Sequence and comparative analysis of

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21 Φεβ 2013 (πριν από 4 χρόνια και 6 μήνες)

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F A C U L T Y O F L I F E S C I E N C E S

U N I V E R S I T Y O F C O P E N H A G E N

Sequence and comparative analysis of
Leuconostoc

dairy bacteriophages



Introduction


Bacteriophages (phages) cause large problems
in the dairy industry, resulting in significant
losses during production. Until now, most work
regarding dairy bacteriophages has focused on
phages of
Lactococcus lactis
. However, the
dairy industry is experiencing increasing
problems with
Leuconostoc

phages. This group
of phages has only been described very
limitedly in the literature, with only one full
genomic sequence of a lytic
Leuconostoc
phage, 1
-
A4, publicly available (Lu
et al
. 2010,
Applied And Environmental Microbiology, 76(6),
1955
-
1966).

In this poster we present 9 full genomes of dairy
Leuconostoc

phages.



Materials and methods



Bacteriophages

Leuconostoc

phages were isolated from dairy
environments. Host ranges were determined
using a set of 28 strains belonging to the
species
Ln. mesenteroides
,
Ln.
pseudomesenteroides

or
Ln. lactis.

Phages were finally purified by cesium chloride
gradient centrifugation.


















Electron microscopy

Transmission electron microscopy (TEM)
micrographs were taken from purified phages
for morphology analysis.


DNA preparation and sequencing

Phage DNA was isolated from purified phages,
purified by phenol
-
chloroform extraction and
characterized by restriction digest analysis.

Genomic DNA of nine phages with unique
restriction digest pattern were fragmented and
tagged with multiplex identifiers (MID).
Sequencing was performed on a 454 GS FLX
Titanium platform. Assembly process was
confirmed by PCRs.

Additional Sanger sequencing of ligated and
unligated phage DNA was performed in order to
analyze the
cos

site region.



Data analysis

Reads were assembled into contigs using 454
Newbler Assembler software v.2.3 or CLC
Genomics Workbench 4.6.1.

The genome sequences were analyzed using
Genmark.hmm gene prediction software,
adjusted for a novel phage gene prediction.


Comparative genome analysis

Genomic sequences were subjected to analysis
using TBlastX program. Comparison files and
sequences were visualized using Artemis
Comparison Tool.





Results



Selected phages were lytic to
Ln.
mesenteroides
(phages LN25, LN34, LNTR2
and LNTR3) or
Ln. pseudomesenteroides
(phages LN03, LN04, LN12, LN18 and LN6B).

No phages attacking
Ln. lactis

strains were
isolated. Phages selected for sequencing
revealed three different host
-
ranges. The
conserved host patterns corresponded with the
2 distinct morphotypes I and II except for type I
phage LN25 with a third unique host range.

All phages belongs to the
Siphoviridae

family
(
Caudovirales)
. Furthermore, phages could be
classified into two major groups based on the
baseplate structure (Fig.1). All
Ln.
mesenteroides
phages were morphotype I
phages while all
Ln. pseudomesenteroides

were morphotype II phages.



The full genomic sequences were determined,
with 44 to 876
-
fold coverage on average.
Phages had dsDNA genome with size ranging
from 25.7 to 28.4 kb. The genomic

G+C content was approximately 36% in all
cases.

Analysis of the sequences using
Genemark.hmm reveled 38
-
43 putative ORFs.

Genomes were modularly organized. No
lysogeny modules were detected.


Sanger sequencing of ligated and unligated
phage DNA reveled presence of
cos
-

sites in all
phage genomes, suggesting that all phages
analyzed show
cos
-
type DNA packaging.
















The conserved
cos
-
site of group I phage
genomes (CGGTTAGTAGTA) differed from
group II phage
cos
-
site (TCGTGCAATAGTA)
(Fig. 2)
.

Comparative genome analysis showed high
percentage of similarity within the groups on the
protein level.
Group I phages shared a high
degree of similarity to phage 1
-
A4 (
Lu
et al.
2010
), while group II showed only limited 30
-
60% similarities to this phage (Fig.3).





Witold Kot
1
, Lars H. Hansen
2
, Horst Neve
3
, Karin Hammer
4
,
Per D. Pedersen
5
,

Søren J. Sørensen
2
, Knut J. Heller
3

and Finn K. Vogensen
1
.


1
Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Denmark.

2
Department of Biology, Faculty of Natural Sciences, University of Copenhagen, Denmark.

3
Department of Microbiology and Biotechnology, Max Rubner
-
Institut, Kiel, Germany.

4
Department of System Biology, Technical University of Denmark, Lyngby, Denmark.

5

Clerici
-
Sacco Group, Cadorago, Italy.


































































Figure

1
.

Two

major

morphotypes

of

analyzed

Leuconostoc

phages
.

Globular

baseplate

structures

of

morphotype

I

phages

are

visible

on

micrograph

A
.

Morphotype

II

phages

can

be

divided

into

those

with

visible

NPS

(B,

see

arrow)

and

those

where

NPS

was

not

detected

(C,

see

arrow)
.

The

phage

presented

on

the

picture

is

indicated

with

red

color
.

Other

phages

with

a

similar

morphotypes

are

labeled

with

black
.

A

B

C

Figure

3
.

Comparative

genome

analysis

of

9

dairy

Leuconostoc

phages

and

reference

phage

1
-
A
4

(
Lu

et

al
.

2010
)
.

The

intensity

of

the

red

colour

indicates

the

degree

of

similarities

between

protein

sequences

in

predicted

ORFs
.

Two

clusters

of

highly

similar

sequences

can

be

distinguished,

which

correlates

with

the

TEM

picture

analysis

and

the

host

range

of

phages
.




Conclusions




Complete genomic sequences of 4 lytic
Ln.
mesenteroides

and 5
Ln. pseudomesenteroides

phages were obtained
.



TEM and comparative genomics of phages allow
to classify them into two major groups.



Genome
sequences provide deeper insight into
the biology of
Leuconostoc

phages.



Information
obtained during this study may
serve as the basis for developing new strategies
for
Leuconostoc

phage control.

LN34

LN25

LNTR2

LNTR3

LN12

LN03

LN18


LN6B

LN04


Group I Group II

Figure

2
.

Determination

of

cos
-
site

regions

in

Leuconostoc

phages
.

Sanger

sequencing

of

ligated

(A)

and

unligated,

linear

phage

DNA

(B

and

C)

reveled

presence

of

cos
-
sites

in

all

phage

genomes
.

The

conserved

cos
-
site

of

group

I

phage

genomes

(CGGTTAGTAGTA)

differed

from

group

II

phage

cos
-
site

(TCGTGCAATAGTA)
.


A

B

C