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Plasmids of Carotenoid-Producing Paracoccus spp.
(Alphaproteobacteria) - Structure, Diversity and Evolution
Anna Maj, Lukasz Dziewit, Jakub Czarnecki, Miroslawa Wlodarczyk, Jadwiga Baj, Grazyna Skrzypczyk,
Dorota Giersz, Dariusz Bartosik*
Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland
Abstract
Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information
via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in
Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the
genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus
(Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P.
marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of
diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the
relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding
relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of
AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved
in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse
transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of
Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems
not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer
of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in
lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors
constructed in this study will facilitate genetic studies of these bacteria.
Citation: Maj A, Dziewit L, Czarnecki J, Wlodarczyk M, Baj J, et al. (2013) Plasmids of Carotenoid-Producing Paracoccus spp. (Alphaproteobacteria) -
Structure, Diversity and Evolution. PLoS ONE 8(11): e80258. doi:10.1371/journal.pone.0080258
Editor: Axel Cloeckaert, Institut National de la Recherche Agronomique, France
Received August 9, 2013; Accepted October 11, 2013; Published November 8, 2013
Copyright: © 2013 Maj et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Ministry of Science and Higher Education, Poland – grants 2 P04A 028 29 (characterization of pMARC
plasmids), PBZ-MNiSW-04/I/2007 (characterization of pAES, pHAE, and pMOS plasmids) and N N302 224638 (construction of vector cassettes and
shuttle vectors). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: bartosik@biol.uw.edu.pl
Introduction
Bacterial plasmids have a modular structure: their genomes
can be separated into several DNA cassettes encoding specific
functions. Besides the conserved backbone, composed of
genetic modules encoding replication (REP), stabilization and
transfer functions, plasmids can contain an additional “genetic
load”, which may significantly influence the metabolic
properties of any recipient strain. Many plasmids are giant
molecules that can even exceed the size of some bacterial
chromosomes. Such mega-sized replicons (megaplasmids) are
particularly common in Alphaproteobacteria.
Alphaproteobacteria constitute interesting models for
studying the complexity and diversity of bacterial genomes.
Many strains within the genera Rhizobium, Agrobacterium and
Paracoccus contain chromosomes, chromids, megaplasmids
and sometimes several smaller plasmids (e.g. [1,2]). Analysis
of the genomic data collected by The National Center for
Biotechnology Information (NCBI) revealed that the sequenced
genomes of 240 alphaproteobacterial strains include a total of
315 plasmids. Twenty six of these strains are multi-plasmid
containing, with at least five extrachromosomal replicons.
For many years, knowledge of plasmids of the
Alphaproteobacteria was mainly limited to the repABC and
repC families of replicons, which are specific for megaplasmids
of this group of bacteria (repA and repB of repABC replicons
encode partitioning proteins, while repC encodes replication
initiator) (e.g. [3,4]). Recent studies have revealed the
presence of plasmids classified into the repA and repB families
as well as a dnaA-like family, encoding replication proteins with
similarity to the DnaA proteins involved in the initiation of
replication of bacterial chromosomes [1]. Detailed analysis of
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the REP regions allowed the following incompatibility (inc)
groups to be distinguished: (i) 9 groups of repABC replicons,
(ii) 5 groups of repA-family replicons and (iii) 4 groups of repB-
family replicons [1]. Most of the analyzed REPs are harbored
by megaplasmids and much less is known about smaller
alphaproteobacterial plasmids.
Several years ago we initiated a project aimed at identifying
and characterizing the pool of mobile DNA in bacteria
belonging to the genus Paracoccus (Alphaproteobacteria). This
genus currently comprises 42 species and hundreds of strains
(not identified at species level), which are known for their
versatile physiological properties and ability to perform a
number of different growth modes. We focused our interest on
mobile genetic elements (MGE) of Paracoccus spp., especially
plasmids and transposable elements (TE) (e.g. [5]). As a result
of this approach we have identified and analyzed (i) four
related repABC as well as several pTAV3-type megaplasmids
– both groups residing in P. versutus UW1 and four strains of
Paracoccus pantotrophus [6,7], (ii) plasmid pALC1 of
Paracoccus alcaliphilus JCM 7364, with an iteron-containing
replication system [8], (iii) plasmid pMTH1 of Paracoccus
methylutens DM12, whose genome is predominantly (80%)
composed of transposable modules (TMos) [9], (iv) three
plasmids of Paracoccus aminophilus JCM 7686, whose REP
modules were used for the construction of versatile DIY
cassettes [10,11], as well as (v) plasmid pWKS1 of P.
pantotrophus DSM 11072 – the smallest replicon identified so
far in Paracoccus spp. [12].
In this study we identified four plasmid-rich strains of
Paracoccus spp. Genomic analysis of their plasmids revealed
that the genetic organization and structure of many of them
differ significantly from that previously described in
Alphaproteobacteria.
Results and Discussion
Plasmids of carotenoid-producing strains of
Paracoccus spp.
At the initial stage of this study we analyzed the plasmid
content of 22 strains representing 20 Paracoccus spp. (listed in
Methods section). Plasmid screening revealed that the majority
of the tested strains contained megaplasmids (above 100 kb)
(data not shown). Only four of the strains (P. aestuarii DSM
19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574
and P. marcusii OS22 – all able to produce beta-carotenoid
pigments) contained numerous smaller replicons ranging in
size from approx. 2.5 kb to 85 kb.
To analyze plasmid diversity in these strains we obtained the
nucleotide sequences of 14 randomly selected replicons (listed
in Table 1): (i) 5 pAES plasmids of P. aestuarii DSM 19484, (ii)
2 pHAE plasmids of P. haeundaensis LMG P-21903, (iii) 4
pMARC plasmids of P. marcusii DSM 11574 and (iv) 3 pMOS
plasmids of P. marcusii OS22.
The results of the overall characterization of the plasmids are
presented in Table 1. A summary of the distinguished open
reading frames (ORFs), including their position, the size of the
putative proteins they encode and their closest homologs, is
presented in Table S1 in the supplemental material.
Comparative bioinformatic analysis was used to distinguish
the plasmid backbones, composed of different combinations of
genetic modules responsible for plasmid replication (REP),
stabilization (toxin-antitoxin – TA; partitioning – PAR) and
mobilization for conjugal transfer (MOB), and accessory
genetic information, which may potentially influence the
phenotype of the host (Figure 1). Detailed characterization of
the predicted modules is presented below.
Replication modules
Replication systems of the vast majority of plasmids residing
in gram-negative bacteria consist of two elements: (i) a gene
encoding a Rep protein, which initiates DNA replication, and (ii)
the cis-required origin (oriV; equivalent to chromosomal oriC)
where replication begins. Most Rep proteins are highly
conserved and they can be grouped into several families on the
basis of amino acid (aa) sequence similarities. In contrast,
oriVs are more divergent and they are usually placed in close
proximity to the rep genes. In many cases, the location of the
oriVs can be predicted in silico by the presence of (i) directly
repeated sequences (including iterons), which constitute the
Rep protein binding sites (iterons, being key elements in the
control of replication initiation, determine plasmid
incompatibility [13]), (ii) A+T-rich DNA regions, where strand
opening and assembly of host replication initiation factors
Table 1. Basic characterization of the Paracoccus spp.
plasmid genomes.
GC content (%)
Plasmid Host Size (bp)
Plasmid
DNA
Host
DNA
Number
of ORFs
Genetic
modules
pAES1 P. aestuarii DSM
19484 2925 64.4 62
[43] 2 REP, MOB
pAES2 4502 57.9 5 REP, R-M
pAES3 5434 51.7 7 REP, TA
pAES4 5850 58.6 9 REP, MOB,
TA
pAES7 13,005 60.1 15 REP, MOB,
TA(2)
pHAE1 P. haeundaensis
LG P-21903 5301 58.8 66.9
[44] 4 REP
pHAE2 5777 53.6 5 REP, TA
pMARC1 P. marcusii DSM
11574 5122 49.0 66
[45] 6 REP, TA
pMARC2 5789 51.6 6 REP, MOB
pMARC3 10,672 59.0 10 REP, PAR,
MOB, TA
pMARC4 15,289 53.4 15 REP, MOB,
TA
pMOS2 P. marcusii
OS22 6410 54.0 66
[45] 5 REP, MOB
pMOS6 7672 63.1 12 REP, MOB,
TA
pMOS7 5979 50.0 7 REP, TA
doi: 10.1371/journal.pone.0080258.t001
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occurs, and (iii) conserved DNA boxes representing sites of
interaction with chromosomally-encoded proteins, e.g. DnaA or
integration host factor (IHF) [14].
Comparative sequence analysis revealed that the analyzed
plasmids of Paracoccus spp. contain different types of
replication systems. Based on aa sequence similarities of the
predicted Rep proteins, the presence of conserved motifs and
the oriV structures, the REP modules could be classified into
four groups, some representing novel replication system types.
The genetic structure of these REP modules is illustrated in
Figure 2 and the nucleotide sequences of the predicted oriVs
are presented in Additional file 2: Figure S1.
Figure 1. The genetic organization of the Paracoccus spp. plasmids analyzed in this study. Arrows indicate the
transcriptional orientation of the ORF2. The color-coded keys show the species and strain of origin of each plasmid (circles) and the
likely plasmid maintenance/transfer processes in which the genes are involved (squares). Plasmid islets (PI) of lower than average
G+C content, insertion sequences (IS) and restriction and modification systems (R-M) are indicated by the use of different boxes
(see figure). Shaded areas connect genes of plasmids that encode orthologous proteins. For comparative analysis, two other
related plasmids of Paracoccus spp. have been included: pAMI3 of P. aminophilus JCM 7686 [10] and pWKS1 of P. pantotrophus
DSM 11072 [12].
doi: 10.1371/journal.pone.0080258.g001
Plasmids of Carotenoid-Producing Paracoccus spp.
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REP modules of pAES2, pAES3, pHAE1, pHAE2,
pMARC1, pMARC4 and pMOS7
The REP modules of plasmids pAES2, pAES3 (P. aestuarii
DSM 19484), pHAE1, pHAE2 (P. haeundaensis LMG
P-21903), pMARC1, pMARC4 (P. marcusii DSM 11574) and
pMOS7 (P. marcusii OS22) encode replication initiation
proteins (Rep) of the Rep_3 superfamily (PFAM: PF01051),
which show significant level of aa sequence identity with RepB-
type proteins from Rhodobacterales, whose phylogenetic
analysis was presented by Petersen et al. [15]. Comparative
analysis of the sequences and structures of these modules
identified three distinct subgroups, present in the following
plasmids: (i) pAES2, pAES3, pHAE1, pMARC4, (ii) pMARC1,
pMOS7 and (iii) pHAE2.
pAES2, pAES3, pHAE1 and pMARC4. The Rep proteins of
the first subgroup show aa sequence identity ranging from 54
to 81% (highest identity observed between Reps of pHAE1 and
pAES2). Database searches revealed that related proteins are
commonly encoded by alphaproteobacterial plasmids. The best
BLAST hits were obtained for replication initiator proteins of
small plasmids: pMG160 of Rhodobacter blasticus (accession
no NP_775696) [16] (70-78% aa sequence identity) and
pWKS1 of Paracoccus pantotrophus DSM 11072 (accession
no NP_775696) [12] (66-72% identity).
The predicted oriVs of pAES2, pAES3, pHAE1 and pMARC4
were identified upstream of the rep genes (Figure 2). These
highly conserved regions contain (i) three long iteron-like
directly repeated sequences (DR1-DR3) of 19 bp, separated
one from another by 2 bp spacers, and (ii) a single inverted
partial iteron situated adjacent to the start codon of the rep
gene (see Figure S1 in the supplemental material). As shown
in Figure 2, the iterons of the analyzed plasmids are not
identical, which suggests that these replicons belong to
different inc groups. The highest level of sequence identity was
observed between DRs of plasmids pHAE1 and pAES2, while
DRs of pMARC4 were most divergent (Figure 2). A common
Figure 2. Schematic structure of the REP modules analyzed in this study. The color-coded keys show the species and strain
of origin of each plasmid (circles) and identified direct repeats (DRs), inverted repeats (IRs) as well as predicted DnaA and IHF
binding sites (mixed shapes). The sequences of the iteron-like DRs are presented next to the relevant diagrams with a consensus
sequence shown for DRs of plasmids with related REP modules. Blue arrows indicate the rep genes and their transcriptional
orientation. Specific motifs identified within the aa sequences of the Rep proteins are indicated by colored rounded bars. A+T and G
+C indicate DNA regions of lower or higher than average G+C content, respectively. The components of the REP modules are not
shown to scale.
doi: 10.1371/journal.pone.0080258.g002
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feature of all distinguished iterons is the presence of a 3’-end G
+C-rich sequence. Within the oriV-containing region of
pMARC4 we also identified a palindromic sequence (5'-
AGCCTTGCAAGGCT-3'), located upstream of the identified
iterons.
pMARC1 and pMOS7. Both REP modules are highly
related and their Rep proteins share 96% aa sequence identity.
Homologous replication initiator proteins are encoded by many
plasmids residing in strains classified within the Beta- and
Gammaproteobacteria. The highest level of identity (46%) was
with the Rep of the small plasmid pHLHK19 of Laribacter
hongkongensis HLHK19 (Betaproteobacteria) (accession no
ABC70160).
The predicted oriVs of pMOS7 and pMARC1 are placed
upstream of the rep genes and in order they contain (i) three
putative 16-bp iterons (DR1-DR3) separated by spacer
sequences of 5 bp (Figure 2), (ii) a putative DnaA-box situated
2 bp downstream of DR3 (5’-TcATCCACA-3’ in pMARC1 and
5’-TTATCCACA-3’ in pMOS7; nucleotide not matching the
consensus DnaA box, 5'-TTT/ATNCACA-3' [17], shown in
lowercase), and (iii) 27-bp-long IR sequences (separated by 14
bp), which cover the predicted promoter, ribosome binding site
(rbs) and ATG start codon of the rep gene (Figure S1).
pHAE2. Plasmid pHAE2 also contains an iteron-type oriV,
composed of (i) five 19-bp repeats (DR1-DR5), separated by
spacer sequences of 3 bp, and (ii) identical IRs of 16 bp
(separated by 22 bp), which precede the predicted iterons
(Figure 2 and Figure S1). A BLAST search with the amino acid
sequence of the pHAE2 Rep protein identified only two proteins
with significant sequence similarity, encoded by Sphingobium
yanoikuyae XLDN2-5 (accession no ZP_09907537) (60% aa
sequence identity) and Citreicella sp. 357 (accession no
ZP_10022073) (62% aa sequence identity) – both strains
belonging to the Alphaproteobacteria. Weak homology to
replication proteins of several Acinetobacter spp. plasmids
(Gammaproteobacteria) was also observed.
REP modules of pAES1, pAES7, pMARC3 and pMOS2
Another group of REP modules (present in plasmids pAES1,
pAES7, pMARC3 and pMOS2) encode replication initiation
proteins containing predicted helix-turn-helix (HTH) domains
(HTH_36) conserved in many transcription regulators (PFAM:
13730). Based on aa sequence comparisons, these REP
modules were divided into two subclasses comprising (i)
pAES7, pMARC3, pMOS2, and (ii) pAES1.
pAES7, pMARC3 and pMOS2. The Rep proteins of these
three plasmids have aa sequence identities ranging from 57 to
62%. Homologous proteins are commonly encoded by many
alphaproteobacterial strains, e.g. Sphingobium xenophagum
QYY (accession no YP_195758), Acetobacter pomorum
DM001 (accession no ZP_08242880) and Acidiphilium cryptum
JF-5 (accession no YP_001220371) (approx. 60% aa
sequence identity).
These plasmids possess oriVs with different structures
(Figure 2). However, all contain 2 iteron-like DRs (of 39 bp in
pAES7, 18 bp in pMARC3 and 19 bp in pMOS2). The DRs of
pMOS2 are overlapped by IRs of 14 bp (Figure S1).
Interestingly, within the pAES7 oriV, an incomplete inverted
iteron (14 bp) was identified, partially overlapping the start
codon of the rep gene, which is analogous to the location of the
partial iterons observed within the oriVs of pAES2, pAES3,
pHAE1 and pMARC4.
pAES1. The REP module of pAES1 is unique among
Alphaproteobacteria, although related replication systems are
common in enteric bacteria (Gammaproteobacteria). The
pAES1-encoded Rep protein (ReppAES1) shows highest aa
sequence identity (46%) to the protein encoded by plasmid
pKL1 of Escherichia coli KL4 (accession no NP_053155)
(Gammaproteobacteria). The oriV of pKL1 is placed upstream
of the rep gene and contains an IHF-box and several Rep
binding sites [18]. The oriV-containing regions of pKL1 and
pAES1 do not show significant nucleotide sequence similarity,
but they contain analogously placed IR sequences: 16-bp long
in pKL1 (perfectly matching repeats) and 28-bp long in pAES1
(5 mismatches) (Figure S1).
Within the left and right IR of pAES1, two shorter (15 bp)
directly repeated sequences were identified (DR1 and DR2),
and a third copy of this repeat was detected elsewhere (DR3,
located 70 bp downstream of DR2) (Figure 2 and Figure S1).
REP modules of plasmids pAES4 and pMARC2
The highly homologous REP modules of pAES4 and
pMARC2 do not possess iteron-like DRs. Upstream of the rep
genes they contain only long inverted repeats, covering the
predicted rep promoters (Figure 2 and Figure S1). The IRs of
pAES4 form a long (46 bp) imperfect (3 mismatches)
palindromic sequence, while those of pMARC2 (18 bp) are
identical and are separated by a 4-bp spacer sequence (Figure
S1).
The Rep proteins of these plasmids show 92% aa sequence
identity. Both contain a predicted HTH motif typical of
transcriptional regulators of the MarR family. Homologous Rep
proteins are not common in other bacteria. Only four proteins
with significant aa sequence identity (47-55%) (annotated as
hypothetical proteins) were identified in the NCBI databases –
all from strains belonging to the Alphaproteobacteria. These
are encoded by (i-ii) plasmids pACMV6 and pACMV8 of
Acidiphilium multivorum AIU301 (accession nos
YP_004277313 and YP_004277317, respectively), (iii) plasmid
pAPA01-060 of Acetobacter pasteurianus IFO 3283-01
(accession no YP_003189587) and (iv) Rhodospirillum
photometricum DSM 122 (sequence contig; accession no
YP_005418451). All the aforementioned proteins are annotated
as hypothetical proteins and they do not show sequence
similarity to any other plasmid encoded Rep proteins.
Therefore, the results of the comparative analysis strongly
suggest that the Rep proteins of pAES4 and pMARC2 may be
considered as the archetypes of a novel group of plasmid
replication initiators.
REP module of pMOS6
The REP module of pMOS6 encodes a predicted Rep
protein (ReppMOS6), which is related to proteins encoded by
strains belonging to the Alpha-, Beta- and
Gammaproteobacteria as well as the CFB group of bacteria
(genus Bacteroides). The majority of these proteins are
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PLOS ONE | www.plosone.org 5 November 2013 | Volume 8 | Issue 11 | e80258
annotated as conserved hypothetical proteins of unknown
function. However, more detailed analysis revealed that all of
them contain domains conserved in the plasmid replication
proteins of the RepL family from Firmicutes (PFAM: PF05732).
Closely related Rep protein sequences were identified within a
few plasmids, i.e. pTINT02 of Thiomonas intermedia K12, pML
of Bartonella schoenbuchensis m07a (accession no
ENN90461) and pPsv48C of Pseudomonas savastanoi, and
some chromosomes, e.g. Bartonella vinsonii (accession no
YP_007462201). Interestingly, besides ReppMOS6 homologs, the
three aforementioned plasmids encode other proteins that may
be involved in replication initiation (RepC in pML and a
replication protein with a primase domain in pTINT02 and
pPsv48C). Plasmid pMOS6 encodes only a single Rep protein,
which is sufficient for initiation of replication, as proved by the
construction of a pMOS6 minimal replicon (data not shown).
The oriV-containing region of pMOS6, identified upstream of
the rep gene, contains a sequence (5’-gAACcTCTGTCTTG-3’)
with similarity to the IHF-box distinguished in Paracoccus
methylutens DM12 plasmid pMTH4 [19] and two identical 18-
bp iteron-like repeated sequences, separated by 3 bp (Figure 2
and Figure S1).
Toxin-antitoxin modules
Toxin-antitoxin modules confer plasmid stabilization in a
population by eliminating plasmid-less cells at the post
segregational level. Such genetic modules are composed of
two elements: (i) a toxin protein that binds a specific cellular
target and (ii) an antitoxin (protein or antisense RNA), which
counteracts the toxin. BLAST searches revealed that seven of
the analyzed plasmids (pAES4, pAES7, pMARC1, pMARC3,
pMARC4, pMOS6 and pMOS7) contain putative toxin-antitoxin
modules representing five TA groups: (i) relBE/parDE, (ii) tad-
ata, (iii) higBA, (iv) mazEF and (v) toxBA.
Plasmids pAES3 and pAES7 (P. aestuarii DSM 19484)
contain highly related TA modules (94% nucleotide sequence
identity) of the RelBE/parDE superfamily [20], composed of two
short overlapping ORFs (4 bp overlap). The first ORFs of the
predicted operons (orf6 of pAES3 and orf3 of pAES7) encode
putative proteins with significant similarity to a number of
plasmid-encoded antitoxins, classified within a large family of
transcriptional regulators containing a CopG/Arc/MetJ DNA-
binding domain (cluster of orthologous groups COG3609). The
highest similarity was to the TA module antitoxin of plasmid
pAMI3 of Paracoccus aminophilus JCM 7686 [10] (78 and 79%
aa sequence identity, respectively). The downstream ORFs
(orf5 of pAES3 and orf4 of pAES7) encode putative proteins
with substantial homology to toxins of the ParE family
(COG3668), with the highest similarity to the toxin of the
aforementioned pAMI3 TA module [10] (72 and 75% aa
sequence identity, respectively).
Interestingly, plasmid pAES7 also carries another TA module
(orf14-orf15) representing the tad-ata group, whose archetype
was identified in plasmid pAMI2 of Paracoccus aminophilus
JCM 7686 [21]. A related module is also present within plasmid
pMOS6 of P. marcusii OS22 (orf11-orf12) (Figure 1). The Tad-
related toxins are encoded by the first genes of the predicted
TA operons. They belong to a large family of proteins
(COG4679), exhibiting significant sequence similarity to the
RelE toxins (relBE-type TA modules), which act as mRNA-
cleaving RNAses [22]. Comparative sequence analysis of the
antitoxins of pAES7 and pMOS6 (encoded downstream of the
tad-homologs) revealed that these proteins belong to
COG5606 and COG1396, respectively, and they contain a
HTH motif typical of the Xre/Cro family.
An analogous genetic organization (toxin gene upstream of
the antitoxin gene) was also observed in the TA system of
pAES4 (P. aestuarii DSM 19484), classified within the higBA
family [23]. This TA module is composed of two ORFs (orf8
and orf9), separated by a 10-bp intergenic region, encoding
proteins with the highest level of aa identity to the killer
chromosomal protein of Rhodopseudomonas palustris CGA009
(accession no NP_947628) (68%) and the antitoxin of the Xre
family encoded by another strain of R. palustris – BisB18
(accession no YP_534586) (70%), respectively.
Plasmid pMARC4 of P. marcusii DSM 11574 carries two
overlapping (4 bp) ORFs: orf3 and orf4. The latter encodes a
predicted toxin with substantial similarity (63% aa sequence
identity) to the PemK-like protein of Chlorobium
phaeobacteroides BS1 (mazEF family of TA systems).
Interestingly, the orf3-encoded protein displays 65% identity to
a hypothetical protein of plasmid pDSHI01 of Dinoroseobacter
shibae DFL 12, and is quite different from typical MazE-type
antitoxins. BLAST searches revealed that gene pairs
homologous to orf3-orf4 are conserved in many bacterial
genomes (mainly in plasmids). Our analysis suggests that the
TA hybrid module of pMARC4 might be considered the
prototype of a new subgroup within the MazEF TA family.
Plasmids pMARC1, pMARC3 (P. marcusii DSM 11574) and
pMOS7 (P. marcusii OS22) contain related pairs of genes
(orf5-orf6, orf8-orf9, orf6-orf7, respectively), which represent a
novel group of TA modules, that we designate the toxAB family
(Figure 1). Homologous loci were distinguished by Leplae et al.
[24], but none of them were analyzed at the molecular level.
The first genes of the predicted TA operons encode DUF497
proteins (predicted toxin; ToxB), while the genes in the second
position were classified into the COG3680 (pMOS7 and
pMARC1) or COG3514 (pMARC3) orthologous groups
(putative antitoxins; ToxA). The predicted secondary structure
of the COG3680 and COG3514 antitoxins is highly conserved.
Proteins of both groups contain, in their C-terminal regions, a
RHH_1 domain related to a domain of the CcdA antitoxin of the
ccdAB TA system [25]. The most closely related TA module
was identified within Desulfomicrobium baculatum DSM 4028
(accession nos YP_003157266 and YP_003157267).
Restriction-modification module
One of the analyzed plasmids (pAES2 of P. aestuarii DSM
19484) contains a putative type II restriction-modification (R-M)
system (Figure 1). Similarly to TA, such systems may increase
the stability of plasmids by killing plasmid-less cells [26]. The
RM module of pAES2 is composed of overlapping orf3 and orf4
(1-bp overlap). The orf3 protein shares substantial similarity
with a large number of proteins annotated as m5C
methyltransferases (MTases) (PFAM: PF00145). The predicted
pAES2 MTase contains six (I, IV, VI, VIII, IX and X) of the ten
Plasmids of Carotenoid-Producing Paracoccus spp.
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amino acid sequence motifs (placed in conserved order)
characteristic of m5C MTases, including an invariant Pro-Cys
dipeptide in the catalytic motif IV [27] (data not shown). The
orf4-encoded protein is similar to restriction endonuclease
NgoMIV, which recognizes the sequence 5’-GCCGGC-3’. The
most closely related R-M module was identified in plasmid
pAOVO02 of Acidovorax sp. JS42 (accession nos YP_974088
and YP_974088) with 89% and 93% aa sequence identity of
the MTases and endonucleases, respectively.
Partitioning module
Partitioning systems (PAR), which allow proper segregation
of plasmid copies upon cell division, are components of the
vast majority of large low copy number plasmids. Only one of
the plasmids analyzed in this study (pMARC3 of P. marcusii
DSM 11574; 10,672 bp) contains a predicted PAR module of
typical structure, composed of two par genes (orf2 and orf3)
and a centromere-like partitioning site (Figure 1).
BLAST searches revealed that the deduced aa sequence of
Orf2 is similar over its entire length to a large number of
partitioning ATPases (ParA), with highest identity (44%) to the
putative ParA protein of plasmid pMRAD03 of the
alphaproteobacterium Methylobacterium radiotolerans JCM
2831 (accession no YP_001776801).
Detailed analysis of Orf2/ParA revealed the presence of a
sequence motif [KGGSGKS] matching the canonical sequence
[KGG(T/N/V)GKT] of a deviant Walker A motif, which is
characteristic for ATPases of type I partitioning modules [28].
The orf3-encoded polypeptide of pMARC3 (putative ParB)
displays only slight homology to a hypothetical protein of
Bacillus sp. 2_A_57_CT2 (accession no ZP_08007698) (30%
aa sequence identity), whose gene (as in pMARC3) is
associated with that encoding a partitioning ATPase.
The putative centromere-like site (parS) of the pMARC3 PAR
module is located within the promoter region of parA, which
consists of three non-identical 13-bp-long repeated sequences.
Taking into account its structure (i.e. the presence of a small
parB gene and the location of the parS site) and the results of
detailed comparative analyses, the predicted PAR module of
pMARC3 was classified into the Ib group of partitioning
systems [28].
Modules for mobilization for conjugal transfer
Many plasmids are capable of horizontal transfer by
conjugation. According to their transfer ability, they may be
grouped into two categories comprising self-transmissible and
mobilizable replicons. The latter grouping contains MOB DNA
regions, which carry genetic information essential for the
processing of conjugative DNA. The MOBs are usually
composed of two elements: an origin of transfer (oriT) and a
gene coding for relaxase, which nicks DNA at the oriT sites.
The transfer of mobilizable plasmids requires a membrane-
associated mating pair formation complex, which may be
provided by self-transmissible plasmids or integrative and
conjugative elements (ICE) co-residing in the cell [29].
None of the 14 Paracoccus spp. plasmids was a self-
transmissible replicon, but nine of them contained predicted
MOB modules (Figure 1). Based on comparative analysis of
the relaxase aa sequences, these proteins (and the MOB
modules encoding them) were classified within the MobQ, MobP
or MobV families [30].
MOBQ modules. The MOBQ family constitutes a diverse
group, comprising several subgroups (clades) [30]. The MOBs
of paracoccal plasmids represent the MOBQ1 (pHAE1) and
MOBQ3 (pAES7, pMARC3 and pMOS6) clades. The overall
genetic organization of the MOB modules and the conserved
sequence motifs of their relaxases are shown in Figure S2 in
the supplemental material.
BLAST searches revealed that plasmid pHAE1 (P.
haeundensis LMG P-21903) encodes a protein with 34% aa
sequence identity to the MobA relaxase of a broad-host-range
(BHR) plasmid RSF1010 – the archetype of the MOBQ family. A
putative origin of transfer was identified upstream of the pHAE1
mobA gene (5’-AAAtaCATAAGTGCGCCCTCCC-3’), showing
similarity to the MOBQ family oriT consensus sequence (5’-
NWACCNNTAAGTGCGCCCTYNN-3’) [31] (residues matching
the consensus are shown in uppercase). Closely related MOB
modules are encoded by several mobilizable plasmids,
including pAB6 of Neisseria meningitidis, pP of Salmonella
enteritidis and ColE2-P9 of E. coli [31].
The MOBs of pMARC3 (P. marcusii DSM 11574) and
pMOS6 (P. marcusii OS22) (MOBQ3 clade; Additional file 3:
Figure S2) are composed of two non-overlaping ORFs
encoding, respectively, MobA relaxase and mobilization protein
C (MobC), while the MOB of pAES7 is defective (it carries a
truncated mobA gene, lacking its proximal part). The pMARC3
and pMOS6 relaxases exhibit the highest aa sequence identity
(86%) to the MobA protein encoded by plasmid pAMI3 of
Paracoccus aminophilus JCM 7686 [10]. The predicted oriTs of
pMARC3 and pMOS6 were identified between divergently
oriented mobA and mobC genes. These sequences are nearly
identical (5’-ATAAGTGGGCACTTCGTGTCTTGCACCCTAt/
c-3’; non-conserved nucleotides are shown in lowercase) and
they show significant similarity to the putative oriT of pAMI3
[10]. Plasmid pAES7 does not contain related sequences.
MOBP modules. Two of the analyzed plasmids, pMARC2
(P. marcusii DSM 11574) and pAES4 (P. aestuarii DSM
19484), encode relaxases of the MOBP family, classified within
the MOBP5(MOBHEN) clade (Figure S2). MOBP encodes the
largest group of relaxases, which are closely related to those of
the MOBQ family. The prototype of the MOBP5(MOBHEN)
relaxases is protein MbeA of plasmid ColE1 [30].
The MOB modules of pMARC2 and pAES4 are composed of
three overlapping and convergently oriented ORFs: mobA
(encoding the relaxase), mobB and mobC (Figure 1). The
predicted MobA, MobB and MobC polypeptides exhibit
significant similarity to the corresponding proteins encoded by
plasmid pAsal2 (MOBP5 family) of Aeromonas salmonicida
subsp. salmonicida [30]. The predicted oriT sequences of
pMARC2 and pAES4 are placed upstream of the mobC gene
and are highly conserved – they differ in only 3 nucleotides (5’-
GGGGGATTGAAGGGGGCCAa/ca/ta/
gGCCCCCTCACAAGC-3’; non-conserved nucleotides are
shown in lowercase). A homologous DNA region (80% identity)
is also conserved in plasmid pAsal2 (accession no AJ508383;
nt position 590-621).
Plasmids of Carotenoid-Producing Paracoccus spp.
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MOBV modules. Plasmids pMARC4 (P. marcusii DSM
11574), pMOS2 (P. marcusii OS22) and pAES1 (P. aestuarii
DSM 19484) encode relaxases that are members of the MOBV
family (MOBV2 clade) (Figure S2). Most of the plasmids
encoding MOBV relaxases were identified in Firmicutes and
Bacteroidetes, with the exception of the MOBV2 clade, which
comprises plasmids of Proteobacteria and Cyanobacteria
(pBBR1 of Bordetella bronchiseptica is an archetype of the
group) [30]. Plasmids pMARC4, pMOS2 and pAES1 encode
single mobilization proteins (MobA), which share the highest
level of aa sequence identity (40 to 60%) with the MobA protein
encoded by the small cryptic plasmid pWKS1 of Paracoccus
pantotrophus DSM 11072 [12]. Significant aa sequence identity
(from 30 to 40%) with the pBBR1 relaxase was also observed
(Figure S2). In each case the oriT sites are situated upstream
of the mobA genes. The predicted oriTs of pMOS2
(AATTTGGaCgcagGaCAAATTGTCTAGTaAGTgcACATttttCTc
aaaT-3’) and pMARC4 are highly related (62% nucleotide
sequence identity; nucleotides conserved in pMARC4 are
shown in uppercase), while the oriT of pAES1 is more
divergent (nucleotides conserved in all three oriTs are
underlined).
Additional genetic load
Besides the REP, TA, PAR, R-M and MOB modules, the
analyzed plasmids contain diverse accessory genetic
information, including two insertion sequences (ISs) in pAES7
and pMARC3 (Figure 1). Plasmid pAES7 carries the functional
element ISPaes2 (IS427 group, IS5 family), which was
identified previously by its transposition into the trap plasmid
pMAT1 [5]. ISPaes2 is bordered by dinucleotide (TA) direct
repeats (DR), which represent the duplicated target site of
transposition.
The IS of pMARC3 is a novel element that we have
designated as ISPmar4 (1343 bp). It contains 12-bp-long
imperfect terminal IR sequences (5'-ATGGc/tCCGCCCC-3')
and carries a single ORF (orf5), encoding a predicted protein
with similarity to transposases of the IS110 family (IS1111
group). ISPmar4 is not flanked by DRs in the pMARC3
genome, which is a typical feature of members of the IS110
family.
An intriguing feature of all but one of the plasmids (pMARC3)
is the presence of related ORFs (orf2), placed downstream of
the predicted rep genes (Figure 1). These ORFs encode
putative proteins (Orf2) with low, but significant similarity [at
least 30% aa identity (E value 1e-04)] to entry exclusion-like
proteins 1 (Exc1). All of these predicted proteins contain a
conserved helix-turn-helix domain (HTH_17) in their N-terminal
region (Figure S3 in the supplemental material).
Entry exclusion (EEX) systems prevent the entry of
exogenous plasmids into a host cell carrying an identical or
related EEX system. It is thought that EEX is a specific feature
of all conjugative plasmids [32]. The exclusion phenotype has
been observed for mobilizable plasmid ColE1, whose exc1 and
exc2 genes were predicted to encode the EEX system [33].
Although subsequent studies [34] excluded the possibility of
exc1 and exc2 involvement in plasmid exclusion, homologous
genes are still being annotated and described in the literature
as entry exclusion components, e.g. [35].
The examined plasmids of Paracoccus spp. were found to
contain only one of these genes – exc1. This is not a unique
feature, since related “orphan” exc1-like genes have been
identified in several other plasmids, including pKlebB-K17/80 of
Klebsiella pneumoniae [36] and pMWHK1 of Pedobacter
cryoconitis BG5 [35]. The conserved position of this gene,
accompanying different REP modules, strongly suggests that
the Exc1 proteins may play an important role in the biology of
these plasmids. However, their specific function has yet to be
determined.
Downstream of the exc1 genes in several plasmids (pAES3,
pAES4, pHAE1, pHAE2, pMARC1, pMARC2, pMOS2 and
pMOS7), putative plasmids islets (PI) were identified, i.e.
horizontally-acquired DNA regions of lower than average GC
content (Figure 1). The ORFs encoded within these PI (listed in
Table 2) show similarity to genes (mainly of unknown function)
conserved in the chromosomes of many bacteria.
Table 2. Plasmid islets (PI) identified in Paracoccus spp.
plasmids.
GC content (%)
Plasmid
PI size (bp)
(position) PI
Remaining
part of the
plasmid ORF (aa)
Hypothetical
function
pAES3 2273
(1509-3781) 42.3 58.5 orf3
(374) Unknown
orf4 (93) Unknown
pAES4 2218
(1303-3521) 52.3 62.4
pHAE1 1142
(1400-2542) 47.7 61.8 orf3 (87) Unknown
pHAE2 2695
(1365-4059) 46.5 59.8 orf3
(151) Unknown
orf4
(473) Unknown
pMARC1 1831
(1312-3143) 35.7 55.1 orf3
(131) Unknown
orf4
(389)
GTP
pyrophosphokinase
(RelA/SpoT domain-
containing protein)
pMARC2 2218
(1303-3521) 39.5 57.7 orf3
(415)
ATPase (SMC
domain-containing
protein)
orf4
(211) Unknown
pMOS2 1949
(1042-2991) 43.4 58.6 orf3
(113) Unknown
pMOS7 2425
(1329-3754) 38.5 57.8 orf3
(113) Unknown
orf4
(314)
Reverse transcriptase
(retron-like)
doi: 10.1371/journal.pone.0080258.t002
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The significant role of PI in shaping plasmid genomes was
revealed by comparative analysis of the related plasmids
pMARC1 and pMOS7 of P. marcusii (strains DSM 11574 and
OS22, respectively). These plasmids have highly conserved
backbones (REP and TA modules showing approx. 86%
nucleotide sequence identity), but they contain different PIs
(Figure 3). Plasmids pMARC2 and pHAE4 represent an
analogous pair of related replicons containing different PIs
(Figure 1). The PI of pMOS7 carries two ORFs, including orf4
encoding a predicted retron-type reverse transcriptase, while
that of pMARC1 is composed of orf3, encoding a putative
DUF805 transmembrane protein and orf4, encoding a protein
containing a conserved domain of RelA and SpoT proteins
(both proteins are involved in the metabolism of the regulatory
compound guanosine 3',5'-bis-pyrophosphate, ppGpp, which
plays a crucial role in the bacterial stringent response).
The PI of pMARC2, another plasmid of P. marcusii (strain
DSM 11574), contains a pair of overlapping genes (1-bp
overlap), orf3 and orf4, conserved (in synteny) in several
bacterial chromosomes. The first gene of this predicted module
encodes a putative ATPase with significant similarity to the
SMC proteins, which play an important role in chromosome
condensation, packaging, partitioning and DNA repair [37]. The
downstream orf4 encodes a hypothetical protein of unknown
function. The closest homologs of orf3 and orf4 were identified
within the chromosome of Pedobacter heparinus DSM 2366
(accession nos YP_003093112 and YP_003093113,
respectively).
The largest of the plasmids analyzed in this study, pMARC4,
does not contain a PI, but it does encode proteins possibly
involved in carbohydrate metabolism. The predicted proteins
show significant similarity to (i-ii) acyltransferases (Orf5 and
Orf7), (iii) polysaccharide biosynthesis protein (Orf10), (iv)
phosphoribosyltransferase (Orf11), (v) dolichyl-phosphate
mannose synthase (Orf12), and (vi) glycosyltransferase
(Orf13). Two other ORFs (orf6 and orf14) encode a putative
undecaprenyl-diphosphate phosphatase and UDP-glucose 6-
dehydrogenase (EC 1.1.1.22), respectively. It has been
demonstrated that Orf6 homologs are involved in the synthesis
and recycling of undecaprenyl phosphate (Und-P), a lipid
carrier of glycan biosynthetic intermediates of carbohydrate
polymers exported to the bacterial cell envelope [38], while
Orf14 relatives are responsible for the NAD-dependent
oxidation of UDP-glucose to UDP-glucuronic acid, a key
component in the biosynthesis of gellan (extracellular
polysaccharide of biotechnological value) [39]. Based on these
similarities, it is likely that the pMARC4-encoded gene cluster
may be involved in the biosynthesis of envelope-associated
polysaccharides.
Another plasmid, pMOS6 of P. marcusii OS22, besides
several ORFs of unknown function, carries orf4 encoding a
putative zinc-dependent alcohol dehydrogenase (ADH_ZINC),
Figure 3. Comparison of the structure and G+C sequence profile of P. marcusii plasmids pMARC1 and pMOS7. Arrows
show the transcriptional orientation of the genes and the color code indicates their predicted functions (as shown in Figure 1).
Shaded areas connect homologous DNA regions. The PI regions (with indicated G+C content) are marked by yellow rectangles and
dashed lines. The plot shows the G+C content of pMARC1 and pMOS7 sequences (the average values are given to the right).
doi: 10.1371/journal.pone.0080258.g003
Plasmids of Carotenoid-Producing Paracoccus spp.
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containing a conserved signature sequence: G-H-E-x(2)-G-
x(5)-[GA]-x(2)-[IVSAC] (where x indicates any amino acid; H is
a zinc ligand) [40]. Related enzymes catalyze the oxidation of
alcohols, with the concomitant reduction of nicotinamide
adenine dinucleotide (NAD) [41]. The most similar alcohol
dehydrogenase (99% aa sequence identity) is encoded by
Methylobacterium chloromethanicum CM4 (accession no
YP_002424246).
Distribution of related plasmids in genus Paracoccus
We analyzed the distribution of the replication modules of the
pAES, pHAE, pMARC and pMOS plasmids in the genomes of
20 strains representing 19 species of Paracoccus spp. To do
this, a specific DNA probe for each plasmid (rep gene fragment
amplified by PCR and DIG-labeled) was used in dot blot
hybridization to screen total DNAs isolated from the paracoccal
strains.
This analysis revealed that the majority of the analyzed
plasmids occur exclusively in the multireplicon carotenoid-
producing species (LMG P-21093, OS22, DSM 11574, DSM
19484), although none of the replicons was present in all four
strains (Figure 4). The strains P. haeundaensis LMG P-21093
and P. aestuarii DSM 19484 displayed an almost identical
hybridization pattern (7 common replicons), which was also
similar to that of P. marcusii OS22 (4 common plasmids). In
contrast, the hybridization pattern of two strains of P. marcusii
(OS22 and DSM 11574) was significantly different, indicating
the presence of four strain-specific replicons (pMARC2,
pMARC3, pMARC4 and pMOS2), which were unique among
Paracoccus spp. (Figure 4).
A few related plasmids were also detected in other species,
located on distant branches of the phylogenetic tree, which
points to the role of horizontal gene transfer in the
dissemination of these replicons. The most ubiquitous replicons
were plasmids related to pAES4, which were detected in 6
strains, including P. zeaxanthinifaciens ATCC 21588T, P.
thiocyanatus JCM 20756 and P. yeei CCUG 46822 (Figure 4).
Host range of the Paracoccus spp. plasmids
The host range of the paracoccal plasmids in several
bacterial strains belonging to the Alpha-, Beta- or
Gammaproteobacteria was examined. For this analysis we
employed REP regions representing different groups of
plasmids (Figure 2): (i-ii) pAES1 and pMOS7, (iii) pAES7, (iv)
pMARC2 and (v) pMOS6. The REPs were cloned within the
multiple cloning site (MCS) of the mobilizable vector pABW1
(Kmr, oriV ColE1, oriT RK2) (see Methods for details) and the
resulting plasmids were introduced into 9 strains of
Alphaproteobacteria (Rifr derivatives of Paracoccus spp. strains
P. alcaliphilus JCM 7364, P. aminophilus JCM 7686, P.
aminovorans JCM 7685, P. kondratievae NCIMB 13773, P.
pantotrophus DSM 11072, P. versutus UW225, as well as
Rhizobium etli CE3 and Ochrobactrum sp. LM19R, one strain
of Betaproteobacteria (Alcaligenes sp. LM16R) and two strains
of Gammaproteobacteria (Pseudomonas sp. LM7R and E. coli
BR825
The constructed shuttle plasmids contained an E. coli-
specific pMB1 (ColE1-type) replication system, which is
functional in neither Alpha- and Betaproteobacteria nor in
Pseudomonas sp. LM7R and E. coli BR825
(Gammaproteobacteria; the BR825 strain carries a mutation
within the DNA polymerase I gene that blocks pMB1
replication). Therefore, the functions required for replication
and maintenance of these plasmids in the tested hosts have to
be provided by the paracoccal REP modules. It is important to
mention that the Paracoccus spp. strains, in which the
plasmids were tested for their ability to replicate, were not the
original hosts of any of these plasmids, and most of these
strains did not render a positive hybridization with the REP
probes of such plasmids (Figure 4).
All of the shuttle plasmids were found to replicate exclusively
in strains of Alphaproteobacteria, which indicates the relatively
narrow host range of the tested REP modules. This is in
agreement with the results of our previous studies, which
showed that the plasmids of Paracoccus spp. are not
promiscuous (e.g. [10]).
Members of the genus Paracoccus are not naturally
competent for transformation, therefore conjugal transfer is the
only efficient way of introducing of foreign DNA into their cells.
Unfortunately, conjugative plasmids (or ICE elements) have not
yet been identified in these bacteria. We also do not know
whether such replicons are present in the natural host strains
of the plasmids analyzed in this study (P. aestuarii, P.
haeundensis and P. marcusii). However all of them (as well as
many other Paracoccus spp. and numerous
Alphaproteobacteria) carry mega-sized replicons, some of
which could be self-transmissible.
Until now, the complete nucleotide sequences of only three
Paracoccus spp. genomes (with defined physical maps of
chromosomes and plasmids) have been deposited in the NCBI
database (P. denitrificans PD1222, P. aminophilus JCM 7686
and Paracoccus sp. N5). Our detailed in silico analysis
revealed that each of the strains carries one replicon
(chromosome II, megaplasmid pAMI8 and a replicon referred to
as contig 3, respectively) containing a complete predicted type
IV secretion system, with a potential to support the conjugal
transfer of mobilizable plasmids. Plasmid mobilization might be
therefore a frequent phenomenon in Paracoccus spp.,
especially when the fact that the majority of the plasmids
characterized in this study (9 replicons) contained the MOB
modules is taken into account.
Vector cassette construction
To facilitate genetic manipulation of the carotenoid-producing
strains of Paracoccus spp. two vector cassettes were
constructed (see Methods for details). We used REP regions of
plasmids pMOS6 (P. marcusii OS22) and pAES7 (P. aestuarii
DSM 19484), which were free of the majority of restriction sites
commonly found in the MCSs of many cloning vectors. The
construction of these cassettes was performed according to the
general scheme used for the generation of the DIY (Do It
Yourself) cassettes, described in our previous study [10].
The cassettes contain (i) the REP regions, (ii) a kanamycin
resistance gene, providing a selectable marker convenient for
Paracoccus spp. (and other Alphaproteobacteria), and (iii) a
MOB module, which enables conjugal transfer of the plasmids
Plasmids of Carotenoid-Producing Paracoccus spp.
PLOS ONE | www.plosone.org 10 November 2013 | Volume 8 | Issue 11 | e80258
Figure 4. Distribution of the REP modules analyzed in this study in the Paracoccus spp. genomes. A specific DNA probe
(fragment of a rep gene amplified by PCR and DIG-labeled) was prepared for each analyzed REP module and used in dot blot
hybridization analysis with total DNA isolated from 20 strains of Paracoccus spp. The results are presented as a matrix. The
relatedness of the tested Paracoccus strains is shown beneath by a phylogenetic tree based on their 16S rDNA sequences. The
tree was constructed by the neighbor-joining algorithm with Kimura corrected distances. The statistical support for the internal nodes
was determined by 1000 bootstrap replicates and values of >50% are shown. The Paracoccus strains from which the plasmids were
isolated are denoted by red text.
doi: 10.1371/journal.pone.0080258.g004
Plasmids of Carotenoid-Producing Paracoccus spp.
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in the presence of a helper, functional transfer system (the
MOB module originated from BHR conjugative plasmid RK2,
commonly used in the construction of vectors for gram-
negative bacteria). Both cassettes, designated DIYpMOS6 and
DIYpAES7, also contain polylinkers with a number of restriction
sites to facilitate their insertion into different locations (plasmids
pKRP-DYIpMOS6 and pKRP-DIYpAES7; Figure 5). The pKRP-DIY
plasmids are not cloning vectors and they serve exclusively as
a source of the DIY cassettes. Insertion of a single cassette
into any E. coli plasmid can create a mobilizable shuttle vector.
The cassettes were used to construct two mobilizable E. coli-
Paracoccus spp. shuttle vectors pVIV6 and pVIV7, whose
genetic organization is shown in Figure 5. These vectors also
carry a MluI restriction fragment of E. coli-specific plasmid
pBGS18 containing (i) a replication system originating from
pMB1 (non functional in Paracoccus spp.) and (ii) a selection
cassette (MCS and the lacZ’ gene) enabling the identification of
recombinant molecules by "blue-white" screening. These
shuttle vectors proved to be good cloning vectors with specific
features determined by the DIY cassettes (data not shown).
There are also available some other cloning vectors based
on the REP modules of Paracoccus spp. plasmids. They
contain replication systems of pTAV1 (P. versutus UW1) [42] or
pAMI plasmids of P. aminophilus JCM 7686 [10]. Although all
of them are functional in Alphaproteobacteria, their stability
significantly varies in different hosts [10]. Therefore there is a
need to enrich the pool of such vectors, which will enable
selection of the most convenient one for a given host and task.
Figure 5. The plasmids containing the DIY cassettes constructed in this study. A. pKRP-DIY plasmids. B. pVIV mobilizable
shuttle vectors. The plasmids contain DIYAES7 and DIYMOS6 cassettes composed of REP modules (of plasmids pAES7 and pMOS6,
respectively), a Kmr gene and a MOB module derived from BHR plasmid RK2. B – BamHI, E – EcoRI, H – HindIII, K – KpnI, P –
PstI, Sc – SacI, Sh – SphI, Sl – SalI, Sm – SmaI, X – XbaI.
doi: 10.1371/journal.pone.0080258.g005
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Conclusions
The findings of this study provide a molecular insight into the
genomes of a pool of small plasmids occurring in four
carotenoid-producing strains of the genus Paracoccus
(Alphaproteobacteria). Three of these strains (P. haeundaensis
LG P-21903, P. marcusii OS22 and patented P. marcusii DSM
11574) synthesize astaxantin, which is a commercially
produced carotenoid used in a range of industrial applications.
The DIY cassettes and shuttle vectors constructed in this study
may facilitate further genetic analysis of these
biotechnologically important bacteria.
Until now, small alphaproteobacterial plasmids have been
neglected by researchers, e.g. complete nucleotide sequences
of only four such plasmids of Paracoccus spp. were available -
pWKS1 of P. pantotrophus DSM 11072 [12] and pAMI2,
pAMI3, pAMI7 of P. aminophilus JCM 7686 [10,11]. Interest
has almost exclusively been focused on mega-sized replicons,
which appear to be less diverse than the plasmids
characterized in this study. Comparative analysis has revealed
that the plasticity and diversity of Paracoccus spp. plasmids
result from (i) recombinational shuffling of genetic modules of
the plasmid backbones, (ii) insertion of exogenous foreign
DNA, including commonly identified plasmid islets (PIs), as well
as (iii) the acquisition of novel replicons. Our detailed
description of the genetic content of these plasmids allows
prediction of the possible origin of individual genes (or sets of
genes) and the direction of horizontal gene flow in this group of
bacteria.
Some of the plasmids characterized in this study (pAES1,
pAES2, pAES3, pEAS4, pHAE1 and pHAE2).carry replication
systems which occur exclusively in the phylogenetically closely
related orange-pigmented strains of Paracoccus spp. (Figure
4). This suggests that their ancestor replicons might have been
acquired a long time ago (from an evolutionary point of view)
by a progenitor strain. In contrast, a few plasmids (pMARC2,
pMARC3, pMARC4, pMOS2), present only in single strains,
are unique among Paracoccus spp., which suggests their
relatively recent acquisition. Interestingly, two of the
aforementioned replicons (pAES4 and pMARC2) encode
related Rep proteins, which may be considered as archetypes
of a novel group of plasmid replication initiators.
Analysis of plasmid host range strongly suggests that the
Alphaproteobacteria is a kind of “isolated island”, since all
plasmids identified so far in these bacteria (including those
analyzed in this study) are narrow-host-range class-specific
replicons. They do not replicate in Beta- or
Gammaproteobacteria, and vice versa, foreign plasmids (with
the exception of broad host range replicons) do not replicate in
alphaproteobacterial hosts. This isolation seems to be the main
factor limiting plasmid diversity in Alphaproteobacteria. These
observations also suggest that Alphaproteobacteria may
encode as yet unidentified host-specific factors that are crucial
for the maintenance of certain types of plasmids. The
identification of such factors is an immediate goal of our future
studies.
Materials and Methods
Bacterial strains and culture conditions
The following strains of the genus Paracoccus were used in
this study: (i) P. aestuarii [43], (ii) P. haeundaensis LG P-21903
[44], (iii) P. marcusii [45], (iv) P. marcusii OS22 [46] as well as
(v-xviii) P. alcaliphilus JCM 7364 [47], P. aminophilus JCM
7686 [48], P. aminovorans JCM 7685 [48], P. halophilus JCM
14014 [49], P. homiensis DSM 17862 [50], P. kondratievae
NCIMB 13773 [51], P. koreensis JCM 21670 [52], P.
methylutens DM12 [53], P. pantotrophus DSM 11072 [54], P.
pantotrophus KL100 [6], P. seriniphilus DSM 14827 [55], P.
solventivorans DSM 11592 [56], P. sulfuroxidans DSM 14013
[57], P. thiocyanatus JCM 7364 [58], P. versutus UW1 [59], P.
versutus UW225 [60], P. yeei CCUG 46822 [61] and P.
zeaxanthinifaciens ATCC 21588 [62]. Other strains used in this
study were (i) Rhizobium etli CE3 [63], Ochrobactrum sp.
LM19R [10], (Alphaproteobacteria) (ii) Alcaligenes sp. LM16R
[10] (Betaproteobacteria), and (iii) Eschericha coli – strains
TG1 [64], BR825 [65] and Pseudomonas sp. LM7R [66]
(Gammaproteobacteria). As recipients in triparental matings,
rifampicin-resistant (Rifr) derivatives of the wild-type strains
were used. Bacteria were grown in LB (Luria Bertani) medium
[67], at 37°C (E. coli) or 30°C (other strains). P. homiensis
DSM 17862 was cultivated in Marine Broth (Difco) and R. etli
CE3 in TY medium [68]. Where necessary, the medium was
supplemented with kanamycin (50 μg/ml), streptomycin (50
μg/ml) and rifampicin (50 μg/ml).
Plasmids used and constructed in this study
The following plasmid vectors were used: (i) pABW1 (Kmr; ori
pMB1; oriT RK2; MCS-lacZ') [69], pBGS18 (Kmr; ori pMB1;
MCS) [70], pBluescript KSII (Apr; ori pMB1; MCS-lacZ') [71],
pGEM-T Easy (Promega), pKRP12 (Apr; Kmr; ori pMB1) [72],
pDIY-KM (Apr; Kmr; ori pMB1) [10]. Plasmids constructed in
this study were (i-v) pABW-AES1, pABW-AES7, pABW-
MARC2, pABW-MOS6 and pABW-MOS7 – shuttle plasmids
used for host range analysis, (vi-ix) pKRP-DIYAES7, pKRP-
DIYMOS6, pBS-DIYAES7 and pBS-DIYMOS6 – containing DIY
cassettes, and (x-xi) shuttle vectors pVIV6 and pVIV7.
Shuttle plasmids pABW-AES1, pABW-AES7, pABW-
MARC2, pABW-MOS6 and pABW-MOS7 (containing REP
regions of plasmids pAES1, pAES7, pMARC2, pMOS6 and
pMOS7, respectively) were constructed by cloning of REP-
containing plasmid restriction fragments (pAES7, pMARC2,
pMOS6 and pMOS7) or DNA fragment amplified by PCR
(pAES1; primers used are listed in Table S2 in the
supplemental material) into the MCS of mobilizable vector
pABW1.
The plasmids representing the source of the DIY cassettes
were constructed in several steps. First, the REP modules of
pAES7 and pMOS6 were amplified by PCR (pMOS6; primers
listed in Table S2) or recovered within 1.9 kb SacI-NotI
restriction fragment (pAES7) and cloned into vector pGEM-T
Easy. These modules were then excised from the recombinant
plasmids with restriction endonuclease NotI and cloned into
compatible sites of plasmid pBS-MOBKm (pBluescript SKII
containing the Kmr cassette from pMBS1 and the BHR plasmid
Plasmids of Carotenoid-Producing Paracoccus spp.
PLOS ONE | www.plosone.org 13 November 2013 | Volume 8 | Issue 11 | e80258
RK2 MOB module within its MCS). The REP-Kmr-MOB
cassettes were excised from the recombinant plasmids and
inserted (i) between HindIII sites of plasmid pKRP12 (HindIII
digestion of pKRP12 removed the original resistance gene
cassette of this plasmid), yielding plasmids pKRP-DIYAES7 and
pKRP-DIYMOS6, or (ii) cloned into the MCS of Bluescript SKII,
yielding plasmids pBS-DIYAES7 and pBS-DIYMOS6.
The mobilizable E. coli-Paracoccus spp. shuttle plasmids
pVIV6 and pVIV7 were constructed by ligation of the DIYpAES7 or
DIYpMOS6 cassettes (excised from pBS-DIYAES7 and pBS-
DIYMOS6, respectively) with MluI-cleaved pBGS18.
Plasmid DNA isolation, genetic manipulations and PCR
conditions
Plasmid DNA was isolated using a standard alkaline lysis
procedure [73] and when required, purified by CsCl-ethidium
bromide density gradient centrifugation. Total DNA was
isolated from Paracoccus spp. using the procedure described
by Chen and Kuo [74]. Southern hybridization analysis and
common DNA manipulation methods were performed
according to Sambrook and Russell [67]. Oligonucleotides
used to generate molecular probes are listed in Table S2. PCR
was performed in a Mastercycler (Eppendorf) using HiFi
polymerase (Qiagen; with supplied buffer), dNTP mixture and
total DNA of Paracoccus spp. with appropriate primer pairs
(listed in Table S2).
Introduction of plasmid DNA into bacterial cells and
plasmid stability assay
DNA was introduced into Rifr (Alcaligenes sp. LM16R,
Ochrobactrum sp. LM19R, Paracoccus spp. and Pseudomonas
sp. LM7R) or Strr (R. etli CE3) recipient strains by triparental
mating as previously described [42]. Chemical transformation
of E. coli cells was performed according to the method of
Kushner [75]. The stability of plasmids was tested during
growth under non-selective conditions. Stationary-phase
cultures of plasmid-containing strains were diluted in fresh
medium without antibiotic selection and cultivated for
approximately 10, 20 and 30 generations. Samples were
diluted and plated onto solid medium lacking selective
antibiotics. One hundred colonies were tested for the presence
of the Kmr marker by replica plating. Plasmid retention was
determined from the percentage of kanamycin-resistant
colonies.
DNA sequencing
The nucleotide sequences of plasmids pMARC and pMOS
were determined in the DNA Sequencing and Oligonucleotide
Synthesis Laboratory at the Institute of Biochemistry and
Biophysics, Polish Academy of Sciences, using a dye
terminator sequencing kit and an automated sequencer (ABI
377 Perkin Elmer). Primer walking was used to complete the
sequences. In the case of pMARC2, pMARC3 and pMOS2,
unidirectional nested deletions within the cloned plasmid
restriction fragments were generated by the use of
exonuclease III and S1 nuclease (ExoIII/S1 deletion kit; MBI
Fermentas). The nucleotide sequences of plasmids pAES and
pHAE were determined by pyrosequencing performed by
Genomed.
Bioinformatic analysis
Plasmid nucleotide sequences were analyzed using Clone
Manager (Sci-Ed8) and Artemis software [76]. Similarity
searches were performed using the BLAST programs [77]
provided by the National Center for Biotechnology Information
(NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Comparison
searches of insertion sequences were performed with ISfinder
[78]. Protein homology detection and structure prediction were
performed using the HHpred program [79]. Protein families
were identified using the PFAM database [80]. Helix-turn-helix
motifs were predicted using the HELIX-TURN-HELIX MOTIF
PREDICTION program [81]. Phylogenetic analyses were
performed using the Phylogeny Inference Package – PHYLIP
v3.69 [82], applying the neighbor-joining algorithm with Kimura
corrected distances and 1000 bootstrap replicates. DNA
sequence alignments obtained using ClustalW [83] were
manually refined using the T-Coffee Multiple Sequence
Alignment program [84]. The tree was rendered with TreeView
version 1.6.6 [85].
Nucleotide sequence accession numbers
Plasmid nucleotide sequences have been annotated and
deposited in the GenBank database (accession numbers are
given in parenthesis): pAES1 (JQ041633), pAES2 (JQ065021),
pAES3 (JQ066766), pAES4 (JQ684025), pAES7 (JQ796370),
pHAE1 (JQ066767), pHAE2 (JQ684024), pMARC1
(KC542384), pMARC2 (KC561053), pMARC3 (KC561054),
pMARC4 (KC561055), pMOS2 (JQ664550), pMOS6
(JQ678602) and pMOS7 (JQ684023).
Supporting Information
Figure S1. Nucleotide sequence of DNA regions
containing the predicted origin of replication of the
Paracoccus spp. plasmids analyzed in this study. Iterons
(DRs) are shown against orange background, while DnaA-
boxes and IHF-box have violet and blue backgrounds,
respectively. Inverted, repeated sequences are indicated by
blue arrows. Predicted -35 and -10 promoter sequences are
indicated by black frame.
(TIF)
Figure S2. Comparison of sequence motifs identified in
relaxases encoded within MOB modules of the Paracoccus
spp. plasmids analyzed in this study. The conserved motifs
identified within the relaxase (MobA) proteins of analyzed
plasmids were present in a form of alignments. Conserved
amino acids, characteristic for each motif (according to Francia
et al. [31]; Garcillan-Barcia et al. [30]), were shown against the
blue background. Other conserved amino acids common in
more than 50% of analyzed sequences are shown against
black background, and those common in less than 50% have
gray background. For the alignments additional MobA
sequences of various mobilization plasmids, classified into
Plasmids of Carotenoid-Producing Paracoccus spp.
PLOS ONE | www.plosone.org 14 November 2013 | Volume 8 | Issue 11 | e80258
appropriate category (according Francia et al. [31]; Garcillan-
Barcia et al. [30]) were used.
(TIF)
Figure S3. Multiple alignment of amino acid sequences of
Exc1-like proteins encoded by Paracoccus spp. plasmids
analyzed in this study. For the alignment the Exc1-like
proteins of the following plasmids were used: pMARC1,
pMARC2, pMARC4 of P. marcusii DSM 11574, pMOS2,
pMOS4, pMOS7 of P. marcusii OS22, pAES2, pAES3, pAES4,
pAES7 of P. aestuarii DSM 19484, pHAE1, pHAE2 of P.
heaundaensis LMG P-21903, pAMI3 of P. aminophilus JCM
7686 (YP_003305342), pSX-Qyy of Sphingobium xenophagum
QYY (sequence distinguished in this work), pYAN-1 of
Sphingobium yanoikuyae JCM 7371 (sequence distinguished
in this work), pUT2 of Sphingobium japonicum UT26S
(YP_003550321), as well as protein sequence annotated within
a contig of an unfinished genomic project of Sulfitobacter sp.
NAS-14.1 (ZP_00964870). Amino acids identical in at least
50% of the analyzed sequences are shown against a black
background, while those common to at least 15% of the
analyzed sequences have a gray background. The HTH motifs
were distinguished by blue frame.
(TIF)
Table S1. ORFs located within the Paracoccus spp.
plasmids analyzed in this study.
(DOC)
Table S2. Oligonucleotide primers used in this study.
(DOCX)
Acknowledgements
We acknowledge E. Piechucka for technical assistance. We
also acknowledge J.-W. Bae for providing P. aestuarii DSM
19484, J. Hirschberg for providing P. marcusii DSM 11574, A.
Sklodowska and L. Drewniak for providing P. marcusii OS22.
Author Contributions
Conceived and designed the experiments: MW JB DB.
Performed the experiments: AM LD JC GS DG. Analyzed the
data: AM LD DB. Contributed reagents/materials/analysis tools:
DB. Wrote the manuscript: DB.
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