15
See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/6213084 Three small, cryptic plasmids from Aeromonas salmonicida subsp. salmonicida A449 ARTICLE in PLASMID · OCTOBER 2003 Impact Factor: 1.76 · DOI: 10.1016/S0147-619X(03)00058-1 · Source: PubMed CITATIONS 25 6 AUTHORS, INCLUDING: Jessica May Boyd American University of Nigeria 37 PUBLICATIONS 929 CITATIONS SEE PROFILE Bruce A Curtis Dalhousie University 25 PUBLICATIONS 1,092 CITATIONS SEE PROFILE Rama Singh National Research Council Canada 2 PUBLICATIONS 128 CITATIONS SEE PROFILE Available from: Jessica May Boyd Retrieved on: 02 September 2015

Aeromonas plasmids

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Seediscussions,stats,andauthorprofilesforthispublicationat:http://www.researchgate.net/publication/6213084

Threesmall,crypticplasmidsfromAeromonassalmonicidasubsp.salmonicidaA449

ARTICLEinPLASMID·OCTOBER2003

ImpactFactor:1.76·DOI:10.1016/S0147-619X(03)00058-1·Source:PubMed

CITATIONS

25

6AUTHORS,INCLUDING:

JessicaMayBoyd

AmericanUniversityofNigeria

37PUBLICATIONS929CITATIONS

SEEPROFILE

BruceACurtis

DalhousieUniversity

25PUBLICATIONS1,092CITATIONS

SEEPROFILE

RamaSingh

NationalResearchCouncilCanada

2PUBLICATIONS128CITATIONS

SEEPROFILE

Availablefrom:JessicaMayBoyd

Retrievedon:02September2015

Plasmid 50 (2003) 131–144

www.elsevier.com/locate/yplas

Three small, cryptic plasmids from Aeromonas salmonicidasubsp. salmonicida A449

Jessica Boyd,* Jason Williams, Bruce Curtis, Catherine Kozera, Rama Singh,and Michael Reith

National Research Council Institute for Marine Biosciences, 1411 Oxford Street, Halifax, NS, Canada B3H 3Z1

Received 21 February 2003, revised 2 June 2003

Abstract

The nucleotide sequences of three small (5.2–5.6 kb) plasmids from Aeromonas salmonicida subsp. salmonicida A449

are described. Two of the plasmids (pAsa1 and pAsa3) use a ColE2-type replication mechanism while the third (pAsa2)

is a ColE1-type replicon. Insertions in the Rep protein and oriV region of the ColE2-type plasmids provide subtle

differences that allow them to be maintained compatibly. All three plasmids carry genes for mobilization (mobABCD),

but transfer genes are absent and are presumably provided in trans. Two of the plasmids, pAsa1 and pAsa3, carry

toxin–antitoxin gene pairs, most probably to ensure plasmid stability. One open reading frame (ORF), orf1, is con-

served in all three plasmids, while other ORFs are plasmid-specific. A survey of A. salmonicida strains indicates that

pAsa1 and pAsa2 are present in all 12 strains investigated, while pAsa3 is present in 11 and a fourth plasmid, pAsal1, is

present in 7.

Crown Copyright � 2003 Published by Elsevier Inc. All rights reserved.

Keywords: Furunculosis; Plasmid addiction; ColE1-type replicon; ColE2-type replicon

1. Introduction

Aeromonas salmonicida subsp. salmonicida is the

aetiological agent of the salmonid disease furun-

culosis, a disease with both high mortality andmorbidity (Smith, 1997). Furunculosis is a signifi-

cant cause of economic loss in salmonid aquacul-

ture throughout the world. Furunculosis is a

complex disease and exists in different forms

* Corresponding author. Fax: +902-426-9413.

E-mail address: [email protected] (J. Boyd).

0147-619X/$ - see front matter. Crown Copyright � 2003 Published

doi:10.1016/S0147-619X(03)00058-1

depending on the health, age, and species of fish

and the conditions of their environment, particu-

larly temperature. The acute form is characterized

by rapid onset of general septicaemia, melanosis,

inappetence, lethargy, and haemorrhage at thebase of the fins. The chronic form is characterized

by slow-onset with low mortality, affected animals

often have raised skin lesions called furuncles

which are considered pathognomic for the disease.

Conversion from the chronic to the acute form can

be caused by environmental stressors.

Aeromonas salmonicida belongs to the fam-

ily Aeromonadaceae of the c proteobacteria.

by Elsevier Inc. All rights reserved.

132 J. Boyd et al. / Plasmid 50 (2003) 131–144

A. salmonicida is further subdivided into typical(A. salmonicida subsp. salmonicida) and atypical

strains. Many groups have surveyed plasmid car-

riage in both typical and atypical A. salmonicida

(Bast et al., 1988; Belland and Trust, 1989; Han-

ninen et al., 1995; Pedersen et al., 1996). A. sal-

monicida carries multiple plasmids of various sizes:

small, multi-copy plasmids ranging from 1 to 6 kb,

and larger low-copy plasmids from 11 to 150 kb.Most strains of A. salmonicida carry at least four

plasmids, and some, as many as six. The variation

in the number and size of plasmids is greater for

atypical than for typical strains. Some of the large

plasmids are known to carry antibiotic resistance

genes, but other than this no correlation can be

drawn between plasmid carriage and virulence

(Brown et al., 1997).Most typical A. salmonicida strains carry a

group of three small plasmids of 5.0, 5.2, and

5.4 kb. In the typical A. salmonicida strain A449,

their copy number has been estimated to be very

high, between 50 and 55 per cell (Belland and

Trust, 1989). Further characterization of these

plasmids demonstrated that they expressed very

few genes and, despite great effort, it was notpossible to cure the strain of any of these three

plasmids (Belland and Trust, 1989). In addition to

the three small plasmids, Belland and Trust (1989)

identified a large, 145 kb plasmid in A. salmonicida

A449.

As part of the process of sequencing the entire

genome of A. salmonicida A449, which is estimated

to be 4.6 Mb (Umelo and Trust, 1998), we have

Table 1

Aeromonas salmonicida subsp. salmonicida strains used in this study

Strain Characteristics and origin

A449 Virulent, Brown trout, Eure, France

A450 Virulent, Brown trout, Tarn, France

A450-1 Lab-derived avirulent variant of A450

A450-3 Lab-derived avirulent variant of A450

80204 Virulent, Atlantic salmon, New Brunswick, Can

80204-1S Lab-derived avirulent variant of 80204

SS70.1 Lab-derived avirulent, originally from Coho salm

NG10 Virulent, Atlantic salmon, New Brunswick, Can

97132 Virulent, Atlantic salmon, New Brunswick, Can

84222-S Lab-derived avirulent, originally from Atlantic s

OAR Virulent, oxolinic acid resistant, Atlantic salmon

MT004 Lab-derived avirulent, originally from Atlantic s

identified contigs corresponding to the three smallplasmids. This paper describes the complete se-

quence and annotation of these plasmids from

A. salmonicida A449.

2. Materials and methods

2.1. Bacterial strains

Aeromonas salmonicida subsp. salmonicida

strains used were kindly donated by Dr. Gilles

Olivier of the Department of Fisheries and Oceans

(DFO) in Moncton, N.B., Dr. Trevor Trust, Mi-

crotek International, Saanichton, B.C., and by Dr.

Rafael Garduno at Dalhousie University, Halifax,

N.S. They are described in Table 1. A. salmonicida

strains were grown in Tryptic Soy broth (TSB,

Difco) for 3 days at 17 �C with shaking. Plasmids

were isolated from A. salmonicida using the Nu-

cleobond Mini Prep kit (Clontech, Palo Alto, CA).

Genomic DNA was isolated using the PureGene

DNA isolation kit (Gentra Systems, Minneapolis,

MN).

2.2. DNA sequencing

Aeromonas salmonicida plasmid DNA was di-

gested with BamHI and BamHI/EcoRI and cloned

into pre-digested and dephosphorylated pTrue-

Blue vector (Genomics One, Laval, PQ) and

transformed into Escherichia coli XL-1 Blue MRF�(Stratagene, La Jolla, CA). E. coli clones were

Source

T. Trust

R. Garduno

R. Garduno

R. Garduno

ada G. Olivier

G. Olivier

on, Oregon, USA G. Olivier

ada G. Olivier

ada G. Olivier

almon, New Brunswick, Canada G. Olivier

, New Brunswick, Canada G. Olivier

almon, Scotland G. Olivier

J. Boyd et al. / Plasmid 50 (2003) 131–144 133

grown and plasmid DNA isolated using standardtechniques. Clones containing the appropriate-

sized inserts were sequenced from both ends. These

end-sequences were used as a tag to identify con-

tigs from the main A. salmonicida genome assem-

bly that had been generated by sequencing random

clones and assembled using the Staden package

(Bonfield et al., 1995). Primer-walking on the

plasmid clones was done to fill any gaps in therespective contigs as well as to confirm the se-

quences. Total sequencing coverage on these

plasmids is approximately 12-fold. These se-

quences have been deposited in GenBank under

Accession Nos.: pAsa1, AY301063; pAsa2,

AY301064; and pAsa3, AY301065.

2.3. PCR primers and methods

PCR conditions to amplify specific regions of

the plasmids were: 45 s at 94 �C; 30 cycles of 45 s at

94 �C, 45 s at 55 �C, and 90 s at 72 �C; followed by

a 10min extension at 72 �C. The reaction mix

contained 100 ng genomic DNA, 0.3mM each

primer and 1.25U of rTaq (Amersham–Pharmacia

Biotech, Uppsala, Sweden). Primers used were:pAsa1F, 50GGACGATTAACCTTCGCATC 30;

pAsa1R, 50 GTATCGCCCAACTTCTTCCA 30;

pAsa2F, 50AAAAGAGCGTGCAACCCTAA 30;

pAsa2R, 50 GCGATGCTACTTCATTCACC 30;

pAsa3F, 50TCATGGAGAATGTTCGCAAG 30;

pAsa3R, 50 GCCCAATTATCACAGCAACA 30;

pAsal1F, 50TAACATGGGTGAGTCAGGA30;

and pAsal1R, 50 TGCATGTTTGTAAAAAGTAGGTG 30.

1 Abbreviations used: ORF, open reading frame; DFO,

Department of Fisheries and Oceans; TSB, Tryptic Soy broth;

rm, restriction/modification system.

3. Results and discussion

3.1. General description of plasmids

The restriction patterns of the completed plas-mid sequences were compared to those generated

by Belland and Trust (1989) who characterized but

did not sequence three small plasmids from the

same strain of A. salmonicida. The restriction maps

of our plasmids were very similar to those previ-

ously reported, so we chose to use their nomen-

clature. The maps are not identical in size (pAsa1

and pAsa3) and restriction site pattern (pAsa2 andpAsa3) but this can be explained by the simulta-

neous mapping of all three plasmids by these au-

thors, while we were able to sequence them

individually.

Plasmids pAsa1, pAsa2, and pAsa3 (Fig. 1) are

5424, 5247, and 5616 bp in length, respectively,

with G+C contents of 57, 52, and 55%. The G+C

content of the A. salmonicida chromosome is58.3% (unpublished data). pAsa1 and pAsa3 ap-

pear to be ColE2-type replicons while pAsa2 is a

ColE1-type plasmid. Both pAsa1 and pAsa3 carry

toxin–antitoxin genes and all three plasmids en-

code genes for plasmid mobilization. Other than

genes for plasmid replication, stability, and mo-

bilization, each plasmid contains only one to three

other open reading frames (ORFs)1 that encodeproteins of unknown function.

During the preparation of this manuscript,

three A. salmonicida plasmid sequences were de-

posited in GenBank by D. Fehr, S.E. Burr, and

J. Frey. One of these sequences, pAsal2 (Accession

No: NC_004339.1), is identical to the pAsa1 se-

quence while pAsal3 (NC_004340.1) differs from

the pAsa2 sequence at 4 positions and has 2 ad-ditional bases. Plasmid pAsal1 (NC_004338.1),

another ColE2 plasmid, is not present in A. sal-

monicida A449, while pAsa3 was apparently not

sequenced by Fehr et al.

3.2. Replication of plasmids pAsa1 and pAsa3

On the basis of similarity to other rep genes inthe GenBank database, those of plasmids pAsa1

and pAsa3 are members of the ColE2 family

(Table 2). Plasmids with ColE2-type replicons are

usually small, have a high copy-number and

replicate using the theta mechanism (del Solar

et al., 1998; Espinosa et al., 2000). The minimum

replicating unit consists of the rep gene; a short

antisense RNA, RNAI, that is complementary tothe 50 untranslated region of rep; and a cis acting

origin, oriV, where the Rep protein binds. ColE2-

type Rep proteins are primases and thus they

Fig. 1. Maps of plasmids pAsa1, pAsa2, and pAsa3. Gene position and direction of transcription are indicated by arrows.

134 J. Boyd et al. / Plasmid 50 (2003) 131–144

both bind to their cognate origin and synthesizea small primer RNA, ppApGpA, that is required

for initiation of the leading-strand DNA synthe-

sis by the chromosomally encoded DNA poly-merase I. The RNAI antisense RNA negatively

regulates rep expression post-transcriptionally,

Table 2

Percent similarity (lower left) and identity (upper right) of the Rep proteins

pAsa1 pAsa3 pAsal1 ColE2 ColE3 ColE5

pAsa1 — 73 72 38 39 38

pAsa3 82 — 90 38 36 36

pAsal1 82 94 — 39 36 36

ColE2 49 50 51 — 88 75

ColE3 49 47 48 91 — 83

ColE5 48 47 46 82 88 —

Similarity was calculated using the BLOSUM 62 matrix. GenBank Accession Nos. are as follows: pAsal1 (A. salmonicida),

23897236; ColE2-P9 (Shigella sp.), 808894; ColE3-CA38 (Escherichia coli), 808865; and ColE5-099 (Shigella sonnei), 809524.

J. Boyd et al. / Plasmid 50 (2003) 131–144 135

although the exact mechanism of the regulation

is unclear.

ColE2-type plasmids are often mutually com-

patible, thus allowing some bacteria, including

A. salmonicida, to carry more than one suchplasmid. Incompatibility among ColE2-type plas-

mids is controlled by two factors: the RNAI

molecule which specifically binds to its own sense

transcript and controls copy number (Takechi

et al., 1994) and the Rep protein which specifically

binds to its cognate origin. Subtle variations in

the sequence of the Rep protein and the origin of

replication provide the possibility for mutualcompatibility among ColE2-type plasmids (Hiraga

et al., 1994; Shinohara and Itoh, 1996).

Fig. 2 shows the predicted position of the an-

tisense RNAI between the rep promoter and start

codon in the A. salmonicida plasmids pAsa1,

pAsa3, pAsal1, and three ColE2-type plasmids

from E. coli and Shigella species. In the latter

ColE2-type plasmids the RNAI promoters overlapthe rep start codon on the opposite strand (grey

boxes). The aligned sequences of the A. salmoni-

cida plasmids are quite dissimilar from those of E.

coli and Shigella and do not seem to carry RNAI

promoter sequences in the same position. Instead

putative )10 and )35 regions appear to reside

about 30 bp upstream of the rep start codon (also

shown with grey boxes). The difference in the po-sition of the promoters reflects the different sizes of

the A. salmonicida and ColE2 RNAI molecules.

The RNAI molecules from the A. salmonicida

plasmids are shorter and appear to form only a

single stem–loop structure (solid arrows in Fig. 2),

as does that of ColE5-099 and other ColE2 RNAI

molecules (Hiraga et al., 1994). In contrast, the

RNAI molecules of ColE2-P9 and ColE3-CA38

form two stem–loop structures (solid and dashed

arrows). The conserved stem–loop is very similar

in all six plasmids, except that the loop sequences

in the A. salmonicida plasmids are exactly com-plementary to those of the other ColE2 plasmids.

A run of thymidines at the 50 end of the repmRNA

likely acts as a rho-independent transcriptional

terminator for RNAI.

In addition to the RNAI molecule, incompati-

bility among members of the ColE2 group has

been shown to be controlled by three insertions in

the C-terminal region of the Rep protein andcorresponding insertions in the oriV sequence

(Hiraga et al., 1994; Shinohara and Itoh, 1996).

The three insertions in the Rep protein, termed A,

B, and C are 9, 2, and 4–6 amino acids in length,

respectively, and occur in the C-terminal 50 amino

acids of the protein. Each is associated with a

single nucleotide insertion in the origin region, a,b, and c; that occur at positions 5, 20, and 9 bpupstream of the ppApGpA primer, respectively.

Chimeric rep and origin constructs (Shinohara and

Itoh, 1996) demonstrate the specificity of these

Rep/oriV insertions and their involvement in

plasmid incompatibility.

Alignment of the A. salmonicida pAsa1, pAsa3

and pAsal1 Rep, and oriV sequences with those of

other ColE2 plasmids (Fig. 3A) reveals the pres-ence of all three types of Rep/oriV insertions in the

A. salmonicida plasmids. All three plasmids appear

to have the B/b insertions, while pAsa1 addition-

ally has the A/a insertions and pAsa3 has the C/cinsertions. This variation in Rep/oriV type be-

tween the three A. salmonicida ColE2-type plas-

mids demonstrates how Rep/oriV specificity is

Fig. 2. Alignment of the DNA sequences upstream of the rep genes of three sequenced A. salmonicida ColE2-type plasmids and three

ColE2-type plasmids from E. coli and Shigella species. The rep-35 and rep-10 promoter region is indicated by boxes and is approx-

imately 160 bases upstream from the rep start codon. Two sets of grey boxes indicate the most likely promoters for the antisense RNAI

molecules. The promoters for the A. salmonicida RNAI molecules are not the same as those for the E. coli and Salmonella plasmids.

Arrows indicate stem–loop structures formed in the RNAI molecule with the conserved loop region boxed. Dashed arrows indicate the

second stem–loop structure found only in plasmids ColE2-P9 and ColE3-CA38. The RNAI terminator is likely the run of A�s indicatedby the solid line. Accession Nos. are: pAsal1, NC_004338.1; ColE2-P9, 487322; ColE3-CA38, 487267; and ColE5-099, 487324.

136 J. Boyd et al. / Plasmid 50 (2003) 131–144

determined for each plasmid and indicates that all

three ColE2-type plasmids would be compatible in

a single strain. In addition, the A. salmonicida Rep

proteins, while overall very similar to other ColE2-type Rep proteins, have several conserved inser-

tions throughout the length of the protein, as well

as a single insertion unique to pAsa1 (Fig. 3A).

The A. salmonicida Rep proteins thus appear to be

a distinct subgroup of the ColE2-type Rep family.

3.3. Replication of pAsa2

On the basis of similarity to other plasmids,

pAsa2 is a member of the ColE1-type group, andthus is a theta replicating, DNA polymerase de-

pendent plasmid (Chan et al., 1985; del Solar et al.,

1998; Espinosa et al., 2000). ColE1-type replication

requires no plasmid-encoded proteins; instead

it uses two RNA molecules. RNAII acts as the

Fig. 3. (A) Alignment of the Rep proteins from the three sequenced A. salmonicida ColE2-type plasmids and three ColE2-type

plasmids from E. coli and Shigella species. The conserved leucine zipper and helix–turn–helix motifs are boxed and shaded. The three

insertions that determine compatibility are labeled A, B, and C. (B) Alignment of the origin of replication (oriV) of the three sequenced

A. salmonicida ColE2-type plasmids and three ColE2-type plasmids from E. coli and Shigella species. The Rep stop codons are boxed

and the ppApGpA primer site is indicated by a box and an arrow. The three insertions that determine compatibility are labeled a, b,and c. Rep protein Accession Nos. are: pAsal1, NP_710167.1;ColE2-P9, 808894; ColE3-CA38, 808865; and ColE5-099, 809524.

J. Boyd et al. / Plasmid 50 (2003) 131–144 137

138 J. Boyd et al. / Plasmid 50 (2003) 131–144

primer for DNA synthesis while RNAI is a shorter,antisense RNA complementary to the 50 end of

RNAII. RNAI is constitutively expressed, but is

rapidly turned over, resulting in tight control of

plasmid copy number. RNAI is also the main in-

compatibility determinant of ColE1-type plasmids

(Tomizawa and Itoh, 1981). RNAI and RNAII

molecules have both been predicted to form three

stem–loop structures. RNAI forms structures I, II,and III, while RNAII forms the complementary

structures of I and II, and a different larger struc-

ture, IV (Tamm and Polisky, 1983; Tomizawa,

1990). The initial interactions between RNAI and

RNAII takes place at these loops. Alignment of the

origin region of pAsa2 with those of other ColE1-

type plasmids shows a high degree of similarity

(Fig. 4). Furthermore, as predicted by m-fold(Mathews et al., 1999; Zuker et al., 1999) the RNA

molecules from pAsa2 also form similar stem–loop

structures, (indicated by inverted arrows in Fig. 4).

The stem structures are very highly conserved while

the loop structures, which are responsible for the

initial binding interactions and therefore incom-

patibility, are quite variable.

Some ColE1-type plasmids encode a smallprotein, Rom or Rop that stabilizes the interaction

between RNAI and RNAII; however pAsa2 does

not appear to encode a Rom homologue.

We have been able to show that pAsa2 can

replicate stably in E. coli (data not shown). It is

also interesting that cloning vectors based on

E. coli ColE1 can replicate in A. salmonicida for a

few generations only. Indeed we have successfullyused pBluescript (Stratagene, La Jolla, CA) de-

rivatives as suicide vectors in A. salmonicida. We

have no information about whether the other two

A. salmonicida plasmids can replicate in E. coli.

3.4. Mobilization and oriT of pAsa1, pAsa2, and

pAsa3

All three A. salmonicida plasmids carry genes,

mobABCD, encoding proteins similar to those of

ColE1 that are involved in plasmid mobilization.

MobA proteins are relaxases that nick the double-

stranded plasmid DNA at a specific site, nic, in the

origin of transfer (oriT). MobA becomes cova-

lently attached to the plasmid DNA and the

MobA–DNA complex then moves through themating bridge into the donor cell (Zechner et al.,

2000). MobA and other conjugative relaxases

are not highly similar (Table 3) but they do have

three recognizable motifs in their N-termini. Motif

I includes a conserved tyrosine residue that

remains covalently attached to the DNA at the

50 end of nic, motif II includes a serine that is

implicated in interacting with the 30 end of nic,motif III contains either three histidines, (HHH) or

a histidine, a glutamate and an asparagine (HEN)

(Varsaki et al., 2003). The MobA proteins of the

A. salmonicida plasmids all have motifs I and II

and the HEN sequence at motif III (data not

shown). MobB, C, and D are accessory proteins

that facilitate the action of MobA. As in ColE1,

these genes are organized in an apparent operonwith mobC upstream of mobA, while mobB and

mobD are encoded on the same DNA segment as

mobA, but in different reading frames (Fig. 1). The

MobA, C, and D proteins of plasmid pAsa2 are

very similar to those of ColE1 (Table 3), while

those of pAsa1 and pAsa3 are similar to each

other, but less similar to ColE1. The A. salmonicida

MobB proteins are not very similar to each otheror to that of ColE1.

Putative oriT regions have been found upstream

of the mobC genes in plasmids pAsa1 and pAsa3,

and upstream of orf1 in pAsa2 (Fig. 1). These oriT

regions are similar to those of ColE1-type plas-

mids, which reflects the similarity of the mobA

genes to that of ColE1-type plasmids (Lanka and

Wilkins, 1995; Zechner et al., 2000). Fig. 5 showsan alignment of the putative oriT regions from the

A. salmonicida plasmids with those of ColE1-type

plasmids. We have no evidence that the various

MobA proteins can specifically identify their cog-

nate oriTs, but the sequence diversity of both the

oriTs and the MobA proteins suggests that this is

the case.

While the presence of the mob genes and oriT

regions in these three plasmids suggests they are

mobilizable, they are lacking genes encoding pro-

teins required for transfer. To be mobilized, these

plasmids must rely on transfer proteins encoded

elsewhere. It is worth noting that the two large

plasmids found in A. salmonicida A449 each carry

transfer gene operons (unpublished results).

Fig. 4. Alignment of the replication control region (oriV) of pAsa2 with that of other ColE1-type plasmids from E. coli. Stem

structures are indicated by solid and dashed line arrows, loops are clear boxes. Structures I, II, and III are found in RNAI; structures I,

II and IV are found in RNAII. Promoter elements are indicated by grey boxes. Open arrows indicate the site and direction of

transcription and replication. Accession Nos. are: ColE1, J01566.1; ColA, 144670; and ColD, 144289.

J. Boyd et al. / Plasmid 50 (2003) 131–144 139

Table

3

Percentsimilarity

(lower

left)andidentity

(upper

right)

oftheMobproteins

MobA

MobB

MobC

MobD

pAsa1

pAsa2

pAsa3

pColE1

pAsa1

pAsa2

pAsa3

pColE1

pAsa1

pAsa2

pAsa3

pColE1

pAsa1

pAsa2

pAsa3

pColE1

pAsa1

—21

60

22

—15

54

26

—22

80

26

—18

41

16

pAsa2

34

—23

44

27

—9

22

39

—21

44

31

—24

51

pAsa3

70

35

—21

76

23

—18

88

38

—24

57

35

—24

pColE1

34

57

34

—41

39

36

—40

58

39

—29

60

36

Sim

ilarity

wascalculatedusingtheBLOSUM

62matrix.GenBankAccessionNo.ofColE1isJ01566.1.

Fig. 5. Alignment of the origins of transfer (oriTs) of four

A. salmonicida plasmids with that of ColE1 and ColA. The nick

site (nic) is indicated by an arrow. Accession Nos. are: pAsal1,

NP_710167.1; ColE1, J01566.1; and ColA, 144670.

140 J. Boyd et al. / Plasmid 50 (2003) 131–144

3.5. Toxin–antitoxin systems in pAsa1 and pAsa3

Plasmids pAsa1 and pAsa3 both have proteic

toxin–antitoxin genes that presumably act as

plasmid stability mechanisms by post-segrega-tional killing. These systems, also known as ad-

diction modules, consist of two proteins, one a

toxin and the other the specific antidote (Engel-

berg-Kulka and Glaser, 1999). The antidote is

more labile than the toxin and if a cell stops

making the antidote, as in the case of a plasmid-

less daughter cell, the toxin will be able to kill the

cell. These genes are usually transcribed as anoperon with the antitoxin gene upstream and

overlapping the toxin gene. The systems on pAsa1

and pAsa3 also follow this general rule.

Plasmids pAsa1 and pAsa3 carry genes ho-

mologous to the relBE system (Gronlund and

Gerdes, 1999), in which RelB is the antitoxin and

RelE is the toxin. RelE has recently been shown to

cleave mRNAs bound to ribosomes in a codon-specific manner (Pedersen et al., 2003). This toxin–

antitoxin system is widespread among prokaryotes

and is found in Gram-negative and Gram-positive

bacteria and archaea (Gerdes, 2000). Many of

these RelBE systems are found on the chromo-

some where they act as a global regulator of

translation (Christensen et al., 2001).

The homologues most similar to the pAsa1system are those of the E. coli and Salmonella

enterica chromosomes, while those of pAsa3 are

most similar to genes called pasAB in plasmids of

Acidithiobacillus caldus (Gardner et al., 2001;

Smith and Rawlings, 1997) and Pseudomonas flu-

orescens (Peters et al., 2001). While the RelE toxins

J. Boyd et al. / Plasmid 50 (2003) 131–144 141

from pAsa1 and pAsa3 do not show high identity(26%) or similarity (47%) to each other, the pasAB

genes do belong to the relBE family (Gerdes,

2000), therefore an alignment of all these proteins

is shown in Fig. 6.

To our knowledge, the presence of two plasmid-

encoded, toxin–antitoxin systems in the same

bacterial strain has not been detected. However,

there are instances of strains carrying more thanone plasmid with other types of plasmid stability

systems, most notably restriction/modification

(rm) systems. In this situation, two plasmids car-

rying rm systems will both be stably maintained as

long as the target rm sites on the chromosome are

different. If both methylases can protect the same

site then one or the other restriction enzyme can be

lost without cell death (Kusano et al., 1995). In theA. salmonicida case as long as each RelB antitoxin

recognizes only its own toxin, neither plasmid can

be lost without cell death. The low degree of se-

Fig. 6. Alignment of the toxin and antitoxin proteins from plasmids p

are: RelE E. coli, 76201; RelE S. enterica, 16763250; RelB E. coli, 132

10834752; PasB Acidithiobacillus caldus, 14209913; PasA P. fluorescen

quence conservation among the antitoxin proteins(Fig. 6) may be indicative of their specificity for

their cognate toxin.

No other known form of plasmid stability sys-

tem is found on these plasmids. Most notably no

cer or xis sites are obvious. These are sites of site-

specific recombination that are used by the plas-

mid to resolve dimers created during replication.

Dimer and higher multiple forms of all threeplasmids are commonly seen in plasmid prepara-

tions (data not shown).

3.6. Other ORFs of unknown function

There are several ORFs in these plasmids that

share little or no sequence similarity with genes in

the databases. The most notable is orf1, which isfound on all three plasmids. orf1 shares limited

identity with a putative gene of unknown function

from a Ralstonia solanacearum plasmid, pJTPS1

Asa1 and pAsa3 with their closest homologues. Accession Nos.

283; RelB S. enterica, 16763249; PasB Pseudomonas fluorescens,

s, 10834752; and PasA A. caldus, 14209912.

Table 4

Percent similarity (lower left) and identity (upper right) of the

ORF1 proteins

pAsa1 pAsa2 pAsa3 pJTPS1

pAsa1 — 36 77 20

pAsa2 50 — 33 15

pAsa3 86 50 — 21

pJTPS1 29 24 30 —

Similarity was calculated using the BLOSUM 62 matrix.

GenBank Accession No. of pJTPS1 OrfC1 (Ralstonia solana-

cearum) is NP_052314.

142 J. Boyd et al. / Plasmid 50 (2003) 131–144

(Table 4). In addition to the sequence similarity,the orf1 genes are all positioned upstream of the

mobC genes. In pAsa2, orf1 is positioned between

mobC and oriT. This close proximity to the mob

cluster and the fact that the mob and orf1 genes are

the only genes shared among all the three plasmids

suggests orf1 may be involved in mobilization of

these plasmids. pAsa2 has two more ORFs of

unknown function (orf2 and orf3) and pAsa3 hasone (orf4). Each plasmid also has a large region

(500–1000 bp) with no obvious features.

Fig. 7. Plasmids from A. salmonicida subsp. salmonicida strains. (A) P

EcoRI cuts once in pAsal1, pAsa1, pAsa2, and four times in pAsa3

visible). Plasmid names are indicated to the right and MW standards a

amplified with primers specific to each of the four small plasmids usi

3.7. Presence of pAsa1, pAsa2, and pAsa3 in other

strains of A. salmonicida

Previous analyses of the plasmid profiles of

A. salmonicida strains (Belland and Trust, 1989;

Giles et al., 1995; Pedersen et al., 1996; Sorum et

al., 2000) suggested that different strains often

carried different sets of plasmids. To investigate

the plasmid profiles of the strains present in our A.salmonicida strain collection, we designed primers

to specific regions of the plasmids and used them

to amplify total genomic DNA from the strains.

For pAsa1 and pAsa3 the toxin–antitoxin regions

were amplified, and for pAsa2, orf3 was chosen.

We also amplified the aopP gene from plasmid

pAsal1 that was sequenced by Fehr et al. (Acces-

sion No: NC_004338). Additionally, plasmidDNA was isolated from all the strains and analy-

sed on an agarose gel following digestion with

EcoRI. EcoRI cuts all plasmids only once, except

pAsa3, in which case a diagnostic 4.8 kb fragment

is generated. In all cases the results of the PCR and

agarose gel were identical (Fig. 7). All of the

lasmids prepared from A. salmonicida were digested with EcoRI.

creating one large 4800 bp fragment and three small ones (not

re on the left. (B) Presence (+) or absence ()) of PCR products

ng total genomic DNA as the template.

J. Boyd et al. / Plasmid 50 (2003) 131–144 143

strains in our collection carried pAsa1 and pAsa2.All but one carried pAsa3. Many of the strains,

but not A449, also carried another larger plasmid

of approximately 6.5 kb, which is similar to the

size of pAsal1. These strains also gave positive

results in PCR with primers to aopP, suggesting

that the 6.5 kb plasmid in these strains is indeed

pAsal1. As predicted by the different Rep/oriV

insertions in the ColE2 plasmids, pAsa1, pAsa3,and pAsal1, all three can indeed be compatibly

maintained in a single A. salmonicida strain.

The virulence of the strains in our collection is

quite varied, as is their plasmid profile. However,

as others have shown (Bast et al., 1988; Brown

et al., 1997), there does not appear to be any

correlation between carriage of these small plas-

mids and virulence. We now know this is because,at least for the small plasmids, there are no known

virulence factors except for the type III secretion

system effector, AopP, encoded by pAsal1.

4. Summary

The sequences of the three small plasmids fromA. salmonicida subsp. salmonicida A449 demon-

strate that one is a ColE1-type while the other two

are ColE2-type plasmids. The genes encoded by

these plasmids function primarily in replication,

mobilization and plasmid stability. Differences in

the Rep/oriV regions of the ColE2-type plasmids

provide specificity for replication and the ability

for multiple ColE2-type plasmids to be maintainedin a single strain. The presence of two different

plasmid addiction systems in a single bacterial

strain supports the idea that these plasmids exist

primarily to promote their own replication and

spread.

Acknowledgments

We thank the IMB DNA sequencing team for

carrying out the DNA sequencing and Drs. S.

Douglas and A. Patrzykat for comments on the

manuscript. This work was supported by the NRC

Genomics and Health Initiative. This is NRCC

Publication No. 42381.

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Communicated by I. Kobayashi