
°
C
.

All

propionibacterial

strains

were

grown

on

mNLB
.


Genomic DNA isolation, extraction from gel, primers designing, and specific PCR

Genomic

DNA

was

isolated

using

Fermentas

g

DNA

purification

kit,

whereas

DNA

bands

were

extracted

using

AxyPrep
TM

DNA

Gel

Extraction

Kit
.

Suitable

primers

[PLG
1
BAMHI
2

(G

G

A

T

C

C

A

A

T

G

T

C

G

A

T

G

C

C

A

G)

and

PLG
1
R
3
-
13

(T

G

G

G

G

T

C

G

A

G

T

T

G

C

A

G

A

C

C

C

C

A

A

T)]

were

designed

using

Geneious

software

4
.
0
.
2

to

isolate

plg
-
1

gene

of

P
.

thoenii

P
127
.

PCR

experiments

were

carried

out

with

a

thermal

cycler

[
Techne
,

UK]
;

Gold

Master
-
Mix

Beads

were

used

as

recommended

by

the

manufacturer

[
Bioron
,

Germany]
.

The

reactions

[volume,

20

µl]

were

performed

with

100

pmol

of

each

primer
.

The

PCR

conditions

used

for

amplification

of

DNA

fragments

containing

the

plg
-
1

gene

included

a

hot

start

at

94

°
C

for

3

min,

followed

by

35

cycles

of

denaturation

at

94

°
C

for

30

s,

annealing

at

59

°
C

for

30

s,

and

polymerization

at

72

°
C

for

6

min
.

Fig

1

Different

PCR

trials,

lane

5

is

the

most

perfect

trial
.

Table
1
The antimicrobial activity of different LAB transformants.